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10.5114/aoms.2015.53289
|
Systematic review/Meta-analysis The role of gender in patients with diffuse large B cell lymphoma treated with rituximab-containing regimens: a meta-analysis
|
Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL). Although gender has not been included in prognostic systems, male gender has been found as a bad prognostic indicator in Hodgkin lymphoma, follicular lymphoma and chronic lymphocytic leukemia. The relationship between gender and prognosis is not clear in patients with DLBCL treated with rituximab-containing regimens. The aim of this meta-analysis is to determine the prognostic/predictive role of gender in patients with DLBCL treated with rituximab-containing regimens.We systematically searched for studies investigating the relationships between gender and prognosis in DLBCL treated with rituximab-containing regimens. After careful review, survival data were extracted from eligible studies. A meta-analysis was performed to generate combined hazard ratios for overall survival, disease-free survival (DFS) and event-free survival (EFS).A total of 5635 patients from 20 studies were included in the analysis. Our results showed that male gender was associated with poor prognosis in terms of overall survival (OS) (hazard ratio (HR) = 1.155; 95% confidence interval (CI): 1.037-1.286; p < 0.009). The pooled hazard ratio for DFS and EFS showed that male gender was not statistically significant (HR = 1.219; 95% CI: 0.782-1.899; p = 0.382, HR = 0.809; 95% CI: 0.577-1.133; p = 0.217).The present meta-analysis indicated male gender to be associated with a poor prognosis in patients with DLBCL treated with rituximab-containing regimens.
|
[
{
"section_content": "age, performance score, stage, proliferation fraction and gene expression profiles [3, 4]. Today, the International Prognostic Index (IPI) and age-adjusted International Prognostic Index (aaIPI) are the most important scores used in daily practice to determine the prognosis and treatment strategies. The IPI scoring system includes age, performance status, serum lactate dehydrogenase (LDH) level, stage and extranodal involvement; the aaIPI includes stage, LDH, performance status and age older than 60 years. \n\nIn the last decade, the standard of care in patients with DLBCL has been the addition of anti-CD20 antibody-rituximab to classic cytotoxic chemotherapy. More sophisticated methods and drugs targeting oncogenic pathways and gene expression profiles have been used to predict the prognosis and response to therapy in recent years [5] [6] [7]. Although gender has not been included in prognostic systems, male gender has been found to be a bad prognostic indicator in Hodgkin lymphoma, follicular lymphoma and chronic lymphocytic leukemia [8] [9] [10]. However, the prognostic significance of gender has not been shown in all studies [11, 12]. \n\nThe aim of this meta-analysis was to determine the prognostic/predictive role of gender in patients with DLBCL treated with rituximab-containing regimens.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Material and methods",
"section_num": null
},
{
"section_content": "A computer-based literature search using the PubMed/Medline database was performed by two independent researchers (VK, OD). The initial PubMed search using the combined term (rituximab) and (lymphoma, large B-cell, diffuse) resulted in 1520 returns up to August 7, 2013. Only English language and human studies were included in this analysis. Full text articles of all selected studies were retrieved. If a paper was selected for inclusion, the bibliographic references were carefully investigated to look for additional studies.",
"section_name": "Research strategy",
"section_num": null
},
{
"section_content": "Prospective and retrospective randomized controlled studies involved patients older than 18 years who were treated according to rituximab-containing regimens. Case reports and series, letters, commentaries, lymphoma series including those other than DLBCLs and studies not containing an effect size for survival according to gender were not included.",
"section_name": "Inclusion and exclusion criteria",
"section_num": null
},
{
"section_content": "Two independent reviewers decided which studies to include (MY, SP). The abstracts of all papers found to be appropriate for meta-analysis were read. The full texts of the candidate papers for this analysis were evaluated. Patients with DLBCL treated with rituximab-containing regimens were included in this analysis. If the patients had been included in different studies by the same author, the higher quality study was considered for this meta-analysis.",
"section_name": "Selection of studies",
"section_num": null
},
{
"section_content": "The Newcastle-Ottawa Quality Assessment Scale was used for the evaluation of non-randomized controlled studies and the Jadad scoring system was used for the evaluation of randomized controlled studies by two independent reviewers (MY, VK). The Newcastle-Ottawa Quality Assessment Scale is used to determine the choice of patient population, comparability, follow-up and results of the studies. For these criteria studies are scored with stars between 0 and 9. A score of nine stars indicates the highest quality [13]. The Jadad scoring system is based on 5 stars [14]. Discrepancies between the authors after evaluations were re-evaluated and consensus was reached for all data.",
"section_name": "Assessment of study quality",
"section_num": null
},
{
"section_content": "For studies evaluating gender and rituximabcontaining regimens in cases of NHL treated with rituximab-containing regimens, the following variables were extracted: essential data about study, author, publication year, country of study, design of the study, demographic data, gender distribution, treatment schedules, stage, and effect size of gender on the overall survival (OS), disease-free survival (DFS) and event-free survival (EFS).",
"section_name": "Data extraction",
"section_num": null
},
{
"section_content": "The primary aim of this study was to analyze the effect of gender on the OS in patients with DL-BCL treated with rituximab-containing regimens. Disease-free survival and EFS were also analyzed. Hazard ratio (HR) was calculated with a 95% confidence interval (CI). Hr > 1 and not including 95% coincidence interval 1 were considered as significant. If there was no HR, summary statistics were used. Homogeneity was evaluated using the χ 2 -based test of homogeneity and inconsistency index. A p-value < 0. 10 for χ 2 or I 2 > 50% was accepted as heterogeneity. Results were given using a fixed model. A p-value for summary HR of less than 0. 05 was considered statistically significant. Publication bias was examined using Egger's regression intercept, Begg-Mazumdar rank correlation analysis, and a visual inspection of a funnel plot [15, 16]. Statistical analyses were performed using Comprehensive Metaanalysis V 2. 0 (Biostat, Englewood, NJ).",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The computer-based literature search using PubMed/Medline resulted in 1520 articles. Evaluation of the title and abstract of these articles revealed 377 case reports, 188 reviews, 32 letters, 34 comments, 63 non-English language studies, 13 pediatric studies, 11 cell studies, and 3 animal studies, and these were excluded from further analysis, leaving 799 papers. Of these 11 were cases series, 69 had included other lymphoma subtypes, 120 papers had not included rituximab-containing regimens, 3 did not report subgroup survival analyses, and 564 did not include the data effect size for gender. All these papers were excluded, leaving 26 papers. However, among these 26 papers, there were no effect size data in cases receiving rituximab according to gender in 5 papers and these papers were thus excluded from further analysis. Figure 1 shows the flowchart of articles included in this meta-analysis. Ultimately, 20 studies were included in this meta-analysis (Table I [11, [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] ).",
"section_name": "Study eligibility",
"section_num": null
},
{
"section_content": "Two of 20 studies included in this analysis were prospective and 18 were retrospective. The quality of retrospective studies was evaluated using the Newcastle-Ottawa Scale. In this scale 1-3 is considered as a low quality study, 4-6 as an intermediate quality study and 7-9 as a high quality study. The median Jadad score was 6 and 5 for retrospective and prospective studies, respectively.",
"section_name": "Quality of papers included in this metaanalysis",
"section_num": null
},
{
"section_content": "The total number of patients included in this meta-analysis was 5635: 2879 (54. 4%) men and Overall survival, disease-free survival, event-free survival Pooled HR for OS was evaluated in 16 studies and male gender was found to be associated with OS (HR = 1. 155; 95% CI: 1. 037-1. 286; p < 0. 009) (Figure 2 ). The association between gender and progression-free survival (PFS) was evaluated in eight studies and there was no statistically significant association (HR = 0. 849; 95% CI: 0. 671-1. 074; p = 0. 171) (Figure 3 ). Event-free survival was evaluated in two studies and there was no association between gender and EFS (HR = 0. 809; 95% CI: 0. 577-1. 133; p = 0. 217) (Figure 4 ).",
"section_name": "Patients",
"section_num": null
},
{
"section_content": "There was no publication bias for OS (Begg's test, p = 0. 564; Egger test, p = 0. 557). The funnel plot did not show publication bias for OS (Figure 5 A ). There was no publication bias for PFS (Begg's test, p = 0. 286; Egger test, p = 0. 254). The funnel plot was symmetric and did not show publication bias for OS (Figure 5 B ). Publication bias could not be determined for EFS because only two studies were evaluated.",
"section_name": "Publication bias",
"section_num": null
},
{
"section_content": "Male gender has been found to be a bad prognostic indicator for OS in cases treated with rituximab-containing regimens in some studies [17, 18]. An OS advantage has been shown in patients with DLBCL treated with rituximab-containing regimens for every age and risk group [36, 37]. \n\nImmunochemotherapy is the standard of care in patients with DLBCL; however, variable responses have been documented. This means that more sensitive prognostic and/or predictive factors are required. Although it has not been evaluated in all studies, male gender has been found to be a prognostic factor at least in some studies [38]. The relationship between gender and prognosis has been considered in recent years. Although this analysis covers studies between 2002 and 2013, the majority of the studies in this meta-analysis were published in 2013. Male sex has been shown to be a bad prognostic indicator in patients with DLBCL treated with rituximab-containing regimens [11, 27]. \n\nPrognostic factors in 700 cases from eight centers in Asian and Western countries treated by R-CHOP were compared by Castillo et al. They found no difference for PFS and OS, but male sex was found to be a bad prognostic indicator in multivariate analysis of patients of Asian origin [24]. This point is important, and more studies from different countries will be required to evaluate racial differences. \n\nThe prognostic role of gender in DLBCL has been reported in only a limited number of papers. In one of these papers, Carella et gender was found to be a bad prognostic indicator and the HR was 1. 52. A higher dose of rituximab in males was suggested by the authors [27]. Gender was also found to be an independent prognostic factor by Gisselbrecht et al. in relapsed/refractory DLBCL treated with rituximab maintenance after autologous stem cell transplantation [39]. \n\nLower serum rituximab levels, shorter exposure times and worse outcome in men were observed in the RICOVER-60, RICOVERnoRTh and pegfilgrastim trials (R1, R2, R3), and the SEXIE-R-CHOP 14 trial has been planned and performed. In the last ASCO conference this study was presented and a significantly improved outcome in male patients was observed with higher rituximab doses (500 mg/m 2 instead of 375 mg/m 2 ) [40]. These results confirm the worse outcome with standard R-CHOP in men. \n\nThe mechanism underlying the different prognosis in men and women is not clear. Pharmacokinetic studies suggest that higher serum levels are associated with lower drug clearance in females [41]. Additionally, gender-associated gene polymorphisms may be a contributing factor in the higher response to immune-chemotherapy in females. GSTT1 deletion has been suggested as a causative factor for resistance to R-CHOP and also poor prognosis in male Korean patients [42]. \n\nIn conclusion, OS was found to be better in female cases than males with DLBCL treated with immunochemotherapy in this meta-analysis. Higher doses of rituximab could be more useful in males; the ongoing SEXI-R-CHOP-14 study will help clarify this point.",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Conflict of interest",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest.",
"section_name": "Conflict of interest",
"section_num": null
},
{
"section_content": "",
"section_name": "R e f e r e n c e s",
"section_num": null
}
] |
10.31557/apjcp.2022.23.9.3229
|
Interleukin-10-1082A>G (rs1800896) Single Nucleotide Polymorphism is Not a Risk Factor of Chronic Lymphocytic Leukemia in Sudanese Population
|
Objectif : La présente étude a été menée pour examiner l'association entre le polymorphismeIL-10-1082A >G (rs 1800896) et le risque de leucémie lymphoïde chronique et pour évaluer la corrélation entre ce polymorphisme et les caractères clinicopathologiques. Méthodes : Une étude cas-témoins a été menée dans l'État de Khartoum, au Soudan, entre avril 2017 et avril 2018, auprès de 110 patients atteints de LLC et de 80 volontaires sains en tant que groupe témoin. Un examen physique, une numération formule sanguine complète et un immunophénotype ont été effectués chez tous les patients pour confirmer le diagnostic. La stadification clinique telle que Rai et Binet a été étudiée. CD38 et ZAP70 ont été réalisés par cytométrie de flux. Des échantillons de sang ont été prélevés chez tous les participants ; l'ADN a été extrait à l'aide du kit d'extraction d'ADN sanguin ANALYTIKJENA et analysé le polymorphisme IL-10-1082A >G à l'aide de la réaction en chaîne de la polymérase spécifique de l'allèle. L'analyse statistique a été réalisée à l'aide d'un progiciel statistique pour la version 23.0 du logiciel des sciences sociales. Résultats : La fréquence des génotypes AA, AG et GG était de 32,7 %, 55,5 % et 11,8 % pour le groupe de patients et de 31,25 %, 51,25 % et 17,5 % dans le groupe témoin, respectivement. Le génotype de l'IL-10 (-1082A>G) n'était pas associé à la sensibilité de la LLC dans notre population. L'étude a montré que l'allèle G du gène IL-10 (-1082A >G) est associé au sexe masculin. Cependant, aucune association significative n'a été trouvée entre le génotype -1082A >G et les caractères clinicopathologiques. Conclusion : Nos résultats ne supportent pas l'implication du polymorphisme du gène promoteur IL-10 − 1082A >G dans la susceptibilité accrue à la LLC. L'allèle IL-10-1082G (IL-10-1082AG ou IL-10-1082GG) a été trouvé plus fréquemment chez les hommes. De plus, aucune association n'a été observée entre le polymorphisme IL-10-1082A >G et les systèmes de stades cliniques ainsi que les marqueurs de mauvais pronostic établis. Enfin, au sein du groupe de patients atteints de LLC, il n'y avait pas de différence entre l'âge au diagnostic et les paramètres hématologiques en fonction des distributions génotypiques.
|
[
{
"section_content": "Chronic lymphocytic leukemia is characterized by the progressive accumulation of monoclonal, small, mature-appearing CD5 + B-cells in the peripheral blood, bone marrow, and secondary lymphoid tissue (Caligaris-Cappio and Hamblin, 1999). Genetic variation of some immunomodulatory cytokine may lead to different subtypes of lymphoma predisposition (Chatterjee et al., 2004). \n\nInterleukin-10 is an anti-inflammatory cytokine",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Interleukin-10-1082A>G (rs1800896) Single Nucleotide Polymorphism is Not a Risk Factor of Chronic Lymphocytic Leukemia in Sudanese Population important for proper immune system function (Iyer and Cheng, 2012). IL-10 which acts as a growth factor for normal activated human B and T lymphocytes stimulation and proliferation (El Far et al., 2004). \n\nThe gene encode IL-10 is located on chromosome 1(1q31-1q32) (Eskdale et al., 1997). Some evidence suggest of IL-10 gene that polymorphic variations in the promoter sequences of IL-10 gene may influence in the gene expression (Turner et al., 1997; Gibson et al., 2001). Moreover, SNPs in IL-10 gene promoter play a role in pathogenesis of lymphoid disorders, and may increase a risk of NHL development, especially diffuse large B-cell lymphoma subtype (Dai et al., 2014). \n\nRecently, a series of epidemiological studies have focused on the association between these SNPs of IL-10 (-3575T>A, -1082A>G,-819C>T and -592C>A) and the risk of CLL (Domingo-Domènech et al., 2007; Ennas et al., 2008; Lech-Maranda et al., 2008; Lech-Maranda et al., 2013; Ovsepyan et al., 2015). \n\nIL-10-1082G allele were characterized by more advanced CLL (Rai stages III or IV and elevated LDH) (Lech-Maranda et al., 2013). Furthermore, this allele may be a marker which correlates with worse prognosis, fast progression of the disease and short TFS (Lech-Maranda et al., 2013). On contrast, Ovsepyan et al. shown single nucleotide replacement-1082G>A in IL-10 gene promoter region is probable genetic factor of CLL risk and of disease manifestation at advanced stages (Ovsepyan et al., 2015). \n\nTo the best of our knowledge, this is the first study with a large sample size conducted in Sudan to investigate the association between the IL-10-1082A>G polymorphism (rs1800896) and susceptibility of CLL and correlate with clinical presentation, hematological parameter, and some biological prognostic markers. The present study was conducted to examine the association between the IL-10-1082A>G polymorphism and the risk of CLL and to assess the correlation between IL-10-1082A>G polymorphism and clinical presentation, hematological parameter, and some biological prognostic markers among Sudanese patients with CLL.",
"section_name": "RESEARCH ARTICLE",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "This study is a case-control study, conducted in Khartoum state, Sudan, in the period from April 2017 to April 2018, a total of 110 patients with CLL and 80 apparently healthy volunteers as a control group were recruited to participate in this study. Patients were obtained at Flow Cytometry Laboratory for Leukemia & Lymphoma Diagnosis, Khartoum; they were referred for Immunophenotype diagnosis. \n\nAll patients were diagnosed based on clinical history, physical examination and complete blood count. The peripheral blood is important to show morphological abnormalities and immunophenotypic criteria. All our patients have ≥5000×10 9 /L B lymphocyte, considered in our diagnosis according to International Workshop on Chronic Lymphocytic Leukemia (Hallek et al., 2008). The stage of the CLL was assessed by Rai and Binet classification (Rai et al., 1975; Binet et al., 1981). All patients were newly diagnosed without any previous treatment, whereas patients with other lymphoid neoplasms (both B and T-cell Lineage) were excluded.",
"section_name": "Study Population",
"section_num": null
},
{
"section_content": "The peripheral blood was collected as samples from both groups included in the study (the patient group and control group). For the patient group, an amount of four milliliters (ml) of blood was collected from each patient in (EDTA) and divided equally into two tubes; one tube for complete blood count and immunophenotype test and the other tube for molecular analysis. For the control group, two (ml) of blood was collected from each healthy individual in (EDTA) for molecular analysis.",
"section_name": "Sample Collection",
"section_num": null
},
{
"section_content": "Two ml of peripheral blood were withdrawn from each patient; these samples were collected in EDTA tubes and preserved at room temperature (22-24 O C) then processed within 6-24h from the collection. Complete blood count was analyzed by using automated hematology analyzer (SYSMEX KX-21N, Japan). All results such total White Blood Cells, Absolute lymphocyte count, Hemoglobin level, Red Blood Cells and platelets were recorded.",
"section_name": "Determination of Blood Count",
"section_num": null
},
{
"section_content": "The diagnosis of CLL was confirmed in each patient by flow cytometry (EPICS XL Beckman Coulter Flow Cytometry, Miami, FL, USA), standard protocol of Beckman Coulter (COULTER, 2010) was used in fluorescent dye-labelled monoclonal antibody for CD45, CD3 CD5, CD10, CD19, CD20, CD22, CD23, FMC7, CD79b, kappa, and lambda light chain. A marker was considered positive at cutoff ≥30%. Cutoff point of 30% was selected as recommended by British Committee for Standards in Haematology guideline (Oscier et al., 2012). However, in order to confirm diagnostic CLL, a scoring system was applied depending on Moreau et al. 1997 (Moreau et al., 1997). ZAP-70 and CD38 were used as prognostic markers, with a cutoff point of 20% and 30%, respectively, as previously described (Basabaeen et al., 2019).",
"section_name": "Determination of Immunophenotyping and (CD38 & ZAP-70 expressions)",
"section_num": null
},
{
"section_content": "DNA was extracted from all blood samples of patients and control groups by using ANALYTIKJENA Blood DNA Extraction Kit (Germany) (REF-845-KS-1020050), according to the manufacturer's instructions. The β-globin gene was used to assess the quality of DNA in all extracted samples, the primer sequences as previously described (Kerr et al., 2000). All specimens for β-globin gene were successful amplification. To evaluate the DNA quantification after DNA extraction, we measured DNA by using a NanoDrop spectrophotometer. Then DNA samples were routinely stored at -20 O C.",
"section_name": "DNA extraction",
"section_num": null
},
{
"section_content": "Genotyping was carried out by using the allele specific polymerase chain reaction (AS-PCR), primers as described in our previous work (Sharif et al., 2019). Two separated PCR reaction mixtures of 20 µl were prepared for each sample. PCR was performed by using Maxime PCR Premix Kit (i-Taq), (iNtRON BIOTECHNOLOGY, South Korea), Cat. No. 25025), 4µl of genomic DNA, 0. 5 µl of each primer, and 15 µl distilled water. PCR started at 94°C for 5 min, followed by denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 40 s, with a final extension at 72°C for 5 min. Thermocycling was using TECHNE Tc-412-UK PCR Thermal Cycler 96 well. The amplified products were run on 1. 5% DOI:10. 31557/APJCP. 2022. 23. 9. 3229 IL-10-1082A>G in Sudanese Patients with CLL gene (Tables 1 and 2) Moreover, CLL patients and healthy individuals presented with similar frequencies of -1082A alleles (60. 45% and 56. 87% for -1082A allele in patients and controls, respectively; (Table 1 ). IL-10-1082A>G polymorphism appeared not to be associated either with susceptibility to CLL. TheIL-10-1082A>G genotypes were then grouped into G+ alleles (combining the AG and GG genotypes into one group) and alleles encompassing the AA genotype appeared not to be associated either with susceptibility to CLL. The allele and genotype frequencies among patients with CLL and control subjects are summarized in (Table 1 ). The genotype distributions for the IL-10-1082A>G polymorphism in both 110 CLL patients and 80 control subjects were in Hardy-Weinberg equilibrium (HWE) and normally distributed with P-value > 0. 05.",
"section_name": "Genotyping of Interleukin-10-1082A>G (rs1800896)",
"section_num": null
},
{
"section_content": "There was no difference in mean age at diagnosis for patients with the AA, AG, and GG genotypes (59. 63, 63. 98, and 67. 46 years, respectively). However, among the patients with CLL, IL-10-1082G allele (IL-10-1082AG or IL-10-1082GG) was found more frequently in males. We also compared the distribution of IL-10-1082 genotypes in patients concerning Rai and Binet classification. However, no significant associations were observed. Also, did not observe any influence of the IL-10-1082 genotype distribution on CD38 expression and ZAP-70 expression as well as hematological parameters (Tables 2 and 3 ).",
"section_name": "IL-10-1082A>G polymorphism and relation to some established prognostic markers",
"section_num": null
},
{
"section_content": "CLL is characterized by a complicated etiology (Rogalinska and M Kilianska, 2010). It is suggested that CLL heterogeneity may be associated with the single nucleotide polymorphic variations within the genes governing leukocyte differentiation, life/death control, as well as cell-cell interactions (Żołnierczyk and Kiliańska, 2015). Many cytokines are known to be involved in the agarose gel, and then stained with ethidium bromide for visualization under ultraviolet gel documentation system (Figure 1 ).",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Data was analyzed using the statistical package for social sciences version 23. 0 (Chicago, IL, USA). Numerical data were summarized as mean and standard deviation and n (%) of study participants, respectively. Logistic regression was used for calculation of odds ratio (OR) with confidence interval (CI) for risk estimation. The Hardy-Weinberg equilibrium was tested by goodness of fit X2 test to compare the observed genotypic frequencies in normal individual to the expected genotypic frequencies, and then calculated from the observed allelic frequencies. Chi-Square test was used for analyzing associations between categorical variables. One-Way ANOVA were used to compare the means of two groups. All P-values were two-sided, and < 0. 05 was considered as the significance level.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "A total of 190 DNA samples were included in this study, namely, 110 CLL patients and 80 healthy controls. The CLL cases included 79 (71. 8%) males and 31 (28. 2%) females; the mean age was 62. 97 ± 12. 06 years. The controls included 57 (71. 3%) males and 23 (28. 7%) females; the mean age was62. 9±11. 88 years. There were no differences between the case and control groups on age, sex (P-value>0. 05). \n\nOut of 110 CLL cases included in the study, 36 (32. 7%) showed the AA genotype of the IL-10-1082A>G polymorphism, whereas 61 (55. 5%) displayed a heterozygous genotype and 13 (11. 8%) were found to carry the GG genotype. Comparison of the total sample of patients with CLL and control group did not reveal any significant differences in genotype frequencies of polymorphic locus -1082A>G of IL-10 Figure 1. -1082A>G Genotyping Using AS-PCR. Lane L, 100 bp DNA molecular weight marker. Lanes 1 and 3, allele A is represented by the presence of a 550 bp PCR fragment. Lanes 2 and 6, allele G is represented by the presence of a 550 bp PCR fragment. Each two lanes represent one sample, lanes 1 and 2, heterozygous (AG); lanes 3 and 4, homozygous (AA); lanes 5 and 6, homozygous (GG). pathogenesis of CLL (Allegra et al., 2020). IL-10 is a multifunctional cytokine; is key cytokine involved in the balance between cell-mediated and humoral immunity and lymphoid development (Moore et al., 2001; Mocellin et al., 2004). IL-10 stimulates the proliferation and differentiation of B and Th2 cells (Moore et al., 2001; Mocellin et al., 2004). There is, however, conflicting data regarding the effect of IL-10 on CLL cells, with some reports suggesting that IL-10 inhibits proliferation and induces apoptosis of malignant cells (Fluckiger et al., 1994), while others postulating that IL-10 protects against apoptotic cell death ( Kitabayashi et al., 1995). The possible mechanism of involving IL-10 in the lymphoid development and disease pathogenesis are still debated (Lech-Maranda et al., 2012). \n\nSeveral studies have investigated the association of IL-10-1082A>G gene polymorphism and CLL susceptibility, reporting conflicting results. In the present study, allelic frequencies and genotype distributions of IL-10-1082A>G SNP were similar in the CLL patients' group and matched controls group. It may suggest that this polymorphism has no impact on the susceptibility for the CLL occurrence. Our results are consistent with the authors who did not find any associations between IL-10-1082A>G polymorphisms and CLL risk occurrence. (Ennas et al., 2008; Lech-Maranda et al., 2013). In contrast, a contradictory result was obtained by Ovsepyan et al.,( 2015) in Russia found Allele -1082A was significantly more incident in the CLL patients group in comparison with the control group, also found associations between allele -1082A and genotypes (-1082AA/-1082AG) and risk of CLL (Ovsepyan et al., 2015). The reasons for the discrepancies between different studies are not clear. This might be due to ethnicity. \n\nSharif et al. analyzed the association of IL-10-1082A>G polymorphism with Acute Myeloid Leukemia and concluded that the frequency of GA genotype was significantly higher in AML patients than in control subjects and they resulted that IL-10-1082A>G polymorphism was associated with AML in the Sudanese population (Sharif et al., 2019). Sharif et al., (2019) revealed that the allelic frequency of AA, GG, and GA genotypes was 53. 3%, 36. 7%, and 10% for the control group, respectively. The frequency IL-10-1082G>A genotype in the control group in this study displayed dissimilar frequencies to those in the control group reported by (Sharif et al., 2019). This may be due to different sample sizes, also may due to differences in ethnic populations from Sudan indicted significant intra-population differences in genotype distribution. \n\nIn the current study, genotype distribution revealed a higher frequency of heterozygous (AG) 55. 5% than homozygous (AA, GG; 32. 7, 11. 8, respectively) in CLL patients. These frequencies agree with those previously reported in the literature (Guzowski et al., 2005; Domingo-Domènech et al., 2007; Ennas et al., 2008; Lech-Maranda et al., 2013; Ovsepyan et al., 2015). \n\nIn the past several years, some meta-analysis studies have been conducted on the association of IL-10 SNPs with NHL risk (Dai et al., 2014; Zhang et al., 2015; Li and Li, 2016). When stratified by NHL subtypes, all these meta-analysis studies revealed that IL-10 -1082A>G polymorphism was not associated with increased CLL risk (Dai et al., 2014; Zhang et al., 2015; Li and Li, 2016). \n\nRegarding the association between this SNP and established prognostic markers in the present study, comparison of age at diagnosis between patients with different genotype IL-10 -1082A>G did not reveal any significant difference. Also previous studies have been unable to detect any significant difference in mean age among patients with different genotypes (Domingo-Domènech et al., 2007; Lech-Maranda et al., 2008; Lech-Maranda et al., 2013). \n\nNevertheless, data in this study suggested the CLL patients were carriers of G allele in IL-10-1082A>G promoter gene polymorphism is associated with the male sex, which can be partially explained by the fact that CLL is more prevalent in males than in females. This prognostic association has not been reported in previous studies (Domingo-Domènech et al., 2007; Ennas et al., 2008; Lech-Maranda et al., 2008; Lech-Maranda et al., 2013; Ovsepyan et al., 2015). But this supports that male to have carriers G allele than women (Lio et al., 2002). Thus, it is intriguing that the possession of -1082G genotype, suggested being associated with IL-10 high production. \n\nWe were unable to show any significant association between this polymorphism and clinical-stage systems (Rai and Binet) on CLL patients. A similar result was obtained by (Lech-Maranda et al., 2008) found no significant effect of this polymorphism on the Rai stage system. Moreover, Domingo-Domenech et al. explained IL-10-1082A>G was not associated with established prognostic factors such as the Rai stage system (Domingo-Domènech et al., 2007). While another study conducted by Lech-Maranda et al found the presence of IL-10-1082G allele (IL-10-1082AG or GG) was associated with the Rai stage III or IV (Lech-Maranda et al., 2013). In contrast, Ovsepyan et al. explained -1082AA genotype is significantly more incidents in patients with more advanced Binet stages (B+C) than patients with earlystage (Ovsepyan et al., 2015). This discrepancy may be the result of racial differences. Besides, in this study, about 90% of patients displayed advanced Rai stages and 70% were at Binet stage B or C. As expected, reverse patterns with highest patient percentages at stage (Rai 0 and Binet A) and lowest percentages at advanced stages (III, IV, and C) were reported in developed countries. \n\nOur study demonstrated no associations between IL-10-1082A>G polymorphism and CD38 expression or ZAP-70 expression. The results of the current study are consistent with those of Lech-Maranda et al. and suggest that no association was found between IL-10: -1082A>G genotype or haplotype distribution and clinical characteristics of CLL patients at diagnosis, including CD38 expression (Lech-Maranda et al., 2008). More recently Lech-Maranda et al., (2013) unable to detect any association between IL-10-1082A>G and prognostic factors except the presence of IL-10-1082G allele (IL-10-1082AG or GG) was associated with the elevated serum levels of lactate dehydrogenase and advanced Rai stage system. In conclusion, our results do not support the involvement of the IL-10-1082A>G promoter gene polymorphism in the increased CLL susceptibility. Also, in our group of CLL patients no significant differences were found in the distribution of IL-10-1082A>G alleles and genotypes as compared to controls. IL-10-1082G allele (IL-10-1082AG or IL-10-1082GG) was found more frequently in males. Furthermore, no association was observed between the IL-10 -1082A>G SNP and clinical stages systems as well as established poor prognostic markers. Finally, within the group of patients with CLL there was no difference in the age at diagnosis and hematological parameters according to genotype distributions.",
"section_name": "IL-10-1082A>G polymorphism in CLL patients group and control group",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "We would like to thank the staff of the Hematology Department at Al Neelain University for facilities and supporting and we are grateful to the staff of Flow Cytometry Laboratory for Leukemia & Lymphoma for their collaboration. Finally special thanks to the patients for being cooperative, despite their pains.",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The research did not receive any fund or financial support.",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Author Contribution Statement",
"section_num": null
},
{
"section_content": "AAB & EAA & IKI conceived the study design, participated in data collection, performed the statistical analysis, interpreted the results, and revised the manuscript. EAB, NMA, & SOA participated in the statistical analysis and drafted the manuscript. OAA and EAF participated in the data collection, carried out the laboratory work, and prepared the results. AAB& OMS were performed the molecular analysis. All authors read and approved the final manuscript.",
"section_name": "Author Contribution Statement",
"section_num": null
},
{
"section_content": "The individual data are available in the archives of the Flow Cytometry for Leukemia & Lymphoma Diagnosis, Khartoum, Sudan and can be obtained from the corresponding author on request.",
"section_name": "Availability of Data and Materials",
"section_num": null
},
{
"section_content": "Not applicable.",
"section_name": "Consent for Publication",
"section_num": null
},
{
"section_content": "Ethical clearance was obtained from the Institutional Review Board at Al Neelain University. The principal investigator obtained written informed consent from all participants prior to their inclusion in the study.",
"section_name": "Ethics Approval and Consent to Participate",
"section_num": null
},
{
"section_content": "The authors declare that they have no conflict interests.",
"section_name": "Conflict of interest",
"section_num": null
}
] |
10.3892/ijo.2012.1528
|
Epimutation and cancer: A new carcinogenic mechanism of Lynch syndrome
|
Epimutation is defined as abnormal transcriptional repression of active genes and/or abnormal activation of usually repressed genes caused by errors in epigenetic gene repression. Epimutation arises in somatic cells and the germline, and constitutional epimutation may also occur. Epimutation is the first step of tumorigenesis and can be a direct cause of carcinogenesis. Cancers associated with epimutation include Lynch syndrome (hereditary non-polyposis colorectal cancer, HNPCC), chronic lymphocytic leukemia, breast cancer and ovarian cancer. Epimutation has been shown for many tumor suppressor genes, including RB, VHL, hMLH1, APC and BRCA1, in sporadic cancers. Methylation has recently been shown in DNA from normal tissues and peripheral blood in cases of sporadic colorectal cancer and many studies show constitutive epimutation in cancers. Epimutation of DNA mismatch repair (MMR) genes (BRCA1, hMLH1 and hMSH2) involved in development familial cancers has also been found. These results have led to a focus on epimutation as a novel oncogenic mechanism.
|
[
{
"section_content": "",
"section_name": "Recommendation",
"section_num": null
},
{
"section_content": "",
"section_name": "Reasons for Recommendation",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Additional Comments",
"section_num": null
},
{
"section_content": "Submission of this form allows Spandidos Publications to contact your institution regarding this recommendation. If you do not want Spandidos Publications to contact your institution, this form can be printed and submitted to your librarian. For more information regardingour library subscription charges, please contact: contact@spandidos--publications. com",
"section_name": "Additional Comments",
"section_num": null
}
] |
10.7554/elife.98747.3
|
The Jag2/Notch1 signaling axis promotes sebaceous gland differentiation and controls progenitor proliferation
|
<jats:p>The sebaceous gland (SG) is a vital appendage of the epidermis, and its normal homeostasis and function is crucial for effective maintenance of the skin barrier. Notch signaling is a well-known regulator of epidermal differentiation, and has also been shown to be involved in postnatal maintenance of SGs. However, the precise role of Notch signaling in regulating SG differentiation in the adult homeostatic skin remains unclear. While there is evidence to suggest that Notch1 is the primary Notch receptor involved in regulating the differentiation process, the ligand remains unknown. Using monoclonal therapeutic antibodies designed to specifically inhibit of each of the Notch ligands or receptors, we have identified the Jag2/Notch1 signaling axis as the primary regulator of sebocyte differentiation in homeostatic skin. Mature sebocytes are lost upon specific inhibition of the Jag2 ligand or Notch1 receptor, resulting in the accumulation of proliferative stem/progenitor cells in the SG. Strikingly, this phenotype is reversible, as these stem/progenitor cells re-enter differentiation when the inhibition of Notch activity is lifted. Thus, Notch activity promotes correct sebocyte differentiation, and is required to restrict progenitor proliferation.</jats:p>
|
[
{
"section_content": "The skin is a vital organ that acts as a protective barrier against the external environment, and safeguards against fluid loss. An important component of this barrier function is the presence of a complex mixture of oils, known as sebum, which is produced by the SGs. SGs are part of the epidermis and are typically associated with the hair follicle. These acinar structures have two cell types: basal stem or progenitor cells which encase the differentiated sebocytes (Figure 1a ). Sebocyte differentiation begins at the proximal tip of the of the SG, with maturing sebocytes moving upwards, enlarging, accumulating lipids, and ultimately undergoing a highly regulated and specialized form of cell death in which they release their lipid contents into the sebaceous duct (Figure 1b ; Kretzschmar et al., 2014; Schneider and Paus, 2010). This process requires the constant turnover of sebocytes, which occurs over a period of 7-14 d in mice (Jung et al., 2015). Both over-and underproduction of sebum have been linked to various skin disorders including acne or dry skin (Al-Zaid et al., 2011; Binczek et al., 2007; Karnik et al., 2009; Lovászi et al., 2017; Rittié et al., 2016; Seiffert et al., 2007; Shi et al., 2015; Smith and Thiboutot, 2008; Stenn et al., 1999), and rare sebaceous carcinomas constitute aggressive tumors leading to high mortality (Buitrago and Joseph, 2008; Nelson et al., 1995), thus SG number and function have to be tightly regulated for proper skin function. \n\nThe Notch signaling pathway is one of the most studied regulators of cell fate decisions, and is known to be widely involved in epidermal differentiation. The pathway consists of multiple ligands and receptors that typically form a signaling axis in pairs. Notch signaling can regulate cell fate by either inducing or inhibiting differentiation, or by making binary cell fate decisions (Wilson and Radtke, 2006). Classically, these functions of Notch signaling have been studied during development, but increasing evidence suggests that the Notch pathway is also involved in regulating cell fate and cell states in adult homeostatic tissues (Ables et al., 2011; Lafkas et al., 2015; Mosteiro et al., 2023; Sato et al., 2012; Siebel and Lendahl, 2017). \n\nWhile it is known that Notch signaling is not required for embryonic development of the epidermis, it is essential for the postnatal maintenance of the hair follicles and the SGs (Watt et al., 2008). However, the precise role of Notch signaling in adult sebocyte differentiation has not been comprehensively investigated, with most studies examining irreversible deletions of the Notch pathway components in the embryonic ectodermal lineages. While these studies report SG defects, it remains unclear whether these defects are due to a direct effect on the SGs, or whether they are a consequence of general skin defects also observed in these models. For example, SGs are absent in mice with embryonic pan-Notch deletions such as Rbpj (Blanpain et al., 2006), gamma-secretase, Notch1Notch2, and Notch1Notch2Notch3, and are severely reduced in Notch1 and Notch1Notch3 embryonically-deleted skin (Pan et al., 2004), while deletion of Notch2 alone does not affect the SG (Pan et al., 2004). Consistent with the constitutive deletions, loss of Rbpj in adult SGs also results in missing sebocytes, while loss of Notch1 in the adult SGs results in miniaturized lobes that still contain some differentiated sebocytes (Veniaminova et al., 2019). Interestingly, activation of Notch1 in the adult skin results in enlarged SGs (Estrach et al., 2006). Conversely, for the Notch pathway ligands, embryonic and adult deletion of Jag1 (Estrach et al., 2006), and embryonic deletion of Dll1 (Estrach et al., 2008) results in normal SG morphology. Collectively, these data suggest that Notch1 is the dominant Notch receptor involved in regulating sebocyte differentiation, however, it remains unclear which ligand is required. \n\nIn our previous work, we observed that systemic inhibition of Jag2 using monoclonal therapeutic antibodies resulted in SG defects in adult mice (Lafkas et al., 2015). Given that our therapeutic antibodies selectively, potently, and transiently inhibit each of the distinct Notch receptors and ligands (Tran et al., 2013; Wu et al., 2010; Yu et al., 2020), they constitute ideal tools to dissect the contribution of each of these pathway members to sebocyte differentiation in adult homeostatic skin. Leveraging the use of these antibodies, we demonstrate that specific inhibition of the Jag2 ligand or Notch1 receptor both result in the loss of mature sebocytes in the SG, establishing the Jag2/Notch1 signaling axis as a crucial regulator of sebocyte differentiation in adult homeostatic skin. The loss of mature sebocytes in the SG is concomitant with an accumulation of cells with a basal phenotype, forming epithelial remnants in the SG. Cells in these epithelial remnants are actively proliferating and express stem/progenitor markers indicating that sebocyte differentiation is halted, while stem/ progenitor numbers are increased. Importantly, this phenotype is reversible, as these epithelial cells re-enter differentiation with the return of Notch activity. Thus, Notch activity is required in sebocyte stem/progenitor cells for their proper differentiation, and its inhibition locks these cells in a reversible progenitor state.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To investigate the role of Notch signaling in the homeostatic skin, we treated 8-wk-old mice with a single dose of the various antagonizing antibodies (anti-Notch1, anti-Notch2, anti-Jag1, anti-Jag2,",
"section_name": "Jag2 is the dominant Notch signaling ligand involved in regulating sebocyte differentiation",
"section_num": null
},
{
"section_content": "Basal stem cell Lrig1",
"section_name": "Progenitor cells AR,Lrig1",
"section_num": null
},
{
"section_content": "Proximal tip and the isotype control antibody anti-Ragweed) alone or in combination. We then examined the dorsal, resting phase (telogen) skin at 3, 7, and 14 d post-treatment. We first confirmed that our antibodies could reproduce the requirement of Notch1 in regulating sebocyte differentiation. To this end, we treated mice with Notch1 (aN1) and Notch2 (aN2) blocking antibodies and examined the SGs at 7 d post-treatment. Loss of mature sebocytes was observed specifically after aN1 treatment, while the aN2-treated SGs showed normal morphology (Figure 1c and d ). However, the combined treatment of aN1N2 had a more pronounced effect on SG morphology as compared to aN1 alone (Figure 1c and d ), indicating that Notch2 also contributes to regulating sebocyte differentiation, possibly as a compensatory mechanism after inhibition of the dominant Notch1 receptor. Next, we investigated which ligand formed the signaling pair with Notch1. Loss of mature sebocytes was observed specifically after treatment with the Jag2 blocking antibody (aJ2), but not after treatment with the Jag1 blocking antibody (aJ1) at 7 d post-treatment (Figure 1e and f ). For aJ2 treatment, the loss of sebocytes began at 3 d post treatment, but affected only a small proportion of the SGs at this time point (Figure 1g ). However, by 7 ds post-treatment, most SGs had either completely lost all sebocytes, or had only some sebocytes remaining (Figure 1g ). The loss-of-sebocyte phenotype was most pronounced with a combined treatment of Jag1/Jag2 blocking antibodies (aJ1J2) (Figure 1g ), suggesting that while Jag2 is the primary Notch ligand involved in regulating sebocyte differentiation, Jag1 also plays a minor role in this process, but is unable to produce a phenotype on its own. In conclusion, these data indicate that the Jag2-Notch1 signaling axis is the dominant Notch ligand-receptor pair required for sebocyte differentiation in the adult skin.",
"section_name": "N1ICD,Lrig1",
"section_num": null
},
{
"section_content": "We also examined the percentage of SGs consisting of bursting sebocytes releasing sebum as a proxy of a functional sebaceous duct. There were no significant differences between treatments (Figure 1 -figure supplement 1a and b), hinting at a functionally intact sebaceous duct.",
"section_name": "Hair follicle",
"section_num": null
},
{
"section_content": "We next characterized the expression of the relevant receptor and ligand in the SG to develop a spatial map of Notch activity in this tissue. Both the Notch receptors and their ligands are transmembrane proteins that interact with each other in neighboring cells to activate the pathway. To examine this interaction, we performed a triple stain for the cleaved (active) form of the Notch1 intracellular domain (ICD), and Notch1 and Jag2 in situ hybridization (ISH) probes at 3 d post antibody treatment. In the control treated mice, Notch1 ICD was observed in the basal stem cell compartment of the SG, but not in the differentiating sebocytes (Figure 2a-c, cell types based on morphology). We saw a similar pattern of N1ICD-positive cells for aJ1 treatment (Figure 2d-f ). Notch1 signaling was not active in all basal cells, as only ~50% of them were positive for N1ICD after control and aJ1 treatment (Figure 2g ). Consistent with previous studies, the majority of these N1ICD + cells were present near the proximal tip of the SG (Figure 2 -figure supplement 1a-c; Veniaminova et al., 2019), where the initial sebocyte differentiation has been proposed to occur (Kretzschmar et al., 2014). However, after aJ2 treatment, Notch1 activity (ICD staining) was absent, or observed only at very low levels in the basal stem cells, while they still expressed Notch1 and Jag2 mRNA (Figure 2h-j ). Strikingly, we noticed the expression of both Notch1 ISH and Jag2 ISH in the same cell on most N1ICD + cells (Figure 2k ). Interestingly, a majority of all basal cells (including N1ICD-cells), expressed both N1 and Jag2 mRNA (Figure 2 -figure supplement 1d). Our data shows that the vast majority of SG basal stem cells express both the ligand and receptor, but only some basal stem cells experience active Notch signaling at any one point in time, as evidenced by the presence of N1ICD. This could be a technical limitation as the triple staining only captures a temporal snapshot of the basal stem cells, with the N1 + J2 mRNA positive cells going on to express N1ICD later, or it could hint towards a more complex regulatory mechanism involved in activating Notch signaling.",
"section_name": "Notch is active in the sebaceous gland stem cells",
"section_num": null
},
{
"section_content": "Notch activity, as well as Notch1 and Jag2 mRNA, were also observed in the interfollicular epidermis (IFE) and other cells of the hair follicle (Figure 2 -figure supplement 1e and f). While there is a reduction in Notch-active cells in these other regions, it does not appear to significantly impact the rest of the skin. There were no significant differences in the width of the IFE or the adipocyte layer between treatments (Figure 2 -figure supplement 1g and h). This together with the histological appearance (Figure 2 -figure supplement 1i and j) of these regions suggests that proliferation and differentiation in these compartments remain unaffected.",
"section_name": "Figure 1 continued",
"section_num": null
},
{
"section_content": "To further detail the effect of Notch inhibition on sebocyte differentiation, we examined the expression of mature sebocyte markers. Adipophilin (Adipo) is expressed in all mature sebocytes (Frances and Niemann, 2012; Ostler et al., 2010) and Fatty acid synthase (FASN) is expressed in mid-and late-differentiating sebocytes, with FASN levels decreasing in the most mature sebocytes (Cottle et al., 2013; Figure 1b ). Since N1ICD staining disappears at 3 d post antibody treatment, we examined the mature sebocyte markers at this time point. While N1ICD staining was specifically lost in the SG basal stem cells of aJ2-treated skin, mature sebocytes expressing FASN were still observed at this timepoint (Figure 2j, Figure 3a and b and Figure 3-figure supplement 1a ). All cells that express FASN also express adipophilin, but since FASN levels decrease with sebocyte maturity, we also examined and focused on adipophilin to mark all sebocytes (Figure 3c and d and Figure 3 -figure supplement 1b-e). The number of cells that expressed adipophilin was not significantly different between treatments at 3 d post antibody treatment (Figure 3h ). At 7 d post antibody treatment, control and aJ1 treated skin showed normal SG morphology and sebocyte marker expression (Figure 3e and Figure 3 -figure supplement 1f, h and i), but both aJ2 and aJ1J2 SGs had lost sebocyte marker expression, and the SG was filled with cells with a basal phenotype (basal-like cells) (Figure 3f and Figure 3 -figure supplement 1g, j and k). Consistently, the number of cells expressing adipophilin were significantly lower for aJ2 and aJ1J2 treatment (Figure 3i ). Interestingly, we noticed that some of these affected SGs still contained a few mature sebocytes (Figure 3f and Figure 3 -figure supplement 1g), which were found at the distal end near the sebaceous duct. The location of these remaining sebocytes suggests that existing mature sebocytes are not affected by the Notch blockade, and go through their normal differentiation process, eventually bursting and releasing the sebum (Figure 3g ). Thus, we propose that Notch blockade inhibits differentiation at the basal stem cell level or in a sebocyte progenitor.",
"section_name": "Loss of Notch activity in the SG stem cells inhibits sebocyte differentiation",
"section_num": null
},
{
"section_content": "To determine whether the epithelial cells that filled the affected SGs at 7 d post-treatment were stem/ progenitor cells, we examined them for stem and early differentiation markers. Lrig1-positive cells form a distinct stem cell compartment that maintains the SG and the upper part of the hair follicle (Frances and Niemann, 2012; Niemann and Horsley, 2012; Page et al., 2013), allowing us to use Lrig1 as a marker for the basal stem cells of the SG, while Androgen Receptor (AR) can be used as an early marker of sebocyte differentiation (Cottle et al., 2013; Figure 4a and b and Figure 4 -figure supplement 1a and b). We noticed that the AR-expressing cell population could be divided into two groups: a basal stem cell population that co-expressed Lrig1, but did not express FASN (arrowheads in Figure 4a and c ), and an early differentiating sebocyte population that expressed FASN, but did not express Lrig1 (arrows in Figure 4a and c ). We hypothesize that the Lrig1+/AR + population is a progenitor cell population, in addition to the Lrig1 + stem cells, similar to the recently identified transitional basal cell population in the SG (Veniaminova et al., 2023). There were no significant differences in the number of Lrig1 positive stem cells per SG, or the AR-expressing progenitor population between treatments at 3 d post-treatment (Figure 4d and e ). By 7 d post-treatment, however, the basal-like cells that filled the SG were all positive for Lrig1 (Figure 4f and g and Figure 4 figure supplement 1e), with the total number of Lrig1 positive cells per SG increasing significantly for aJ2 and aJ1J2 treated SGs (Figure 4h ), while SGs after aJ1 treatment showed normal morphology and marker expression (Figure 4 -figure supplement 1c and d ). The proportion of AR-expressing cells was not significantly different between treatments (Figure 4i ). These results indicate that the basal-like cells that fill the SG after blocking Notch signaling by aJ2 and aJ1J2 treatment, are stem/ progenitor cells. Additionally, we examined the SGs for their proliferative capacity to confirm stem/ progenitor function. In a normal SG, proliferation is restricted to the basal stem cells (Figure 4j ). As expected, proliferation was also restricted to the basal stem cells in aJ1-treated SGs (Figure 4 -figure supplement 1f and g). Remarkably, most of the basal-like cells in the aJ2-treated SGs were proliferative, while the rare mature sebocytes remaining in the SG were non-cycling (Figure 4k ). As the SGs are mostly filled with basal-like cells at this time (Figure 4h ), the total number of proliferating cells per SG was significantly higher (Figure 4l ). Overall, these data suggest that inhibition of Notch activity by aJ2 treatment retains the stem and progenitor (Lrig1 + and Lrig1+/AR+, respectively) cells in their immature proliferative state and prevents differentiation.",
"section_name": "Notch activity in the SG stem cells is required to prevent unregulated progenitor proliferation",
"section_num": null
},
{
"section_content": "The therapeutic antibodies employed do not inhibit Notch signaling permanently, as the antibodies eventually become cleared from the animal's system. To determine whether the loss of sebocyte phenotype was reversible, we examined the SGs 14 d post single-dose treatment. Intriguingly, mature sebocytes begin to recover at this time point (Figure 5a and b, compared with Figure 1f and g ). We hypothesized that the sebocyte recovery must be due to the return of Notch activity after antibody washout. To test this, we examined the SGs for Notch activity at 7 d post-treatment, since the return of Notch activity must precede the recovery of mature sebocytes. Fittingly, we observed N1ICD expression return in some of the basal-like cells at this time (Figure 5c and d and Figure 5 -figure supplement 1a-d ). The percent of N1ICD positive cells per SG strongly increased from 1% at 3 d post-treatment to 29% at 7 d post-treatment (Figure 5e ), even though the percentage of N1ICD positive cells per SG remained significantly lower than control for aJ2 and aJ1J2 treated SGs at this time (Figure 5 -figure supplement 1d ). Next, we examined the SGs at 14 d post-treatment for mature sebocyte markers to confirm the sebocyte recovery. Indeed, we saw the return of cells expressing adipophilin in the aJ2 and aJ1J2 treated SGs (Figure 5f and g and Figure 5 -figure supplement 1e-h). There was an overall increase in the number of these cells from day 7 to day 14 (Figure 5h ), even though the number of these cells remained significantly lower in these SGs compared to controls (Figure 5 -figure supplement 1h). Interestingly, the majority of the adipophilin-expressing cells were found in the proximal third of the SG (51% for aJ2 treatment and 66% for aJ1J2 treatment), consistent with the initiation of sebocyte differentiation at the proximal tip. However, there was a significant proportion of these cells found in the middle third (22% and 25%, respectively), and distal third (27% and 9%, respectively) of the SG. This could be due to the newly differentiated cells moving through the SG in a proximal to distal direction, as is the case during normal homeostasis. Alternatively, the stem cells could also be differentiating at sites other than just the proximal tip, as previously demonstrated by multi-color lineage tracing (Andersen et al., 2019). We further examined the SGs at 14 d post-treatment for their AR expression, and confirmed that it had also been restored to its normal pattern (Figure 5i and j and Figure 5 -figure supplement 1i-l). We also found that the average number of AR-expressing cells decreased from day 7 to day 14 post aJ2 and aJ1J2 treatment (10-2. 95, and 8. 56-5. 55, respectively) (Figure 5k ), returning to a more homeostatic state. Together, these data indicate that Notch inhibition does not result in a permanent cell fate switch, but maintains the stem/progenitor state, allowing the recovery of the differentiation process with the restoration of Notch activity.",
"section_name": "The block in sebocyte differentiation is lifted upon recovery of Notch activity",
"section_num": null
},
{
"section_content": "Based on our findings, we propose that the Jag2/Notch1 signaling axis is essential for correct sebocyte differentiation in homeostatic dorsal skin, and that inhibition of this signaling retains the basal stem and progenitor cells in a proliferative state, and blocks further differentiation. Thus, Notch signaling is required to prevent unregulated stem/progenitor proliferation, and induction of the sebocyte differentiation program. Moreover, Notch inhibition resulting in complete loss of mature sebocyte differentiation is a reversible phenotype, indicating that there remains a functional progenitor pool present during the studied timeframe. \n\nHere, we have leveraged the use of monoclonal therapeutic antibodies designed to inhibit each of the distinct Notch receptors or ligands to study the role of Notch signaling in adult homeostatic tissue. We have shown that Jag2 is the hitherto unknown ligand involved in regulating sebocyte differentiation. Inhibition of Notch signaling using Jag2 blocking antibodies results in the loss of mature sebocytes, with the resulting SG being filled with basal-like cells forming 'finger-like' epithelial remnants. \n\nA similar phenotype has been described by a previous study (Veniaminova et al., 2019) that used Lrig1-CreERT2 to irreversibly knock out RBPJ in adult homeostatic skin. This strategy enabled inhibition of Notch signaling specifically in the Lrig1 + stem cell population that normally maintains the SGs, but preserved it in the rest of the hair follicle. Interestingly, the authors saw two contradictory phenotypes as a result: overall loss of SG lobes, replaced by the finger-like epithelial remnants, as well as the presence of persistent SGs that were enlarged. The authors showed that over time, patches of Rbpj mutant cells extended out of their niche into the IFE, where Lrig1 is not expressed, and that loss of RBPJ in the IFE led to enlarged SGs. They argue that while Notch signaling promotes sebocyte differentiation in the SG stem cells, it indirectly suppresses these glands from the IFE. Our results did not show the enlarged SG phenotype, and we only observed the finger-like epithelial remnants. While injections of the Notch blocking antibodies are systemic, we only observed a reduction in the number of Notch-active cells in the IFE, but not a complete loss. This could explain why we didn't observe the enlarged SG phenotype. Additionally, we observed the phenotype as early as 3 d post antibody treatment, while Veniaminova et al., observed the SGs 10 wk after tamoxifen injection. These differences are likely due to the different methodologies used in the two studies. \n\nSeveral studies indicate that Notch signaling is essential for the postnatal maintenance of SGs (Blanpain et al., 2006; Estrach et al., 2008; Estrach et al., 2006; Pan et al., 2004; Watt et al., 2008), but so far only Veniaminova et al., have specifically examined SGs in the adult homeostatic skin. They observed that the stem/progenitor and differentiation markers were intermingled in the finger-like remnants forming an unnatural hybrid state, neither staying in a true progenitor state nor differentiating. In contrast, we observed a complete block in differentiation, with the stem/progenitor cells being locked in an immature proliferative state. These observations suggest that antagonistic antibodies may be able to achieve a more complete inhibition of Notch signaling in the SG stem/ progenitor compartment. \n\nImportantly, we were able to show that the loss-of-sebocyte phenotype in the SG is reversible. Having used therapeutic antibodies to block Notch signaling, the inhibition of the signaling pathway was not permanent. As Notch activity returned to the SG after antibody washout, sebocyte differentiation also recovered. These data indicate that the loss of Notch signaling does not impact the stem/ progenitor potential of the SG, but prevents further differentiation. Indeed, a functional progenitor pool accumulates in the SG, primed for differentiation, as soon as Notch signaling becomes active. A recent study by Veniaminova et al., has shown that Lrig1-CreERT2 used to irreversibly knock out Pparg in adult homeostatic skin ablates 99% of the SGs (Veniaminova et al., 2023). Interestingly, they showed that non-recombined cells from other parts of the hair follicle migrate to the SG zone and regenerate the genetically ablated SGs. This regeneration process is dependent on the hair growth cycle, with the SGs primarily regenerating during the anagen (active growth) phase and not in the telogen phase. While our studies cannot rule out the contribution of non-SG cells to the SG recovery seen upon the return of Notch activity, this recovery is not dependent on the hair growth cycle. Sebocytes are able to differentiate while the hair follicle is still in the telogen phase, indicating a lift of the An important open question is how Notch signaling regulates sebocyte differentiation. FASN, which can be used as a readout for sebocyte differentiation, is a downstream target of AR (Schirra et al., 2005). We observed a Lrig1+/AR+/FASN-population in the normal SGs, similar to the basallike cells that fill the finger-like remnants seen after Notch inhibition. This expression pattern indicates that AR requires a co-activator to activate downstream gene expression in the SG. Previous studies have reported that Notch effectors such as HEY1, HEY2, and HEYL can act as co-repressors of AR in prostate cells (Belandia et al., 2005; Kamińska et al., 2020; Lavery et al., 2011). It is possible that certain Notch effectors can act as either co-activators or co-repressors of AR in a context-dependent manner. Further investigation will be needed to understand the exact molecular role Notch signaling plays in regulating sebocyte differentiation.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "aJ2=2. 90E-87, aJ1J2=1. 73E-92. Chi-square test used for statistical analysis. All treatments were compared against aRW. Total n of SGs quantified per treatment: aRW = 485, aJ1=430, aJ2=394, aJ1J2=364. Scale bars are 100 μm. (c,d) Representative co-stain images for N1ICD and fatty acid synthase (FASN) in SGs from mice (n=5 each) treated with aRW (c) and aJ2 (d), 7 d post-treatment. (e) Quantification of the percentage of N1ICD + cells in SGs from mice (n=5 each) treated with aRW and aJ2, 3 and 7 d post-treatment. Percentage was calculated by dividing the number of N1ICD+ cells by the total number of basal-like cells in each SG. p-values: for comparison between day 3 and day 7 for aRW treatment = 0. 297, for comparison between day 3 and day 7 for aJ2 treatment = 1. 00E-14. Total n of SGs quantified per treatment: aRW at 3 d = 13, aRW at 7 d = 13, aJ2 at 3 d = 15, aJ2 at 7 d=22. (f,g) Representative adipophilin staining in SGs from mice (n=5 each) treated with aRW (f) and aJ2 (g), 14 d post treatment. (h) Quantification of the number of cells expressing adipophiln in each SG, 7 and 14 d after treatment with aRW, and aJ2. p-values: for comparison between day 7 and day 14 for aRW treatment = 0. 292, for comparison between day 7 and day 14 for aJ2 treatment = 1. 38E-4. Total n of SGs quantified per treatment: aRW at 7 d = 18, aRW at 14 d = 19, aJ2 at 7 d = 22, aJ2 at 14 d=29. (i,j) Representative co-stain images for androgen receptor (AR) and FASN in SGs from mice (n=5 each) treated with aRW (i) and aJ2 (j), 14 d post-treatment. (k) Quantification of the number of cells expressing AR in each SG, 7 and 14 d after treatment with aRW and aJ2. p-values: for comparison between day 7 and day 14 for aRW treatment = 0. 273, for comparison between day 7 and day 14 for aJ2 treatment=9. 53E-09. Total n of SGs quantified per treatment: aRW at 7 d=12, aRW at 14 d=10, aJ2 at 7 d=18, aJ2 at 14 d=23. Student's t-test used for statistical analysis. C57BL/6 mice were obtained from Charles River-Hollister, and were used for all experiments. Mice were housed under specific-pathogen-free conditions, and were 8 wk old upon treatment. This time point was chosen to correspond to the resting (telogen) phase of the hair growth cycle, as SG size can vary by hair cycle stage. All mice were injected intraperitoneally with a single dose of blocking antibodies diluted in sterile saline at the following concentrations: anti-Jag1 at 20 mg/kg, anti-Jag2 at 20 mg/kg, anti-Jag1 + anti-Jag2 at 20 mg/kg + 20 mg/kg=40 mg/kg, anti-Notch1 at 5 mg/kg, anti-Notch2 at 10 mg/kg and anti-Notch1 + anti-Notch2 at 5 mg/kg + 10 mg/kg=15 mg/kg. Anti-Ragweed isotype control antibody was injected at concentrations to match the maximum dose of treatment antibodies.",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Telogen mouse dorsal skin was collected at 3, 7, and 14 d post antibody injection. For frozen sections, skin samples were fixed in 4% paraformaldehyde in PBS, for 40 min at 4 °C, washed, and then immersed in 30% sucrose in PBS overnight at 4 °C. The tissue was then embedded in OCT (TissueTek), frozen immediately on dry ice, and stored at -80 °C. Additional skin tissue from the same animals was also fixed with 10% neutral buffered formalin and then used to create paraffin-embedded sections. Hematoxylin and eosin (H&E) staining was performed by the Pathology core at Genentech. The triple immunofluorescence stain for Notch1 ICD (IHC), and Notch1 mRNA (ISH), and Jag2 mRNA (ISH) was performed by the histopathology development group at Genentech. ACD LS 2. 5 probes were ordered from Advanced Cell Diagnostics. RNAScope LS 2. 5 Murine-Jag2_C1 (417518) nucleotides spanning from nt 552-1480 of reference sequence NM_010588. 2, and Murine-Notch1_ C2 (404648-C2) nucleotides spanning from nt 1153-1960 of reference sequence NM_008714. 3. For positive control, RNAScope LS 2. 0 Murine-PPIB probe (313917) nucleotides spanning from nt 98~856 of reference sequence NM_011149. 2 were used. For negative control RNAScope LS 2. 0 DapB probe (312038) nucleotides spanning from nt 414~862 reference sequence EF191515 were used. For Immunohistochemistry, we used N1ICD (Cell Signaling, 4147), at 20 ug/ml working concentration. \n\nThe triple immunofluorescence for murine Jag2_Notch1 (dual ISH) with anti_Notch1 ICD (IHC) 3 Plex ISH_ISH_IHC in murine tissues using formalin-fixed, paraffin-embedded sections was performed on the Leica Bond-RX complete automation system using the RNAscope LS Multiplex Reagent Kit (322800). Slides were baked and dewaxed on Leica Bond-RX and pretreated with Bond Epitope Retrieval Solution 2 (ER2) (AR9640) from Leica at 100 °C for 40 min. After pretreatment, the probes were cocktailed, and hybridization was performed at 42 °C for 120 min followed by amplification steps and developed with Opal 570 fluor at 1:1000 and Opal 690 fluor at 1:1500 from Akoya using the Opal Polaris 7 Auto Detection Kit (NEL811001KT). The immunohistochemistry was performed upon completion of ISH. The primary antibody incubation was 60 min at room temperature, followed by secondary antibody incubation for 30 min at room temperature with HRP conjugated Goat anti-Rabbit (Perkin Elmer, NEF812001EA). The slides were then developed with Opal 780 fluor at 1:25 (Akoya, SKU FP1501001KT) following the manufacturer's instructions. Slides were imaged using Olympus VS200. \n\nAll other immunohistochemistry was performed using the following antibodies: N1ICD at 1:500 (Cell Signaling, 4147), FASN at 1:100 (BD, 610963), Adipophilin at 1:500 (Fitzgerald, 20R-AP002), Ki67 at 1:100 (Thermo Fisher Scientific, SP6 RM-9106-SO), Lrig1 at 1:200 (R&D Systems, AF3688-SP), and AR at 1:250 (Abcam, ab133273 [EPR1535(2)]). Slides were imaged using the Leica Thunder microscope. \n\nImaging parameters were identical for all images. Images were processed using Fiji and Adobe Illustrator.",
"section_name": "Histopathological analysis and immunochemistry",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We are grateful to C Cottonham, S Hankeova, and G Hernandez for their helpful discussions. We thank the Genentech Research Pathology, Necropsy, and Histology laboratories for their experimental contributions. We appreciate the insightful feedback and comments on the paper from L Mosteiro, E Reyes, and B Biehs.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "No external funding was received for this work.",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "All data generated or analysed during this study are included in the manuscript and supporting files; source data files have been provided for Figure 1 ; Figure 2; Figures 3-5 and Figure 1-figure supplement 1, Figure 2-figure supplement 1 and Figure 5-figure supplement 1.",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional information",
"section_num": null
},
{
"section_content": "Competing interests Syeda Nayab Fatima Abidi: S. N. F. A. is an employee of Genentech. Sara Chan: S. C. is an employee of Genentech and holds shares in Roche. Kerstin Seidel: K. S. is an employee of Genentech and holds shares in Roche. Daniel Lafkas: D. L. was a Genentech employee, and is currently employed at Roche, and holds shares in Roche. Louis Vermeulen: L. V. is an employee of Genentech and holds shares in Roche. Frank Peale: F. P. is an employee of Genentech and holds shares in Roche. Christian W Siebel: C. W. S. was a Genentech employee, and holds shares in Roche. C. W. S is currently employed at Gilead Sciences.",
"section_name": "Additional information",
"section_num": null
},
{
"section_content": "",
"section_name": "Author contributions",
"section_num": null
}
] |
10.1186/s13000-017-0634-3
|
Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay
|
Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management.We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization.Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL).We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an effective alternative to lengthy conventional diagnostic procedures requiring both cytogenetic analysis followed by targeted quantitative reverse transcription (qRT-PCR) methods, thus allowing timely patient management.
|
[
{
"section_content": "Fusion genes are genetic chromosomal aberrations formed by the juxtaposition of two disparate gene loci through chromosomal translocation, interstitial deletion, or inversion and is most frequently detected through cytogenetic abnormalities. Although hundreds of fusion genes have been associated with leukemia, only a small portion of these fusion events are consistently recurrent and clinically characterized to the extent where they can be diagnosed and treated effectively in leukemia patients [1] [2] [3]. The prevalence of characterized gene fusions with clinical implications varies amongst differing hematological malignancies. Approximately 25-50% of acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) cases comprise at least one fusion gene [2, 4], while over 90% chronic myeloid leukemia (CML) cases comprise BCR-ABL1 gene fusions [5]. Importantly, the detection of certain fusion gene events in certain hematological malignancies can act as diagnostic indicators in the selection of an effective targeted therapeutic regimen [1] [2] [3]. For example, prevalent detection of BCR-ABL1 fusions in CML cases may be useful in prescribing tyrosine kinase inhibitor (TKI) treatment regimens such as imatinib [6], while detection of PML-RARa fusions may require treatments designed to treat the M3 AML subtype, acute promyelocytic leukemia (APL) [7]. In other instances, detection of fusion genes can classify malignancies into prognostic subgroups where diagnostic outcomes were not associated with targeted therapies [2, 4]. Moreover, a patient may concurrently comprise multiple oncogenic fusion genes, thereby requiring comprehensive treatment strategies targeting multiple tumorigenic components. \n\nCurrently, the detection of specific gene fusion events requires prior characterization by cytogenetic analysis to narrow possible fusion gene events to be determined by secondary screening procedures. However, cytogenetic analysis is time-consuming and may take up to 2 weeks, thus preventing immediate patient management. Further, cytogenetic analysis requires extensive training and exceptional expertise, leaving room for clinical errors that may hinder rapid and accurate diagnosis and proper treatment. Thus, there is a need to develop screening methods to bypass time constraints and clinical errors that may be associated with, cytogenetic analysis. In this study, we describe effective use of a fluorescent real-time quantitative reverse transcription PCR (F-qRT-PCR)-based multi-fusion gene screening method with the capacity to accurately screen 22 common fusion genes concurrently without prior characterization of chromosome translocation phenotypes by cytogenetic analysis. Moreover, this method can also accurately detect co-existing fusion genes, while conventional qRT-PCR methods commonly detect single gene fusions. Our method described here would greatly improve diagnostic efficiency and accuracy in leukemia, allowing immediate therapeutic management to achieve optimal outcome.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "A total of 345 patients from Henan Cancer Hospital participated in this study. These patients were diagnosed with AML (108 patients), ALL (60), Chronic lymphocytic leukemia (CLL) (15), CML (41), Myelodysplastic Syndromes MDS (33), or other non-leukemia hematological diseases including lymphoma and cytopenia (88) from April 2014 to September 2015. Bone marrow and peripheral blood samples were collected from all participating patients for disease diagnosis and experiments proposed in this study, and written informed consent was obtained from all participants in this study. This study was approved by the Institutional Ethics Committee of Henan Cancer Hospital (2103ys34). The group of 108 patients with AML were used an \"evaluation cohort\" to evaluate the efficiency of the F-qRT-PCR method (described below), while the remaining patients were used as the \"validation cohort. \" The clinicopathological features of the 108 AML patients from the evaluation cohort are listed in Table 1.",
"section_name": "Patients",
"section_num": null
},
{
"section_content": "F-qRT-PCR was performed to detect 22 fusion genes (Table 2 ) using a Leukemia Related Fusion Gene Detection ing leukemia cDNA fragments were used to replace cDNA templates to serve as negative and positive controls, respectively. Three PCR reactions were simultaneously conducted in each PCR reaction tube using three fluorescently-labeled (FAM, HEX and CY5) probes. A multi-PCR reaction tube system using a set of 6 reaction tube was used to detect up to 22 fusion genes in a single assay set (Table 3 ). PCR amplification was performed with a protocol modified from a published study [8, 9], using initial incubation at 42 °C for 5 min and at 95 °C for 10 min followed by 40 cycles of consecutive amplification at 94 °C for 15 s and 60 °C for 1 min using a 7500 Real Time PCR System (ABI, USA). Using a plasmid DNA template, the detection limit was determined to be 1000 DNA copies per reaction, while the detection specificity for both positive and negative controls were assayed at 100%. PCR products successfully amplified from gene fusion products were subcloned into plasmid vectors (pCDNA3 or equivalent) with T7 or SP6 promoter sequencing primer sites and subjected to Sanger sequencing using a 3130 Genetic Analyzer (ABI, USA) system according to the manufacturer's instructions to verify the sequence of the amplified fusion fragment. Diagnostic results obtained by F-qRT-PCR were compared to results obtained from cytogenetic analysis. The concordance rate was calculated as the number of samples positively diagnosed by both detection methods, plus the number of samples with negative diagnoses by both detection methods, as a fraction of the total sample number.",
"section_name": "Detection of 22 fusion genes using fluorescent real-time RT-PCR",
"section_num": null
},
{
"section_content": "Conventional cytogenetic analysis of bone marrow was conducted in the cytogenetic laboratory at the Affiliated Cancer Hospital of Zhengzhou University using standard protocol as described elsewhere [10]. Chromosomes were prepared using a direct preparation method or a short-term culture (24 h) of bone marrow cells and visualized with R-Table 2 22 fusion genes screened using the multi-fusion gene F-qRT-PCR assay system Fusion genes Also known as Frequently associated diseases\n\nAML acute myeloid leukemia, ALL acute lymphocytic leukemia, CML chronic lymphocytic leukemia, MDS myelodysplastic syndromes, APL acute promyelocytic leukemia, CMML chronic myelomonocytic leukaemia, CEL chronic eosinophilic leukemia banding. At least 20 cells were examined where possible, although the experiments with less than 20 cells will not be excluded. The karyotype was described according to the International System for Human Cytogenetic Nomenclature (ISCN).",
"section_name": "Cytogenetic analysis",
"section_num": null
},
{
"section_content": "We first evaluated the efficiency of the F-qRT-PCR diagnostic system using the 108 AML patients comprising the evaluative cohort. All patients were characterized for cytogenetic abnormalities to include chromosome translocations by cytogenetic analysis (Fig. 1a ). F-qRT-PCR was performed on all patients in a blinded manner without prior knowledge of translocation classification obtained from cytogenetic characterization (Fig. 1b ). mRNA gene fusion products successfully detected by F-qRT-PCR were purified and subjected to Sanger sequencing analysis to validate amplification of the chromosomal mRNA fusion product (Fig. 1c ). Results obtained from cytogenetic and F-qRT-PCR analysis were compared to gauge diagnostic accuracy (Table 4 ). From the AML patient cohort, 34 patients (Cyto-) showed no chromosomal rearrangement with 28 patients assayed with normal karyotypes and 6 patients observed with abnormal cytogenetic morphologies. Chromosomal rearrangements were identified in the remaining 74 cases (Cyto+), implicating the occurrence of the following chromosomal gene fusions: RUNX1-RUNX1T1, AML1-MDS/EVII/MTG16, FIP1L1-PDGFRA, PML-RARa, CBFb-MYH11, SIL-TAL1, or MLL-AF6/AF9/AF10/ELL/ENL. We successfully identified all predicted gene fusion events in the 74 cases characterized with chromosomal arrangements using the F-qRT-PCR diagnostic system. In addition, we detected a RUNX1-RUNX1T1 gene fusion by F-qRT-PCR in a Cyto-case, indicating that use of our F-qRT-PCR method may have superior sensitivity in comparison to cytogenetic analysis. Comparing F-qRT-PCR to cytogenetics, the sensitivity and specificity were 100. 0 and 97. 1%, respectively and the overall concordance rate between the two methods was determined to be 99. 1% (Table 5 ). \n\nWe next expanded our study to other leukemic subtypes to include 60 ALL, 15 CLL, 41 CML and 33 MDS, or non-leukemic disorders. Combined with the aforementioned AML cases, a total of 345 patients were characterized (Table 6 ). Within this new cohort, cytogenetic analysis predicted chromosomal rearrangements (Cyto+) in 144 patients, where chromosome rearrangements were not detected in (Cyto-) in 201 patients. A total of 4 cases (including 1 patient described in the AML evaluation cohort) showed discrepancies between F-qRT-PCR and cytogenetic diagnostic assays. In addition to the aforementioned Cyto-patient in the AML cohort, gene fusions were detected in two additional Cyto-cases, including an ALL patient with BCR-ABL rearrangements and a CML patient with AML-ETO fusions. However, we failed to detect expression of a BCR-ABL fusion in a CML patient with cytogenetic 46,xy,t(9;22) (q34;q11) [1] /46,xy [9] rearrangements by F-qRT-PCR. Nonetheless, we determined the concordance rate between F-qRT-PCR and cytogenetic analysis methods to be 98. 8%, as determined by the sum of PCR-/Cyto-(198) and PCR+/Cyto + (143) concordant cases as a ratio of the total number of cases (345). \n\nWe detected gene fusions in 138 out of 257 leukemia cases by F-qRT-PCR, accounting for 53. 7% of the patients assayed. Gene fusions were observed most abundantly in CML patients at a frequency of 97. 6% patients assayed, followed by AML (69. 4%), ALL (33. 3%), and MDS (9. 1%). No fusion genes were detected in any of the 15 CLL patients characterized in this study. Together, these results indicate that leukemia rearrangements can be rapidly and accurately diagnosed by the F-qRT-PCR method described here, which may facilitate timely application of proper treatment strategies to optimize patient outcome.",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Accurate and efficient diagnosis in leukemia using reliable detection methods is essential in prescribing effective tumor treatment strategies and relevant targeted therapeutic regimens. Although the characterization of chromosomal rearrangements and gene fusion events is crucial to successful diagnosis and treatment in leukemia, clinical diagnosis may be complicated by factors such as limitations in sample availability, error-prone, lengthy analytic procedures and costly procedural expenses. In this study, we describe the use of a F-qRT-PCR-based multi-fusion gene screening method to rapidly and accurately characterize chromosomal gene fusions in leukemia patients. The efficiency of this method is exemplified by a rapid turnaround time, and presents a cost-effective diagnostic alternative to conventional methods that require multiple procedural assays, such as cytogenetic karyotyping and FISH, followed by qRT-PCR to characterize a single oncogenic chromosomal aberration. Moreover, this method can detect a comprehensive set of 22 frequently recurrent fusion genes in a single experiment with superior accuracy and rapid turnaround times. Conventional diagnostic tests in leukemic patients include comprehensive blood analysis, peripheral blood smear analysis, bone marrow tests, immunophenotyping (CD antigen expression) and intensive chromosomal characterization assays (cytogenetics, FISH and RT-PCR). These labor-intensive measures, however, are necessary to accurately determine oncogenic chromosomal gene rearrangements which give indication to an optimal treatment strategy. Specific cytogenetic abnormalities in over 25-50% of patients with ALL or AML [2, 4] and over 90% of patients with CML [5] have been previously described, giving further indication that specific leukemic gene rearrangements require accurate diagnosis to determine a proper treatment regimen. For decades, characterization of leukemic subtypes involved initial analysis by cytogenetic examination to pre-determine chromosomal translocation or rearrangements, which would then be subsequently defined by a combination of analysis by FISH, immunohistochemistry, RT-PCR and Sanger sequencing. Since both\n\nE2A-PBX1 7 7 7 FISH and conventional RT-PCR analytic methods are limited by their ability to detect one particular chromosomal translocation, inversion, or deletion event, prior cytogenetic analysis was required to predetermine which particular chromosomal aberrations required further characterization by FISH or RT-PCR. As cytogenetic analysis may take up to 2 weeks, F-qRT-PCR screening procedures as described here may bypass lengthy cytogenetic pre-screening requirements, with the capacity to identify 22 fusion genes commonly associated with leukemia, which can expedite patient management strategies to optimize patient outcome. It should be noted that cytogenetic analysis is still of importance in characterizing other types of chromosomal abnormalities, including aneuploidy, in hypodiploid or hyperdiploid ALL and other leukemic subtypes. \n\nFISH is frequently used to detect and confirm predefined gene fusion events in leukemia. The standard turnaround time for FISH analysis is 3 days in our hospital, while it can be done within 24-h by rapid hybridization. Similar to qRT-PCR, FISH can also be used to document the disease burden and detect residual disease [11, 12], while qRT-PCR may exhibit better efficiency and sensitivity [13]. However, successful application of FISH analysis is dependent on the predetermination of chromosomal translocation events as defined by cytogenetic assay and other clinical assay methods in order to select and use appropriate DNA probes to detect specific chromosomal rearrangements [14]. Although FISH panels have been employed to detect more than one type of chromosomal rearrangement, this procedure is often costly, labor-intensive and technically difficult which may prevent a universal capacity to produce reliable and affordable diagnoses using this method. In contrast, RT-PCR-based methods require less training and experience, and application of RT-PCR analyses have become standard in clinical diagnostic procedures. As shown in Table 7, the cost and turnaround time in our hospital are higher for FISH or FISH panels than for single qRT-PCR or F-qRT-PCR, respectively. Moreover, our F-qRT-PCR covers a much broader spectrum of diseases than individual FISH panels, which are usually designed to cover one particular subtype of leukemia disease with very limited types of chromosomal rearrangements. It would require multiple FISH panels to cover the same number of fusion genes covered by our F-qRT-PCR method. Moreover, qRT-PCR has achieved greater sensitivity with enhanced flexibility in sample quantity and quality [15]. In the study described above, we used both target-specific primers and fluorescent probes to enhance signal specificity and quantitative fidelity. To overcome the lack of multiplexing capability [16, 17], we used a multi-PCR reaction tube system by simultaneously amplifying three PCR reaction sets using three fluorescently-labeled (FAM, HEX and CY5) probes. Using this method, we were able to detect up to 22 fusion genes in a single assay set. \n\nThis advanced screening method allowed us to detect gene fusions in 53. 7% of leukemia patients. In agreement with previous reports, we detected fusion genes in 69. 4% patients with AML, and 33. 3% with ALL [2, 4]. Additionally, we detected fusion genes in 97. 6% or BCR-ABL in 90. 2% of all CML patients characterized, which is also in good agreement to CML-associated gene fusions reported previously [3]. In this study, 4 cases revealed inconsistent outcomes using cytogenetic and F-qRT-PCR methods. Although three of these cases failed to reveal abnormalities by cytogenetic analysis, gene fusions were identified in these individuals by F-qRT-PCR, which was confirmed by direct sequencing of the resulting PCR products. These results were verified immediately by subsequent duplicating assays. The failure to detect genetic abnormalities by cytogenetic analysis is likely due to but not limited to one or more of the following reasons: (1) cytogenetic analysis is less sensitive than F-qRT-PCR; (2) The chromosomal rearrangements occurred in an extremely low percentage of tumor cells due to the high genetic heterogeneity in these cases; (3) Technical errors may occur by chance in certain cases, especially since this method demands tremendous skill and experience; (4) The quality of the samples was poor due to poor preservation, due to various reasons; (5) cytogenetic analytic procedures were inconsistent, perhaps due to sample limitations and technically difficult repeatability, which may be exacerbated by the laborintensive and costly nature of the procedure. Chromosomal abnormalities were revealed in one case by cytogenetic analysis, which failed to reveal any gene fusions by F-qRT-PCR analysis. This incidence is likely an extremely rare event as leukemia is extremely heterogeneous with only 1 of 10 cells harboring a t(9,22) translocation have been detected by a subsequent cytogenetic assay. \n\nIn conclusion, we describe an advanced F-qRT-PCRbased multi-fusion gene screening method using targetspecific primers and fluorescent-labeled hybridization probes to detect 22 common leukemic fusion genes in a single assay system. This method is extremely efficient with reduced turnaround times, and shows enhanced sensitivity and specificity for a wide target range. Accurate detection and characterization of gene fusions using this method can be comprehensively accomplished within 1 to 2 days without prior chromosomal characterization by cytogenetic analysis. The methods described in this study may potentially enhance the efficiency and accuracy of diagnosis of leukemic patients, which may greatly affect current procedural costs and therapeutic management.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Use of a F-qRT-PCR multi-gene fusion detection system described here can rapidly and accurately detect and characterize 22 gene fusion events commonly found in leukemia with comparable accuracy to conventional cytogenetic methods. As conventional diagnostic methods require pre-characterization of chromosomal abnormalities by cytogenetic analysis, this system bypasses the need for cytogenetic pre-characterization, thus expediting diagnosis and patient treatment management in leukemia subtypes characterized with gene fusions.",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Not applicable.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This work was partly supported by the National Natural Science Foundation of China (NSFC), grant 81470287.",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "The datasets supporting the conclusions of this article are included within the article.",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Abbreviations ALL: Acute lymphoblastic leukemia; AML: Acute myeloid leukemia; APL: Acute promyelocytic leukemia; CLL: Chronic lymphocytic leukemia; CLL: Chronic lymphocytic leukemia; FISH: Fluorescent in situ hybridization; F-qRT-PCR: Fluorescent real-time RT-PCR; MDS: Myelodysplastic syndromes Authors' contributions XL, YS and LZ designed the study and wrote the manuscript. XW, ZC, YZ, JH and RF performed the experiments and data analysis. All authors reviewed the manuscript. All authors read and approved the final manuscript.",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Not applicable.",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "This study was approved by the Institutional Ethics Committee of Henan Cancer Hospital (2103ys34). Written informed consent was obtained from all participants.",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.1038/s41467-024-46547-7
|
Accelerated DNA replication fork speed due to loss of R-loops in myelodysplastic syndromes with SF3B1 mutation
|
<jats:title>Abstract</jats:title><jats:p>Myelodysplastic syndromes (MDS) with mutated <jats:italic>SF3B1</jats:italic> gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes <jats:italic>SRSF2</jats:italic> or <jats:italic>U2AF1</jats:italic>. Molecular bases of these divergences are poorly understood. Here we find that <jats:italic>SF3B1</jats:italic>-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in <jats:italic>U2AF1</jats:italic>- or <jats:italic>SRSF2</jats:italic>-mutated MDS. Compared to erythroblasts from <jats:italic>SRSF2-</jats:italic> or <jats:italic>U2AF1</jats:italic>-mutated patients, <jats:italic>SF3B1</jats:italic>-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves <jats:italic>SF3B1</jats:italic>-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of <jats:italic>SF3B1</jats:italic>-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.</jats:p>
|
[
{
"section_content": "term treatment [6] [7] [8]. Deciphering the mechanisms of anemia is needed to generate treatments. \n\nSF3B1 mutation causes multiple alterations in mRNA processing. The use of alternative 3′ or 5′ splice site produces transcripts containing short intronic sequences that are degraded by the non-sense mediated decay (NMD) or are translated into a variant protein [9] [10] [11]. These splicing changes drive transcriptional reprogramming that shapes disease phenotype. For example, down-regulation of Fe-S cluster transporter ABCB7 by transcript isoform-specific degradation, and reduced translation efficiency of mitochondrial iron transporter TMEM14C, contribute to mitochondrial iron accumulation 12, 13. Overproduction of alternative and canonical transcripts of ERFE gene encoding hepcidin transcriptional repressor erythroferrone, leads to systemic iron overload 11. SF3B1 mutation also targets mitochondrial respiration and serine synthesis pathway 14. While mutations in serine and arginine-rich splicing factor 2 (SRSF2) and U2 small nuclear RNA auxiliary factor 1 (U2AF1), predominantly alter cassette exon 15, 16, SF3B1 MUT splicing pattern is dominated by intron retention reduction (IRR) 17. \n\nAn increasing number of genomic alterations associated with MDS progression to acute myeloid leukemia (AML) suggests a genomic instability of stem and progenitor cells 18. DNA damage and activation of ataxia telangiectasia and Rad3-related protein (ATR) pathway were detected in SF-mutated MDS 19, 20. More specifically, SRSF2 and U2AF1 mutations induce the formation of unscheduled RNA:DNA hybrids or R-loops, triggering DNA replication stress [19] [20] [21]. Mechanistically, mutant SRSF2 impairs the RNA polymerase II transcription pause release, allowing nascent RNA forming a R-loop at promoter 21. SF3B1 has been involved in the pathways of DNA repair 22, 23. However, SF3B1 MUT MDS patients have a lower risk of AML than other MDS 24, 25 suggesting that SF3B1 MUT MDS are less prone to genomic instability. \n\nIn the present study, we report that, on the contrary to SRSF2 MUT or U2AF1 MUT cells, SF3B1 MUT erythroblasts demonstrate a significant loss of R-loops. These cells endure a DNA replication stress consisting in accelerated fork progression and single-stranded (ss)DNA exposure, and correlating with increased erythroid cell proliferation and impaired differentiation. The ability of low doses of histone deacetylase inhibitor (HDACi) vorinostat to restore R-loops without DNA damage, and to improve erythroid differentiation could serve as a therapeutic approach.",
"section_name": "",
"section_num": ""
},
{
"section_content": "Reduction of intron retention correlates with transcriptomic changes of SF3B1-mutated bone marrow mononuclear cells A total of 143 subjects were enrolled in this study including 70 MDS with SF3B1 mutation, 49 SF3B1 WT MDS and 24 healthy controls (Table 1 ). To investigate the molecular pathways whose deregulation drives the phenotype of SF3B1 MUT -MDS, BM mononuclear cells (MNC) RNA-sequencing data available from 21 SF3B1 MUT -MDS and 6 SF3B1 WT -MDS were re-analyzed 11. With a mean number of 87 to 97 million reads per sample, DESeq2 analysis identified 1764 differentially expressed genes (DEGs) including 812 up-and 952 down-regulated genes (log 2 fold-change (FC) > | 1 |, Benjamini-Hochberg (BH)-adjusted P value < 0. 05) (Fig. 1a ; Supplementary Data 1). Gene ontology (GO) enrichment analysis showed that up-and down-regulated genes were involved in several pathways such as DNA replication, DNA repair, chromatid segregation, and cell cycle checkpoint signaling (Fig. 1b ). These pathways were over-represented among up-regulated genes (Supplementary Fig. 1a ). Eighty genes associated with these pathways allowed the clustering of SF3B1 MUT -samples (Fig. 1c ; Supplementary Data 1). Using KisSplice with a variation of percent splice in (ΔPSI) > | 0. 10| and a BHadjusted P value < 0. 05, we detected 3937 differential splicing events (DSEs) in SF3B1 MUT samples, including 1256 abnormal intron retention events, consisting in a majority of IRRs (n = 1027) in SF3B1 MUT -samples (Fig. 1d ). IRRs were the most frequent event in SF3B1 MUT -MDS, uncommon in the SRSF2 MUT or U2AF1 MUT -MDS of a large cohort of 189 lower-risk patients (Supplementary Fig. 1b ). A GO analysis of the 822 genes affected by IRR revealed their over-representation in DNA replication, DNA repair, cell cycle regulation, and mRNA splicing (Supplementary Fig. 1c ). Combined DEG and DSE analyses showed that 296 DEGs targeted by 384 IRR events referred to DNA repair, DNA replication, cell cycle process and regulation of chromosome separation (Supplementary Data 1; Fig. 1e ). Thus, IRR might contribute to gene expression changes of SF3B1 MUT -BM MNC.",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To decipher how transcriptional reprogramming driven by SF3B1 mutation affected erythroid lineage, we expanded erythroblasts from CD34 + hematopoietic stem and progenitor cells (HSPCs) collected from 14 SF3B1 MUT, 10 without SF (SF3B1, SRSF2, U2AF1) mutation (SF WT ) MDS and 6 healthy controls (Fig. 2a ). May-Grünwald-Giemsa (MGG)stained cytospins showed the predominance of basophilic erythroblasts (basoE) at days 11-13 and polychromatophilic erythroblasts (polyE) at days 14-15. Compared to healthy controls, the proportion of immature erythroblasts was significantly higher in MDS samples (Fig. 2b ). Despite a significant increase of apoptotic cells, the rate of proliferation of SF3B1 MUT erythroblasts was similar to control and SF3B1 WT erythroblasts (Supplementary Fig. 2 ). In SF3B1 MUT -cultures, the range of SF3B1 variant allele frequency was 23-54% at d11-13 and 17-51% at d14-15 (Fig. 2c ). \n\nRNA-sequencing provided an equivalent mean number of million reads per sample in basoE (116 ± 18) and polyE (97 ± 19) (t-test; P = 0. 543). With a log 2 (FC) > | 1| (BH-adj P value < 0. 05), a total of 675 DEGs (514 up and 161 down) separated mutant and wild-type basoE. The number of DEGs was 765 (390 up, 375 down) in polyE with an overlap of 179 genes with basoE (Fig. 2d, Fig. 2e ; Supplementary Data 2). GO enrichment analysis showed that up-and down-regulated genes in SF3B1 MUT -basoE or -polyE were involved in several pathways such as DNA repair, regulation of MAP kinase cascade, ubiquitindependent protein catabolism, cellular response to DNA damage and oxygen-containing compound (Fig. 2f ; Supplementary Fig. 3a ). In basoE, a GeneSet Enrichment Analysis (GSEA) refined these results showing the deregulation of specific DNA repair pathways such as base excision repair, and a trend for nucleotide excision repair, but neither homologous recombination nor non-homologous end-joining or mismatch repair. Genes involved in DNA replication and G1/S phase checkpoint were also significantly deregulated (Fig. 2g ; Supplementary Fig. 3b, c ). The DSE profiles identified in basoE and polyE were similar to that of BM MNC (Fig. 2h ). IRR represented 194/829 (23. 4%) DSEs in SF3B1 MUT -basoE and 383/1182 (32. 4%) DSEs in SF3B1 MUT -polyE, respectively, showing that IRR frequency increased with cell differentiation (Fig. 2i ; Supplementary Data 2). Genes affected by IRR regardless of the stage of erythroid differentiation were involved in the response to DNA damage, mitotic cell cycle regulation, nucleocytoplasmic transport and in the positive regulation of histone deacetylation (Fig. 2j ). As the nuclear retention or cytoplasmic degradation of IR-containing transcripts participate in the differentiation and specialization of normal erythroid cells [26] [27] [28], we reasoned that IRR-transcripts detected in SF3B1 MUT -erythroblasts may reshape the transcriptome contributing to defective maturation.",
"section_name": "SF3B1-mutated erythroid precursors demonstrate characteristic transcriptomic signatures",
"section_num": null
},
{
"section_content": "We concomitantly performed a proteomic analysis of proE, basoE and polyE with label-free quantification (LFQ). Principal component analysis showed that proE and basoE clustered together and separately from polyE. In each group, the second dimension discriminated SF3B1 MUT from SF3B1 WT -samples (Supplementary Fig. 4a ). In subsequent analyses, proE and basoE were grouped. The mean number of identified proteins with LFQ values per sample was 4231 ( ± 263), without significant difference between SF3B1 MUT and SF3B1 WT -samples, or between proE/basoE and polyE samples of each genetic group. A total of 443 and 290 differentially expressed proteins were detected between SF3B1 MUT and SF3B1 WT -samples in proE/basoE and in polyE, respectively (t-test, Log 2 (LFQ intensity)>|0. 20|, P value < 0. 05) (Fig. 2k ; Supplementary Fig. 4b, c ). \n\nIn SF3B1 MUT -proE/basoE proteome, mitochondrial ABCB7 (ATPbinding cassette B member 7), SUCLA2 (succinyl coA ligase), NNT (NAD(P) transhydrogenase), PPOX (protoporphyrinogen oxidase) were decreased, while SOD2 (superoxide dismutase) and ATP5A1 (ATP synthetase subunit alpha) were increased, further indicating mitochondrial dysfunction. Several key components of DNA replication pathway, such as DNA ligases LIG1 and LIG3 were decreased (Supplementary Fig. 4d ; Supplementary Data 3). In SF3B1 MUT -polyE, FEN1 (5'FLAP-endonuclease and gap endonuclease) and POLE (polymerase ε) expression was decreased while XRCC3 (X-ray repair crosscomplementing 3), and ORC1 (Origin recognition complex subunit 1) expression was increased (Supplementary Data 3). Ingenuity Pathway Analysis showed that changes of SF3B1 MUT -proE/basoE and polyE proteomes overlapped with changes of their transcriptomes (Fig. 2f, g ) such as base excision repair, nucleotide excision repair, oxidative stress response, protein ubiquitination pathways. MAP kinase pathway, pyrimidine de novo biosynthesis and iron metabolism were specifically deregulated in proE/basoE, while heme biosynthesis, cell cycle checkpoint control and cell cycle control of replication were deregulated in polyE (Fig. 2i ). Together, proteomic changes, notably those affecting components of DNA damage response kept the imprint of IRR-transcripts associated with SF3B1 mutation in erythroid precursors (Fig. 2j ).",
"section_name": "SF3B1-mutated erythroid precursors demonstrate characteristic proteomic signatures",
"section_num": null
},
{
"section_content": "RNA splicing may attenuate the probability of forming R-loops by reducing the homology between nascent RNAs and their DNA templates and/or by recruiting splicing factor that antagonize RNA:DNA hybrid formation [29] [30] [31] [32] [33]. We examined the profiles of R-loops genomewide in human primary basoE by performing DNA-RNA immunoprecipitation (DRIP) using S9. 6 antibody followed by sequencing in 5 SF3B1 MUT, 6 SF3B1 WT including 3 with low variant allele frequency, 3 with high risk-mutations (1 SRSF2 mutation, 1 SRSF2 and bi-allelic TET2 (biTET2) co-mutations, 1 NRAS and biTET2 co-mutations) and 4 controls. DRIP specificity was assessed by a pre-treatment with RNase H1 (RNH1). Stringent calling identified true-positive peaks (BH-adj P value < 0. 05) and we considered shared peaks between samples of each group. The number of shared peaks in SF3B1 MUT erythroblasts was significantly lower than SF3B1 WT or control cells (Fig. 3a ). Visualization of R-loop profiles of a 50 kb region on chromosome 7 demonstrated the overall reduction of the peaks in SF3B1 MUT sample compared to SF3B1 WT and controls (Fig. 3b ). We then compared the localization of the peaks to gene features between SF3B1 MUT and SF3B1 WT cells. In SF3B1 MUT, the proportion of R-loops decreased at 5'UTR, promotertranscription start site (TSS) and gene body (Fig. 3c ). R-loops remaining in SF3B1 MUT cells, were more frequently detected in intergenic regions and at 3'UTR of the genes (Fig. 3d ). As an example, the SUZ2 gene exhibited a reduced R-loop near its promoter in a SF3B1 MUTsample (Fig. 3e ). By contrast, at mitochondrial DNA loci, rDNA repeats, or at some loci like CPNE7 gene, peak intensity was found elevated in SF3B1 MUT -samples showing that their R-loop profiles were selectively changed (Supplementary Fig. 5a ). R-loops assemble dynamically at TSS and TTS where they contribute to the regulation of transcription [34] [35] [36]. Thus, we compared the mRNA level of genes overlapping shared R-loops at TSS, gene body, TTS and 3'UTR in SF3B1 WT and SF3B1 MUT -samples. The expression of genes in which R-loops were detected at TTS or 3'UTR in SF3B1 WT samples was similar in SF3B1 WT and SF3B1 MUT -cells while the expression of genes in which R-loops were detected at gene body in SF3B1 WTsamples increased in SF3B1 MUT -samples. By contrast, the expression of genes in which R-loops were detected at TSS in SF3B1 WT but not in SF3B1 MUT samples dramatically decreased in SF3B1 MUT -samples (Fig. 3f ). \n\nTo look for differential peaks between the groups, the count of restriction fragments overlapping with significant peaks was normalized using DESeq2. We confirmed a global decrease in the number of R-loops in SF3B1 MUT versus controls or SF3B1 WT basoE. In these analyses, the number of differential peaks (with log 2 (FC) > | 1| and BH-adj P value < 0. 05), was 4589 between SF3B1 MUT and controls with only 52 up Fig. 1 | Intron retention reduction correlates with transcriptomic changes of human SF3B1 MUT bone marrow mononuclear cells. RNA-sequencing data of 21 SF3B1 MUT and 6 SF3B1 WT (4 SRSF2 MUT and 2 SF WT ) bone marrow mononuclear cell samples were re-analyzed. a Volcano plot showing 1764 up or down-regulated genes in SF3B1 MUT samples (Log 2 (FC) <|1 | ; Two-sided Wald test and Benjamini-Hochberg (BH)-adjusted P value < 0. 05). b Gene Ontology (GO) enrichment analysis of the up and downregulated genes showing significantly enriched terms according to -log10(adjusted P value). Fisher's exact test corrected by false discovery rate (FDR) < 0. 05. Terms of interest are in bold. c Heatmap representing the clustering of samples by the variations of expression of a subset of 80 genes belonging to GO terms highlighted in (b). Genes affected by 1 to 8 differential splicing events are marked with an asterisk. d Barplot representing the number and types of differential splicing events in SF3B1 MUT in comparison to SF3B1 WT with ΔPSI > | 0. 10| using two-sided Wald test and BH-adjusted P value < 0. 05. The bars over 0 indicate the events upregulated in mutant cases and the bars under 0 indicate the events downregulated in wild type cases. e GO over-representation analysis of 296 significantly deregulated genes affected by 383 intron retention reductions. Fisher's exact test corrected by FDR < 0. 05. FC: fold-change. Source data are provided as a Source Data file. in SF3B1 MUT samples, and 19,394 between SF3B1 MUT and SF3B1 WT samples, of which only 33 were up-regulated in SF3B1 MUT cells (Fig. 3g ; Supplementary Data 4). In comparison to controls, 53% and 20% of the peaks lost in SF3B1 MUT cells were located in gene bodies (mostly centered on intronic sequences), and promoter-TSS, respectively (Supplementary Fig. 5b ). Similarly, in comparison to SF3B1 WT cells, 63% and 12% of the peaks lost in SF3B1 MUT cells were located in gene bodies and promoter-TSS (Fig. 3h ). In this latter comparison, we identified 7,039 unique genes affected by losses of R-loops either in their UTRs, TSS, TTS or gene bodies, which overlapped 345/498 (69%) of the recurrent IRRs shared by SF3B1 MUT samples (Fig. 3i ). For example, at RAD9A and IQGAP3 loci (Fig. 3j ) or at COASY locus (Supplementary Fig. 5c ), all occupied by an IRR, R-loops were significantly reduced in SF3B1 MUT cells. To validate these findings, we selected two genes (ABCC5 and TCIRG1) with IRR and two genes (IREB2 and TMX2) without IRR in SF3B1 MUT -erythroblasts. We performed DRIP-qPCR in basoE generated from 4 SF3B1 MUT MDS, 3 SF3B1 WT MDS including one MDS with U2AF1 mutation, and 4 healthy donors. Positive controls for R-loops detection were RPL13A and TFPT genes and for each sample, the specificity of the signal was assessed by RNH1 pre-treatment (Supplementary Fig. 6a, b ). R-loops detected at ABCC5 and TCIRG1 loci in healthy donor and SF3B1 WT cells were not or hardly detected in SF3B1 MUT -samples. By contrast, at IREB2 and TMX2 loci, the enrichment signal was faint, suggesting the absence of R-loop, whatever the sample (Fig. 3k ). \n\nAltogether, these results validate a link between SF3B1 mutation, decreased R-loop formation, intron retention reduction and deregulated gene expression.",
"section_name": "SF3B1 mutation induces a significant loss of R-loops in erythroid cells",
"section_num": null
},
{
"section_content": "Since the accumulation of R-loops is reported to slow down the DNA replication fork velocity and cell proliferation 37, loss of R-loops in SF3B1 MUT erythroblasts may reduce obstacles to fork progression, promote DNA replication and cell proliferation. To explore this, we expanded erythroblasts from CD34 + HSPCs of 5 SF3B1 MUT, 3 SRSF2 MUT or U2AF1 MUT, 3 SF WT -MDS and 4 healthy donors. Flow cytometry analysis showed significant increase in the BrdU intensity of S-phase cells (Fig. 4a, b ) and percentage of S-phase cells (Fig. 4c ) in SF3B1 MUT -basoE. \n\nTo further monitor DNA replication, we performed DNA combing in primary erythroblasts. We labelled basoE from 4 SF3B1 MUT, 2 SRSF2 MUT and 1 U2AF1 MUT (SF MUT ), 2 SF WT MDS and 2 healthy control samples with 5-iodo-2′-deoxyuridine (IdU) and then with 5-chloro-2′deoxyuridine (CldU) for 30 min each (Fig. 4d ). Incorporation of thymidine analogs allowed measurement of DNA fibers length and symmetry. We observed a significant increase of replication fork speed in SF3B1 MUT -erythroblasts (0. 75 ± 0. 35 kb/min) compared to SF MUT -(0. 60 ± 0. 25 kb/min), SF WT -(0. 53 ± 0. 21 kb/min) and healthy donor (0. 64 ± 0. 25 kb/min) erythroblasts while fork symmetry of SF3B1 MUTcells measured by IdU/CldU ratio remained similar to that in controls. Compared to SF3B1 MUT -cells, SF MUT -erythroblasts exhibited a slower and asymmetric fork progression (Fig. 4e, f ). Altogether, an accelerated replication fork speed defines the DNA replication stress of SF3B1 MUT -erythroblasts, whereas, it is associated with fork stalling in SRSF2 MUT or U2AF1 MUT cells 19, 21. \n\nTo address whether SF3B1 MUT -erythroblasts endure a DNA damage, we investigated the expression by immunofluorescence of phospho(p)-RPA32 serine 33 that is recruited on single-stranded (ss) DNA during DNA replication and phosphorylated by ATR, and of p-RPA32 serine 4/8 that is phosphorylated by DNA-PK to regulate replication stress checkpoint activation 38, 39. We also used pan DNAdamage markers γH 2 AX or 53BP1 in human basoE from SF3B1 MUT, SF MUT (SRSF2 MUT or U2AF1 MUT ), SF WT MDS and controls, at d11 of the culture (Fig. 4g-k ) or at different timepoints d9, d11, d13 and d15 (Supplementary Fig. 7a-c ). p-RPA s33, p-RPA s4/8, γH 2 AX and 53BP1 foci were detected in control erythroblasts treated with hydroxyurea (HU), SRSF2 MUT and U2AF1 MUT -erythroblasts. By contrast, γH 2 AX and 53BP1 foci were undetectable in SF3B1 MUT erythroblasts, while these cells were positive for p-RPA32s33 and p-RPAs4/s8 indicating ssDNA exposure. Of note, p-RPA32 s33 foci were significantly less abundant in SF3B1 MUT than in SRSF2 MUT cells. \n\nAltogether, these results show that accelerated fork velocity was observed when R-loops were lost in SF3B1 MUT -erythroblasts. Exposure of ssDNA without evidence for DNA damage marked by γH 2 AX/53BP1 indicates that SF3B1 MUT -erythroblasts endured a milder replication stress than SRSF2 MUT and U2AF1 MUT -erythroblasts.",
"section_name": "SF3B1 mutation promotes a DNA replication stress in human erythroblasts",
"section_num": null
},
{
"section_content": "To validate these findings in another model, we used the murine proerythroblastic cell lines G1E-ER4 CRISPR-Cas9 Sf3b1 K700E/+ and its isogenic G1E-ER4 Sf3b1 +/+, which could differentiate into basoE upon induction with estradiol of GATA1, with no excess of apoptosis (Supplementary Fig. 8a-f ) 11. RNA sequencing of the murine Sf3b1 K700E/+ and Sf3b1 +/+ erythroblasts identified 1226 (719 up and 507 down) and 1434 (574 up and 860 down) DEGs before (t0) and after (t24) induction of GATA1, with log2 (FC) > | 1| and BH-adj P value < 0. 05, respectively (Supplementary Data 5; Supplementary Fig. 9a, b ). We detected 1116 and 1301 genes affected by DSEs, mainly IRR, in Sf3b1 K700E/+ -proE and basoE, respectively (Supplementary Data 5; Fig. 5a ). Despite the substantial species specificity of RNA splicing, the deregulated pathways associated with DSE in murine cells seemed similar to those identified in human cells (Fig. 5b, upper panel; Fig. 2f ). Notably, DNA repair, cellular response to DNA damage and nucleic acid metabolic process GO terms gathered genes presenting IRR in Sf3b1 K700E/+ -proE (n = 272) or -basoE (n = 355) (Fig. 5b, bottom panel). Such deregulated pathways were conserved at protein level with deregulated expression of Lig1, Lig3, Pnkp, Parp1 in murine and human cells while DNA damage checkpoint proteins like Atm, Gmnn, Tp53bp1 were specifically deregulated in murine cells highlighting some differences between the two models (Fig. 5c ). \n\nTo address the functional consequences of these dysregulations, we compared the proliferation of the G1E-ER4 clones. Before GATA1 induction, the proliferation of Sf3b1 K700E/+ proerythroblasts was significantly higher than that of Sf3b1 +/+ cells and remained higher upon induction (P < 0. 0001, Fig. 5d ). The differentiation to basoE was significantly lower in Sf3b1 K700E/+ cells (Fig. 5e, f ). In accordance with this, Sf3b1 K700E/+ cells showed a higher BrdU incorporation and a higher G1/S fraction than Sf3b1 +/+ cells, before and after induction (Fig. 5g ). Concomitantly to GATA1 induction, when a mild replication stress was imposed by inducing a cell cycle arrest in early S-phase with Erythroid precursors were expanded in culture from SF3B1 MUT, SF WT MDS and controls samples. a Schematic representation of the protocol. b Erythroid differentiation evaluated on May-Grünwald Giemsa-stained cytospins. Histograms representing the proportion of erythroid precursors in up to 7 controls, 11 SF3B1 MUT and 7 SF WT independent samples at days (d)7-8, 9-10, 11-13 and 14-16. Results are expressed as means ± standard error of the mean. 2-way ANOVA for multiple comparisons. Controls versus SF3B1 MUT, P = 0. 017; controls versus SF WT, P = 0. 012. c Variant allele frequencies of SF3B1 mutation in erythroblasts at d7, d11-13 and d14-15 of 14 independent SF3B1 MUT samples. d Volcano plot representing up-and downregulated transcripts in SF3B1 MUT basoE and polyE compared to SF WT ones. Twosided Wald-test and BH-adjusted P value < 0. 05. e Venn diagram representing the numbers of differentially expressed genes between SF3B1 MUT and SF WT samples at basoE and polyE stages. f Gene Ontology (GO) enrichment analysis of up-and down-regulated genes in SF3B1 MUT versus SF WT erythroblasts. Fisher's exact test corrected by false discovery rate (FDR) < 0. 05. Specific terms to basoE or polyE as blue or red bars, respectively, shared terms as violet bars. g Gene set Enrichment Analysis (GSEA) showing terms deregulated in SF3B1 MUT basoE. h Barplots representing numbers and types of differential splicing events in SF3B1 MUT versus SF3B1 WT basoE and polyE with ΔPSI > | 0. 10| using two-sided Wald-test and BH-adjusted P value < 0. 05. Bars over 0 indicate events upregulated and bars under 0 indicate events downregulated in SF WT erythroblasts. i Venn diagram of intron retention reductions (IRR) in SF3B1 MUT basoE and polyE. j. GO terms overrepresented among genes with IRR in SF3B1 MUT basoE and polyE. Fisher's exact-test corrected by FDR < 0. 05. k Volcano plots representing differentially expressed proteins in SF3B1 MUT versus SF WT samples at proE/basoE and polyE stages (Wald-test, BHadjusted P value < 0. 05). l Cytoscape representation of Ingenuity Pathway Analysis showing deregulated pathways in SF3B1 MUT versus SF3B1 WT samples (P values < 0. 05 by Student t-test) either basoE-specific (blue dots), polyE-specific (red dots) or shared (violet dots). Scale: dot size proportional to -log10 (adjusted-P value). Source data are provided as a Source Data file. \n\naphidicolin, Sf3b1 K700E/+ compared to Sf3b1 +/+ cells exhibited a significantly higher G1/S phase fraction (Fig. 5h, right panel). Inhibition of dNTP biosynthesis by 0. 2 mM hydroxyurea (HU) for 16 h, normalized BrdU incorporation in Sf3b1 K700E/+ cells after induction (Fig. 5i ). In metabolomic analysis, the quantities of dATP, dCTP and dTTP before induction were equivalent in Sf3b1 K700E/+ to Sf3b1 +/+ cells, showing that DNA replication stress was not related to nucleotide pool depletion (Fig. 5j ). After induction of differentiation with estradiol, dNTP quantities increased in Sf3b1 +/+ cells, consistent with a slowdown of DNA synthesis. In Sf3b1 K700E/+ cells, lower dNTP quantities argued for persistent DNA synthesis (Fig. 5j ; Supplementary Fig. 9c ). \n\nUpon induction of differentiation, more Sf3b1 K700E/+ cells were positive to p-Rpa32s4/s8 labelling with a higher sensitivity to HU treatment than Sf3b1 +/+ -cells (Fig. 5k ). In estradiol-treated cells, Western blot confirmed the engagement of Rpa, but did not show phosphorylation of Chk1 suggesting that the Atr-Chk1 pathway was not activated (Fig. 5l ). Finally, the delayed differentiation of Sf3b1 K700E/+ murine erythroblasts can be partially rescued by lowering their high rate of DNA synthesis with HU at the expense of cell viability (Supplementary Fig. 9d, e ).",
"section_name": "Sf3b1 K700E/+ in murine erythroblasts reproduces DNA replication stress",
"section_num": null
},
{
"section_content": "To establish a link between the loss of R-loops and the phenotypic characteristics of fork velocity and replication stress in human primary SF3B1 MUT erythroblasts, we thought to modulate the level of R-loops in the cell. Previous works have established that the THO complex which contributes to prevent R-loop accumulation interacts with SIN3Ahistone deacetylase complex. Furthermore, inhibiting histone deacetylase activity by depleting SIN3A or treating the cells with trichostatin A stabilizes R-loops 40. We used pan-HDAC inhibitor Suberoylanilide hydroxamic acid (SAHA)/vorinostat (further denoted HDACi) in DRIPseq experiment. Human erythroblasts from 3 SF3B1 MUT, 3 SF3B1 WT (1 SRSF2, 1 SRSF2/biTET2 or 1 NRAS/biTET2 mutations), and 4 controls samples were pre-treated with HDACi 0. 5 μM for 20 h, at day11 of culture. The numbers of shared peaks increased in 3/4 controls, even not significantly. HDACi treatment restored the level of R-loops of 2/3 SF3B1 MUT erythroblast samples up to normal (Fig. 6a ; Supplementary Data 6). Unexpectedly, the numbers of R-loops counted in SRSF2 or TET2/NRAS mutated samples collapsed almost entirely (P < 0. 001). In SF3B1 MUT samples, the number of shared R-loops was quantitatively important in the gene bodies. The augmentation of R-loops also affected intergenic regions, 3'UTR and TTS more than 5′UTR or promoter-TSS (Fig. 6b ). We visualized the changes in R-loop profiles at specific loci. As shown in Fig. 6c and Supplementary Fig. 10, HDACi treatment produced large R-loops near the promoter of BCL2L1, PTPN11, ARPC3 and NCOA4 genes specifically in SF3B1 MUT cells. By contrast, HDACi did not change the profile of R-loops in SF3B1 MUT cells at HK1 locus (Supplementary Fig. 10 ). To verify whether, by rescuing Rloops, gene expression may change, we performed RT-qPCR at these 4 loci. Upon treatment with HDACi, the expression of BCL2L1 increased significantly in SF3B1 MUT. While the expression of NCOA4 and PTPN11 also tended to increase, HK1 did not (Fig. 6d ). These data suggest the relationship between R-loops and gene expression in these cells. \n\nThen we wondered whether the effect of HDACi could be at least partly due to a modification of IRR profiles. To explore this hypothesis, we selected 5 genes with known IRR (PPOX, PPM1A, COASY, S100A4, BCL2L1) and performed RT-PCR to visualize the IRR-transcripts and the spliced isoforms in 4 SF3B1 MUT, 6 SF3B1 WT and 2 control erythroblast samples. HDACi did not modify the pattern of transcripts in controls and SF3B1 WT samples. The abundance of IRR-transcripts in SF3B1 MUT remained similar in the presence or absence of HDACi suggesting that the restoration of R-loops observed under this treatment did not depend on intron retention (Fig. 6e ). We confirmed these results for BCL2L1 and COASY transcripts by fluorescent PCR fragment analysis (Supplementary Fig. 11a, b ). \n\nTo evaluate the impact of R-loop restoration on fork progression, we performed DNA combing of 3 samples of erythroblasts treated or not with 0. 2 μM HDACi (1 control, 2 SF3B1 MUT including one with a frameshift mutation in the histone acetyltransferase EP300). The fork velocity in SF3B1 MUT erythroblasts was high and decreased significantly after HDACi treatment. By contrast, neither the SF3B1 MUT /EP300 MUT sample nor the control showed variations of fork velocity after HDACi (Fig. 7a ). To assess DNA synthesis in a larger number of patients, we performed BrdU assays. The percentage of S-phase cells decreased significantly in SF3B1 MUT cells. However, the BrdU intensity higher in SF3B1 MUT samples did not change (Fig. 7b ). Then, we verified the impact of HDACi (0. 5 μM 20 h) on the frequency of p-RPA32 s33 and γH2AX foci. No increase of positive cells was observed in control, SF3B1 MUT or SF3B1 WT erythroblast samples suggesting that HDACi at the concentration used did not provide DNA damage to these cells (Fig. 7c-e ). \n\nFinally, we studied the consequences of HDACi treatment on erythroid cell differentiation. At progenitor level, HDACi did not change the number or size of the BFU-E type colonies formed in methylcellulose (Fig. 7f ). Looking at mutations in single colonies we did not observe any clonal selection during the 14 days of semi-solid culture (Supplementary Fig. 11c ). By contrast, HDACi drastically forced the maturation of erythroblasts at late stage as shown by a significant increase of mature erythroblasts (Fig. 7g, h ). This effect was also confirmed by the increase of GPA + CD49d low cell proportion corresponding to orthochromatic erythroblasts (Fig. 7i ). Interestingly we did not observe any inhibitory effect of HDACi at 0. 5 μM on the overall rate of expansion of SF3B1 MUT erythroid precursors compared to SF3B1 WT or control erythroblasts (Supplementary Fig. 11d ), or increase of cell death in SF3B1 MUT, SF3B1 WT, and control cell cultures (Fig. 7j ). Altogether, these results showed that HDACi by producing R-loops, slowed DNA replication and facilitated erythroid cell differentiation without altering cell proliferation.",
"section_name": "Targeting of R-loops improves the differentiation of human SF3B1 MUT erythroblasts",
"section_num": null
},
{
"section_content": "The present study shows that SF3B1 mutations causing an increased proliferation of immature erythroblasts with a reduced capacity to terminal differentiation trigger a replication stress with ssDNA exposure in erythroid cells. Accelerated DNA replication fork velocity is observed when R-loops are lost. HDAC inhibition restores R-loops and decreases fork speed, which correlates with erythroid differentiation improvement. These features distinguish SF3B1 mutation from other splicing factor mutations. \n\nAlternative splicing of pre-mRNA contributes to physiological hematopoiesis [26] [27] [28] 41. Int on retentions increase along terminal steps of erythroid differentiation 28. Intron retention modulates gene fragments overlapping with peaks between SF3B1 MUT and control samples (left panel) and SF3B1 MUT and SF3B1 WT samples (right panel) with log 2 (FC) >|1| using twosided Wald-test and a BH-adjusted P value < 0. 05. h istribution to gene features of differential R-loops in SF3B1 WT samples and lost in SF3B1 MUT samples. i Venn diagram showing overlap between genes that lost one R-loop and genes with intron retention reduction (IRR) in SF3B1 MUT erythroblasts. j DRIP-seq and RNA-seq overlays at RAD9A and IQGAP3 loci showing R-loop losses and IRR events in SF3B1 MUT erythroblasts. Gene structures using GENCODE GRCh37. k DRIP-qPCR analysis of 4 controls, 3 SF3B1 WT including 1 U2AF1 MUT designated as green dot and 4 SF3B1 MUT samples. Enrichment signals (normalized to input) at specific loci were normalized to EGR1 (no R-loop). RPL13A and TFPT as positive controls. In box plots, central lines represent medians, bounds represent lower and upper quartiles and whiskers correspond to min-max values. Two-sided unpaired t-test for P values (see Suppl informations). b, e, j RPM: reads per million. * P < 0. 05; ** P < 0. 01; **** P < 0. 0001; ns: not significant. Source data are provided as a Source Data file. \n\nexpression by generating transcripts that are either detained in the nucleus or degraded in the cytoplasm by the NMD 41, 42. Here, we show that SF3B1 MUT promotes a reduction of intron retention in erythroblasts and that the number of retained introns lost in SF3B1 MUT -erythroid precursors increases between basoE and polyE. By reversing a physiological process, SF3B1 mutation changes gene expression profile and reshapes the proteome, affecting several pathways such as DNA replication, DNA repair mainly base excision repair and nucleotide biosynthesis. SF3B1 MUT -erythroblasts retain proliferative capacities, which contributes to enrichment of the bone marrow in immature erythroblasts and to defective production of mature erythroblasts, defining ineffective erythropoiesis. \n\nTo synthesize DNA properly, the replication machinery must overcome several obstacles, including R-loops and transcription complexes 37. Most of the R-loops located at promoter-TSS, in gene bodies and intergenic regions in SF3B1 WT -erythroblasts were lost in SF3B1 MUT -erythroblasts, in contrast with the augmented R-loops detected in SRSF2 MUT erythroblasts. Previous studies in cell lines or in primary CD34 + SF MUT progenitors using single-cell imaging with S9. 6 antibody have shown that not only SRSF2 MUT or U2AF1 MUT, but also SF3B1 MUT cells may produce undesirable R-loops [19] [20] [21]. However, S9. 6 foci may indicate RNA:DNA hybrids and also double-stranded RNA which are more abundant, making difficult the quantification of R-loops even when RNaseH1 is overexpressed in cell lines to assess the specificity of the signals 19, 43, 44. Here we used DRIP-seq to obtain a genome-wide landscape of R-loops. This technique allowed us to identify short type I R-loops located at GC-skewed promoter-proximal regions, or in intergenic regions that may contain active enhancers 21, which are preferentially detected by R-ChIP, and also, large type II R-loops (spanning over 300 bp to 1 kb) distributed along the body of transcribed genes 45, 46. The decreased number of R-loops at promoters-TSS and gene bodies in SF3B1 MUT cells suggest that both large and short R-loops are lost. As the GC skew characteristic of R-loops at promoter progressively diminishes after the first exon/intron junction, the mechanism of R-loop formation may be different in gene bodies 45. Intron retention, by increasing homology between nascent RNA and its DNA template can initiate the co-transcriptional formation of large R-loops spreading over the gene coding sequence 22, 29, 47, 48. Conversely, the binding of splicing machinery making intron excision hinders R-loop accumulation 30, 33, 49. Increased intron excision occurring when SF3B1 is mutated may suppress R-loops along the gene bodies. We report here that most of IRR overlap with lost R-loops. However, because R-loops were lost also in promoter-TSS, intergenic regions and TTS, IRR is not the unique mechanism for R-loop loss in these cells. \n\nThe presence of unscheduled R-loops at promoter-TSS as a consequence of RNA polymerase II pausing usually correlates with high gene expression 45. R-loop-positive regions overlap with DNase I-hypersensitive regions, indicative of open chromatin 21, 45. Our integrative analysis of R-loops and gene expression associated the loss of R-loops with low transcript level in SF3B1 MUT erythroblasts. Importantly, we identified BCL2L1, a GATA1 and STAT5 target gene, which expression increased, without splicing changes, when R-loops formed near its promoter under HDACi treatment. This is consistent with the interference of R-loops with transcription. In addition, since SF3B1 protein together with U2AF1 and SRSF2 were detected in the vicinity of R-loops at promoters 50, the SF3B1 mutant protein, could notably modify the kinetics of transcription elongation as generally reported for splicing factors 51 Replication stress that appears at the early stages of malignant cell transformation, has been linked to events that impair DNA synthesis integrity such as reduced or increased origin firing, nucleotides or replication factor depletion and replication-transcription conflicts 52, 53. Replication stress can produce stalled forks, under-replicated DNA, or supra-acceleration of forks 54. As opposed to SRSF2 MUT or U2AF1 MUT cells in which fork progression is slowed down, we detected an accelerated fork speed when R-loops were lost in SF3B1 MUT -erythroblast. Alternative causes of increased fork velocity were associated with overexpression of oncogenes, downregulation of mRNA biogenesis, or PARP1 inhibition [55] [56] [57] [58]. Moreover, activation of oncogenic HRAS in presenescent cells was shown to accelerate forks by inducing overexpression of topoisomerase 1 (TOP1), which is known to resolve unwanted R-loops 59. TOP1 was heavily expressed in SF3B1 MUT and SF3B1 WT erythroblasts, while senataxin and THO complex proteins, other R-loop modulators were specifically upregulated in SF3B1 MUT erythroblasts and could contribute to fork velocity 60. Furthermore, if the transcription rate decreases when R-loops are resolved, not only R-loop loss but also the reduction of transcription-replication conflicts could prevent replication fork stalling 61, 62. \n\nThe detection of p-RPA32 foci without DNA damage marks γH2AX or 53BP1 suggested replicative ssDNA gaps. ATR continuously monitors the recruitment of RPA32 on ssDNA within the replisomes and its phosphorylation on serine 33, independently of CHK1 63. SF3B1 MUTerythroblasts endure a mild replication stress without engagement of the CHK1 pathway (Fig. 5l ). In cancer cells, the tolerance to replication stress is supported by the overexpression of the upstream components of ATR-CHK1 pathway, Clapsin and Timeless, independently of ATR signaling 64. As SF3B1 MUT -cells overexpressed Claspin and Timeless genes and Timeless protein, we cannot exclude their role in the mechanism of replication stress tolerance. Alternatively, RPA could bind the displaced ssDNA of R-loops and recruit RNaseH1 to facilitate its resolution 65. Further studies are needed to test these hypotheses. \n\nScreening for synthetic lethal approaches using SF3B1 MUT -cell lines have revealed potential vulnerabilities by targeting of ATR, PARP1 or NMD 19, [65] [66] [67]. An alternative, more conservative approach to improve ineffective erythropoiesis could be slightly restoring R-loops. The formation and degradation of R-loops involve multiple actors that include splicing factors, topoisomerases, DNA/RNA helicases, DNA repair molecules (BRCA1, BRCA2 or FANCD2/A/M), RNaseH1 or H2 which degrade R-loops directly, or SAMHD1 (alpha motif and HDdomain containing protein 1) that promotes ssDNA degradation at stalled forks 68, 69. Chromatin accessibility also interferes with R-loop generation 62. In accordance, R-loops were shown to increase in HDACitreated or SIN3A-depleted cells 40. We show here that R-loops reappeared in SF3B1 MUT cells treated with HDACi, which correlated with significant increase of BCL2L1 expression and improvement of erythroid differentiation without evidence of DNA damage. Since clinical trials have shown a limited hematotoxicity of vorinostat alone compared to its combination with azacitidine 70, 71, our results provide a rationale for testing if low doses of vorinostat could improve erythropoiesis of patients with SF3B1 MUT MDS. SF MUT vs SF3B1 MUT, P < 0. 0001; SF WT vs SF MUT, P = 0. 0009; SF WT vs controls, P < 0. 0001; SF MUT vs controls, P = 0. 002. f Scatter plot showing fork symmetry as ratios of IdU/CldU length expressed in means ± SD. Two-sided Mann-Whitney test. Controls vs SF3B1 MUT, P = 0. 302; SF WT vs SF3B1 MUT, P < 0. 0001; SF MUT vs SF3B1 MUT, P = 0. 005; SF WT vs SF MUT, P = 0. 0003; SF WT vs controls, P < 0. 0001; SF MUT vs controls, P = 0. 0004. g-k Immunofluorescence experiments in MDS or control erythroblasts treated or not with 5 mM hydroxyurea (HU). g Representative images of phospho(p)-RPA32s33, p-RPA32s4/s8, γH2AX and 53BP1 at d11. Nuclei were labeled with DAPI. Magnification 100X (scale: 20 µm). h-k Quantification of positive cells with >5 intranuclear foci. h p-RPA32s33 (2 controls, 6 SF3B1 MUT, 7 SF MUT, 5 SF WT ). i p-RPA32s4/8. j γH2AX. k 53BP1 (4 controls, 9 SF3B1 MUT, 7 SF MUT, 3 SF WT ). Results are expressed as mean percentages of positive cells ± SD. Two-sided unpaired ttests; * P < 0. 05; ** P < 0. 01; *** P < 0. 001; **** P < 0. 0001; ns: not significant. Source data are provided as a Source Data file.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "7 U2AF1 MUT, 25 triple-negative as SF WT ) and 24 age-matched healthy subjects as controls was enrolled between 2015 and 2023. Bone marrow (BM) aspirates were collected after each patient gave his informed consent for biological investigations according to the recommendations of institutional review board (IRB) and ethics committee (IRB numbers: IdFV 212-A01395-38 EudraCT 2012-002990-7338; OncoCCH 2015-08-11-DC). BM aspirates or femoral head samples were collected from healthy subjects. Patient characteristics including age, gender, WHO, hemogram, BM blast, erythroblast and ring sideroblast percentages, karyotype, IPSS-R are indicated in (Table 1 ).",
"section_name": "Patients",
"section_num": null
},
{
"section_content": "BM mononuclear cells (MNC) were purified on Ficoll gradient and were processed for DNA extraction using the DNA/RNA Kit (Qiagen, Hilden, Germany). Mutations in SF3B1 were screened by Sanger sequencing or next generation sequencing of a panel of 37 genes (ASXL1, ATM, BCOR, BCORL1, BRAF, CBL, CEBPA, CUX1, DDX41, DNMT3A, EP300, ETV6, EZH2, FLT3, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, NRAS, MPL, NPM1, PHF6, PTPN11, RAD21, RIT1, RUNX1, SETBP1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, ZRSR2).",
"section_name": "Genomic studies",
"section_num": null
},
{
"section_content": "BM CD34 + -derived erythroblasts were expanded from 92 MDS (49 SF3B1 MUT and 43 SF3B1 WT including 24 SF WT, 13 SRSF2 MUT and 6 U2AF1 MUT ) and 24 healthy donors as controls. For erythroid cell expansion, CD34 + cells were isolated from the MNC fraction of BM samples using the MidiMacs system (Miltenyi Biotec, Bergisch Gladbach, Germany). CD34 + cells, which purity was higher than 80% were then cultured at 0. 8 × 10 6 per mL for 4 days in Iscove's modification of Dulbecco medium (IMDM; Thermo Fisher Scientific, Waltham, MA) containing 15% BIT9500 (Miltenyi Biotech), 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine (all from Thermo Fisher Scientific), 2 UI/mL recombinant human erythropoietin (rHu Epo; Roche, Basel, Switzerland), 100 ng/mL stem cell factor (SCF; Miltenyi Biotech), 10 ng/mL interleukin 6 (IL6; Miltenyi Biotech) and 2. 10 -7 M dexamethasone (Merck, Darmstadt, Germany). Cells were diluted every day in the same medium until day 4. From day 4, IL6 was removed. From days 10 to 16 cells were switched to rHu Epo (2 UI/mL) to obtain terminal erythroid differentiation. \n\nThe murine G1E-ER4 cell line expressing a GATA1-estrogen receptor fusion gene 72 was cultured in IMDM, containing 20% fetal calf serum (FCS; GE Healthcare, Chicago, IL), 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 2 U/mL Epo, SCF in Chinese Hamster Ovary cell conditioned medium, monothioglycerol and 0. 5 µg/mL puromycin (Merck, Darmstadt, Germany) to select cells expressing the GATA1-ER fusion gene. This cell line was used to edit mutant Sf3b1 K700E and isogenic Sf3b1 WT using CRISPR-Cas9 strategy 11. \n\nFor some experiments, cells were arrested in G1/S phase with 0. 6 µg/mL aphidicolin (APH, Merck, Darmstadt, Germany, cat no. 38966-21-1) or with 0. 2 mM hydroxyurea (HU, Merck, cat no. H8627), or treated with 0. 1 to 1 μM histone deacetylase inhibitor SAHA/vorinostat (HDACi, Merck, cat no. #SML0061) for 20 h.",
"section_name": "Primary cells and cell lines cultures",
"section_num": null
},
{
"section_content": "RNA-seq data from a first cohort of BM MNC from 27 lower-risk (LR)-MDS (21SF3B1 MUT and 6 SF3B1 WT (4 SRSF2 MUT and 2 SF WT ) previously published 2 were re-analyzed. RNA-sequencing of two additional cohorts was performed: one cohort of BM MNC from 185 LR-MDS (74 SF3B1 MUT, 30 SRSF2 MUT, 11 U2AF1 MUT, 70 triple-negative samples) and one cohort of basoE and/or polyE obtained from 13 MDS (8 SF3B1 MUT and 5 SF3B1 WT ). RNA integrity (RNA integrity number ⩾ 7. 0) was checked on the Agilent Fragment Analyzer (Agilent, Santa Clara, CA) and quantities were determined using Qubit (Invitrogen, Waltham, CA). 50-100 ng of total RNA sample was used for poly-A mRNA selection using oligo(dT) beads and subjected to thermal fragmentation. For BM MNC, MuLV Reverse Transcriptase (Invitrogen) was used for cDNA synthesis. Libraries were constructed using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA) and sequenced on an Illumina HiSeq 2500 platform using a 100-bp pairedend sequencing strategy. For RNA-sequencing of erythroblasts, fragmented mRNA samples were subjected to cDNA synthesis, converted into double stranded DNA using SureSelect Automated Strand Specific RNA Library Preparation Kit. Libraries were bar-coded, and subjected to 100-bp paired-end sequencing on Novaseq-6000 sequencer (Illumina, San Diego, CA). For RNA-sequencing of murine G1E-ER4 erythroblasts, libraries were constructed using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina) and sequenced on an Illumina NextSeq500 platform (Illumina) using a 75-bp paired-end sequencing strategy.",
"section_name": "RNA-sequencing",
"section_num": null
},
{
"section_content": "FASTQ files were mapped using STAR (v2. 7. 9) to align the reads against the human reference genome GRCh37 (UCSC version hg19) downloaded from the GENCODE project website. Reference genome and annotations are available on Gencode (https://www. gencodegenes. org/ human/release_19. html) 73. Read count normalizations and groups comparisons were performed using DESeq2 (v1. 30. 1), with the Wald test for significance testing. Genes with low counts were filtered, if at least half the samples have less than 20 normalized reads. Outliers removal with Cook's distance was left at default. For differential expression study, the results obtained after DESeq2 comparison were selected for further analysis and filtered at Benjamini-Hochberg (BH)adjusted P value < 0. 05 and log2(FC) > | 1 | 74. To identify differentially expressed splicing events, we used KisSplice (v2. 6. 2), KisSplice2refgenome (v2. 0. 7) and KissDE (v1. 15. 3) 75, 76, and filtered results at BHadjusted P value < 0. 05 and ΔPSI > |0. 10 |. Biological processes associated with genes that were differentially expressed or spliced were determined using the Gene Ontology enrichment or overrepresentation analyses and Gene Set Eenrichment analysis with reference to specific gene sets (Supplementary Data 7).",
"section_name": "Bioinformatic analysis of RNA-sequencing",
"section_num": null
},
{
"section_content": "Sample preparation was done using the FASP procedure 77. Briefly, cells were solubilized in 100 μL of Tris/HCl 100 mM pH8. 5 buffer containing 2% sodium dodecyl sulfate (SDS). Total protein amounts were quantified using BiCinchoninic acid Assay (BCA; Thermo Fischer Scientific). Then, 10 mM TCEP and 40 mM chloroacetamide were added and the samples were boiled for 5 min. 50 μg of proteins were sampled and treated with urea to remove SDS. After urea removal, proteins were digested overnight with 1 μg of sequencing-grade modified trypsin Fig. 5 | DNA replication stress in murine G1E-ER4 Crispr-Cas9 Sf3b1 K700E/+ proerythroblasts. a Barplots representing numbers and types of differential splicing events in Sf3b1 K700E/+ (clone 5. 13) versus Sf3b1 +/+ (clone 9. 82) cells at t0 (proE) and t24 (basoE) after induction of differentiation with estradiol (ΔPSI > | 0. 10| using two-sided Wald test and BH-adjusted P value < 0. 05). b Gene Ontology (GO) overrepresentation analysis. Upper panel: Pathways involving differentially spliced genes in Sf3b1 K700E/+ cells shared at t0 and t24 (violet bars), specific to t0 (blue bars) or t24 (red bars). Bottom panel: Pathways involving genes with IRR in Sf3b1 K700E/+ cells. Shared GO terms at t0 and t24. Fisher's exact test corrected by false discovery rate (FDR) < 0. 05. c Ingenuity Pathway Analysis of differential proteins at t0 (Student t-test, P values < 0. 05. Canonical Pathways (hexagons), Diseases and Functions pathways (circles). d Live cell imaging. Mean percentages (± SD) of confluence (n = 3). 2-way ANOVA test for multiple comparisons. e Differentiation of Sf3b1 K700E/+ (n = 4) and Sf3b1 +/+ cells (n = 3) by flow cytometry. Mean percentages of Ter119 + Kit low cells ± SEM. Unpaired t-test for multiple comparisons. t24h: q = 0. 033; t36h: q = 0. 027; t48h: q = 0. 007. f May-Grünwald-Giemsa-stained cytospins. g BrdU incorporation in S-phase ± estradiol 24 h. Medians ± 95% confidence intervals (CI) of RFI anti-BrdU antibody/control Ig (5 independent experiments). Sf3b1 +/+ vs Sf3b1 K700E/+, P = 0. 002; Sf3b1 +/+ +estradiol vs Sf3b1 K700E/+ +estradiol, P = 0. 001; Sf3b1 +/+ vs Sf3b1 K+/+ +estradiol, P = 0. 011. h Cell cycle analysis by BrdU incorporation ± aphidicolin (APH). Median percentages ( ± 95% CI) of G1/S-phase cells (4 independent experiments). Left: Sf3b1 +/+ vs Sf3b1 K700E/+, P = 0. 035; Sf3b1 +/+ vs Sf3b1 +/+ +APH, P = 0. 002. Right: Sf3b1 +/+ vs Sf3b1 K700E/+, P = 0. 007; Sf3b1 +/+ +APH vs Sf3b1 K700E/+ +APH, P = 0. 002; Sf3b1 +/+ vs Sf3b1 +/+ +APH, P = 0. 029; Sf3b1 K700E/+ vs Sf3b1 K700E/++ APH, P = 0. 029. i BrdU incorporation in S-phase ± hydroxyurea (HU). Medians ± 95% CI of RFI estradiol-treated/untreated cells (5 independent experiments). j dNTP relative quantities. Medians ± 95%CI (4 independent experiments). g-j Two-sided unpaired t-test. k Immunofluorescence of pRpa32s4/8 ( ± estradiol 24h, HU 16 h). l Western blot of pRpa32s4/s8, Rpa32, pChk1s345 and Chk1. Actin as loading control. k, l Representative of 3 independent experiments. **** P < 0. 0001, *** P < 0. 001, ** P < 0. 01, * P < 0. 05; ns not significant. Source data are provided as a Source Data file. \n\n(Promega, Madison, Wi) in 50 mM Tris/HCl pH8. 5 buffer. Peptides were recovered by filtration, desalted on C18 reverse phase StageTips and dried. They were then separated in 5 fractions by strong cationic exchange StageTips and analyzed using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Peptides from each fraction were separated on a C18 reverse phase column (2 μm particle size, 100 A pore size, 75 μm inner diameter, 25 cm length) with a 170 min gradient starting from 99% of solvent A containing 0. 1% formic acid in milliQ-H 2 O and ending in 55% of solvent B containing 80% acetonitrile and 0. 085% formic acid in milliQ-H 2 O. The mass spectrometer acquired data throughout the elution process. The MS1 scans spanned from 350 to 1500 Th with 1. 10 6 Automatic Gain Control (AGC) target, 60 ms maximum ion injection time (MIIT) and resolution of 60 000. MS Spectra were recorded in profile mode. High energy Collision Dissociation (HCD) fragmentations were performed from the most abundant ions in top speed mode for 3 seconds with a dynamic exclusion time of 30 s. Precursor selection window was set at 1. 6Th. HCD Normalized Collision Energy was set at 30% and MS/MS scan resolution was set at 30000 with AGC target 1. 10 5 within 60 ms MIIT.",
"section_name": "Sample preparation and mass spectrometry analysis",
"section_num": null
},
{
"section_content": "The mass spectrometry data were analyzed using Maxquant version 2. 1. 1. 0 78. The database used was a concatenation of Human sequences from the Uniprot-Swissprot database (Uniprot, release 2022-05) and the list of contaminant sequences from Maxquant. Cystein carbamidomethylation was set as constant modification and acetylation of protein N-terminus and oxidation of methionine were set as variable modifications. Second peptide search and the \"match between runs\" (MBR) options were allowed. False discovery rate (FDR) was kept below 1% on both peptides and proteins. Label-free protein quantification (LFQ) was done using both unique and razor peptides with at least 2 peptides ratios required for LFQ. \n\nStatistical analysis was done using \"R\". Among identified proteins, those with at least 70% of values in at least one condition were selected. Then, proteins with t-test P value < 0. 05 were defined as significantly differentially expressed, and proteins with 100% of valid values in one condition and 0% of valid values in the other condition were defined as \"Appeared\" or \"Disappeared\". Log2(LFQ intensity) matrix were filtered before imputation. For heatmaps, Z-score were calculated after imputation. \n\nFunctional analyses were generated through Ingenuity Pathway Analysis (QIAGEN Inc., https://www. qiagenbioinformatics. com/ products/ingenuitypathway-analysis) version 76765 844 for each list of differential proteins. Significantly over-represented biological terms (Canonical pathways or Diseases and functions) were identified with a right-tailed Fisher's Exact test that calculates an overlap P value determining the probability that each term associated with our lists of differential proteins was due to chance alone. The z-score is a statistical measure of correlation between relationship direction and experimental protein expression. Its calculation assessed the activation (positive z-score) or repression (negative one) of each term. To be considered significant the z-score has to be greater than 2 in absolute value. Overlap of selected pathways and functions was designed using Cytoscape version 3. 9. 1.",
"section_name": "Proteomic data analysis",
"section_num": null
},
{
"section_content": "DRIP-qPCR or DRIP-seq was performed on human CD34-derived proE/ basoE from 6 MDS with SF3B1 mutation, 6 MDS without SF3B1 mutation and 4 healthy controls 79. Briefly, 8×10 6 cells were harvested and processed for genomic DNA extraction by SDS/ Proteinase K treatment at 37 °C followed by phenol-chloroform extraction and ethanol precipitation. 8 μg of genomic DNA was fragmented using HindIII, EcoRI, BsrGI, XbaI, and SspI. Then DNA pre-treated or not with 16 UI/mL RNase H1 (New Engl Biolabs, Ipswitch, MA) overnight was immunoprecipitated using S9. 6 antibody at 40 μg/mL (Kerafast, Shirley, MA or ATCC mouse hybridoma) overnight at 4 °C (Supplementary Methods 1). Quality control of immunoprecipitation was performed by qPCR, at R-loop-positive loci (RPL13A, CALM3, TFPT) and R-loopnegative loci (EGR1, SNRP1). DRIP-qPCR was also performed at specific loci (ABCC5, IREB2, TCIRG1, TMX2) (Supplementary Method 2). \n\nPercentage of input expected between 1-15% %input = 100 × 2 ðCt input corrected À Ct DRIPed DNAÞ where Ct ðcycle thresholdÞ input ðcorrectedÞ = ðCt input À log 2ð10ÞÞ and fold enrichment (expected between 20-300)\n\nFold enrichement = ½2 ðCt input ðpositive locus correctedÞÞ À Ct DRIPed ðDNA positive locusÞ = ½2 ðCt input negative locus ðcorrectedÞÞ À Ct DRIPed DNA ðnegative locusÞ were calculated to assess the immunoprecipitation efficiency and specificity, respectively. For DRIP-seq, good quality DRIPed DNA samples (with and without RNase H1-treatment) were sonicated to get an average length of 200-300 bp. Then samples were subjected to endrepair, dATP tailing, adaptor ligation and library indexing. Lastly, the libraries were cleaned up using AMPure beads, and amplified. After checking library quality on Agilent Bioanalyzer using Agilent High Sensitivity DNA 1000 kit, libraries were sequenced on Illumina Nova-Seq instruments.",
"section_name": "DRIP-qPCR and DRIP-sequencing",
"section_num": null
},
{
"section_content": "100 bp paired-end reads were trimmed and filtered using fastp (v0. 23. 4) to remove low quality reads, low complexity sequences, as well as polyG tails (corresponding to no signal in the Illumina two-color systems, in NovaSeq data). Reads were mapped to the human reference genome (GENCODE, GRCh37; https://www. gencodegenes. org/ human/release_19. html) with Bowtie2 (v2. 3. 5. 1), using parametersnodiscordant andno-mixed. Resulting SAM files were piped through Samblaster (v0. 1. 24) to remove duplicate reads, then through Samtools (v1. 10) to generate sorted (by coordinates) BAM files, with a cutoff for MAPQ score of 10. Peaks were called for each replicate with the MACS algorithm (MACS3) in broad mode, with the input DNA as control as well as the same sample treated with RNase H1, at q value < 0. 1. Resulting peaks were analysed by groups of biological replicates with MSPC (v5. 5. 0) 80. Firstly, by categorizing them as either background, weak, or stringent (with both cut-off on P value at 1e-4 and 1e-8). Weak peaks were rescued if stringent in other biological replicates. Peaks were then confirmed or discarded based on the combined stringency test supported by enough replicates and if their combined stringency, using Fisher's combined probability test, satisfies the threshold of 1e-8. Confirmed peaks are qualified as true positive if they pass the Benjamini-Hochberg (BH) multiple testing correction at level 0. 05. \n\nTo identify differentially expressed R-loops, we intersected the peaks from MACS3 calling with the 5. 5 million of restriction fragments generated before S9. 6 immunoprecipitation, using the resulting matrix as a reference frame for featureCounts (v2. 0. 0). Normalization and differential R-loop expression analysis between wild-type, mutant and control samples was performed with DESeq2, using BH-adjusted P value < 0. 05 and log2 (FC) > | 1 |. Bedtools (v2. 27. 1) intersect was used for overlap analyses.",
"section_name": "DRIP-seq bioinformatic analysis",
"section_num": null
},
{
"section_content": "Erythroid differentiation of human primary erythroblasts and murine G1E-ER4 clones was followed by May-Grünwald Giemsa staining of cytopsins. Cell viability was assessed by the scatter profile (FSC/SSC) using flow cytometry. For erythroid differentiation, 5×10 4 cells are washed in 1X phosphate buffered saline (PBS) supplemented with 2% FCS and incubated for 20 min at 4 °C with fluorescent antibodies to SD of positive cells with > 5 intranuclear foci. f. Burst forming unit-erythroid (BFU-E) colony assays in 9 SF3B1 MUT, 8 SF3B1 WT and 5 controls. Mean ratios between HDACi and DMSO conditions ± SD. g May-Grünwald-Giemsa-stained cytospins (d12). h Proportions of erythroid precursors in 7 SF3B1 MUT, 3 SF3B1 WT and 4 controls at d7-10 and d14-16. Means ± SEM and 2-way ANOVA multiple comparisons for q values. i Scatter plots showing differentiation by flow cytometry expressed as mean percentages ± SD of GPA + CD49d low cells. j Scatter plots showing mean percentages ± SD of dead cells (FSC/SSC) at d12-14. i, j Two-sided paired t-test for P values. **** P < 0. 0001, *** P < 0. 001, ** P < 0. 01, * P < 0. 05. Source data are provided as a Source Data file. \n\nGPA (CD235a), CD49d and CD71 for human primary cells (Beckman Coulter, Brea, CA) or Kit (CD117) and Ter-119 for murine cells (BD Biosciences, Franklin Lane, NJ). Analysis was performed on LSRFortessa apparatus (BD Biosciences) with Kaluza software (Beckman Coulter) (Supplementary Methods 3).",
"section_name": "May-Grunwald Giemsa staining and flow cytometry",
"section_num": null
},
{
"section_content": "Cell cycle was analysed by double labelling using a fluorescent anti-BrdU antibody and 7-aminoactinomycine D (7-AAD, BD Biosciences). Cells were incubated with 10 µM BrdU for 30 min at 37 °C (BD Biosciences). Then cells were pelleted, fixed in 500 µL 70% ethanol for 20 min at room temperature and washed in 1X PBS supplemented with 0. 5% bovine serum albumin (BSA). Cell pellet is resuspended in 2 M chlorhydric acid for 20 min at room temperature and washed in 1X PBS. Acid was neutralized by a 0. 1 M borate solution pH 8. 5 for 2 min (Borax Na 2 B 4 O 7, Merck). Cells were washed 3 times and transferred to 96-well plates for incubation with 10 µL anti-BrdU antibody or isotype control for 20 min at room temperature either FITC-anti-BrdU (BD Biosciences, clone 3D4) or APC-anti-BrdU (BD Biosciences, clone 3D4) at a final concentration of 0. 5 μg/mL. Cells were washed 3 times and incubated with 50 µL of a 10 µg/mL 7-AAD and 50 µg/mL RNase A (Macherey-Nagel, Hoerdt, France) solution for 30 min at room temperature. Finally, cells were transferred into Eppendorf vials containing 100 µL 1X PBS and analysed on BD Accuri C6 flow cytometer using CFlow Plus software (BD Biosciences) (Supplementary Method 4). The proliferation capacities of G1E-ER4 cells treated or not with β-estradiol at 10 -7 M (Merck) were measured using IncuCyte live-cell imaging system (Essen Instruments, Ann Arbor, MI). 48-well plates were coated with 0. 01% Poly-L-lysine sterile-filtered solution (Merck). Cells were seeded at 30,000 cells/well and monitored over time with 9 images per well every 3 hours for 72 h. Results were expressed as cell confluence (in % of occupied space). \n\nDNA fiber combing DNA replication was analyzed by pulse labelling with fluorescent thymidine analogs of human erythroblasts. Cells were first labelled with 20 µM iododesoxyuridine (IdU) for 30 min at 37 °C and then with 100 µM chlorodesoxyuridine (CldU) for 30 min at 37 °C. DNA replication was blocked by the addition of an excess of thymidine (300 µM) on ice and cells were washed in 1X PBS and counted. DNA combing was performed after DNA extraction in 1% low melting agarose plugs (0. 3 × 10 6 cells in 90 µL per plug). Briefly, low melting agarose was maintained at 45 °C. Cells were resuspended at 6. 66 × 10 6 in one volume of 1X PBS, mixed in the same volume of 2% agarose and distributed in plug mold. Then proteins in agarose plugs were digested with 1 mg/mL proteinase K in 0. 25 M EDTA pH 8/ 1% SDS at 42 °C for 48 h. Finally, plugs were washed 3 times in 10 mM Tris-HCl pH 8/1 mM EDTA (TE), and agarose was eliminated by digestion using β-agarase for 48 h. For DNA fiber stretching, extracted DNA was resuspended in 0. 25 M 2-(N-morpholino)-ethanosulfonic acid pH 5. 5 in Eppendorf vials for 30 min at 65 °C, 30 min at room temperature and 2 weeks at 4 °C. Before combing, vials were placed at room temperature for 30 min. DNA was then placed in FiberComb reservoir (Genomic Vision, Bagneux, France) and stretched on silane treated coverslips. Coverslips were sticked on glass slides and incubated for 2 h at 60 °C and stored at -20 °C. For hybridization, DNA was denatured in a 1 N NaOH solution, rinsed in cold 1X PBS and dehydrated in 70%, 85% and 100% ethanol. Aspecific sites were blocked for 30 min at 37 °C. DNA was first incubated with primary anti-IdU (BD Biosciences, cat no. 347580, dilution: 1/50) mouse antibody and anti-CldU (Abcam, cat no. Ab 6326, dilution: 1/50) rat antibody, and then with fluorescent secondary antibodies. Finally, DNA was labelled with a primary anti-ssDNA antibody (mouse), and with a first fluorescent secondary anti-mouse antibody (goat), and with a second fluorescent secondary anti-goat antibody (donkey). Slides were mounted in Vec-taShield medium (Vector Laboratories, Burlingame, CA). All antibodies and reagents are described in Supplementary Methods 5. Fiber length and symmetry of a minimum of 200 fibers per group were measured. Fork symmetry was expressed as the IdU/CldU ratio. The speed of the replication fork was calculated by the ratio (d I + d Cl ) / (t I + t Cl ), where d I and t I represent respectively the measured distance (in kb) and labelling time (in min) for IdU incorporation, and d Cl and t Cl denote the corresponding parameters for CldU incorporation.",
"section_name": "Cell cycle analysis and live cell imaging",
"section_num": null
},
{
"section_content": "Immunofluorescence was used to detect phospho-RPA32 (p-RPA32, serine 33 and serine 4/8), phospho-H2AX (γ-H2AX, serine 139) and 53BP1 nuclear foci. Cells treated with 5 mM hydroxyurea for 3 h were used as positive controls (Merck). Briefly, cytospins were prepared with 10 5 cells in 1X PBS, fixed in 1X PBS/2% paraformaldehyde (Santa Cruz Biotechnologies, Santa Cruz, CA) for 20 min at room temperature and washed. Cells were permeabilized in 1X PBS/0. 5% Triton X-100 for 10 min and washed in cold 1X PBS. Saturation was performed using 1X PBS/3% BSA (Euromedex, Souffelweyersheim, France) for 30 min and cells were incubated with primary antibodies for 1 h at 37 °C (Supplementary Methods 6). After washing, cells were incubated with secondary antibody for 30 min at 37 °C in the dark. DNA was counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 5 min and cytospins were rinsed in 1X PBS and mounted with Fluoromount-G (Clinisciences, Nanterre, France). Analysis was conducted using an inverted DMI600 microscope at 100X magnification (Leica, Wetzlar, Germany). Images were analyzed using the ImageJ software (NIH, Bethesda, MD).",
"section_name": "Immunofluorescence experiments",
"section_num": null
},
{
"section_content": "Cell lysates were solubilized for 5 minutes at 95 °C in Laemmli buffer (65 mM Tris [pH 6. 8], 20% glycerol, 5% β-mercaptoethanol, 0. 01% bromophenol blue, and 2% sodium dodecyl sulfate [SDS]) Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (VWR, Radnor, PA). Membranes were blocked with 5% of dry milk in TBS-T buffer (10 mM Tris-HCl, pH 7. 5, 150 mM NaCl, 0. 15% Tween 20) for 1 h and incubated in specific antibody overnight at 4 °C (Supplementary Methods 7). Membranes were washed in TBS-T buffer and incubated for 1 h at room temperature with secondary horseradish peroxidase (HRP)-linked antibody (horse anti-mouse HRP-linked antibody 7076 S or goat antirabbit HRP-linked antibody 7074 V, Cell Signaling, Danvers, MA). Enzyme activity was visualized by an ECL-based detection system (VWR, Radnor, PA). Blot imaging was performed on the Fujifilm LAS-3000 Imager (Fujifilm, Tokyo, Japan) and images were analysed using the Multi Gauge software (Fujifilm). \n\nTargeted LC-MS metabolomics analyses 3. 10 5 Sf3b1 K700E/+ or Sf3b1 +/+ G1E-ER4 cells were collected after 0 h or 24 h of β-estradiol treatment and with or without 0. 2 mM HU for 16 h (Merck). For metabolomics analysis, extraction was performed in 30 μL of 50% methanol, 30% acetonitrile (ACN) and 20% water. After centrifugation at 16,000 g for 15 min at 4 °C, supernatants were collected and stored at -80 °C until analysis. LC/MS analyses were conducted on a QExactive Plus Orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe coupled to a Dionex UltiMate 3000 uHPLC system (Thermo). Samples (5 µL) were injected onto a ZIC-pHILIC column with a guard column (Millipore) for LC separation in a gradient of buffer A (20 mM ammonium carbonate, 0. 1% ammonium hydroxide pH 9. 2), and buffer B (ACN) with a flow rate of 0. 200 µL. min -1 as follows: 0-20 min, linear gradient from 80% to 20% of buffer B; 20-20. 5 min, linear gradient from 20% to 80% of buffer B; 20. 5-28 min, 80% buffer B. The mass spectrometer was operated in full scan, polarity switching mode with the spray voltage set to 2. 5 kV and the heated capillary held at 320 °C. The sheath gas flow was set to 20 units, the auxiliary gas flow to 5 units and the sweep gas flow to 0 units. The metabolites were detected across a mass range of 75-1000 m/z at a resolution of 35,000 (at 200 m/z) with the automatic gain control target at 10 6 and the maximum injection time at 250 ms. Lock masses were used to ensure mass accuracy below 5 ppm. Data were acquired with Thermo Xcalibur software (Thermo Fisher Scientific). The peak areas of metabolites were determined using Thermo TraceFinder software (Thermo Fisher Scientific), identified by the exact mass of each singly charged ion and by the known retention time on the HPLC column. Each metabolite was quantified as the area under the curve and results were expressed as arbitrary unit (A. U).",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "For RT-PCR, RNA was extracted using RNAeasy Mini Kit (Qiagen) and retrotranscribed with the Maxima First Strand cDNA synthesis kit (Thermo Fischer Scientific). cDNA was amplified using Phire Hot Start II DNA polymerase (Thermo Fischer Scientific). Amplicons were analysed by electrophoresis on a 2% agarose gel. For fluorescent RT-PCR, the PCR was performed using the same primers except that the forward primers were labelled with 6-carboxyfluorescein (6-FAM) at 5' end. For capillary electrophoresis, 1 μL of diluted PCR product was added to 0. 2 μL of GeneScan 500 ROX dye standard and 18 μL of RNAse-free water. After denaturation for 5 min at 95 °C, fragments were separated using the 3730xl DNA analyzer and analyses was performed using GeneMapper Software 5 (Thermo Fischer Scientific). For qPCR, the primers were used with the SYBRGreen Master Mix (Meridian Bioscience) in a LightCycler480 (Roche). Expression levels were normalized to actin (ACT) and cyclophilin A (PPIA) expression using geometric averaging, and analyzed using ΔΔCt method. Primers for RT-PCR, fluorescent RT-PCR and qPCR are listed in Supplementary Methods 8. \n\nFor the detection of mutants by PCR, colonies were picked and transferred in 50 μL of lysis buffer (KCl 50 mM, Tris HCl pH8 10 mM, 0. 4%NP-40, 0. 4% Tween-20, 0. 2 mg/mL proteinase K). After 1hincubation at 55 °C, DNA was treated for 10 min at 95 °C. Samples (5 μL) was used for 40 cycles of PCR cycles (30 s at 95 °C, 30 s at 60 °C, 1 min at 72 °C) using specific primers (Supplementary Data 2). Amplicons were analysed by gel electrophoresis.",
"section_name": "RT-PCR and PCR on colonies",
"section_num": null
},
{
"section_content": "For quantitative variables, values were expressed as median and interquartile range (IQR) or means and standard error of the mean (SEM) and compared using the Student t-test or non parametric Mann-Whitney or Kruskal-Wallis tests. Chi-squared or Fisher exact tests were used to compare categorical variables. For transcript quantification, the Mann-Whitney test was used to assign a statistical significance for each group comparison. P values < 0. 05 were considered significant (JMP version 10. 0. 2, SAS Institute Inc, Cary, NC).",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. \n\nOpen Access This article is licensed under a Creative Commons Attribution 4. 0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/ licenses/by/4. 0/. © The Author(s) 2024",
"section_name": "Reporting summary",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The authors dedicate this work to the memory of their colleague Dr Angelos Constantinou whose discoveries inspired this work. The authors acknowledge Pr Seishi Ogawa ( Kyoto University, Japan ) for very helpful discussion and comments on the manuscript, Dr Evelyne Lauret for support and discussion, Institut Cochin, Paris ; Dr Sarah Lambert ( Institut Curie, Orsay ), Dr Jean-Charles Cadoret ( Institut Jacques Monod, Paris ), Dr Valeria Naim ( Institut Gustave Roussy, Villejuif ), Pr Raphaël Itzykson ( Institut de Recherche Saint-Louis, Paris ) for discussion. The authors thank Dr Carole Almire, Laboratory of Hematology, Cochin Hospital, Dr Emilie-Fleur Gautier, Platform Proteom'IC, Institut Cochin, Dr Franck Letourneur, Platform Genom'IC, Institut Cochin, Ms Stella Hartono, Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Dr. Ivan Nemazanyy, Platform for Metabolic Analyses ( INSERM US24/CNRS UAR 3633, Necker, Paris, France ) for their expertise, Ms Katherine Wetmore, Ms Ania Alik, Mrs Angélique Marcon, Mrs Marlène Dejean, Ms Camille Knops, Ms Laïla Zaroili, Ms Alice Rousseau ( Laboratory of Hematology, Cochin Hospital, Paris ) for technical help. Grants: This work was funded by the Institut National du Cancer INCa PLBio 2015-129 (M. F., M-H. S., A. C. ), the Fondation pour la Recherche Médicale Equipe labellisée FRM202003010191 (M. F. ), the Laboratoire d'Excellence GR-Ex and the MDS-RIGHT European Union 's Horizon 2020 research and innovation programme under grant 634789 (P. F., M. F. ).",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "The RNA-sequencing and DRIP-sequencing data generated in this study have been deposited in the NCBI's Gene Expression Omnibus (GEO) database. Accession codes are provided below. The processed RNA-sequencing and DRIP-sequencing data are available in the Supplementary information as Supplementary Data indicated for each of them. RNA-seq BM MNC cohort of 27 cases (Supplementary Data 1): GSE220525, RNA-seq of human basophilic erythroblasts and polychromatophilic erythroblasts (Supplementary Data 2): GSE220523. DRIP-seq of human basophilic erythroblasts (Supplementary Data 4): GSE220271. RNA-seq of mouse G1EER erythroblasts (Supplementary Data 5): GSE220516. RNA-seq data of the BM MNC cohort of 185 MDS patients (Supplementary Fig. 1b ) are available at GSE220518 under restricted access since these data are considered sensitive personal data according to the European Union General Data Protection Regulation (GDPR) and thus cannot be shared with third-parties without prior approval. Access can only be granted for research purposes. An application must be sent to michaela. fontenay@inserm. fr. The proteomic data are available on ProteomeXchange Consortium via the PRIDE partner repository: Human erythroblast proteome (Supplementary Data 3) and mouse erythroblast proteome (Supplementary Data 5): PXD038700. Source data are provided with this paper.",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional information",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi. org/10. 1038/s41467-024-46547-7. \n\nCorrespondence and requests for materials should be addressed to Michaela Fontenay.",
"section_name": "Supplementary information",
"section_num": null
},
{
"section_content": "Halene and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available. \n\nReprints and permissions information is available at http://www. nature. com/reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Peer review information Nature Communications thanks Stephanie",
"section_num": null
}
] |
10.1186/2045-824x-3-17
|
Therapeutic promise and challenges of targeting DLL4/NOTCH1
|
DLL4-mediated NOTCH1 signaling represents an essential pathway for vascular development and has emerged as an attractive target for angiogenesis-based cancer therapies. However, newly reported toxicity findings raise safety concerns of chronic pathway blockade. Lessons learned from the development of γ-secretase inhibitors (GSIs) might offer insights into how to safely harness this important signaling pathway.
|
[
{
"section_content": "In metazoans, the evolutionarily conserved NOTCH pathway functions as an essential mechanism to regulate numerous cell fate/lineage decisions during embryogenesis, postnatal development, and in the maintenance of adult tissue homeostasis. NOTCH receptors are normally constrained in a dormant state. Ligand binding exposes the ADAM protease cleavage site that is normally buried within the negative regulatory region (NRR) [1]. Subsequent intramembrane cleavage catalyzed by γ-secretase, a multisubunit protein complex, permits the release of the intracellular portion (NICD) from the cell membrane and its entry into the nucleus where it forms a transcriptional activation complex. In mammals, the NOTCH signaling apparatus consists of four single-pass transmembrane receptors (NOTCH1-4) and at least five membrane-anchored ligands (Jagged1, 2 and Delta-like or DLL1, 3 and 4). Despite the apparent redundancy of multiple NOTCH ligands and receptors expressed in the vascular system, recent studies have revealed that the DLL4-NOTCH 1 interaction appears to be the dominant functioning component in the vascular system. DLL4 was initially identified as an endothelium-specific NOTCH ligand [2] [3] [4] [5]. Haploinsufficiency of Dll 4 in mice results in early embryonic lethality due to severe vascular defects including impaired arteriogenesis, disrupted vascular hierarchy, and enhanced vascular density with reduced vessel caliber [2, 6, 7]. This uniquely non-redundant role of DLL4 goes beyond early embryonic development. Studies utilizing a DLL4-selective antagonistic antibody demonstrate that DLL4 is also essential for early postnatal vascular development, angiogenesis during pathological wound healing (unpublished observations) and tumor angiogenesis [8]. Compared to DLL4, NOTCH1 is more broadly expressed and targeted disruption of Notch1 results in vascular defects similar to what has been observed with Dll4 deficiency [9, 10]. Moreover, results from recent work using paralogue-specific antibodies indicate that NOTCH1 inhibition alone is sufficient to disrupt angiogenesis [11]. \n\nStudies in multiple model systems have revealed important insights into the function of Dll4/NOTCH1 signaling in angiogenesis and the underlying mechanism of vascular defects resulting from attenuated DLL4/NOTCH1 activity [11, 12]. Excessive angiogenic sprouting, branching and increased endothelial cell proliferation are commonly associated with blockade of DLL4/NOTCH 1 signaling. Signaling induced by DLL4 through NOTCH1 causes downregulation of VEGFR2, whereas blocking DLL4 signaling leads to increased expression of VEGFR2 and VEGFR3 [13]. Therefore, DLL4/NOTCH1 signaling is apparently required to restrain the magnitude of response of endothelial cells to angiogenic stimuli. The chaotic angiogenesis resulting from DLL4/NOTCH1 blockade may reflect unrestricted VEGF signaling and disruption of a dynamic balance between tip cells and stalk cells during angiogenic sprouting [14].",
"section_name": "DLL4/NOTCH1 signaling pathway in angiogenesis",
"section_num": null
},
{
"section_content": "The initial speculation of DLL4 signaling involvement in tumor angiogenesis came from expression analyses of DLL4 [12]. Results from a flurry of recent studies confirmed that targeting DLL4/NOTCH1 signaling has a profound impact on tumor angiogenesis and growth. Pathway blockade was achieved using either a DLL4-selective neutralizing antibody [8, 15, 16], a NOTCH1-selective antagonistic antibody [11], a soluble DLL4 fusion protein [17, 18], or a soluble NOTCH fusion protein [19]. These reagents demonstrated broad and robust anti-tumor activity in a wide variety of tumor models. \n\nWhile most of the current anti-angiogensis approaches act through a reduction or elimination of tumor blood vessels, DLL4 blockade results in the formation of a non-functional vasculature incapable of supporting tumor growth (Figure 1 ). Histological analysis of tumors treated with DLL4/NOTCH1 inhibitors has revealed that the reduced tumor growth is associated with an apparent increase in tumor vascular density. However, labeling with an intravascular tracer has demonstrated these vascular structures to be poorly perfused [8, 11, 17, 18]. Inadequate vascular function was also reflected by the increased hypoxia observed in tumors treated with soluble DLL4 [17]. Two major changes following DLL4/NOTCH1 blockade might contribute to the defective function of the tumor vasculature: impairment of lumen formation and promotion of a chaotic vascular network. Impaired lumen formation was described in the aortas of Dll4 +/-embryos [2]. Antagonizing DLL4/NOTCH1 also caused a morphological change of endothelial sprouts formed in an in vitro 3D culture system, with the luminal space being absent or greatly reduced [8]. Conceivably, a similar change in tumor vessels may lead to a reduction in vessel lumen size that is incompatible with the passage of red blood cells. Indeed, tumor vascular histology and imaging studies have revealed a shift to smaller vessel calibers and inefficient blood flow in anti-DLL4 treated tumors [20] (unpublished observations). Since inhibition of DLL4 leads to excessive branching of tumor vessels, the other plausible cause of inefficient blood flow could be the manifestation of a chaotic vascular network lacking a functional hierarchy. DLL4 blockade could worsen the already impaired vascular communication in the tumor microvascular network and lead to exacerbated functional shunting, a suspected primary cause of dysfunctional microcirculation and local hypoxia in cancer [21].",
"section_name": "Therapeutic promise",
"section_num": null
},
{
"section_content": "In addition to tumor studies using anti-DLL4 as a single agent, additive anti-tumor activity was observed in combination with anti-VEGF therapy in a majority of tested tumor models. Since angiogenic sprouting after DLL4 blockade remains a VEGFdependent process [8], DLL4 inhibition may increase the dependency of the tumor microvasculature on a VEGF-mediated survival signal. In anti-Dll4 treated neonatal mouse retinas, there was a defect in arteriogenesis with a complete absence of pericyte coverage of the retinal vessels [8]. Soluble DLL4 was also able to reduce the recruitment of pericytes in a murine xenograft tumor model [18]. Therefore, DLL4 blockade may impair the remodeling of the tumor vasculature to become more mature and stable, resulting in increased vulnerability of tumor vessels to VEGF blockade. In tumors targeted by DLL4/ NOTCH1 inhibition, the hyperproliferative state of endothelial cells, together with the reduced protection of the tumor endothelium by supporting cells, may render the tumor vasculature more susceptible to agents that selectively target proliferating cells. Indeed, anti-DLL4 in combination with chemotherapy shows enhanced anti-tumor activity in preclinical tumor models despite the concern that reduced perfusion of tumor vessels might interfere with the delivery of therapeutic agents [15, 16]. \n\nExisting data support the endothelial cellautonomous role for DLL4/NOTCH1 signaling in restricting the angiogenic response [22]. The NOTCH pathway has been implicated in a variety of human cancers in connection with the genetic alterations and epigenetic events that lead to either constitutive NOTCH activation or sensitized response to ligand-induced activation [12]. Interestingly, a recent study has suggested that DLL4 blockade may reduce tumor-initiating cell frequency in certain xenograft models [15, 16]. At present, however, the mechanism underlying DLL4-mediated tumor initiation and/or progression remains unclear.",
"section_name": "Figure 1",
"section_num": null
},
{
"section_content": "Targeting a key step in the generation of amyloidogenic peptides and the proteolytic activation of NOTCH signaling, a number of GSIs are currently in preclinical and clinical development for indications ranging from Alzheimer disease to T-cell acute lymphoblastic leukemia (T-ALL) [23]. A major hurdle to the therapeutic development of GSIs has been the on-target toxicity in the gastrointestinal tract. Inhibition of NOTCH signaling results in goblet cell metaplasia due to the skewed differentiation of epithelial cells in the intestinal crypts away from an enterocyte fate and towards that of a secretory goblet cell [24]. Development of goblet cell metaplasia requires simultaneous disruption of both Notch1 and Notch2 [25]. Because GSIs indiscriminately block all NOTCH receptors, targeted inhibition of individual receptors might help to alleviate the observed gut toxicity. Using paralogue-specific anti-NOTCH antibodies, Wu et al recently showed that selective NOTCH1 inhibition resulted in only mild goblet cell metaplasia, representing an important progress in overcoming the toxicity associated with pan-Notch inhibitors [11]. Recently, Radtke's group identified DLL1 and DLL4 as the key physiological ligands that mediate NOTCH signaling in the mouse intestinal epithelium [26]. Intestine-specific inactivation of either Dll4 or Jag1 had no obvious phenotype, while loss of Dll1 resulted in a moderate increase in goblet cell numbers. \n\nDll1-Dll4 double knockout mice developed intestinal abnormalities to a degree similar to Notch1-Notch2 compound mutant mice [26]. These findings are consistent with earlier work showing that neutralizing anti-DLL4 antibodies had no discernable impact on goblet cell differentiation and/or the proliferation of crypt progenitor cells [8]. \n\nWhile selective inhibition of DLL4 apparently avoids the gut toxicity that plagued the therapeutic application of GSIs, other safety concerns have been raised [27]. Administration of a DLL4-specific neutralizing antibody caused a rapid and significant change in mouse liver gene expression, including the upregulation of endothelium-specific genes as well as genes implicated in proliferation and cell cycle regulation. More importantly, mice, rats and cynomolgus monkeys exposed to anti-DLL4 antibody developed histopathological changes in the liver, including profound sinusoidal dilation and centrilobular hepatocyte atrophy [27]. These changes are believed to be a class effect of inhibiting DLL4 signaling through NOTCH1, since similar changes were also observed when DLL4/ NOTCH1 signaling was inhibited by agents targeting NOTCH1 or γ-secretase. Along with the histological findings, hepatic clinical pathology changes were observed in treated animals. These findings suggest that DLL4 signaling is essential for maintaining the structural and functional integrity of the liver sinusoidal endothelium as well as hepatocyte homeostasis. Since it has been documented that some chemotherapy agents could cause liver sinusoidal dilation or other types of liver damage [28], extra precaution should be taken when Besides the liver clinical-and histopathology changes observed upon exposure to anti-DLL4 antibody, other concerning safety signals were seen following prolonged treatment. Most strikingly, skin lesions with features of vascular neoplasms were observed in male rats. In addition, rare tumors with similar features were identified in heart and lung [27]. Among the species tested, which included mice, rats and monkeys, the proliferative vascular lesions were reported only in rats after 8 weeks of continuous anti-DLL4 exposure. However, the potential consequences of longer drug exposure in other species remain uncertain. In clinical trials, patients treated with semagacestat (LY450139), a γ-secretase inhibitor, had an increased risk of skin cancer [29]. A recent study employing an elegant genetic model showed that loss of Notch1 caused widespread vascular tumors, particularly in the liver, further underscoring the potential safety concerns associated with continuous blockade of NOTCH1 signaling [30]. \n\nAccumulating evidence suggests that DLL4-mediated NOTCH signaling is not restricted to the vascular compartment. Recent genetic studies have identified DLL4 expressed by thymic epithelial cells as the essential and nonredundant NOTCH1 ligand responsible for intrathymic T cell development [31, 32]. Consistent with DLL4 genetic inactivation, anti-DLL4 treatment resulted in a complete blockade of T cell development coupled with ectopic appearance of immature B cells in the thymus (unpublished observations). In addition, DLL4 together with DLL1 regulate NOTCH signaling in the intestinal crypt [26]. The recognition of a broader role for DLL4-mediated NOTCH signaling raises the concern that on-target toxicity of DLL4 inhibition could extend beyond the endothelium. \n\nWhile preclinical models have revealed the potential toxicities associated with DLL4/NOTCH1 blockade, safety findings in humans have just begun to emerge from recent clinical trials. DLL4 targeting antibodies, OMP-21M18 and REGN421, have entered clinical development in recent years. In a Phase I study of OMP-21M18, 28% of patients experienced grade III asymptomatic hypertension, a condition that has not been described in preclinical studies [33]. Since hypertension is a known doselimiting side effect of anti-VEGF therapy [34, 35], patients receiving combination therapy of anti-DLL4 and anti-VEGF should be carefully monitored.",
"section_name": "Safety challenges",
"section_num": null
},
{
"section_content": "Preclinical studies have suggested that DLL4/ NOTCH1 blockade could augment the efficacy of anti-VEGF in \"sensitive\" tumors. DLL4/NOTCH1 blockade may also have the potential to enhance the effects of chemotherapy or other targeted therapies in tumors that are either intrinsically less dependent on VEGF or have progressed due to a shift to dependency on other angiogenic pathways. While significant safety concerns have been raised in recent preclinical studies, the full therapeutic potential of targeting DLL4/NOTCH1 should be further explored given its remarkable impact on the tumor vasculature and tumor growth in preclinical models. Lessons learned from the preclinical and clinical development of GSIs may offer some clues for a potential path forward. Since it is generally believed that the outcome of Notch signaling is dependent on context and the degree of activation, it is possible that partial pathway inhibition might ameliorate the toxicities associated with stringent and continuous pathway blockade while maintaining efficacy. Indeed, Cullion et al were able to show that a GSI dosing schedule with drug holidays largely avoided the gut toxicity while maintaining significant efficacy in mouse T-ALL models [36, 37]. It will be interesting and exciting to determine if strategies based on the same principle could expand the therapeutic window of selective targeting of DLL4/NOTCH1. The other remarkable observation from Ferrando's group was that dexamethasone protected mice against GSIinduced gut toxicity by shifting differentiation away from goblet cell development [38]. Since the effects of DLL4/NOTCH1 blockade might be partly attributed to enhanced VEGF signaling, anti-VEGF may have the potential to protect against anti-DLL4 mediated toxicity. Interestingly, it has been reported that bevacizumab protects against injury in patients treated with oxaliplatin-based chemotherapy, which is known to cause liver sinusoidal dilation [39]. Finally, it is important to point out that, given the potent effect of DLL4/ NOTCH1 blockade and its potential safety implications, future development of anti-DLL4/ NOTCH1 therapeutics will require careful monitoring of patient safety until the relevance and/or translatability of observed preclinical toxicity concerns are better understood in the clinical setting.",
"section_name": "Potential path forward for targeting DLL4/NOTCH1",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The author would like to thank Weilan Ye, Jessica Couch and Kevin Leong for valuable suggestions and critical reading of the manuscript.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Authors' original submitted files for images",
"section_num": null
},
{
"section_content": "Below are the links to the authors' original submitted files for images. \n\nAuthors' original file for figure 1 Click here to view.",
"section_name": "Authors' original submitted files for images",
"section_num": null
}
] |
10.18632/oncotarget.13712
|
Humoral immune responses toward tumor-derived antigens in previously untreated patients with chronic lymphocytic leukemia
|
In chronic lymphocytic leukemia (CLL) the occurrence and the impact of antibody responses toward tumor-derived antigens are largely unexplored. Our serological proteomic data show that antibodies toward 47 identified antigens are detectable in 29 out of 35 patients (83%) with untreated CLL. The glycolytic enzyme alpha-enolase (ENO1) is the most frequently recognized antigen (i.e. 54% of CLL sera). We show that ENO1 is upregulated in the proliferating B-cell fraction of CLL lymph nodes. In CLL cells of the peripheral blood, ENO1 is exclusively expressed at the intracellular level, whereas it is exposed on the surface of apoptotic leukemic cells.From the clinical standpoint, patients with progressive CLL show a higher number of antigen recognitions compared to patients with stable disease. Consistently, the anti-ENO1 antibodies are prevalent in sera from patients with progressive disease and their presence is predictive of a shorter time to first treatment. This clinical inefficacy associates with the inability of patients' sera to trigger complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity against leukemic cells.Together, these results indicate that antibody responses toward tumor-derived antigens are frequently detectable in sera from patients with CLL, but they are expression of a disrupted immune system and unable to hamper disease progression.
|
[
{
"section_content": "Immune dysfunctions are a key feature of chronic lymphocytic leukemia (CLL) and frequently result in clinically manifest complications contributing to patients' morbidity and mortality, such as opportunistic infections and autoimmune conditions. The impairment of the host immune system also correlates with disease progression and may reflect the attempt of the leukemic cells to evade immune control [1]. \n\nPerturbations of both cellular and humoral immunity are observed in CLL patients. The T-cell compartment is highly abnormal, with changes in the phenotype and absolute number of circulating CD4+ and CD8+ T cells and with the accumulation of terminally differentiated effector memory T cells [2]. T lymphocytes express Research Paper markers of exhaustion, such as upregulated PD-1, leading to a pronounced Th2 skewing of the T-cell responses [3], have reduced cytotoxic functions and an impaired formation of immune synapses with antigen (Ag) presenting cells [4]. Regulatory T cells are increased in the peripheral blood (PB) of patients with advanced disease [5], and Vγ9Vδ2 T cells display features of exhaustion and are negative prognosticator of a shorter time to first treatment (TTFT) [6]. \n\nQuantitative and qualitative changes of the humoral responses are also very common in patients with CLL. Hypogammaglobulinemia affects up to 85% of patients and its severity correlates with stage and duration of the disease and with the susceptibility to recurrent infections [1, 7]. On the other hand, haematopoieticspecific auto-antibodies (Ab) are frequently observed and autoimmune haemolitic anemia (AIHA) and/or immune thrombocytopenia are rather frequent complications. Autoimmunity is usually due to high affinity polyclonal immunoglobulin (Ig) G auto-Ab that are produced by the non-malignant B lymphocytes and recognize red cells-or platelets-derived auto-Ag [4, 8]. \n\nSo far, little is known on the occurrence of humoral immune responses toward tumor-derived Ag in CLL. In a previous report, Krackhardt et al. detected circulating Ab recognizing 14 Ag derived from primary tumor samples in sera from patients with CLL [9]. However, the biologic functions and the clinical impact of spontaneously occurring humoral responses directed toward tumorderived Ag have not been investigated. \n\nSerological Proteome Analysis (SERPA) is a valuable method to detect Ag-specific Ab responses in human malignancies. SERPA combines electrophoretic separation of proteins from tumor cells, western blotting and mass spectrometric (MS) identification of Ag recognized by sera. SERPA allowed the identification of tumor-derived Ag able to trigger humoral immune responses in several solid [10] [11] [12] [13] [14] [15] [16] and hematologic tumors [17] [18] [19] [20]. Dubovsky et al. have recently applied a modified SERPA to CLL to define new membrane-associated targets, and identified lymphocyte cytosolic protein 1 as an important factor in chemokine-induced migration of leukemic cells [21]. \n\nThe aim of our study was to investigate the occurrence of humoral responses toward Ag derived from primary tumor cells in sera from previously untreated patients with CLL, also evaluating their cytotoxic function and the correlation with parameters of disease evolution.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "Sera obtained from 35 patients with previously untreated CLL were individually screened for the presence of IgG-based immunoreactivity against proteins derived from autologous CLL cells (Figure 1A and 1B ). To verify the CLL-specificity of the detection, 8 CLL proteomic maps (patients 05, 06, 10, 11, 12, 13, 16, 29) were individually probed with a different healthy donor's (HD) serum (data not shown). Overall, the 35 CLL sera recognized 144 Ag (mean:4. 1, min:0, max:20), whereas the 8 HD sera recognized only 3 Ag (mean:0. 4, min:0, max:1) (p=0. 007). \n\nThe MS analyses of the 144 Ag spots produced by CLL sera led to the identification of 47 proteins that were recurrently recognized by patients' sera. Table 1 summarizes the chemical features, spectrometric data, and frequency of recognition of 18 Ag, which Sera from progressive CLL are more immunoreactive than sera from stable CLL\n\nThe immunoreactive status of patients was not associated with gender, age, Rai or Binet stage, lymphocytosis, monocyte and platelet counts, hemoglobin, β2-microglobulin or lactate dehydrogenase values, percentage of CD38+ or ZAP70+ cells, immunoglobulin heavy chain variable regions (IGHV)mutational status, fluorescence in situ hybridization data, hypogammaglobulinemia, autoimmunity, concurrent infections or allergies. Our cohort included 10 patients in active disease progression and 22 patients with a stable disease. For the remaining 3 patients, the disease status at the time of SERPA was not available. Interestingly, we found that patients with progressive CLL showed a significantly higher number of Ag recognitions compared to patients with stable disease (p=0. 01) (Figure 2 ). Overall, median TTFT of patients was 29 months and median overall survival (OS) was not reached at the median follow-up of 81 months. The immunoreactivity status was not a statistically significant TTFT or OS predictor.",
"section_name": "Immunoreactivity of CLL sera toward CLLderived Ag and protein identification",
"section_num": null
},
{
"section_content": "To determine whether the circulating Ab detected by SERPA in patients' sera were partly produced by a class-switched sub-clone derived from the leukemic clone, we first analyzed the IGHV repertoire of patients who shared the same Ag recognitions. Patients with similar immunoreactivities did not exhibit the same IGHV rearrangements. In addition, the analysis of the complementarity determining region 3 (CDR3) revealed that 6 out of 35 CLL patients displayed stereotyped B-cell receptor (BCR) and belonged to 4 subsets, with no association between stereotypy and Ag recognition. As a confirmatory evidence, we cloned and produced a soluble derivative of the leukemic BCR (ScFv-Fc) from patient 10 and blotted it in parallel with the whole patient's serum on two identical autologous proteomic maps. The patient's serum recognized 5 Ag, but none of these was also recognized by the autologous tumor-derived ScFv-Fc (Supplementary Figure S1 ). Taken together, these results indicate that the Ab detected by SERPA are entirely produced by the normal B-cell population, and do not include a soluble fraction of the leukemic BCR.",
"section_name": "Circulating Ab are produced by the polyclonal B-cell population and not by the leukemic clone",
"section_num": null
},
{
"section_content": "ENO1 was the most relevant Ag recognized by patients' sera, since ENO1-specific circulating Ab were detectable in 19 out of 35 (54%) CLL sera and in none of the HD sera (p=0. 006) (Table 1 ). To confirm the MS identification of the protein, we incubated the proteomic maps obtained from the tumor cells of 10 anti-ENO1 Ab+ (patients 22, 23, 24, 25, 26, 27, 29, 31, 32, 33) and 4 anti-ENO1 Ab-patients (patients 28, 30, 34, 35) with a commercially available anti-ENO1 polyclonal Ab. The anti-ENO1 Ab generated the same Ag spots produced by the sera of patients with CLL (Supplementary Figure S2 ). \n\nENO1 expression pattern was investigated by immunohistochemistry and multicolor immunofluorescence confocal microscopy in lymph node (LN) sections obtained from 3 CLL and 3 reactive (R) LN. The CLL LN displayed an almost complete effacement of the normal architecture by CLL cells and evidence of morphologically distinct pseudo-follicles, comprising areas rich in prolymphocytes and paraimmunoblasts. The anti-ENO1 Ab stained all CLL and R LN, and higher magnification indicated an increased expression in correspondence of proliferating cells of both CLL and R LN sections, at least on the basis of cell morphology (Figure 3A and 3B ). Multicolor tissue immunofluorescence was then used to determine which cells were mostly ENO1+. The anti-ENO1 Ab was combined with anti-CD2 and anti-CD23 Ab to detect T and B cells, respectively. In the CLL LN ENO1 reactivity was mostly associated to the CD23+ population, while in the R LN it was mostly associated to the CD2+ T cells (Figure 3C-3E ). Ki67 was then used to identify proliferating cells in both tissues. Within the CLL LN a significant proportion of proliferating B cells was apparent and these cells were intensely ENO1+. Conversely, in the reactive LN the most of the proliferating cells were CD2+, and they also showed ENO1 reactivity, suggesting that ENO1 is highly expressed by proliferating cells, independently of the lineage (Figure 3D-3K ).",
"section_name": "Alpha-enolase (ENO1) is the most frequently recognized Ag and is overexpressed by proliferating CLL cells of the LN",
"section_num": null
},
{
"section_content": "ENO1 expression was also evaluated in the PB compartment. First, we compared the intracellular expression of ENO1 in PB mononuclear cells (PBMC) subpopulations from CLL patients. Cytofluorimetric analysis showed that CD19+/CD5+ CLL cells, CD14+ 4A ). We then evaluated the membrane expression of ENO1. ENO1 was not expressed on the surface of CD19+/ CD5+ leukemic cells, whereas it was expressed to higher extent by CD14+ monocytes, and in a proportion of CD3+ T lymphocytes (Figure 4B ). After 4 days of in vitro culture, the mean fold increase in the percentage of cells expressing ENO1 on the surface was significantly higher in the apoptotic (Annexin-V+ [AnnV+]) than in the viable (AnnV-) fraction of CD19+ cells (p=0. 02) (Figure 4C ). This difference in ENO1 surface expression (mean fold increase) was not observed between the apoptotic and viable fraction of CD19-cells (Figure 4D ).",
"section_name": "ENO1 is transferred on the surface of CLL cells undergoing apoptosis",
"section_num": null
},
{
"section_content": "The presence of circulating anti-ENO1 Ab was not associated with the occurrence of clinically evident autoimmune manifestations. By contrast, ENO1 reactivity was significantly associated to parameters of disease progression such as higher lymphocyte counts (p=0. 02) and lower platelet numbers (p=0. 007) (Supplementary Figure S3A and S3B ). Similarly, anti-ENO1 Ab (Figure 5A ) were more frequently detected in sera from patients with progressive disease than in sera from patients with stable disease (p<0. 0001). As expected, log-rank test indicated that the presence of anti-ENO1 Ab in patients' sera was a negative TTFT predictor in univariate analysis: the median TTFT of anti-ENO1 Ab+ CLL patients was 7 months, whereas that of anti-ENO1 Ab-CLL patients was 107 months (p=0. 01) (Figure 5B ). Lastly, we observed that the total level of serum IgG was significantly lower in anti-ENO1 Ab+ compared to anti-ENO1 Ab-CLL sera (p=0. 02) (Supplementary Figure S4 ).",
"section_name": "Immunoreactivity toward ENO1 associates with CLL progression and shorter TTFT",
"section_num": null
},
{
"section_content": "Next, we asked whether the circulating Ab identified in patients sera were effective in triggering CDC. We first tested the efficacy of our samples of CLL sera as source of complement. As expected, the viability of CLL cells exposed to the monoclonal Ab alemtuzumab was significantly reduced compared to untreated controls (p<0. 01). By contrast, we did not observe any significant decrease in cell viability when CLL cells were exposed to patients' sera, in the absence of alemtuzumab (Figure 6A-6D ). Flow cytometry revealed that there was no deposition of the complement component 4 on the surface of CLL cells after incubation with patients' sera (data not shown). Overall, these data show that the circulating Ab detected by SERPA in CLL sera are not able to induce CDC toward CLL cells. \n\nWe then performed CDC assays with sera from anti-ENO1 Ab+ patients and the U937 cell line, which expresses high levels of surface ENO1 (Figure 6E ). Again, no modification of cell viability was observed when U937 cells were treated with anti-ENO1 Ab+ sera, even when sera were used at 100% concentration (Figure 6F and 6G ). These data demonstrate that the circulating Ab do not trigger CDC at the in vivo concentrations, even when the (I, J ). Cumulative analysis of ENO1 mean pixel intensity (arbitrary units) confirmed that the CD3+/Ki67+ proliferating fraction of the T-cell compartment expressed significantly higher levels of ENO1 than CD3+/Ki67-resting fraction, in both CLL and R LN (p<0. 001 and 0. 03 respectively) (K). For cumulative data, at least 3 randomly chosen fields from 3 different samples were analyzed. Samples were analyzed using a TCS SP5 laser scanning confocal microscope (Leica Microsystems) with an oil immersion 63x/1. 5 objective lens, images were acquired with LAS AF Version Lite 2. 4 software and processed with Photoshop (Adobe Systems). ENO1 mean pixel intensity was calculated with ImageJ software (http://rsbweb. nih. gov/ij/). Box and whiskers plots represent median values, first and third quartiles, and minimum and maximum values for each dataset. Statistical analysis was performed using Mann-Whitney U test. ENO1 was not expressed on the surface of CD19+/CD5+ CLL cells, whereas it was widely expressed by CD14+ monocytes and in a proportion of CD3+ lymphocytes. C, D. Surface ENO1 fold increase expression in CD19+ and CD19-cell fractions. Fold increase was calculated for each patient as a ratio between the percentage of CD19+/ENO1+ detected after 4-day in vitro culture and the percentage of CD19+/ENO1+ cells at baseline. After 4 days of culture, the fold increase in ENO1 expression was significantly higher in the apoptotic (AnnV+) than in the viable (AnnV-) fraction of CD19+ cells (p=0. 02) (C). By contrast, there was no difference in the fold increase of ENO1 expression between the apoptotic (AnnV+) and the viable (AnnV-) fraction of CD19-cells (D). Box and whiskers plots represent median values, first and third quartiles, and minimum and maximum values for each dataset. cognate Ag is highly expressed on the surface of the target cells. \n\nIn the last set of experiments, we tested the ability of anti-ENO1 Ab+ sera to trigger ADCC toward both the U937 cell line and primary CLL cells. We found that the % of ADCC of U937 cells cultured for 18 hours in the presence of 10% diluted anti-ENO1 Ab+ serum and HD PBMC, used as effector cells, was not significantly increased compared to target cells cultured with PBMC alone, in the absence of serum. Similar results were obtained using CLL cells as targets (Figure 7A-7D ). Therefore, we can conclude that the circulating Ab detected by SERPA are unable to trigger ADCC against leukemic cells.",
"section_name": "Serum Ab are not able to trigger complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC)",
"section_num": null
},
{
"section_content": "In CLL, cellular immune responses are unable to respond to the tumor and they can even support disease evolution [22]. The impact of humoral responses toward tumor-derived Ag in CLL is largely unexplored. Through the use of SERPA we show that 83% of CLL sera have circulating Ab toward at least one CLL-derived Ag. Interestingly, we observe that sera from progressive CLL are significantly more immunoreactive than sera from stable CLL. \n\nThe circulating Ab detected by SERPA are expression of an immune response generated by the residual polyclonal B-cell population toward self-Ag. We do not observe shared Ag recognition between a representative CLL serum and the ScFv-Fc derived from the autologous leukemic BCR. On the other hand, patients who share the same Ag recognitions do not show recurrent IGHV rearrangements or BCR stereotypies. Autoimmune phenomena are rather frequent complications of CLL and mostly result in autoimmune cytopenias [23]. A clear association between autoimmune cytopenias and poor prognostic variables, such as high β2-microglobulin, CD38 and ZAP70 expression, and unmutated IGHV status, has already been described [24] [25] [26]. Moreover, direct antiglobulin test positivity, even in the absence of overt AIHA, associates with adverse prognostic factors and shorter OS [27]. In our cohort, we observe a significantly higher degree of immunoreactivity in patients with progressive CLL than in patients with stable disease, even though the immunoreactive status is not related to adverse prognostic factors. \n\nMS analyses show that the Ab detected by SERPA mostly recognize intracellular Ag, such as glycolytic enzymes, cytoskeletal elements and ribonucleoproteins. Among others, ENO1, a glycolytic enzyme, is the most relevant Ag, detected in 19 out of 35 (54%) CLL sera. The overexpression of ENO1 is associated with tumor development through a process known as Warburg effect [28]. The Warburg effect is a metabolic reprogramming, which consists in a positive regulation exerted by the hypoxia-inducible factor (HIF) on the expression of glycolytic enzymes, such as ENO1, occurring when cancer cells are exposed to hypoxic conditions [29, 30]. Interestingly, CLL cells express the oxygen-regulated HIF-1α subunit even under normoxia [31], and the expression and the transcriptional activity of HIF-1α are further upregulated by exposure of CLL cells to stromal cells [32]. In the LN, proliferating CLL cells are confined to specialized structures called pseudo-follicles, where they interact with T lymphocytes and stromal cells. Our data show that ENO1 is more expressed in the leukemic cells of the CLL LN than in the normal B cells of the R LN, and confirm that it is more expressed by the proliferating B cells of the pseudo-follicles than by the resting B-cell fraction. \n\nDue to its tumor-related overexpression and ability to induce humoral and/or cellular immune responses, ENO1 has already been classified as a tumor-associated Ag in solid cancers, such as pancreatic ductal adenocarcinoma (PDA) [12, 33]. Cappello et al. have reported that a DNA vaccine coding for ENO1 can elicit anti-ENO1 IgG Ab which are able to induce the killing of murine PDA cells by CDC [33]. Unexpectedly, in our cohort of CLL patients, the presence of anti-ENO1 Ab is prevalent in sera from patients with progressive CLL and is predictive of a shorter TTFT. One possible reason of the different clinical impact exerted by anti-ENO1 Ab in solid tumors and in CLL is that ENO1 is highly expressed at the intracellular level, but is not expressed on the cell membrane. It is not clear how intracellular self-Ag can become immunogenic and trigger humoral responses. One possible explanation is that overexpressed self-proteins are exposed during the apoptotic turnover of the leukemic cells, thus becoming visible and capable of inducing autoreactive immune responses. This hypothesis is corroborated by our data showing that after 4 days of in vitro culture, the mean fold increase in the percentage of cells expressing ENO1 on the surface is significantly higher in the apoptotic than in the viable fraction of CD19+ cells. The higher frequency of immunoreactivities detected by SERPA in the sera of patients with progressive CLL can be explained by the observation that in advanced-stage CLL the malignant clone, which in early-stage is arrested in G0/G1, evolve into an autonomously proliferating cell population showing a greater ability to enter spontaneous apoptosis [34]. In the appropriate context, such as in the spleen and LN, where T cells may induce the activation and Ag presentation by B cells, this turnover potentially leads to a greater exposition of intracellular overexpressed Ag and to the production of autoreactive Ab. \n\nTo functionally characterize the humoral responses detected by SERPA we evaluated the in vitro ability of patients' sera to kill primary CLL cells by CDC or ADCC. Deficiencies or reduced levels in one or more complement components have already been reported in CLL patients [35, 36]. However, we demonstrated that CLL sera behave as proper source of complement, in inducing alemtuzumab-mediated CDC as effectively as HD sera. By contrast, in the absence of alemtuzumab, polyreactive CLL sera are not able to induce complement deposition on the surface of CLL cells and do not trigger CDC toward primary tumor cells. Similarly, CLL sera do not trigger ADCC against CLL cells, in the presence of PBMC isolated from HD used as effector cells. A further demonstration of the functional incompetence of circulating Ab detected by SERPA is the observation that CLL patients with immunoreacitive sera, even those with polyreactive sera, lack clinically manifest autoimmune phenomena. A number of reasons may underlie this functional incompetence. First of all, the tumor Ag recognized by the circulating Ab are mainly intracellular proteins and therefore are not optimal targets for an Abmediated immune response. However, we do not observe any cytotoxicity even when the U937 cell line, which expresses high levels of surface ENO1, is used as target for the CDC and ADCC assays. In line with these results is the observation that the absolute number of monocytes, which also express high levels of surface ENO1, is not decreased in the PB of CLL patients with anti-ENO1 circulating Ab (data not shown). These findings show that Ab-related issues, such as low sera concentrations of the circulating Ab and/or low binding affinity toward the corresponding Ag, may also contribute to the functional incompetence of the humoral responses detected by SERPA. \n\nOverall, our results show that the Ab responses detected by SERPA are an epiphenomenon of a disrupted immune system, which is unable to control disease evolution. It has already been reported that the chronic inflammation state determined by an enhanced and persistent activation of humoral immune responses, in combination with the suppressed cellular anti-tumor immunity, may favour the tumor progression and support disease evolution [37]. In this context, it will be of interest to determine the immunomodulatory properties of the new BCR inhibitors, which have now been introduced in the clinical practice for the treatment of CLL. Current studies already show a recovery of humoral immunity and normal B-cell numbers in patients on ibrutinib, leading to a decrease in the rate of infections [38]. This observation, together with the promising results obtained in experimental models of autoimmune diseases [39, 40], support the hypothesis of an ibrutinib-induced immunomodulation which may contribute to tumor control.",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "PB samples were collected from a total of 86 patients with untreated CLL, after their informed consent, in accordance with the Declaration of Helsinki and approval by the local institutional review board. Sections of LN infiltrated by CLL cells and reactive R LN were obtained from the Department of Medical Sciences of the University of Torino, Italy. The diagnosis and progression of CLL were defined according to International Workshop on CLL-National Cancer Institute (IWCLL/NCI-WG) guidelines [41]. The disease was defined stable in absence of signs of progression. A cohort of 35 patients with CLL was analyzed by SERPA. Patients' biological and clinical features were collected, when available, by chart review (Supplementary Table S2 ). The median follow-up of all patients was 81 months. The IGVH mutational status and the CDR3 clustering analyses were performed as previously reported [42, 43]. The control group consisted of 12 HD kindly provided by the local blood bank.",
"section_name": "Patient population",
"section_num": null
},
{
"section_content": "PBMC and purified B lymphocytes were prepared as described [32]. Serum samples were isolated from PB by centrifugation and stored at -80°C until use. In selected experiments, the human cell line U937 (ATCC, CRL 1593. 2) was used.",
"section_name": "Cells and serum samples",
"section_num": null
},
{
"section_content": "Pellets obtained from at least 10 7 purified CLL cells were solubilized in lysis buffer (urea 9M, CHAPS 4%, Na 3 VO 4 1mM, DTT 80mM, protease inhibitors and nuclease). Samples were spun down at 13800 g for 10 minutes at 4°C. The clear supernatant was quantified with DC Protein assay kit (Bio-Rad, Hercules, CA, USA). 2-DE on ready-made IPG strip (7-cm IPG strips, pH 3-10NL; Bio-Rad) was performed essentially as described [44]. The 2-DE maps were obtained in duplicate and stained with Coomassie Blue or transferred onto a nitrocellulose membrane (GE Healthcare Biosciences GmbH, Uppsala, Sweden) for serological analysis. The 2-D gel images were acquired using the ChemiDoc MP system (Bio-Rad).",
"section_name": "Sample preparation and 2-Dimension Electrophoresis (2-DE)",
"section_num": null
},
{
"section_content": "The membrane was incubated overnight at 4°C with serum, as primary Ab at working dilution of 1:100, with a single chain variable fragment-fragment crystallisable region (ScFv-Fc) or with an anti-ENO1 Ab (Santa Cruz Biotechnology, Inc; CA, USA). Then the membrane was incubated with an anti-human (Santa Cruz Biotechnology, Inc) or anti-mouse (GE Healthcare Biosciences GmbH) IgG HRP-conjugated Abs. Images were acquired by the ChemiDoc MP system (Bio-Rad).",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "MS analysis was performed as described [44]. The 25 most intense masses were used for database searches against the Swiss-Prot database using the free search program Mascot (http://www. matrixscience. com). Only proteins with a Mascot score greater than 56 were taken into consideration.",
"section_name": "Protein identification by MS",
"section_num": null
},
{
"section_content": "VH and VL genes were amplified with specific primers from CLL derived cDNA as already described [45]. V genes were assembled as ScFv and cloned in fusion with the human Fc region (as scFv-Fc) in the pUCOE vector [46]. CHO-S cell line was transfected and stable clones was obtained for ScFv-Fc expression. Ab produced in cell culture supernatants were purified using a protein A/G column.",
"section_name": "ScFv-Fc production",
"section_num": null
},
{
"section_content": "ENO1 surface and intracytoplasmic expression was evaluated cytofluorimetrically on B cells, T cells and monocytes in PB samples as reported in Supplemental Methods.",
"section_name": "Flow cytometry",
"section_num": null
},
{
"section_content": "PBMC from patients with CLL were cultured as described [32]. The percentage of viable and apoptotic cells was determined by Annexin-V (AnnV) or AnnV/ Propidium Iodide (PI) staining with the MEBCYTO-Apoptosis Kit (MBL Medical and Biological Laboratories, Naka-ku Nagoya, Japan).",
"section_name": "Cell culture and viability assay",
"section_num": null
},
{
"section_content": "Cell morphology and numbers were studied by Giemsa staining. For immunocytochemistry, coverslips were stained as detailed in Supplemental Methods. Formalin-fixed, paraffin-embedded sections of CLL or R LN were stained and analyzed by light microscopy as already described [47] (details in Supplemental Methods).",
"section_name": "Immunohistochemistry and confocal immunofluorescent microscopy",
"section_num": null
},
{
"section_content": "For CDC, purified CLL cells (2 x 10 5 ) were incubated in presence or absence of alemtuzumab (10 μg/ml) for 30 minutes at room temperature. CLL cells were then washed with PBS and incubated with selected patients' sera at different dilutions for 60 minutes at 37°C. U937 cells were incubated only with CLL patients' sera. Cell viability was evaluated using AnnV/PI staining (details in Supplemental Methods). For ADCC, purified CLL cells or U937 cells, used as target cells (T), were labeled with CFSE (5 μM) for 30 minutes at 37 °C. CLL cells and U937 cells were then incubated with PBMC from healthy donors, used as effector cells (E) for 18 hours at 30:1 E:T ratio, in the presence or absence of 10% diluted CLL patients' sera. At the end of the co-culture target cells viability was evaluated by PI staining. % of ADCC was calculated as follow: % PI (T + E + serum) -% PI (T) / % PI (triton X treated T) -% PI (T).",
"section_name": "CDC and ADCC assays",
"section_num": null
},
{
"section_content": "Statistical analyses were performed with GraphPad Prism (San Diego, CA, USA). Continuous variables were compared by Mann-Whitney U (unpaired data) or Wilcoxon signed rank (paired data) tests. The χ 2 test was used in case of dichotomous variables. OS and TTFT were defined as the time between the date of SERPA and, respectively, the date of death or last follow-up and the date of first-line treatment or the last follow-up. OS and TTFT were estimated by the Kaplan-Meier method and the difference between groups was assessed by log-rank test. A p value <. 05 was considered significant.",
"section_name": "Statistical analysis",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
},
{
"section_content": "",
"section_name": "Authorship contributions",
"section_num": null
}
] |
10.1371/journal.pone.0004434
|
Tumorigenic Potential of Olfactory Bulb-Derived Human Adult Neural Stem Cells Associates with Activation of TERT and NOTCH1
|
Multipotent neural stem cells (NSCs) have been isolated from neurogenic regions of the adult brain. Reportedly, these cells can be expanded in vitro under prolonged mitogen stimulation without propensity to transform. However, the constitutive activation of the cellular machinery required to bypass apoptosis and senescence places these cells at risk for malignant transformation.Using serum-free medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), we established clonally derived NS/progenitor cell (NS/PC) cultures from the olfactory bulb (OB) of five adult patients. The NS/PC cultures obtained from one OB specimen lost growth factor dependence and neuronal differentiation at early passage. These cells developed glioblastoma tumors upon xenografting in immunosuppressed mice. The remaining NS/PC cultures were propagated either as floating neurospheres or as adherent monolayers with maintenance of growth factor dependence and multipotentiality at late passage. These cells were engrafted onto the CNS of immunosuppressed rodents. Overall, the grafted NS/PCs homed in the host parenchyma showing ramified morphology and neuronal marker expression. However, a group of animals transplanted with NS/PCs obtained from an adherent culture developed fast growing tumors histologically resembling neuroesthesioblastoma. Cytogenetic and molecular analyses showed that the NS/PC undergo chromosomal changes with repeated in vitro passages under mitogen stimulation, and that up-regulation of hTERT and NOTCH1 associates with in vivo tumorigenicity.Using culturing techniques described in current literature, NS/PCs arise from the OB of adult patients which in vivo either integrate in the CNS parenchyma showing neuron-like features or initiate tumor formation. Extensive xenografting studies on each human derived NS cell line appear mandatory before any use of these cells in the clinical setting.
|
[
{
"section_content": "Due to their ability to self-renew and to differentiate towards the neuronal phenoype, human adult neural stem cells (NSCs) provide an attractive tool for transplantation-based therapy of neurodegenerative diseases that avoids the ethical issues raised by the use of human embryos. However, proliferation and self-renewal properties make NSCs sensitive targets for malignant transformation [1]. Some evidence suggests that adult mouse NSCs are quite resistant to transform even in high-passage cultures under mitogen stimulation [2]. In contrast, neural precursors from the adult rat subventricular zone (SVZ) have recently been shown to transform into tumorigenic cell lines after expansion in vitro [3]. Moreover, several arguments advise caution before grafting NSCs in patients that include, a) evidence that glioblastoma may arise de novo from the oncogenic transformation of NSCs [1, 4], b) common molecular determinants regulating neurogenesis and tumorigenesis [5] [6] [7], and c) generation of glioma-like lesions following growth factor stimulation of the adult SVZ [8]. \n\nThe forebrain SVZ and the dentate girus of the hippocampus are two areas of persistent neurogenesis in the adult brain. These regions contain dividing cell populations that have been recognized as NSCs and transit amplifying progenitors (TAPs). The former are relatively quiescent cells with the capacity of self-renewal. TAPs proliferate more rapidly and differentiate into migratory neuroblasts and oligodendrocyte precursors. In rodents, TAPs move along the rostral migratory stream to the olfactory bulb (OB). In humans, a lateral ventricular extension of the migratory stream to the OB has recently been demonstrated and NS/progenitor cells (NS/PCs) have successfully been isolated from the OB, which therefore represents an accessible source of neural precursors [9] [10] [11]. Using xenograft models, we found that human adult OB-derived NS/PCs are capable of initiating tumor formation. Although an oncogenic potential has previously been described in rodent NSCs [3, [12] [13] and in human adult mesenchymal stem cells [14], the present work provides the first demonstration that human adult NS/PCs arising from normal brain may be tumorigenic in vivo.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Tumorigenic human adult NS/PCs arising from an OB adjacent to meningioma\n\nThe OB was harvested from five adult patients who had undergone surgery for extracerebral benign lesions (Table S1 ). Unilateral division of the OB, which is often necessary for surgical exposure, is well tolerated by the patients because the olfactory function is preserved. Immunohistochemistry showed that the human adult OB contains a few hundreds of putative NS/PCs (Fig. 1 ). Dissociated OB specimens were cultured in serum-free medium supplemented with the mitogens EGF and bFGF. Under these conditions, the OB cells generated primary neurospheres with latencies that ranged from 6 to 8 weeks (Table S2 ). An exception was Case OB3 where primary neurosphere formation was observed as early as 3 weeks of culturing. Primary neurospheres were dissociated into single cells and plated one cell per mini-well (Fig. 1 ). Clonal cell cultures were established by dissociation of secondary neurospheres and passaged up to P30 in mitogens. The ability to form spheres after serial passaging, the number and diameter of spheres produced during each passage, and the cloning efficiency were similar among different cultures (Table S2 ). Upon removal of mitogens and serum exposure, the NS/PC cultures obtained from four of the OB specimens arrested their growth and gave rise to adherent cells that expressed neuronal, astrocytic, and oligodendrocytic markers (Figs. 2A-2B ). In contrast, OB3 NS/PC cultures lost both growth factor dependence and potential to differentiate as neurons between P4 and P6. Notably, the OB3 patient harbored a meningioma adjacent to the OB. Losing growth factor dependence and capacity to differentiate by NSCs may indicate transformation. On soft agar assay, an in vitro correlate of transformation, the OB3 NS/PCs developed colonies (Fig. S2 ). Then, we assessed tumorigenicity in vivo using hetero-and orthotopic xenografts in immunodeficient mice. Two to 3 weeks after grafting, NS/PCs from all OB3 cultures developed subcutaneous tumors with a 88. 6 percent take (Fig. 2C and Table 1 ). Histologically, these tumors showed glioblastoma features, like perinecrotic pseudo-palisading and vascular proliferation. Tumorigenicity of OB3 NS/PCs was demonstrated both at early (P6) and at late passages (P30). Subcutaneous injection of OB1, OB2, OB4, and OB5 NS/PCs resulted in amorphous tissue grafts with embedded scarce cells showing heterogenous morphology and occasional GFAP staining without neoplastic features (not shown). Intracerebral injection of OB3 NS/PCs also produced tumors which developed at 63. 1 percent of injection sites by 4 to 6 weeks after grafting (Fig. 2D and Table 1 ). Histologically, these tumors featured anaplastic astrocytoma with predilection for growing into the ventricles. Intracerebral injection of OB1, OB2, OB4, and OB5-derived NS/PCs did not result in tumor formation (Table 1 ). \n\nIn principle, taking NS/PCs from patients with pre-existing tumors nearby the organ where the cells are obtained is inappropriate. For example, human adult non-tumorigenic NSCs surrounding low-grade glioma tissue transform in vitro into highly tumorigenic cancer stem cells [15]. In patient OB3, errant meningioma cells infiltrating the OB or adhering to its surface might have overwhelmed the NS/PCs in culture. This hypothesis, however, seems unlikely because of the following, 1) the phenotype of meningioma cells (EMA+/GFAP2/NG22/O42) differed both from that of OB3 NS/PCs (EMA2/GFAP+/NG2+/O4+) and from OB3-derived tumor xenografts (EMA2/GFAP+/ NG22/O42); 2) sphere generation in serum-free cultures occurs in glioblastoma, anaplastic astrocytoma, medulloblastoma, and ependymoma but not in meningioma, and 3) meningioma-derived NS/PCs are expected to develop xenografts with the histological appearance of meningioma or sarcoma not of glioblastoma. In brain pathology, concurrent adjacent meningioma and astrocytic tumors have been described raising the hypothesis that meningioma-released agents may work as growth factors for the glial cells of surrounding brain tissue [16]. Thus, the NS/PCs resident in the OB adjacent to meningioma may undergo chronic pressure for growth becoming highly sensitive to mitogens in vitro.",
"section_name": "Results and Discussion",
"section_num": null
},
{
"section_content": "Transplantation technologies of adult human NS/PCs imply strategies where minimal donor material is highly expanded in vitro to the adequate cell number before implantation. In general, NSCs can be expanded either as floating neurospheres in serumfree medium supplemented with mitogens or as adherent monolayers in medium containing both mitogens and serum [17]. Neuronal and oligodendroglial differentiation of adherently growing NSCs can be enhanced by growth factor withdrawal and exposure to triiodothyronine (T3) and ascorbic acid [18]. Then, we propagated GFP-positive OB1, OB2, OB4, and OB5 NS/PCs between P7 and P10 either under mitogens or under mitogens and 5% serum (Fig. 1 ). In mitogens and serum, the NS/PCs became adherent, continued to proliferate, and either maintained an undifferentiated phenotype or differentiated, mainly as astrocytes (Fig. 3A ). When such adherent serum-stimulated (SS) NS/PCs were returned to serum-free medium with mitogens, they formed floating neurospheres within one week maintaining their clonal efficiency. Upon removal of mitogens and exposure to 1% serum supplemented with T3 and retinoic acid, the SS-NS/PCs slowered down their growth and further differentiated towards the neuronal, astrocytic, and oligodendrocytic lineages (Fig. 3B ). Aberrant coexpression of neuronal and glial markers by the SS-NS/PCs was not seen. \n\nTo examine the behavior of NS/PCs in the CNS environment, we engrafted GFP-positive NS/PCs, which had been expanded either as neurospheres or as adherent monolayers, onto the spinal cord of ciclosporine treated rats or onto the striatum of SCID mice. Surprisingly, 85. 7 percent of the rats engrafted onto the spinal cord with the clonal SS-OB2a NS/PCs showed progressive palsy of their hindlimbs by 2 to 4 weeks after grafting. These animals developed highly infiltrating intramedullary tumors that histologically were reminiscent of neuroesthesioblastoma, a malignant neoplasm of the OB that is supposed to arise from an ancestral neuroblast (Fig. 3C and Table 1 ). The tumor xenografts expressed markers for neuronal, astrocyte, and oligodendrocyte cells. Intracerebral grafting of the SS-OB2a NS/PCs also resulted in tumor formation with 76. 9 percent take (Fig. 3D and Table 1 ). Importantly, the clonally-derived OB2a NS/PCs which had been expanded as neurospheres homed in the host parenchyma showing ramified morphology and neuronal marker expression without generating any tumor (Figs. 3C-3D ). Similar findings were seen in animals engrafted with OB1, OB4, and OB5 NS/PCs irrespective the technique used for their propagation in vitro. Thus, the oncogenic transformation of human adult NSCs may occur whether a combination of expansion/ selection stimuli, like mitogens and serum, are simultaneously applied to these cells in vitro. Consistently, mouse embryonic NS The OB was obtained from adult patients who underwent neurosurgical operations. On immunohistochemical analysis, the human OB was found to contain about 700 to 1000 cells expressing the NS markers nestin and CD133. The nestinexpressing cells colocalize glial fibrillary acid protein (GFAP). These cells are located either within the inner plexiform layer close to the lateral olfactory tract where they show an astrocyte-like morphology, or in the external plexiform layer where they mainly appear as small rounded or unipolar cells. In the external plexiform layer, a few proliferating cells (n, 200-300) are detected by Ki67 labeling. Dissociated OB specimens were cultured in serumfree medium supplemented with the mitogens EGF and bFGF. Primary neurospheres were dissociated into single cells and plated one cell per miniwell. Clonal cell cultures were established by dissociation of secondary neurospheres. Clonal cultures from each OB were passaged up to P30 in mitogens. NS/PC cultures which lost growth factor dependence and multipotentiality were assessed for tumorigenicity in vivo. At P6, the NS/PCs that maintained growth factor dependence and multipotentiality were transduced to express GFP. The GFP-positive NS/PCs were expanded either as neurospheres in serum-free medium supplemented with mitogens or as adherent monolayers in medium containing mitogens and serum and then engrafted onto the striatum or spinal cord of immunocompromised rodents. doi:10. 1371/journal. pone. 0004434. g001",
"section_name": "Transformation of human adult NS/PCs following propagation in mitogens and serum",
"section_num": null
},
{
"section_content": "Somatic stem cells are thought to possess proficient mechanisms that allow replicative potential and chromosomal stability. However, chromosomal rearrangements have been detected in long-term expanded adult murine NSCs which apparently do not result in a malignant phenotype [2]. We performed kariotype analysis on NS/PCs at regular time points and found chromosomal rearrangements with repeated passages under mitogen stimulation (Figs. 4A and S1 ). Chromosomal changes were found both in tumorigenic and in non-tumorigenic NS/PCs suggesting that these cells need additional requirements to achieve tumorigenicity in vivo. It has been reported that the tumor-like growth properties of the stem cells associate with changes either in oncosuppressors or in oncogenes [2, [5] [6] 15]. Then, we set up a custom real-time RT-PCR array to analyze the expression of 92 mRNAs related with cell proliferation and cancer in tumorigenic relative to non tumorigenic NS/PCs both under proliferating culture conditions and under serum-induced differentiation (Fig. 4B ). Relative to the non tumorigenic OB1a and SS-OB1a NS/PCs, tumorigenic OB3a and SS-OB2a NS/PCs showed upregulation of genes related to cell proliferation and inhibiting apoptosis, though solely hTERT and NOTCH1 were overexpressed independently from mitogen stimulation. Tumorigenic OB3a and SS-OB2a NS/PCs did express the hTERT protein, which was undetectable in non tumorigenic NS/PCs, consistent with that reported in normal NS cells (Fig. 4C ) [19]. Immunofluorescence with anti-NOTCH1 antibody on tumorigenic OB3a and SS-OB2a NS/PCs demonstrated either increased cytoplasmic staining or abnormal nuclear staining (Fig. 4D ). Following NOTCH1 blockade with the c-secretase inhibitor X (GSI), OB3a and SS-OB2a NS/PCs lost their ability to form soft-agar colonies suggesting a functional role of NOTCH1 in tumorigenicity of these cells (Figs. 4C and S2 ). Although the xenografts grown after injection of OB3a and SS-OB2a NS/PCs were histologically reminiscent of different tumors, in both of them molecular analyses pointed to hTERT and NOTCH1 as critical pathways. Telomerase is highly expressed in the majority of human cancers including glioblastoma, where it is believed to contribute to tumor progression because telomerase-dependent telomere maintenance provides cells with an extended proliferative potential [20]. Glioblastoma stem cells, which express telomerase under proliferating serum-free conditions, transiently lose telomerase activity in serum-containing media; however, these cells regain telomerase at passages coincident with their exponential growth phase [21]. NOTCH is known to promote the proliferation of nonneoplastic NSCs and to inhibit their differentiation; it is also highly activated in embryonal brain tumors, such as medulloblastoma, where it is required both for maintaining the stem cell fraction in vitro and for tumor formation in vivo [22]. Up-regulation of hTERT and NOTCH1 in both tumorigenic OB3a and SS-OB2a NS/PCs suggests that a common mechanism may underly the malignant transformation of these cells, and that the histological differences between the OB3a-derived glioblastoma and the SS-OB2a-derived neuroesthesioblastoma may reflect different stages at which the NS/PCs have undergone neoplastic transformation in culture. In the OB3a-derived glioblastoma, the tumorigenic hit may have occurred in an astrocytic-committed precursor cell, whilst in the SS-OB2a-derived neuroesthesioblastoma the cell of origin may be a less differentiated NS/PC that has retained its multipotentiality. \n\nGene therapy trials using human hematopoietic stem cells after retroviral transduction have demonstrated a risk of insertional mutagenesis and oncogenic transformation [23]. However, we do not believe that that the tumorigenic transformation of the OBderived NS/PCs may be a consequence of the use of the lentiviral vector that integrated the GFP gene into the genome of these cells. The following arguments do not favour this hypothesis, 1) the OB3 cells, which in vivo gave origin to glioblastoma-like tumors, were not transduced with lentivirus to express GFP; 2) both the OB2 cells and the SS-OB2 cells were transduced with lentivirus, however, only the latter cells developed tumor in vivo, whilst the GFP-positive OB2 cells did not; and 3) the lentivirally transduced OB1, OB4, and OB5 NS/PCs were not tumorigenic in vivo. \n\nTo conclude, human adult NS/PCs cultured under mitogen stimulation are prone to develop chromosomal rearrangements. In vivo tumorigenicity is heralded by, 1) short latency in primary neurosphere formation, 2) persistent growth after removal of mitogens, 3) loss of serum-induced neuronal differentiation, and 4) up-regulation of hTERT and NOTCH1. The tumorigenic transformation of human adult NS/PCs isolated from an OB adjacent to meningioma raises the possibility that unusual levels of growth factors in the in situ condition, i. e. prior to ex vivo culture, may prime tumorigenicity. This indicates that the tumorigenic potential of the OB3 NS/PCs may be a specific feature of this cell line and not generalizable. In the case of SS-OB2 NS/PCs, however, specific culture conditions seem critical to transformation. Mitogens used simultaneously with factors favouring cell specification may disrupt the regulatory mechanisms that control self-renewal of NSCs and differentiation of TAPs. Therefore, culturing techniques where both proliferation and differentiation of NS/PCs are simultaneously enhanced should be evaluated further in future and discouraged if confirmed as linked to in vivo tumorigenicity.",
"section_name": "Cytological and molecular characteristics of tumorigenic NS/PCs",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "The OB was harvested from adult patients undergoing craniotomy at the Institute of Neurosurgery, Catholic University, Rome (Table S1 ). Informed consent was obtained according to protocols approved by the Ethical Commettee of the Catholic University. Immediately after removal, the OBs were dissociated in Papain 0,1% (Sigma-Aldrich, St. Louis, MO) for 30 minutes at PCs (P6) cultured in serum-free medium supplemented with mitogens (left) and in medium containing 1% serum without mitogens (right). Doublepositive cells for nestin and GFAP were counted positive for each antigen and also for both antigens (nestin/GFAP). The OB3a cells do not differentiate towards the neuronal lineage in response to serum stimulation. C, Subcutaneous xenografs of OB3a cells in nude athymic mice. a, Subcutaneous nodules two weeks after grafting (arrows in a). Histological features of glioblastoma (b, H&E). Expression of astrocytic cell marker GFAP (c) and negative staining for the neuronal cell marker neurofilament (d). D, Intracerebral tumor xenografts of OB3a cells in SCID mice. Pattern of brain invasion by OB3a cells one week after grafting into the striatum (a, H&E). Low (b) and high (c and d) magnifications of intraventricular anaplastic astrocytoma-like tumor by two weeks after grafting (b and c, H&E d, anti-HNA immunoreaction). A, Scale bar 250 mm; b, Scale bar 80 mm; c, Scale bar 50 mm; d, Scale bar 30 mm. doi:10. 1371/journal. pone. 0004434. g002 37uC. Dissociated cells were cultured in the presence of human recombinant EGF (20 ng/ml; PeproTech, Rocky Hill, NJ), human recombinant bFGF (10 ng/ml; PeproTech), and LIF (20 ng/ml; Immunological Sciences, Rome, Italy) in DMEM/F12 (1:1) serum-free medium (Invitrogen, Carlsband, CA) containing Lglutamine 2 mM, glucose 0. 6%, putrescine 9. 6 ug/ml, progesterone 0. 025 mg/ml, sodium selenite 5. 2 ng/ml, insulin 0. 025 mg/ ml, apo-transferrin sodium salt 0. 1 mg/ml, sodium bicarbonate 3 mM, Hepes 5 mM, BSA 4 mg/ml, heparin 4 ug/ml [24]. Primary neurospheres were dissociated with Accutase (Invitrogen) for 4 minutes at 37uC, serially diluted, and plated one cell per mini-well onto 96-well plates. Mini-wells containg one single cell were marked after microscopic confirmation and assessed for secondary neurosphere generation after one week. Secondary neurospheres were subsequently dissociated, plated at the density of 10 3 cells/cm 2 in serum-free medium containing EGF and bFGF, and passaged up to P30. All experiments were done on at least two clonal cultures from each OB. Between P7 and P10, parallel cultures were established in which cells were grown as adherent monolayers in medium containing EGF and bFGF supplemented with 5% fetal calf serum (Hyclone, Logan, UT). For cell growth experiments, dissociated cells were plated on Matrigel at the density of 10 3 cells/cm 2 either in serum-free medium containing EGF and bFGF or in medium where mitogens were replaced with 1% serum or in medium containing mitogens and 5% serum (Hyclone). Cells were counted with hemacytometer every 48 hours. Cell viability was determined colorimetrically by MTS-assay (Supplementary Methods S1). Differentiation assays were performed by 14 days after plating on Matrigel coated glass coverslips in the absence of EGF and bFGF and in the presence of 1% fetal calf serum (Hyclone) supplemented with 39-59-cyclic adenosine monophosphate (cAMP) 50 mM, all-trans retinoic acid 5 mM (Sigma Aldrich), and triiodothyronine (T3) 30 nM (Sigma Aldrich). Immunostaining of NS/PCs was performed as described [15]. We used antibodies against nestin (Chemicon, Temecula, CA), CD133 (CD133/2; Miltenyi, Bergisch, Germany), GFAP (Dako, Glostrup, Denmark), b tubulin III (Chemicon), neurofilament RT-97 (Developmental Studies Hybridoma Bank, Iowa City, IA), MAP2 (Chemicon), NG2 (Chemicon), O4 (Chemicon), hTERT (Novocastra Laboratories), and NOTCH1 (Chemicon).",
"section_name": "Isolation, Culturing, and Immunophenotyping of NS/PCs",
"section_num": null
},
{
"section_content": "Enhanced green fluorescent protein (GFP) gene transfer in the NS/PCs was performed at P6 using a variant of third generation lentiviral vectors as described [25].",
"section_name": "Generation of Fluorescent NS/PCs",
"section_num": null
},
{
"section_content": "Studies involving animals were approved by the Ethical Committee of the Catholic University School of Medicine, Rome. The NS/PCs were grafted either subcutaneously in nude athymic mice, or into the brain of severe combined immunodeficient (SCID) mice, or onto the spinal cord of ciclosporine treated rats (Supplementary Methods S1). For implantation, the NS/PC cultures were splitted 24-48 hours prior to transplant and injected as single cell suspensions. After two to 6-week survival, the animals were sacrificed with an overdose of barbiturate. Either the subcutaneous graft or brain or spinal cord was removed and processed for histology as described [25].",
"section_name": "Grafting of NS/PCs in Immunodeficient Rodents",
"section_num": null
},
{
"section_content": "Immunohistochemistry was performed on deparaffinized sections using the avidin-biotin-peroxidase complex methods as ). Gene expression in tumorigenic OB3a and SS-OB2a cells is presented relative to the non tumorigenic OB1a and SS-OB1a cells, respectively, both under proliferating culture conditions and under serum-induced differentiation. Upregulated genes (2 folds, red), downregulated genes (2 folds, green), unchanged genes (gray). C, Expression of hTERT protein in OB-derived NS/PCs. Western blot analysis of HUVEC (lane 1), OB1a (lane 2), OB2a (lane 3), SS-OB2a (lane 4), OB3a (lane 5), and TB10 human glioblastoma (lane 6) cells. Immunohistochemical analysis of hTERT expression in the human adult OB and OB-derived NS/PCs. The hTERT protein is absent in the adult OB (a) as well as in the non-tumorigenic OB1a (b), OB2a (c), and SS-OB1a (e) cells. hTERT is strongly expressed in the nuclei of both tumorigenic OB3a (d) and SS-OB2a (f) NS/PCs. Scale bar 40 mm. D, Immunofluorescence analysis of NOTCH1 expression (left) in OB1a (a), OB3a (b), OB2a (c), and SS-OB2a (d) NS/PCs. NOTCH1 signaling is required for the formation of colonies in soft agar (right). Exposure to c-secretase inhibior X (GSI) after seeding in soft agar significantly reduced clonigenic potential of tumorigenic OB3a and SS-OB2a NS/PCs (P,0. 0001, Student t-test). doi:10. 1371/journal. pone. 0004434. g004\n\ndescribed [25]. The following primary antibodies were used, anti-GFAP (Ylem, Avezzano, Italy), anti-neurofilament (Ylem), anti-NG2 (Chemicon), anti-CD133/1 (Miltenyi), anti-nestin (Santa Cruz Biotecnology), anti-human nuclei antigen, (HNA; Chemicon), anti-epithelial membrane antigen (EMA; Ylem), anti-Ki67 (Dakocytomation), anti-hTERT (Novocastra Laboratories). Endogenous biotin was saturated by biotin blocking kit (Vector). For antigen retrieval, paraffin sections were microwave-treated in 0. 01 M citric acid buffer at pH 6. 0 for 10 min. For hTERT antigen retrieval, paraffin section were microwave-treated in EDTA buffer at pH 8. 0 for 10 min.",
"section_name": "Immunohistochemistry",
"section_num": null
},
{
"section_content": "NS/PC cultures at P3-P8 were incubated in medium containing 10 ng/ml colcemid for 18 hours. The cultures were then lifted and centrifuged. Pellets were osmotically shocked with 0. 075 M KCl and fixed with 3:1 methanol:glacial acetic acid. Standard cytogenetic G bands were performed and a mean of 20 methaphases per cell lines were analyzed.",
"section_name": "Chromosome analysis",
"section_num": null
},
{
"section_content": "We used a 7900HT instrument equipped with SDS2. 2 software to perform a custom real-time RT-PCR array (Microfluidic Card, Applied Biosystems, CA). Briefly, cells were plated on Matrigel precoated 100 mm dishes and processed as described above. Preparation of total RNA and cDNA was performed using Ribo Pure kit (Ambion, Austin, TX) and high capacity cDNA Reverse Transcriptase kit (Applied Biosystems), respectively. For data analysis, the mathematical process for deriving relative quantification values was used as described by the manufacturer's guide (Applied Biosystems).",
"section_name": "Macroarray Analysis",
"section_num": null
},
{
"section_content": "Cell pellets were lysated in a modified RIPA buffer (Tris-HCl 10 mM pH 7. 5, NaCl 10 mM, NP-40 0. 2%, EGTA 1 mM, EDTA 1 mM, DTT 1 mM and protease inhibitor cocktail; Sigma-Aldrich) on ice for 5-10 min. Nuclear extracts were resuspended in Urea buffer (10 M Urea, Tris-HCl 50 mM pH 7. 5, DTT 25 mM) sonicated and normalized using Bradford Assay (Promega Corp). Protein extracts were analyzed by polyacrylamide gel electrophoresis and Western blot. Proteins were probed with rabbit polyclonal anti-TERT (1:1000; Santa Cruz Biotecnology) and monoclonal anti-b-actin (1:5000; Sigma-Aldrich). As control, HUVEC cells at passages 4 to 5 (Bio-Wittaker, Walkersville, MD) and TB10 glioblastoma cells were used.",
"section_name": "Western Blot",
"section_num": null
},
{
"section_content": "Table S1 Found at: doi:10. 1371/journal. pone. 0004434. s001 (0. 02 MB DOC). The OB2a, SS-OB2a, and OB3a NS/PCs were seeded with a mixture of Top Agar (0,5%)-proliferation medium on top of the base layer. The plates were then incubated at 37u in humidified incubator for 3-4 weeks and colonies were counted. Every week fresh medium mixed with Top-agar was added together with 5 mmol/L csecretase inhibitor X (GSI; L-685. 458) or DMSO as control. Three plates for each NSC/PC culture were used. Found at: doi:10. 1371/journal. pone. 0004434. s005 (9. 58 MB TIF)",
"section_name": "Supporting Information",
"section_num": null
},
{
"section_content": "",
"section_name": "Table S2",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "We thank Teresa M. Natale for her contribution in karyotype analysis.",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This work was supported by grants from Fondi d9Ateneo ( Progetti D1 ), from ATENA Onlus, and Nando Peretti Foundations, and from Mrs. Paola Krajnik. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "Conceived and designed the experiments: PC GM LML RP. Performed the experiments: PC MB LRV GP NM ET. Analyzed the data: LM. Contributed reagents/materials/analysis tools: CC. Wrote the paper: RP.",
"section_name": "Author Contributions",
"section_num": null
}
] |
10.1038/s41467-018-04283-9
|
NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence
|
<jats:title>Abstract</jats:title><jats:p>Senescent cells interact with the surrounding microenvironment achieving diverse functional outcomes. We have recently identified that NOTCH1 can drive ‘lateral induction’ of a unique senescence phenotype in adjacent cells by specifically upregulating the NOTCH ligand JAG1. Here we show that NOTCH signalling can modulate chromatin structure autonomously and non-autonomously. In addition to senescence-associated heterochromatic foci (SAHF), oncogenic RAS-induced senescent (RIS) cells exhibit a massive increase in chromatin accessibility. NOTCH signalling suppresses SAHF and increased chromatin accessibility in this context. Strikingly, NOTCH-induced senescent cells, or cancer cells with high JAG1 expression, drive similar chromatin architectural changes in adjacent cells through cell–cell contact. Mechanistically, we show that NOTCH signalling represses the chromatin architectural protein HMGA1, an association found in multiple human cancers. Thus, HMGA1 is involved not only in SAHFs but also in RIS-driven chromatin accessibility. In conclusion, this study identifies that the JAG1–NOTCH–HMGA1 axis mediates the juxtacrine regulation of chromatin architecture.</jats:p>
|
[
{
"section_content": "ellular senescence is an autonomous tumour-suppressor mechanism that can be triggered by pathophysiological stimuli including replicative exhaustion, exposure to chemotherapeutic drugs and hyper-activation of oncogenes, such as RAS 1. Persistent cell cycle arrest is accompanied by diverse transcriptional, biochemical and morphological alterations. These senescence hallmarks include increased expression and secretion of soluble factors (senescence-associated secretory phenotype (SASP)) 2, 3 and dramatic alterations to chromatin structure 1, 4, 5. Importantly, the combination, quantity and quality of these features can vary depending on the type of senescence. Senescent cells have profound non-cell autonomous functionality. The SASP can have either protumorigenic or antitumorigenic effects and act in an autocrine or paracrine fashion 2, [6] [7] [8]. In addition, we have recently identified that NOTCH signalling can drive a cellcontact-dependent juxtacrine senescence 9. \n\nThe NOTCH signalling pathway is involved in a wide array of developmental and (patho-)physiological processes. NOTCH has roles in differentiation and stem cell fate 10 and perturbations have been linked to tumorigenesis where NOTCH can have either oncogenic or tumour-suppressive functionality 11. The pathway involves proteolytic cleavage of the NOTCH receptor upon contact-mediated activation by a ligand of the JAGGED (JAG) or DELTA family on the surface of an adjacent cell. The cleaved NOTCH-intracellular domain translocates to the nucleus where, together with transcriptional co-activators such as mastermindlike 1 (MAML1), it drives transcription of canonical target genes, including the HES and HEY family of transcription factors 10. NOTCH signalling has also been shown to induce a type of senescence, NOTCH-induced senescence (NIS), where cells are characterised by distinct SASP components 9, 12. Recently, we showed that during NIS there is a dramatic and specific upregulation of JAG1 that can activate NOTCH1 signalling and drive NIS in adjacent cells ('lateral induction') 9. \n\nDuring senescence, particularly in oncogenic RAS-induced senescent (RIS) fibroblasts, characteristic changes to chromatin culminate in the formation of senescence-associated heterochromatic foci (SAHFs) 13 rearrangement of existing heterochromatin 14. Other alterations include the formation of senescence-associated distention of satellites (SADS) 15. SAHF formation is dependent on chromatin-bound highmobility group A (HMGA) proteins, particularly HMGA1 16. These are a family of architectural proteins, consisting of HMGA1 and HMGA2, which bind to the minor groove of ATrich DNA via three AT-hook domains to alter chromatin structure 17, 18. Despite a critical role in the formation of SAHFs during senescence, HMGA proteins are also important during development where they promote tissue growth 19, 20 and regulate differentiation [21] [22] [23] [24]. Furthermore, many studies have demonstrated an association between high HMGA1 expression and aggressive tumour biology 25, 26. \n\nChromatin accessibility at regulatory elements including promoters and enhancers is highly correlated with biological activity 27. High-throughput sequencing using FAIRE-seq, a method that identifies open and closed chromatin based on phenol separation 28, has revealed that, in cells that have undergone replicative senescence, previously heterochromatic domains enriched for various repeat elements become more accessible while euchromatic domains undergo condensation 29. However, it remains unknown how chromatin accessibility is altered in RIS and NIS cells. \n\nHere we characterise the chromatin phenotype in RIS and NIS cells. We demonstrate that these two types of senescent cells exhibit distinct chromatin structures at microscopic and nucleosome scales. \n\nBoth gain multiple chromatin accessible regions, which are often exclusive between RIS and NIS. Strikingly, we find that autonomous and non-cell autonomous activation of the NOTCH signalling pathway in RIS cells can repress SAHFs and the formation of RISdriven chromatin-accessible regions, partially by transcriptional repression of HMGA1. Our study demonstrates that chromatin structure and the nucleosome landscape can be regulated through juxtacrine signalling. The relationship between these two prominent tumour-associated genes, HMGA1 and NOTCH1, may also have prognostic value in vivo.",
"section_name": "C",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "SAHFs. We have previously demonstrated that ectopic NOTCH1-intracellular domain (N1ICD), an active form of NOTCH1 (Fig. 1a ), can drive NIS that is distinct from RIS in terms of SASP composition 9 and noticed that NIS cells also have a unique chromatin structure. \n\nTo examine the relationship between NOTCH1 and chromatin structure, we introduced ectopic N1ICD into IMR90 human diploid fibroblasts (HDFs) stably expressing a 4-hydroxytamoxifen (4OHT)-inducible oestrogen receptor-oncogenic HRAS fusion protein (IMR90 ER:HRAS G12V cells) 30. Ectopic expression of N1ICD alone induced senescence with dramatically enlarged nuclei, even larger than in RIS (Fig. 1b, c ). Similarly to RIS, NIS cells formed SADS, a more common chromatin feature of senescence than SAHFs (Supplementary Fig. 1a ) 15. However, in marked contrast to RIS, NIS cells lacked SAHFs (Fig. 1b, d ). \n\nTo ask whether NIS cells simply lack SAHFs or whether N1ICD actively modulates chromatin structure, we expressed N1ICD in the presence of HRAS G12V induced using 100 nM of 4OHT for 6 days. Interestingly, N1ICD in the context of RIS also resulted in a dramatic enlargement of nuclei but a complete ablation of SAHF formation (Fig. 1b-d ). This was emphasised by a 'smoothening' of chromatin as indicated by a marked reduction in the standard deviation of 4,6-diamidino-2-phenylindole (DAPI) signal measured within individual nuclei (Fig. 1b, c ; Supplementary Fig. 1b-d ), We have previously shown that ectopic N1ICD in the RIS context results in senescence with SASP composition broadly similar to NIS 9. Thus our data indicate that NOTCH is dominant over RIS in terms of chromatin phenotype as well as SASP composition. \n\nIn IMR90 ER:HRAS G12V cells, RIS develops progressively over a time period of ~6 days following the addition of 4OHT 30. NOTCH1 signalling is temporally regulated during RIS, where cleaved and active N1ICD is transiently upregulated before downregulation at full senescence 9. To examine the temporal effects of NOTCH1 signalling on SAHF formation, we performed a time course experiment in IMR90 ER:HRAS G12V cells. Cells were retrovirally infected with a dominant-negative form of MAML1 fused to mVenus (dnMAML1-mVenus) or treated with the γ-secretase inhibitor N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) to repress downstream signalling by N1ICD (Fig. 1a ). We found that a greater number of SAHF-positive cells were formed and that these accumulated at earlier time points when NOTCH1 signalling was repressed (Fig. 1e ). Furthermore, a dose-dependent effect was evident where higher concentrations of DAPT resulted in a greater proportion of cells developing SAHF during RIS (Supplementary Fig. 1e ). SAHFs are not typically prominent in DNA damage-induced senescence (DDIS) in IMR90 cells 1. However, DAPT significantly promoted SAHF formation in DDIS (by etoposide) (Supplementary Fig. 1f ). To determine whether NOTCH1 activity can reverse SAHF after they have formed, we infected IMR90 cells with doxycycline (DOX)inducible N1ICD-FLAG and constitutive HRAS G12V. The addition of DOX after the establishment of senescence was sufficient to reduce the number of SAHF-positive cells and the standard deviation of DAPI signal, suggesting some degree of reversibility (Supplementary Fig. 1g ). Together, our data suggest that NOTCH signalling has a chromatin 'smoothening' effect that antagonises SAHF formation. \n\nNon-cell autonomous regulation of SAHFs. N1ICD-expressing cells can induce NIS in adjacent normal cells, at least in the case of IMR90 fibroblasts 9. To determine whether N1ICD-expressing cells can also alter chromatin structure in adjacent cells, we performed co-cultures between mRFP1-expressing IMR90 ER: HRAS G12V and IMR90 cells expressing DOX-inducible N1ICD-FLAG in the presence and absence of 4OHT and DOX (Fig. 2a ). Strikingly, co-culture with N1ICD-expressing IMR90 cells was sufficient to repress SAHF formation in adjacent RIS (red) cells (Fig. 2b, c ). \n\nOf the canonical NOTCH1 ligands, we have previously observed a strong and unique upregulation of JAG1 following ectopic N1ICD expression, which we found to be responsible for the juxtacrine transmission of NIS 9. We reasoned that N1ICDmediated upregulation of JAG1 and subsequent 'lateral induction' of NOTCH1 signalling is a likely mechanism by which SAHFs are regulated non-autonomously. To test this hypothesis, we expressed ectopic JAG1 fused to mVenus (JAG1-mVenus) in retinal pigment epithelial (RPE1) cells. We confirmed cell surface expression of ectopic JAG1 by flow cytometry (Supplementary Fig. 2a ) before co-culturing with mRFP1-expressing IMR90 ER: HRAS G12V cells. RPE1 JAG1-mVenus cells, but not control RPE1 cells, significantly repressed the formation of SAHFs (Fig. 2e, f ). Note that this repression did not occur when these two types of cells were co-cultured without physical contact in a transwell format (Supplementary Fig. 2b ). Our data suggest a mechanism by which lateral induction of NOTCH signalling by JAG1 can block SAHFs in the context of RIS; i. e. higherorder chromatin structure can be regulated through cell-cell contact.",
"section_name": "NOTCH1 reprogrammes chromatin structure and abrogates",
"section_num": null
},
{
"section_content": "To unravel the mechanisms underpinning NOTCH1-dependent repression of SAHFs, we re-analysed previously published RNAseq data generated from IMR90 cells expressing HRAS G12V and N1ICD 9. We found that N1ICD dramatically represses the expression of HMGA1 and HMGA2 (Supplementary Fig. 3a ), critical components of SAHF structure 16. \n\nTo validate that NOTCH1 signalling represses HMGAs, we introduced constitutive N1ICD into IMR90 ER:HRAS G12V cells. Ectopic N1ICD significantly repressed HMGA1 and HMGA2 at an mRNA and protein level in both the presence and absence of 4OHT-induced HRAS G12V (Fig. 3a, b ). The enforced expression of N1ICD after senescence establishment also resulted in the reduction of HMGA1 albeit to a lesser extent than pre-senescence N1ICD expression (Supplementary Fig. 1g ). N1ICD has a similar effect on HMGA1 and 2 protein levels when expressed in other cell lines in the absence of HRAS G12V, suggesting a conserved mechanism (Supplementary Fig. 3b ). In the DOX-inducible N1ICD-FLAG system, inhibition of NOTCH1 signalling by coexpression of dnMAML1-mVenus was sufficient to rescue N1ICD-mediated repression of HMGA1 and HMGA2 (Fig. 3c, d ), suggesting the effect is dependent on the canonical pathway of NOTCH signalling. \n\nFinally, we used IMR90 ER:HRAS G12V cells expressing DOXinducible N1ICD-FLAG to investigate whether ectopic reexpression of EGFP-tagged HMGA1 is sufficient to rescue SAHFs. The introduction of EGFP-HMGA1 resulted in a partial, but significant, rescue of SAHF-positive cells when cells were treated with DOX and 4OHT (Fig. 3e ). \n\nCollectively, our data suggest that NOTCH1 signalling represses the formation of SAHFs at least partially by inhibiting HMGAs. \n\nNon-cell autonomous inhibition of HMGAs. To determine whether HMGAs are repressed non-autonomously by JAG1 expressing cells, we performed further co-cultures between RPE1 cells retrovirally infected with JAG1-mVenus and IMR90 cells ectopically expressing a cell surface marker, rat-Thy1, allowing for subsequent isolation using magnetic-activated cell sorting (MACS) (Fig. 3f ). As expected, IMR90 cells co-cultured with JAG1-expressing cells upregulated canonical NOTCH1 target genes, HEY1 and HEYL. Both HMGA1 and HMGA2 were significantly repressed in the same IMR90 cells (Fig. 3f ), demonstrating that HMGA proteins can be repressed non-cell autonomously. \n\nAltered chromatin accessibility in RIS and NIS. To investigate whether NOTCH1 influences chromatin structure at a higher resolution, we employed ATAC-seq (assay for transposaseaccessible chromatin using sequencing) 31. This method exploits a hyperactive Tn5 transposase that inserts sequencing adapters into regions of accessible chromatin. Following adapter-primed PCR amplification, these regions were sequenced to identify accessible regions of chromatin genome wide (Fig. 4a ). \n\nWe generated at least three replicates from IMR90 ER: HRAS G12V cells expressing N1ICD-FLAG or a control vector and induced with 4OHT or not. For simplicity, these conditions were labelled as 'Growing', 'RIS', 'NIS' and 'N+RIS' (expressing both N1ICD and RAS). Using a previously published normalisation approach 32, we generated normalised coverage files that appeared comparable to each other, especially around housekeeping genes (Fig. 4b ). Most of the samples, excluding a single replicate from the NIS and N+RIS conditions (which were excluded from downstream analysis), were of high quality with a 'reads in peaks' percentage (RiP%) of >10% (Supplementary Fig. 4a ). Replicates clustered well by unbiased principal component analysis (PCA) (Supplementary Fig. 4b ). Moreover, our samples clustered with publically available ATAC-seq and DNase-seq data generated from IMR90 cells (Supplementary Fig. 4c ), but separated from other cell types (BJ, HaCaT, MCF710A and HEKn cells). \n\nUsing MACS peak calling, we found that the number of peaks identified in each replicate of a condition was similar and that, in general, chromatin accessibility was dramatically increased in RIS (145,649 consensus peaks detected in ≥2 replicates) and NIS cells (149,877 peaks) relative to growing cells (83,920 peaks) (Supplementary Fig. 5a ). To quantitatively identify regions of altered accessibility in RIS and NIS cells relative to growing cells, we performed differential binding analysis using both edgeR 33, 34 and THOR 35 before taking only regions identified by both methods for downstream analysis (Supplementary Data 1). Using this stringent approach, we identified 44,556 regions that become significantly more accessible (opened) and 9603 regions that become significantly less accessible (closed) in RIS cells relative to growing cells. In NIS cells, 20,499 regions became more accessible and 15,444 regions less accessible (Fig. 4c ). Despite the robust gain of chromatin accessibility in both types of senescence, there were relatively few shared sites (Fig. 4d ). \n\nA previous study mapping chromatin accessibility in replicatively senescent cells using FAIRE-Seq found that gene-distal regions, especially repeat regions, become relatively more open whereas genic regions become closed compared to growing fibroblasts 29. Consistently, regions of increased accessibility in RIS and NIS cells were enriched at gene-distal sites (Supplementary Fig. 5b ) with the majority of opened regions mapping to enhancer, intergenic, intronic and repeat regions (Fig. 4e ). Many of these repeat regions were further annotated as long interspersed elements, long-terminal repeats, short interspersed elements and simple repeat regions (Supplementary Fig. 5c, d ), although these values may be underestimated due to the exclusion of multi-mapping reads from our data. Many of the regions that became less accessible in RIS cells relative to growing cells were closer to transcriptional start sites (TSSs) (Supplementary Fig. 5b ) and mapped to exons, CpG-islands and untranslated regions (UTRs) (Fig. 4e ). In contrast to replicative senescence 29 gene-distal elements (Supplementary Fig. 5b, Fig. 4e ). Therefore, while RIS largely mirrors replicative senescence, NIS is characterised by remodelling (both opening and closing) of genedistal regions. \n\nAltered accessibility of genes reflects expression. Chromatin accessibility at regulatory elements has been correlated with gene expression 27. To determine whether genic alterations to chromatin accessibility in RIS and NIS reflects gene expression, we assigned regions of altered accessibility (opened or closed in RIS or NIS) to genes if within 500 bp of a TSS (Fig. 4f ). On average, genes that were opened in RIS relative to growing cells were also transcriptionally upregulated by mRNA-seq in RIS relative to growing cells (Fig. 4f, top). Genes that were opened in NIS cells were transcriptionally upregulated in NIS cells, while less accessible genes were transcriptionally repressed (Fig. 4f, bottom). Consistent with our previous RNA-seq data 9, genes that became more accessible in RIS were significantly enriched within gene ontology (GO) terms such as 'inflammatory response' and 'cytokine secretion', reflecting the inflammatory secretome produced by RIS cells (Fig. 4g ). Genes that became less accessible in RIS were enriched within GO terms such as 'regulation of cell cycle' (Fig. 4g ), perhaps reflecting non-proliferative features of RIS (although average gene expression of this gene set was not significantly altered). Unbiased motif enrichment analysis revealed that regions opened in RIS were highly enriched for the C/EBPβ-binding motif (Supplementary Fig. 5e ), consistent with the important role of C/EBPβ in regulating the inflammatory SASP 3. Regions opened in NIS were enriched with the RBP-Jbinding motif (Supplementary Fig. 5 f ), a critical DNA-binding factor downstream of NOTCH signalling 11. Normalised ATACseq coverage files, when viewed using a genome browser (Fig. 4b, Supplementary Fig. 6a-d ), demonstrated increased accessibility around transcriptionally activated genes. We also noted that, while the accessibility at many promoters was unaltered in RIS cells, some transcriptionally activated genes, such as IL1A and HMGA1, were proximal to enhancer elements that became more accessible (Fig. 4b, Supplementary Fig. 6a ). Together, these data demonstrate that RIS and NIS cells have unique open chromatin landscapes and that (gene proximal) alterations reflect their transcriptional landscapes. \n\nNOTCH signalling antagonises chromatin opening in RIS. By unbiased clustering of ATAC-seq data, we observed a greater correlation between NIS and N+RIS cells than between RIS and N+RIS cells (Fig. 5a ). This suggests a dominant effect of N1ICD over RAS on the nucleosome scale, consistent with our previous observations for SASP components 9 and SAHFs (Fig. 1d ). \n\nTo determine whether NOTCH1 signalling can repress the chromatin alterations observed in RIS in favour of a 'NIS-like' chromatin landscape, we focussed on the 44,556 regions that became significantly more accessible in RIS cells relative to growing cells (referred to as 'RIS-driven accessible regions', Fig. 4c ) and the 20,499 regions that became significantly more accessible in NIS cells relative to growing cells (referred to as 'NIS-driven accessible regions', Fig. 4c ). By comparing chromatin accessibility of N+RIS cells with RIS cells we found that formation of many RIS-driven accessible regions (62. 7%) were repressed by N1ICD expression (Fig. 5b ). N1ICD expression also increased the accessibility of NIS-driven accessible regions (Fig. 5b ). When viewed in the genome browser, it was evident that N1ICD expression can repress the formation of accessible regions located at enhancer elements upstream of the HMGA1 promoter in RIS cells (Supplementary Fig. 6a ), although we failed to detect any alterations at the HMGA2 locus (Supplementary Fig. 6b ), providing a potential mechanism for NOTCH1mediated repression of HMGA1. \n\nHMGA proteins have previously been shown to affect chromatin compaction. To determine whether repression of HMGA1 is a mechanism by which N1ICD can repress formation of RIS-driven accessible regions, we generated additional ATACseq samples from IMR90 ER:HRAS G12V cells expressing a short hairpin against HMGA1 16 and treated with 4OHT, hereafter referred to as 'RIS+shHMGA1'. By comparing RIS+shHMGA1 with RIS, we identified 8909 RIS-driven accessible regions that were dependent on HMGA1 (Fig. 5c ). Of these, 69. 9% (6168) were also repressed by N1ICD (Fig. 5d ). These analyses illustrate that a subset of RIS-driven accessible regions can be repressed by N1ICD, possibly by HMGA downregulation. However, HMGA1 knockdown was not sufficient to induce the formation of NIS-driven accessible regions (Fig. 5c ), suggesting an HMGA1-independent mechanism in the formation of these sites. RIS-driven accessible regions (opened in RIS) were significantly more AT-rich than NISdriven accessible regions (opened in NIS) or regions with reduced accessibility (Fig. 5e ), supporting the involvement of HMGA1 in the formation of RIS-driven accessible regions. \n\nTo validate the above approach, we used our normalised coverage files to perform unbiased k-means clustering centred around accessible regions that were altered in either RIS or NIS cells (opened or closed relative to growing cells) (Fig. 5f ). Accessible regions separated into clusters that were dominated by either the RIS (clusters 1 and 2) or NIS (clusters 3 and 4) conditions. Strikingly, the signal in RIS-dominated clusters, cluster 2 in particular, was reduced in the N+RIS and RIS +shHMGA1 conditions when compared to the RIS condition (Fig. 5f ). Consistently, cluster 2 was more AT-rich than cluster 1 (Fig. 5g ), supporting a role for HMGA1. Notably, while peaks in clusters 3 and 4 were increased in the N+RIS condition, they did not increase in the RIS+shHMGA1 condition, reinforcing an HMGA1-independent mechanism of chromatin opening in NIS (Fig. 5f ). Therefore, in line with microscopic SAHF structures, N1ICD alters chromatin structure in RIS at the nucleosome scale in part by repressing HMGA1 expression. . n = 3 biological replicates except for Hep3B cultures where n = 6. c SAHF quantification in IMR90 ER:HRAS G12V cells expressing mRFP+100 nM 4OHT±10 µM DAPT. d Schematic showing experimental set-up. IMR90 ER:HRAS G12V cells expressing mRFP were cultured with tumour cell lines +100 nM 4OHT before flow sorting to isolate red cells. e qRT-PCR of mRNA isolated from the cells described in c. n = 3 biological replicates. f Immunoblotting of JAG1 in the cells indicated. g Quantification of SAHFs in IMR90 ER:HRAS G12V expressing mRFP co-cultured with the tumour cells indicated for 6 days +100 nM 4OHT. n = 3 biological replicates. h Volcano plots showing regions of altered accessibility in RIS cells co-cultured with MCF7, A549 and Hep3B cells (as in c) relative to RIS cells cultured alone. Regions that also become more accessible in RIS (red) and NIS (purple) vs. growing are indicated where numbers indicate the total number of significant alterations (log 2 fold change <-0. 58 or >0. 58 and FDR < 0. 01) and <-1 indicates the number with a log 2 fold change of <-1. i Number of more accessible regions in RIS (identified in Fig. 4c ) that are repressed by co-culture with MCF7, A549 and Hep3B. b, c, e, g Statistical significance calculated using one-way ANOVA with Tukey's correction for multiple comparisons; *p ≤ 0. 05, **p ≤ 0. 01, ***p ≤ 0. 001, NS = not significant Non-cell autonomous regulation of SAHFs by tumour cells. Both HMGA1 and NOTCH1 can act as oncogenes or tumour suppressors in a context-dependent manner. We reasoned that the relationship between these two genes might also be important in the tumour microenvironment and asked whether tumour cells expressing JAG1 can affect HMGA1 expression and chromatin structure in adjacent fibroblasts. \n\nTo answer this question, we used the Cancer Cell Line Encyclopedia 36 to identify tumour cell lines that express low (MCF7), medium (A549) and high (Hep3B) levels of JAG1, which we confirmed by immunoblotting (Fig. 6a ). Co-culture of tumour cell lines with IMR90 cells expressing both ER:HRAS G12V and mRFP1 in the presence of 4OHT was sufficient to repress SAHF formation in red (RIS) cells in a contact-dependent manner (Fig. 6b ; Supplementary Fig. 7a ). The number of SAHF-positive red cells inversely correlated with the level of JAG1 expressed by the tumour cell lines (Fig. 6b ). Non-autonomous inhibition of SAHF formation in the co-culture system was completely abrogated by DAPT, suggesting the effect is dependent on the canonical NOTCH pathway (Fig. 6b, c ). Consistent with our previous experiments (Fig. 1e, Supplementary Fig. 1e ), the addition of DAPT was sufficient to increase the percentage of SAHF-positive IMR90 cells above basal levels both in monoculture (Fig. 6c ) and co-culture (Fig. 6b ). \n\nTo determine whether tumour cell lines can induce NOTCH1 signalling and repress HMGAs non-autonomously, we repeated the co-cultures and isolated the IMR90 ER: HRAS G12V mRFP1 cells using flow cytometry (Fig. 6d, Supplementary Fig. 7b ). We found a dramatic upregulation of the canonical NOTCH1 target gene HEYL and a concurrent downregulation of HMGA1 and HMGA2 in fibroblasts cocultured with JAG1-expressing tumour cells, particularly A549 and Hep3B cells (Fig. 6e ). Two other canonical target genes, HES1 and HEY1, were not dramatically upregulated by JAG1expressing cell lines (Supplementary Fig. 7c ). Although HEYL, HEY1 and HES1 are known as 'canonical targets' of NOTCH, their transcriptional regulation by NOTCH signalling is highly complex: for example, unique combinations of, or interactions between, NOTCH ligands and receptors can provide preferential induction of certain targets 37, 38. Different tumour cell lines might differentially express other NOTCH ligands (in addition to JAG1) or other NOTCH pathway modulators, conferring additional complexity. \n\nTo further determine whether the effect described above is JAG1-dependent, we used CRISPR-Cas9 technology to generate A549 cells with bi-allelic knockout of endogenous JAG1. Two knockout clones were isolated; clone 1 had a 5 bp deletion in the first allele and a 1 bp deletion in the second allele while clone 2 had a 1 bp deletion in the first allele and a 14 bp deletion in the second allele (Supplementary Fig. 7d ). A control clone was generated by transfecting cells with Cas9 but omitting guide RNA (-gRNA control cells). We found that both clones 1 and 2 had reduced JAG1 levels by immunoblotting (Fig. 6f ) and cell-surface JAG1 (plus JAG2) levels by flow cytometry (Supplementary Fig. 7e ). Proliferation was not substantially altered in JAG1knockout cells relative to control or parental cells (Supplementary 7f ). In contrast to control or parental A549 cells, co-culture of JAG1-knockout A549 cells with red IMR90 ER:HRAS G12V cells in the presence of 4OHT had little effect on SAHF formation in red (RIS) cells (Fig. 6g ). \n\nIn addition to JAG1-knockout A549 cells, we generated MCF7 cells containing DOX-inducible JAG1 fused to mVenus (JAG1-mVenus). By immunoblotting, we observed low-level expression of JAG1-mVenus even in the absence of DOX, likely caused by 'leaky' transcription (Supplementary Fig. 7g ). Addition of 10 ng/ mL of DOX to the culture was sufficient to induce JAG1 to comparable levels as those observed endogenously in Hep3B cells (Supplementary Fig. 7g ). Co-culture of MCF7 cells containing inducible JAG1 with red IMR90 ER:HRAS G12V cells was sufficient to reduce the number of SAHF-positive red cells even in the absence of DOX (reflecting the slightly increased levels of JAG1) and completely repress SAHF formation in red cells in the presence of DOX (Supplementary Fig. 7h ). While we cannot exclude the effects of other cell-contact-mediated signalling pathways on chromatin structure, our data together demonstrate that JAG1-expressing tumour cells can repress SAHF formation in adjacent senescent cells in a JAG1-dependent manner. \n\nNon-cell autonomous regulation of chromatin accessibility. Next, we asked whether tumour cell lines could repress the formation of RIS-driven accessible regions in fibroblasts, as was the case for ectopic N1ICD (Fig. 5b ). Utilising flow cytometry, we isolated 4OHT-induced IMR90 ER:HRAS G12V mRFP1 cells after co-culture with tumour cell lines and performed ATAC-seq (Fig. 6d ). We found that 66% (29,507), 72% (32,364) and 75% (33,581) of RIS-driven accessible regions were significantly repressed by co-culture with MCF7, A549 and Hep3B cells, respectively (Fig. 6h, i; Supplementary Fig. 8a, b ). Co-culture with MCF7, A549 or Hep3B cells induced opening of 6948, 10,303 and 14,064 NIS-driven accessible regions, respectively (Fig. 6h ). These data correlated well with the ability of the tumour cell lines to repress SAHFs in adjacent IMR90 (Fig. 6b ) and the JAG1 levels expressed by each line (Fig. 6a ). RIS-driven accessible regions repressed by co-culture with tumour cell lines overlapped well with each other and with regions repressed by ectopic N1ICD (Supplementary Fig. 8c, d ). These data suggest that tumour cells expressing JAG1 can dramatically alter the chromatin landscape of adjacent stromal cells at the nucleosome level. \n\nHEYL and HMGA1 anti-correlate in multiple tumour types. If NOTCH1 signalling inhibits HMGA1 in vivo, we would expect an anti-correlation between NOTCH1 activity and HMGA1 expression in human tumour samples. To test this, we first performed a pan-tissue-type analysis using expression microarray data from the R2 database (http://r2. amc. nl) by comparing the expression of HMGA1 and canonical NOTCH1 target genes. When Z-score expression values were analysed in 36,846 human samples, we observed a significant negative correlation between HMGA1 and HEYL (R = -0. 356, p < 0. 0001) and HMGA1 and HEY1 (R = -0. 281, p < 0. 0001), but no correlation between HMGA1 and HES1 (Supplementary Fig. 9a, b, c ). Interestingly, HEYL and HEY1, but not HES1, were also significantly upregulated in IMR90 fibroblasts co-cultured with JAG1-expressing RPE1 cells (Fig. 3g ). To study the prognostic importance of this relationship, we used the web-based tool KM-plotter 39, 40 and found that patients with low HMGA1 or high HEYL have a significantly better prognosis in lung adenocarcinoma, but not in lung squamous cell carcinoma (SCC) (Supplementary Fig. 9d, e ), suggesting that the relationship between these proteins may have prognostic value in certain types of cancer. High HEY1 levels were prognostic of better overall survival in both types of lung cancer patient (Supplementary Fig. 9d, e ). \n\nAs microarray data can be dependent on the quality of the probe used, we analysed the co-expression of HMGA1 and HEYL or HEY1 using RNA-seq data generated by The Cancer Genome Atlas (TCGA) Research Network 41 (http://cancergenome. nih. gov). There was a significant negative correlation between HMGA1 and HEYL in the majority of tumour types analysed (Fig. 7a ) and a particularly strong anti-correlation in lung SCC (Fig. 7b ) (R = -0. 4842; p = < 0. 0001). When TCGA patients with lung SCC were categorised based on expression into 'HMGA1 high-HEYL low' and 'HMGA1 low-HEYL high' tumours, patients in the former category had a better overall survival (Fig. 7c ) (p = 0. 00316). We also found a significant negative correlation between HMGA1 and HEY1 in various cancer types, which were not completely overlapping with those where HMGA1 and HEYL anti-correlate (Supplementary Fig. 10a ). For example, they were not negatively correlated in lung SCC and the expression of these two genes was not prognostic of patient survival (Supplementary Fig. 10b, c ). However, kidney renal clear cell carcinoma showed the strongest negative correlation between HMGA1 and HEY1 and their expression patterns were indicative of prognosis (Supplementary Fig. 10a, d, e ). Together, these data demonstrate that an anti-correlation between HMGA1 expression and NOTCH1 activity is evident in cancer and that this correlation can be prognostic of patient outcome.",
"section_name": "NOTCH signalling represses the expression of HMGA genes.",
"section_num": null
},
{
"section_content": "In the current study, we provide evidence for NOTCH-mediated 'lateral modulation' of chromatin structure at the microscopic and nucleosome scales. While RIS cells form prominent SAHFs at the microscopic scale 13, 42, at the nucleosome scale we observed a robust increase in chromatin accessibility. Both SAHFs and RIS-",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Fig. 8 NOTCH1 signalling mediates non-cell autonomous regulation of chromatin structure at the microscopic and nucleosome scale. Lateral induction of NOTCH1 activity in a signal-receiving cell by JAG1 on the surface of an adjacent cell (including cancer cells) can drive NIS. NIS cells form unique chromatin-accessible regions and microscopically 'smoothened' chromatin. In the context of RIS, non-cell autonomous activation of NOTCH1 signalling can repress the formation of AT-rich RASdriven accessible regions at the nucleosome level and SAHF formation at the microscopic level. Mechanistically, N1ICD represses HMGA1, which is responsible for SAHF formation and at least partially for the formation of ectopic-accessible chromatin in RIS cells driven accessibility can be inhibited by N1ICD-mediated repression of HMGA1 (Fig. 8 ). While the essential and structural role for HMGA1 in SAHF formation is well established 16, its role in chromatin accessibility is unclear. HMGA proteins compete with Histone-H1 for linker DNA and thus affect chromatin compaction, as demonstrated by techniques such as fluorescence recovery after photo bleaching and MNase digestion assays 43, 44. Our data, using sequencing technology, demonstrate that HMGA1 effects the formation of ectopic accessible regions, potentially by facilitating the binding of other transcription factors, such as C/EBPβ, identified here through motif analysis of RIS-driven accessible regions. It is known that chromatin accessibility is an indicator of developmental maturity 45 and that cancer cells acquire ectopic accessible regions 45, 46. For example, during the metastasis of small cell lung cancer, a dramatic increase in chromatin accessibility at distal regulatory elements allows tumour cells to co-opt pre-programmed gene expression programmes, providing a growth advantage 32. Thus our data raise a possibility that HMGA1 can drive pluripotency and cancer in part by modulating chromatin accessibility. It will be important to understand how HMGA1 facilitates both chromatin 'opening' at the nucleosome scale and the formation of SAHFs and to determine whether the two are related. We wonder whether the subset of HMGA1-dependent regions that are gene distal could have structural rather than regulatory functionality. \n\nChromatin accessibility was also increased in NIS cells although these were often at distinct loci. Unlike RIS cells, NIS cells do not form SAHFs and are instead characterised by chromatin 'smoothening'. The mechanisms of chromatin smoothening and formation of NIS-driven accessible regions, and whether these events are related, remains unclear. Note, although knockdown of HMGA1 blocked formation of SAHFs and many RISdriven accessible regions, it was not sufficient to induce NIS-like chromatin smoothening or NIS-like chromatin accessibility, thus NOTCH signalling modulates chromatin by both HMGA1dependent and -independent mechanisms (Fig. 5b, c ). One possible mechanism through which NOTCH modulates chromatin is through directed histone acetylation [47] [48] [49] [50]. N1ICD activates gene transcription by recruiting histone acetyl-transferases 11 and was more recently shown to drive rapid and widespread deposition of H3K56ac 51, which is known to be associated with nucleosome assembly, particularly in DNA replication and repair 52. \n\nNOTCH signalling can be transiently activated during stressinduced senescence (e. g. oncogene-and DNA damage-induced senescence) 9 but also plays important roles during development and in cancer, thus 'lateral induction' of NOTCH activity through JAG1 could affect chromatin structure in various biologically relevant scenarios involving epithelial and/or fibroblast cells. Here we extend our analysis to the more specific 'epithelial-fibroblast' scenario that might mirror the cancer microenvironment where epithelial tumour cells are in active communication with stromal cells through the NOTCH1-JAG1-HMGA1 signalling axis. Consistently, using the Pten-null mouse model of prostate cancer, Su and colleagues 53 demonstrated that JAG1 expression in tumour cells facilitates the formation of a 'reactive stroma', which plays an important role in tumour development. It will be important to test whether chromatin structure is altered in the stroma of such tumours and whether this is dependent on HMGA1 repression. In NOTCH-ligand-expressing tumours, targeting chromatin-modifying enzymes in the stromal compartment may present a unique therapeutic opportunity to alter the tumour niche.",
"section_name": "SAHF positive",
"section_num": null
},
{
"section_content": "Cell culture. IMR90 HDFs (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM)/10% foetal calf serum (FCS) in a 5% O 2 /5% CO 2 atmosphere. \n\nhTERT-RPE1 cells (ATCC) were grown in DMEM-F12/10% FCS in a 5% O 2 /5% CO 2 atmosphere. MCF7, H1299, A549 and Hep3B cells (ATCC) were grown in DMEM/10% FCS in a 5% CO 2 atmosphere. Cell identity was confirmed by STR (short tandem repeats) genotyping. Cells were regularly tested for mycoplasma contamination and always found to be negative. \n\nCo-cultures were set-up at a cell number ratio of 1:1 and performed in DMEM/ 10% FCS in a 5% O 2 /5% CO 2 atmosphere. For transwell experiments, IMR90 cells were plated in the bottom chamber and hTERT-RPE1 or tumour cells were plated into the top chamber of a Corning 12-well Transwell plate (CLS3460 Sigma). \n\nThe following compounds were used in cultures: 100 nM 4-hydroxytamoxifen (4OHT) (Sigma), 10 µM DAPT (Sigma), 100 µM etoposide (Sigma), between 10 and 1000 ng/mL doxycycline (DOX) (Sigma) as indicated in individual figures. \n\nVectors. The following retroviral vectors were used: pLNCX (clontech) ER: HRAS G12V30 ; pWZL-hygro for N1ICD-FLAG (residues 1758-2556 of human NOTCH1 9 ) and mRFP1; pLPC-puro for dnMAML1-mVenus (residues 12-74 of human MAML1) 9, mRFP1, rThy1-mRFP1, JAGGED1-mVenus and mVenus; pQCXIH-i for DOX-inducible N1ICD-FLAG 9 ; MSCV-puro for miR30 shHMGA1 (shHMGA1 target sequence 5′-ATGAGACGAAATGCTGATGTAT-3' 16 ); and pCLIIPi 54 for pCLIIPi JAGGED1-mVenus. \n\nTo generate pLPC-puro rThy1-mRFP1, we first PCR cloned mRFP into pLPCpuro (pLPC-puro-x-mRFP, where x denotes cloning sites to express mRFP-fusion proteins). The CDS of rat-Thy1 was PCR amplified from cDNA (a gift from M. de la Roche, CRUK CI, UK), removing the stop codon, before cloning into pLPCpuro-x-mRFP. To generate pLPC-JAGGED1-mVenus, the CDS of human JAGGED1 was amplified using cDNA derived from N1ICD-expressing IMR90 cells, removing the stop codon, before cloning into pLPC-puro-x-mVenus. To generate pCLIIPi (DOX-inducible) JAG1-mVenus, JAGGED1-mVenus was subcloned using PCR into pCLIIPi. \n\nFlow cytometry. Analysis of ectopic JAG1-mVenus expression was conducted by flow cytometry 9. Cells were fixed using 4% paraformaldehyde (PFA) in phosphatebuffered saline (PBS) and stained with anti-JAG1-APC (FAB1726A, R&D Systems, 1:10) or isotype control antibody (IC0041A, R&D Systems, 1:10) before analysis on a FACSCalibur flow cytometer (Becton Dickenson). Flow data were further analysed using FlowJo v10. \n\nMACS and FACS. MACS of rThy1-expressing cells was performed using CD90. 1 microbeads (130-094-523, Miltenyl Biotec) according to the manufacturer's instructions. Fluorescence-activated cell sorting (FACS) was performed using an Influx (Becton Dickenson) flow cytometer. \n\nFluorescence microscopy. Analysis was performed as previously described 16. Briefly, cells were plated onto #1. 5 glass coverslips the day before fixation to achieve approximately 60% confluence. Cells were fixed in 4% (v/v) PFA and permeabilised with 0. 2% (v/v) Triton X-100 in PBS with DAPI. Coverslips were mounted onto Superfrost Plus slides (4951, Thermo Fisher) with Vectashield Antifade mounting medium (H-1000, Vector Laboratories Ltd. ). Images were obtained using a Leica TCS SP8 microscope with a HC PL APO CS2 1. 4NA 100× oil objective (Leica Microsystems). At least 30 nuclei were captured per biological replicate and condition before Fiji 55 was used to calculate nuclear area, standard deviation and maximum intensity of DAPI signal per nucleus. Specifically, the DAPI channel was duplicated, desaturated and a threshold applied using the Otsu method before holes were filled and the 'analyse particles' function was used to create a region of interest per nucleus for measurement in the original DAPI-stained image. SADS were visualised by DNA-fluorescence in situ hybridisation as previously described 15 using fluorescent probes that target the α-satellite repeat sequence (5′-CTTTTGATAGAGCAGTTTTGAAACACTCTTTTTGTA-GAATCTGCAAGTGGATATTTGG-3′). The percentage of SAHF-and SADSpositive cells was counted by scoring at least 200 cells per replicate and condition. \n\nQuantitative reverse transcription-PCR. RNA was prepared using the Qiagen RNeasy Plus Kit (74136, Qiagen) according to the manufacturer's instructions and reverse-transcribed to cDNA using the Applied Biosystems High-Capacity Reverse Transcription Kit (43-688-13, Thermo Fisher). Relative expression was calculated as previously described 16 on an Applied Biosystems Quantstudio 6 by the 2 -ΔΔCt method 56 using β-actin (ACTB) as an internal control. The following primers were used:\n\nACTB forward: 5′-GGACTTCGAGCAAGAGATGG-3′ ACTB reverse: 5′-AGGAAGGAAGGCTGGAAGAG-3′ HEYL forward: 5′-CTCCAAAGAATCTGTGATGCCAC-3′ HEYL reverse: 5′-CCAGGGACAATGAAAGCAAGTTC-3′ HEY1 forward: 5′-CCGCTGATAGGTTAGGTCTCATTTG-3′ HEY1 reverse: 5′-TCTTTGTGTTGCTGGGGCTG-3′ HES1 forward: 5′-ACGTGCGAGGGCGTTAATAC-3′ HES1 reverse: 5′-ATTGATCTGGGTCATGCAGTTG-3′ HMGA1 forward: 5′-GAAAAGGACGGCACTGAGAA-3′ HMGA1 reverse: 5′-TGGTTTCCTTCCTGGAGTTG-3′ HMGA2 forward: 5′-AGCGCCTCAGAAGAGAGGA-3′\n\nHMGA2 reverse: 5′-AACTTGTTGTGGCCATTTCC-3′\n\nProtein quantification by immunoblotting. Immunoblotting was performed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels using the following antibodies: anti-β-actin (Sigma, A5441, 1:10,000); anti-HRAS (Calbiochem, OP-23, 1:500); anti-NOTCH1 (Cell Signaling, 4380, 1:500); anti-HES1 (Cell Signalling, 11988, 1:1000); anti-FLAG (Cell Signaling, 2368, 1:1000), anti-HMGA1 (Cold Spring Harbor Labs, #37, 1:1000); anti-HMGA1 (Abcam, Ab4078, 1:1000); anti-HMGA2 (Cold Spring Harbor Labs, #24, 1:1000); anti-GFP (Clontech 632377, 1:1000); and anti-JAG1 (Cell Signaling, 2155, 1:1000). Images of uncropped immunoblots are included in Supplementary Fig. 11. \n\nATAC-seq. ATAC-seq samples were generated as previously 31 using 100,000 IMR90 cells and 13 cycles of PCR amplification. Samples were size selected between 170 and 400 bp (in order to isolate 'nucleosome free' and 'mono-nucleosome' fragments) using SPRIselect beads (B23319, Beckman Coulter) before single-end sequencing to generate 75 bp reads on the NextSeq-500 platform (Illumina). \n\nChIP-seq. Chromatin immunoprecipitation (ChIP) was performed as previously described using 20 µg of sonicated chromatin 57 from growing and RIS IMR90 ER: HRAS G12V cells and 5 µg of anti-H3K27ac antibody (Clone CMA309 58 ) or 5 µg of H3K4me1 antibody (Clone CMA302 58 ). Libraries were prepared using the NEB-Next Ultra II DNA Library Prep Kit for Illumina (37645, New England Biolabs) according to the manufacturer's instructions except that size selection was performed after PCR amplification using SPRIselect beads (B23319, Beckman Coulter). Samples were sequenced single-end using 50 bp reads on the HiSeq-2500 platform (Illumina). \n\nRNA-seq. RNA-seq data was generated from IMR90 ER:HRAS G12V cells expressing a short-hairpin targeting the 3′ UTR of human HMGA1 (RIS+shHMGA1). RNA was purified as above and quality checked using the Bioanalyser eukaryotic total RNA nano series II chip (Agilent). mRNA-seq libraries were prepared from six biological replicates of each condition using the TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer's instructions and sequenced using the HiSeq-2500 platform (Illumina). \n\nGeneration of genome-edited JAG1 knockout clones. The following CRISPR guides were designed against Exon 2 of JAG1 (NM_000214. 2) (Supplementary Fig. 7d ): sgJAG1_2. 1: 5′-AGTCCCGCGTCACGGCCGGG-3′ (PAM:GGG) and sgJAG1_2. 2: 5′-CGCGGGACTGATACTCCTTG-3′ (PAM:AGG). Oligonucleotides (Sigma Aldrich) were cloned into pSpCas9(BB)-2A-GFP 59. pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138). Guide cutting efficiency was determined in A549 cells using the T7 assay (New England Biolabs, following manufacturer's instructions). To generate independent, non-sister clonal cell lines, A549 cells were transiently transfected (Lipofectamine 3000, Thermo Fisher Scientific) with PX458-empty (control), PX458-sgJAG1_2. 1 and PX458-sgJAG2. 2, and single cell was cloned 96 h posttransfection by FACS (BD FACSAria II). gDNA was extracted from each clone (Extracta DNA Prep, VWR, 95091-025) and Exon 2 of JAG1 was amplified by PCR (FastStart HF System (Sigma Aldrich, 3553361001)) using the following primers (universal Fluidigm tag in lower case, JAG1-specific sequence in upper case)):\n\nForward: 5′-acactgacgacatggttctaca-GAGCTGCAGAACGGGAACT-3′; Reverse: 5′-tacggtagcagagacttggtct-CTTGAGGTTGAAGGTGTTGC-3′. Amplicons were diluted 1:150 and re-amplified with Fluidigm barcoding primers (incorporating a unique sample barcode and Illumina P5 and P7 adapter sequences), pooled and subjected to sequencing (Illumina MiSeq platform). The AmpliconSeq analysis pipeline was used for data processing and variant calling. Briefly, reads were aligned against the reference genome (GRCh38) using BWA-MEM 60 and variants were called using two methods (VarDict 61 and GATK HaplotypeCaller 62 ). Consensus variants and their effects on CRISPR clones were then calculated. All clones used in this paper were STR genotyped and confirmed as free from mycoplasma. \n\nRNA-seq analysis. Reads were mapped to the human reference genome hg19 with the STAR (version 2. 5. 0b) aligner 63. Low-quality reads (mapping quality <20) as well as known adapter contaminations were filtered out using Cutadapt (version 1. 10. 0) 64. Read counting was performed using Bioconductor packages Rsubread 34 and differential expression analysis with edgeR 33, 34. The conditions were contrasted against the growing samples. Genes were identified as differentially expressed with a FDR (false discovery rate) cut-off of 0. 01 and an absolute value of logFC (log 2 of the fold change) >0. 58. \n\nChIP-seq and ATAC-seq analysis. ChIP-seq and ATAC-seq reads were mapped to the human reference genome (hg19) with BWA (version 0. 7. 12) 60. Low-quality reads (mapping quality <20) as well as known adapter contaminations were filtered using Cutadapt (version 1. 10. 0) 64, and reads mapping to the 'blacklisted' regions identified by ENCODE 65 were further removed. Average fragment size was determined using the ChIPQC Bioconductor package 66, and peak calling was performed with MACS2 (version 2. 1. 0) 67, using fragment size as an extension size (--extsize) parameter. High-confidence peak sets for each condition were identified separately using only those peak regions that were present in at least two replicates. Differential accessibility analysis. THOR 35 and edgeR 33, 34 were used to identify differentially accessible regions between conditions. For the comparisons 'NIS vs. growing' and 'RIS vs. growing', the intersect of regions detected by THOR and edgeR was taken. This approach gave us a robust set of regions that are altered in RIS and NIS conditions. For other comparisons where volcano plots were generated, edgeR was used to interrogate how different genetic and cell-culture manipulations effect the alterations detected in RIS. \n\nedgeR 33, 34 was used on a merged set of growing, RIS, NIS, N+RIS, shHMGA1 and RIS/shHMGA1 high-confidence ATAC-seq peak sets (present in at least two replicates of a single condition) to identify regions of differential accessibility between conditions. We utilised the TMM method implemented in edgeR for normalisation and dispersion calculation of the replicated samples. The results were further filtered based on FDR < 0. 05 and logFC ≥ 0. 58 or ≤-0. 58. \n\nTHOR is a Hidden Markov Model based approach that utilises all mapped reads and identifies the differentially accessible regions between two conditions. THOR was used in parallel with edgeR to identify the differences between the conditions using our pre-computed normalisation factors (see section 'Generation of normalised coverage files') to normalise between samples. Regions were further filtered using a -log(p-value) cut-off of 10. \n\nAnnotating differentially accessible regions. Bedtools intersect (v2. 26. 0) 68 was used to identify regions annotated as 'unchanged' by extracting high-confidence peaks in the 'growing' condition that did not intersect with regions that become more or less accessible in the NIS and/or RIS conditions (relative to the growing condition). The rest of the categories (open or closed in RIS or NIS) were identified based on the differential accessibility analysis compared to growing using the intersect of THOR and edgeR as described above. Regions displaying altered chromatin accessibility were mapped to genomic annotations or repeat regions using bedtools v2. 26. 0 68. For the genomic annotations, we used TSSs from the FANTOM database 69, repeats from repeatMasker (UCSC genome browser) and other genomic features (exons, introns, UTRs, etc. ) were extracted from the UCSC Table Browser. The enhancers were identified based on our own H3K4me1 and H3K27ac histone mark ChIP-seq data sets; all regions that had peaks in both of these marks in either growing or RIS cells were considered as enhancers. \n\nIntersecting consensus peaks and generating Venn diagrams. The Homer (v3. 12) 70 command 'mergePeaks' with default settings and the output options '-venn' and '-prefix' were used to generate values for plotting Venn diagrams and associated bed files for further analysis. Only literal overlaps (overlapping by 1 bp) were considered. Venn diagrams were plotted using the R package 'Venneuler' (https://cran. rproject. org/web/packages/venneuler/index. html). \n\nCalculating proximity to genes and GC percentage. To calculate the distance of consensus peaks from TSSs and GC percentage of accessible regions, the Homer (v3. 12) 70 command 'annotatePeaks. pl' was used with default settings and the output option '-CpG'. \n\nGene enrichment analysis. Altered accessible regions within 500 bp of a gene TSS were identified using Homer as described above. Gene enrichment analysis was performed using the GO Biological Process 2015 annotation provided on the webtool 'Enrichr' 71 (http://amp. pharm. mssm. edu/Enrichr/). Generation of normalised coverage files. A previously described approach was used to generate scaling factors for each ATAC-seq condition relative to others 32. Briefly, we reasoned that the enrichment of reads within ATAC-seq peaks containing TSSs of genes that are both expressed (logCPM > mean logCPM) and have low variance between conditions (-0. 14 < logFC < 0. 14) by RNA-seq should not vary, unless there are differences in ATAC-seq sample quality, preparation or sequencing. By reanalysing our previously published IMR90 RNA-seq data 9 together with newly generated RNA-seq samples for RIS+shHMGA1 cells, we identified 589 genes that fit these criteria. We counted the reads from the ATACseq samples that map to these specific genes using Rsubread 34 and computed scaling factors based on the mean counts for each condition separately. Normalised coverage files (bigWig) were generated by pooling reads from all of the replicates and applying the calculated scaling factors using the 'genomecov' function in bedtools, sorting the resulting normalised bedGraph files and then converting them to bigWigs using the 'bedGraphToBigWig' function from UCSC. \n\nGeneration of clustered heatmaps. Heatmaps were generated using normalised coverage of peaks (+/-2. 5 kb) representing novel accessible regions (regions with significantly altered chromatin accessibility in RIS or NIS relative to growing cells) with k-means clustering using the deepTools package 72. \n\nPCA analysis and correlation heatmaps. Samples were normalised with the precalculated normalisation factors (as described above in 'Generation of normalised coverage files'), and reads from all growing, RIS, NIS, N+RIS, shHMGA1 and RIS/ shHMGA1 consensus peak sets (present in at least two replicates across all of the samples) were extracted and used in the PCA analysis and the correlation analysis of data sets. Pearson correlation was calculated between samples based on these normalised read counts and correlation heatmaps were generated with pheatmap (http://CRAN. R-project. org/package=pheatmap) and WPGMA clustering. PCA plots were generated using ggplot2 73. \n\nReads from publicly available ATAC-seq and DNase-seq data sets [74] [75] [76] [77] (references and NIH Epigenomics Roadmap Initiative) were extracted from the same regions; however, since these were not included in normalisation factor calculation, standard CPM normalisation was used for Supplementary Fig. 4c. \n\nVolcano plots. edgeR calculated statistical parameters (logFC and logFDR) were used to visualise differentially accessible regions in RIS (red) and NIS (purple) compared to growing cells in the comparisons indicated. Plots were generated using ggplot2 73. \n\nMotif enrichment analysis. Meme-ChIP suite (version 4. 12. 0), together with Hocomoco (version 11) human and mouse PWMs, was used to detect motif enrichment in a 600 bp region centred at the peak summit. \n\nTCGA analysis. We analysed the expression levels of NOTCH-associated genes in the publicly available RNA sequencing data generated by the TCGA Research Network: http://cancergenome. nih. gov/ 41. \n\nComputational analysis and statistical testing of the Next-Generation Sequencing data was conducted using the R statistical programming language 78. Filtered and log 2 -normalised RNA expression data along with all available clinical data were downloaded from the GDAC firehose database (run: stddata_2015_06_01) for each gene of interest from the relevant cancer-specific collections. \n\nCorrelation testing for associations between expressed genes was performed using the cor. test function in R to calculate the Pearson's product moment correlation coefficient and test for significant deviation from no correlation. Plotting of TCGA data was performed using the ggplot2 R package 73. Survival analysis was performed using the survminer and survival 79 R packages. Kaplan-Meier estimated survival curves were constructed using the TCGA clinical data. Statistical testing of differences between survival curves used the G-rho family of tests, as implemented in the survdiff function of the survival package.",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "No statistical method was used to predetermine sample size and experiments were not randomised. Statistical analyses were conducted using the Graphpad Prism 6 and R statistical software, except for TCGA data analysis (which was as described in the methods above). One-way analysis of variance with Tukey's correction for multiple comparisons was used for data sets with >2 conditions. Two-sample t-tests were used for two-condition comparisons. The statistical tests were justified as appropriate based on the number of samples compared and the assumed variance within populations. A p-value of <0. 05 was used to indicate statistical significance.",
"section_name": "Statistics and reproducibility.",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We thank all members of the Narita laboratory for helpful discussions, M. de la Roche for reagents and staff of the Cancer Research UK Cambridge Institute core facilities for technical support. The University of Cambridge, Cancer Research UK and Hutchison Whampoa supported this work. M. N., S. B. and I. A. R laboratories are funded by a Cancer Research UK Cambridge Institute Core Grant ( C14303/A17197 ). M. N. is also supported by a Cancer Research UK Early Detection Pump Priming award ( C20/A20976 ), Medical Research Council ( MR/M013049/1 ) and Tokyo Tech World Research Hub Initiative (WRHI). M. H. is supported by a CRUK Clinician Scientist Fellowship ( C52489/A19924 ). R. H. -H. is funded by an EMBO Long-Term fellowship. S. A. S. and D. B. were supported by Medical Research Council core funding. H. K. was supported by JSPS KAKENHI JP25116005, JP26291071, 15K21730 and 17H01417.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Data availability. The RNA-seq, ChIP-seq and ATAC-seq data generated for this study have been deposited at the Gene Expression Omnibus (GEO) with the accession number GSE103590. Gene expression data from RIS, NIS and N+RIS is previously published 9 and available under GEO accession number GSE72404.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "Supplementary Information accompanies this paper at https://doi. org/10. 1038/s41467-018-04283-9. \n\nCompeting interests: The authors declare no competing interests. \n\nReprints and permission information is available online at http://npg. nature. com/ reprintsandpermissions/ Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Additional information",
"section_num": null
}
] |
10.1186/s40164-016-0036-3
|
New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies
|
CD20 is a B cell lineage-specific marker expressed by normal and leukemic B cells and targeted by several antibody immunotherapies. We have previously shown that the protein from a CD20 mRNA splice variant (D393-CD20) is expressed at various levels in leukemic B cells or lymphoma B cells but not in resting, sorted B cells from the peripheral blood of healthy donors.Western blot (WB) analysis of B malignancy primary samples showed additional CD20 signals. Deep molecular PCR analysis revealed four new sequences corresponding to in-frame CD20 splice variants (D657-CD20, D618-CD20, D480-CD20, and D177-CD20) matching the length of WB signals. We demonstrated that the cell spliceosome machinery can process ex vivo D480-, D657-, and D618-CD20 transcript variants by involving canonical sites associated with cryptic splice sites. Results of specific and quantitative RT-PCR assays showed that these CD20 splice variants are differentially expressed in B malignancies. Moreover, Epstein-Barr virus (EBV) transformation modified the CD20 splicing profile and mainly increased the D393-CD20 variant transcripts. Finally, investigation of three cohorts of chronic lymphocytic leukemia (CLL) patients showed that the total CD20 splice variant expression was higher in a stage B and C sample collection compared to routinely collected CLL samples or relapsed refractory stage A, B, or C CLL.The involvement of these newly discovered alternative CD20 transcript variants in EBV transformation makes them interesting molecular indicators, as does their association with oncogenesis rather than non-oncogenic B cell diseases, differential expression in B cell malignancies, and correlation with CLL stage and some predictive CLL markers. This potential should be investigated in further studies.
|
[
{
"section_content": "CD20 protein was highlighted in 1980 as a B lymphocyte-specific cell-surface antigen expressed in all stages of B cell ontogenesis except for early pro-B cells and plasma cells [1]. Despite no identified ligands, CD20 functions were investigated, and studies assigned it a role in cell differentiation [2] and calcium flux pathways [3]. \n\nThe anchorage within the membrane of the 33 kDa protein makes it a good candidate as an ion channel [3], especially when organized into tetramers [4]. Moreover, the presence of two extracellular loops allows for targeting by monoclonal antibodies (MAbs) to induce B cell depletion. The most well-known MAb is rituximab (RTX), which has greatly improved treatment of B cell malignancies [5], in association or not with chemotherapy [6]. After RTX, numerous other MAbs (such as obinutuzumab and ofatumumab) were subsequently developed to improve B cell depletion but also to treat RTX resistance to or escape from treatment [7]. \n\nCD20 is encoded by a MS4A family gene located on chromosome 11. Multiple transcription initiation sites have been identified, and the translated region of this gene is located between the third (193th nucleotide) and eighth exons (216th nucleotide), resulting in a coding sequence of 894 bp distributed into six exons [8]. Moreover, alternative splicing of the CD20 gene has been highlighted, occurring in the 5′ untranslated region and resulting in translation of three alternative CD20 mRNAs encoding the same protein in human B lymphocytes. \n\nAlternative splicing remains a key process of pre-RNA maturation and allows an increase in protein translation and phenotype diversity [9]. Different patterns of splicing have been described, based on two families of regulatory proteins (constituting the spliceosome), the serine-rich (SR) and heterogeneous nuclear ribonucleoproteins (hnRNP) (for review, see [10] ). \n\nAberrant splicing, caused by mutation in splice site sequences within cancer-related genes or in genes encoding splicing regulation proteins [11], has a dominant role in tumor establishment, progression, and response to treatment [12]. Abnormal splicing mechanisms produce numerous cancer-associated alternatively spliced variants that could promote angiogenesis, invasion, and drug resistance, conferring a more aggressive tumoral profile [13]. These alternative variants are differentially expressed in tumors [14] and thus may be used as diagnostic tools and prognostic markers [15]. Moreover, emerging treatments target new isoform proteins encoded from aberrant splicing [16] or modify splice site selection by oligonucleotide approaches to prevent abnormal splicing [17]. \n\nIn oncohematology, numerous spliceosome gene mutations have been identified in chronic lymphocytic leukemia (CLL), myelodysplastic syndromes, and lymphomas; among the most well-known of these are those involving SF3B1, U2AF1, and SRSF2 [18] [19] [20]. Alternative splicing occurring in B cells could also be modified by Epstein-Barr virus (EBV) infection in which the BMLF1 viral protein modifies STAT1 splicing after binding with the spliceosome component SRp20 [21] and thus may influence immortalization of target B cells. \n\nWe [22] and others [23] have identified novel alternative CD20 transcripts, fully matching the MS4A1 sequence, except for 501 bp (from nucleotides 111-612, starting +1 of the ATG codon) flanked by the cryptic acceptor (AS) and donor (DS) splice sites. The resulting in-frame cDNA sequence encodes a truncated CD20 protein, called D393-CD20 (previously named ΔCD20 [22] ), that is missing the major part of the transmembrane and extracellular domains, including the RTX epitope. Interestingly, this protein has been observed in malignant or EBV-transformed B cells but not in peripheral blood mononuclear cells (PBMCs), bone marrow-derived mast cells, or plasmocytes from healthy donors. \n\nAdditional investigations of D393-CD20 protein expression by western blotting on different hematologic samples have allowed us to detect extra signals that we followed up in the current work, extended to autoimmune or EBV-infected samples. Our molecular analysis has led to the description and characterization of new alternative CD20 transcripts that are differentially expressed in hematologic malignancies.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "As expected, western blot analysis using a carboxy terminus CD20 antibody targeted to circulating PBMCs from patients with B cell hematologic malignancies (CLL and NHL), CBL, B cell lines, or healthy donors revealed immunoreactive bands at 35 kDa corresponding to the full-length CD20 protein, indicating the presence of B lymphocytes in each sample (Fig. 1 ). As previously described, a band at 19 kDa, encoded by the CD20 alternative transcript D393-CD20 [22], was detected on CLL (5/5) and NHL samples (3/3), as well as on leukemic B cell lines (3/3). In contrast, the three CBL (without tumoral circulating B cells, as detected by B cell clonality analysis) and the four healthy donor samples were all negative for the 19 kDa band. \n\nWe clearly detected an unexpected additional immunoreactive band at approximately 27 kDa in all CLL and NHL samples. This band was also detected on MCL samples (data not shown). Surprisingly, this band was not detected on the three B cell lines. Moreover, western blot allowed detection of a supplementary signal at 33 and 17 kDa, respectively, close to the 35 kDa (full-length CD20 protein) or the 19 kDa (D393-CD20) bands.",
"section_name": "Additional band signal is detected by c-terminal CD20 western blotting on blood samples collected from patients with hematologic malignancies",
"section_num": null
},
{
"section_content": "After RT-PCR of the full-length CD20 (fl-CD20) coding sequence, agarose gel electrophoresis allowed us to detect the expected two 894 and 393 bp PCR products corresponding respectively to the wt-and D393-CD20 cDNA sequences. None of these visible amplified DNA fragments matched in size to products that could correspond to a sequence encoding the 27 kDa or other additional signals. \n\nAll fl-CD20 PCR fragments between <894 and 100 bp in length, excluding the major 393 bp PCR product, were gel purified, TA cloned, amplified, and Sanger sequenced. Sequencing of more than 150 individual bacterial colonies allowed identification, in addition to the D393-CD20 sequence, of four new nucleotide sequences partially homologous to the wtCD20 reference nucleotide sequence published in GenBank (NM152866. 2) (Additional file 1: Figures S2 and S3 ). The four sequences are named according to the length of the nucleotide deletion compared to the CD20 reference. Thus, D657-CD20, D618-CD20, D480-CD20, and D177-CD20 indicate deletions of 237, 276, 414, and 717 bp, respectively.",
"section_name": "Both CD20 homologous and truncated nucleotide sequences are identified in B cell lines",
"section_num": null
},
{
"section_content": "Bioedit© alignments revealed that all of the new sequences matched perfectly at the 5′ and 3′ regions with the conservation of start and stop codons of the wtCD20 whereas we detected a missing central area, generating a new sequence junction (Fig. 2a, b ). A deeper analysis of the fusion sequences allowed highlighting of an alternative splicing phenomenon, bringing in a combination of cryptic or canonical DS or AS sites. Five splicing sites corresponded to canonical and three to cryptic sites, either DS or AS. Identification of the three cryptic-DS or cryptic-AS was confirm using the online splicing prediction tools SplicePort Prediction [24] and ASSP Prediction [25] (Fig. 2b ). \n\nBased on [26], two patterns of splicing involving both canonical DS and AS were identified as exon or multiple exon skipping for D480-and D657-CD20, respectively. One splicing pattern involving cryptic and canonical sites was qualified as an alternative 3′ splice site (D618-CD20); lastly, two patterns (including the known D393-CD20) concerned alternative 5′ and 3′ cryptic splice sites. The characteristics of the splice variant transcripts are reported in Fig. 2b and summarized in Table 1. All sequences were in frame, and translation generated new amino acid fusion sequences (Fig. 2c ).",
"section_name": "All newly identified sequences code for in-frame CD20 transcript variants resulting in MS4A1 alternative splicing",
"section_num": null
},
{
"section_content": "To study the presence of transcripts and their level of expression, we designed RT-PCR and RT-qPCR assays (Additional file 1: Figure S1 ). As shown in Fig. 3a, fl-CD20 PCR allowed amplification of all CD20 alternative transcripts from either genomic DNA or cDNA extracted or synthesized from transfected PG13 cell lines. Moreover, transcript-specific RT-PCR allowed detection specifically of the respective CD20 alternative transcripts without crossreactivity with the others, as shown when the target used was gDNA (Fig. 3b ). Interestingly, D393spe-PCR amplified cDNA synthesized from ARN extracted from wt-, D657-, D480, and D393-CD20 cell lines. Positive signals detected with D177spe-PCR from all cell lines meant that all constructs could produce the D177-CD20 transcript. \n\nFinally, RT-qPCR assays allowed specific detection without cross-reactivity (data not shown) from one CD20 Standard deviations for values from each RT-qPCR for all four cell lines were (min-max) (0. 52-0. 8), (0. 05-0. 2), (0. 1-0. 52), (0. 001-0. 39), and (0. 28-0. 62), respectively, for D657-, D618-, D480-, D393-, and D177-CD20 PCR. Interestingly, we noted that B cell lines resulting from different B cell malignancies present specific CD20 splicing profiles.",
"section_name": "Design of RT-PCR and RT-qPCR molecular tools allowed for specific detection and quantification of all newly identified spliced CD20 sequences",
"section_num": null
},
{
"section_content": "To confirm that canonical sites associated with cryptic splicing sites may be involved in CD20 variant transcription, as hypothesized from sequencing identification, some intron (3, 5, 6) sequences were used to generate artificial constructs carrying intron sequences within the wtCD20 coding sequence (Fig. 4a ). D393-and D177-CD20 were produced by all three constructs independently of the presence of canonical sites because splicing involved only cryptic DS and AS. However, reintroduction of int5 alone in addition produced D618-CD20 transcripts. Dual reintroduction of int3 and 6 produced D480-CD20 whereas the presence of int5 and 6 allowed expression of D657-and D618-CD20 mRNA (Fig. 4b ). \n\nAll of these results confirmed that the cell spliceosome machinery can process the ex vivo D480-, D657-, and D618-CD20 transcript variants by involving canonical sites associated with cryptic splice sites.",
"section_name": "Reintroduction of intron sequences within the coding CD20 sequence confirms involvement of canonical DS or AS splicing sites in D657-, D618, and D480-CD20 splice variant transcription",
"section_num": null
},
{
"section_content": "Among the four B cell lines, CD20 splicing quantification showed a higher and significant increase in D393-CD20 variants in the EBV-transformed cell line SKW6. 4. For this reason, the impact of EBV infection or transformation on CD20 splicing was investigated within different kinds of EBV samples. \n\nSix EBV-transformed BLCLs were derived from the PBMCs of six healthy donors. As shown in Fig. 5a, total CD20 splicing was significantly increased in BLCL (3. 4fold, p < 0. 01) compared to their respective PBMCs. Separate CD20 splice variant analysis revealed that increased total CD20 splices involved D657-and D618-CD20 but mainly and statistically significantly D393-CD20 (110 time, p < 0. 001). D393-CD20 represented the major part (76. 5 %) of the total CD20 splice increase compared to both D657-and D618-CD20 (23. 4 %). \n\nIn contrast, total CD20 splicing did not vary significantly either for IMN samples compared to healthy PBMCs (Fig. 5b ) or for EBV-reactivated samples after allograft (Fig. 5c ), although we noted an increase in D657 and D618-CD20 splicing. Interestingly, D393-CD20 transcripts did not increase in these EBV-infected cases compared to EBV-transformed cell lines.",
"section_name": "EBV transformation modifies the CD20 splicing profile and increases mainly D393-CD20 variant transcripts",
"section_num": null
},
{
"section_content": "Using the different CD20 variant profiles in the different B cell lines, Raji, Mec, Rec, and SKW6. 1 (Fig. 3c ), derived from different hematologic diseases (respectively, CLL, Burkitt lymphoma, MCL, B lymphoblastoid), we investigated the RT-qCPR expression of the different CD20 variant transcripts in different B cell malignancies. The percentage of total alternative CD20 transcripts from all four B cell lines but also from the primary cells of FLs and DLBCLs were significantly different from healthy PBMCs (p = 0. 01 and <0. 01 respectively) (Fig. 6a ). \n\nWhen analyzed separately, D657 was found to be mainly involved in the increase of total CD20 splicing (81. 93 %) whereas D618 and D393 represented, respectively, 7. 04 and 10. 66 % of the increase in FL. In contrast, in DLBCLs, the increase was due in part to D657-CD20 (43. 87 %) but also to D393-CD20 (40. 26 %) whereas D618 participated only at 15. 72 % in the increase (Fig. 6b ).",
"section_name": "CD20 splice variant profile expression can discriminate B cell malignancies",
"section_num": null
},
{
"section_content": "",
"section_name": "b a",
"section_num": null
},
{
"section_content": "",
"section_name": "D177-CD20 D480-CD20 D393-CD20 D618-CD20 D657-CD20",
"section_num": null
},
{
"section_content": "C-ter N-ter",
"section_name": "Ex3 Ex8 Ex7",
"section_num": null
},
{
"section_content": "Ex7 Ex8",
"section_name": "Ex3",
"section_num": null
},
{
"section_content": "",
"section_name": "C-ter N-ter",
"section_num": null
},
{
"section_content": "C-ter N-ter Table S2. Considering the percentage in CLL2007-SA, the median of total CD20 splicing (1. 26 ± 1. 23 %) was significantly higher than in routine CLL (0. 65 ± 0. 5 %, p < 0. 02) or BOMP (0. 76 ± 1. 02 %, p < 0. 001) (Figs. 7a, 8 ). The increase was mainly due to the D657-and D618-CD20 transcript variants and to a lesser degree to the D393-CD20 transcripts (Fig. 7b ).",
"section_name": "Ex3 Ex8",
"section_num": null
},
{
"section_content": "We have previously identified a novel alternative CD20 transcript, fully matching the MS4A1 sequence, except for a 501-bp region flanked by cryptic AS and DS [1]. The resulting in-frame cDNA sequence encodes a truncated CD20 protein revealed by a C-terminal CD20 polyclonal antibody. Interestingly, this protein has been observed in malignant or EBV-transformed B cells but not in PBMCs, bone marrow-derived mast cells, or plasmocytes from healthy donors. Detection of unexpected additional western blot signals with an anti-carboxy terminus CD20 antibody led us to conduct a deep molecular analysis to characterize potential transcripts that could match the protein signal. Starting from nucleic acid material extracted from B cell lines or, interestingly, from primary samples of patients with B cell hematologic diseases such as CLL, MCL, or DLBCL, we identified and characterized more precisely, in addition to the D393-CD20, four additional CD20 splice variants. Two of these variants are the result of exon skipping (D657-and D480-CD20), and the other two result from the use of alternative splice sites: a canonical DS and a cryptic AS for D618-CD20 and two cryptic splice sites for D177-CD20, by previously well-described mechanisms [10]. In addition to the D393-CD20 transcript, Small et al. [23] have already detected the D618and D657-CD20 transcripts only in lymphoblastoid B cell lines. In the current work, we detected expression of these two transcripts in primary samples of human B cell diseases and reported for the first time two novel additional D480-and D177-CD20 transcripts. We also demonstrated ex vivo the involvement of canonical sites associated with cryptic splice sites that produce these transcripts. Although it should be formally demonstrated, except for D177, the lengths of the D393, D618-, D657-, and D480-CD20 transcripts matched the immunoreactive bands on western blot. \n\nWe designed quantitative molecular tools for studying alternative CD20 transcript expression in different B autoimmune, malignant B diseases or EBV-infected samples. \n\nThe comparison of splicing profiles revealed a more important CD20 alternative splicing in B diseases compared to healthy donors, suggesting a splicing deregulation in these pathologies. \n\nWhereas a slight increase of CD20 alternative splicing was detected in CBCL, LZM, MM, and some CLL samples, a significantly higher amount of alternative transcripts was observed in FL, HG-NHL, and EBVtransformed B cell lines. In all cases, the increase compared to healthy donors results from a higher proportion of D618-and D657-CD20 transcripts. In addition, this increase is associated with a D393-CD20 expression induction in lymphomas (FL, DLBCL, Burkitt and MCL) and EBV transformation. Interestingly, in autoimmune diseases (rheumatoid arthritis and pemphigus), we never detected D393-CD20 transcripts [28, 29]. These observations suggest a splicing deregulation during oncogenesis leading to D393-CD20 expression, which could be an interesting molecular marker of B malignancies. \n\nFrom another side, the slight increase of D657-and D618-CD20 expression could be the result of splicing deregulation associated with an enhanced proliferation and activation [30] during cancer but also during autoimmune disease and virus infection [31]. \n\nIncrease of D393-CD20 occurs mainly in post-germinal center (GC) lymphomas (FL, Burkitt, and DLBCL). In GC, BCR maturation requires activation-induced cytidine deaminase (AID) intervention to introduce mismatches, which are then repaired by a mismatch repair complex. This process is known to be responsible for genetic abnormalities involved in oncogenesis but could also disturb splicing. Indeed, interactions and associations have been identified between AID and splicing factor SnRNPs such as U2AF65 [32], PTB2, and SRSF2 [33]. This link may explain in part how the AID activation pathway could lead to deregulation of splicing factors that disturb CD20 splicing, thus producing alternative CD20 variant expression in post-GC lymphomas. In post GC CLL, the fact that D393-CD20 increase was not observed may be explained by a lower AID expression [34] and activation. Splicing pattern of immortalized B cell after EBV virus infection (DBCL) revealed a significantly greater increase in total CD20 splice variants, mainly because of an expression of D393-CD20. Interestingly, CD20 splicing was not statistically increased in IMN or in reactivated EBV samples: although we noted an increase of D618and D657-, no D393-CD20 expression was measured, contrasting with DBCL. These results suggested a CD20 splicing modulation caused by an oncogenesis process rather than viral infection itself. This strengthens the hypothesis of an association between D393-CD20 and oncogenesis. It is known that the SM (Mta, EB2, BMLF1) EBV protein, a viral oncogenic nuclear protein bound to RNA, influences RNA stability, splicing, nuclear export, and translation. This influence facilitates virus replication and persistence in vivo [35]. SM protein is associated with three splice regulators, SF2/ASF (SRSF1), 9G8 (SRSF7), and SRp20 (SRSF3), and antagonizes SRSF3 [36]. Thus, the SM EBV protein may be an actor that regulates CD20 cellular gene expression at the level of alternative splicing. \n\nThis work shows a deregulated expression of CD20 variant transcripts in B malignancies that may be useful as a molecular marker to study splicing patterns in order to better classify malignancies, predict resolution of disease, or monitor treatment [12]. In this way, we took advantage of the availability of sf3b1 mutational status of the BOMP relapsed CLL cohort to evaluate if there is a correlation with CD20 splicing. CLL disease is an interesting model because mutations of sf3b1, which encode a critical component of the splicing machinery, are associated with progression and fludarabin-refractoriness [37]. Interestingly, we noticed that patient group with more cd20 alternative splicing correspond to those with higher sf3b1 mutation frequency (data not shown). These results should be confirmed with other CLL cohorts, and a potential correlation with other gold standard biomarkers of CLL should be investigated. Moreover, a significant difference of total CD20 splicing between the 2 CLL cohorts at diagnosis (routine CLL cohort vs elderly CLL2007-SA, respectively 50 and 11 % stage A) could make this marker an indicator of the stage of the disease progression, which could be useful for CLL stratification. Alternative CD20 splicing may have consequences on CD20 protein function that may influence BCR/CD20 cell signaling and finally B cell functions. We previously described that D393-CD20 transcript encoded a truncated CD20 protein [22]. Using CD20 immunoprecipitation with an antibody targeting the extracellular domain followed by western blot with the C-terminal CD20 specific antibody, we have already demonstrated that D393-CD20 protein is associated with wtCD20. According to the predicted sizes of other putative proteins, they could also be associated with wtCD20 since the sizes match additional bands observed on western blot (Additional file 1: Figure S4 ). Subcellular division of transfected cells with the D393-CD20 coding sequence revealed that the variant protein is found mainly in the membrane fraction, although the main part of the transmembrane coding sequence is missing. This result strongly suggests an association between wtCD20 and D393-CD20 protein. Finally, lipid raft isolation showed the presence of D393-CD20 and wtCD20 already within the lipid rafts. All of these observations suggest a possible involvement of proteins encoded by cd20 alternative variants in BCR signaling or calcium flux, both putative functions of CD20 protein [38]. Another consequence of the CD20 splicing is the production of in-frame mRNA that could be translated into new proteins and could thus participate in the tumoral edition by generating neo-epitopes that could be targeted in anti-tumoral vaccine strategies [39, 40]. Concerning CD20 alternative splice variants, we have demonstrated that the 20mer D393-CD20 peptide spanning the splicing site might be targeted by the immune system, and we have shown that D393-CD20-specific CD4 Th1 clones could directly recognize malignant B cell lines and kill autologous lymphoma B cells, indicating that D393-CD20-derived epitopes are naturally processed and presented on tumor cells [41]. Additional CD20 alternative variants may also be new tumoral antigens that could be targeted by a redirected immune system, such as transgenic T cell receptors. \n\nThese observations may be useful for the development of new immunotherapies applied to patients refractory to conventional (chemotherapy) or targeted treatments (anti-CD20, Ibrutinib, iBTK). \n\nIn conclusion, the discovery of new alternative CD20 transcript variants makes them of interest as molecular indicators to investigate in further studies, particularly given the involvement of some of them in EBV transformation, their association with oncogenesis rather than non-oncogenic B diseases, their differential expression in B malignancies, and correlation with CLL stage and some predictive CLL markers. Overall, these findings need to be confirmed by larger prospective trials in order to fully validate CD20 transcript variant as molecular markers of oncogenesis.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Master cell banks of human and mouse cell lines were prepared from cells from the DSMZ or ATCC cell banks. Working cell cultures were then established, and cells were cultured in RPMI 1640 or DMEM with 10 % fetal calf serum. STR profiling identification was performed regularly. \n\nPeripheral blood samples were selected from cases of hematologic B cell disease: B-CLL, follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL) and cutaneous B cell lymphoma (CBCL), multiple myeloma (MM), marginal zone lymphoma, non-Hodgkin lymphoma (NHL), or autoimmune disease (rheumatoid arthritis), as well as infectious mononucleosis (IMN). In addition, EBV-reactivated samples collected from renal, lung, or hematopoietic allografts were screened. Samples were collected from diagnostic assessment or clinical trials or from a blood bank for the healthy PBMCs. \n\nEBV-derived B lymphoblastoid cell lines (BLCLs) were established from healthy donor PBMCs. PBMCs were transformed with EBV supernatant in X-VIVO medium with cyclosporine A at 1 µg/ml for 2 days and maintained in culture for at least 10 days, until an immortalized B cell line was obtained. \n\nCLL samples were collected from three different cohorts of patients: PBMCs collected at diagnosis for routine analysis (CHU Toulouse, France); CD19+ immunomagnetic-purified B cells (whole human blood CD19 MicroBeads, Miltenyi Biotec) from CLL patient samples, stage B and C, included within the CLL2007-SA (for elderly patients older than 65 years); and patients included in the ICLL01 BOMP clinical trial (relapsed or refractory CLL stages A, B, or C with active disease or after 1-3 previous lines including at least one line with fludarabine), both initiated by the GOELAMS/GCFLLC-MW intergroup. Written informed consent was obtained according to institutional protocol and approbation of the Ethic Committee (Comité de protection des personnes: CPP-Est, France).",
"section_name": "Patients, biological samples, and cell lines",
"section_num": null
},
{
"section_content": "Cells were lysed in sample buffer (2 % sodium dodecyl sulfate (SDS) in 125 mM Tris HCl, pH 6. 8). An equivalent protein amount, extracted from 1 × 10 7 to 8 × 10 7 cells, was separated by electrophoresis on 12 % SDS-polyacrylamide gels and transferred to Polyvinylidene difluoride (PDVF) membranes (GE Healthcare). \n\nBlots were then blocked for 1 h in 6 % milk before incubation with specific antibodies as follows: rabbit antihuman CD20 specific to the COOH-terminal region [22] (Thermo Scientific) and rabbit anti-actin (#8457L, Cell Signaling). Blotted proteins were detected and quantified on a bioluminescence imager and BIO-1D advanced software (Vilber-Lourmat) after blots were incubated with a horseradish peroxidase-conjugated appropriate secondary antibody (Beckman Coulter).",
"section_name": "Western blotting",
"section_num": null
},
{
"section_content": "Total RNA was extracted using the RNeasy Total RNA Isolation kit (Qiagen, Courtaboeuf, France), following manufacturer protocols. One microgram of total RNA was used as template for cDNA synthesis performed using a high-capacity RNA to cDNA kit (Applied Biosystem, Courtaboeuf, France). \n\nGenomic DNA was extracted using a DNeasy blood or tissue kit (Qiagen, Courtaboeuf, France) or the salting out method. Briefly, cells were lysed by TES buffer supplemented by SDS 20 % and proteinase K 0. 5 mg/",
"section_name": "Molecular studies: RNA isolation, reverse transcription, cloning, real-time quantification, and Sanger cycle sequencing",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The authors thank Dr. Alain Coaquette ( Service de virology, CHU Besançon, France ) for providing post-transplant EBV samples, Dr. Bernard Royer ( INSERM UMR1098 ) for assistance with statistical analysis, Roselyne Delepine for extracting the biological data from the CLL2007 SA clinical trial, Sarah Odrion for reading the manuscript as well as the scientific committee and clinical investigators of the French cooperative group of CLL/MW.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Cell transfection and packaging cell line production",
"section_num": null
},
{
"section_content": "ml. Proteins were then precipitated in a saturated NaCl solution and centrifuged, and DNA then was precipitated using ethanol. \n\nQualitative RT-PCR was performed using the MyTaq DNA polymerase ready-to-use master mix (Bioline, France) and specific primers. PCR products were analyzed by agarose gel electrophoresis followed by ultraviolet detection. When useful, PCR products were gel purified, cloned within pCR ® 2. 1-TOPO ® TA vector (Life Technologies), and Sanger forward and reverse sequenced using M13 primers. Purified sequencing products were run on an ABI-3130 DNA analyzer and analyzed using sequencing analysis v5. 2 software (Applied Biosystems). Sequences were aligned against the wildtype (wt)CD20 coding sequence using the Bioedit v7. 1 software. \n\nQuantitative RT-PCR (RT-qPCR) was performed using splice variant-specific primers and bi-fluorescence probes. cDNA was amplified with TaqMan Universal Master Mix with UNG (Applied Biosystem, Courtaboeuf, France) using a standard two-step amplification program (10 s at 95° and 1 min at 60°). CD20 variant transcript copy number was assessed by RT-qPCR against a plasmid dilution curve. All PCR samples were normalized to ABL copy number. The proportion of each CD20 transcript variant was calculated against all CD20 isoforms. \n\nPCR conditions, sizes of PCR products, and names and sequences of primers are described in Additional file 1: Table S1. Schematic localizations of all PCR primers and bi-fluorescent probes are provided in Additional file 1: Figure S1.",
"section_name": "",
"section_num": ""
},
{
"section_content": "The wtCD20 coding sequence was first cloned into a pcDNA3. 1-GFP (green fluorescent protein) mammalian expression vector. To restore canonical splice sites within the cDNA coding sequence, intron 5 (int5) was previously reintroduced into pcDNA3. 1-GFP-wtCD20 to generate pcDNA3. 1-GFP-int5-CD20, theoretically producing D618-CD20 transcripts. Intron 6 (int6) was then inserted into pcDNA3. 1-GFP-int5-CD20 to generate pcDNA3. 1-GFP-int5-and 6-CD20-expressing D657-CD20 transcripts. Finally, reintroduction of introns 3 (int3) and 6 within the wtCD20 sequence allowed expression of D480-CD20 mRNA from pcDNA3. 1-GFP-int3 and the 6-CD20 vector. All of these vectors were amplified into JM105 bacteria. The HT1080 cell line was then transfected by these vectors using the Lipofectamine transfection kit (Life Technologies) to produce transiently expressing cell lines. \n\nIn addition to the D393-CD20 packaging cell line, previously produced, wtCD20, D657-CD20, and D618-CD20 coding sequences were inserted into the retroviral pBABE-GFP vector (Addgene, UK). The PG13 packaging cell line was transfected by the pBABE-GFP-wtCD20, pBABE-GFP-D657-CD20, and pBABE-GFP-D618-CD20 vectors using the Lipofectamine transfection kit (Life Technologies). Supernatants were then collected at 48, 72, and 96 h to produce HT1080-transduced cells, cultured and selected in puromycin-containing medium. The percentage of stably transduced cells was controlled by assessing GFP expression by flow cytometry.",
"section_name": "Cell transfection and packaging cell line production",
"section_num": null
},
{
"section_content": "Splicing sites were identified using online splice site prediction tools such as SpliceProt prediction [24] (http:// www. spliceport. org. ) or ASSP Prediction [25] (http:// wangcomputing. com/assp/). Statistical analysis was performed using the Χ 2 test.",
"section_name": "Splice site prediction and statistical computational analysis",
"section_num": null
},
{
"section_content": "CG executed all experiments, including cell cultures, cytometry, western blotting, and molecular biology, and wrote the original draft of the manuscript. AD performed all PCR, RT-PCR, and RT-qPCR set-up. EBR helped with cell transfection and retroviral transduction. LY, OT, CD, EVDN, FL, ED, FA, and ET provided biological samples from their respective clinical trials or clinical experience. FGO, YG, PT, PS, and CB contributed to improving the manuscript and gave final approval. CF and MD initiated and designed the study, participated in every step of the study, managed the whole project, and wrote the manuscript. All authors read and approved the final manuscript.",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "Additional file 1: Table S1. Table of primers used for wtCD20 and transcript variant detection (RT-PCR) as well as realtime PCR quantification (RT-qPCR). Specific annealing temperature and PCR product size in bp are given for RT-PCR. ABL PCR was used for control gene expression quantification. Table S2 : Characteristics (n, genders, Binet score, biological parameters, mutational status) of the three CLL patient cohorts. NA: not available. Figure S1 : Schematic representation of wtCD20 and transcript variants. Qualitative PCR primers as well as quantitative primers forward (→) and reverse (←) and bi-fluorescent probes (•-•) are localized up and down, respectively, on the different transcripts. Figure S2 : wtCD20 coding sequence (NCBI-GenBank NM152866. 2) given as reference as well as D393-CD20, previously described [22] shown in blue. The 4 new identified coding sequences of the CD20 alternative transcripts are also in blue. Figure S3 : Alignment of the newly discovered sequences against the wtCD20 coding sequence using the BioEdit v7. 1 software, which allowed precise identification of junction sequence regions. Figure S4 : a/CD20 immunoprecipitation (IP) was performed using an antibody specific to an extracellular epitope of human CD20 (#302302, Biolegend) and western blot detection with the cterminal human CD20 Rabbit Polyclonal antibody (#E2562, Thermofischer) b/Subcellular fractions [Membrane (M), Cytoplasm (C), Nucleus (N)] obtained from 293 cells transfected with a lentiviral vector pFIV-D393-CD20 or pFIV-wtCD20 were subjected to western blot analysis using c-terminal CD20 or actin (for protein loading control) antibodies. Blotted proteins were detected and quantified on a bioluminescence imager with BIO-1D advanced software (Wilber-Lourmat) after incubation of blots with a horseradish peroxidase-conjugated appropriate secondary antibody (Beckman Coulter). c/Lipid raft isolation by ultra-centrifugation on sucrose density gradient. Fractions 1 to 4 (10 % to 40 % of sucrose density) respectively harvested after centrifugation were subjected to c-terminal anti-CD20 western blotting. Actin and Flotillin-2 antibody staining was used as protein-loading and lipid-raft control, respectively.",
"section_name": "Additional file",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "Competing interests",
"section_num": null
}
] |
10.21203/rs.3.rs-2175987/v1
|
The stromal-tumor amplifying STC1-Notch1 feedforward signal promotes the stemness of hepatocellular carcinoma
|
<jats:title>Abstract</jats:title> <jats:p>Background Cancer associated fibroblasts (CAFs), an important component of the tumor microenvironment (TME), play crucial roles in tumor stemness. Stanniocalcin-1 (STC1) was found secreted by CAFs in various cancers, but its main source and its role in hepatocellular carcinoma (HCC) was still unclear. Methods The serum and intracellular expression levels of STC1 were detected by ELISA and western blot. The role of CAFs-derived STC1 in HCC stemness was probed by sphere formation, sorafenib resistance, colony formation, and transwell migration and invasion assays in vitro and orthotopic liver xenograft tumor model in vivo. An HCC tissue microarray containing 72 samples was used to identify the STC1 and the Notch1 in HCC tissues. Co-immunoprecipitation (CoIP) and dual-luciferase reporter assay were performed to further explore the underlying mechanisms. ELISA assays were used to detect the serum concentration of STC1 in HCC patients. Results We demonstrated that CAFs were the main source of STC1 in HCC and that CAFs-derived STC1 promoted HCC stemness through the activation of the Notch signaling pathway. In HCC patients, the expression of STC1 was positively correlated with poor prognosis and the Nocth1 expression. Co-IP assay showed that STC1 directly bound to Notch1 receptors to activate the Notch signaling pathway, thereby promoting the stemness of HCC. Our data further demonstrated that STC1 was a direct transcriptional target of CSL in HCC cells. Furthermore, ELISA revealed that the serum STC1 concentration was higher in patients with advanced liver cancer than patients with early liver cancer. Conclusions CAFs-derived STC1 promoted HCC stemness via the Notch signaling pathway. STC1 might serve as a potential biomarker for the prognostic assessment of HCC, and the stromal-tumor amplifying STC1-Notch1 feedforward signal could provide an effective therapeutic target for HCC patients.</jats:p>
|
[
{
"section_content": "As the fourth leading cause of cancer death, liver cancer causes a great burden on global health [1]. Hepatocellular carcinoma (HCC), which accounts for about 80% of all primary liver cancer, is a highly heterogeneous disease caused by multiple etiologies and has a low long-term survival rate [2, 3]. HCC generally develops with brosis or cirrhosis [4] and is usually accompanied by an accumulation of activated broblasts. The intricate cross-talk between the stroma and the tumor has a key impact on tumor progression in various cancers, including HCC [5] [6] [7] [8]. Further clari cation on the mechanisms of the tumor-stromal crosstalk is therefore needed to develop therapeutic strategies targeting both cancer cells and cancer-associated broblasts (CAFs) [9] [10] [11]. \n\nCAFs, an abundant stromal-activated broblast type of HCC TME [12], play a diverse role in the tumor progression of various cancers [13], including HCC [8, 14, 15]. Well-documented evidence links the multifaceted function of CAFs with the stemness phenotypes of cancer cells [5, 16]. Cancer stem cells, the special stem-like cell subpopulations of tumor cells, could fuel and maintain tumor growth at low cell numbers [17], and are responsible for the development, treatment resistance, and recurrence of tumors [18, 19]. \n\nCAFs not only exist around tumor cells but can also secrete various paracrine factors to interact with tumor cells. In prior reports, stanniocalcin-1 (STC1), a secreted glycoprotein hormone, was shown to be secreted by CAFs and could promote cancer progression in a variety of cancers, such as colorectal cancer, lung adenocarcinoma, and breast cancer [20] [21] [22]. STC1was also overexpressed in various cancers [23] [24] [25], and was reported to promote cancer stemness [26] [27] [28]. A recent study demonstrated that secretory STC1 promoted HCC metastasis via activation of the JNK pathway in HCC [29]. However, the main source of STC1 and its relationship with cancer stemness in HCC remains unclear. \n\nThe Notch signaling pathway participates in various biological processes and is a major signaling pathway to regulate cancer stemness [30]. It is a highly conservative signaling pathway, and the core Notch signaling pathway consists of relatively few molecules. After Notch receptor activation, the Notch intracellular domain (NICD), is translocated into the nucleus and interacts with the DNA-bound protein CSL (RBP-J) to regulate the expression levels of downstream molecules [31, 32]. Our previous studies have demonstrated that Notch signaling is one key signal promoting HCC stemness [33] [34] [35] [36]. \n\nIn this study, we con rmed that CAF was the main source of STC1 in HCC and provided evidence for an amplifying STC1-Notch1 axis in the HCC stromal-tumor crosstalk, which might be a new therapeutic target of HCC. In addition, STC1 might be a promising biomarker for the prognostic evaluation of HCC.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "We downloaded the transcriptomic data and the clinicopathological data of the TCGA-LIHC project from The Cancer Genome Atlas database at http://portal. gdc. cancer. gov/. Relevant survival data were gotten from previous literature [37]. Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) enrichment analysis were performed on these data using \"clusterPro ler\" R packages [38]. For survival analysis, we excluded samples with an overall survival time of less than 30 days to exclude non-tumor causes of death. Kaplan-Meier survival analysis was performed with log-rank test.",
"section_name": "Bioinformatics analysis",
"section_num": null
},
{
"section_content": "Samples of HCC tissue and the corresponding adjacent normal HCC tissues were collected from HCC patients who had undergone surgical resection and had not received radiotherapy and chemotherapy. We also collected 5 samples of hepatic hemangioma. The collection and usage of human specimens were approved by the Ethics Committee of Tongji Hospital, HUST, Wuhan, China (IRB ID: TJ-IRB20220562).",
"section_name": "Clinical Liver Samples",
"section_num": null
},
{
"section_content": "IHC staining with the STC1 antibody (HPA 023918) and Notch1 (Abcam, ab8925) was performed to detect the protein expression level in HCC tissues. The IHC staining score was carried by ImageJ IHC pro ler (http://sourceforge. net/projects/ ihcpro ler).",
"section_name": "Immunohistochemistry (Ihc)",
"section_num": null
},
{
"section_content": "The primary CAFs and NFs were isolated from the clinical HCC tissues and liver tissues adjacent to hepatic hemangioma respectively. The fresh liver tissues were washed with D-Hank's solution, cut into 2-3mm fragments and then plated in a culture dish with DMEM (GIBCO) comprising 15% fetal bovine serum (FBS) for attachment. Fibroblasts were allowed to grow out of tumor fragments for 1-2 weeks. 95% puri ed broblasts were obtained after 2-3 generations and veri ed by the broblast marker α-SMA.",
"section_name": "Isolation And Puri cation Of Cafs And Normal Fibroblasts (Nfs)",
"section_num": null
},
{
"section_content": "MHCC-97H was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM comprising 10% FBS. SNU-398 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultivated in RPMI 1640 Medium (GIBCO) with 10% FBS.",
"section_name": "Cell Culture",
"section_num": null
},
{
"section_content": "Cells were xed with 4% paraformaldehyde, washed with PBS for 3 times, permeabilized with 0. 1% Triton X-100 for 10 min, and incubated in 10% normal fetal sheep serum in PBS for 40 min at room temperature. Then the CAFs and NFs were co-incubated with α-SMA (Boster, BM0002) and STC1 (Santa Cruz, sc-293435) primary antibodies at 4°C for overnight. The MHCC-97H were incubated with Notch1 (Abcam, ab52627) and/or STC1 primary antibodies. Observed and photographed cells under uorescence microscope.",
"section_name": "Immuno uorescence (If)",
"section_num": null
},
{
"section_content": "The culture medium supernatants from CAFs, NFs, and HCC cell lines (MHCC-97H and SNU-398) were collected and centrifuged at 1500 rpm for 5 min. For plasma samples, cells were removed by centrifugation at 3000 rpm for 10 minutes. Subsequently, the secretion of STC1 was detected according to the manufacturer's instructions using the ELISA kit (Boster, EK1404).",
"section_name": "ELISA",
"section_num": null
},
{
"section_content": "The cells were given an appropriate amount of RIPA /PMSF/cocktail buffer and centrifuged to extract the protein. Then the total proteins were separated on SDS-PAGE and transferred onto the PVDF membrane (Merck Millipore, USA). After blocking with 5% milk for 1h, the PVDF membranes were incubated with primary antibodies overnight at 4°C. Anti-β-actin (Proteintech, 66009-1-ig) was used as a loading control. The primary antibodies were as follows: STC1 (Santa Cruz), NANOG (Cell Signaling Technology, 4903), OCT4 (CST, 2750), SOX2 (CST, 3579), Notch1 (CST, 3608), cleaved Notch1 (CST Cat# 4147), HES1 (CST, 11988), and HEY1 (ABclonal, A16110). The ImageJ software (NIH, America) was used to quantify the gray value of the protein bands. \n\nTrizol reagent (Invitrogen, Carlsbad, CA, USA) and HiScript® III Reverse Transcriptase (Vazyme, Jiangsu, China, R302-01) were used to extract the ribonucleic acid (RNA) and reversed to cDNA following the manufacturer's instructions. Then quantitative real-time PCR was carried out using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02). The data was analyzed by the 2 -ΔΔCt (Ct, cycle threshold) method. Primer sequences were provided in Table S1.",
"section_name": "Western Blot And Qrt-pcr",
"section_num": null
},
{
"section_content": "The cells were seeded in 24-well low attachment plates (Corning, NY, USA) with tumor sphere medium, composed of DMEM/F12 medium with 1% penicillin/streptomycin, 1× B27 supplement (cat# 17504-044; GIBCO), 20 ng/ml EGF (cat# PHG0311; GIBCO), 10 ng/ml bFGF (cat# PHG0266; GIBCO), and 1% methyl cellulose (cat# M0262; Sigma-Aldrich). Fresh medium was added every 3-4 days. Spheres were visualized and counted by microscopy.",
"section_name": "Sphere Formation Assay",
"section_num": null
},
{
"section_content": "The Cell Counting Kit (CCK-8) (40203ES92, Yeasen, Shanghai, China) was used to measure the sensitivity of the cells to Sorafenib. 1000 cells per well were seeded in 96-well plates, and after cell adhesion, treated with different concentrations (2. 5, 5, 10 or 20 µM) of Sorafenib (Sigma-Aldrich). After incubating for 24h at 37℃, CCK-8 reagent was added to each well. 2 hours later, absorption value at 450 nm was detected at the microplate reader (Thermo Fisher, Shanghai, China).",
"section_name": "Cck8 Toxic Assay",
"section_num": null
},
{
"section_content": "Cells were seeded at a density of 1000 cells per well in 6-well plates. Approximately 10 days later, colonies were dyed with crystal violet (C0121, Beyotime, China) and counted after xing the cells with 4% methanol for 20 minutes. The results are presented as the mean ± SD.",
"section_name": "Colony-formation Assay",
"section_num": null
},
{
"section_content": "For the migration assay, serum-free cell suspension was added into the upper transwell chambers (Millipore, Billerica, MA, USA) without FBS, and 600 ul of DMEM with 10% FBS was added as a chemoattractant. And cell invasion assay was performed with Matrigel (1:8 diluent, BD Biosciences, San Jose, CA, USA) coating the transwell chambers. After 24h (migration) or 32h (invasion) of incubation, the membranes were xed and imaged. The number of cells in 3 randomly selected was counted, and the experiments were repeated three times.",
"section_name": "Transwell Migration And Invasion Assays",
"section_num": null
},
{
"section_content": "Viral particles expressed STC1 shRNA, STC1, or Notch1 shRNA were obtained from DesignGene Biotechnology(shanghai, China) and Genechem Corporation (Shanghai, China). The sequence for virusbased RNAi were provided in Table S1. CAFs or HCC cells were infected with lentivirus. 48h after infection, puromycin was added to screen cells for establishing stable cell lines.",
"section_name": "Virus Infection",
"section_num": null
},
{
"section_content": "Cells were collected and lysed in an appropriate precooled IP buffer. The cell lysate was centrifuged after being frozen for 30min. Took 50ul lysate supernatant as input. And the rest was incubated with indicated antibody on a rotating device overnight at 4 ℃. The next day, protein A/G Magnetic Beads (MCE, HY-K0202) were washed 3 times with PBST and added to the cell supernatant-antibody system. After incubation on a rotating device at 4 ℃ for 4h, the magnetic beads were washed 6 times and boiled. Samples were conducted by Western blot analysis. The primary antibodies had been listed in Western blot.",
"section_name": "Co-immunoprecipitation (Co-ip)",
"section_num": null
},
{
"section_content": "The STC1 promoter fragment was inserted into the pGL3-Basic vector. The stable lines overexpressed NICD and the control cells were co-transfected with STC1 reporter-gene plasmid and pGMLR-TK. Following a 48h incubation, a Dual-Luciferase Reporter Assay Kit (Yeasen, 11402ES60) was employed to measure re y luciferase activity, and Renilla luciferase activity was implemented for normalization.",
"section_name": "Dual-luciferase Reporter Assay",
"section_num": null
},
{
"section_content": "Four-week-old male nude mice were bought from GemPharmatech company and raised in a pathogenfree barrier environment. HCC cells with or without CAFs were resuspended in PBS, and slowly injected into the left lobe of the liver. Animals were sacri ced about 4-5 weeks after implantation. Bioluminescence was measured after an intraperitoneal injection of 100ul of potassium D-luciferin salt (30 mg/mL, per animal). All experiments were approved by Tongji Hospital Institutional Review Board (IRB ID: TJH-202206023)",
"section_name": "Mouse Orthotopic Liver Xenograft Tumor Model",
"section_num": null
},
{
"section_content": "Each assay was carefully performed with three independent experiments, and the data were shown as the mean and standard deviation (mean ± SD) unless otherwise indicated. Statistical analysis was processed using R (v4. 0. 2) and GraphPad Prism 9. 0 software. We chose appropriate statistical tests according to the types of data. For the data approximately normally distributed, student t-test and one-way ANOVA were performed. Wilcoxon signed-rank test was applied to analyze the nonnormal distribution data. Survival analysis was evaluated using a log-rank test. P < 0. 05 was regarded as statistically signi cant.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "CAFs -derived STC1 promotes HCC stemness. \n\nCAFs were isolated from HCC patient tumor tissue samples, and NFs were puri ed from liver tissues adjacent to hepatic hemangioma. To check whether CAFs were the main source of STC1 in HCC, we rst analyzed the published single-cell sequencing data of HCC [39], and found that STC1 was higher expressed in broblasts than in HCC cells (Fig. 1A ). IF showed more α-smooth muscle actin (α-SMA) (green) and STC1 (red) colocalization in CAFs than those in NFs. α-SMA (green) and STC1 (red) colocalized in the cytoplasm and nucleus (Fig. 1B ). Next, we detected the secreted and intracellular levels of STC1 in the CAFs, NFs, MHCC-97h, and SNU-398 HCC cell lines, individually. ELISA assay showed that the CAF secreted the highest level of STC1 (Fig. 1C ). And the protein levels of STC1 were dramatically upregulated in CAFs relative to the levels in other cells (Fig. 1D ). Collectively, these data indicated that CAFs were the main source of STC1 in the HCC TME. \n\nOur previous study demonstrated that CAFs could promote or maintain stem-like properties in HCC [40]. To investigate whether CAF-derived STC1 affects HCC stemness, MHCC-97h and SNU-398 were cultured in different media: DMEM, the conditional medium of CAFs (CAF-CM), CAF-CM supplemented with 1µg/ml IgG (CAF-CM + IgG), CAF-CM supplemented with 1µg/ml STC1 neutralizing antibody (CAF-CM + STC1-Ab), and DMEM supplemented with human STC1 recombinant protein (rhSTC1, 20ng/ml). Then a series of assays were performed to test the stem-like properties of HCC. Sphere formation assay showed that CAF-CM or rhSTC1 signi cantly promoted the self-renewal ability of MHCC-97h and SNU-398 cells, while STC1 neutralizing antibody (STC1-Ab) dramatically reversed the increase in the self-renewal ability induced by CAF-CM (Fig. 1E ). Different concentrations of sorafenib were used to treat HCC cells for 24 h. The data indicated that CAF-CM, CAF-CM + IgG, and rhSTC1 groups possessed higher sorafenib resistance than DMEM and CAF-CM + STC1-Ab groups (Fig. 1F and S1A ). Colony formation assays demonstrated that CAF-CM or rhSTC1 could promote the proliferation ability, while STC1 neutralizing antibody attenuated the promoting effects of CAF-CM (Fig. 1G ). Moreover, transwell migration and invasion assays showed that after being treated with CAF-CM or rhSTC1 (20ng/ml), MHCC-97h and SNU-398 owned higher migration and invasion abilities than treated with DMEM. And compared with the CAF-CM group, the CAF-CM + IgG group still could promote migration or invasion, while the CAF-CM + STC1-Ab group had diminished migration and invasion abilities (Fig. 1H and S1B ). Western blot analysis revealed that the cancer stemness markers, including NANOG, SOX2, and OCT4, were upregulated in CAF-CM, CAF-CM + IgG, and rhSTC1 groups compared with DMEM. Meanwhile, compared with the DMEM group, cells exposed to CAF-CM + STC1-Ab showed no signi cant changes in the levels of NANOG, SOX2, and OCT4 (Fig. 1I ). These results suggested that CAFs were the main source of STC1 in the HCC TME, and the CAFsderived STC1 could promote HCC stemness. \n\nKnocking out of STC1 in CAFs attenuated its ability to promote HCC stemness. \n\nPrimary CAFs were used to establish stable cell lines, CAFs-shcontrol, CAFs-shSTC1-1, and CAFs-shSTC1-2, with lentivirus infection (Fig. 2A ). The data showed that down-regulation of STC1 in CAFs signi cantly decreased CAFs-enhanced spheroids ability in HCC cells (Fig. 2B ). The resistance ability to sorafenib of HCC cells was reduced when cultured in CAFs knocking out STC1 conditioned medium, comparing with cultured in CAF-CM (Fig. 2C and S1C ). In addition, cells treated with CAF-shSTC1-1-CM and CAF-shSTC1-2-CM possessed lower proliferation, migration, and invasion abilities than those treated with CAFshcontrol -CM (Fig. 2D-F ). Western blot revealed that down-regulation of STC1 in CAFs signi cantly reversed CAFs-upregulated the expression of NANOG, SOX2, and OCT4 in MHCC-97H and SNU-398 (Fig. 2G ). Additionally, we explored the effect of CAFs-derived STC1 in vivo by the mouse orthotopic liver xenograft tumor model. SNU-398 luciferase-tagged (SNU-398-LUC) HCC cells mixed with or without CAFs of different treatments were injected into the livers of nude mice. Mice were randomly divided into four groups: SNU-398-LUC cells group, SNU-398-LUC cells + CAFs-shcontrol group, SNU-398-LUC cells + CAFs-shSTC1-1 group, SNU-398-LUC cells + CAFs-shSTC1-2 group. 28 days after cell injection, the tumor size was measured by bioluminescence imaging. Interestingly, we observed that that luminescence intensity from SNU-398-LUC cells + CAFs-shcontrol group was higher than that derived from SNU-398-LUC cells group, while knockdown of STC1 in CAFs attenuated the luminescence intensity (Fig. 2H ). Taken together, these data indicated STC1 played a vital role in CAFs-mediated promotion of HCC stemness in vitro and in vivo. \n\nCAFs -derived STC1 promoted HCC stemness in a Notch1-dependent manner Our previous studies have demonstrated that the Notch1 signaling pathway is important for promoting the stem-like properties in HCC [33] [34] [35]. To explore the underlying mechanism of how CAFs-derived STC1 promoted HCC stemness, we performed GSEA pathway enrichment analysis and GO analysis among the HCC samples in the TCGA database. The results indicated that high expression of STC1 was signi cantly related to positive regulation of the Notch signaling pathway (Fig. 3A and 3B ). IF showed more Notch1/NICD (green) expression in CAF-CM or rhSTC1 treated MHCC-97h cells than DMEM treated cells (Fig. 3C ). Next, we detected the expression of downstream molecules of the Notch1 signaling pathway and we found that in the CAF-CM, CAF-CM + IgG, and rhSTC1 group cells, the NICD, HES1, and HEY1 expression levels were signi cantly higher than that of the DMEM and CAF-CM + STC1 Ab groups (Fig. 3D ). The western blot assay was carried out to further verify the effect of STC1 on the Notch signaling pathway. We found that the expression of NICD, HES1 and HEY1 were signi cantly decreased in MHCC-97H and SNU-398 cells treated with CAF-shSTC1-1-CM and CAF-shSTC1-2-CM relative to those treated with CAF-shcontrol-CM (Fig. 3E ). These data suggested that the Notch signal pathway is downstream of STC1. \n\nTo explore whether Notch1 was involved in CAFs-derived STC1-medicated HCC stemness, we knocked down the expression of Notch1 in MHCC-97H and SNU-398 with lentivirus transfection. Our previous research has proved that CAF-induced promotion of HCC stemness was weakened after Notch1 was knocked out, so in this part, we focused our attention mainly on the effect of Notch1 on CAFs-derived STC1 induced stem-cell like properties. The data revealed that the self-renewal ability of shNotch1 cells was attenuated regardless of being treated with rhSTC1 (Fig. 3F ). Similarly, after Notch1 knockdown, despite being treated with rhSTC1, the resistance to sorafenib ability of HCC cells were diminished (Fig. 3G and S2A ). And also, knockdown of Notch1 in MHCC-97H and SNU-398 cells decreased proliferation, migration, and invasion abilities despite being pretreated with rhSTC1 (Fig. 3H, 3I, and S2B-D). In addition, the expression of cancer stemness markers NANOG, OCT4, and SOX2 were decreased regardless of whether treated with or without rhSTC1 (Fig. S2E ). The in vivo orthotopic liver xenograft tumor model showed that knockdown of Notch1 lowered the luminescence intensity of the CAF-STC1 group (Fig. 3J ). Taken together, these data suggested that the effect of CAFs-derived STC1 on HCC stemness was prominently restrained through Notch1 knockdown, and the CAF/STC1/Notch1 axis might play a crucial role in the HCC stemness.",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To investigate the underlying mechanism of STC1-mediated activation of Notch1 signaling, we rst analyzed the correlation between STC1 and Notch1 in the HCC TCGA database at the GEPIA website (http://gepia. cancer-pku. cn/). IF showed the co-localization of STC1 (green) and Notch1 (red) in MHCC-97H (Fig. 4B ). The co-IP experiments were performed in MHCC-97H and SNU-398. The results proved there is a physical interaction between the STC1 and Notch1 (Fig. 4C-D ), and were consistent with the results of a previous study, which con rmed that STC1 could directly bind to Notch1 as a non-canonical Notch ligand to activate the Notch1 signaling pathway [28]. To evaluate the possible correlation of STC1 and Notch1 in human HCC tissues, protein levels of STC1 and Notch1 were detected by immunohistochemical (IHC) staining on a tissue microarray (Fig. 4E ). Importantly, we observed that STC1 expression was positively correlated with Nocth1 expression in HCC patients (Fig. 4F ; n = 72, r = 0. 6443, **** P < 0. 0001, Pearson's correlation). These data implied that CAFs-secreted STC1 could activate the Notch signaling pathway by directly binding to Notch1 in HCC cells, and in the HCC samples we collected, STC1 was positively correlated with Notch1.",
"section_name": "CAFs-secreted STC1 directly bound to Notch1 receptors and activated the Notch signaling pathway",
"section_num": null
},
{
"section_content": "In the previous experiment, we surprisingly found that CAF-CM treated MHCC-97H had a higher STC1 expression than DMEM-treated cells (Fig. 4B ). In addition, we observed that Notch1 depletion reduced the protein expression of STC1 in MHCC-97H and SNU-398 (Fig. 5A ). This nding drove us to examine whether the STC1 gene was potentially regulated by NICD at the transcriptional level in HCC cells. STC1 protein and mRNA levels were up-regulated by overexpressing NICD with lentivirus transfection in MHCC-97H and SNU-398 (Fig. 5B and 5C ). NICD, the activated composition of the Notch receptor, is inserted into the nucleus as a transcriptional coactivator and interacts with CSL (RBP-J), the DNA-binding protein. We got the CSL binding motif from the JASPAR database (http://jaspardev. genereg. net/) (Fig. 5D ). Then, the STC1 promoter region for possible CSL binding sites was screened with the JASPAR, and we chose 3 most possible CSL binding sites for the mutation (Fig. 5E ). Dual-luciferase reporter assays suggested the mutation of R1 (binding region 1) in STC1 promoter abolished the promoter inducibility that was mediated by NICD overexpression (Fig. 5F and 5G ). Collectively, these ndings indicated that STC1 was regulated by Notch1 at the transcriptional level, which formed an amplifying STC1-Notch1 feedforward signal in tumor-stromal crosstalk. STC1 was correlated with poor prognosis of HCC patients and might serve as a biomarker. \n\nThe effect of CAF-derived STC1 on HCC cells' stem-cell properties motivated us to explore the importance of STC1 expression in patients with HCC. By using TCGA public databases (GEPIA), we found that the HCC tumor tissues had a higher expression level of STC1 than normal liver samples (Fig. 6A ). In addition, high expression of STC1 predicted poor survival of HCC patients by Kaplan-Meier analysis (Fig. 6B ). During our analysis, samples with an overall survival time of fewer than 30 days were excluded to exclude non-tumor causes of death. As STC1 is a secretory protein, we further investigated whether the concentration of STC1 in serum could serve as a clinical biomarker for HCC. We measured the level of STC1 in the serum of patients with advanced liver cancer (n = 17) and patients with early liver cancer (n = 42) by ELISA. Patients with advanced liver cancer possessed higher serum STC1 concentration than patients with early liver cancer (Fig. 6C ), which suggested that STC1 could be a biomarker in HCC with poor prognosis. The STC1 expression level was assessed by IHC staining in a total of 72 HCC tissues and matched adjacent normal liver tissues (Fig. 6D ). The data indicated that HCC patients with high STC1 expression had shorter median overall survival than that of patients with low STC1 expression (19 months vs. 37 months, HR = 2. 63, 95% CI: 1. 23-5. 64, P = 0. 0127) (Fig. 6E ). We next examined the correlations between STC1 expression and multiple clinical-pathologic characteristics. Our data indicated that high STC1 expression was signi cantly correlated with the advanced TNM stage (Table 1 ; P = 0. 024; χ2 test). Together, these ndings suggest that STC1 was up-regulated and correlated with poor survival of HCC patients, and might be a biomarker for strati ed analysis of HCC patient prognosis. \n\nTable 1 The expression of STC1 related to clinicopathological features in HCC patients.",
"section_name": "Notch1 directly regulated STC1 expression to establish a paracrine amplifying STC1-Notch1 feedforward signal",
"section_num": null
},
{
"section_content": "CAFs, one of the most important activating components of TME, differs from the normal resident broblasts and directly regulates tumor progression, metastasis, therapy resistance, tumor immune, and the cancer stem-like properties [41]. Previous studies had shown that CAFs were closely related to the stemlike properties of HCC cells, but the mechanism by which CAFs in regulating HCC stemness was not fully clari ed. Our previous studies indicated the impact of HCC stemness on HCC progression and the effect of the Notch signaling pathway on the stem-like properties of HCC [34]. However, the impact of CAFs on HCC stemness is not well understood yet. Herein, we focused on understanding the impact and the underlying mechanism of the CAFs in stemness facilitation and their association with the poor prognosis of HCC. \n\nIn this study, we demonstrated that CAFs secreted STC1 and facilitated the stem-like properties of HCC. In the process of tumor progression, CAFs secreted cytokines, chemokines, secreted proteins, exosomes, and other media in the TME and promoted cancer stemness. CLCF1, which was secreted by CAFs, could promote tumor stemness in HCC [14]. Our previous studies demonstrated that CAFs promoted the stem-cell like properties of HCC through the secretion of IL6 and HGF [40]. Through the analysis of the single-cell sequencing data of HCC [39], we found that STC1 was mainly derived from broblasts rather than HCC cells. ELISA and western blot assay respectively proved that STC1 had higher secretion and intracellular expression levels in CAFs. STC1 has been reported to distribute in multiple biological activities such as tumorigenesis [42], angiogenesis [43], wound healing, and tumor immune resistance [23]. Long-term exposure of HCC cells to CAF-CM or rhSTC1 resulted in an increase in HCC stemness, while STC1-Ab dramatically reversed the promotion. The results showed that CAFs-derived STC1 could promote the stem-cell like properties of HCC. In addition, STC1 has recently been reported to facilitate stem-like traits in glioblastoma cells [28]. Silencing STC1 in CAFs decreased CAFs-mediated promotion of HCC stemness in vitro and tumorigenicity in vivo. These ndings strongly suggested the role of STC1 in the CAFs-induced promotion of HCC stemness. \n\nThe Notch signaling pathway plays a crucial role in the regulation of stem cell properties in HCC [44],\n\ncolorectal cancer [45], and other tumors [46, 47]. Here, our data indicated that CAFs-derived STC1 could activate the Notch signaling pathway in HCC. GSEA pathway enrichment analysis and GO analysis implied that the Notch signaling pathway was positively enriched in tumor tissues highly expressing STC1. We observed that CAFs-derived STC1 activated the Notch signaling pathway of HCC cells and knockdown of STC1 in CAFs signi cantly inhibited this effect. In breast cancer, the Notch signaling driven crosstalk between tumor cells and CAFs to promote the radioresistance of tumor [48]. In our previous study, the Notch signaling pathway participated in the CAFs mediating stem-cell like properties, which was reversed when Notch1 was knocked out [36]. In this study, we found that the silence of Notch1 in HCC cells signi cantly inhibited CAFs-derived STC1 induced stemness in vitro and in vivo. These data supported the important role of the Notch signaling pathway in facilitating CAF-derived STC1 induced stem-cell like properties in HCC. \n\nLigands binding to Notch receptors leads to the activation of Notch signaling. During this process, NICD is released after receptors undergo three cleavages and is inserted into the nucleus to regulate the transcription of downstream genes [30]. Recently, some new molecules have been reported as noncanonical ligands, for example, OSM-11 [49]. In glioblastoma, STC1 could bind directly to 1-24 EGF like domains in the Notch extracellular domain (NECD), which are the binding sites of Notch receptors [28]. Our data showed the direct binding between STC1 and Notch1, which suggested that STC1 might be a noncanonical ligand of Notch1 receptors. Moreover, STC1 and Nocth1 were positively correlated in clinical HCC samples. In addition, we found that the Su (H) motif of CSL, responsible for DNA binding, could bind to the promoter of STC1. CSL recruits histone deacetylases (HDACs) to inhibit the transcription of downstream genes when there is no NICD binding, and promotes the transcription of target genes after NICD binding [32]. The data revealed that the STC1 was a direct target of Notch1 in HCC cells. Taken together, we demonstrated that the stromal-tumor amplifying STC1-Notch1 feedforward signal promoted the HCC stemness, and provided new therapeutic targets targeting both cancer cells and CAFs. Besides, we believed that more therapeutic targets pointed at both cancer cells and CAFs could be developed in cancer, which could have a strategic advantage over targeting tumor cells or CAFs only. \n\nSTC1 was found to be a biomarker in many diseases [52] [53] [54] [55]. In this study, we found that STC1 was highly expressed in HCC and was closely correlated with poor prognosis in HCC patients. Furthermore, we looked into 59 serum samples of HCC patients and demonstrated that high serum concentration of STC1 correlated with advanced stage in HCC. These data supported the important role of STC1 in HCC and showed that STC1 might be a promising biomarker for the diagnostic and prognostic assessment of HCC.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In summary, we identi ed that CAFs were the main source of STC1 and STC1 was positively related to",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "We sincerely thank Menghan Sha and Mengjia Jing from the Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology for their help of animal experiments and mechanism diagram.",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This was supported by the National Natural Science Foundation of China [grant numbers 81974380, 82173318 and 81802418 ].",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Data to this paper may be requested from the corresponding author.",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Supplementary Files",
"section_num": null
},
{
"section_content": "feedforward signal, a new mechanism leading to cancer stemness and progression, could provide an effective therapeutic target for HCC patients.",
"section_name": "",
"section_num": ""
},
{
"section_content": "This is a list of supplementary les associated with this preprint. Click to download. SupplementaryFigure1. tif SupplementaryFigure2. tif SupplementaryTable1. docx",
"section_name": "Supplementary Files",
"section_num": null
}
] |
10.5334/jbsr.2475
|
Neurotoxoplasmosis in a Patient with Chronic Lymphocytic Leukemia
|
Teaching Point: Neurotoxoplasmosis should be part of the differential diagnosis for single or multiple cerebral lesions in hematologic patients.
|
[
{
"section_content": "Neurotoxoplasmosis is the most aggressive presentation of toxoplasmosis and is associated with high mortality. It usually occurs in immunocompromised patients, with hematologic patients representing an emerging group at risk. We describe a case of neurotoxoplasmosis in a HIV-negative patient with chronic lymphocytic leukemia (CLL).",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "A 76-year-old man was admitted to the hospital after he developed dysarthria and confusion over the last three weeks. His medical history was remarkable for CLL, diagnosed 12 years prior. Recent history included neutropenia, thrombopenia, hypogammaglobulinemia, and obinutuzumab-chlorambucil chemotherapy (suspected cause of the cytopenia [1] ). The patient was afebrile. Neurological examination revealed leftsided hemiparesis and aphasia, but no meningeal signs. Laboratory results revealed a leukocyte count of 1. 01 × 10³/µl (normal range, 3. 7-9. 5 × 10³) and increased C-reactive protein (14. 6 mg/L). Toxoplasma gondii IgG was positive (IgM negative), indicating past infection. HIV test was negative. Brain MRI was performed, and gadolinium-enhanced T1-weighted images (WI) revealed three rim-enhancing lesions, the main one measuring 27. 5 mm (Figure 1 ). Five focal areas of enhancement were scattered throughout the cortex. There was important vasogenic edema appearing as large areas of hyperintense signal involving the white matter around the main three lesions on both fluidattenuated inversion recovery (FLAIR) and T2-WI. There was no restricted diffusion within the lesions, making the diagnosis of multiple cerebral pyogenic abscesses unlikely. Metastatic disease was suspected, but whole-body 18-fluorodeoxyglucose positron emission computed tomography did not detect any distant primary malignancy. Cerebrospinal fluid (CSF) examination disclosed mild pleocytosis and elevated protein level, but PCR for Toxoplasma gondii was negative. Surgical brain biopsy was performed, and histopathological examination revealed Toxoplasma pseudocysts consistent with cerebral toxoplasmosis as a result of reactivation of latent infection (Figure 2 ). Intravenous co-trimoxazole was prescribed, and the patient's neurological status improved rapidly.",
"section_name": "CASE REPORT",
"section_num": null
},
{
"section_content": "Neurotoxoplasmosis remains a common cerebral opportunistic infection in patients with acquired immune deficiency syndrome. This case emphasizes the need to consider toxoplasmosis in immunocompromised patients with hematological malignancies, representing an emerging group at risk [2] [3] [4] [5]. Early diagnosis is essential to ensure early treatment, but clinical presentation of cerebral toxoplasmosis is unspecific [4]. Immunosuppression and delay of antibodies' appearance can be responsible for false-negative serologic results in immunocompromised hematologic patients [2, 6]. Brain MRI often demonstrates rim-enhancing cerebral masses on contrast-enhanced T1-WI [7, 8]. When present, the \"eccentric target sign\", a ring-shaped zone of peripheral enhancement with an eccentric nodule along the wall on post-contrast T1-WI is considered highly suggestive of toxoplasmosis [9]. Perilesional edema is common [7, 10]. A T2-WI/FLAIR target sign with a hypointense core and an hyperintense intermediate region and a peripheral hypointense rim has been described [8]. The core of a rim-enhancing Toxoplasma abscess shows no restriction of water diffusion-unlike pyogenic abscess-and may resemble a metastasis or a primary brain tumor on diffusion-weighted MRI [7]. Intralesional susceptibility signal foci on susceptibility-weighted imaging, likely representing hemorrhage, have been reported to be present in most patients [11]. Brain biopsy with histological examination can provide a definitive diagnosis [2, 12].",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "In summary, brain MRI can show rim-enhancing Toxoplasma lesions with no restriction of water diffusionunlike pyogenic abscess-mimicking metastases or primary brain tumors. While commonly encountered in HIV-positive patients, neurotoxoplasmosis should also be considered in immunocompromised patients with hematological diseases.",
"section_name": "CONCLUSION",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "We thank Delrée Paul ( Department of Pathology, Institute of Pathology and Genetics, Gosselies ) who provided histopathological illustration.",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "",
"section_name": "COMPETING INTERESTS",
"section_num": null
},
{
"section_content": "The authors have no competing interests to declare.",
"section_name": "COMPETING INTERESTS",
"section_num": null
},
{
"section_content": "",
"section_name": "AUTHOR AFFILIATIONS",
"section_num": null
}
] |
10.1038/s41467-020-16276-8
|
Whole genome landscapes of uveal melanoma show an ultraviolet radiation signature in iris tumours
|
<jats:title>Abstract</jats:title><jats:p>Uveal melanoma (UM) is the most common intraocular tumour in adults and despite surgical or radiation treatment of primary tumours, ~50% of patients progress to metastatic disease. Therapeutic options for metastatic UM are limited, with clinical trials having little impact. Here we perform whole-genome sequencing (WGS) of 103 UM from all sites of the uveal tract (choroid, ciliary body, iris). While most UM have low tumour mutation burden (TMB), two subsets with high TMB are seen; one driven by germline <jats:italic>MBD4</jats:italic> mutation, and another by ultraviolet radiation (UVR) exposure, which is restricted to iris UM. All but one tumour have a known UM driver gene mutation (<jats:italic>GNAQ, GNA11, BAP1, PLCB4, CYSLTR2, SF3B1, EIF1AX</jats:italic>). We identify three other significantly mutated genes (<jats:italic>TP53</jats:italic>, <jats:italic>RPL5</jats:italic> and <jats:italic>CENPE</jats:italic>).</jats:p>
|
[
{
"section_content": "veal melanoma (UM) arises from melanocytes in the uveal tract, and though less common than cutaneous melanoma, a higher proportion of UM patients die from the disease [1] [2] [3]. Risk determination of metastatic spread can be obtained through assessment of specific chromosome copy number alterations (CNAs) 4, gene expression profiles 5 and mutation status of known UM driver genes 6. \n\nPrevious genomic studies have pointed to the existence of four UM categories, strongly linked to prognosis [7] [8] [9]. Similarly we segregate our tumours into four categories based on CNAs: category 1 are chromosome 3 disomy (D3) tumours lacking chromosome 8q copy-number gain and frequently possessing EIF1AX mutations; category 2 are D3 UM with chromosome 6p and 8q gain and a high proportion of SF3B1 mutations; category 3 are chromosome 3 monosomy (M3) tumours lacking chromosome 8q gain dominated by BAP1 mutations; category 4 UMs are M3 with chromosome 8q gain and BAP1 mutations. These genomic stratifications, while prognostic, are not indicative of treatment responses once progression has occurred. \n\nTo improve knowledge of UM genomics and to identify potential therapeutic targets, we conduct a whole-genome sequencing (WGS) study of 103 UM, comprised of 91 primary tumours and 12 metastases, with matched germline DNA. Eightyfour tumours originate from the choroid, eight from the iris, four from the ciliary body, and seven without known primary uveal site (Supplementary Data 1).",
"section_name": "U",
"section_num": null
},
{
"section_content": "Recurrent copy number aberrations. In line with previous studies [8] [9] [10] [11], TMB was low in the majority of UM (median 0. 50 mutations per megabase, range 248-42,669, Supplementary Data 2) and tumours generally displayed low counts of structural variations (SVs) (median: 13; range 0-213) (Fig. 1 ). One sample had noticeably more SVs, of which the majority (71%) were midsized (<100 kb) deletions, suggesting this was not due to chromothripsis. There were no additional notable features or known driver mutations in this sample. Commonly observed losses of chromosome 1p, 3, 8p, 6q and 16q were present, as were gains of 1q, 6p and 8q (Fig. 2 ) 4, 12. Two samples presented with whole-genome duplication (WGD). Tumours were grouped into the four categories described. Category 4 was predominant, with 55 samples (53%) displaying M3 and copy-number gain of chromosome 8q. In previous studies 8, 9, the vast majority (93-95%) of M3 tumours also displayed 8q gain, whereas in this cohort a notable proportion (13/68, 19%) of the M3 samples showed no 8q gain (category 3) (two-sided Fisher's exact test, P = 0. 04). Some D3 samples (11/35, 31%) had gain of 8q (category 2), as previously observed 8, 9. Chromosome 8p loss was only observed in samples with 8q gain (categories 2 and 4), reflecting the formation of isochromosome 8q, whereas the other alterations were spread across categories 2, 3 and 4. The majority of category 1 tumours lacked these common, large CNAs, instead displaying either few or more dispersed rearrangements. \n\nUV mutation signatures in iris tumours. Assessment of single base substitution (SBS) signatures revealed SBS5 to predominate in most cases, with strong representation of SBS3, SBS39 and SBS40 in some samples (Fig. 1 ) 13. Two samples were dominated by mutation signature SBS1 (associated with spontaneous deamination) and had correspondingly high TMB (>3 mutations per Mb). As previously observed 14, 15, these features corresponded to the presence of germline loss-of-function (LOF) MBD4 mutations; this takes the published tally of germline MBD4 mutant UM cases to six 14, 15, strengthening its role as a UM predisposition gene. The two UMs with germline LOF BAP1 mutations displayed no unique features. Strikingly, all iris melanomas displayed the genomic features associated with UVR damage (mutation signatures SBS7a, SBS7b and DBS1 13 combined with a high TMB). While exposure to UVR has been suggested as a cause of the elevated UM risk among arc-welders 16, no molecular evidence of UVR as an aetiological factor has yet been observed in UM sequencing studies. The iris is located anteriorly within the uveal tract and is directly exposed to sunlight that breaches the cornea; we now show that UVR-associated DNA damage results from this exposure and is a unique genomic feature of iris UM. \n\nPatterns of driver mutations and chromosomal aberrations. Assessment of known UM driver genes revealed an oncogenic driver mutation in 102 of 103 tumours: 51 in GNAQ (48 p. Q209P/L, two p. R183Q, one p. G48L), 46 in GNA11 (44 p. Q209L/ P, two p. R183C), five in PLCB4 (three p. D630Y, two p. D630N) and two in CYSLTR2 (p. L129Q). These mutations were generally mutually exclusive except for two PLCB4 p. D630 mutations that co-occurred with GNAQ/GNA11 p. R183H mutations. This cooccurrence between PLCB4 mutation and the minor hotspot p. R183, rather than the stronger oncogenic p. Q209 hotspot mutations, has previously been described in the UM TCGA data 8. Though not previously highlighted, GNAQ p. G48L mutations have been reported in two UM samples from two separate studies 8, 17, as well as in two hepatic small vessel neoplasms, which are driven by activating GNAQ/GNA14 mutations 18. This suggests that GNAQ p. G48L is another minor UM oncogenic hotspot mutation. Similar to previous observations 19, 20, BAP1 was the most altered gene in M3 samples (75%), including eight splice site mutations, two germline and 32 somatic LOF mutations, and three cases with disrupted BAP1 due to SV breakpoints. In addition, two D3 tumours carried BAP1 mutations, indicating that although BAP1 inactivation typically occurs after M3 8, BAP1 aberration can also occur in D3 tumours, which may or may not later undergo loss of chromosome 3. Of note, one of these D3 tumours (MELA_0800) had a low BAP1 variant allele frequency (VAF = 9/80) suggesting it was only present in a subclone, and as copy number tools are not as sensitive as mutation callers, it is possible that the subclone had loss of heterozygosity (LOH) that was not detected by the algorithm. Five tumours had BAP1 mutations and copy-neutral LOH, suggesting that the mutations occurred before WGD in the two tetraploid UMs and before the LOH event in the three diploid UMs. SF3B1 mutations were present in 15 tumours, the majority occurring in category 2, in line with other studies [7] [8] [9]. EIF1AX hotspot mutations were observed in 19% of tumours. EIF1AX mutations were first discovered in D3 UMs 21 and in the TCGA cohort they were restricted to category 1 tumours (D3 and no 8q gain) 8, while in the cohort presented by Royer-Bertrand and colleagues two of seven mutations were seen in tumours with M3 and/or 8q gain 9. Similarly, here six of the 20 EIF1AX mutations (30%) were seen in UM with M3 (n = 5) or 8q gain (n = 1) (Fig. 3a ). \n\nSignificantly mutated genes. In addition to these known UM genes, three other statistically significantly mutated genes (SMGs) were identified (CENPE, TP53, RPL5; Supplementary Table 1 ). Three missense mutations and two LOF SVs were identified in CENPE, along with one LOF germline mutation (late truncating) (collectively ~5% of UM). An additional six samples were hemizygous for CENPE (Fig. 3a ). CENPE is a plus end-directed kinetochore motor protein which plays a critical role in mitosis and chromosome segregation. Knockdown of CENPE has been shown to cause chromosome misalignment and lagging 22, 23. Two of the missense mutations (p. R14W and p. R251W) occurred in the kinesin motor domain (Fig. 3c ) at highly evolutionarily conserved regions (ECRs) and the third (p. Q1098P) occurred in a reasonably conserved residue (Supplementary Fig. 1 ). An additional two missense mutations were identified in the UM TCGA cohort at p. I1038T (weak ECR) and p. K1821N (reasonable ECR), both also in a coiled-coil domain 24. CENPE has been shown to interact with CENPF, BUB1B and Aurora B, the latter two being critical in the activation of the spindle assembly checkpoint [25] [26] [27]. In the UM cohort described here, one sample had a BUB1B missense substitution (p. R691H) within the region reported to directly interact with CENPE 25 ; another had a p. D303E substitution in a highly ECR of the Aurora B catalytic domain. It is possible that disruption of this pathway is responsible for creating genomic instability allowing for chromosome aberrations to occur. Indeed, the twelve UM with CENPE alterations had significantly higher genome percentages with CNAs (Mann-Whitney, P = 0. 028, median 23% vs 15%), though this association is confounded since tumours with high CNA generally have more genome-wide regions of LOH. Studies both in cell line and mouse models have demonstrated that CENPE/Cenpe functions in a haploinsufficient manner, with elevated levels of chromosome missegregation observed in heterozygous cells and animals 28, 29. Functional work on CENPE missense mutations is required to determine their impact in UM. \n\nTP53 is commonly disrupted in uveal melanoma. High expression of p53 has been reported in some UM, often associated with histological and clinicopathological features correlated with poor prognosis, but the potential genetic basis of these observations was not assessed [30] [31] [32]. Somatic mutations in TP53 have been described in two UM. A hotspot mutation p. R175H (n = 1216 in IARC TP53 database) was observed in a hypermutated metastatic UM with deficient MBD4 33 and another hotspot mutation p. M237I (n = 196 in IARC TP53 database) was observed in a UM in a pan-cancer study of metastatic tumours 17. Here we identified TP53 as an SMG and report six somatic TP53 mutations across four tumours in addition to eight cases of LOH (Figs. 3a, b ). One LOH case overlapped with an LOF mutation (p. C277*) resulting in a double-hit in TP53. Another double-hit was seen in a sample with two missense mutations (p. H193R and p. T155I) confirmed as occurring on different alleles by assessing read pairs spanning both mutations. To evaluate the consequence of these mutations, we applied a computational prediction tool, FATHMM 34, and assessed the results of two comprehensive characterisation studies of TP53 mutations (Table 1 ) 35, 36. p. H193R is a recurrent hotspot classified as pathogenic by PHANTM, RFS and FATHMM, while the consequence of p. T155I is more uncertain, as the variant is classified pathogenic by PHANTM but predicted to have neutral impact by RFS and b Burden of structural variation in each tumour displayed as numbr of events. Variants are coloured according to their event category. One outlier was observed with >200 events, mostly mid-sized (<100 kb) deletions. c Spectrum of single base substitution (SBS) signatures shown as percentage. Most samples were dominated by signature 5, which has been observed in most cancers. Signatures SBS7a and SBS7b, only seen in iris melanomas here, are commonly observed in cutaneous melanoma and associated with exposure to ultraviolet radiation (UVR). SBS1 was predominantly observed in melanomas with loss of MBD4 and is associated with deamination of 5-methylcytosine. Spectrum of double base substitutions shown as number of substitutions in each tumour. DBS1 is characterised by CC>TT transition and associated with exposure to UVR. \n\nFATHMM. Finally, one UM had a LOF mutation (p. R342*, COSM11073) and a missense p. R248Q mutation, both of which frequently occur in malignancies and are classified as pathogenic (Table 1 ). RNA-seq data revealed one read pair spanning both positions, which contained p. R248Q and was wildtype for p. R342; furthermore, there was a significantly lower VAF at p. R342 (4/78) than at p. R248 (25/68) (two-sided Fisher's exact test, P = 2 × 10 -6 ). These data suggest the two mutations occurred on different alleles, with the majority of the transcripts from the p. R342* allele undergoing nonsense mediated decay. TP53 is a tumour suppressor gene frequently deleted or mutated, resulting in either no production of p53 or the expression of a truncated and unstable protein. The spectrum of a few highly recurrent missense mutations, including p. R248Q, has, however, given rise to hypotheses that these hotspot mutations translate to mutant p53 with gained oncogenic functions 37. For example, the p. R248Q mutation reported here has been shown to increase the migratory potential of cells in an in vitro model 38. \n\nRPL5 is significantly mutated in uveal melanoma. SMG analysis also identified RPL5, with truncating mutations in three cases and LOH in an additional 30 cases (Fig. 3a ). No UM had a double hit in RPL5, in line with previous studies suggesting RPL5 is a haploinsufficient tumour suppressor, with heterozygous inactivation in glioblastoma (11%), breast cancer (34%) and cutaneous melanoma (28%) 39. In UM the majority of RPL5 LOH occurred through loss of chromosome 1p, where the gene is located, but in four cases, focal loss occurred, suggesting that RPL5 may drive positive selection for 1p loss. Chromosome 1p loss in UM has been reported as a marker of poor prognosis, independent of M3 12. RPL5 encodes ribosomal protein L5, which complexes with 5S rRNA and forms an important part of the impaired ribosome biogenesis checkpoint (IRBC). Together with RPL11 and ARF, RPL5 binds to and inhibits MDM2, resulting in p53 stabilisation in response to blocks in ribosome biogenesis and nucleolar stress [40] [41] [42]. Of note, one truncating mutation in RPL11 was observed in the UM TCGA cohort 8 further supporting the importance of this pathway in UM. Given the link between RPL5 and p53, we tested for an association between aberrations in RPL5 and TP53. Indeed, mutations in these genes were mutually exclusive, but when also considering copy-number loss there was no association. However, as p53 functions in multiple pathways, it may be inactivated in some tumours with defective RPL5 due to selective pressures outside the IRBC response. Interestingly, the IRBC is often triggered by oncogene-induced translational stress [40] [41] [42], with oncogenic MYC being shown to significantly increase IRBC activation [41] [42] [43] [44]. In addition to their IRBC role, RPL5 and RPL11 have also been shown to bind to MYC transcripts, mediating RNA-induced silencing and interfering with c-Myc driven transcription 45, 46. Overexpression of MYC is thought to be the driving force behind positive selection of chromosome 8q gains in UM. It is possible that c-Myc overexpression leads to ribosomal stress and IRBC activation, likely inhibiting tumour growth. To overcome this, it may be necessary to disrupt this pathway through mutation/loss of either RPL5, RPL11 or TP53. Supporting this notion, RPL5 and TP53 disruption (including mutations, SV breakpoints and chromosomal copy loss) was more common in cases with 8q gain (48%) than in cases without 8q gain (22%) (two-sided Fisher's exact test, P = 0. 01). Tumours with alterations in this pathway were predominantly M3, making it difficult to disentangle the prognostic impact; however, these individuals had poorer prognosis (Supplementary Fig. 2 ).",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To correlate UM genomic categories with prognosis, time from first presentation to metastasis was examined for all patients. Comparing the four TCGA categories, there was a trend that category 4 tumours had shorter relapse-free survival (RFS) (median: 2. 5 years) than UMs in category 3 (median: 7. 5 years) and similarly category 2 had shorter RFS (median: 7. 0 years) than category 1 (median not reached), but the differences were not statistically significant (P 3vs4 = 0. 11, P 1vs2 = 0. 28) (Fig. 4a ). However, M3 UM had significantly shorter RFS (median: 2. 9 years) than D3 UM (median 7. 0 years, log-rank test, P = 0. 001, Fig. 4b ), confirming the prognostic strength of M3/D3 status. Interestingly, while iris melanomas are associated with earlier detection and favourable prognosis, 4/8 iris cases had M3, with two already having progressed to metastatic disease. The high TMB in iris UM reported here suggests they are more likely to respond to immunotherapy, given the observations for MBD4 germline UM patients 14, 15, who have high TMB, a predictor of response to immunotherapy response in cutaneous melanoma and other cancers 47. In combination with previous reports of metastatic iris UM 48, these data suggest a subset of iris UM are at high risk for disease progression and will likely respond to immunotherapy in the event of progression and perhaps even in the adjuvant setting. DNA extractions. Tumour DNA was extracted using the AllPrep DNA/RNA Kit (80204, Qiagen Ltd, Hilden, Germany), blood DNA using standard salting out methods, and saliva DNA was collected and extracted using the Oragene DNA kit (OG-500, DNA Genotek, Ottawa, Canada) according to the manufacturer's instructions. All samples were quantified using a NanoDrop (ND1000; Thermo Fisher Scientific, Waltham, Massachusetts, USA) and Qubit dsDNA HS Assay Q32851; Life Technologies, Carlsbad, California, USA).",
"section_name": "Genomic categories correlate with prognosis.",
"section_num": null
},
{
"section_content": "Whole-genome sequencing. Sequencing libraries were constructed using TruSeq DNA Sample Preparation kits (Illumina, San Diego, California, USA) according to the manufacturer's instructions. WGS was performed on Novaseq or HiSeq X Ten instruments (Illumina) by Macrogen (Seoul, South Korea). Sequence data were adapter trimmed using Cutadapt v1. 9 49 and aligned to the GRCh37 assembly using BWA-MEM v0. 7. 12 and SAMtools v1. 8 50, 51. Duplicate reads were marked with Picard MarkDuplicates v1. 129 (https://broadinstitute. github. io/picard). Similarly, RNA sequence reads were adapter adapter trimmed using Cutadapt and aligned using STAR v2. 5. 2a to the GRCh37 assembly with the gene, transcript, and exon features of Ensembl gene model v70 52. \n\nSomatic mutations. Somatic SNV and indels were detected using an established pipeline 53 in which SNVs were called with qSNP 54 and GATK HaplotypeCaller and indels were detected with GATK 55. The contribution from different mutation signatures was inferred by approximating (minimising the squared error) the distribution of mutations as a linear combination of COSMIC signature v3 13 with the constraint that contributions were non-negative. SMGs were identified using MutSigCV 1. 3. 5 (via GenePattern) as well as Oncodrive-fm and OncodriveClust (via IntOGen) 56. A Benjamini-Hochberg adjusted p-value (q-value) below 0. 05 was considered significant. To avoid false negatives in hotspots of known UM genes, these regions (GNAQ p. 48, p. 183, and p. 209; GNA11 p. 183 and p. 209; SF3B1 codons p. 625, p. 666 and p. 700; EIF1AX codons p. 1-20, PLCB4 p630; and CYSLTR2 p. 129) were called with higher sensitivity. For each sample and genomic position the variant and reference read counts were compared with the variant and reference read counts in the pool of all 103 normal/germline samples at that specific position. Fisher's exact test was used to identify somatic mutations and a Bonferroni corrected p-value below 0. 001 was considered statically significant. \n\nClassification of TP53 mutations. To evaluate the TP53 mutations, they were compared with two comprehensive characterisation studies. The Phenotypic Annotation of TP53 Mutations (PHANTM) score v1. 0 is a weighted sum of zscores for which common (i. e. benign) germline variants have values around 0 and recurrent somatic hotspot mutations have scores around 1 35. The relative fitness score (RFS) is on average -2. 50 for synonymous variants, while the average score for protein truncating variants is 0. 42 36. FATHMM is an in silico tool predicting the probability that variants are deleterious 34. Scores above 0. 5 are considered deleterious. The IARC TP53 mutation database (R20, July 2019) was used to evaluate how frequent mutations are 57. Fig. 4 Kaplan-Meier estimates of relapse-free survival (RFS) for UM patients. Higher risk categories have shorter RFS. UM patients with monosomy of chromosome 3 has significantly shorter RFS than patients with disomy 3 (blue). Difference between curves were assessed using a two-sided log-rank (Mantel-Cox) test; p-values were not adjusted for multiple testing.",
"section_name": "Methods",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We are indebted to the patients and their families for their participation and support of this study, and the many clinicians and allied health professionals involved in their management. We gratefully acknowledge the participation of Sharon Morris, Nicholas O'Rourke, David Cavallucci Thomas O'Rourke, Helen Marfan and Rachel Susman. As well, we thank Kevin Whitehead, Gary Quagliotto, and Sullivan Nicolaides Pathology staff for processing the Queensland samples. This project was funded by the National Health and Medical Research Council (NHMRC ; 1093017 ), the Walking On Sunshine Foundation, Anne Stanton, Nicola Laws and Lloyd Owen in Memorial and Civic Solutions. This study was also funded by Fight for Sight, Denmark. A. L. P. is supported by Highland Island Enterprise ( HMS9353763 ). This work was supported by an NHMRC Program Grant (G. V. L., G. J. M., R. A. S. and N. K. H. ). G. V. L. is supported by an NHMRC Practitioner Fellowship and The University of Sydney, Medical Foundation. R. A. S. is supported by an NHMRC Practitioner Fellowship. Support from Melanoma Institute Australia and The Ainsworth Foundation is also gratefully acknowledged. J. S. W. is supported by a NHMRC early career fellowship ( 1111678 ). N. W. is supported by an NHMRC Senior Research Fellowship ( 1139071 ). N. K. H. is supported by an NHMRC Senior Principal Research Fellowship ( 1117663 ).",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Code availability",
"section_num": null
},
{
"section_content": "Tools used in this publication that were developed in-house are available from the SourceForge public code repository under the AdamaJava project ([http://sourceforge. net/projects/adamajava/]). Updated versions of software are available at [https://github. com/AdamaJava].",
"section_name": "Code availability",
"section_num": null
},
{
"section_content": "",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "Copy number aberrations and SV. Copy number aberrations were identified using ascatNgs 58. Copy number of at least 6 was considered high gain. The underlying model of ascatNgs assumes the data come from two clones of cells: the tumour and normal contamination. For a heterogeneous tumour it may therefore overestimate copy numbers; to distinguish heterogeneity from WGD, the distribution of VAF for somatic mutations within regions with allelic balance were used. For a tetraploid tumour, two peaks in VAF are expected corresponding to mutations occurring before and after the copy number gain, with the latter having half the VAF of the former. Statistical models for the data coming from a homogeneous tetraploid tumour and from a heterogeneous diploid tumour, respectively, were inferred using maximum-likelihood and if the heterogeneity was significantly more likely (P < 0. 001), copy numbers were adjusted. \n\nStructural variants were identified using an in-house tool, qSV, as previously described 53. Gene truncating breakpoints and consequence of the SVs were determined using in-house scripts and transcript annotation from Ensembl. \n\nSurvival analysis. Relapse-free curves were estimated using the Kaplan-Meier method. Difference between curves was assessed with the log-rank (Mantel-Cox) test. \n\nReporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.",
"section_name": "",
"section_num": ""
},
{
"section_content": "The BAM files are deposited in the European Genome-phenome Archive ([https://www. ebi. ac. uk/ega/]) with accession number EGAS00001001552. The source data underlying Fig. 2 are provided as a data source file. All other data are available in the Article, Supplementary Information or available from the author upon reasonable request.",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "P. A. J. and K. B. were responsible for data interpretation and writing the manuscript. N. K. H., R. A. S. and G. J. M. conceptualised the study. P. A. J., F. N., C. L., S. W., N. B., L. T. K., J. V. P. and N. W. performed data analysis. K. B. N. B., V. N., C. W. S., R. D. and A. B. B. were responsible for sample processing and extraction. R. A. S., N. K. H., R. A. S., G. V. L., H. R., E. G. acted in a supervisory capacity. A. L. P., J. M. P., J. S. W., M. S. C., M. H., H. R., G. V. L., H. H., J. J. P., O. J. R., J. F. K., T. I., N. vB., K. W. W., L. A. M., A. S., S. K. W., W. G. were responsible for patient data curation, recruitment and tumour acquisition. All authors reviewed and edited the manuscript.",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "J. V. P. and N. W. are founders and shareholders of genomiQa Pty Ltd, and members of its Board. K. W. W. participated in one Advisory board meeting for MSD and AstraZeneca. R. A. S. receives fees for professional services from Merck Sharp & Dohme, Glax-oSmithKline Australia, Bristol-Myers Squibb, Dermpedia, Novartis Pharmaceuticals Australia Pty Ltd, Myriad, NeraCare and Amgen. G. V. L. is consulant advisor for Aduro, Amgen, Array, BMS, MERCK MSD, Novartis, Pierre-Fabre, Roche. None of these relationships involve the work described in this manuscript. The remaining authors declare no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Supplementary information is available for this paper at https://doi. org/10. 1038/s41467-020-16276-8. \n\nCorrespondence and requests for materials should be addressed to N. K. H. \n\nPeer review information Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. \n\nReprints and permission information is available at http://www. nature. com/reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Additional information",
"section_num": null
}
] |
10.1007/s12471-015-0649-x
|
Bicuspid aortic valve; optimal diagnosis and latest interventional treatment
|
Bicuspid aortic valve (BAV) is one of the most common congenital heart defects with a population prevalence of 0.5 to 1.3 % [1]. The defect is considered to be a heritable disorder with a family recurrence rate of approximately 35 %. Recent studies show that mutations in the NOTCH1 gene are associated with BAV [2]. BAV progresses more rapidly into regurgitation or stenosis of the valve [3]. This results in a higher occurrence of aortic valve replacement, especially at younger age. Additionally, BAV is more susceptible than a tricuspid aortic valve (TAV) to nest bacteria or other organisms, leading to endocarditis. BAV is not only a peculiar valve morphology leading to specific valve pathology, it is also frequently associated with (asymptomatic) ascending aorta dilatation which leads to an increased susceptibility to ascending aortic aneurysms and aortic dissection [4]. Aortic elasticity measurements of BAV patients suggest that diminished aortic elasticity is at least part of its causation.
|
[
{
"section_content": "in BAV patients more frequently than echocardiography (96 versus 73 %); CMR appeared to be more sensitive for detecting of BAV whereas echocardiography appeared to be more specific. Among unselected patients with severe aortic valve stenosis, a high percentage of patients with BAV was found (40 %). Patients with BAV are significantly younger and more frequently male. Typically, the ascending aorta was larger in patients with BAV. From a cost-effectiveness perspective, echocardiography will still be the first choice in BAV patients. When the echocardiograms are difficult to analyse or when in doubt, CMR can be useful to come to a diagnosis. In a recent study, CMR was found to be superior to transthoracic echocardiography for imaging of the aorta in patients with congenital aortic stenosis and BAV, especially at the level of the proximal ascending aorta when an aortic aneurysm is present [6]. In particular, when the ascending aorta appears large at echocardiography, it is important to evaluate progression of the aortic diameters with CMR as standard care [7, 8]. At present, multi-slice computed tomography (MSCT) is increasingly used for sizing TAV and BAV through noninvasive evaluation of the aortic root [9] [10] [11]. However, the true gold standard for assessing these stenotic valves appears to be the appraisal of the surgeon.",
"section_name": "",
"section_num": ""
},
{
"section_content": "Currently, BAV stenosis and/or regurgitation is the most common indication for surgical aortic valve replacement in patients < 70 years of age. Nonetheless, 20 % of patients > 80 years of age have underlying bicuspid pathology. Over the past 10 years, transcatheter aortic valve replacement (TAVR) has become a standard procedure in elderly patients with severe inoperable aortic stenosis [12]. Recently, a study by Mylotte et al., published in JACC [13], evaluated Bicuspid aortic valve (BAV) is one of the most common congenital heart defects with a population prevalence of 0. 5 to 1. 3 % [1]. The defect is considered to be a heritable disorder with a family recurrence rate of approximately 35 %. Recent studies show that mutations in the NOTCH1 gene are associated with BAV [2]. BAV progresses more rapidly into regurgitation or stenosis of the valve [3]. This results in a higher occurrence of aortic valve replacement, especially at younger age. Additionally, BAV is more susceptible than a tricuspid aortic valve (TAV) to nest bacteria or other organisms, leading to endocarditis. BAV is not only a peculiar valve morphology leading to specific valve pathology, it is also frequently associated with (asymptomatic) ascending aorta dilatation which leads to an increased susceptibility to ascending aortic aneurysms and aortic dissection [4]. Aortic elasticity measurements of BAV patients suggest that diminished aortic elasticity is at least part of its causation.",
"section_name": "Latest interventional treatment",
"section_num": null
},
{
"section_content": "Patients with BAV frequently remain undiagnosed until the manifestation of symptoms. Therefore, early screening and detection of patients is warranted. Imaging of a severely stenotic aortic valve is challenging. Due to the severity of stenosis and calcified nature of the aortic valves, echocardiography is frequently unable to differentiate between TAV and BAV. In a previous study published in the Netherlands Heart Journal in 2011 [5], cardiovascular magnetic resonance (CMR) was able to assess aortic valve morphology E. E. van der Wall, MD () Holland Heart House/Netherlands Society of Cardiology, Moreelsepark 1, 3511 EP Utrecht, The Netherlands e-mail: eevanderwall@hotmail. com 1 3\n\nNeth Heart J (2015) 23:149-150 the clinical value of TAVR in 139 BAV patients (mean age 78. 0 ± 8. 9 years) from 12 centres in Europe and Canada, being the largest collection of BAV patients treated with TAVR. Evaluation of the morphology of the aortic valve was performed using transoesophageal echocardiography in all patients; MSCT-based TAV sizing was used in 63. 5 % of patients. Thirty-day device safety, success, and efficacy were noted in 79. 1, 89. 9, and 84. 9 % of patients, respectively. There was a 30-day mortality rate of 5 %, a 30-day stoke rate of 2 %, and a device success rate of 90 %. One-year mortality was 17. 5 %, and the patients were New York Hear Association functional class I or II. It was concluded that TAV-in-BAV is feasible with encouraging shortand intermediate-term clinical outcomes. However, a high incidence of post-implantation aortic regurgitation was observed of 28 %, which appears to be mitigated by MSCTbased TAV sizing (17 %). Since MSCT-based TAV sizing was clearly associated with reduced para-valvular regurgitation, MSCT should be considered a mandatory element of patient screening for TAV-in-BAV, certainly in view of the suboptimal echocardiographic results. In an accompanying Editorial by Colombo and Latib [14], it was stated that the incidence of significant aortic regurgitation, even with full MSCT evaluation, is still too high to extend TAVR to BAV unless the patient is truly inoperable or has an unacceptably high surgical risk. On the other hand, the Editorial reports that the current study sets a benchmark for next-generation TAVR devices demonstrating the feasibility of TAV-in-BAV. \n\nTo summarise, to diagnose patients with BAV, echocardiography remains the first choice. However, when the echocardiograms are difficult to analyse or for careful evaluation of the progression of aortic diameters, CMR is very useful to come to a diagnosis. MSCT is increasingly being used to accurately size the aortic root diameters. The recent study by Mylotte et al. [12] is the first large multicentre analysis of TAV implantation in patients with significant BAV stenosis or regurgitation. TAV-in-BAV proved to be feasible with encouraging short-and intermediate-term clinical outcomes, but the relatively high incidence (28 %) of post-implantation aortic regurgitation is of serious concern. Therefore, longer-term follow-up of a larger cohort of patients is required to more completely assess the efficacy and durability of TAV implantation in patients with bicuspid disease. \n\nOpen Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.",
"section_name": "Optimal diagnosis",
"section_num": null
}
] |
[] |
10.12669/pjms.36.3.541
|
Prognostic markers in Chronic Lymphocytic Leukaemia - A flow cytometric analysis
|
<jats:p>Objective: To find out the frequency of ZAP-70, CD38 and CD49d in patients diagnosed with CLL in our population.
 Methods: This is a cross sectional study conducted in Army Medical College in collaboration with Armed Forces Institute of Pathology and Military Hospital Rawalpindi from 1st January 2018 to 30th November 2018. Permission from Institutional Ethical Committee was obtained. Blood samples were collected by non-probability consecutive sampling technique and analyzed for blood counts and flow cytometry was done for ZAP-70, CD38 and CD49d. Manufacturer’s instructions for the kits were strictly followed.
 Results: Fifty-one newly diagnosed patients with CLL were studied for the prognostic markers in CLL. CD 38 was expressed in 25(49%) and CD49d in 21(41.2%). ZAP-70 expression was not detected in our series of patients.
 Conclusion: We conclude that CD38 and CD49d expression was detected in almost half of the patients of CLL in our series. CD49d showed statistically positive correlation with CD38, showing that it is a more pragmatic choice for reliable prognostication of CLL along with CD38.
 doi: https://doi.org/10.12669/pjms.36.3.541
 How to cite this:Haq H, Uddin N, Khan SA, Sunia Ghaffar4. Prognostic markers in Chronic Lymphocytic Leukaemia - A flow cytometric analysis. Pak J Med Sci. 2020;36(3):338-343. doi: https://doi.org/10.12669/pjms.36.3.541
 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</jats:p>
|
[
{
"section_content": "Chronic Lymphocytic Leukemia (CLL) is characterized by accumulation of small mature looking, ineffectual, CD5+ B-cells in the peripheral blood, bone marrow and secondary lymphoid tissues. 1 These cells have a characteristic immunophenotype i. e. CD19+, CD20+ and CD23+ with relatively low expression of CD22 and CD79b. 2 The peak incidence of CLL is between 60 to 80 years, and only 10% of patients are younger than 55 years of age. In Pakistan it accounts for 20. 1% of all leukemias. 3 It is more common in the West with an incidence of 4. 2:100,000 per year. Most cases are diagnosed during routine complete blood counts done for other reasons. 4 iginal Article",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Leukaemia -A flow cytometric analysis\n\nIts diagnosis is based upon clinical presentation and laboratory features. 5 Peripheral blood counts with absolute lymphocyte count more than 5 x 10 9 /L, which may reach to 300 x 10 9 /L. The peripheral smear shows small mature looking lymphocytes with numerous smudge cells. A bone marrow biopsy, if done, shows predominance of mature lymphocytes, replacing 95% of normal hemopoietic tissue. Immunophenotyping is done to confirm the diagnosis. 6 he Rai and Binet staging systems are well recognized systems which serve as standard for assessing treatment requirements and overall survival in the patients diagnosed with CLL. However, both these systems are unable to categorize the patients into groups requiring treatment from those in whom the disease remains indolent. 7 The outcome of patients can be predicted by a number of immunophenotypic markers (ZAP-70, CD38 and CD49d), Immunoglobin heavy variable (IGHV) gene mutation and chemical analysis. 8 AP-70 and CD38 are well-established immunophenotypic markers indicating poor prognosis in CLL. ZAP-70 is a protein rarely present on normal B-lymphocytes whereas CD38 is expressed on them. Patients who are positive for ZAP-70 and CD38 have a poor prognosis with aggressive disease course and a shorter overall survival. On the other hand those who are negative for ZAP-70 and CD38 have much better results. 7, 9 D49d is a recently added prognostic marker. It belongs to the integrin family with an important function in leukocyte activation, trafficking and survival. 10 Its expression promotes unfavorable progressive disease course and can identify patients with poor disease outcome, independent of CD38 and ZAP-70. 8 Studies show that CD49d positive patients have increased risk of death and lower overall survival, independent of ZAP-70 and CD38. Comparing models of these three prognostic markers, with and without CD49d by several prediction performance measures indicated that excluding CD49d significantly reduced the prognostic power of the model. 11 nother study compared the combined expression of CD38 and CD49d. They showed that all those patients who require treatment had strong double expression of both. On the contrary, those with double negative expression had good prognosis and long treatment free survival. 12 Patients with > 30% of CLL B-cell expressing CD49d are labeled positive. 11 Till to date no study has been done in Pakistan to study presence and frequency of CD49d in Pakistani patients with CLL. \n\nThis study helps us to evaluate the prognosis of CLL at the time of diagnosis in our population. ZAP-70, CD38 and CD49d are prognostic markers of CLL. They help in segregating those patients of CLL which will need treatment from those that can be placed in the \"Wait and watch\" group. Globally, studies show that CD49d is gaining acceptance as an independent prognostic marker and may replace the combination of ZAP-70 and CD38. Foregoing in view, we planned a study to find out the frequency of ZAP-70, CD38 and CD49d in CLL patients our setup.",
"section_name": "Prognostic markers in Chronic Lymphocytic",
"section_num": null
},
{
"section_content": "This cross sectional study was conducted in Department of Hematology, Army Medical College in collaboration with Armed Forces Institute of Pathology Rawalpindi from 1 st January, 2018 to 31 st October, 2018 after the approval of Institutional Review Board dated on January 24, 2019. \n\nTotal 51 newly diagnosed cases of CLL were included in our study. Sample size (n=44) was calculated by WHO calculator (Confidence interval at 95% and Anticipated population as 13% with absolute precision required as 5%). Sample collection was done by non-probability purposive sampling. \n\nThree (3) ml of venous blood was drawn under aseptic conditions. It was transferred to Ethylenediaminetetraacetic acid (EDTA) tube. Complete blood counts were generated through Sysmex KX-21TM automated hematology analyzer after adequate quality control. Immunophenotyping was performed by flow cytometry to analyze for ZAP-70, CD38 and CD49d by BD FACS caliber and BD FACS CANTO. Known negative samples were used as normal controls. Manufacturer's instructions for the kits used were strictly followed. Statistical analysis: Data was analyzed by statistical package for social sciences (SPSS 23). For qualitative variables frequency and percentages were calculated and quantitative variables Mean and Standard Deviation (SD) were calculated. Correlation between CD38 and CD49d was calculated by applying the Pearson Chi-Square. P-value <0. 05 was considered statistically significant.",
"section_name": "METHODS",
"section_num": null
},
{
"section_content": "Our study included a total of 51 newly diagnosed cases of CLL. Out of these, 40 (78%) were male and 11 (21. 5%) were females. The mean age of the patients was 65 years± 10. 64 (mean ± SD) years with a range of 39 -86 years. The mean hemoglobin, white cell and platelets counts were 11. 1 ± 2. 7 g/dl (range 3. 6 -16. 1); 80. 8 ± 74. 2 x 10 9 /L (range 11. 7 -538x10 9 /L) and 169 ± 67 x 10 9 /L(range 08 -383x10 9 /L) respectively. The mean absolute lymphocyte count was 68. 2 ± 71. 6 x 10 9 / L (range 7. 9 -505. 7 x 10 9 /L) (Table-I). \n\nCD38 expression was detected in 25 (49%) cases with the mean value of 34. 4% (range 0-96) while CD49d was expressed in 21 (41. 2%) cases with a mean of 35. 4 (range 0-99). ZAP-70 expression was not detected in any case (Fig. 1 ). The mean of positive expression of CD38 was 70. 9 ± 18. 2 with range of 42%-96% and CD49d was 76. 2 ± 16. 2 with a range of 42%-99% (Table-II). \n\nWe calculated the correlation between CD38 and CD49d by applying the Pearson Chi-Square. The p-value was <0. 05 showing that the two variables are statistically significant (Fig. 2 ). We divided our data into two groups i. e. Group-1 (below 60 years of age) and Group-2 (61 and above). In Group-1, 3/13 cases were positive for CD49d and CD38. In Group-2, 18/38 cases were positive for both CD38 and CD49d. No positive association was found between CD markers and age group (p-value >0. 05).",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "Chronic lymphocytic leukemia is the most frequently occurring chronic leukemia in the Western world. 7 The progression and response to treatment is variable. Previously, prognosis of patients with CLL was based on the clinical features alone. However with the advent of new techniques, significant progress has been seen like identification of immunophenotypic markers and molecular genetics which help us to predict the progression of the disease. 13, 14 e studied 51 newly diagnosed patients of CLL. Among these, 25 (49%) were positive for CD38. Our study is consistent with other studies conducted in China, India and Iraq showing almost similar results i. e. 6] [17] However, our values are slightly higher than those reported by Wiestner A et al. in UK and D'Arena G et al. in Italy which were 30% and 29% respectively. 18, 19 D49d was positive in 21 (41. 2%) cases which is comparable to results by Bulian P et al. in Italy, Uzay A et al in Turkey and Gattei V et al. in Italy who reported CD49d positivity in 52%, 47% and 39% of cases respectively. 11, 20, 21 Our results were slightly lower than study done by Al-Rubaie HA et al. 15 in Iraq who showed positivity for CD49d (60%). This might be explained by the smaller sample size they studied (n=30). We could not find any similar studies conducted in our region with which our statistics could be compared. So we believe that this is the first study of this kind in Pakistan. \n\nThe expression of ZAP-70 was not recorded in our cases. A study conducted by Zeeshan R et al. showed ZAP-70 expression in only 13% cases. 7 n the contrary, an Indian study revealed that expression of ZAP-70 by flow cytometry was weak in a vast majority of cases (n=60) with small shifts above the baseline threshold thus, was not a robust assay. This may explain why ZAP-70 could not be detected in any of our studied Prognostic markers in Chronic Lymphocytic Leukaemia cases. And hence low frequency in South Asian population. 22 here was a significant correlation between CD38 and CD49d (p-value <0. 05). No correlation was found between ZAP-70 and either of the other two markers (CD38 & CD49d). We divided our data into two groups i. e. Group-1 (below 60 years of age) and Group-2 (60 and above). No association was found between the immunophenotypic markers and age. \n\nIn an analysis of 3000 patients done in Italy for flow cytometric based prediction of overall survival (OS) in CLL, CD49d+ had a significantly high risk of death and lower poor survival rate as compared to CD49d-patients. By a Cox analysis for OS, they showed that CD49d+ patients have a two-fold increase risk of death and it was the only flow cytometry based marker with independent prognostic relevance for OS. They also compared models for prognostic markers with and without CD49d by several prediction performance measures indicating that excluding CD49d significantly reduced the prognostic power of the model. 11 n 2017, a study conducted by Ahmed S et al. 12 compared the expression of CD38 and CD49d in patients with CLL. They concluded that patients with strong double expression of CD38 and CD49d required treatment. Whereas, patients with negative expression of CD38/CD49d had a good prognosis and long treatment free survival.",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "The limitation in our study was that only a small number of patients were studied. It is strongly recommended that all the three prognostic markers i. e. CD38, ZAP-70 and CD49d are studied in all the newly diagnosed patients of CLL. As the study was single centered, that's why it's difficult to extrapolate it to whole Pakistani population. Therefore, it is highly recommended that these immunophenotypic markers should be studied on a large cohort of patients in centers which extend diagnostic and therapeutic facilities to patients of Chronic Lymphocytic Leukemia in Pakistan. Thus, this pilot study can form a foundation for larger studies on the subject.",
"section_name": "Limitation of the study:",
"section_num": null
},
{
"section_content": "We conclude that assessing prognosis of CLL at the time of diagnosis is essential for all patients to segregate patients into groups who need urgent treatment because of presence of adverse prognostic markers from those who can be placed in watch and wait group. CD38 and CD49d expression was detected in almost half of the patients and were significantly correlated. CD49 is gaining acceptance internationally as an independent prognostic marker and is more reliable for prognostication of CLL along with CD38.",
"section_name": "CONCLUSION",
"section_num": null
},
{
"section_content": "We recommend that along with the conventionally used biomarkers i. e. ZAP-70 and CD38, CD49d should also be added to the immunophenotyping panel for stratification of prognostic groups in CLL. Further studies should also be done on larger groups of patients to evaluate the frequency of these biomarkers in our population and re-evaluation should be done for the inclusion of ZAP-70 in the immunophenotyping panel.",
"section_name": "Recommendation:",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Conflict of interest:",
"section_num": null
},
{
"section_content": "There is no conflict of interest among the authors.",
"section_name": "Conflict of interest:",
"section_num": null
},
{
"section_content": "",
"section_name": "Source of funding: Higher Education Committee.",
"section_num": null
},
{
"section_content": "HH conceived, designed and did statistical analysis & manuscript writing. NUD did overall supervision, proof reading and final drafting. SAK did the conceptualization of the study and critical revision of article. HH, SG did data collection and statistical analysis. HH takes the responsibility and is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.",
"section_name": "Authors' Contribution:",
"section_num": null
}
] |
10.1186/1742-6405-11-38
|
Expression of activating receptors on natural killer cells from AIDS-related lymphoma patients
|
Abnormal NK phenotype and cytotoxic functions have been described in acute myeloid leukemia, chronic lymphocytic leukemia, myeloma and myelodysplastic syndromes. Defective NK cytotoxicity is due to decreased expression of the Natural Cytotoxicity Receptors (NCRs), 2B4/CD244/p38, or NKG2D. This prompted us to test the expression of these molecules on circulating NK cells from patients with AIDS-related lymphomas (RL) in comparison with HIV + patients without lymphoma, healthy subjects and HIV-negative patients with lymphoma.Blood samples were analyzed by flow cytometry for NCRs, 2B4/CD244/p38 and NKG2D expression on NK cells defined as CD3-/CD56+ lymphocytes. We also analyzed by quantitative PCR specific RNA for NKp30/NCR3 and NKp46/NCR1.We could not detect any defect in NKp46/NCR1 expression between all groups. NKp44/NCR2, NKp30/NCR3 and NKG2D had lower expression in AIDS-RL in comparison with HIV + patients without lymphoma when compared to patients with similar (>0.3 G/L) CD4+ lymphocyte levels. Expression of 2B4/CD244/p38 was lower in AIDS-RL than in HIV-negative lymphoma. Comparison of specific NKp30/NCR3 and NKp46/NCR1 RNA showed increased steady state levels, despite decreased surface expression for NKp30/NCR3, suggesting abnormal post-transcriptional regulatory mechanisms.We show a more pronounced defect in NK activating molecule when HIV infection is associated with lymphoma than when only one condition (HIV positivity or lymphoma) is present. Defective NK phenotype, in addition to CD4+ depletion and dysfunction, may participate to the increased incidence of lymphoma in HIV patients.
|
[
{
"section_content": "Advances in lymphoma treatment prolong progressionfree survival. Nonetheless, many patients relapse. Deficient cytotoxic functions of natural killer (NK) cells [1], which can be infected by HIV [2], may participate in the failure to cure AIDS-related lymphomas (AIDS-RL). Engagement of inhibitory receptors by human leukocyte antigen (HLA)-class-I molecules inhibits NK cytotoxicity. Thus, according to the \"missing self hypothesis\", absent or deficient expression of HLA-class-I molecule activates NK if an additional activating signal is delivered by the natural cytotoxicity receptors (NCR) NKp30/ NCR3, NKp44/NCR2 or NKp46/NCR1, 2B4/CD244/p38 and NKG2D. Deficient NK functions interfere with the anti-tumor response: 1) during treatment, via decreased efficiency of anti-CD20 antibody-driven cell cytotoxicity (ADCC) [3] [4] [5] 2) during the complete remission phase by favoring residual HLA-class-I negative lymphoma cells to escape from NK-mediated immunity [6]. Abnormal NK functions have been described in hematological malignancies such as acute myeloid leukemia, chronic lymphocytic leukemia, myeloma and myelodysplastic syndromes [7] [8] [9] [10]. Of note, down-regulation of NCRs is associated with HIV infection [11]. We compared the NK cell surface activating molecules expression between patients with AIDS-RL, HIV-positive patients without lymphoma, lymphoma patients not infected by the HIV, and healthy subjects.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Among the 31 AIDS-RL (mean age: 43 ± 8 years) of the study, 20 had CD4+ lymphocytes <300/mm 3 (mean 133 ± 70/mm 3 ), 11 had CD4+ lymphocytes >300/mm 3 (mean 630 ± 260/mm 3 ). Only 2 patients (6. 4%) were not treated by highly active antiretroviral therapy (HAART) at study inclusion. The control cohort included 56 HIV-positive patients without lymphoma (mean age: 44 ± 9 years) selected to be matched for the CD4+ lymphocyte count with the AIDS-RL: among this population, 9 patients [16%] were not treated by HAART. Two groups were designed: HIV patients with <300 CD4+ lymphocytes/mm 3 (n = 12; mean age 44 ± 8. 4 years, 1 patient without HAART) and HIV patients with >300 CD4+ lymphocytes/mm 3 (n = 44; mean age 44 ± 9. 8 years patients without HAART). Two other control cohorts of 33 HIV-negative lymphoma patients (mean age: 62 ± 14 years) and 19 healthy subjects (HS, mean age: 41 ± 16 years) were included.",
"section_name": "Population characteristics",
"section_num": null
},
{
"section_content": "There was no significant difference in total lymphocytes and T-lymphocytes count among the different groups (p > 0. 05). Lymphoma groups had more B lymphocytes (1700 ± 7000/mm 3 ) than HIV (200 ± 200/mm 3 ) and HS (300 ± 100/mm 3 ) because of lymphoma circulating cells. Total NK cells counts in AIDS-RL with <300 CD4+ lymphocytes/mm 3 and HIV + patients without lymphoma but with <300 CD4+ lymphocytes/mm 3 were lower than in the other groups (mean 41 ± 35/mm 3 for AIDS-RL <300 CD4+ lymphocytes/mm 3 and 51 ± 52/mm 3 for HIV + without lymphoma with <300 CD4+ lymphocytes/mm 3 vs 137 ± 257/mm 3 for AIDS-RL with >300 CD4+ lymphocytes/mm 3, 131 ± 148/mm 3 for HIV + with >300 CD4+ lymphocytes/mm 3, 276 ± 510/mm 3 for HIV-negative lymphoma patients and 150 ± 70/mm 3 for HS (p = 0. 04 and 0. 02). No difference in NKp46/NCR1 expression was observed (Figure 1, panel B, p > 0. 05, NS). The analysis of NKp44/ NCR2 expression, an activating receptor only expressed on activated NKs (Figure 1, panel C) showed no difference between AIDS-RL and HIV-negative lymphoma patients (p > 0. 05, NS) and between AIDS-RL and HIV patients without lymphoma but with <300 CD4/mm 3 patients (p > 0. 05, NS). However, AIDS-RL with >300 CD4+ lymphocytes/mm 3 expressed lower NKp44/NCR2 than HIV + patients without lymphoma with >300 CD4+ lymphocytes/mm 3 (MFIr median (M) = 1. 52; interquartile range (IQR)[1. 24-1. 850] vs M = 1. 80 IQR[1. 540-2. 110], p = 0. 011) and than HS (M = 1. 75 IQR[1. 650-2. 000], p = 0. 006). In spite of a very high NKp44/NCR2 expression by one HIV + lymphoma with <300 CD4+ lymphocytes/mm 3 (MFIr =6. 21), this group tended to express lower NKp44/NCR2 than HIV + patients with >300 CD4+ lymphocytes/mm 3 (M = 1. 42 IQR[1. 320-1. 765] vs 1. 80[1. 543-2. 115], p = 0. 06). HIV-negative lymphoma patients expressed more NKp44/NCR2 than HIV + patients without lymphoma and >300 CD4+ lymphocytes/ mm 3 (M = 1. 48 IQR[1. 330-1. 690] vs 1. 80[1. 543-2. 115], p = 0. 02) or HS (M = 1. 75[1. 650-2. 000], p = 0. 002). There was no difference in NKp30/NCR3 expression (Figure 1, panel D) between AIDS-RL and HIV-negative lymphoma patients (p > 0. 05, NS) or between AIDS-RL and HIV + patients without lymphoma and <300 CD4+ lymphocytes/mm 3 (p > 0. 05, NS). NKp30/NCR3 expression was lower in AIDS-RL with >300 CD4+ lymphocytes/mm 3 than in HIV + patients without lymphoma and >300 CD4+ lymphocytes/mm 3 Regarding NKG2D (Figure 1, panel E), AIDS-RL patients with <300 CD4+ lymphocytes/mm 3 had a lower expression of NKG2D than AIDS-RL patients with >300 CD4+ lymphocytes/mm 3 We failed to detect 2B4/CD244/P38 expression difference (Figure 1, panel F) between AIDS-RL and HIV + patients without lymphoma (NS, p > 0. 05). 2B4/CD244/P38 expression was lower in AIDS-RL with <300 CD4+ lymphocytes/ mm 3 Quantitative RT-PCR of NKp30/NCR3 and NKp46/NCR1 (Figure 2 )",
"section_name": "Lymphoid cells repartition (Figure 1-Panel A)",
"section_num": null
},
{
"section_content": "In line with cell surface expression, AIDS-RL had no modification of NKp46/NCR1 specific RNA level (0. 5 < normalized ratio <2 for the 6 of the 7 patients analyzed) compared with HIV + population without lymphoma (Panel A). We did not find any correlation between NKp46/NCR1 expression and CD4+ lymphocyte count. On the contrary, NKp30/NCR3 RNA (Panel B) was overexpressed in AIDS-RL patients (normalized ratio >2 for the 7 patients analyzed). No significant RNA level was detected for NKp44/NCR2, a results in line with normal physiology since this molecule is only expressed by stimulated NKs (data not shown).",
"section_name": "NK activating receptors expression (",
"section_num": null
},
{
"section_content": "The total NK cells count in patients with <300 CD4+ lymphocytes/mm 3 was lower in AIDS-RL in comparison with the other groups, suggesting a poor prognosis as shown in low or high grade HIV-negative lymphomas [12, 13]. Low circulating counts concerned the NK CD56 bright and CD56 low subsets, while the ineffective CD56 negative subpopulation was elevated in AIDS-RL (data not shown). Regarding the NCR we found no difference in NKp46/NCR1 expression in the different groups but, in contrast, a significant decrease in both NKp44/NCR2 and NKp30/NCR3 was observed in AIDS-RL with >300 CD4/mm 3 in comparison with HIV + patients with comparable CD4+ lymphocytes. Thus in moderately immune-suppressed patients the development of lymphoma is associated with low expression of two activating molecules. Regarding NKp44/NCR2, low levels should be of good prognosis since NK cells in HIV-controller patients do not up-regulate NKp44/ NCR2 thus protecting uninfected CD4+ lymphocytes from inadequate NK killing [14]. Regarding the mechanism of NCR regulation, quantitative RT-PCR measured comparable NKp46/NCR1 levels in AIDS-RL and controls, suggesting an identical regulation at both transcriptional and post-transcriptional levels. However elevated level of NKp30/NCR3 specific RNA was detected in AIDS-RL in comparison with HIV patients without lymphoma, despite identical surface expression of NKp30/ NCR3. This suggests that a post-transcriptional mechanism negatively interferes with NKp30/NCR3 RNA traduction or protein stability, leading to identical surface expression despite higher specific RNA levels. Regarding NKG2D, we observed a gradient of expression from lower level (AIDS-RL <300 CD4/mm 3 ) to HS/HIV-negative lymphoma patients, with intermediary levels for AIDS-RL with >0. 3 G/L CD4+ lymphocytes followed by HIV + patients without lymphoma. The NKG2D ligands MICA/ MICB/ULBP are stress molecules expressed on tumor cells, and secreted at high levels in HIV patients, leading to down-regulated NKG2D expression on NK and impaired anti-lymphoma cytotoxicity [15]. Expression of 2B4/CD244/p38 was lower in AIDS-RL than in HIVnegative lymphoma patients. The 2B4/CD244/p38 ligand is the CD48 molecule [16, 17] which is expressed on B normal and neoplastic lymphocyte and is drastically up-regulated by Ebstein-Barr virus (EBV) infection. The (See figure on previous page. ) Figure 1 Flow cytometry analysis of whole PBMC population (panel A) and of NK cells (panels B to F). Results are expressed as absolute numbers of cells per volume unit, i. e. Giga/Liter in panel A. Results are expressed as mean fluorescence intensity ratio (in comparison with isotype controls, cf. Material and Methods). When significant, statistical results are indicated with the corresponding p-value. The number of analyzed patients was: AIDS-RL/CD4 < 300/mm3 = 20, AIDS-RL/CD4 > 300/mm3 = 11, HIV + <300 CD4/mm3 = 12, HIV+ > 400 CD4/mm3 = 44, non AIDS-RL lymphoma = 33, control HS = 19. \n\ndown-regulation of 2B4/CD244/p38 could thus impair the cytotoxicity against EBV-positive B-cell lymphomas.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "The AIDS-RL patients had decreased levels of 2 out of 3 NCRs, of NKG2D and of 2B4/CD244/p38. The most significant difference concerned NKG2D, which expression was significantly decreased regarding both HIV patients without lymphoma, non-HIV lymphoma patients and HS. This specific abnormality is of great interest since lymphoma cells express the stress ligands MICA/B and ULBP, but may escape to NK cytotoxicity due to impaired NKG2D expression. Of note, HAART was not sufficient to restore a normal phenotype since most of our patients were already treated at the time of NK phenotype analysis, with NK abnormalities also detected in patient with CD4+ lymphocytes >300/mm 3. Since defects in NK immune surveillance may also impair the anti-infectious immunity, they could also partly explain the susceptibility to infection of HIV patients during chemotherapy, even in patients with high CD4+ T-lymphocytes levels. Altogether our data suggest than immune intervention aiming at NK cell function restoration could be of interest in AIDS-RL patients.",
"section_name": "Conclusion",
"section_num": null
},
{
"section_content": "",
"section_name": "Material and methods",
"section_num": null
},
{
"section_content": "According to previous data [8], we hypothesized that 75% ±10% of AIDS-RL patients and 15% ±10% of HIV patients without lymphoma had low NCR expression (NCR dull ). In order to show a statistically significant difference between the 2 groups with a risk α = 5% and β = 75%, we included in our study 31 AIDS-RL patients and 56 HIV positive patients without lymphoma.",
"section_name": "Study design",
"section_num": null
},
{
"section_content": "From July 2006 to June 2011 patients from Marseille, Nice and Paris were included in first line of therapy. Inclusion criteria were the co-existence of HIV infection with biopsy-proven lymphoma. The 56 HIV positive patients without lymphoma were recruited from Service des Maladies Infectieuses (Hôpital Nord, Marseille). According to Helsinki declaration, patients were informed and signed a consent form. Biological samples were collected at diagnosis time, before lymphoma treatment. Additional comparison of our data was also performed with 33 non-HIV patients with lymphoma and 19 healthy subjects (HS). \n\nThis study was approved by the Comité de Protection des Personnes (CPP) Aix-Marseille II.",
"section_name": "Patients",
"section_num": null
},
{
"section_content": "Blood samples were collected on EDTA and analyzed by flow cytometry. Dry pellets of PBMC were frozen at -80°C for subsequent quantitative RT-PCR analysis. \n\nqRT-PCR analysis qRT-PCR analysis concerned 7 AIDS-RL patients and 32 HIV-positive patients without lymphoma. qRT-PCR analysis was performed with the Applied Biosystems 7900HT Fast Real-Time PCR system using Taqman detection. Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies). Capture of fluorescence was recorded on the ABI Prism 7900HT scanner and the Ct (threshold cycle) was calculated for each assay (Sequence Detection System Software 2. 3, Applied Biosystems). We used GAPDH as endogenous control (ΔCt = Ct target gene -Ct GAPDH). GAPDH TaqMan Gene Expression assays were from Applied Biosystems. Since the NCR expression is almost exclusively restricted to NK, the PCR was performed on the whole PBMC population, but the values were adjusted to the percentage of NK present in each sample. We compared ΔCt with the mean of VIH ΔCt using a ratio (ΔCt HIV + Lymphoma/ΔCt HIV), considering that a ratio >2 corresponded to RNA overexpression.",
"section_name": "Blood samples and cell separation",
"section_num": null
},
{
"section_content": "Flow cytometry was performed on an Epics XL R flow cytometer (Beckman Coulter). The NK cells were defined as CD3-/CD56+/CD16+. The following mAbs were used (Beckman-Coulter, Marseille, France): anti-CD3 FITC (UCHT1), anti-CD56 PC5 (N901-NKH1), anti-NCR1/ NKp46 PE (BAB281), anti-NCR2/ NKp44PE (Z231), anti-NCR3/NKp30 PE (Z25), anti-NKG2D-PE (ON72), anti-P38 (C1. 7), anti-IgG1 -FITC, anti-CD19 -PC5 (J4. 119), CD4 -PE / CD8 -ECD /CD3 -PC5, anti-CD3 -FITC /CD16 -PE (UCHT1/3G8). All our results were expressed as the mean fluorescent intensity ratio molecule of interest/isotypic control (MFIr).",
"section_name": "Phenotypic analysis",
"section_num": null
},
{
"section_content": "Data were compared between the 4 groups using a nonparametric Kruskal-Wallis test; post hoc tests for multiple comparisons were performed when the test was significant (macro Marta Garcia-Granero [07/2008] for SPSS). The statistical analyses were performed using the SPSS software package, version 17. 0 (SPSS Inc., Chicago, IL, USA). All tests were two-sided. Statistical significance was defined as p <0. 05.",
"section_name": "Statistical analysis",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Special thanks to Bernadette Barbarat for kind help for qRT-PCR, and special thanks to Marta Garcia-Granero for additional help in statistical analysis. Special thanks to Jean Gabarre for helpful discussion.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Abbreviations NK: Natural killer; NCR: Natural cytotoxicity receptors; AIDS-RL: AIDS-related lymphoma; HS: Healthy sublects; MFI: Mean fluorescent intensity; PBMC: Peripheral blood mononucleated cells; PCR: Polymerase chain reaction.",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare that they have no competing interests. \n\nAuthors' contributions DMB, CS, TLT, CB, RC performed or contributed to the flow cytometry experiments, DMB performed the qPCR experiments, RC, DMB, CS, TLT, CB, DO, GS, CB, NM, SM, KB, GS participated to the interpretation of data and to the revising of the manuscript, RC, SM, NM contributed to subject recruitment, KB, RC contributed to the conception of the study and performed statistical analysis. All authors have given final approval of the version to be published, and are accountable for all aspects of the work.",
"section_name": "Competing interests",
"section_num": null
}
] |
10.3390/molecules24132367
|
The Metabolomic Profile of Lymphoma Subtypes: A Pilot Study
|
<jats:p>Lymphoma defines a group of different diseases. This study examined pre-treatment plasma samples from 66 adult patients (aged 20–74) newly diagnosed with any lymphoma subtype, and 96 frequency matched population controls. We used gas chromatography-mass spectrometry (GC-MS) to compare the metabolic profile by case/control status and across the major lymphoma subtypes. We conducted univariate and multivariate analyses, and partial least square discriminant analysis (PLS-DA). When compared to the controls, statistically validated models were obtained for diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), and Hodgkin lymphoma (HL), but not follicular lymphoma (FL). The metabolomic analysis highlighted interesting differences between lymphoma patients and population controls, allowing the discrimination between pathologic and healthy subjects: Important metabolites, such as hypoxanthine and elaidic acid, were more abundant in all lymphoma subtypes. The small sample size of the individual lymphoma subtypes prevented obtaining PLS-DA validated models, although specific peculiar features of each subtype were observed; for instance, fatty acids were most represented in MM and HL patients, while 2-aminoadipic acid, 2-aminoheptanedioic acid, erythritol, and threitol characterized DLBCL and CLL. Metabolomic analysis was able to highlight interesting differences between lymphoma patients and population controls, allowing the discrimination between pathologic and healthy subjects. Further studies are warranted to understand whether the peculiar metabolic patterns observed might serve as early biomarkers of lymphoma.</jats:p>
|
[
{
"section_content": "Lymphomas represent a heterogeneous group of lymphoid malignancies with varied patterns of clinical behavior and responses to treatment. Lymphomas rank the fifth most common cancer in the developed world [1]. Prognosis depends on the histologic type, clinical factors, and molecular characteristics. Lymphomas are classified based upon their histological characteristics, and the stage of maturation of the lymphocytes from which they originate [2]. B-cell lymphomas are the most frequently represented, and they include diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), multiple myeloma (MM), and other less frequent subtypes. \n\nLymphoma classification keeps evolving thanks to new molecular tools, such as metabolomics. Metabolomics is one of the most recent innovative technologies aiming to understand the metabolic processes within cells, tissues, organs, and organisms. It focuses on the quantitative analysis of a large number of metabolites, representing the end-products of genes, transcripts, and protein functions. The strong interest in metabolomics relates to the fact that even subtle changes in genes, abundance of Molecules 2019, 24, 2367 2 of 11 transcripts, or levels of protein can substantially change the quantity and dynamics of metabolites. Therefore, the analysis of metabolites represents a sensitive measure of the biological status in health or disease [3]. Altered metabolic fingerprints of lymphoma patients offer novel opportunities to detect or identify potential risks, and ultimately help achieve the goal of \"personalized medicine\" [4]. In this regard, a sizable number of findings have been tested for translational applications, focusing on lymphoma ranging from early detection to therapy prediction and prognosis [5, 6]. \n\nRecently, a metabolomic approach has been proposed to identify possible biomarkers for characterization and early diagnosis of the different lymphoma subtypes [6]. The metabolomic reports published thus far employed different techniques, such as liquid chromatography-mass spectrometry (LC-MS) [7] [8] [9] [10], both gas chromatography-mass spectrometry (GC-MS) and LC-MS [11, 12], or nuclear magnetic resonance (NMR) [13] [14] [15] [16], and different bio specimen [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]. In this study, a GC-MS technique was used to analyze plasma samples from patients affected by different lymphoma subtypes, and from age (10-year groups) and gender frequency matched population controls. The aim of the study was to identify possible metabolic biomarkers allowing early diagnosis, and possibly differential diagnosis between the subtypes.",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "Table 1 shows the gender distribution and mean age of the study population by case-control status. Cases are subdivided by histotypes. We compared the metabolomic profile of patients affected by the five major B-cell lymphoma subtypes to that detected in healthy controls using univariate t-test analysis, multivariate analysis, and partial least square-discriminant analysis (PLS-DA). The following analyses were conducted: Diffuse large B-cell lymphoma (DLBCL) (13 samples vs 42 controls), follicular lymphoma (FL) (8 samples vs 34 controls), chronic lymphocytic leukemia (CLL) (6 samples vs 29 controls), multiple myeloma (MM) (9 samples vs 36 controls), and Hodgkin lymphoma (HL) (10 samples vs 36 controls). Table 2 shows the results of the univariate analysis. The PLS-DA identified four cross-validated models. Table 3 shows the results, and Figure 1 reports the corresponding score plots. The variable importance in projection (VIP) score plots are reported as Supplementary Figures S1-S4. As shown in Table 3, the PLS-DA discriminating ability from the controls was maximum for CLL (Q 2 = 0. 734). The comparison between FL and control samples did not result in significant differences in respect to the controls (Q 2 = 0. 131), and therefore will not be discussed further. For each comparison, the PLS-DA analysis identified the most important metabolites in the class discrimination. Table 4 shows the relative abundance differences of the most important metabolites for the different comparisons. The PLS-DA identified four cross-validated models. Table 3 shows the results, and Figure 1 reports the corresponding score plots. The variable importance in projection (VIP) score plots are reported as Supplementary Figure S1-S4. As shown in Table 3, the PLS-DA discriminating ability from the controls was maximum for CLL (Q 2 = 0. 734). The comparison between FL and control samples did not result in significant differences in respect to the controls (Q 2 = 0. 131), and therefore will not be discussed further. For each comparison, the PLS-DA analysis identified the most important metabolites in the class discrimination. Table 4 shows the relative abundance differences of the most important metabolites for the different comparisons. Two metabolites were more abundant in all lymphoma subtypes compared to the controls: Hypoxanthine and elaidic acid. Another interesting feature was the number of metabolites showing a common behavior across the different lymphoma subtypes. In particular, eight metabolites showed a similar upward or downward change in DLBCL and CLL cases compared to the controls, namely 2-aminoadipic acid, 2-aminoheptanedioic acid, 4-hydroxyproline, erythritol, glucoheptonic acid, inositol-like (an inositol isomer other than myo-, scyllo-and chiro-inositol), threitol, and unknown 1910. Among these, 2-aminoadipic acid/2-aminoheptanedioic acid (common name 2-aminopimelic acid), and erythritol/threitol are chemically closely related (Figure 2 ). \n\na Identified by NIST (matching factor >70%). b Identified by GMD (matching factor >70%). c Identified by in-house library. d Inositol structural isomer other than myo-inositol, chiro-inositol, scyllo-inositol. e Chemical class: AA (Amino acid), HA (Hydroxy acid), A (Acid), FA (Fatty acid), PO (Polyol), Am (Amine), S (Sugar), P (Purine), I (Inorganic). \n\nTwo metabolites were more abundant in all lymphoma subtypes compared to the controls: Hypoxanthine and elaidic acid. Another interesting feature was the number of metabolites showing a common behavior across the different lymphoma subtypes. In particular, eight metabolites showed a similar upward or downward change in DLBCL and CLL cases compared to the controls, namely 2-aminoadipic acid, 2-aminoheptanedioic acid, 4-hydroxyproline, erythritol, glucoheptonic acid, inositol-like (an inositol isomer other than myo-, scyllo-and chiro-inositol), threitol, and unknown 1910. Among these, 2-aminoadipic acid/2-aminoheptanedioic acid (common name 2-aminopimelic acid), and erythritol/threitol are chemically closely related (Figure 2 ). In fact, 2-aminoadipic and 2-aminoheptanedioic acids are α-amino bicarboxylic acids differing by only one carbon (i. e., they are homologous), and both were less abundant in DLBCL and CLL patients compared to the controls. Threitol and erythritol are four-carbon polyols differing by the configuration of only one chiral carbon (i. e., they are diastereomers), and both were more abundant in DLBCL and CLL cases compared to the controls. In fact, 2-aminoadipic and 2-aminoheptanedioic acids are α-amino bicarboxylic acids differing by only one carbon (i. e., they are homologous), and both were less abundant in DLBCL and CLL patients compared to the controls. Threitol and erythritol are four-carbon polyols differing by the configuration of only one chiral carbon (i. e., they are diastereomers), and both were more abundant in DLBCL and CLL cases compared to the controls. \n\nEight other metabolites showed similar changes in MM and HL cases compared to the controls, namely cis-aconitic acid, glutamic acid, hippuric acid, myristic acid, oleic acid, palmitoleic acid, and stearic acid. All these metabolites are carboxylic acids; four are fatty acids, two saturated and two unsaturated. All the four fatty acids were more abundant in MM and HL patients compared to the controls.",
"section_name": "Results",
"section_num": "2."
},
{
"section_content": "We analyzed the metabolome of plasma samples from patients of five lymphoma subtypes and healthy controls by untargeted GC-MS. We obtained a significant PLS-DA model for four out of the five major lymphoma subtypes. A common feature of the four significant models was the relative abundance of hypoxanthine and elaidic acid among the patients in respect to the controls. Hypoxanthine is a purine involved in adenine and guanine metabolism and, therefore, in the synthesis of the corresponding nucleosides. In this regard, Yoo found low amounts of hypoxanthine in the urine of non-Hodgkin lymphoma (NHL) patients [7], while plasma levels were elevated in children with acute lymphoblastic leukemia (ALL) or NHL: In these patients, treatment with high-dose methotrexate lowered hypoxanthine levels [17]. Serum hypoxanthine levels were also elevated in a heterogeneous group of hemolymphatic malignancies, including acute myeloid leukemia, NHL and CLL [14], and in rectal cancer patients who underwent chemoradiotherapy [18]. Uric acid, another purine metabolite, showed higher levels in CLL and MM, and lower in DLBCL and HL when compared to the controls. Uric acid is the end-product of the purine oxidative degradation, deriving from hypoxanthine through xanthine by a NAD-dependent oxidoreductase (https://www. genome. jp/dbget-bin/www_bget?rn: R01768; https://www. genome. jp/dbget-bin/www_bget?rn:R02103). \n\nElaidic acid is the trans isomer of monounsaturated C18 oleic acid, naturally present in ruminant fat, meat, margarine, and baked products [19] ; its plasma level has been associated with an increase in total mortality and in cardiovascular mortality [20], and a diet high in trans fatty acids has been associated with an increase in NHL risk [21]. Herein, for the first time, we report that elaidic acid plasma level is more elevated in lymphoma patients, likewise in the four subtypes we could investigate, compared to the controls. \n\nOther fatty acids, such as myristic, oleic, palmitoleic, and stearic acid were more represented in both MM and HL, and plasma samples from HL patients were also characterized by an increased amount of linoleic and palmitic acid. Dysregulation of fatty acid metabolism in cancer cells is well known [22, 23] as it is the potential of fatty acid synthase (FAS) as a drug target; in fact, FAS was expressed above normal in MM [24] and CLL [25, 26]. \n\nGlycine was more abundant in plasma samples of DLBCL and HL cases compared to the controls. How this observation matches the reported impairment in intracellular glycine transport in DLBCL patients [9] is still unclear. A connection has been suggested between defective intracellular glycine import and increase in tetrahydrofolate-bound one-carbon unit production resulting from conversion from serine to glycine by serine hydroxymethyltransferase (SHMT) [9] ; the hypothesis is worth exploring, as previous studies have shown the relevance of one-carbon metabolism and changes in the methylation pattern in the etiology of lymphoma subtypes [27, 28]. \n\n2-aminoadipic acid was reported at increased levels in patients with carcinoma of the prostate [29], and it was tentatively proposed as a biomarker of glioblastoma aggressiveness [30]. The finding of a higher level of its homologous 2-aminoheptanedioic acid in the cerebrospinal fluid of glioblastoma patients, compared to that of grade I-II and grade III glioma patients [31], and in fecal samples from colorectal cancer patients [32] would support the proposal. On the contrary, levels of the same fatty acids were lower in plasma samples of DLBCL and CLL patients than in controls, and 2-aminoadipic acid was lower in colorectal cancer tissue in respect to the adjacent normal mucosa [33]. \n\nRecently, erythritol, a four-carbon bacterial metabolite [34], has been identified as an endogenous human metabolite derived from glucose-6-phosphate in the pentose phosphate pathway (PPP) [35], which would link its production to obesity in young adults. In the present study, erythritol and threitol were more abundant in DLBCL and CLL cases: The links between these metabolites and the PPP would suggest a disorder of the glucose catabolic pathway in these lymphoma subtypes. \n\nConsistent with previous reports [14], CLL cases had an elevated level of 2-hydroxybutyric acid, a by-product in the synthesis of glutathione from cystathionine under oxidative stress condition. This four-carbon hydroxy acid was also increased in plasma from hepatocellular carcinoma cases [36], and it was suggested as a potential biomarker of insulin resistance and impaired glucose regulation [37, 38]. \n\nOur study has several limitations. First, the small sample size did not allow discrimination between the individual major lymphoma subtypes based on their peculiar metabolic features, although we could identify specific metabolic imprints for each in respect to the healthy controls. All patients donated their blood before undergoing treatment, so that we could be reasonably confident that what we observed was in fact a disease effect. Only large-scale follow-up studies in the general population might help in understanding whether the metabolic changes observed could also be predictive of a developing lymphoma in its early stage. Secondly, we performed a large number of comparisons, which might have resulted in a proportionally elevated number of chance findings. However, we corrected p-values using the false discovery rate technique, and we interpreted our results consequently, based also on their consistency with previous literature reports. \n\nIn spite of such limitations, we think our findings warrant replication in larger pooled analyses.",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "4."
},
{
"section_content": "During 2012-16, we recruited incident adult patients (aged 20-74) with a first diagnosis of lymphoma at the hematology unit of the A. Businco Hospital in Cagliari-the main referral center for oncohematology in southern Sardinia, Italy-to participate in a case-control study on gene-environment interactions in the etiology of lymphoma. The pathologists collaborating to the study reviewed the clinical diagnosis of lymphoma using the 2008 World Health Organization (WHO) classification of lymphoma. All lymphoma subtypes, including B-cell and T-cell lymphomas, and Hodgkin lymphoma were included. Controls were a random sample of the resident population in southern Sardinia, the referral area of the hematology department of the oncology hospital. Controls were frequency matched to the cases by gender, 10-year age group, and local health unit of residence. Patients affected by infectious diseases and suffering from immune system disorders were ineligible to serve as controls. \n\nFollowing the Helsinki protocol, all study subjects provided written consent to the use of their biological samples before participation, in which they acknowledged that their samples would have been fully anonymized, and their identity could not be identified via the papers or in the databases. The study protocol included an in-person interview, conducted by trained interviewers at the hospital or the residence home; at the end of the interview, subjects were requested to donate a 40 mL blood sample to investigate genetic and epigenetic determinants of disease. Overall, samples were available for 196 cases and 151 controls; after storing plasma samples for the main analyses originally planned, aliquots for 66 cases and 96 controls remained available to study the metabolic profile of lymphoma subtypes, with reference to the controls. After collection, blood samples were centrifuged, and plasma samples were aliquoted and stored at -80 • C until metabolomic analysis.",
"section_name": "Study Population",
"section_num": "4.1."
},
{
"section_content": "The analytical method has been described elsewhere [39], but it was slightly modified for the purposes of the present study. In brief, 400 µL plasma aliquots were treated with 1200 µL of cold methanol in 2 mL Eppendorf tubes, vortex mixed, and centrifuged 10 min at 14,000 rpm (16. 9 G). 400 µL of the upper phase were transferred in glass vials (1. 5 mL) and evaporated to dryness overnight in an Eppendorf vacuum centrifuge. 50 µL of a 0. 24 M (20 mg/mL) solution of methoxylamine hydrochloride in pyridine was added to each vial, samples were vortex mixed, and left to react for 17 h at room temperature in the dark. Then 50 µL of MSTFA (N-Methyl-N-trimethyl-silyltrifluoroacetamide) were added and left to react for 1 h at room temperature. Samples were subsequently diluted with hexane (100 µL), with tetracosane (0. 01 mg/mL) as the internal standard, just before GC-MS analysis. Analyses were performed on an Agilent 5977B GC/MS interfaced to the GC 7890B (Agilent Technologies, Palo Alto, CA, USA), equipped with a DB-5ms column (Agilent J&W Scientific, Folsom, CA, USA). Injector temperature was 230 • C, detector temperature 280 • C, helium carrier gas flow rate of 1 mL/min. GC oven temperature program was the following: 90 • C initial temperature, 1 min hold time, increasing 10 • C/min to a final temperature of 270 • C, 7 min hold time. Samples (1 µL) were injected in split (1:4) mode. After a solvent delay of 3 min, mass spectra were acquired in full scan mode using 2. 28 scans/s with a mass range of 50-700 Amu. Each acquired chromatogram was analyzed by means of the free software AMDIS (Automated Mass spectral Deconvolution and Identification System) (http://chemdata. nist. gov/mass-spc/amdis), that identifies each chromatographic peak by comparison of the relative mass spectra and the retention times with those stored in an in-house library comprising 255 metabolites. Other metabolites were identified using NIST08 (National Institute of Standards and Technology's mass spectral database) and the Golm Metabolome Database (GMD) (http://gmd. mpimp-golm. mpg. de/). Through this approach, 108 compounds were detected and quantified: 97 were accurately identified and 11 compounds were not identified and were defined as unknown.",
"section_name": "Samples Preparation and GC-MS Analysis",
"section_num": "4.2."
},
{
"section_content": "For the metabolomic analysis, the AMDIS data matrix including 108 metabolites was processed with the integrated web-based platform MetaboAnalyst 4. 0 [http://www. metaboanalyst. ca/] [40]. Missing values were replaced with half of the minimum positive values in the original data, and after normalization by sum, data were log transformed and categorized using Pareto scaling for the purposes of analysis, including univariate analysis, partial least square discriminant analysis (PLS-DA), and its associated variable importance in projection (VIP) score. PLS-DA models were tested with the leave-one-out cross validation (LOOCV) method for the evaluation of statistical parameters (correlation coefficient R 2, cross validation coefficient Q 2 ) [41], which allowed us to determine the optimal number of components for the model description.",
"section_name": "Statistical Analysis",
"section_num": "4.3."
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "The authors are thankful to the patients and the population controls who participated in the study.",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "The following are available online.",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "",
"section_name": "Author",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest.",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.1186/s13073-015-0252-1
|
Landscape of gene fusions in epithelial cancers: seq and ye shall find
|
Enabled by high-throughput sequencing approaches, epithelial cancers across a range of tissue types are seen to harbor gene fusions as integral to their landscape of somatic aberrations. Although many gene fusions are found at high frequency in several rare solid cancers, apart from fusions involving the ETS family of transcription factors which have been seen in approximately 50% of prostate cancers, several other common solid cancers have been shown to harbor recurrent gene fusions at low frequencies. On the other hand, many gene fusions involving oncogenes, such as those encoding ALK, RAF or FGFR kinase families, have been detected across multiple different epithelial carcinomas. Tumor-specific gene fusions can serve as diagnostic biomarkers or help define molecular subtypes of tumors; for example, gene fusions involving oncogenes such as ERG, ETV1, TFE3, NUT, POU5F1, NFIB, PLAG1, and PAX8 are diagnostically useful. Tumors with fusions involving therapeutically targetable genes such as ALK, RET, BRAF, RAF1, FGFR1-4, and NOTCH1-3 have immediate implications for precision medicine across tissue types. Thus, ongoing cancer genomic and transcriptomic analyses for clinical sequencing need to delineate the landscape of gene fusions. Prioritization of potential oncogenic "drivers" from "passenger" fusions, and functional characterization of potentially actionable gene fusions across diverse tissue types, will help translate these findings into clinical applications. Here, we review recent advances in gene fusion discovery and the prospects for medicine.
|
[
{
"section_content": "Recurrent chromosomal rearrangements in cancers have been described for over half a century [1, 2]. The characterization of the oncogenic fusion BCR-ABL1 at t (9, 22) translocation loci in chronic myeloid leukemia, which culminated in the development of a molecularly targeted therapy, provides a compelling \"bench to bedside\" paradigm for cancers [3, 4]. Numerous gene fusions have since been defined at cytogenetically distinct loci of recurrent chromosomal aberrations in hematological malignancies and sarcomas, as well as in solid cancers, albeit much less frequently, arguably owing to technical limitations in resolving karyotypically complex, heterogeneous sub-clones in solid tumor tissues [5, 6]. The serendipitous discovery of ETS family gene fusions in common prostate carcinoma [7, 8], and of ALK and ROS kinase fusions in lung cancer [9, 10] through transcriptomic and proteomic approaches, bypassing chromosomal analyses, provided a strong fillip to the search for gene fusions in common solid cancers and pointed to alternative approaches to gene fusion discovery. Developments in high-throughput sequencing techniques over the past decade [11] have made possible a direct, systematic discovery of gene fusions in solid cancers [12] [13] [14], rapidly revealing a diverse genomic landscape. Gene fusions have now been identified in several common carcinomas, including those of the prostate, lung, breast, head and neck, brain, skin, gastrointestinal tract, and kidney, which alongside the widely documented gene fusions in thyroid and salivary gland tumors support the notion that gene fusions are integral to the genomic landscape of most cancers. \n\nHere, we review the emerging landscape of gene fusions across solid cancers, focusing on the recent spurt of discoveries made through sequencing. We review common features of \"driver\" fusions (those that contribute to tumor progression), the major functional classes of fusions that have been described, and their clinical, diagnostic and/or therapeutic implications.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "The first gene fusions to be defined in solid cancers, RET/PTC [15] and NTRK1 [16] rearrangements in papillary thyroid carcinoma were identified through a \"transformation assay\" using cancer genomic DNA transfected into murine NIH3T3 cells, followed by retrieval and analysis of human genomic DNA from transformed cells [17]. More typically, karyotyping and cytogenetic analysis of recurrent translocations helped define early gene fusions in solid cancers, such as CTNNB1-PLAG1 [18] and HMGA2 fusions [19] in salivary gland pleomorphic adenomas, PRCC-TFE3 in renal cell carcinomas [20], and ETV6-NTRK3 fusion in secretory breast carcinoma [21]. Incorporating more molecular approaches, a recurrent 2q13 breakpoint locus, t(2;3) (q13;p25), in follicular thyroid carcinoma was fine mapped using yeast artificial chromosomes, and cloned through 3′ rapid amplification of cDNA ends (RACE) of the candidate PAX8 cDNA, leading to characterization of the PAX8-PPARγ gene fusion [22]. Anticipating high-throughput genomics approaches, an expressed sequence tag (EST) mapping to the recurrent chromosomal breakpoint at t(15;19) (q13;13. 1) in midline carcinoma was identified from an EST database and cloned through RACE to identify the pathognomonic gene fusion BRD4-NUT [23]. The gene fusions defined in solid cancers thus far were localized at cytogenetically distinct, recurrent chromosomal aberrations, and were largely confined to relatively rare subtypes of solid cancers [5]. \n\nHowever, between 2005 and 2007, independent of a priori evidence of genomic rearrangements, recurrent gene fusions involving ETS family genes were discovered in prostate cancer, based on analysis of genes displaying outlier expression [7, 8, 24]. Around the same time, a transformation assay with a cDNA expression library (not genomic libraries [17] ) from a lung adenocarcinoma sample led to the discovery of EML4-ALK fusions [10], and a high-throughput phosphotyrosine signaling screen of lung cancer cell lines and tumors identified SLC34A2-ROS1 fusions in non-small-cell lung carcinoma (NSCLC) [9]. Thus, analyses of cancer RNA and proteins provided a critical breakthrough in the identification of oncogenic gene fusions in common carcinoma. In Fig. 1, we summarize the timeline of gene fusion discoveries, 100 years since Boveri's prescient hypothesis that malignant tumor growth is a consequence of chromosomal abnormalities, including \"combinations of chromosomes\" [25].",
"section_name": "Detection of gene fusions in carcinoma",
"section_num": null
},
{
"section_content": "High-throughput sequencing of tumor samples provides a direct readout of chimeric sequences corresponding to putative gene fusions, and the available depth of coverage helps uncover even relatively minor, sub-clonal events. In a proof of principle study, high-throughput genomic sequencing was used to identify several gene fusions in a panel of breast cancer cell lines and tissues [14]. However, considering that only a small subset of genomic breakpoints correspond to gene fusions encoding fusion transcripts or proteins, alternative approaches were explored. In a directed approach, focusing on chimeric transcripts as the readout of \"expressed\" gene fusions, Maher and colleagues used coupled short-and long-read transcriptome sequencing [12] and paired-end transcriptome sequencing [13] to detect chimeric RNAs that could be analyzed to characterize gene fusions. RNA sequencing has since been widely used in the discovery of numerous gene fusions in diverse epithelial cancers. Additionally, paired-end tag [26] and chromatin interaction analysis by paired-end-tag sequencing have been employed for gene fusion discovery [27], as well as phosphoproteome analysis, as in the discovery of a SND1-BRAF fusion in a gastric carcinoma sample [28].",
"section_name": "Next-generation sequencing",
"section_num": null
},
{
"section_content": "1. Gene fusions are an integral component of the landscape of somatic aberrations in all cancers. \n\n2. Recurrent 5′ fusion genes are generally lineage-and/or cell-type specific. \n\n3. Recurrent 3′ fusion genes in epithelial cancers are usually kinases or transcription factors, similar to the situation in hematological and soft tissue cancers. \n\n4. High-throughput sequencing enables systematic discovery of gene fusions with high sensitivity and precision. \n\n5. High-throughput sequencing often identifies multiple gene fusions in individual samples, presenting a challenge to distinguish oncogenic \"driver\" from unimportant \"passenger\" aberrations. \n\n6. Chimeric RNAs expressed independent of chromosomal rearrangements are frequently observed in cancer (and benign) tissues. \n\n7. Functionally recurrent gene fusions provide clinically relevant molecular subclassifications of existing morphological categories of tumors. 8. Functionally recurrent gene fusions that are seen across tissue types define functionally distinct molecular subtypes of cancers. 9. Gene fusions represent personalized therapeutic targets and prognostic and diagnostic markers. \n\nThe DNA-or protein-based methods, however, are not as commonly used as RNA sequencing, likely owing to several additional, specialized steps that are involved. \n\nInterestingly, RNA sequencing has also identified a class of chimeric RNAs that do not involve chromosomal aberrations. For example, \"read-through\" chimeric SLC45A3-ELK4 transcripts, such as those detected in prostate cancer, result from runaway transcription of the androgen-inducible, prostate-specific gene SLC45A3 into ELK4, the adjacent ETS family gene in the same orientation [12, [29] [30] [31]. Similarly, the VTI1A-TCF7L2 fusion, originally identified through genomic sequencing of colorectal carcinoma (CRC) samples [32], was found in a follow-up study using RNA analyses to be quite prevalent in other cancers, as well as in benign samples [33]. Chimeric transcripts not associated with genomic translocation have also been observed between non-contiguous genes. Guerra and colleagues identified CCND1-TACSTD2 (TROP2) chimeric mRNA that involves genes located on different chromosomes in subsets of ovarian, breast, gastrointestinal, and endometrial cancers [34]. The functional significance of these RNA chimeras is not clear at present, as their expression is typically seen to be relatively non-specific. \n\nFig. 1 Timeline of gene fusion discoveries. A timeline representation of salient gene fusion discoveries starting with 1914, the year that marked the publication of Boveri's monograph \"Zur Frage der Entstehung maligner Tumoren\", in which he proposed that aberrant \"combinations of chromosomes\" underlie malignant transformation [25]. The top bar shows recurrent chromosomal rearrangements or gene fusions in hematological (purple) and soft tissue (green) malignancies, and the bottom bar shows gene fusions in relatively rare (blue) and those in common (red) epithelial cancers. ACC adenoid cystic carcinoma, AML acute myeloid leukemia, ALL acute lymphoblastic leukemia, APL acute promyelocytic leukemia, cholangio cholangiocarcinoma, CML chronic myeloid leukemia, CRC colorectal carcinoma, MLL mixed lineage leukemia, PLGA pediatric low grade astrocytoma, Ph Philadelphia chromosome",
"section_name": "Box 1. Summary points",
"section_num": null
},
{
"section_content": "High-throughput sequencing of cancer samples frequently identifies multiple gene fusions in individual samples, often presenting a challenge for identifying potentially oncogenic driver fusions among irrelevant passenger aberrations. Some useful generalizations have emerged from multiple analyses: first, driver fusions are typically marked by a continuous open reading frame (ORF) that retains functional domains, such as the kinase domain in gene fusions involving oncogenic kinases, or DNA-binding domains in the case of transcription factors; second, some fusions display loss of auto-inhibitory domains (for example, loss of the N-terminal inhibitory domain in the product of BRAF fusions, or loss of 3′ UTR sequences in FGFR or HMGA2 fusions that serve as binding sites for inhibitory microRNAs). Yet other types of fusions juxtapose the promoter of certain tissue-specific, inducible or highly expressed genes; for example, the prostate-specific, androgen-inducible genes TMPRSS2 or SLC45A3 fused in frame with the proto-oncogenes ERG or BRAF, respectively, generate the TMPRSS2-ERG and SLC45A3-BRAF gene fusions in prostate cancer. \n\nIn the case of novel gene fusions involving less characterized genes, distinguishing candidate driver fusions from random events is complicated by the many false positive candidates resulting from alignment artifacts, such as multi-mapping of reads owing to homologous (pseudogenes) and/or repetitive sequences, and sequencing artifacts due to errors in library generation (particularly ligation and PCR artifacts) and sequencing. Incorporating these considerations, and additional bioinformatics filters, various bioinformatics pipelines have been developed to help prioritize fusion candidates from next-generation sequencing (NGS) data, including Chimerascan [35], FusionSeq [36], DeFuse [37], TopHat-Fusion [38], PRADA [39], and JAFFA [40]. While useful to help reduce the number of false candidates, the output from bioinformatics pipelines needs to be further validated, preferably followed by functional assays, before designating candidate gene fusions as novel driver aberrations. Recurrence of fusions, fusion partners or partner gene families in gene fusion databases also helps to prioritize candidate fusions. Once validated, screening for novel gene fusions in larger cohorts of samples employs quantitative RT-PCR or more recent techniques such as nano-string-based detection [41] [42] [43].",
"section_name": "Driver and passenger gene fusions",
"section_num": null
},
{
"section_content": "From the first reported chromosomal rearrangements in the 1960s up to the year 2000 (roughly marking the advent of high-throughput molecular techniques), the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer catalogued more than 600 \"recurrent balanced neoplasia-associated aberrations\", in which solid cancers accounted for less than 20 % [44] ; in its latest update (7 May 2015), this database lists 10,004 \"gene fusions\" [45], with solid cancers accounting for a much greater proportion, and with a large number of these fusions identified by recent high-throughput gene expression or sequencing analyses. Over the last decade, numerous gene fusions have been characterized in diverse solid cancers, including ETS family gene fusions in prostate cancer [7, 8, 12, 30, [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] ; ALK, ROS1 and RET kinase fusions in lung cancer [9, 10, [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] ; RAF kinase fusions in brain tumors [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80], melanoma [81, 82], gastric cancer [28, 82], and prostate cancer [82, 83] ; R-spondin fusions in colorectal and prostate cancer [83, 84] ; CD44-SLC1A2 gene fusions in gastric cancer [85] ; MAST-and NOTCH-family gene fusions in breast cancer [86] ; MITF gene fusions in renal cancer [87] ; and a number of FGFR family fusions in diverse cancer types [88] (Table 1 ). More than 8000 gene fusions across 16 different tumor types are tabulated in The Cancer Genome Atlas (TCGA) Fusion gene Data Portal (http://www. tumorfusions. org) [89]. The key points regarding gene fusions in epithelial cancers are summarized in Box 1. \n\nThese gene fusions in solid cancers encompass the diversity of fusion architectures, as shown in Fig. 2 and Box 2, and represent a spectrum of functional categories, including those described earlier such as kinases and transcription factors, as well as those involving newer pathways and loss-of-function fusions (discussed later). Notably, even as numerous novel gene fusions are being discovered fairly rapidly, most of these are either non-recurrent singletons, or are seen to recur at exceedingly low frequency in tumor subtypes or to recur across tumor types (Table 1 ). Incidentally, gene fusions displaying molecular recurrence involving both 5′ and 3′ partner genes, as in TMPRSS2-ERG, EML4-ALK, and BRD4-NUT, are relatively few. A large number of fusions display recurrence of a fusion gene in combination with multiple different partners; for example, BRAF/RAF1 [76, 79, 82, 83] and FGFR1/2/3 [88] [89] [90] [91] [92] [93] [94] are fused to several different 5′ partners across different tissue types (Additional file 1). This heterogeneity is likely reflective of the diverse tissue-physiological milieu in which these oncogenes impart selective advantage to the cancer cells. Conversely, some lineage-specific genes are seen to serve as 5′ partners across multiple different 3′ genes; for example, TMPRSS2 and SLC45A3 in prostate cancer have been observed as 5′ partners of ERG, ETV1, ETV4, ETV5, BRAF, and ELK4 (Table 1 and Additional file 1). Another type of observed \"recurrence\" involves isoforms of a gene familyfor example, ETV1/2/3/4/5, FGFR1/2/3, BRAF/RAF1, BRD3/4, CRTC1/CRTC3, and NTRK1/3as fusion partners. Considering that individual fusions may be observed relatively rarely (even uniquely), the potential functional consequences of gene fusions assumes priority over considerations of recurrence.",
"section_name": "Overview of the landscape of gene fusions in epithelial cancers",
"section_num": null
},
{
"section_content": "Functionally distinct molecular classes of gene fusions that are shared across tumor types can be identified in solid cancers.",
"section_name": "Functional consequences of gene fusions",
"section_num": null
},
{
"section_content": "Given their therapeutic importance, identification of gene fusions involving kinases can often signify a clinically actionable observation. Kinase fusion genes detected across multiple cancer types include RET, NTRK1, NTRK3, ALK, ROS1, FGFR1/2/3, and serine threonine kinases including the RAF family genes BRAF, RAF1, CRAF, and MAST1/2 (Table 1 and Additional file 1). In most gene fusions involving kinases, the kinase domain is retained [95], and this provides a strong filtering criterion in high-throughput sequencing data analysis. Analysis of mRNA sequencing data from the TCGA compendium, comprising 4366 primary tumor samples from 13 tissue types, revealed kinase fusions involving ALK, ROS, RET, NTRK, and FGFR gene families, which were detected in several types of cancer: bladder carcinoma (3. 3 %), glioblastoma (4. 4 %), head and neck cancer (1. 0 %), low-grade glioma (1. 5 %), lung adenocarcinoma (1. 6 %), lung squamous cell carcinoma (2. 3 %), and thyroid carcinoma (8. 7 %) [89].",
"section_name": "Kinases",
"section_num": null
},
{
"section_content": "Gene fusions involving dysregulated expression of transcription factors include ETS family gene fusions, seen in approximately 50 % of all prostate cancers and probably one of the most prevalent transcription factor gene fusions in common epithelial cancers. Among these, ERG represents the most common fusion partner and ETV1 the most promiscuous, with a dozen or more different fusion partners described to date (Additional file 1) [24, 96]. \n\nOther gene fusions involving transcription factors include NUT (or NUTM1), POU5F1, MAML2, NFIB, PLAG1, TFE3, NOTCH, and PAX8 fusions, imparting spatially and/or stochastically dysregulated expression in multiple different cancer types. NOTCH1 and NOTCH2 fusions result in dysregulated transcriptional outcomes, because after ligand activation, the NOTCH intracellular domain (NICD) forms a transcriptional activator complex, activating genes involved in differentiation, proliferation and apoptosis, and those associated with carcinogenesis. MAML2 acts as a transcriptional coactivator for NOTCH proteins by amplifying NOTCHinduced transcription of HES1. TFE3, which belongs to the MITF/TFE family of basic helix-loop-helix leucine zipper transcription factors, is involved in TGF-βinduced transcription, and has important roles in cell",
"section_name": "Transcription factors",
"section_num": null
},
{
"section_content": "An overview of the genomic architecture of gene fusions reveals that fusions may result from insertion, deletion, inversion, or tandem duplication or amplification, and may involve the same chromosome (intra-chromosomal) or different chromosomes (inter-chromosomal) (Fig. 2 ). A majority of chromosomal rearrangements have been associated with intra-chromosomal tandem duplications and amplifications in multiple whole-genome sequencing studies [14, 26, 80, 150]. Micro-homologies and repeat elements have been associated with loci of recurrent break points [151]. In an analysis of RAF family gene fusion breakpoints in low-grade astrocytomas, tandem duplications generated by microhomology-mediated break-induced replication were identified as the mechanism of generation of fusions [74]. \n\nSpatial proximity between distant chromosomal loci has been associated with chromosomal rearrangements, as observed between RET and the H4 genes located 30 megabases (Mb) apart on chromosome 10, involved in RET gene fusions in papillary thyroid carcinoma [152]. This proximity may be induced by genotoxic stress; for example, androgen stimulation coupled with the genotoxic stress of radiation was shown to generate gene fusions through \"induced proximity\" between TMPRSS2 and ERG (located on chromosome 21q22. 2, approximately 3 Mb apart) as well as between TPMRSS2 and ETV1 (located on chromosome 7) [153, 154] (Fig. 3a ). \n\nAnother phenomenon, called chromothripsis, describes the frequent occurrence of massive chromosomal aberrations localized to only one or two chromosomes, with fragments of chromosome joined randomly [155, 156]. Chromothripsis may be responsible for the generation of numerous, apparently random passenger gene fusions that are retained in the multiclonal cells of epithelial cancers, as well as loss-offunction fusions involving tumor suppressors, likely involving the non-homologous end-joining DNA repair system (Fig. 3b ). \n\nSeveral cancer-causing viruses, such as Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human papilloma virus (HPV), hepatitis B and C viruses (HBV and HCV), and Merkel cell polyomavirus (MCV), integrate into human genomic DNA at defined hotspots as well as seemingly randomly [157]. Viral integration events have been associated with chromosomal aberrations, such as MYC amplification in HPVpositive genital carcinoma [158], and not uncommonly, loss of gene function [159, 160] or gene fusions involving viral-human sequences have been reported [161, 162]. The recent report of a recurrent gene fusion of UBR5 on 8q22. 3 and ZNF423 on 16q12. 1 (UBR5-ZNF423) in 8 % of EBVassociated primary nasopharyngeal carcinomas suggests a driver function of this gene fusion in a subset of nasopharyngeal cancers [163]. The fusion retains all the functional domains of HMGA2, and removes the 3′ UTR sequence that contains several inhibitory let7 microRNA binding sites. Absence of the Let-7-regulated 3′ UTR in the fusion transcript results in overexpression of HMGA2 that is sufficient for neoplastic transformation [19] FGFR-PLAG1 FGFR is the 5′ partner, which, without its kinase domain, provides the promoter to drive the expression of the 3′ partner, PLAG1\n\nThis FGFR fusion product does not include the FGFR kinase domain, and therefore is not a target for FGFR inhibitors [91] Adenoid cystic carcinomas (salivary glands, lacrimal glands, ceruminal glands; also breast) FDA Food and Drug Administration, FTC follicular thyroid carcinoma, GBM glioblastoma multiforme, MASC mammary analog secretory carcinoma of salivary glands, MEC mucoepidermoid carcinoma, nccRCC non-clear-cell renal cell carcinoma, NMC NUT midline carcinoma, NSCLC non-small-cell lung carcinoma, PTC papillary thyroid cancer, RCC renal cell carcinoma, RMC renal medullary carcinoma, TF transcription factor growth and proliferation. TFE3 is involved chromosomal translocations that result in various gene fusions (such as PRCC-TFE3, RCC17-TFE3, PSF-TFE3, NON-O(p54nrb)-TFE3 and ASPL-TFE3) in papillary renal cell carcinomas. PLAG1 is an oncogenic transcription factor associated with the neoplastic transformation of pleomorphic adenomas of the salivary gland and lipoblastomas through upregulation of IGF2, CRLF1, CRABP2, CRIP2, and PIGF. NFIB binds viral and cellular promoters activating transcription and replication. POU5F1 and PAX8 are homeobox-containing transcription factors, a family of genes that play a role in cell fate and differentiation programs, and whose role in cancer is well recognized, particularly PAX8 in thyroid cancer [22].",
"section_name": "Box 2. Mechanisms of generation of gene fusions",
"section_num": null
},
{
"section_content": "",
"section_name": "MYB-NFIB",
"section_num": null
},
{
"section_content": "CD44-SLC1A2/EAAT2 gene fusions are detected in 1-2 % of gastric cancers involving the glutamate transporter SLC1A2 [85], and cause intracellular accumulation of glutamate, a growth-promoting amino acid associated with oncogenic functions [97, 98]. Thus, this gene fusion may be establishing a pro-oncogenic metabolic milieu, akin to the increased levels of sarcosine reported in prostate cancer [99]. \n\nWnt/β-catenin signaling pathway RNA sequencing of 68 \"microsatellite stable\" subtype colorectal cancer samples revealed two recurrent fusions The likely mechanisms of chimera generation are indicated. Chr chromosome involving R-spondin family genes, EIF3E-RSPO2 in two cases and PTPRK-RSPO3 in five cases [84]. these gene fusions retained the functional domain of the Rspondins that are known to be agonists of the canonical Wnt/β-catenin signaling pathway. Additionally, the LACTB2-NCOA2 chimeric transcript detected in 6 of 99 (6. 1 %) colorectal cancer cases led to disruption of NCOA2 expression, thus activating the Wnt/β-catenin pathway [100]. Recently, R-spondin fusions such as GRHL2-RSPO2 were described in prostate cancer as well [83].",
"section_name": "Other functional classes Metabolic enzymes",
"section_num": null
},
{
"section_content": "Recently, fusions involving SKIL (which encodes a SMAD inhibitor) 3′ to androgen-regulated promoters such as TMPRSS2, SLC45A3, and ACPP, were found in 6 of 540 (1. 1 %) prostate cancers and one cell line xenograft, LuCaP-77 [101]. SKIL overexpression in these tumors was associated with upregulation of the TGF-β pathway, likely providing the oncogenic mechanism in these tumors.",
"section_name": "TGF-β pathway",
"section_num": null
},
{
"section_content": "In an analysis of fusion transcripts observed in TCGA data across multiple tumor types, fusions involving chromatin modifier genes, including histone methyltransferase and histone demethylase genes, were identified in 111 samples (2. 5 %) [89]. Chromatin modifier genes are potential therapeutic targets and these gene fusions thus represent a novel class of potentially actionable aberrations.",
"section_name": "Chromatin modifier genes",
"section_num": null
},
{
"section_content": "Additional classes of genes represented among recurrent fusions in solid cancers include those encoding growth factor receptors (GABBR2, TACSTD2, ITPR2), adaptors and co-factors (WIF1, GAB2), Ras-Gap proteins (DOCK5, ARHGAP15), and cytoskeletal proteins (SNF8, SEC22B, HIP1R, STXBP4, MYO19, TPR). Although some of these fusions are scored as recurrent, they may represent passenger mutations associated with loci of recurrent chromosomal aberrations, while others may define tissuespecific or cooperative roles.",
"section_name": "Further functional classes",
"section_num": null
},
{
"section_content": "While most reported gene fusions pertain to gain-offunction aberrations imparting neoplastic phenotypes, with high-throughput sequencing, fusions resulting in loss of function of tumor suppressors such as TP53 and PTEN have been identified as well [102]. The LACTB2-NCOA2 fusion in colorectal cancer leads to disruption of NCOA2, which encodes an inhibitor of the Wnt/β-catenin pathway [100], thus acting to promote carcinogenesis.",
"section_name": "Loss-of-function gene fusions",
"section_num": null
},
{
"section_content": "Some gene fusions are associated with distinct subtypes of carcinoma, while others have been detected across different tissues or lineages, defining molecular subsets of cancers transcending morphological distinctions.",
"section_name": "Gene fusion signatures in personalized medicine of epithelial cancers",
"section_num": null
},
{
"section_content": "Some of the salient gene fusions that define molecular subtypes of epithelial within specific organs or tissue types are summarized in Table 1. The ETV6-NTRK3 fusion is a diagnostic biomarker of secretory breast carcinoma, as well as the acinic cell carcinoma or cystadenocarcinoma recently designated as \"mammary analog secretory carcinoma of salivary glands\" (MASC) [21, 103]. BRD-NUT fusions define NUT midline carcinoma [104, 105]. CRTC-MAML2 fusions are the defining molecular aberration of mucoepidermoid carcinoma (MEC) [106, 107] ; translocation-negative MECs are proposed to be designated as a distinct subgroup of adenosquamous carcinoma [108]. CRTC-MAML fusions are also found in MEC of the lung [109] [110] [111] [112], cervix [113], thyroid glands and oral cavity [114], as well as in clear cell hidradenoma of the skin [115, 116]. In all cases, MAML2 fusions characterize benign or low-grade tumors, and for reasons not described so far have been associated with a favorable prognosis [117]. Interestingly, pulmonary MECs have shown clinical response to gefitinib in the absence of sensitizing EGFR mutations, suggesting a potential connection with CRTC-MAML2 and the possibility of therapeutic application in other MECs harboring this fusion [110, 118]. The diagnostic subclass of adenoid cystic carcinomas, including salivary gland and breast cancer, is characterized by MYB-NFIB gene fusions [119, 120]. Fusions defining subtypes within a cancer include RET and NTRK gene fusions in subsets of papillary thyroid carcinoma [121], while PAX8-PPARγ fusions characterize subsets of follicular thyroid carcinoma [22, 122]. ETS family gene fusions, primarily including ERG (and less frequently, ETV1, ETV4, ETV5 or FLI1), are found in approximately 50 % of prostate cancers, the most common fusion being TMPRSS2-ERG. The EWSR1-ATF1 fusion found in hyalinizing clear cell carcinoma of the salivary glands, a rare and indolent tumor, can potentially be used as a molecular marker of this subtype that is histologically similar to the more aggressive MEC [123]. Gene fusions or fusion partners found across tissue types are common in solid cancers. The EML4-ALK fusion, initially identified in lung cancer [9, 10] has since been reported in breast cancer [124], colorectal carcinomas [66, 124], and in pediatric renal medullary carcinoma that affects young African-Americans with the sickle cell trait [125, 126]. Similarly, RET fusions, first characterized in thyroid cancer, are widely observed in lung cancers, and the EWSR1-POU5F1 fusion was detected in two rare epithelial tumors, hidradenoma of the skin and MEC of the salivary glands [127]. \n\nGene fusions involving RAF kinase genes (BRAF, RAF1, CRAF) have been identified in low-grade tumors of the central nervous system (pilocytic astrocytomas and other low-grade gliomas), gastric cancer, melanoma and prostate cancer. RAF family fusions involve truncation of the N-terminal auto-inhibitory domain, thus generating constitutively active RAF protein. Curiously, BRAF gene fusions in low-grade astrocytomas have been associated with a tendency to growth arrest, conferring a less aggressive clinical phenotype and a better clinical outcome [75, 128]. Additionally, RAF family fusions have been defined across diverse solid cancers, including prostate, gastric, and skin cancers [82, 83]. A screen for BRAF gene fusions in 20,573 solid tumors, using the Foun-dationOne™ targeted gene panel, identified BRAF fusions involving 29 unique 5′ fusion partners in 55 (0. 3 %) cases across 12 different tumor types, including 3 % (14/531) of melanomas, 2 % (15/701) of gliomas, 1. 0 % (3/294) of thyroid cancers, 0. 3 % (3/ 1,062) of pancreatic carcinomas, 0. 2 % (8/4,013) of non-small cell lung cancers and 0. 2 % (4/2,154) of colorectal cancers, as well as single cases of head and neck cancer, prostate cancer, rectal adenocarcinoma, ovarian, uterine endometrial, and mesothelioma [70]. \n\nFusions involving FGFR tyrosine kinase family genes have also been observed across diverse cancers [88]. The first FGFR fusion observed in epithelial cancers, FGFR1-PLAG1, was found in a subset of pleomorphic salivary gland adenomas, and involves FGFR1 as the 5′ partner upstream of PLAG1, the known driver of salivary gland tumors [91]. Curiously, this fusion excludes the tyrosine kinase domain of FGFR. Fusions that retain the tyrosine kinase domain of FGFR include FGFR3-TACC3 in glioblastoma [92, 129]. Subsequently, diverse FGFR fusions, all retaining the tyrosine kinase domain, have been observed in bladder, lung, breast, thyroid, oral, and prostate cancers, involving FGFR1, 2, or 3 either as the 5′ or 3′ partners [88, 94].",
"section_name": "Recurrent gene fusions as biomarkers of subtypes of solid cancers",
"section_num": null
},
{
"section_content": "In Additional file 2 we summarize recent clinical trials involving gene fusions in epithelial cancers. The RET inhibitor vandetanib shows antiproliferative activity in RET-mutant medullary thyroid cancer (MTC) [130], and was recently approved by the US Food and Drug Administration for treatment of metastatic MTC. Sensitivity to vandetanib was also observed in RET-fusionpositive papillary thyroid carcinoma [131] and lung cancer cells [68, 132]. Treatment with Pfizer's kinase inhibitor crizotinib (PF02341066) led to a dramatic clinical response in EML4-ALK-positive NSCLC patients [133, 134], as well as in one patient with an SLC34A2-ROS1fusion-positive tumor [58]. Unfortunately, resistance is inevitably observed, owing mutations in the kinase domain [134, 135], or ALK gene fusion amplification, KIT amplification or increased auto-phosphorylation of EGFR [136]. This is representative of the challenge of treating solid cancers and argues for the development of combinatorial therapeutic approaches from the start rather than sequentially, as is the practice currently. RAF or MEK inhibitors represent potential precision therapeutic options for several solid cancers with the diverse RAF family gene fusions described earlier. Several FGFR inhibitors currently in clinical trials represent potential therapeutics for cancers harboring FGFR fusions across multiple cancer types, including bladder cancer, prostate cancer, and others [88, 90, 94, 137]. The rare PIK3C family gene fusions in prostate cancer (for example, TBXLR1-PIK3CA and ACPP-PIK3CB) show overexpression of the PI3KC genes and may be sensitive to PIK3CA inhibitors [83]. \n\nFor treatment of secretory breast carcinoma expressing the ETV6-NTRK3 fusion, therapeutic targeting of the downstream signaling axis of IGF1R, using the IGIFR/INSR kinase inhibitors BMS-536924 and BMS-754807 that are currently in clinical trials, was found to be effective [138]. Breast cancer cells expressing NOTCH fusion products that retain the γ-secretase cleavage site were sensitive to γ-secretase inhibitor (GSI) in culture, and treatment with GSI reduced tumor growth in vivo [86]. On the other hand, breast cancer cells harboring NOTCH fusions that encode NICD independent of the γ-secretase cleavage site were insensitive to GSI. \n\nIn a recent clinical sequencing study of 102 pediatric cancers, among 37 non-sarcoma solid cancers, several functional gene fusions were identified, including TFE3 fusions in a colorectal cancer (SFPQ-TFE3) and renal cell cancer (ASPSCR1-TFE3)both cases were treated with pazopanib, the latter displaying stable disease for 10 months [139]. \n\nEfforts to target several other gene fusions are underway. The newly developed bromodomain inhibitors that have shown dramatic efficacy in hematological malignancies [140, 141] are now being tested in multiple clinical trials for NUT midline carcinoma characterized by BRD3/4-NUT gene fusions, which represent a rare but highly aggressive class of tumors with no effective treatment currently available [104]. Also, the R-spondin fusions observed in colorectal and prostate cancer may be sensitive to Wnt pathway antagonist porcupine inhibitors [142]. \n\nGene fusions involving ETS transcription factors have been utilized in diagnostic applications. A non-invasive assay system has been developed based on the detection of TMPRSS2-ERG fusion transcripts in urine samples from patients, which in combination with the detection of urine PCA3 improved the performance of the multivariate Prostate Cancer Prevention Trial risk calculator in predicting cancer on biopsy [143]. Detection of TMPRSS2-ERG in circulating tumor cells in therapynaive patients and in castration-resistant prostate cancer patients following treatment suggests potential applications in non-invasive monitoring of the therapeutic response [144]. While therapeutic targeting of transcription factor oncogenes is intrinsically challenging, on the basis of the interaction of ERG with the DNA repair enzyme PARP1 and DNA protein kinase DNA-PKc, use of PARP inhibitors was shown to inhibit growth of TMPRSS2-ERG-positive prostate cancer xenografts [145]. Additionally, PARP inhibition was associated with radiosensitization of TMPRSS2-ERG-positive prostate cancer cells [146, 147]. These experimental leads point to possible therapeutic avenues targeting a prevalent gene fusion in a common carcinoma.",
"section_name": "Some gene fusions provide personalized therapeutic targets",
"section_num": null
},
{
"section_content": "Genomic or transcriptomic sequencing has virtually supplanted molecular and cytogenetic techniques as the primary modality for discovery of gene fusions, and detection of gene fusions is increasingly incorporated into the standard workflow for genomic characterization of tumors in both research and clinical settings. Transcriptome sequencing has been useful in helping to identify expressed gene fusions based on evidence of the fusion of exon boundaries, but putative promoter fusions that do not generate chimeric transcripts are likely to go undetected. Furthermore, typically recurrent gene fusions characterized in cancers represent gain-of-function events arising from the juxtaposition of cell-type-or lineage-specific regulatory elements and proto-oncogenes, or novel combinations of functional domains derived from two proteins that provide combinatorial or additive functionalities to normal genes. However, NGS data also reveal less frequently described loss-of-function chimeras involving tumor suppressor genes such as TP53, PTEN, and others. A systematic analysis of loss-of-function gene fusions could identify additional cancer samples with loss of tumor suppressors that might be currently going unreported, and could help broaden our understanding of the role of gene fusions in cancer. \n\nThe rapid increase in detection of gene fusions across cancers has spawned multiple discovery and prioritization pipelines to help distinguish bona fide functional gene fusions from random chimeras (and experimental artifacts). However, the development of diverse pipelines following different analysis parameters underscores a need for standardization of the vocabulary and information content in recording and reporting gene fusions, along the lines of the Minimum Information About a Microarray Experiment [148, 149]. Furthermore, even as bioinformatics analyses help prioritize fusion candidates, the \"recurrence\" of fusion genes and/or retention of functional domains provide the most compelling rationale for functional characterization. \n\nThe detection of distinct gene fusions across subtypes of common carcinoma also provides a basis for molecular subclassification of these cancers. Recurrent gene fusions that characterize distinct subtypes of cancers include BRD4-NUT in NUT midline carcinoma, ETV6-NTRK3 in secretory breast carcinoma, CRTC-MAML2 fusions in mucoepidermoid carcinoma, and RAF family fusions in pilocytic astrocytomas. It is expected that as more and more carcinomas are analyzed by sequencing, additional subclasses may be recognized on the basis of whether the detected molecular aberrations are driver fusions. Importantly, the emerging landscape of gene fusions in solid cancers also reveals many gene fusions involving oncogene families or isoforms that are seen across multiple tumor types or subtypes, for example, fusions involving RAF and FGFR family genes. This supports the notion that a molecular classification of tumors in terms of driver fusions (or SNVs) may complement histopathological descriptions. \n\nMany oncogenes involved in gene fusions (for example, RET, BRAF, ALK, NOTCH or PIK3CA/B) are also known to harbor activating mutations. However, fusions and mutations tend to be mutually exclusive. This indicates that either fusions or activating mutations can independently provide oncogenic function, and that either of these aberrations may render the tumors sensitive to therapeutic targeting. Thus, for example, MEK inhibitors that have been found to be useful for tumors with a BRAF activating mutation may also benefit tumors with the BRAF fusion. \n\nThe development of technologies that enable the systematic detection of molecular aberrations in cancer has profound clinical implications, as high-throughput sequencing of individual tumor samples is expected to become available as a routine diagnostic modality (as for whole-body PET scans or MRI) in the not-too-distant future. Considering the important diagnostic and therapeutic implications, the integration of approaches for the detection of driver gene fusions into cancer genomics pipelines is crucial for precision cancer medicine.",
"section_name": "Perspectives and discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We thank Robin Kunkel for help with the artwork for the figures. AMC is supported by the Doris Duke Charitable Foundation Clinical Scientist Award and the Prostate Cancer Foundation. AMC is an American Cancer Society Research Professor and A. Alfred Taubman Scholar. This work was supported in part by the US National Institutes of Health ( R01CA132874 ), Early Detection Research Network grant UO1 CA111275, Prostate SPORE grant P50CA69568, the Department of Defense Era of Hope grant BC075023 (AMC).",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "Additional file 1: Recurrent gene fusions in epithelial cancers. Summary of recurrent gene fusions in epithelial carcinoma across different tissues. a Gene fusions with common 5′ and 3′ genes.",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "The University of Michigan has filed for a patent on recurrent gene fusions in prostate cancer and AMC is named as a co-inventor. The technology has been licensed to Hologic Inc. to develop a molecular diagnostic.",
"section_name": "Competing interests",
"section_num": null
}
] |
10.3389/fmed.2018.00335
|
From Vascular Smooth Muscle Cells to Folliculogenesis: What About Vasorin?
|
First described in 1988, vasorin (VASN) is a transmembrane glycoprotein expressed during early mouse development, and with a less extent, in various organs and tissues (e.g., kidney, aorta, and brain) postnatally. Vasn KO mice die after 3 weeks of life from unknown cause(s). No human disease has been associated with variants of this gene so far, but VASN seems to be a potential biomarker for nephropathies and tumorigenesis. Its interactions with the TGF-β and Notch1 pathways offer the most serious assumptions regarding VASN functions. In this review, we will describe current knowledge about this glycoprotein and discuss its implication in various organ pathophysiology.
|
[
{
"section_content": "Vasorin (VASN), a cell surface glycoprotein of 673 amino acids (aa), is encoded by the VASN gene also named Slit-like 2 (Slitl2) due to its strong homologies with the slit family of important signaling molecules. Vasorin was identified by various screens in several vertebrates including rodents (Mus musculus and Rattus norvegicus) (UniProt Q9CZT5 and D3ZAE6), zebrafish (Danio rerio) (UniProt A4QNV9), and humans (Homo sapiens) (UniProt Q6EMK4). Located on chromosome 16 in human and mouse, it is encoded by two exons separated by a large intronic sequence (Supplementary Figure 1A ). Vasorin is highly conserved at the DNA and protein levels; alignments of the coding region reveal an overall identity of more than 95 and 83% at the amino acid level between rodent and human homologs, respectively (Supplementary Figure 1B ) (1). This high degree of similarity suggests a highly conserved function of the protein throughout evolution, but until now, no human disease or phenotype has been directly associated to variants of the VASN gene. In 2004, Ikeda et al. published the first study about VASN reporting its localization in adult human tissues and suggesting a role in the TGF-β pathway (2). Fourteen years later, the roles of VASN/Vasn in development or in the pathophysiology of adult tissues or organs remains unelucidated although some clues are emerging. This review aims at reporting current knowledge on Vasn/VASN and more specifically at describing the main pathways that have been associated to this transmembrane protein.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Vasorin (VASN/Vasn) is a typical, single-pass, type I transmembrane protein of ∼110 kDa. Based on its sequence analysis by Blast and by the RaptorX modeling program (3), a prediction of its tri-dimensional structure has been performed, using mainly neuronal adhesion molecules (Netrin-G, LRRTM2, and NCAM2) as probes, leading to the following description. Its extracellular amino-terminal domain contains a putative hydrophobic signal peptide, one leucine-rich repeat (LRR) region, comprising 11 LRR repeats, flanked by an amino-and a carboxy-terminal LRR-flank motif, one epidermal growth factor (EGF)-like repeat, and one fibronectin type III (FNIII) domain (Figure 1A ; Supplementary Figure 1 ). These regions are followed by a highly hydrophobic stretch of amino acids that are predicted to be a single pass transmembrane segment. The intracellular carboxy-terminal peptide, of ∼80 amino-acid residues, shows no similarity to any known structure so far. The combination of LRR regions (4-7 repeats depending on the family's members) and EGF domain is conserved within the Slit family of proteins (7). Interestingly, this serial repetition of the LRR motifs is known to form nonglobular, horseshoe structure, allowing tight protein-protein interaction. The concave sides of those structures are specifically involved in the binding with the Ig domains of the Robos receptors (8, 9). It is important to note that, with its 11 LRR repetition motifs, Vasn offers a large extracellular binding site. \n\nDiscussing Vasn structure also raises the possibility of the existence of a cleavage site close to the EGF region. In Slit proteins, this cleavage occurs within the EGF repeats, after the arginine (R) of a VLPR motif, releasing the N-terminal active isoform (8). Malapeira et al. could show that in breast cancer cell lines, the N-terminal part of Vasn was cleaved by ADAM-17 and that only this cleaved form of Vasn was active for TGFβ trapping (5). This observation was confirmed in the human hepatocellular carcinoma (HCC) cell line (Hep G2) by Li et al. (10). Indeed, many arginine residues are present within or around the EGF motif, especially a VTPR motif, the most similar motif to the VLPR, located close to the FNIII domain in the Vasn sequence.",
"section_name": "VASORIN STRUCTURE AND FEATURE PREDICTION",
"section_num": null
},
{
"section_content": "Vasn/VASN expression pattern suggests major role(s) from embryonic development to adulthood (Table 1 ). Whole mount in situ hybridization in zebrafish, embryos revealed that Vasorin mRNA is detected as early as the gastrula stage and rapidly spreads along the dorsal-ventral axis, and later along the anteroposterior axis with a prominent expression observed in the brain primordium during the protruding mouth stage, and in the central neural system (1). In rodents, a similar pattern is observed between E8. 5 and E11. 5 in murine embryos, with strong expression in the hindbrain and the midline of the neural tube. In addition, a strong expression is observed in the first branchial arch and the forelimb and hindlimb buds (20). In this latest study, a reporter gene expression was used in Vasn LacZ and in Vasn Venus transgenic mice and confirmed the endogenous Vasn expression. From E10. 5 onwards, the reporter signal is mainly found around the developing heart, lungs, kidney, testis/ovary, and skeletal system and increases at subsequent stages. At E17. 5, an intense reporter signal is precisely observed in the ossification region of long bones, in vascular smooth muscle cell-rich arterial vessels, in the mucosa lining the gastric fundus and in the spleen capsule, along with previously described organs/tissues. (B) Vasorin major pathways: Vasn was first described as an inhibitor of TGF-β pathway: the metalloprotease ADAM 17 cleaves VASN and releases the soluble extracellular part of vasorin (sVASN). TGF-β binds to sVASN instead of TGFβRII. The hetero-tetrameric complex between TGF-β1 and-2 is not formed and the sequence of intra-cellular phosphorylation does not occur [adapted from (5) ]. A second pathway was recently discovered showing that Notch1 can be stabilized at the membrane by VASN, thus escaping Numb (an inhibitor of the activation of Notch signaling) mediated endocytosis and lysosomal degradation. This bonding allows both extracellular cleavage by ADAM 10/17 and intracellular cleavage by the gamma secretase to cleave Notch1 intracellular peptide NICD1, which translocates to the nucleus to form a transcription complex [adapted from (6) ]. \n\nThese approaches revealed a widespread expression of Vasn from early developmental stage narrowing as development progresses. In situ hybridization on adult mice confirmed Vasn expression in the coronary artery, aorta, kidney (2), and in ovarian follicles (21). Northern blot analysis on adult mice tissues further confirmed Vasn expression in heart, kidney, lung, liver, and testis (19). \n\nIn human, VASN was identified by Northern blot in aorta, with a high expression, compared to moderate levels in kidney, liver, and placenta (2). It was also detected in vascular smooth muscle cells (VSMCs) (2), human umbilical vein endothelial cells (HUVECs) (15), and in permanent periodontal ligament cells (18). Finally, less relevant but still notable, a proteomic approach on healthy fertile women ovum donors identified VASN in the human follicular fluid (17). \n\nAt the cellular level, Ikeda et al. have first reported Vasn expression at the cell-surface membrane, using Chines Hamster Ovary (CHO) cells expressing a human VASN-flag-tagged protein (2). This localization was confirmed in breast cancer cell line MCF7 (5), in Hep G2 (10, 16), and in human glioma stem like cells (GSCs) (6). In parallel, the presence of a secreted extracellular peptide has been highlighted by Malapeira 6 ) but the precise function of this secreted peptides remains to be elucidated. Only one study described an intracellular form of vasorin (called ATIA for Anti TNFα Induced Apoptosis in this study) translocating to the mitochondria in mouse embryonic fibroblasts (19). \n\nOne notable point confirmed by most of studies is the molecular weight of VASN/Vasn around 110 kDa revealed by western blot (2, 5, 10). Some authors propose that the N-or Oglycosylation of the protein could explain such a difference with the expected molecular weight at 72 kDa (2, 19). Only one study presented a different result with a total VASN expression band at around 80 kDa after western blotting (11) ; the extracellular soluble form has been reported to be around 90 kDa (5).",
"section_name": "VASORIN EXPRESSION AND LOCALIZATION",
"section_num": null
},
{
"section_content": "In the primary study, Ikeda et al. have investigated VSMCs and a rat carotid arterial balloon-injury model to demonstrate that Vasn directly bound to transforming growth factor TGF-β1, TGF-β2, and TGF-β3 at the membrane surface or at the extracellular level and inhibited TGF-β signaling in vitro (2). In vivo, Vasn expression was down-regulated after vascular injury whereas the expression of several cytokines, including TGF-β, was up-regulated and the ratio of TGF-β to Vasn was increased. In addition, Vasn administration after injury dramatically reduced the neointimal formation, at least in part by modulating TGF-β signaling in the vessel wall as shown by the decrease of Smad2 phosphorylation. Taken together, these data suggested that the down-regulation of Vasn induced by acute vascular injury contributed to the fibroproliferative response to vascular injury. \n\nFollowing these findings, Malapeira et al. reported that only the soluble extracellular part of vasorin (sVASN) functioned as a trap for TGFβ and confirmed a direct inhibiting effect of sVASN on TGFβ signaling pathway, manifesting by a decrease in pSmad levels in response to TGFβ treatment (5). In addition, they demonstrated that the metalloprotease ADAM17 was able to cleave VASN-although not the only one able to do soand that in their CHO cells model it was the major protease involved in this shedding (Figure 1B ). These results were reproduced in the breast cancer cell lines MCF7 and A459 ( 6 ) and in addition it was shown that inhibition of ADAM17 by a metalloprotease inhibitor-BB94-led to the upregulation of TGF-β signaling. \n\nIn 2011, Choksi et al. explored vasorin in a hypoxia and TNFα-induced apoptosis context (19) and demonstrated that ATIA (VASN) was highly expressed in human glioblastoma and its inhibition allowed hypoxia-mediated apoptosis of cells. They hypothesized that ATIA would be a hypoxia inducible factor (HIF-1) target and generated a partial ATIA KO model targeting the supposed mitochondrial addressing peptide of intracellular VASN. In their mouse and MEF models, ATIA appeared to protect cells against TNFα-induced apoptosis, at least partially through mitochondrial-ATIA and thioredoxin (TRX2) interactions. Looking further on this HIF1 pathway and using GSCs, Man et al. confirmed that Vasorin expression was upregulated by hypoxia, as shown by its co-activation with many other hypoxia response genes, and that Vasorin was mandatory to maintain GSCs under hypoxic conditions (6). This study demonstrated that Notch1 was one major partner in this function and that Vasorin competed Numb (the Notch pathway inhibitor) for Notch1 binding, thus preventing its lysosomal degradation. Hence, when vasorin is present, it appears to decrease Notch1 turnover and increase Notch1 nuclear signaling, thus allowing GSCs renewal and tumorigenic properties.",
"section_name": "VASORIN PARTNERS AND PATHWAYS",
"section_num": null
},
{
"section_content": "Nowadays, diagnosing early stages of pathologies is a major challenge. Numerous studies are searching for non-invasive methods to discriminate healthy vs. ill individuals or to follow, on an individual based strategy, disease/treatments evolution (23). \n\nAlong this line, vasorin has been identified as a potential biomarker of severe progressive nephropathies such as Immunoglobulin A nephropathy (IgAN), thin basement membrane nephropathy (TBMN), or diabetic nephropathy (DN). Vasorin was detected in higher amount by nano LC-MS/MS in the urinary exosome of patients with TBMN compared to that of patients with IgAN as well as control patients, and confirmed by western blot analysis (11). Using nano LC-MS/MS but on total urine sample proteome extracts, Samavat et al. also reported the presence of VASN in the urine of patients with IgAN (24). VASN has also been isolated as a glycoprotein in plasma of patients with diabetic nephropathies. Interestingly, VASN was upregulated in diabetic patients with nephropathy in contrast to diabetic patients without nephropathy (12, 24). In addition, a study also described VASN in synovial fluid of patients with osteoarthritis (14). \n\nFollowing this idea but in another domain, Vasorin was also identified in a few studies looking for cancer biomarkers. In 2011, Caccia et al. reported the analysis of 12 different tumor cell lines, aiming to identify by proteomic assay specific proteins under-or over-expressed in cell secretomes (13). Using the thyroid cancer cell line TPC-1, they identified VASN as one of the 3 specifically under-expressed proteins after inhibiting proliferation treatment in the cell cultures. \n\nIn 2011 also, Choksi et al. proposed that ATIA could be a biomarker for brain cancer due its significant overexpression in brain tumor cell-lines (glioblastoma and astrocytoma) (19). In 2018, the same team showed that VASN expression was increasing along with gliomas aggressiveness (6). \n\nIn 2015, Li et al. reported that VASN was a prospective biomarker of HCC and a potential therapeutic target for this cancer (16). Using subtractive-EMSA-SELEX and MALDI-TOF MS assay they verified that VASN was highly expressed in alpha-fetoprotein (AFP, a classical serological biomarker for liver cancer) negative sera of 100 cases of HCC patients with extrahepatic metastases compared with 97 cases of healthy controls, and 129 cases of hepatitis patients. In addition, using a siRNA based VASN knockdown approach in the cancerous Hep-G2 cell line, they showed a decreased cell proliferation, an increased apoptosis and a reduced migration compared within normal L02 cells. Finally, they identified 2 miRNA on the 7 targeting vasorin sequence (miR145 and miR146a), which were expressed in Hep G2, SMMC7721, and L02 cell lines but were negatively correlated to VASN mRNA level, and whose transfection in Hep G2 cells down-regulated VASN expression, and promoted apoptosis with decreased cell proliferation and migration.",
"section_name": "Vasorin as a Biomarker",
"section_num": null
},
{
"section_content": "Since the first publication in 2004, the major roles of vasorin appear to be related to cell migration and proliferation/differentiation. This fits well with its structural homologies with the Slit family and its expression in specialized cells and in remodeling organs/tissues. \n\nConsidering VASN strong expression in the vascular system and its role in tumorigenesis, Huang et al. hypothesized that vasorin might play a pivotal role in tumorigenesis and vasculogenesis (15). They explored vasorin transfer from Hep G2 to (HUVECs) via an exosome/endocytosis process and demonstrated not only the existence of this transfer, but also that VASN enhanced HUVEC migration. This indicates that VASN could act as a mediator for cancer cells toward their environment, promoting endothelial cells invasion and consecutively tumor development and metastasis. \n\nAlong the idea of TGF-β pathway central role, Rimon-Dahari et al. explored the role of Vasn in folliculogenesis (21). They first reported vasorin expression in ovaries at all the follicular stages and used Vasn systemic KO mice to analyse mice fertility and folliculogenesis. They noticed an enhanced ovulation under hormonal stimulation in KO mice as well as in transplanted KO ovaries in wild type females. However, there was no difference regarding fertility. Vasn-deficient females displayed a significantly lower number of primordial follicles as well as atretic antral follicles compared to wild type mice. The absence of vasorin was also correlated with an overactivated TGF-β pathway in KO mice, evidenced by an increase in pSmad2 levels in KO ovaries after hormonal stimulation. Although exploring a new aspect in vasorin functions, this study did not show the existence of a direct link between the absence of Vasn and TGF-β modulation.",
"section_name": "A Little Bit Further Into Vasorin Role(s) in Pathophysiology",
"section_num": null
},
{
"section_content": "Considering data on the expression of Vasorin in humans or in animal models, one may not be surprised to encounter an altered expression of this protein in human diseases such as nephropathies or cancers. In this context, the use of sVASN as a biomarker seems promising but requires further investigations. Nevertheless, data reporting the physiological distribution and elimination of this secreted form are still missing. Li et al. as continuity of their research on liver cancer (16), identified two mimotopes of sVASN that might be useful to address these questions, as well as to develop biological therapeutic targets (10). Vasorin links with the TGF-β pathway has also led to an analysis of its involvement in tumorigenesis, with strong evidence for a role in cell migration and proliferation. It seems that the main roles of vasorin are driven by its extracellular domain under the control of extracellular proteases such as ADAM 10 and 17 (5, 6). More recently, an exciting interaction with the Notch1 pathway has been unraveled in the context of cancer (6). Considering this slow and step-by-step progression, one may certainly say that there is still a great deal to discover about vasorin. The study of mutant mouse lines will enable either a global, or an organ-restricted, approach to better understand the impact of Vasorin deletion or activation. Studying total knock-out mice for Vasn may confirm a deleterious vascular and/or skeletal phenotype, which could be expected considering the strong Vasn embryonic expression. These models could also reveal other affected organs/tissues in childhood or adulthood. In addition, the kidney appears as a promising target for specific conditional mouse line, but the exact localization of Vasorin in nephrotic specialized cells (tubules cells, podocytes) remains to be defined. All this knowledge opens and will open many other avenues for further research.",
"section_name": "CONCLUSION",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "Mia Krautzberger whom Ph. D. thesis work has supported this review.",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "CG, A-LB, GR, and CC drafted the manuscript. A-LB wrote the first draft of the manuscript and figures. IB, GR, and CG wrote sections and figures of the manuscript. CC and HS critically revised the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The Supplementary Material for this article can be found online at: https://www. frontiersin. org/articles/10. 3389/fmed. 2018. 00335/full#supplementary-material",
"section_name": "SUPPLEMENTARY MATERIAL",
"section_num": null
},
{
"section_content": "The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.",
"section_name": "Conflict of Interest Statement:",
"section_num": null
}
] |
10.1371/journal.pcbi.1002724
|
MOSAIC: A Multiscale Model of Osteogenesis and Sprouting Angiogenesis with Lateral Inhibition of Endothelial Cells
|
The healing of a fracture depends largely on the development of a new blood vessel network (angiogenesis) in the callus. During angiogenesis tip cells lead the developing sprout in response to extracellular signals, amongst which vascular endothelial growth factor (VEGF) is critical. In order to ensure a correct development of the vasculature, the balance between stalk and tip cell phenotypes must be tightly controlled, which is primarily achieved by the Dll4-Notch1 signaling pathway. This study presents a novel multiscale model of osteogenesis and sprouting angiogenesis, incorporating lateral inhibition of endothelial cells (further denoted MOSAIC model) through Dll4-Notch1 signaling, and applies it to fracture healing. The MOSAIC model correctly predicted the bone regeneration process and recapitulated many experimentally observed aspects of tip cell selection: the salt and pepper pattern seen for cell fates, an increased tip cell density due to the loss of Dll4 and an excessive number of tip cells in high VEGF environments. When VEGF concentration was even further increased, the MOSAIC model predicted the absence of a vascular network and fracture healing, thereby leading to a non-union, which is a direct consequence of the mutual inhibition of neighboring cells through Dll4-Notch1 signaling. This result was not retrieved for a more phenomenological model that only considers extracellular signals for tip cell migration, which illustrates the importance of implementing the actual signaling pathway rather than phenomenological rules. Finally, the MOSAIC model demonstrated the importance of a proper criterion for tip cell selection and the need for experimental data to further explore this. In conclusion, this study demonstrates that the MOSAIC model creates enhanced capabilities for investigating the influence of molecular mechanisms on angiogenesis and its relation to bone formation in a more mechanistic way and across different time and spatial scales.
|
[
{
"section_content": "The process of angiogenesis during fracture healing\n\nThe biological process of fracture healing comprises three main stages: (i) the ''inflammation phase'', where the trauma site becomes hypoxic and is invaded by inflammatory cells, fibroblasts, endothelial cells and mesenchymal stem cells [1] ; (ii) the ''reparative phase'', which starts with the production of cartilaginous and fibrous tissue resulting in a soft callus, later replaced by a hard callus, through the process of endochondral ossification; (iii) in the final ''remodeling phase'' the woven bone is replaced by lamellar bone and the vasculature is reorganized. \n\nThe healing of a fracture depends largely on the development of a new blood vessel network (angiogenesis) in the callus. Sprouting angiogenesis involves the following steps: first a ''tip cell'' is selected; this cell extends filopodia sensing the haptotactic and chemotactic cues in the environment and leads the newly formed ''sprout'' comprised of following, proliferating ''stalk cells''; the newly formed sprout, or ''branch'' then connects with another branch in a process called anastomosis, which results in the formation of a closed loop allowing the initiation of blood flow; finally the newly formed vascular network is stabilized by pericytes [2]. \n\nIn order to ensure a correct development of the vasculature, the balance between stalk and tip cell phenotypes must be tightly controlled. The process of tip cell selection consists of the following main steps. Firstly a gradient of vascular endothelial growth factor (VEGF) is formed by the up-regulation of VEGF-expression and secretion, triggered by hypoxia (low oxygen concentration). The VEGF-mediated activation of the VEGFR-2 receptors induces the up-regulation of Dll4 which activates the Notch1-receptors on the neighboring cells, thereby down-regulating their expression of VEGFR-2. This process of lateral inhibition, with cells battling to inhibit each other leads eventually to a ''salt and pepper'' alternating pattern, where cells with high Dll4 levels remain with high VEGFR-2 receptor levels, allowing them to migrate (and becoming tip cells) whilst their neighbors become inhibited, making them less susceptible to VEGF, and thus these adopt the non-migratory stalk cell phenotype. In this manner the adequate amount of tip cells, required for a correct sprouting pattern, is established [2] [3] [4] [5].",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Both fracture healing as well as angiogenesis are very complex biological processes involving the coordinated action of many different cell types, biochemical and mechanical factors across multiple temporal and spatial scales. The time scales of individual events that underlie biological processes range from seconds for phosphorylation events to hours for mRNA transcription to weeks for tissue formation and remodeling processes [6]. The spatial scales vary from nanometers at the molecular level to millimeters at the tissue level and meters at the level of the organism [6, 7]. Thus, it can be concluded that most biological processes have an intrinsic multiscale nature and must be studied and modeled accordingly. \n\nDepending on the biological spatial scale of interest a variety of experimental and modeling approaches can be used, which are nicely summarized by Meier-Schellersheim et al. [7]. The modeling approaches can be arranged in two broad categories: continuum and discrete modeling techniques. Continuum models use ordinary differential equations (ODEs) or partial differential equations (PDEs) to describe the evolution of cell and tissue densities and protein concentrations. The model variables are averages, which makes it difficult to represent individual cell-cell and cell-matrix interactions [8, 9]. Moreover, since the cells are not individually represented, it is challenging to model the individual intracellular processes. Also, continuum models fail to correctly capture the process of angiogenesis due to the inherent discreteness of vascular networks [10]. Discrete approaches are often used to study small-scale phenomena, e. g. biological processes at the cellular and subcellular levels [11]. However, these techniques often become computationally expensive when used for predictions of larger cell population sizes at the tissue scale [11]. \n\nHere we briefly review hybrid, multiscale models of angiogenesis, i. e. models that combine different modeling techniques for various scales mentioned above into one framework, as this is the approach we have adopted here. For comprehensive reviews on (multiscale) mathematical models of angiogenesis the reader is referred to Mantzaris et al.,. Notably none of the hybrid models to date include Dll4-Notch1 tip cell selection. \n\nMilde et al. presented a deterministic hybrid model of sprouting angiogenesis where a continuum description of VEGF, MMPs, fibronectin and endothelial stalk cell density is combined with a discrete, agent-based particle representation for the tip cells [15]. The hybrid model describes the biological process of sprouting as the division of tip cells depending on chemo-and haptotactic cues in the environment and a phenomenological ''sprout threshold age''. In the model of Lemon et al. the formation of a new branch also occurs at the tip cell position, but is modeled as a random process with an average number of branches per unit length of the capillary [16]. Checa et al. model sprout formation stochastically by making the probability of sprouting from a vessel segment proportional to the segment length. The capillary growth rate is also regulated by the local mechanical stimulus [17]. Peiffer et al. proposed a hybrid bioregulatory model of angiogenesis during bone fracture healing [18], based on the deterministic hybrid model of Sun et al. [19]. The process of angiogenesis is modeled discretely, including sprouting and anastomosis. The selection of tip cells in the growing vascular network is, however, modeled with phenomenological rules. A three dimensional model of cellular sprouting at the onset of angiogenesis was developed by Qutub et al. [20]. Although this framework describes sprout formation as a function of the local VEGF concentration and the presence of Dll4, it is only a first approximation of the complex tip-to-stalk cell communication by Dll4/Notch signaling. \n\nA more detailed model of the lateral inhibition that underlies the tip cell selection process in angiogenic sprout initialization was presented by Bentley et al. [3, 21]. They use an agent-based framework to accurately simulate a small capillary comprising 10 endothelial cells that can change shape and sense the local VEGF concentration by extending very thin filopodia. Moreover, every endothelial cell is characterized by its individual protein levels of VEGFR-2, Dll4 and Notch -and their distribution on the cell membrane, by further subdividing the membrane into separate agents -which does not only allow assessment of the effects of the VEGF environment on tip/stalk cell patterning but also those of Dll4 over-and under-expression and cell shape change. \n\nSprouting angiogenesis involves multiple biological scales: the intracellular scale where gene expression is altered so that different phenotypes (e. g. tip and stalk cells) can arise, the cellular scale that involves proliferation and migration and the tissue scale that encompasses the concentration fields of soluble and insoluble biochemical factors. As these scales are highly coupled, multiscale models are needed to study the mechanisms of sprouting angiogenesis. To the best of the authors' knowledge, there is only one model of sprouting angiogenesis with Dll4-Notch1 rigorously implemented [3] but this model simplified the extracellular environment to a uniform or linearly varying field of VEGF concentration, which is constant in time. While this simplification is justified for a detailed study of the short term phenotypic",
"section_name": "Multiscale models of angiogenesis",
"section_num": null
},
{
"section_content": "The healing of a fracture largely depends on the development of a new blood vessel network (angiogenesis), which can be investigated and simulated with mathematical models. The current mathematical models of angiogenesis during fracture healing do not, however, implement all relevant biological scales (e. g. a tissue, cellular and intracellular level) rigorously in a multiscale framework. This study established a novel multiscale platform of angiogenesis during fracture healing (called MOSAIC) which allowed us to investigate the interactions of several influential factors across the different biological scales. We focused on the biological process of tip cell selection, during which a specific cell of a blood vessel, the ''tip cell'', is selected to migrate away from the original vessel and lead the new branch. After showing that the MOSAIC model is able to correctly predict the bone regeneration process as well as many experimentally observed aspects of tip cell selection, we have used the model to investigate the influence of stimulating signals on the development of the vasculature and the progression of healing. These results raised an important biological question concerning the criterion for tip cell selection. This study demonstrates the potential of multiscale modeling to contribute to the understanding of biological processes like angiogenesis. \n\nchanges of a few neighboring endothelial cells, it is not for more complex, multicellular systems that involve cell-matrix interaction and highly dynamic, extracellular environments. This is certainly true for fracture healing, in which matrix densities and (gradients of) extracellular concentrations of soluble signals, like VEGF, are spatially and temporally changing as a result of cellular activity. While efforts have been done to model the interplay of VEGF diffusion and sprouting angiogenesis in the context of skeletal muscle tissue [6], these multiscale models did not incorporate Dll4-Notch1 signaling. Moreover, in the context of fracture repair multiscale models that consider angiogenesis and that relate tissue, cell and intracellular scales have not been established yet [22].",
"section_name": "Author Summary",
"section_num": null
},
{
"section_content": "In this study, we present a multiscale model of osteogenesis and sprouting angiogenesis with lateral inhibition of endothelial cells (MOSAIC) which extends the bioregulatory framework of Peiffer et al. [18] with an intracellular model based on the work of Bentley et al. on tip cell selection [3]. We hypothesize that the MOSAIC model creates enhanced capabilities for investigating the influence of molecular mechanisms on angiogenesis and its relation to bone formation. Simulation results will illustrate the interplay between molecular signals, in particular VEGF, Dll4 and Notch1, endothelial cell phenotypic behavior and bone formation. They will demonstrate the advantages of multiscale modeling in the context of fracture healing, thereby exploring the importance of the model of Bentley et al. [3] for a much more complex and dynamic extracellular environment. At the same time, by comparison to the more phenomenological model of Peiffer et al. [18] the potential of a more mechanistic treatment of tip cell selection will become clear.",
"section_name": "Objectives of this study",
"section_num": null
},
{
"section_content": "The MOSAIC model presented in this work integrates an intracellular module based on the work of Bentley et al. [3] into the model of Peiffer et al. [18]. Figure 1 gives a schematic overview of the MOSAIC model which consists of (1) a tissue level describing the various key processes of bone regeneration with continuous variables, (2) a cellular level representing the developing vasculature with discrete endothelial cells and (3) the intracellular level that defines the internal dynamics of every endothelial cell. The combination of continuous and discrete modeling techniques results in a hybrid, multiscale model.",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "The discrete variable c v represents the blood vessel network, which is implemented on a lattice. When a grid volume contains an endothelial cell this variable is set to 1, otherwise c v = 0. The vessel diameter is defined by the grid resolution and is always one endothelial cell wide, although the movement of the tip cell is grid independent as explained below. Every endothelial cell (c v = 1) has unique intracellular protein levels, which control the behavior of that specific cell. The intracellular module is adapted from the agent-based model of Bentley et al. [3] and consists of the following variables: the level of VEGFR-2 (V), Notch1 (N), Dll4 (D), active VEGFR-2 (V9), active Notch1 (N9), effective active VEGFR-2 (V0), effective active Notch1 (N0) and the amount of actin (A). The effective active levels (V0 and N0) include the time delay of translocation to the nucleus, thereby representing the levels at the nucleus, influencing gene expression. The amount of actin (A) refers to the polymerized actin levels (F-actin) inside the cell. In particular, it is associated to actin used for filopodia formation, owing to its importance for tip cell migration. As such, an increase in actin levels can be interpreted as filopodia extension, while a decrease as filopodia retraction. Other intracellular signaling pathways that involve actin, such as energy metabolism [23, 24], are not considered. \n\nThe following equations describe the intracellular dynamics. An overview of all the parameters of the intracellular module can be found in Table 1. \n\nThe activation of the VEGFR-2 receptor, described by V9, is given by:\n\nwhere the constant V sink represents the amount of VEGFR-1 receptors that act as a sink (decoy receptor) by removing VEGF from the system, t represents the time and dt the time step of the inner loop (more information on these parameters can be found in the section ''implementation details'' below), V max represents the maximal amount of VEGFR-2 receptors, g v is the local VEGF concentration (at the tissue level) and M tot is the total number of membrane agents (constant for all ECs). Equation 1 is adapted from Bentley et al. [3] where every EC is composed of a varying amount of membrane agents, representing small sections of the cellular membrane. In the current framework, however, every EC is represented by one agent so that M tot was chosen to be constant for all ECs and equal to an intermediate value between the initial and maximal amount of membrane agents in the agent-based framework of Bentley et al. [3]. The level of activated VEGFR-2 remains in a range going from 0 to V. When the VEGFR-2 receptors are activated above a certain threshold (V9*), the actin levels of the endothelial cell are incremented in a constant manner (DA). As mentioned earlier, this represents the extension of filopodia by the endothelial cell, which is shown to be regulated downstream of VEGFR-2 [4]. If the endothelial cell fails to extend its filopodia for a certain amount of time D 3, the filopodia retract which is mathematically translated into a reduction of the actin levels in a constant manner (210. DA). The actin level remains in a range between 0 and A max. The amount of Notch1 is considered to be constant in every EC, representing a balance between the rate of degradation and expression. At the same time, initial Notch activity levels are assumed to be zero and in the model Notch activity can only be increased through binding with Dll4 from neighboring ECs. The model therefore neglects the role of Notch in EC quiescence and the fact that high Notch activity levels have been measured in quiescent ECs [25] [26] [27]. Instead, it only focuses on the role of Dll4-Notch in tip cell selection. The number of activated Notch receptors (N9) will be equal to the total amount of Dll4 available (with an upper bound, given by the total number of Notch receptors N). The amount of Dll4 in the environment of an EC is the sum of the amount of Dll4 at the junctions with its neighboring ECs, whereby every cell is assumed to distribute Dll4 uniformly across its cell-cell junctions (see Figure 2 ). After a delay of D 1 for V9 and D 2 for N9 the active VEGFR-2 and Notch levels become the effective active levels (V0 and N0) representing the levels at the nucleus, influencing gene expression. The delays were taken from Bentley et al. which were fitted to a somite clock Delta-Notch system [3, 28]. Note that there is a delay between receptor activation and gene expression (transcription) but not between gene expression and protein synthesis (translation), which is a simplification of the model. \n\nThe amount of Dll4 is modeled in the following way:\n\nD t{dt represents the previous amount of Dll4, d the change in Dll4 expression due to the activation of the VEGFR-2 receptor [4, 29] and N' t{dt,neighbours is the amount of Dll4 that is removed from the environment due to the activation of Notch-receptors on neighboring ECs. If-conditions are used to ensure that the Dll4 level remains in a range between 0 and D max. When Notch signaling is activated in a cell, the amount of VEGFR-2 receptors is down-regulated, suppressing the tip cell phenotype as follows [4, 5] :\n\nV max represents the maximal amount of VEGFR-2 receptors and s models the VEGFR-2 expression change due to Notch1 activation. If-conditions are used to ensure that the VEGFR-2 level remains in a range going from V min to V max. Since the amount of VEGFR-2 (V) at the previous timestep (V t{dt ) is not present in Equation 3, the amount of VEGFR-2 is continuously in equilibrium with the amount of effective active Notch1 (N'' t{dt ). Equation 3 implies that in quiescent cells the number of VEGFR-2 receptors will be maximal, owing to the absence of any Notch activity. As mentioned earlier, the model neglects the role of Notch activity in quiescence and the fact that it will lead to reduced VEGFR-2 levels in quiescent ECs [25] [26] [27]. Note that Bentley et al. [3] represent every EC by a varying number of agents (to account for changes in cell shape and cell growth), whereas in this study every EC is represented by one agent. However, in order to use the parameter values and equations (in an adapted form) of Bentley et al. [3], M tot was fixed at a constant value for all ECs. Consequently, the values of V, N, V9, N9, V0, N0, D and A are evaluated at the cellular level, not at the level of individual membrane agents. This also implies that here cellular polarity is not captured explicitly as receptor and ligand concentrations are uniformly distributed across the membrane junctions. In the current model cell directional behavior follows from gradients of extracellular signals alone. \n\nThe evolution of the vascular network is determined by tip cell movement, sprouting and anastomosis [18, 19], outlined below. \n\nTip cell movement. The model computes the movement of every tip cell in a lattice-free manner. The cells that trail behind this tip are subsequently considered to be endothelial cells. Consequently, although the movement of a tip cell is grid independent, the vessel diameter is defined by the grid resolution due to the projection of the blood vessels on the grid. The movement of the tip cells is determined by their direction and speed, which is described by the tip cell velocity equations:\n\nwhere x tip ! represents the position, v tip the speed and u tip ! the direction of movement of the tip cell. The tip cell speed depends on the active VEGFR-2 concentration, meaning that both the surrounding VEGF concentration as well as the amount of VEGFreceptors influences the behavior of the tip cell [4, 30]. Below a threshold activation level (V9*) the tip cells do not migrate, above this, the tip cell velocity increases with V9 up to a maximal speed of v max tip. This translates into the following equation, where a third order polynomial was used to ensure a smooth threshold [18] :\n\nThe direction of movement is influenced by chemotactic and haptotactic signals and is modeled in the same way as Peiffer et al. [18]. \n\nSprouting. The tip cell phenotype is induced (formation of a new branch) or maintained (in already existing tip cells) if the following requirements are satisfied:\n\nThis criterion means that the endothelial cell must have enough VEGFR-2 and filopodia (polymerized actin) to sense the environment and direct the trailing branch towards the source of VEGF. \n\nAnastomosis. When a tip cell encounters another blood vessel anastomosis takes place, after which the EC loses its tip cell phenotype.",
"section_name": "Discrete description of angiogenesis",
"section_num": null
},
{
"section_content": "At the tissue level, the fracture healing process is described as a spatiotemporal variation of eleven continuous variables: mesenchymal stem cell density (c m ), fibroblast density (c f ), chondrocyte density (c c ), osteoblast density (c b ), fibrous extracellular matrix density (m f ), cartilaginous matrix density (m c ), bone extracellular matrix density (m b ), generic osteogenic growth factor concentration (g b ), chondrogenic growth factor concentration (g c ), vascular growth factor concentration (g v ) and concentration of oxygen (n). The set of partial differential equations (PDEs) accounts for various key processes of bone regeneration. Initially the callus is filled with granulation tissue and the mesenchymal stem cells and growth factors will quickly occupy the regeneration zone. Subsequently the mesenchymal stem cells differentiate into osteoblasts (intramembranous ossification -close to the cortex away from the fracture site) and chondrocytes (central callus region). This is followed by VEGF expression by (hypertrophic) chondrocytes, which attracts blood vessels and osteoblasts and which is accompanied by cartilage degradation and bone formation (endochondral ossification). The model does not include bone remodeling. The general structure of the set of continuous equations is given by:\n\nwhere t represents time, x x the space and c c m (t,x x) the density of a migrating cell type (mesenchymal stem cells and fibroblasts). c c(t,x x) represents the vector of the other nine concentrations, ECM densities, growth factor concentrations and oxygen concentrations for which no directed migration is modeled. D cm (c c) and D are the diffusion coefficients. f i (c c) represents the taxis coefficients for both chemotaxis and haptotaxis. f 0 (c c m,c c) and g(c c m,c c) are reaction terms describing cell differentiation, proliferation and decay and matrix and growth factor production and decay. Detailed information, including the complete set of equations, boundary and initial conditions, parameter values and implementation details can be found in Peiffer et al. [18] and Geris et al. [31] and are provided here as online supplement.",
"section_name": "Evolution of the continuous variables",
"section_num": null
},
{
"section_content": "The partial differential equations are solved on a 2D grid with a grid cell size of 25 mm. The width of the discrete ECs is determined by the size of a grid cell (25 mm). Since the ECs in the model of Bentley et al. [3] have a width of 10 mm, the parameter values taken from Bentley et al. are multiplied with a factor of 2. 5 (see Table 1 ). The partial differential equations are spatially discretized using a finite volume method assuring the mass conservation and nonnegativity of the continous variables [32]. The resulting ODEs are solved using ROWMAP, a ROW-code of order 4 with Krylov techniques for large stiff ODEs [33]. The MOSAIC model is deterministic and implemented in Matlab (The MathWorks, Natick, MA). \n\nThe flowchart in Figure 1B gives a schematic overview of the computational algorithm used in this study. Firstly the continuous variables are calculated. Then the inner loop is iterated which consists of four subroutines: (1) the current tip cells are evaluated by the tip cell selection criterion and, if necessary, they lose their tip cell phenotype; (2) the new position of every tip cell is calculated using a central difference scheme in space in combination with explicit Euler time integration; (3) the code checks whether sprouting occurs, meaning that other endothelial cells also satisfy the criterion for tip cell selection; (4) the intracellular levels of every endothelial cell are updated. Finally, the inner and outer loops are iterated until the end time of the simulation is reached. \n\nThe outer loop has a maximal time step size of 8. 57 hours (row). Since the tip cells do not move more than one grid cell (25 mm) in this time interval (v max tip = 35 mm/day [19] ), this maximal time step size (row) implies that the 11 PDEs can be solved for a constant vasculature. The inner loop has a maximal step size of 1. 2 hours (ee), similar to Peiffer et al. [18], and was chosen so that the movement of the tip cells within one grid cell could be accurately followed (ee%row). To reduce implementation difficulties, the time step of the inner loop (dt) is determined by calculating how many maximal inner loop time steps (ee) can fit in one outer loop time step (DT) and dividing the outer loop time step by this number. Consequently, the time step of the inner loop is not constant, which means that D 1, D 2 and D 3 vary slightly, but this is a minor trade-off for the computational efficiency. Numerical convergence tests have shown that the average inner time step dt is equal to 155 s. Consequently, D 1, D 2, D 3 approximate the delays chosen by Bentley et al. [3]. Since the time step dt is approximately 10 times the time step of Bentley et al. [3], the parameter values of s and d have been altered to match the dynamics of the Dll4-Notch system. Numerical tests have shown that similar behavior is retrieved when both s and d are multiplied with 3. 16 (see Table 1 ).",
"section_name": "Implementation details",
"section_num": null
},
{
"section_content": "Simulations were conducted using a quad-core IntelH XeonH CPU with 12 GB RAM memory. Initially the callus domain is filled with granulation tissue only (m f,init = 10 mg/ml), all other continuous variables are initialized to zero. Boundary conditions are presented in Figure 3. Further information on the choice of appropriate boundary and initial conditions of the continuous variables can be found in Peiffer et al. [18] and Geris et al. [31]. \n\nNormal fracture healing. The initial values of the endothelial cell variables are summarized in Table 2. The EC starting positions are depicted in Figure 3. \n\nImpaired angiogenesis. The influence of very high VEGF concentrations on tip/stalk cell patterning and angiogenesis was investigated by including an additional, constant source term (constant production rate) in the equation for g v. This term was varied between 0% and 10% of the default production rate (G gvc since the chondrocytes dominate the angiogenic growth factor production; see supplementary material for the explanation of the meaning of G gvc ) representing normal and very high VEGF concentrations in the callus respectively. The effect of the injection of VEGF-antibodies, described by an additional sink term in the equation for g v, was also simulated [34]. The sink term was defined in such a way that no more VEGF than present could be removed thereby ensuring that the VEGF concentration stays positive. The effect of pharmacological inhibition of the VEGFR-2 receptor was investigated by setting V9 to zero. \n\nSensitivity analysis. As Peiffer et al. [18] already performed extensive sensitivity analyses additional sensitivity analyses focused on the parameters related to the newly implemented tip cell selection mechanism (i. e. s, d, V9*, V sink ). Experimental observations indicate that heterozygous Dll4 knockout mice still have some Notch activity but produce too many tip cells due to the lowered inhibition levels [5]. The MOSAIC model was used to simulate various genotypes corresponding to different expression levels of Dll4. The parameter d, which defines the Dll4 expression change due to VEGFR-2 activation, was varied between 50% and 200%, representing a heterozygous knockout genotype and overexpression respectively [3]. The parameter s, which represents the influence of the VEGFR-2 expression change due to Notch1, was also varied, between 33% and 133%. The threshold of active VEGFR-2 for actin production and migration of tip cells (V9*) was varied between 25% and 1000%. The amount of VEGFR-1 acting as a decoy receptor was changed by reducing V sink from 0. 9% to 364%. The effect of the initial values of Dll4 (D 0 ) and actin (A 0 ) was also investigated, the initial amount of Dll4 was varied between D min and D max and the initial amount of actin was varied between 0% and 100%.",
"section_name": "Simulation details",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The MOSAIC model predicts the evolution of the continuous variables as well as the evolution of the intracellular variables during normal fracture healing. The osteoprogenitor cells enter the callus from the surrounding tissues and differentiate into osteoblasts under the influence of osteogenic growth factors. This leads to rapid intramembranous ossification near the cortex and distant from the fracture line. In the endosteal and intercortical callus the bone is formed through the endochondral pathway, starting from a cartilage template that is mineralized as the blood vessel network is formed to supply the complete fracture zone with oxygen. Figure 4 compares the predictions of the Peiffer-model [18] and the MOSAIC model with the experimentally measured tissue fractions of Harrison et al. in a rodent standardized fracture model [35]. Both models capture the general trends in the experimental data equally well: the bone tissue fraction gradually increases throughout the healing process; the fibrous tissue fraction disappears; the cartilage template is first produced and later replaced by bone. \n\nAfter one, two and three weeks of simulated healing time the surface fraction of the blood vessels in the callus is respectively 2. 34%, 18. 20% and 46. 25%. Experimental results also show that the vascular plexus is very dense in the fracture callus, although quantitative results are lacking [36, 37]. Images, illustrating the angiogenic and osteogenic process in the fracture callus can be found in Maes et al. and Lu et al. [38, 39]. These experimental studies report that at the progressing front, there is a tree-like structure of tip cells extending filopodia to sense their environment and to guide the developing sprout. At the back, the vasculature is being remodeled into a more structured network of larger vessels with more quiescent endothelial cells. At present this remodeling phase of the vasculature, which will remove some blunt ends as well as redundant vessels, is not included in the MOSAIC model. \n\nFigure 5 shows that the tip cells have high VEGFR-2 levels. The stalk cells are inhibited and have low VEGFR-2 and actin levels. The Dll4-Notch signaling stops when the VEGF-concentration in the callus drops (the VEGFR-2 levels stay constant) (Equations 1-3). The VEGF concentration goes down since the vasculature brings enough oxygen to the fracture site. The endothelial cells far away from the vascular front all have maximal VEGFR-2 levels. \n\nThe average VEGFR-2 concentration, predicted across all ECs present in the fracture callus, drops at day 7 in the standard condition (Figure 6, standard). Indeed, after 7 days the ECs start to inhibit each other in gaining the tip cell phenotype, resulting in a prediction of enhanced Notch1-signaling and reduction of the average VEGFR-2 levels at the vascular front. At the back, VEGFR-2 levels are predicted to return to their maximal value, which is a direct consequence of Equation 3 (effect of Notch activity on VEFGR-2), and the fact that in the model Notch activity levels of an EC are only governed by VEGF-induced Dll4 expression (in its neighboring cells). As mentioned before, the model only focuses on the lateral inhibition between tip cells and stalk cells through Dll4-Notch. It does not address EC quiescence and the fact that Notch activity in quiescent ECs will be associated with reduced VEGFR-2 receptor levels [25] [26] [27]. Despite this anomaly in terms of the number of VEGFR-2 receptors, the model correctly predicts highly reduced VEGFR-2 activity levels in quiescent cells (i. e. cells at the back of the vasculature), because of the low VEGF concentrations encountered here. This trend in the average VEGFR-2 concentration (Figure 6, standard) was also measured by Reumann et al. [40]. Reumann et al. characterized the time course of VEGFR-2 mRNA expression during endochondral bone formation in a mouse rib fracture model by quantitative RT-PCR [40]. They observed a small drop (although statistically insignificant) in the median value of VEGFR-2 mRNA expression at three days post fracture [40]. The simulated average VEGFR-2 concentration (Figure 6, standard) follows a similar trend but drops at later time points (day 7 versus day 3). The experimental results [40] were, however, determined in a mouse rib fracture model whereas the parameter values of the model presented in this study were derived from a rat femur fracture model [35]. At the same time, it should be mentioned that Figure 6 represents protein values where Reumann et al. [40] measured mRNA levels. Moreover, Reumann et al. [40] measured the total mRNA content, of all cells present in the callus whereas Figure 6 shows the average VEGFR-2 concentration on the cell membranes of the ECs in the callus. Not only ECs but also osteoblasts and other osteogenic cells express VEGFR-2 [41], which might also explain the temporal difference seen between the experimental and simulated data.",
"section_name": "Normal fracture healing",
"section_num": null
},
{
"section_content": "If pharmacological blocking of VEGFR-2 receptors is simulated, the vasculature does not develop since the actin production is inhibited, meaning that the ECs cannot extend filopodia and gain the tip cell phenotype. Due to this impaired vascularization only a small amount of bone is predicted intramembranously, resulting in a non-union between the fractured bone ends. \n\nIf the VEGF concentration is increased (Figure 7B ), the vascular density is initially increased since the VEGFR-2 receptors are being more activated in this stimulating environment (Equation 1 ). This simulation result is confirmed by many experimental studies that reported an increase in vascularity at the site of VEGF application in a murine femoral fracture healing model [34], a lapine mandibular defect model [42], a murine ectopic model [43] and a rat femoral bone drilling defect model [44]. In the simulations the increase in vascular density leads to faster healing, which is also found experimentally [34, 42] (Figure 8 ). The MOSAIC model predicts earlier bone formation, less cartilage formation in both the periosteal, intercortical as well as the endosteal callus. Moreover, the cartilage resorption is predicted to 7B ). Evolution of the average VEGFR-2 concentration (day 0 corresponds to the time of fracture). The threshold line represents the first requirement that needs to be fulfilled to obtain the tip cell phenotype, i. e. V. V max /2. Remark that in very high VEGF concentrations (+10%) the average receptor concentration is far below the threshold. doi:10. 1371/journal. pcbi. 1002724. g006 be accelerated, another trend which has been reported in experimental studies [43, 44]. It is striking, however, that after 35 days, the MOSAIC model predicts that the VEGF-treated callus contains slightly less bone and more remnants of fibrous tissue than the normal condition (Figure 8 ). \n\nA further increase of the VEGF concentration (+2%, Figure 7B (iii)) reduces however the vascular density and bone tissue fraction in the MOSAIC model. This is consistent with the trend seen by Street et al., where an optimal dose of VEGF (250 mg) leads to a maximal amount of callus volume (both total and calcified) in a critical rabbit radius segmental gap model [34]. \n\nIn higher VEGF environments (e. g. Figure 7B (iii)) the ECs strongly inhibit each other; creating a salt and pepper pattern of high and low VEGFR-2 levels (Figure 9 ). In addition, the development of the vasculature ceases after a certain period, as can be seen in Figure 9. The ECs that fulfill the tip cell criteria (Equation 6 ) will sprout and will initially move perpendicular to the vessel from which it is originating. In case this ''mother'' vessel is a growing vessel as well that extends towards the source of the chemotactic and haptotactic signals, this implies that the new sprout will initially move perpendicular to the gradients. In this particular configuration (Figure 7B (iii)), the vascular front was V sink = 100% (standard condition), ii: V sink = 45%, iii: V sink = 9%, iv: V sink = 0. 9%); (B) Variation in the amount of VEGF addition in the multiscale model with the standard tip cell selection criterion (based on V; Equation 6 ) (i: 0% (standard condition), ii: 0. 1%, iii: 2%, iv: 10%); (C) Variation in the amount of VEGF addition in the multiscale model with an altered tip cell selection criterion (based on V9) (i: 0%, ii: 0. 1%, iii: 2%, iv: 10%); (D) Variation in the amount of VEGF addition in the hybrid model (i: 0%, ii: 0. 1%, iii: 2%, iv: 10%). doi:10. 1371/journal. pcbi. 1002724. g007 progressing in a more ''sheet-like'' fashion. Consequently, the tip cells persistently want to sprout towards already occupied grid cells, an action that is not allowed in the computational framework. The period after which the vascular development ceases, is shorter in higher VEGF environments. Note that the average VEGFR-2 concentration of all ECs in the callus reaches a constant level which is reduced in high VEGF environments (Figure 6 ). \n\nWhen a reduction of VEGF concentration by means of the addition of VEGF-antibodies is simulated, results demonstrate that the VEGFR-2 receptor is not sufficiently stimulated. This leads to an impaired vasculature and a non-union between the fractured bone ends (see Table 3, Equation 1 ). The simulated reduction in vascular density is consistent with the experimental findings of Street et al. who incorporated a fracture hematoma supernatant with neutralizing monoclonal antibody to human VEGF in a Matrigel vehicle, which was then implanted in a murine dorsal wound model [45]. There was a significant decrease in the number of blood vessels formed in the Matrigel vehicle with neutralizing monoclonal antibody when compared to the fracture hematoma supernatant alone [45].",
"section_name": "Impaired angiogenesis",
"section_num": null
},
{
"section_content": "The sensitivity analysis indicates that the model results are greatly influenced by the parameters s, d and V sink and that the MOSAIC model is insensitive to the initial conditions of Dll4 (D 0 ) and actin (A 0 ). An overview of the results of the sensitivity analysis can be found in Table 3. \n\nSimulations of the heterozygous knockout genotypes (d = 50%, s = 33%) show an increased sprouting due to a clearly reduced inhibition of the tip cell phenotype (Equations 2-3). Figure 5 demonstrates that the endothelial cells behind the brown (tip) cells with high VEGFR-2 levels are strongly inhibited in the normal case but are weakly inhibited in the simulated knockout. The overexpression of Dll4 (d = 200%) increases the inhibition of the tip cell phenotype resulting in a decrease of the vascular density (Table 3 ) and a delay in the endochondral bone formation process, particularly in the periosteal callus (Figure 8 ). The increase in s also causes a more potent suppression of the tip cell phenotype in the stalk cells, due to an increased down-regulation of VEGFR-2 by Notch1 (Equation 3 ). In turn, this leads to a decrease of the vascular density (see Figure 5B, Table 3 ). Thus, if the tip cell phenotype is more inhibited (by increasing d, which defines the enhancement of Dll4 expression due to VEGFR-2 activation (Equation 2 ) or increasing s, which represents the inhibition of the VEGFR-2 expression due to Notch1 activation (Equation 3 )), the vascular density is reduced. This simulation result corresponds to experimental observations [5, 46, 47]. Hellstro ¨m et al. [5] showed that the inhibition of Notch signaling (by inhibiting Notch receptor cleavage and signaling with c-secretase inhibitors, by heterozygous inactivation of the Notch ligand Dll4 or by endothelial cell specific deletion of Notch1) promotes an increase in the number of tip cells in the retina of newborn mice. Conversely, a 35% decrease in filopodia density and a 45% decrease in vessel density were found in a direct gain of function experiment of the Notch1 receptor. \n\nSimulating an increase of the decoy receptor VEGFR-1 (by decreasing V sink ) results in a reduction of the vascular density since less VEGF remains available for VEGFR-2 activation (see Figure 7A, Equation 1 ). This is consistent with Flt-1 (VEGFR-1) loss-and gain-of-function data in zebrafish embryos [34, 48]. Moreover, Street et al. showed that Flt-IgG treatment decreased the vascularity by 18% and impaired cortical bone defect repair in a murine femoral fracture healing model [34]. Similarly, the in silico results show a delayed or even impaired healing of the fracture due to the reduced vascular density. In Figure 7A (iv) the VEGF concentration is too low to activate the VEGFR-2 receptors which stops the angiogenic process. Since only a small amount of bone will be formed intramembranously in the fracture callus of Figure 7A (iv), the impaired vascularization will result in a nonunion. \n\nIf the threshold of VEGFR-2 activation V9*, below which the actin production and tip cell movement is inhibited, is increased, the vascular density decreases (see Table 3 ). The initial amount of actin (A 0 ) only slightly influences the final vascular density (see Table 3 ). The other variables related to the fracture healing, were not influenced. Similarly, the final vascular density is insensitive to the initial intracellular amount of Dll4 (D 0 ) (see Table 3 ).",
"section_name": "Sensitivity analysis",
"section_num": null
},
{
"section_content": "This study established a novel multiscale model of angiogenesis in the context of fracture healing, by integrating an agent-based model of tip cell selection [3] into a previously developed hybrid model of fracture healing [18]. The bone regeneration process was predicted by the MOSAIC model in accordance with experimental reports and previously validated in silico results [18]. The MOSAIC model was also able to capture many experimentally observed aspects of tip cell selection: the salt and pepper pattern seen in developing vascular structures under normal angiogenic conditions, i. e. a tip cell with high VEGFR-2 and actin levels followed by a stalk cell characterized by strong Notch1 signaling and therefore reduced VEGFR-2 and actin levels [30], an increased tip cell density and a higher vascular density in case of Dll4 heterozygous knockouts [5] and an excessive number of tip cells (leading to a very high vascular density) in high VEGF concentrations [3, 34]. The sensitivity analysis also indicated the most influential parameters of the MOSAIC model (d, s and V sink ). \n\nThis study has addressed some, but not all of the limitations of the Peiffer-model [18]. In the MOSAIC model the tip cell selection is based on Dll4/Notch1 signaling whereas the Peiffermodel [18] implemented sprouting with phenomenological rules such that\n\ni. e. in the Peiffer-model the VEGF concentration needs to be high enough (10 ng/ml), there needs to be a minimal separation of 100 mm between two tip cells and the movement direction of the new tip cell should make an angle of. 24u with the orientation of its mother vessel. Moreover, the Peiffer-model foresees three healing days between subsequent sprouting events, which has no experimental foundation and has been removed in the MOSAIC model. Consequently, the MOSAIC model is more mechanistic, allowing investigation of different mutant and druggable cases in the signaling pathways, leading to real predictions for experimentation, which was not possible in the Peiffer-model. Since the incorporation of the lateral inhibition mechanism leads to a denser plexus in the MOSAIC model than in the Peiffer-model, we have reduced the oxygen production rate by a factor of two so that the final oxygen concentrations are the same in both models. Experimental results show that the vascular plexus is indeed very dense in the fracture callus [36, 37]. \n\nIn the MOSAIC model the tip cell velocity increases with the active VEGFR-2 levels, indicating that both the level of VEGFR-2 and the external VEGF-concentration influence the tip cell speed. This is consistent with the experimental data of Arima et al. [49]. They used time-lapse imaging in a murine aortic ring assay (with and without VEGF) to quantify the behavior of the endothelial cells during angiogenic morphogenesis [49]. Arima et al. reported that VEGF-induced vessel elongation was only due to greater displacement per tip cell [49]. Moreover, treatment with Dll4antibodies also resulted in a greater displacement per tip cell [49]. This is due to the reduced inhibitory actions of Dll4, causing a greater number of cells to have high VEGFR-2 levels. These results are however contested by Jakobsson et al. who quantified the average migration speed of wild-type (DsRed and YFP) and heterozygous Vegfr2 +/egfp endothelial cells in different chimaeric embryoid bodies [50]. They observed no difference in migration speed, indicating that VEGFR-2 levels do not determine EC migration velocity [50]. Clearly, more research is necessary to elucidate the above observations and improve the current implementation of tip cell migration in future versions of the model. \n\nThe MOSAIC model indicates a key role of the decoy receptor VEGFR-1 (modeled via V sink ) (Equation 1 ). Increasing the amount of VEGFR-1, results in a decrease of the vascular density (Figure 7A ) which is also seen in loss-and gain-of-function data [34, 48]. Both the MOSAIC model and the model of Bentley et al. [3], use a constant value to represent the decoy-effect of the VEGFR-1 receptor. There is however experimental evidence that both VEGFR-1 and its soluble form are up-regulated in Notchactivated stalk cells [4, 51]. Hence, the stalk cells phenotype is not only consolidated by a decrease in VEGFR-2 but also by an increase in the competing VEGFR-1 receptor. The results of Krueger et al. also suggest that VEGFR-1 regulates tip cell formation in a Notch-dependent manner [48]. The vascular density is measured at three time points post fracture (7, 21 and 35 days). In the standard condition the parameter values are s = 47. 4, d = 6. 32, V sink = 0. 275, V9* = 200, A 0 = 5000, D 0 = 0, which can also be found in Tables 1 and 2. doi:10. 1371/journal. pcbi. 1002724. t003\n\nThe MOSAIC model displays interesting behavior in high VEGF environments (see Figure 7 ). Initially, the increase in VEGF has a positive effect, resulting in a very dense vasculature since the VEGFR-2 receptors are being more activated in this stimulating environment (+0. 1%; see Equation 1 ). This leads to a faster healing, which is also found experimentally [34, 42]. A further increase (+2%), however, reduces the vascular density in the MOSAIC model. This is consistent with the trend seen by Street et al. [34]. Note that a salt and pepper pattern of high and low VEGFR-2 levels is created and maintained in high VEGF environments (+2%; Figure 7B (iii) and Figure 9 ). To explain this observation, one needs to look at the beginning of the angiogenic process in the fracture callus. Initially, some endothelial cells gain the tip cell phenotype and start to migrate. Gradually sprouts arise in the developing vasculature which increases the network size and alters the local VEGF-levels due to the influence of the oxygen tension on VEGF-production. The original tip cells maintain their advantage (e. g. located in a higher VEGF environment) by strongly inhibiting their neighboring ECs, creating a salt and pepper pattern of VEGFR-2 levels. In standard conditions the vasculature would start to mature, leading to quiescent ECs. In high VEGF environments (+2%), however, this salt and pepper pattern of VEGFR-2 is maintained (Figure 9 ), illustrating that some ECs have (very) high VEGFR-2 levels leading to a persistent inhibition of their neighboring ECs (characterized by low VEGFR-2 levels). Figure 6 shows that these high VEGFR-2 levels are cancelled out by the low VEGFR-2 levels resulting in a ''steady state'' level of the average VEGFR-2 concentration. In high VEGF environments (+2%, +10%) this ''steady state'' level is gradually reduced (Figure 6 ), implying the dominance of the lower VEGFR-2 levels. Mathematically, this result follows from Equations ( 1 ) and ( 3 ), indicating that in high VEGF concentrations both the active VEGFR-2 (V9) and Notch (N9) (and with a delay the effective active VEGFR-2 (V0) and Notch (N0)) are high, resulting in a reduction of the VEGFR-2 receptor (Figure 10 ). Consequently, the average VEGFR-2 concentration is reduced below the threshold for tip cell formation (Figure 6 ). In other words, the majority of the ECs have too little VEGFR-2 receptors to assume the tip cell phenotype. In the extreme case, this finally results in the inhibition of the development of the vasculature since there are no tip cells to lead the sprouts towards the VEGF source (Figure 7B (iv)). \n\nInterestingly, Figure 7D shows that similar results cannot be obtained with the Peiffer-model [18], i. e. the vascular density is not reduced in high VEGF environments (+2%, +10%). This is due to the phenomenological rules that determine the tip cell selection in the Peiffer-model (Equation 9 ). A similar ''non-linear'' EC response to VEGF concentrations would only be possible with the Peiffer-model if another phenomenological rule would be implemented that e. g. down-regulates tip cell selection at high VEGF responses. In contrast, the ''non-linear'' response follows naturally from the mechanistic rules of tip cell selection that were implemented in the MOSAIC model. That is, the down-regulation of the tip cell selection in high VEGF environments (+2%, +10%) arises from the negative feedback loop in the Notch-Dll4 signaling pathway. Moreover, in high VEGF-environments (+10%) and at the back of the developing vasculature, we see an indication that patches of endothelial cells oscillate between cell fates (switching between high and low VEGFR-2 levels). These patches are also predicted by the model of Bentley et al. and are observed during pathological angiogenesis [3]. In the future, we will further investigate the conditions that give rise to these oscillations and their implications on the development of the vasculature. \n\nThe results of the MOSAIC model are based on the assumption that a tip cell phenotype can only be acquired if the levels of VEGFR-2 and actin are sufficiently high (Equation 6 ). If this criterion was changed by replacing the requirement on VEGFR-2 by a similar requirement for the level of active VEGFR-2, some ECs could become tip cells, since V9 is high in high VEGF environments (+2%, +10%), and the vasculature would fully develop (Figure 7C ). These results show the added value of the MOSAIC model: the intracellular module and its related state variables and rules decide on the EC response to the extracellular VEGF environment, in turn determining the healing response at the tissue level (Figure 8 ). In case the criterion for tip cell selection is specified in terms of VEGFR-2 (and actin) the absence of blood vessel formation will result in a non-union or a delayed union of the fracture. However, when this criterion is replaced by one that relies on the levels of active VEGFR-2 (and actin), a vascular and healing response is retrieved, similar to the Peiffer-model. \n\nClearly, these findings give rise to some interesting biological questions on a proper criterion for the tip cell phenotype. Since the VEGFR-2 levels are strongly reduced in high VEGF environments (+10%), the tip cells lose their tip cell phenotype and stop migrating although there is a strong angiogenic signal present. Can tip cells move in high VEGF environments although they do not have enough VEGFR-2 receptors? If so, should the tip cell criterion (Equation 6 ) be based on the active VEGFR-2 levels (V9), since these remain high in high VEGF concentrations? Or is the down-regulation of VEGFR-2 receptors in high VEGF environments compensated by other signaling cascades that have VEGFR-2 as one of their downstream targets (leading to an increase of VEGFR-2)?\n\nIn this study, the model of Peiffer et al. [18] was combined with a detailed model of Dll4-Notch1 signaling [3]. Some simplifications were however made to the model of Bentley et al. due to computational reasons, i. e. the size and shape of the ECs are fixed in the PDE framework of the MOSAIC model. Consequently, every EC is represented by one agent whereas Bentley et al. use a varying amount of membrane agents for every EC [3]. This does not only allow Bentley et al. to model the change in membrane and cell shape in great detail, but also to include cellular polarity (non-uniform distribution of receptors and ligands across the cell membrane and cell-cell junctions). In the MOSAIC model, filopodia extension is modeled implicitly by an increase in the level of the ''actin'' variable upon VEGFR-2 activation. This is consistent with current knowledge that activation of Cdc42 by VEGF triggers filopodia formation [4]. Bentley et al. modelled filopodia extension in more detail by adding membrane agents to the cellular membrane. As a result, the number of VEGFR-2 receptors will alter due to filopodia extension, which is proposed to be a mechanism to consolidate the tip cell fate [3]. In the MOSAIC model the accumulation of actin does not lead to an increase in the amount of VEGFR-2 levels or to a change in the microenvironmental range that can be probed by the tip cell. However, if the molecular mechanisms of filopodia extension and its implications on probing the environment and the directionality of tip cell movement are clearer, these can be readily incorporated in the multiscale framework. \n\nThe mechanism of lateral inhibition is based on Dll4/Notch1 signaling between the endothelial cells of the developing sprout. Delta-Notch signaling is however an evolutionary conserved pathway that is also involved in cell fate specification, tissue patterning and morphogenesis [2, 30, 52, 53]. In angiogenesis specifically, Notch signaling influences endothelial cell specification [4, 5, 30, 54, 55], endothelial proliferation [29, 30], cell migration [2, 30], filopodia formation [30], cell adhesion [30], and postangiogenic vessel remodeling and endothelial cell quiescence [56]. These effects are not only dependent on Dll4 and Notch1 but also on the other ligands (Delta-like 1, Delta-like 3, Jagged-1 and Jagged-2) and receptors (Notch2, Notch3 and Notch4) [57]. Due to the complexity and interdependency of these pathways, only the influence of Dll4-Notch1 signaling on tip cell selection was modeled. Consequently, in the model once the VEGF levels are reduced due to the restoration of the blood flow and tissue oxygenation, the Dll4-Notch1 signaling pathway is not active anymore. This is predicted to occur in the ECs that are located at the back of the vasculature, returning their VEGFR-2 levels to the maximal value. As mentioned before this contradicts the fact that in quiescent cells VEGFR-2 levels will be minimal and smaller than those of migrating cells, which is consistent with high Notch activity in quiescence [25] [26] [27]. Since the MOSAIC model does not include the role of Notch in quiescence, the simulation results are only accurate for the initial formation of the vasculature and not for the maturation and stabilization of the vascular plexus. This does not, however, alter the main findings of this work concerning sprouting angiogenesis. \n\nBesides VEGFR-2, also other VEGF receptors, such as VEGFR-1 and VEGFR-3 play a role in angiogenesis. Although the VEGFR-3 receptor is mainly active in lymphangiogenesis, recent experimental evidence indicates that VEGFR-3 is upregulated in tip cells during pathological angiogenesis [4, 58]. Blocking this receptor reduces the amount of sprouting and EC proliferation. It appears that VEGFR-2 induces VEGFR-3 expression in tip cells, whereas it is down-regulated in stalk cells by Notch [4, 59]. However, when more quantitative experimental data become available on the role of VEGFR-1 and VEGFR-3 in sprouting angiogenesis, this can be incorporated in the MOSAIC model. \n\nThe MOSAIC model only focuses on soluble VEGF, whereas VEGF-isoforms that bind to the extracellular matrix are essential to establish the VEGF gradients required for guided tip cell migration [60]. Some modeling work has already been done in this area [61, 62], e. g. Vempati et al. used a detailed molecular model of VEGF ligand-receptor kinetics and transport to investigate the VEGF-isoform specific spatial distributions observed experimentally [61]. Many other factors, such as neuropilin 1 (NRP-1), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) regulate the angiogenic response as well [2]. Nevertheless, it has been stated repeatedly that VEGF is ''the principal dancer'' during angiogenesis [29, 30]. \n\nThe proposed MOSAIC model incorporates biological processes at various temporal and spatial scales: an intracellular module that includes Dll4/Notch1 signaling to determine tip cell selection, a discrete representation of the ECs allowing an accurate representation of the developing vascular network and a continuum description of oxygen, growth factors and tissues that finally result in the healing of the fracture by the formation of bone. Our simulation results demonstrate the advantages of such a multiscale approach. Firstly, the interplay between molecular signals, in particular VEGF, Dll4 and Notch1, endothelial cell phenotypic behavior and bone formation was explored. In this way, the MOSAIC model could be used to verify to what extent gene knockouts, injection of VEGF-antibodies or blockage of VEGFreceptors leads to a ''bone phenotype'' in terms of rate and amount of bone formation (see e. g. Figure 8 ). While some of these simulation results could be (qualitatively) compared to experimental data, it is clear that future research efforts must be focused on a more comprehensive quantitative validation. Again, the multiscale nature of the simulation results presents an advantage here, as it allows for a validation at different scales (molecular, cellular and tissue scale). Secondly, the proposed multiscale model is more mechanistic since tip cell selection is based on intracellular dynamics (Dll4-Notch1 signaling), rather than the phenomenological rules that were used in Peiffer et al. [18]. As such, the MOSAIC model enabled to extend the model of Bentley et al. [3] to the context of fracture healing, leading to interesting emergent behavior at the macro-scale. More specifically, whereas the Peiffermodel predicts the presence of a vascular network in high VEGF environments (+10%) the MOSAIC model (depending on the tip cell criterion) predicts the absence of a vascular network (see Figure 7 ), which was a direct consequence of the Dll4-Notch feedback mechanism (see explanation related to Figure 10 ). In conclusion, the proposed multiscale method was found to be a useful tool to investigate possible biological mechanisms across different time and spatial scales, thereby contributing to the fundamental knowledge of sprouting angiogenesis and its relation to fracture healing.",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "These authors were supported by the following funding: AC: PhD fellow of the Research Foundation Flanders, LG: Funded by the Special Research Fund of the University of Lie `ge ( FRS. D-10/20 ), KB: Funded by the Artemis network grant, Foundation Leducq, PC: This work of PC is supported by the Belgian Science Policy (IAP # P6-30 ); and by long-term structural funding -Methusalem funding by the Flemish Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Supporting Information",
"section_num": null
}
] |
10.1038/s41408-023-00784-z
|
Risk of second primary malignancies in patients with chronic lymphocytic leukemia: a population-based study in the Netherlands, 1989-2019
|
<jats:title>Abstract</jats:title><jats:p>The longevity of patients with chronic lymphocytic leukemia (CLL) has improved progressively over the past decades, making it essential to understand long-term health outcomes, such as second primary malignancies (SPMs). Therefore, this nationwide, population-based study assessed the risk of SPM development in CLL patients diagnosed during 1989-2019 in the Netherlands compared to the expected number of malignancies in an age-, sex-, and period-matched group from the general Dutch population. In 24,815 CLL patients followed for 162,698.49 person-years, 4369 SPMs were diagnosed with a standardized incidence ratio (SIR) of 1.63 (95% confidence interval [CI] 1.59–1.68). This elevated risk was observed for solid (SIR, 1.67; 95% CI, 1.65–1.75) and hematological SPMs (SIR 1.42; 95% CI, 1.24–1.62). The highest risk for SPMs was noted beyond five years post-diagnosis (SIR, 1.70; 95% CI, 1.62–1.77), for male individuals (SIR, 1.70; 95% CI, 1.64–1.77), and patients aged 18–69 years (SIR, 1.92; 95% CI, 1.79–2.05). The risk of SPMs was higher in CLL patients who received anti-neoplastic therapy (SIR, 2.12; 95% CI, 1.96–2.28), as compared with those who did not (SIR, 1.58; 95% CI, 1.53–1.63). Routine surveillance activities and tailored interventions to counteract the increased morbidity and excess mortality associated with SPMs are essential for improving long-term outcomes in CLL patients.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is the most frequently diagnosed leukemia among adults in the Western world, with an age-standardized incidence rate ranging from 3. 8 to 5. 0 per 100,000 person-years as of the 2000s [1] [2] [3] [4] [5]. The clinical behavior of CLL is heterogeneous, ranging from an indolent disease with a tendency to remain stable for many years without therapy to a more aggressive illness that rapidly relapses after initial treatment. \n\nThe past decades have witnessed significant progress in managing patients with CLL. More specifically, the most notable therapeutic breakthrough was the introduction of chemoimmunotherapy, which translated into improved outcomes for CLL patients at the population level [1, [6] [7] [8]. More recently, novel agents such as ibrutinib and venetoclax entered the therapeutic realm of CLL. Due to the availability of more efficacious therapies in the upfront and relapsed setting, the longevity of CLL patients improved progressively over time [1, 8]. Nevertheless, excess mortality among long-term CLL survivors persists and remains a threat in modern times [8, 9]. \n\nAs the population of long-term CLL survivors is rapidly expanding, it is essential to understand long-term health outcomes. The development of second primary malignancies (SPMs) -i. e., cancers diagnosed after CLL-may contribute to morbidity and offset the improved longevity of CLL patients. Therefore, awareness of the nature and magnitude of SPMs in CLL is essential for health-related planning and surveillance activities [10] [11] [12] [13] [14]. The relative scintilla of population-based studies in CLL has shown an increased risk of SPM development compared to the general population [15] [16] [17] [18]. However, most of these studies have not investigated SPM development with long-term follow-up since the widespread use of chemoimmunotherapy in the 2010s and the most recent availability of novel targeted approaches. Also, most of these studies have included comparatively small cohorts with a short follow-up time. Therefore, to complement and extend the currently sparse literature on SPM development in CLL, this nationwide, population-based study aimed to assess temporal trends in SPM development -compared with an age-, sex-and period-matched group of the general populationin various subgroups of CLL patients in the Netherlands during a 30year period that takes into account the treatment advances of CLL during that period.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Established in 1989, the Netherlands Cancer Registry (NCR), which is maintained and hosted by the Netherlands Comprehensive Cancer Organisation (IKNL), has an overall coverage of at least 95% of all newly diagnosed malignancies in the Netherlands [19]. The NCR relies on comprehensive case notification via the Nationwide Network and Registry of Histopathology and Cytopathology and the National Registry of Hospital Discharges (i. e., inpatient and outpatient discharges). Basic information on dates of birth and diagnosis, sex, primary therapy, and disease stage, topography, and morphology of all newly diagnosed malignancies are routinely ascertained in the NCR by trained registrars of IKNL through retrospective review of medical records. Topography and morphology are coded as per the International Classification of Diseases for Oncology (ICD-O) [20]. Information on vital status (i. e., alive, dead, or emigration) is obtained through an annual linkage with the Nationwide Population Registries Network that holds this information for all residents in the Netherlands.",
"section_name": "PATIENTS AND METHODS The Netherlands Cancer Registry",
"section_num": null
},
{
"section_content": "All patients diagnosed with CLL between January 1, 1989, and December 31, 2019, were selected from the NCR using the ICD-O morphology code 9823. Patients diagnosed at autopsy (n = 71) were excluded from the analysis. Through cross-linkage with the NCR, SPMs diagnosed between 1989 and 2019 were identified. The ICD-O morphology and topography codes used to categorize specific SPM groupings are depicted in Supplemental Table 1 [20]. Basal cell carcinomas of the skin were excluded from the analysis because these malignancies were not standardly ascertained throughout the study period. Also, diffuse large B-cell lymphomas (DLBCL) and Hodgkin lymphomas were excluded since these lymphomas might have been misclassified as SPMs when they may be transformations of CLL (i. e., Richter's syndrome). Finally, synchronous SPMs diagnosed within six months after CLL diagnosis were excluded to minimize surveillance bias since these SPMs might be incidental findings rather than true SPMs. Patients with multiple and metachronous SPMs were counted only once in the analysis for all sites and all solid and hematological SPMs combined. However, these subsequent cancers contributed to the cancer site-specific analysis regardless of whether it was preceded by a malignancy from another site [21]. \n\nAccording to the Central Committee on Research involving Human Subjects (CCMO), this type of observational, non-interventional study does not require approval from an ethics committee in the Netherlands. The Privacy Review Board of the NCR approved the use of anonymous data for this study.",
"section_name": "Study population",
"section_num": null
},
{
"section_content": "The NCR generally ascertains information on primary therapy initiated within one-year postdiagnosis. For the overall analysis (i. e., 1989-2019), primary therapy was grouped into (i) no anti-neoplastic therapy, including a watch-and-wait approach, and (ii) anti-neoplastic therapy. The latter group was subdivided into chemotherapy alone and chemoimmunotherapy. Of note, the NCR ascertains the use of rituximab as of January 1, 2007. However, the use of rituximab before 2007 in the frontline management of CLL is presumed to be neglectable, as rituximab was initially introduced around 2010 for previously untreated CLL patients in combination with fludarabine and cyclophosphamide [22]. \n\nAs of January 1, 2014, information on the exact therapeutic regimens was registered in the NCR. These regimens were categorized as fludarabine, cyclophosphamide and rituximab (FCR), bendamustine and rituximab (BR), rituximab, cyclophosphamide, vincristine and prednisone (R-CVP), rituximab or obinutuzumab with chlorambucil (R-or O-Clb), chlorambucil monotherapy, ibrutinib, venetoclax, and other less frequently applied modalities. Of note, ibrutinib was reimbursed in the Netherlands as of late 2014 and venetoclax as of 2017 for previously untreated CLL patients harboring TP53 aberrations [23] [24] [25] [26].",
"section_name": "Primary therapy",
"section_num": null
},
{
"section_content": "Person-years at risk were calculated from the date of CLL diagnosis until SPM diagnosis, death, or end of follow-up (December 31, 2019), whichever occurred first. The risk time ended at the diagnosis of the first SPM of interest in the case of multiple SPMs within one patient. Standardized incidence ratios (SIRs) were computed as the ratio of observed SPMs to expected SPMs from the general population. The expected number of malignancies was based on age-, sex-, calendar, and site-specific cancer-incidence rates in the Dutch population, which were multiplied by the corresponding person-years at risk. The absolute excess risk (AER) represents the additional incidence of SPMs measured beyond the background incidence of SPMs found in the Dutch general population. The AER was calculated as the expected number of SPMs subtracted by the observed number of SPMs, divided by the person-years at risk and multiplied by 10,000, resulting in an AER per 10,000 person-years [27, 28]. Poisson distribution for the number of observed SPMs was assumed to calculate the 95% confidence intervals (CIs) for the SIR and AER. Unless otherwise stated, the SIRs and AERs were presented overall and according to age category (18-69 and ≥70 years), sex, the latency period for SPM development defined as the years from CLL diagnosis until SPM development (0. 5-5 and ≥5 years), calendar period (1989-1995, 1996-2002, 2003-2009, and 2010-2019) and the receipt of antineoplastic therapy (no versus yes). The calendar periods were used as a proxy for the evolution of therapeutic modalities over time. The criteria of non-overlapping CIs were used to show statistically significant differences between subgroups [27]. \n\nThe cumulative incidence of SPMs was evaluated, with death treated as a competing risk. The expected cumulative incidence in the general population was derived from the expected cancer incidence rates and expected overall mortality rates in the Dutch general population. The cumulative incidence was estimated for all sites, all solid and hematological SPMs, and individual SPM subtypes (Supplemental Table 1 ). \n\nA multivariable analysis was performed using the Fine and Grey method to analyze the effect of age (18-59, 60-69, 70-79, and ≥80 years), sex, calendar period (1989-1995, 1996-2002, 2003-2009, and 2010-2019), and receipt of anti-neoplastic therapy on the cumulative incidence of SPMs [29]. \n\nAll statistical analyses were performed with STATA Statistical Software version 17. 0 (StataCorp, College Station, TX) and SAS version 9. 4 (SAS Institute Inc, Cary, North Carolina, USA).",
"section_name": "Statistical analyses",
"section_num": null
},
{
"section_content": "Our analytic cohort included 24,815 CLL patients (61% males; median age 69 years; interquartile age range [IQR], 61-67 years) diagnosed in the Netherlands between 1989 and 2019. The baseline and primary treatment characteristics are presented in Table 1 according to the calendar period of diagnosis. Overall, the median follow-up period was 6. 2 years (IQR, 3. 2-10. 6 years), with 28% of the patients being followed for at least ten years. This overall follow-up period resulted in a total follow-up of 162,698. 49 person-years. \n\nIn the overall series, most patients did not receive antineoplastic therapy, including a watch-and-wait approach, within one year post-diagnosis (84%; Table 1 ). The use of anti-neoplastic therapy decreased with each successive calendar period, following a broader institution of a watch-and-wait approach. The gradual increase in the age-standardized incidence rate between 1989-1995 (3. 23 per 100,000 person-years) to 2003-2009 (4. 54 per 100,000 person-years) was followed by a stabilization during 2010-2019 (4. 53 per 100,000 person-years). This finding may suggest that the initial increase might be attributed to higher detection of early-stage CLL and consequently might explain the higher proportion of watch-and-wait approaches over time (Table 1 ). Of the patients receiving anti-neoplastic therapy as of 2007, 595 (37%) patients received chemotherapy and 893 (55%) chemoimmunotherapy. For patients diagnosed as of 2014 and receiving anti-neoplastic treatment, the majority of the patients received FCR (27%) followed by R-or O-Clb (26%), chlorambucil monotherapy (10%), R-CVP (9%), BR (9%), other, less frequently applied treatments (9%), venetoclax-based (5%), and ibrutinibbased treatment (4%).",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "During the follow-up period, 4,700 SPMs were diagnosed in 4,369 CLL patients. SPMs were diagnosed after a median follow-up period of 4. 2 years post-CLL diagnosis (IQR, 1. 6-7. 9 years) and at a median age of 74 years (IQR, 68-82 years). The cumulative incidence of SPM development was 29. 45% (95% CI, 28. 66%-30. 26%), 50. 84% (95% CI, 49. 20%-52. 52%) and 69. 18% (95% CI, 63. 50%-74. 71%) at 10, 20, and 30 years respectively (Table 1 ). The cumulative incidence of all SPM subtypes is depicted in Supplemental Fig. 1. \n\nOverall, the risk of developing an SPM was significantly higher among CLL patients compared to the general population (SIR, 1. 63; 95% CI, 1. 59-1. 68), resulting in 125. 06 excess malignancies per 10,000 person-years (Table 2 ). This increased risk of SPM development was observed for both solid (SIR, 1. 67; 95% CI, 1. 65-1. 75) and hematological SPMs (SIR, 1. 42; 95% CI, 1. 24-1. 62; Table 2 ). The risk was more than two-fold increased among CLL patients compared to the general population for squamous cell carcinomas of the skin (SIR, 4. 82; 95% CI, 4. 57-5. 07), acute myeloid leukemia (AML; SIR, 2. 75; 95% CI, 2. 08-3. 58), melanomas of the skin (SIR, 2. 74; 95% CI, 2. 43-3. 08), soft-tissue sarcomas (SIR, 2. 39; 95% CI, 1. 70-3. 27), and thyroid cancers (SIR, 2. 12; 95% CI, 1. 26-3. 35; Table 2 ). Although relative risks were doubled, the absolute risk for AML, soft-tissue sarcomas and thyroid cancers remained comparatively low-reflected by a 30-year cumulative incidence of 1. 90%, 1. 12%, and 0. 17%, respectively-owing to the low background risk for these malignancies in the general population (Table 2 and Supplemental Fig. 1 ). \n\nSquamous cell carcinomas of the skin contributed most to the overall excess risk (AER, 80. 31/10,000 person-years), representing 64% of the total excess risk, followed by melanomas of the skin (9%; AER, 11. 72/10,000 person-years), lung and bronchus cancer (8%; AER, 9. 98/10,000 person-years), colon and rectum cancer (5%; AER, 6. 13/10,000 person-years), and kidney cancer (3%; AER, 3. 36/ 10,000 person-years; Table 2 ).",
"section_name": "Risk of second primary malignancies as compared to the general population",
"section_num": null
},
{
"section_content": "The SIRs for any SPM were statistically higher for males (SIR, 1. 70; 95% CI, 1. 64-1. 77) than for females (SIR, 1. 55; 95% CI, 1. 46-1. 63). Also, the AER was nearly 2-fold higher for males than females (155. 20 versus 85. 10 per 10,000 person-years). The spectrum of SPMs for male and female CLL patients is depicted in Fig. 1. Generally, the spectrum of SPMs was comparable across the sexes except for the risk of soft-tissue sarcomas (SIR, 2. 74; 95% CI, 1. 73-3. 75) and non-Hodgkin lymphomas excluding DLBCLs (SIR, 1. 47; 95% CI, 1. 01-1. 93), which was only heightened in males. Although the SIRs for squamous cell carcinoma of the skin were increased for both sexes, the SIRs for males (SIR, 5. 22; 95% CI, 4. 91-5. 54) were significantly higher than for females (SIR, 3. 97; 95% CI, 3. 57-4. 36).",
"section_name": "Relative and absolute excess risk according to sex",
"section_num": null
},
{
"section_content": "Overall, the SIRs were the highest for patients aged 18-59 years (SIR, 1. 92; 95% CI, 1. 79-2. 06) compared with those aged 60-69 years (SIR, 1. 60; 95% CI, 1. 52-1. 68), 70-79 years (SIR, 1. 57; 95% CI, 1. 49-1. 65) and ≥80 years (SIR, 1. 57; 95% CI, 1. 44-1. 71; Supplemental Fig. 2A ). However, the absolute risk increased with advancing age, ultimately reaching 155. 24 excess malignancies per 10,000 person-years in patients aged ≥80 years (Supplemental Fig. 2B ). This inverse correlation is probably due to a lower background risk for developing SPMs in younger individuals. \n\nFor the site-specific analysis, the SIRs and AERs were reported for patients aged 18-69 years and ≥70 years (Fig. 2 ). The risk of colon and rectum carcinomas (SIR, 1. 30; 95% CI, 1. 01-1. 58) and urinary bladder and renal pelvis carcinomas (SIR, 1. 76; 95% CI, ). This elevation was noticeable from 2003 onwards and was attributed to a significantly higher risk for AML and myelodysplastic syndromes (MDS) in the latter period. Also, the risk of thyroid cancer was significantly higher in the most recent calendar period only. Conversely, the risk of lung and bronchus cancer, kidney cancer, soft-tissue sarcomas, and cancers with an unknown origin was higher among CLL patients in the earlier studied periods and lost significance in the latter calendar period. Of note, the risk of squamous cell carcinomas of the skin and melanomas of the skin remained significantly elevated throughout the entire study period. \n\nTrends of second primary malignancies according to the latency period Overall, the SIRs for any SPMs remained significantly higher throughout different latency periods among CLL patients than in the general population, even after 21-30 years post-diagnosis (SIR, 1. 86; 95% CI, 1. 47-2. 34; Supplemental Fig. 3A ). Also, the SIRs remained comparatively stable throughout different latency periods. However, the absolute risk was significantly lower for patients with a latency time of 0. 5-5 years (AER, 109. 00; 95% CI, 101. 77-116. 90) compared to the overall excess risk (AER, 125. 06; 95% CI, 119. 25-131. 23). The absolute risk increased steadily over the latency time, reaching 212. 04 excess malignancies per 10,000 person-years after more than 20 years of follow-up (Supplemental Fig. 3B ). \n\nFor the site-specific analysis, the SIRs and AERs were reported for a latency period of 0. 5-5 years and ≥5 years (Fig. 3 ). The spectrum of SPM subtypes varied across the latency periods. More specifically, the risk of colon and rectum cancers (SIR, 1. 31; 95% CI, 1. 15-1. 46), urinary bladder and renal pelvis cancers (SIR, 1. 31; 95% CI, 1. 03-1. 60), and thyroid cancers (SIR, 3. 08; 95% CI, 1. 27-4. 90) was significantly higher among patients with CLL that developed an SPM within 0. 5-5 years, as compared with the general population. On the other hand, the risk of developing a soft-tissue sarcoma (SIR, 2. 78; 95% CI, 1. 58-3. 99) was only elevated after a latency time of ≥5 years.",
"section_name": "Relative and absolute excess risk according to age",
"section_num": null
},
{
"section_content": "The SIRs and AERs were significantly higher in the patients who received anti-neoplastic therapy within one year post-diagnosis (SIR, 2. 12; 95% CI, 1. 96-2. 28) as compared to those who did not (SIR, 1. 57; 95% CI, 1. 52-1. 62), attributing to 204. 45 and 113. 36 excess cases per 10,000 person-years, respectively (Fig. 4 ). The risk of developing any hematological cancer (SIR, 3. 11; 95% CI, 2. 34-4. 03), AML (SIR, 7. 04; 95% CI, 3. 45-10. 62), MDS (SIR, 3. 75; 95% CI, 1. 20-6. 30), and non-Hodgkin lymphomas (SIR, 2. 90; 95% CI, 1. 32-4. 48) was significantly higher among patients who received anti-neoplastic therapy as compared to those who did not. Also, the risk of squamous cell carcinomas of the skin was significantly higher and more than 2-fold higher among CLL patients receiving treatment (SIR, 9. 38; 95% CI, 8. 27-10. 49) as compared with those that did not (SIR, 4. 32; 95% CI, 4. 08-4. 57). Of note, the risk of colon and rectum carcinomas was only elevated in untreated patients (SIR, 1. 21; 95% CI, 1. 10-1. 32). \n\nThe SIRs for patients treated with chemotherapy (SIR, 2. 16; 95% CI, 1. 98-2. 35) and chemoimmunotherapy (SIR, 2. 09; 95% CI, 1. 72-2. 52) were comparable. As for patients diagnosed with CLL between 2014 and 2019, the spectrum of SPM development per treatment category is listed in Supplemental Table S3. Of note, these data do not show the SIRs and AERs due to the comparatively low number of SPMs. \n\nMultivariable effects on second primary malignancy development within the cohort Next, we fitted a multivariable competing risk model for evaluating the effect of baseline patient characteristics and primary therapy on the risk of developing an SPM within our cohort (Table 3 ). The sHR of developing an SPM was higher in males (sHR, 1. 45; 95% CI, 1. 37-1. 54) and in patients who received anti-neoplastic therapy within one-year postdiagnosis (sHR, 1. 19; 95% CI, 1. 09-1. 27). The sHR of developing an SPM was higher in individuals aged 60-69 (sHR, 1. 39; 95% CI, 1. 29-1. 50) and 70-79 years (sHR, 1. 40; 95% CI, 1. 29-1. 51) as compared with those aged 18-59 years. Conversely, elderly patients ≥80 years had a Fig. 1 Risk of second primary malignancies among patients with chronic lymphocytic leukemia according to sex. Statistically significant standardized incidence ratios are presented in bold in the table and as solid dark blue dots in the forest plot and are scaled according to their magnitude. Abbreviations: AER, absolute excess risk; CI, confidence interval; and SIR, standardized incidence ratio.",
"section_name": "Effect of primary therapy on standardized incidence ratios",
"section_num": null
},
{
"section_content": "In this large, nationwide, population-based study with long-term follow-up, we observed that CLL patients have a 63% higher risk of developing any SPM than an age-, sex, and calendar periodmatched group from the general Dutch population. This risk for developing solid and hematological SPMs was 67% and 42% higher, respectively. This finding aligns with previously reported estimates from an Australian, Danish and U. S. study [15, 16, 18]. The spectrum of SPMs was also broadly comparable and mainly consisted of squamous cell carcinomas of the skin, melanomas of the skin, lung and bronchus cancer, colon and rectum cancer, softtissue sarcomas, AML, and thyroid cancer [14, 15, 18, 30]. The study from the U. S., which was based on data from the Surveillance, Epidemiology, and End Results (SEER) Program, reported the highest risk for Kaposi sarcomas with a SIR of 3. 82 (95% CI, 2. 19-6. 21). Since the number of patients diagnosed with Kaposi sarcoma within our cohort was less than 10, we did not incorporate it in our analysis. Differences in HIV incidence might cause a lower incidence of Kaposi sarcoma in the Netherlands than in the U. S. Indeed, the incidence of HIV in 2020 was 9. 2 and 2. 3 per 100,000 person-years in the U. S. and the Netherlands, respectively [31, 32]. Also, as shown in the U. S. study, we could not objectify the decreased risk of hepatobiliary, breast, and uterine cancers [15]. Since there is currently no clear pathophysiological explanation for these associations, this warrants further validation in forthcoming studies. \n\nThe SEER-based analysis reported the highest incidence of SPMs to be diagnosed between 2 and 6 months after the CLL diagnosis; thereafter, the incidence of SPMs was lower and remained comparatively stable [15]. Notably, we observed that the magnitude of the SIRs remained stable across the different latency periods. However, a significantly lower AER for SPMs was observed within 0. 5-5 years after the CLL diagnosis. This observation is probably related to excluding synchronous malignancies diagnosed within six months after the CLL diagnosis to minimize surveillance bias due to heightened medical care. Also, this finding highlights that a longer follow-up time is needed to capture the effect of impaired immune surveillance, environmental exposures, and chemotherapeutics on SPM development [33]. \n\nIn line with previous studies, we observed that the SIRs were the highest in younger individuals while the AER increased with advancing age, the latter being attributed to a greater background incidence of SPMs in the general population. On the other hand, we observed an increased cumulative incidence of SPMs in CLL patients aged 60-79 years. This finding suggests that the SPM risk progressively increases with age, which might be explained by the improved longevity and the accumulation of risk factors [15, 34, 35]. \n\nThe SIRs and AERs were higher in male CLL patients than in female CLL patients. The greater SPM risk in males is attributed to a higher risk of squamous cell carcinomas of the skin, which might be explained by a higher likelihood of males working outdoors with concomitant higher UV exposure [36, 37]. \n\nWe noted an increasing cumulative incidence of SPMs in the most recent calendar periods, driven mainly by a higher number of hematological cancers rather than an increase in solid cancers, Fig. 2 Risk of second primary malignancies among patients with chronic lymphocytic leukemia to age. Statistically significant standardized incidence ratios and absolute excess risk are presented in bold in the table and as solid dark blue dots in the forest plot and are scaled according to their magnitude. AER absolute excess risk, CI confidence interval, SIR standardized incidence ratio. \n\nwhich actually remained relatively stable over time. Among the hematological the risk of AML and MDS increased as of the 2000s, likely due to a higher application of fludarabine-based therapies from that time onwards. Indeed, this trend was previously described in the SEER-based analysis and in a singlecenter study in which patients were uniformly treated with FCR [12, 15]. In addition, the German CLL study group (GCLLSG) registry study demonstrated a higher-than-expected incidence of hematological SPMs (SIR, 3. 64; 95% CI, 1. 66-6. 90) in treated versus untreated CLL patients within prospective studies as compared with the German general population [38]. An excess of skin malignancies, including squamous cell carcinomas and melanomas, seems to characterize CLL patients, and the burden is known to increase with therapy [39] [40] [41]. Lastly, apart from surveillance bias, it is proposed that CLL-related therapy and the immune dysfunctional nature of CLL might enhance the effect of common carcinogens, such as UV exposure and smoking, in increasing the probability of skin and respiratory cancers [14, 33]. \n\nThe advent of targeted treatment approaches has transformed CLL management, which, in turn, has improved patient survival. As for the latter, long-term health risks, including SPMs, are becoming increasingly important because the improved longevity of CLL patients may be offset by these unwanted risks [42]. In patients treated with Bruton's tyrosine kinase inhibitors, the risk and the spectrum of SPMs were similar to that reported following chemotherapy or chemoimmunotherapy [11, 43]. In our cohort we could not calculate the SIRs for patients receiving novel approaches due to the detection of only two SPMs and a short follow-up time in the last calendar period (2014-2019) in which these agents have become available in the Netherlands [44]. When using the calendar period as a proxy for the evolution of treatment over time, the risk and the spectrum of SPMs were comparable for the 2003-2009 and 2010-2019 periods, suggesting that both the introduction of chemoimmunotherapy and, in part, targeted therapies did not dramatically alter the SPM landscape [45]. Therefore, future research is warranted to assess whether the broader application of targeted therapies might alter the SPM spectrum of solid and hematological cancers in patients with CLL. Also, future research with more detailed patient-level data on CLL-specific characteristics, the entire treatment landscape, and non-treatment-related exposures that may increase the risk of cancer development (e. g., tobacco use and UV exposure) should adopt multi-state modeling to explore sequences of these exposures on SPM development [46]. \n\nThe strength of our study is the use of population-based data from a comprehensive, long-running, and well-established cancer registry, enabling us to accurately quantify the risk of developing an SPM over a 30-year period post-diagnosis. Limitations concern the lack of information on the exposure to well-known carcinogens and the exact therapeutic regimens during the overall course of the disease. \n\nCollectively, identifying and managing SPMs is an essential part of the longevity of patients with cancer, especially in diseases with therapeutic advances that contribute to a noticeable improvement in survival, such as in CLL. Our findings can be used in shared decision-making about appropriate surveillance activities and interventions to counteract the increased morbidity and excess mortality associated with SPMs. The current study serves as a benchmark to assess how the spectrum of SPMs may alter with a broacher application of targeted therapies. \n\nFig. 3 Risk of second primary malignancies among patients with chronic lymphocytic leukemia according to the latency time. Statistically significant standardized incidence ratios and absolute excess risk are presented in bold in the table and as solid dark blue dots in the forest plot and are scaled according to their magnitude. AER absolute excess risk per 10,000 person-years, CI confidence interval, SIR standardized incidence ratio.",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "The data that support the findings of this study are via The Netherlands Comprehensive Cancer Organisation. These data are not publicly available, and restrictions apply to the availability of the data used for the current study. However, these data are available upon reasonable request and with permission of The Netherlands Comprehensive Cancer Organisation. \n\nFig. 4 Risk of second primary malignancies among patients with chronic lymphocytic leukemia according to the receipt of antineoplastic therapy. Statistically significant standardized incidence ratios and absolute excess risk are presented in bold in the table and as solid dark blue dots in the forest plot and are scaled according to their magnitude. AER absolute excess risk, CI confidence interval, SIR standardized incidence ratio.",
"section_name": "DATA AVAILABILITY",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "The authors would like to thank the registration clerks of the Netherlands Cancer Registry (NCR) for their dedicated data collection. The nationwide population-based NCR is maintained and hosted by the Netherlands Comprehensive Cancer Organisation (IKNL).",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "A. G. D. and Lvd. S. designed the study; Lvd. S. and M. A. W. D. analyzed the data; O. V. was responsible for the data collection; Lvd. S. wrote the manuscript with contributions from all authors, who also interpreted the data, and read, commented, and approved the final version of the manuscript.",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The authors declare no competing interests.",
"section_name": "COMPETING INTERESTS",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi. org/10. 1038/s41408-023-00784-z. \n\nCorrespondence and requests for should addressed to Lina van der Straten. \n\nReprints and permission information is available at http://www. nature. com/ reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "ADDITIONAL INFORMATION Supplementary information",
"section_num": null
}
] |
10.3324/haematol.10317
|
APRIL but not BLyS serum levels are increased in chronic lymphocytic leukemia: prognostic relevance of APRIL for survival
|
APRIL (a proliferation-inducing ligand) and BLyS (B lymphocyte stimulator) expression have been reported in chronic lymphocytic leukemia (CLL) cells. We examined APRIL and BLyS serum levels in CLL patients and evaluated the prognostic significance of APRIL expression on survival.
|
[
{
"section_content": "BLyS is a fundamental survival factor for transitional and mature B cells, whereas APRIL mainly affects B-1 cell activity, humoral responses and immunoglobulin class switching. 1 We recently reported that aging APRIL transgenic mice develop B-1 cell-associated tumors that are highly reminiscent of human CLL; our initial analysis showed increased levels of circulating APRIL in CLL patients (n=22) compared to healthy donors. 2 Two other reports described that BLyS and APRIL can act in an autocrine manner in CLL tumor cells, promoting cell survival. 3, 4 In addition, APRIL produced by inflammatory cells that infiltrate tumor lesions appears to contribute to disease in B cell lymphoma. 5 o further evaluate APRIL and BLyS implication in CLL, we performed a retrospective study of a cohort of 95 patients diagnosed with CLL according to NCI criteria. 6 fter informed consent was obtained, peripheral blood samples were collected from 1991 to 2005, aliquoted and frozen. As a control, 32 sera from age-and sex-matched healthy donors were processed in the same manner. The sera tested were of patients at different stages (A, B and C), but mostly (80%) of untreated patients at either stage A or B. Circulating APRIL was measured by ELISA as described 2 and the mean APRIL serum level was 10. 5 ng/ml in healthy donors (range=1. 8-25. 3 ng/mL; n=32) and 64. 5 ng/mL in CLL patients (range=1. 5-208. 5 ng/mL; n=95; p<0. 0001; Figure 1A ); 75% of all CLL patients showed an increase in circulating APRIL protein compared to controls. We also observed significant differences in APRIL levels in CLL patients grouped by Binet's staging; the mean APRIL serum level was 53. 5 ng/mL in stage A patients (n=57) and 79. 9 ng/mL in B/C patients (n=37; p=0. 02). \n\nBy contrast, the CLL patient sera tested by ELISA (R&D Systems) showed a moderate decrease in circulating BLyS levels compared to healthy controls (Figure 1B, p<0. 0001). This was similar to recent observations by Haiat et al. 7 Another study revealed increased BLyS serum levels only in CLL patients with a familial history of B-cell lymphoproliferative malignancies. 8 In our study, information on patient familial history was not available. Whether the observed decrease of circulating BLyS levels is relevant for the development of CLL awaits further studies. \n\nTo evaluate the clinical relevance of circulating APRIL on CLL, we divided the patient cohort according to the median APRIL serum level. Patients were considered APRIL high when serum APRIL concentration ≥56 ng/mL or APRIL low when APRIL concentration was <56 ng/mL. A description of our CLL patient population and distribution according to the median APRIL levels is shown in Table 1. Fisher's exact test showed no significant association between APRIL levels and Binet's stage or VH mutational status when all patients where considered. Nevertheless, when analyzing the association of APRIL levels and VH mutational status in subgroups defined by Binet's stage, we found that B/C patients with unmutated VH genes were associated with higher serum APRIL levels than B/C patients with VH mutated genes (p=0. 04). \n\nWe used Kaplan-Meier analysis to estimate the prognostic value of APRIL serum levels on overall survival (OS). APRIL high patients had a significantly poorer prognosis than those classified as APRIL low (survival probability of 53% and 94% respectively; p=0. 003) (Figure 1C ). Univariate Cox analysis confirmed that APRIL levels had a significant correlation with survival (p=0. 02) and multivariate Cox analysis using the three variables; VH status,",
"section_name": "",
"section_num": ""
}
] |
[
{
"section_content": "+ These authors share senior authorship *Institut de Génétique Moléculaire de Montpellier, UMR5535, Montpellier, France; °Department of Statistics and Operation Research, University of Jaén, Spain; # Laboratory for Experimental Oncology and Radiobiology, Amsterdam Medical Center, Amsterdam, The Netherlands; @ Service d'Hématologie Biologique, Groupe Hospitalier La Pitié-Salpêtrière, Paris, France Funding: MH and LPC were supported by Action Concertee Incitative Jeunes Chercheurs (ACI), Association pour la Recherche sur le Cancer (ARC), Fondation Recherche Médicale (FRM), Fondation de France and Ramón y Cajal Program. JPM was sponsored by a Dutch Cancer Society grant ( 2003-2812 ). Acknowledgments: we thank Sylvie Baudet, Martine Brissard, Patrick Bonnemye and Myriam Boudjoghra for their invaluable help, and Catherine Mark for her editorial assistance. Key words: LLC, APRIL serum levels, overall survival, prognostic factor. Correspondence: Lourdes Planelles, PhD, Department of Immunology and Oncology, Centro Nacional de Biotecnologia/CSIC, Darwin 3, UAM Campus de Cantoblanco, 28049 Madrid, Spain. Phone: international +34. 915854855. Fax: international +34. 9. 13720493. E-mail: lplanelles@cnb. uam. es",
"section_name": "",
"section_num": ""
}
] |
10.1186/bcr2347
|
The cytotoxicity of γ-secretase inhibitor I to breast cancer cells is mediated by proteasome inhibition, not by γ-secretase inhibition
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Introduction</jats:title> <jats:p>Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by γ-secretase. Since aberrant Notch signaling can induce mammary carcinomas in transgenic mice and high expression levels of Notch receptors and ligands correlates with overall poor clinical outcomes, inhibiting γ-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that γ-secretase inhibitor I (GSI I), Z-LLNle-CHO, is toxic to breast cancer cells both in vitro and in vivo. In this study, we compared the activity and cytotoxicity of Z-LLNle-CHO to that of two highly specific GSIs, DAPT and L-685,458 and three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib in order to study the mechanism underlying the cytotoxicity of Z-LLNle-CHO in breast cancer cells.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>Three estrogen receptor (ER) positive cell lines, MCF-7, BT474, and T47D, and three ER negative cell lines, SKBR3, MDA-MB-231, and MDA-MB-468, were used in this study. Both SKBR3 and BT474 cells also overexpress HER2/neu. Cytotoxicity was measured by using an MTS cell viability/proliferation assay. Inhibition of γ-secretase activity was measured by both immunoblotting and immunofluorescent microscopy in order to detect active Notch1 intracellular domain. Proteasome inhibition was determined by using a cell-based proteasome activity assay kit, by immunoblotting to detect accumulation of polyubiquitylated protein, and by immunofluorescent microscopy to detect redistribution of cellular ubiquitin.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>We found that blocking γ-secretase activity by DAPT and L-685,458 had no effect on the survival and proliferation of a panel of six breast cancer cell lines while Z-LLNle-CHO could cause cell death even at concentrations that inhibited γ-secretase activity less efficiently. Furthermore, we observed that Z-LLNle-CHO could inhibit proteasome activity and the relative cellular sensitivity of these six breast cancer cell lines to Z-LLNle-CHO was the same as observed for three proteasome inhibitors. Finally, we found that the cell killing effect of Z-LLNle-CHO could be reversed by a chemical that restored the proteasome activity.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>We conclude that the cytotoxicity of Z-LLNle-CHO in breast cancer cells is mediated by proteasome inhibition, not by γ-secretase inhibition.</jats:p> </jats:sec>
|
[
{
"section_content": "Notch is a family of single-pass type I transmembrane protein receptors that, in mammals, includes four homologs, Notch1 to 4 [1]. Ligand-induced Notch receptor activation requires at least two cleavages that release the intracellular domain from the cytomembrane and allow it to translocate into the nucleus where it activates its target genes [1]. The final cleavage is performed by γ-secretase, whose substrates include all four Notch receptors and their ligands as well as β-amyloid precursor protein, E-cadherin, CD44, ErbB-4, and ephrin-B1 [2] [3] [4] [5] [6] [7] [8]. \n\nAberrant Notch signaling can induce oncogenesis and may promote the progression of breast cancer. Transgenic mice overexpressing active Notch1, Notch3, or Notch4 homologs all developed mammary carcinoma [9, 10]. Furthermore, a recent clinical study reported that the expression level of DMEM: Dulbecco's modified eagle's medium; DMSO: dimethyl sulfoxide; ER: estrogen receptor; FBS: fetal bovine serum; GSI: γ-secretase inhibitor; N1ICD: Notch1 intracellular domain; N1EXT: Notch1 extracellular truncation; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; SD: standard deviation. \n\nNotch1, Notch3, and JAG-1, one of the Notch ligands, were inversely correlated with the overall clinical outcomes in breast cancer patients [11]. These observations have prompted great interest in targeting Notch signaling in breast cancer for therapeutic benefit. However, it should be noted that Notch2 signaling has been reported to function as a tumor suppressor in breast cancer cells [12]. \n\nAmong the several options to block Notch signaling, inhibition of γ-secretase by small molecules offers a promising approach and has been used extensively to study the downstream targets of the Notch signaling pathway [13, 14]. However, experimental data supporting the concept that γ-secretase inhibitors (GSIs) could inhibit the growth of, or kill, breast cancer cells have been scarce. Two recent reports have provided the strongest evidence by showing that Z-LLNle-CHO, commonly considered to be a GSI, has such an effect both in vitro and in vivo [15, 16]. \n\nProteasome inhibitors are a class of recent developed anticancer drugs. Z-LLNle-CHO, as a derivative of a widely used proteasome inhibitor MG-132, has been reported to inhibit chymotryptic protease activity, a core function of the proteasome [17]. In this study, we compared the activity and cytotoxic effects of Z-LLNle-CHO with those of two other widely used and highly specific GSIs, DAPT and L-685,458, and with those of three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib. Our results suggest that the cell killing effect of Z-LLNle-CHO is not mediated by γsecretase inhibition, but is mediated by proteasome inhibition.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Z-Leu-Leu-Nle-CHO (Z-LLNle-CHO, also called GSI I), N-(N-(3,5-Difluorophenacetyl-L-alanyl))-S-phenylglycine t-Butyl Ester (commonly called DAPT or GSI IX), (1S-Benzyl-4R-(1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl)-2R-hydroxy-5-phenylpentyl) carbamic acid tert-butyl ester (commonly called L-685,458 or GSI X), Z-Leu-Leu-Leualdehyde (Z-LLL-CHO, commonly referred to as MG132), lactacystin, and edaravone were purchased from Calbiochem (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO). Bortezomib was purchased from LC Laboratories (Woburn, MA, USA) and dissolved in DMSO. Tiron was from Sigma (St. Louis, MO, USA) and dissolved in water.",
"section_name": "Reagents",
"section_num": null
},
{
"section_content": "Three estrogen receptor (ER) positive cell lines, MCF-7, T47D, and BT474, and three ER negative cell lines, SKBR3, MDA-MB-231, and MDA-MB-468, were used in this study. Both SKBR3 and BT474 cells also overexpress HER2/neu. The culture medium was DMEM/F-12 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco) and GlutaMAX (Gibco) for all cell lines except SKBR3, which was cultured in McCoy's 5A medium (Gibco) supplemented with 10% FBS and GlutaMAX. In addition, MCF-7 culture medium was supplemented with non-essential amino acids (Gibco), sodium pyruvate (Gibco)and 10 μg/ml of insulin (Sigma). T47D culture medium was also supplemented with insulin (10 μg/ml). All cell lines were maintained at 37°C in a humidified atmosphere of 5% carbon dioxide in air.",
"section_name": "Cell culture",
"section_num": null
},
{
"section_content": "Cell viability and proliferation was measured using the Cell-Titer 96 ® AQ ueous One Solution Cell Proliferation Assay (MTS) kit (Promega, Madison, WI, USA). Cells (3000 to 8000 cells/ well) were seeded into 96-well plates in triplicate and allowed to attach overnight before being treated with increasing concentrations of the drugs. All wells, including the control, were exposed to the same concentration of DMSO to eliminate any possible effect of the vehicle on cell viability and proliferation. MTS reagent (20 μl) was added to each well 72 hours later and, after one to four hours incubation, the absorbance at 490 nm was measured using a microplate reader (FLUOstar OPTIMA from BMG LABTECH, Offenburg, Germany). Relative cell viability and proliferation of individual samples was calculated by normalizing their absorbance to that of the corresponding control sample. The mean and standard deviation (SD) of three independent experiments were used to plot dose-response curves. The concentrations that kill and/or inhibit cell growth by 50% (EC 50 ) were calculated from the equations that best fit the linear range of the dose-response curves.",
"section_name": "Cell viability and proliferation assay",
"section_num": null
},
{
"section_content": "Cells at 80% confluence were treated overnight with drugs at the indicated concentrations and control cultures received DMSO. The next day, cells were incubated with trypsin/EDTA (Gibco) solution for 10 minutes before collection by centrifugation. Cell pellets were then washed once with ice-cold PBS, lysed in lysis buffer (100 mM Tris-HCl (pH 6. 8), 10% glycerol, 2% SDS, 1 mM EDTA, 0. 002% bromophenol blue, 2 mM NaF, 1 mM Na 3 VO 4, 1 × protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA)), boiled for five minutes, and passed through a 21 gauge needle. The positive control samples were prepared in the same way as the GSI-treated samples and the negative control samples were prepared by adding the lysis buffer directly to the culture plates after washing with PBS without trypsin/EDTA incubation. Protein concentrations were quantified using a BCA protein assay (Pierce, Rockland, IL, USA).",
"section_name": "Protein sample preparation",
"section_num": null
},
{
"section_content": "Protein samples (50 μg/lane) were separated in 8% SDS-PAGE gels and transferred to Trans-Blot ® pure nitrocellulose membranes (0. 2 μm, Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% skim milk in TTBS (0. 1% Tween-20, 100 mM Tris-HCl (pH 7. 4), 150 mM NaCl) at room temperature for one hour before being probed overnight at 4°C with primary antibody solution. The primary antibodies used were anti-Notch1 (Val1744; Cell Signaling Technology, Danvers, MA, USA, 1:1000), anti-ubiquitin (clone FK2 from Millipore, Billerica, MA, USA, 1:1000) and anti-actin (Abcam, Cambridge, MA, USA, 1:5000). After washing with TTBS four times for 10 minutes each, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or antimouse (Jackson ImmunoResearch Laboratories, West Grove, PA, USA, 1:15,000) secondary antibody solution at 4°C for three hours. After another round of four washes with TTBS, the membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce), exposed to Fuji (Tokyo, Japan) film, and then developed to visualize the protein signal.",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "Synthetic DNA oligonucleotides corresponding to the cDNA encoding human Notch1 signal peptide flanked by restriction enzyme recognition sequences were integrated into pCMV-Tag4A vector (Stratagene, La Jolla, CA, USA) using Sac II/ BamH I sites. Then the cDNA encoding the amino acid residues 1721 to 2555 (corresponding to the substrate of γsecretase) was amplified using reverse transcription-coupled PCR of MCF-7 total cellular RNA and integrated into the vector containing the Notch1 signal peptide-encoding sequence using BamH I/EcoR I sites. The sequence of the new construct was verified by sequencing using T3/T7 primers.",
"section_name": "Construction of flag-tagged Notch1 extracellular truncation (N1EXT) vector",
"section_num": null
},
{
"section_content": "N1EXT plasmid DNA was transfected into MCF-7 and SKBR3 cells plated on glass coverslips using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Culture medium was replaced six hours after transfection with fresh medium containing 5 μM of DAPT, 2 μM of L-685,458, or Z-LLNle-CHO at the calculated EC 50 values of individual cell lines. After overnight incubation to allow the expression of exogenous protein, cells were fixed with 4% paraformaldehyde solution for indirect immunofluorescent microscopy.",
"section_name": "Transfection and treatment",
"section_num": null
},
{
"section_content": "Fixed cells were first permeabilized with 0. 5% Triton X-100 in PBS at room temperature for five minutes and then probed with anti-Flag monoclonal antibody (clone M2 from Sigma, 1:500) at room temperature for one hour. After five washes with PBS, cells were incubated with Alexa 488-conjugated goat anti-mouse secondary antibody (Molecular Probes, Carlsbad, CA, USA, 1:250) at room temperature for 45 minutes and further counterstained with 0. 5 μg/ml of DAPI after five washes with PBS. Images were taken using LSM 510 laser scanning confocal microscope with a Plan-Neofluar 40X/ 1. 3NA oil-immersion objective lens (Carl Zeiss, Jena, Germany). The optical slice thickness was less than 0. 9 μm.",
"section_name": "Indirect immunofluorescent microscopy",
"section_num": null
},
{
"section_content": "MCF-7 and MDA-MB-231 cells plated on glass coverslips were treated with drugs at the indicated concentrations for four hours before being fixed in 4% paraformaldehyde solution. Fixed cells were immunostained in the same way as above except that anti-ubiquitin monoclonal antibody (clone FK2 from Millipore, 1:1,000) was used as the primary antibody. Images were taken using LSM 710 laser scanning confocal microscope with a Plan-Apochromat 20X/0. 8NA objective lens (Carl Zeiss). The optical slice thickness was 1. 8 μm.",
"section_name": "Determination of ubiquitin distribution",
"section_num": null
},
{
"section_content": "Proteasome activity was measured using the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay kit (Promega, Madison, WI, USA). Briefly, MCF-7 (6000 cells/well) and MDA-MB-231 (10 4 cells/well) cells were plated into whitewalled 96-well plates. After overnight incubation to allow cell attachment, cells were treated with drugs at indicated concentrations for two hours. Equal volumes of Proteasome-Glo™ reagent were then added and the luminescence signal was measured using a microplate reader (FLUOstar OPTIMA).",
"section_name": "Proteasome activity assay",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We first compared the cytotoxicity of Z-LLNle-CHO to two other widely used GSIs, DAPT and L-685,458. Treatment with Z-LLNle-CHO resulted in a dose-dependent decrease in cell viability/proliferation of all six breast cancer cell lines tested with ER-negative cell lines being more sensitive than ER-positive cell lines. The calculated ED 50 values were 3. 25 μM, 2. 5 μM, 2. 4 μM, 1. 8 μM, 1. 6 μM, and 1. 4 μM for MCF-7, BT474, T47D, MDA-MB-231, SKBR3, and MDA-MB-468, respectively. However, DAPT and L-685,458 had no cell killing and/ or growth inhibitory effects at concentrations up to 5 μM and 2 μM, respectively (Figure 1 ).",
"section_name": "Among the three GSIs, only Z-LLNle-CHO induces cell death",
"section_num": null
},
{
"section_content": "We then examined whether the lack of cell killing/growth inhibition by DAPT and L-685,458 was due to their lower potency in inhibiting γ-secretase activity. To address this question, we first performed immunoblot analysis using an antibody that only recognizes cleaved Notch1 intracellular domain (N1ICD) [18, 19]. As N1ICD is a product of γ-secretase, its abundance is a good indicator of γ-secretase activity. However, the endogenous N1ICD level (the negative control lanes in Figure 2a ) is too low to be detected confidently. Therefore, we took advantage of the fact that calcium depletion activates Notch1 in the absence of ligand binding [20]. As shown in Figure 2a, DAPT at 5 μM and L-685,458 at 2 μM could block calcium depletion-induced Notch1 cleavage in all six cell lines. At the same time, Z-LLNle-CHO, at the concentrations that inhibited cell growth/viability by 50%, failed to do so to a comparable level in SKBR3 and MDA-MB-468 cells, although similar inhibition was observed in the other four cell lines treated with this drug. \n\nTo confirm the potency of DAPT and L-685,458 on inhibiting γ-secretase activity in intact cells, we transfected MCF-7 and SKBR3 cells with a plasmid expressing a Flag-tagged N1EXT fragment that mimics the immediate substrate of γ-secretase and then treated them with the same concentrations of GSIs as used for the western blot analysis. Without any intervention, the exogenous protein will be cleaved by γ-secretase as long as it is transported to the plasma membrane to produce N1ICD that can be visualized as nuclear signal when transfected cells are immunostained with an anti-Flag antibody (control panels in Figure 2b ). In contrast, when γ-secretase activity is inhibited, the exogenous protein cannot be cleaved and therefore will accumulate at the plasma membrane. As shown in Figure 2b, all the DAPT-and L-685,458-treated cells and Z-LLNle-CHO-treated MCF-7 cells showed exclusively membrane signal. However, 24% and 58% of Z-LLNle-CHOtreated SKBR3 cells displayed mainly nuclear signal or a mixture of nuclear and plasma membrane signal, respectively. This is consistent with the immunoblotting data demonstrating that DAPT and L-685,458 could completely inhibit γ-secretase activity at tested concentrations in both cell lines but Z-LLNle-CHO could only do so in MCF-7 cells (Figure 2c ). \n\nTaken together, because complete inhibition of γ-secretase activity by two structurally unrelated GSIs had no effect on cell viability and proliferation, it is unlikely that the cell killing/ growth inhibitory effect of Z-LLNle-CHO on breast cancer cell lines was mediated by γ-secretase inhibition.",
"section_name": "All three GSIs inhibit γ-secretase activity",
"section_num": null
},
{
"section_content": "Z-LLNle-CHO is derived from a widely used proteasome inhibitor MG132 (Z-LLL-CHO) and has been reported to be a broad chymotryptic and aspartyl protease inhibitor [17]. Therefore, we examined whether Z-LLNle-CHO also has proteasome inhibitor activity at the concentrations that showed dose-dependent cytotoxicity. We first used a cell-based proteasome activity kit to measure proteasome activity after cells were treated with MG132, Z-LLNle-CHO, or DAPT. As shown in Figure 3b, both Z-LLNle-CHO and MG132 showed a dosedependent inhibition of the proteasome at concentrations that showed cytotoxic effects, although DAPT did not. Next, we examined whether or not inhibition of proteasome activity caused accumulation of polyubiquitinated protein, one of the major causes of proteasome inhibitor-induced cell death [21], by subjecting the protein samples from cells treated with 5 μM (MCF-7) or 2. 5 μM (MDA-MB-231) of Z-LLNle-CHO overnight to immunoblotting with an anti-ubiquitin antibody. We used bortezomib, a specific proteasome inhibitor that has been approved to treat multiple myeloma in patients, as the positive control. The results showed that treatment with Z-LLNle-CHO indeed resulted in the same accumulation of polyubiquitinated protein that was observed with bortezomib (lane 2 and 5 of Figure 3b ). Finally, we took advantage of a recent observation that when proteasome-mediated protein degradation was inhibited, cellular ubiquitin would undergo a nuclear-to-cytoplasmic redistribution that could be detected by anti-ubiquitin FK2 antibody [22]. In untreated MCF-7 and MDA-MB-231 cells, FK2 staining showed dominant nuclear signal (Figure 3c ). After a four hour treatment with either bortezomib or Z-LLNle-CHO but not with DAPT, cells displayed a strong cytoplasmic ubiquitin signal, confirming proteasome activity was inhibited by Z-LLNle-CHO.",
"section_name": "Z-LLNle-CHO has proteasome inhibitory activity",
"section_num": null
},
{
"section_content": "We next asked whether or not the cell killing effect of Z-LLNle-CHO is mediated by its proteasome inhibition activity. If this is The redistribution of nuclear ubiquitin to cytoplasm is a phenomenon that can be induced by proteasome inhibition. \n\nnumber not for citation purposes) the case, the relative cellular sensitivity of different breast cancer cell lines to Z-LLNle-CHO should reflect that produced by other proteasome inhibitors. Therefore, we treated the same six breast cancer cell lines with increasing doses of three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib, and measured the effects on cell viability/proliferation using the MTS assay. Similar to the results shown in Figure 1, ER-positive cell lines were more resistant to all the three proteasome inhibitors than ER-negative cell lines (Figure 4 ). In addition, our results were also consistent with a previous study using bortezomib alone [23]. These data strongly suggest that the cell killing effects of Z-LLNle-CHO in breast cancer cells is mediated by its proteasome inhibitory function.",
"section_name": "The cellular sensitivity of six breast cancer cell lines to Z-LLNle-CHO is the same as that to proteasome inhibitors",
"section_num": null
},
{
"section_content": "Recent studies showed that the proteasome inhibitory activity as well as the cell killing effects of bortezomib and MG132 could be specifically blocked by two antioxidants, tiron and edaravone, respectively [24, 25]. As Z-LLNle-CHO is structurally similar to MG132, we speculated that edaravone might also be able to reverse the cytotoxicity of Z-LLNle-CHO by blocking its proteasome inhibition activity. Therefore, we first treated MCF-7 and MDA-MB-231 cells with different combinations of bortezomib or Z-LLNle-CHO and tiron or edaravone, and then measured cell growth using the MTS assay. Consistent with previous studies, tiron but not edaravone rescued cells from bortezomib-induced cell killing. Most importantly, we found edaravone but not tiron could rescue cells from Z-LLNle-CHO-induced cell killing (Figure 5a ). \n\nNext, we tested whether or not edaravone could rescue proteasome activity from Z-LLNle-CHO-induced inhibition. We exposed cells to edaravone at the concentration that showed best cell growth rescue in the presence of Z-LLNle-CHO and measured proteasome activity using the three approaches we used above. We used tiron to reverse bortezomib-induced proteasome inhibition as a control. We found that edaravone indeed rescued the proteasome activity from Z-LLNle-CHOinduced, but not bortezomib-induced, inhibition. Although the proteasome activity of edaravone rescued from Z-LLNle-CHOinduced inhibition was not to the same extent as tiron rescued bortezomib-induced inhibition in the cell based proteasome assay (Figure 5b ), the rescued proteasome activity was enough to prevent the accumulation of polyubiquitinated proteins (lane 4 compared with lane 2 in Figure 3b ) and redistribution of cellular ubiquitin (Figure 3c, treatment 4 vs. treatment 2). In addition, we found edaravone also partially restored γsecretase activity from Z-LLNle-CHO-induced inhibition (Figure 5c ).",
"section_name": "The cytotoxicity of Z-LLNle-CHO can be reversed by a specific antioxidant that restores proteasome activity",
"section_num": null
},
{
"section_content": "To investigate whether the cytotoxicity of Z-LLNle-CHO to breast cancer cells is due to the summation or synergy of its dual activities, we tested whether a combination of a specific γ-secretase inhibitor with a specific proteasome inhibitor could produce an additive or synergetic effect on cell killing. We subjected cells to increasing concentrations of lactacystin with or without 5 μM of DAPT that completely inhibited γsecretase activity in the cell lines tested. We found the doseresponse curves of individual cell lines treated with or without DAPT was almost identical (Figure 6 ), which suggests there was no additive or synergetic effects of inhibiting both γ-secretase activity and proteasome activity. Therefore, γ-secretase inhibitory activity of Z-LLNle-CHO most likely does not contribute to its cell killing effect in breast cancer cells.",
"section_name": "γ-secretase inhibition activity of Z-LLNle-CHO does not contribute to its cytotoxicity to breast cancer cells",
"section_num": null
},
{
"section_content": "Blocking Notch signaling by inhibiting γ-secretase activity with small molecules has been suggested to be a promising\n\nThe relative sensitivity of six cell lines to three proteasome inhibitors is the same as that to Z-LLNle-CHO The relative sensitivity of six cell lines to three proteasome inhibitors is the same as that to Z-LLNle-CHO. Cells were treated with MG132, bortezomib, or lactacystin at indicated concentrations for 72 hours before cell viability was measured by MTS assay. Results represent the mean ± standard deviation of three independent experiments. Please note the relative cellular sensitivity of the same six breast cancer cell lines to three structurally unrelated proteasome inhibitors was the same as that to Z-LLNle-CHO in Figure 1. \n\napproach to battle breast cancer [13, 14]. In fact, there are three ongoing clinical trials registered at ClinicalTrials. gov using GSIs in the treatment of breast cancer. However, experimental data supporting the effectiveness of GSIs in the inhibition of cell growth or killing of breast cancer cells have been scarce. Two recent reports, however, have now shown that Z-LLNle-CHO, commonly called GSI I, has such an effect both in vitro and in vivo [15, 16]. \n\nIn the present study, we first compared the cytotoxicity and activity of Z-LLNle-CHO with two other popularly used GSIs, DAPT and L-685,458. We found that completely inhibiting γ- The cytotoxicity effect of Z-LLNle-CHO could be reversed by edaravone that blocks its proteasome inhibitory function The cytotoxicity effect of Z-LLNle-CHO could be reversed by edaravone that blocks its proteasome inhibitory function. (a) Cells were treated with indicated drugs for 72 hours before cell growth was measured using MTS assay. Results represent the mean ± standard deviation of independent experiments. (b) Proteasome activity in intact cells was directly measured using a cell-based assay after cells were treated with different combinations of drugs for two hours. The treatment conditions were (from left to right): dimethyl sulfoxide (DMSO) vehicle only; bortezomib alone; bortezomib plus tiron; bortezomib plus edaravone; Z-LLNle-CHO alone; Z-LLNle-CHO plus tiron; and Z-LLNle-CHO plus edaravone. The concentrations of bortezomib, tiron, edavarone, and Z-LLNle-CHO are 100 nM, 2 mM, 100 μM, and 5 μM for MCF-7 cells, and 40 nM, 0. 5 mM, 100 μM, and 2. 5 μM for MDA-MB-231 cells, respectively. Results represent the mean ± SD of three independent experiments. (c) The same protein samples used for immunoblotting in Figure 3b plus another negative control sample were subjected to immunoblotting with anti-Notch1 (V1744) antibody that specifically recognizes active Notch1 intracellular domain. The order of the samples were the same as that in Figure 3b except that lane 1 is the new negative control sample. \n\nnumber not for citation purposes) secretase activity by DAPT and L-685,458 had no effect on cell viability and proliferation of a panel of six breast cancer cell lines with different genetic backgrounds. In contrast, Z-LLNle-CHO could cause cell death even at concentrations that did not completely inhibit γ-secretase activity. Therefore, we conclude that the cell killing effect of Z-LLNle-CHO on breast cancer cells is not mediated by γ-secretase inhibition. \n\nWe next measured the proteasome inhibition potential of Z-LLNle-CHO. In contrast to two previous reports that Z-LLNle-CHO at concentrations that inhibited cell growth did not significantly inhibit proteasome activity (see supplemental materials in [15, 26] ), we found that it could inhibit proteasome activity by about 50% in intact cells even at a concentration that did not show significant cytotoxicity in two cell lines tested. Our result is consistent with a recent study that was published during the revision of this manuscript [27]. The new study showed that Z-LLNle-CHO at about 0. 3 μM (calculated by us based on scale) inhibited proteasome activity by 80% and slowed cell growth by 20% in MCF-7 cells. As the approach the new study used to measure proteasome activity is different from ours, the extent of proteasome activity inhibition cannot be compared between their data and ours. However, both studies show that Z-LLNle-CHO could significantly inhibit proteasome activity at concentrations that showed dose-dependent cytotoxicity. The previous two studies used the same method to measure proteasome activity as the latest study but differed from ours. Therefore, it is easy to explain the discrepancy between their data and ours but we cannot explain the discrepancy between their data and the latest study. \n\nFurthermore, we found that the relative cellular sensitivity of six breast cancer cell lines to Z-LLNle-CHO was the same as that to three widely used but structurally unrelated proteasome inhibitors and was also consistent with a previous study [23]. This consistency strongly suggests that the cell killing effect of Z-LLNle-CHO is due to its proteasome inhibitory function. Most convincingly, we found that the cytotoxic effect of Z-LLNle-CHO could be reversed by a specific antioxidant that blocked its proteasome inhibitory activity. Finally, we tested but did not find any additive effect of the combination of a specific γ-secretase inhibitor and a specific proteasome inhibitor on breast cancer cell growth. Therefore, we conclude that the cytotoxicity of Z-LLNle-CHO to breast cancer cells is mediated by proteasome inhibition. \n\nWe noticed that edaravone treatment also partially rescued γsecretase activity from Z-LLNle-CHO-induced inhibition. However, because inhibition of γ-secretase alone or in combination with proteasome inhibition had no effect on cell survival/proliferation or cellular response to proteasome inhibition, we do not consider partially restored γ-secretase activity as a major contributor to the reversion of the cytotoxicity induced by Z-LLNle-CHO. Likewise, although edaravone has been reported to protect cells from apoptosis by acting as an antioxidant [28, 29], we do not think its free radical scavenging activity is a major contributor because it had no effect on bortezomibinduced cytotoxicity. Therefore, its ability to restore proteasome activity through unknown mechanism(s) most likely accounts for the reversion of the cytotoxicity of Z-LLNle-CHO. \n\nBoth previous studies used transient transfection of N1ICD to rescue the cell death induced by Z-LLNle-CHO treatment and argued that the reversion of the phenotype by N1ICD transfection indicated that Z-LLNle-CHO induced cell death through inhibiting Notch signaling pathway [15, 16]. However, transient overexpression of N1ICD has been reported to inhibit wild-type p53-induced apoptosis in immortalized epidermal cells [30], to inhibit dexamethasone, etoposide, or Fas-ligandinduced apotosis in mature T-cells [31], and to protect H460 (lung cancer), HepG2 (liver cancer), and HT1080 (fibrosarcoma) from several chemotherapy drugs [32]. Therefore, an alternative interpretation of the data from the two previous studies is that N1ICD over-expression provided pro-survival signals that antagonize the pro-apoptotic effects of Z-LLNle-CHO. \n\nIt is worthy noting that many of the effects of Z-LLNle-CHO reported in previous studies, including G2/M arrest and regulation of apoptosis-related protein, are consistent with the reported effects of other proteasome inhibitors [33] [34] [35] [36] [37]. In addition, similar to the additive effects of 4-OH-TAM and Z-LLNle-CHO on the inhibition of T47D:A18 cells growth [15], additive or even synergistic effects have also been reported between tamoxifen and bortezomib in some but not all ER-positive breast cancer cell lines tested [38, 39]. Although the sim- ilarities between the biological effects of Z-LLNle-CHO and those of other proteasome inhibitors do not necessarily mean that they function the same, our finding that Z-LLNle-CHO inhibits breast cancer cell growth as a proteasome inhibitor can explain the data produced with the use of Z-LLNle-CHO in previous studies. \n\nIt should be pointed out that although the latest study by Rasul and colleagues found that Z-LLNle-CHO has proteasome inhibitory function at concentrations that showed dosedependent cytotoxicity [27], the authors did not consider its proteasome inhibitory activity as the major contributor to its cell killing effects because the cytotoxicity of Z-LLNle-CHO and MG132 was 'markedly different', although their proteasome inhibition potential was similar. However, by careful analysis of their data, we found that the proteasome inhibition potentials of Z-LLNle-CHO and MG132 differed by more than two-fold, not less than the difference in cytotoxicity, within the range of concentrations that Z-LLNle-CHO and MG132 showed 'markedly different' cytotoxicity (below 0. 6 μM). Most importantly, Z-LLNle-CHO at 0. 75 μM in their study slowed MCF-7 cell growth by 80%, but only inhibited γ-secretase activity by 25%. Meanwhile, it inhibited proteasome activity by 80%. Therefore, their data is more consistent with our conclusion that the cytotoxicity of Z-LLNle-CHO was not due to γsecretase inhibition, but due to proteasome inhibition. \n\nThe observation that both Z-LLNle-CHO and MG132 at given concentrations inhibited proteasome activities to comparable levels in MCF-7 and MDA-MB-231 cells, but showed different cytotoxicity, is not surprising because this has also been observed for bortezomib [23]. The reduced sensitivity of ERpositive MCF-7 cells may be a consequence of pro-survival signal provided by the ER signaling pathway in these ER-positive breast cancer cells. This hypothesis is consistent with the observed additive or even synergistic effect between tamoxifen and Z-LLNle-CHO or bortezomib. However, this requires further investigation. Regardless of the mechanisms, our results, together with the previous reports, suggest that the future clinical trials testing the effectiveness of proteasome inhibitors in treating breast cancer should take the ER status into consideration when enrolling patients. \n\nThe observation that two specific GSIs, DAPT and L-685,458, had no effect on the survival and proliferation of breast cancer cells does not eliminate the potential use of GSIs or other approaches to block Notch signaling for breast cancer treatment. The results presented here were obtained from in vitro cell culture experiments. The effects of GSIs on the tumors grown in vivo, where the Notch signaling might be more active due to enhanced ligand-receptor interaction, could be different and need further investigation. Alternatively, these drugs might block the signaling pathway of some as yet unidentified substrate(s) which antagonizes the effect of reduced Notch1 signaling on breast cancer cell survival and proliferation. There are at least a dozen known γ-secretase substrates and most of the available GSIs have no preference for specific substrates. Rather than laboriously testing all potential candidates that antagonize Notch1, it might be better to develop substratespecific GSIs. To this end, it is encouraging to note that compounds that can preferentially modulate γ-secretase activity against Aβ42 over Notch have recently been reported [40]. These compounds target the substrate (Aβ42) rather than the γ-secretase active site itself. In principle, it should also be possible to find drugs that target individual Notch homologs. Alternatively, it might be useful to develop neutralizing antibody against individual Notch homologs just as the trastuzumab targets HER2/neu. Furthermore, the results of this study do not diminish the potential use of Z-LLNle-CHO for breast cancer treatment. In fact, we believe that clarifying its role as a proteasome and γsecretase dual inhibitor will help to direct its potential development for clinical use. However, we do caution that results obtained using Z-LLNle-CHO as the sole GSI to study the biological outcomes of blocking Notch signaling [41] [42] [43] should be interpreted cautiously or reproduced using more specific GSIs.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "The present study demonstrated that the cytotoxicity of Z-LLNle-CHO toward breast cancer cells was not mediated by γ-secretase inhibiton as reported previously, but by proteasome inhibition. This clarification might help its potential development as a chemotherapeutic agent. The results presented also call for careful interpretation of data produced with using Z-LLNle-CHO as the sole γ-secretase inhibitor.",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This project has been made possible through a grant from the Alberta Cancer Research Institute (ACRI) and the Alberta Cancer Foundation. Jianxun Han is supported by an ACRI Graduate Studentship and the CIHR Translation Research Training in Cancer program. Michael Hendzel is an Alberta Heritage Foundation for Medical Research Senior Scholar. We would like to thank Dr Gordon Chan for sharing reagents and helpful discussions and Bonnie Andrais for tissue culture. We are grateful to Dr Xuejun Sun and the Cell Imaging Facility at Cross Cancer Institute for technical support.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "JH participated in the conception of the study and its design, performed most of the experiments, and wrote the first draft of the manuscript. IM performed cell viability/proliferation assays. MJH and JAT participated in the conception of the study and its design and drafted the final manuscript. All authors read and approved the final manuscript.",
"section_name": "Authors' contributions",
"section_num": null
}
] |
10.18632/oncotarget.6769
|
Combination therapy of RY10-4 with the γ-secretase inhibitor DAPT shows promise in treating HER2-amplified breast cancer
|
RY10-4, a novel protoapigenone analog, shows potent cytotoxicity against human breast cancer cells. However, breast cancer cell lines overexpressing human epidermal growth factor receptor 2 (HER2), SKBR3 and BT474, showed less sensitivity to RY10-4 when compared to breast cancer cells lines expressing lower levels of HER2, such as MDA-MB-231 and MCF-7 cells. This was associated with aberrant hyperactivity in Notch signaling in cells treated with RY10-4, since treatment with RY10-4 causes an increase in Notch activity by 2-to3.5-fold in SKBR3 and BT474 cell lines. The increase in activity was abrogated with a γ-secretase inhibitor, DAPT, or with Notch1 small-interfering RNA (si-Notch1). Cell proliferation was inhibited more effectively by RY10-4 plus DAPT or si-Notch1 than either agent alone. RY10-4 plus DAPT increases apoptosis in both HER2-overexpressing cell lines by two-fold compared to RY10-4 alone, while DAPT alone has no significant effects on apoptosis. In addition, we previously found RY10-4 could inhibit tumor growth through the PI3K/AKT pathway. Here we report that the combination of RY10-4 and DAPT exhibit additive suppression on AKT phosphorylation, contributing to the anti-cancer effects. In an animal model, this combination therapy inhibits the growth of SKBR3 tumor xenografts in nude mice to a greater extent than treatment with either reagent alone. These results indicate that the aberrant activation of Notch signaling impedes the inhibitory effect of RY10-4 on HER2-amplified cell proliferation. Furthermore, these adverse effects can be prevented by treatment combining RY10-4 with a Notch pathway inhibitor.
|
[
{
"section_content": "Breast cancer is the most commonly diagnosed malignancy among women, and it is a leading cause of cancer death in females from western countries [1]. Novel strategies for prevention and treatment of breast cancer are needed to minimize off-target drug-related toxicities and to ultimately enhance patient outcomes. Protoapigenone (Figure S1A ), isolated from torres's ferns [2, 3] is a flavonoid that displays potent antitumor activity against a broad spectrum of human cancer cell lines [2, 4, 5]. This compound contains an unusual nonaromatic B-ring. Based on this, we developed a novel compound RY10-4 (Figure S1B, Publication Number: CN 102731456 B), which is structurally related to protoapigenone but which exhibits better antitumor activity and fewer side effects compared to protoapigenone in vitro and in vivo [6]. Our recent studies show that different human breast cancer cell lines display variable sensitivity to RY10-4. RY10-4 exhibits comparable growth-inhibitory effects on the Combination therapy of RY10-4 with the γ-secretase inhibitor DAPT shows promise in treating HER2-amplified breast cancer www. impactjournals. com/oncotarget triple-negative cell line MDA-MB-231 and the estrogen receptor (ER)-positive cell line MCF-7. The HER2-positive cell lines SKBR3 and BT474 exhibit similar inhibitory effects but less sensitivity than the other two. \n\nNotch signaling is one of the most important signaling cascades involved in drug resistance in tumor cells. Notch genes encode transmembrane receptors that are highly conserved from invertebrates to mammals. These receptors interact with ligands expressed by adjacent cells to regulate cell fate specification, differentiation, proliferation, and survival [7]. The Notch system in vertebrates is comprised of four receptors (Notch1-4) and at least five ligands from the families Delta and JAG/Serrate (DSL): Deltalike(Dll)-1, Dll-3, Dll-4, JAG1, and JAG2 [8, 9]. In breast cancer patients who received tomoxifen treatment, the activity of Notch signaling in tumor tissue correlates with drug resistance and poor prognosis [10]. Also, in a mouse model, the Notch1 pathway promotes acquired resistance to tamoxifen in serially passaged breast cancer xenografts [11]. Similar drug resistance to Adriamycin, Cisplatin, Etoposide, and Taxol were reported in breast cancer cells and lymphoblastic leukemia cells, both due to intracellular Notch1 signaling [12]. Additionally, treating mice with a Notch inhibitor restores tamoxifen sensitivity, and inhibiting glucocorticoid-resistant T-cell acute lymphoblastic leukemia cell lines sensitized to Notch-1 lead to glucocorticoidinduced apoptosis [10, 13]. Most interestingly, other groups found that inhibition of Notch signaling results in downregulation of HER2 expression, while the expression of activated Notch1 and Hes1 is significantly increased after treatment with trastuzumab, a HER2 inhibitor [14, 15]. This indicates that Notch signaling occurs upstream of HER2 signaling, and HER2 negatively regulates Notch expression. \n\nBased on our previous data reporting that RY10-4 inhibits HER2 expression in SKBR3 cells, we propose that decreased HER2 expression induces hyperactive Notch signaling, a possible mechanism of drug resistance caused by RY10-4 treatment. Here, we report aberrant hyperactive Notch signaling in HER2-overexpressing cells SKBR3 and BT474 in response to RY10-4 treatment, opposing the apoptotic effects of RY10-4. Inhibition of Notch signaling by the γ-secretase blocker DAPT or siNotch1 sensitizes breast cancer cells to RY10-4 in vitro and in vivo. Thus, our data illustrates that RY10-4 holds promising antitumor activity against triple-negative breast cancer and ER-positive breast cancer. In combination with a Notch inhibitor, RY10-4 offers a new opportunity in HER2positive breast cancer therapy.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "In the previous studies [6, 16], we have shown that RY10-4 is a potent anti-tumor compound against breast cancer cells, but the HER-2 overexpressing SKBR3 cell line proves less sensitive to RY10-4 than cell lines expressing lower levels of HER2, such as MDA-MB-231 and MCF-7 cells. To specifically address this question, we treated two commonly used HER2overexpressing breast cancer cell lines, SKBR3 and BT-474, with RY10-4 for 24 hours. We further evaluated the anti-proliferation effects of RY10-4 using two independent colorimetric assays, methylene blue (Figure 1A ) and tetrazolium salt (MTT, Figure 1B ). Both HER2negtive cell lines MDA-MB-231 and MCF-7 were more sensitive to RY10-4. Cell proliferation in this cell lines was inhibited about two-fold compared to the SKBR3 and BT-474 cell lines.",
"section_name": "HER2-negtive breast cancer cells are more sensitive to RY10-4",
"section_num": null
},
{
"section_content": "Since activation of Notch signaling in response to HER2 targeted treatment is responsible for drug resistance [17, 18], we first examined Notch activity in four breast cancer cells lines (SKBR3, BT474, MCF-7, and MDA-MB-231) in response to RY10-4 treatment. The results show that treatment with RY10-4 increases Notch transcriptional activity three-fold in SKBR3 and two-fold in BT-474 compared to MDA-MB-231 and MCF-7 cells (Figure 2A ), as measured by a C protein binding factor 1/Suppressor of Hairless/Lag1 (CSL) reporter assay. \n\nWe then measured expression of Notch and downstream target mRNAs in these cell lines in response to RY10-4 treatment. Both HER2-overexpressing cell lines, SKBR3 and BT474, show increased expression of Notch receptor genes (Notch1 and Notch4) and Notch target genes (Hes1 and Hey1) after RY10-4 treatment. Although RY10-4 increases Notch signaling in both HER2-positive cell lines, the expression of genes for Notch ligands is slightly reduced or unchanged only in BT474 cells. This discrepancy may be explained by the existence of different molecule footprints of different breast cancer subtypes [19]. Expression of Notch signaling genes is decreased or unchanged in MCF-7 cells and MDA-MB-231 cells (Figure 2B -2G and Figure S2 ). Furthermore, RY10-4 treatment causes elevated protein expression of the endogenous Notch target Hes1 and activated Notch1 (cleaved Notch1) proteins by two-to-eightfold in SKBR3 cells and BT474 cells. However, in MDA-MB-231 and MCF-7 cells, protein levels remain unchanged upon RY10-4 treatment (Figure 3 ). These results suggested that RY10-4 increases Notch activity in HER2-positive breast cancer cells. \n\nIn addition, we tested MAPK and Wnt signaling pathways in SKBR3 cells to determine whether RY10-4 affects multiple signaling pathways involved in cell survival. Interestingly, protein levels of phospho-p38, phospho-JNK, phospho-LRP6, and wnt3a do not change in response to RY10-4 treatment (Figure S3 ). These results indicate that RY10-4 specifically increases activity of the Notch signaling pathway.",
"section_name": "RY10-4 increases Notch-1 transcriptional activity and expression of endogenous Notch targets in HER2-amplified breast cancer cells",
"section_num": null
},
{
"section_content": "To determine the role of Notch signaling in mediating drug resistance to RY10-4 in HER2-overexpressing cells, we studied the effect of combining RY10-4 treatment with DAPT, a highly specific γ-secretase inhibitor that blocks Notch endoprotrolysis [20] ), on cell viability using two HER2positive cell lines (SKBR3 and BT-474) and two HER2negative cell lines (MDA-MB-231 and MCF-7). The antiproliferation effect was evaluated using the colony formation assay or the MTT assay. RY10-4 at a concentration of 1 μM markedly suppresses cell growth when compared with vehicle control (Figure 4A, Figure S4, Figure S5A-S5B ). Greater suppression is observed with the combinational use of DAPT (5 μM) and RY10-4 (1 μM) in both SKBR3 and BT-474 cell lines. In contrast, we did not observe a synergistic effect in the RY10-4-sensitive cell lines, MDA-MB-231 and MCF-7, suggesting that the increased anti-proliferation effects of DAPT and RY10-4 in HER2-overexpressing cells are specifically mediated by inhibiting the Notch pathway. \n\nAs expected, untreated cells (vehicle control) exhibit normal shape with clear outlines. Growth of the RY10-4treated cells is inhibited when compared with control cells and DAPT-treated cells. Further, the cells treated with RY10-4 became rounded and detached. Cell shrinkage and membrane blebbing were also observed (Figure 4B, Figure S5C ). These data indicate that cells undergo RY10-4-mediated apoptosis. Notably, RY10-4 markedly induces apoptosis when delivered in combination with DAPT, as measured by Annexin V-PE staining (Figure 4C -4D, Figure S5D ). In sharp contrast, DAPT fails to enhance RY10-4-mediated apoptosis in MDA-MB-231 and MCF-7 cells. \n\nSince DAPT preferentially affects Notch1 activity, we sought to determine whether the RY10-4-induced increase in Notch activation is due to specifically Notch1. To do this, we determined the effect of Notch1-specific knockdown on cell proliferation and apoptosis using si-Notch1. The results show that Notch1 siRNA almost completely abolishes Notch1 protein expression (Figure 5D ) and also prevents the increase in CSL reporter activity initially observed upon treating cells with RY10-4 (Figure 5A ). In cell proliferation experiments, cell growth is reduced by 40% in response to RY10-4 alone and by more than 60% upon combining RY10-4 with si-Notch1 (Figure 5C ). In addition, combination treatment further induces cell apoptosis in SKBR3 cells according to morphological study of cell shape (Figure 5B ). These findings show that the addition of a Notch inhibitor to RY10-4 treatment is significantly more effective than RY10-4 alone in suppressing both HER2-overexpressing cell lines.",
"section_name": "Down-regulation of Notch sensitizes HER-2 positive breast cancer cells to RY10-4",
"section_num": null
},
{
"section_content": "Since Notch activates the PI3K/AKT pathway [21, 22] and since AKT activation is necessary for Notch-conferred resistance to apoptosis [23], we assessed the effect of combining RY10-4 treatment with DAPT on Notch signaling and AKT phosphorylation in SKBR3 cells. First, SKBR3 cells express two-fold higher levels of active Notch1 (cleaved Notch1) and eight-fold higher levels of Hes1 protein in response to RY10-4 treatment compared to vehicle control. The increase in cleaved-Notch1 and Hes1 expression is prevented by treatment with DAPT (Figure 6A ). We next showed that RY10-4 alone, at a concentration of 0. 2 μM, was sufficient to inhibit AKT phosphorylation on Ser473 by 50% in SKBR3 cells, and this increased to 95% with DAPT (Figure 6B ). Similar results are seen in BT474 cells (Figure S6 ). Interestingly, treating either HER2-positive cells with DAPT alone proves ineffective in inhibiting AKT phosphorylation (Figure 6B lane 2 and Figure S6 lane 5 ), which suggests that the Notch signaling is suppressed in the HER2-amplified cells [14]. Thus, DAPT cannot inhibit AKT phosphorylation through the Notch pathway. Taken together, these data show that the combination of RY10-4 and DAPT inhibits Notch signaling and Notch-stimulated AKT activation.",
"section_name": "Combination of RY10-4 and DAPT inhibit Notch signaling and AKT activation",
"section_num": null
},
{
"section_content": "From in vivo studies of the effects of treatment with DAPT, RY10-4, combination of DAPT and RY10-4, or vehicle (Figure 7A ), we found no statistical difference (P = 0. 81 and 0. Interestingly, we found that, in the combination group, the tumors are whiter than the other groups (Figure 7B ), hinting that treatment with DAPT in combination with RY10-4 disturbs angiogenesis in SKBR3 tumors. There was no significant difference in mouse weight between treatment groups in either experiment, suggesting that the combination of DAPT and RY10-4 does not significantly increase systemic toxicity (data not shown). In summary, DAPT treatment enhances the anti-tumor effects of RY10-4, but it exerts no effect if used alone in nude mice bearing SKBR3 tumors. \n\nTo evaluate the effects of combined and singleagent treatments on SKBR3 cells, we examined levels of Notch and caspase-3 activity by immunohistochemistry (Figure 7E ). Protein levels of HES1 in SKBR3 tumors are decreased in mice treated with DAPT. In contrast, treatment with RY10-4 results in a great increase in both Dll4 and Hes1 expression, suggesting that Notch signaling is activated in SKBR3 xenografts exposed to RY10-4 alone. Interestingly, we found that the combination of DAPT and RY10-4 in treatment helps reverse the induction effects caused by treatment with RY10-4. Basal levels of cleaved caspase-3 are low in SKBR3 tumor tissues and remain low in DAPT-treated tumor tissues. However, we saw a trend in increased induction by RY10-4 treatment, and RY10-4 as a single agent was less effective in inducing cleaved caspase-3 expression than the combination of DAPT and RY10-4. These results suggest that aberrant Notch activation by RY10-4 treatment impairs the antitumor effects of RY10-4. Further, combining a Notch inhibitor with RY10-4 in treatment could help to improve the anti-cancer effects of RY10-4.",
"section_name": "DAPT and RY10-4 combination inhibits breast tumor growth in vivo",
"section_num": null
},
{
"section_content": "There are four subtypes of breast cancer based on the expression of ER, progesterone receptor (PR), or HER2: ER, PR+, HER2+; ER, PR+, HER2-; ER, PR-, HER2+; and ER, PR-, HER2- [24]. Among four breast cancer cell lines we tested, the triple negative cell line MDA-MB-231 and the ER+, PR+, HER2-cell line MCF-7 are very susceptible to RY10-4 as measured by MTT and methylene blue methods. To our surprise, The ER -, PR-, HER2+ cell line SKBR3 and ER+, PR+, HER2+ cell line BT474 are less sensitive. HER2 positive breast cancer occurs in 25% of breast cancer patients, and poor prognosis is due to activation of the PI3K/AKT survival pathway [25]. Although two HER2-targeted drugs (trastuzumab and lapatinib) have been approved for clinical use by the FDA, most patients still relapse after initially responding to treatment [26] [27] [28]. Activation of Notch signaling is a common result of using HER2 inhibitors or a dual EGFR/HER2 inhibitor, such as a tyrosine kinase inhibitor [14, 15]. The Notch pathway, an essential upstream regulator of HER2 signaling, is positively correlated with drug resistance and has a tumorpromoting function in breast cancer [29]. Activation and subsequent nuclear localization of the Notch intracellular domain induces the transcription of the target HER2 gene, which, in turn, activates the PI3K/AKT pathway [30]. Furthermore, other studies have confirmed that AKT activation is necessary for Notch-conferred resistance to apoptosis [23]. \n\nSince RY10-4 inhibits the HER2/PI3K/AKT axis, we investigated whether Notch signaling contributes to RY10-4-induced drug resistance in HER2-positive cells. Our results demonstrate that treatment with RY10-4 increases expression of Notch ligands, activating Notch-1 ICD and targets by increasing both mRNA and protein levels. The increased activity is abrogated by DAPT, a Notch inhibitor. Importantly, cell proliferation is inhibited more effectively by RY10-4 plus DAPT or Notch1 siRNA than by either agent alone. RY10-4 at a concentration of 0. 5 μM alone was sufficient to decrease cell growth by more than 50% in MDA-MB-231 and MCF-7 cells, but only around 30% in HER2-amplified SKBR3 and BT47 cells. This growth inhibition rises to 50% upon combination with DAPT or Notch1 siRNA. However, we did not observe a synergistic effect between DAPT and RY10-4 in MDA-MB231 and MCF-7 cell lines. This suggests that the increased anti-proliferative effects of DAPT when combined with RY10-4 are specifically mediated by reversal of RY10-4-indued drug resistance. In addition, we show that RY10-4 alone is sufficient to inhibit AKT phosphorylation on Ser473 by 50%, which dramatically increases to 95% with DAPT. \n\nOur in vivo data also demonstrates the synergistic effect of combinationtreatment, which decreases tumor growth 20-30% more than compared to RY10-4 alone. Furthermore, we found the decrease in cell proliferation is due to a significant increase in apoptosis as RY10-4 plus DAPT increases annexin V staining in SKBR3 cells and induces cleaved-caspase3 expression in tumor tissue. \n\nThe mechanisms responsible for Notch-related drug resistance are complex and still poorly understood. Some studies have demonstrated that Notch signaling is involved in the epithelial-mesenchymal transition in drug resistant cancer cells [31]. Other studies showed that abnormal Notch signaling might contribute to carcinogenesis by regulating the formation of cancer stem cells [32]. However, the mechanisms by which Notch signaling regulates the sensitivity of breast cancer cells to RY10-4 are still under study. \n\nOur results reveal that RY10-4 is a promising drug candidate for the treatment of different types of breast cancer. Cell proliferation of triple-negative and ER-positive breast cancer is significantly inhibited upon treatment with RY10-4. For HER2-positive cell lines, we found that increased activation of Notch signaling upon RY10-4 treatment can cause drug resistance. Combination treatment with a Notch inhibitor increases the efficacy of RY10-4 and prevents resistance. This work helps us gain important insight into the molecular pathways involved in the sensitivity of breast cancer cells to RY10-4, and it allows us to rationally design successful combination therapies for breast cancer treatment.",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "RY10-4 (Figure S1 ) was synthesized previously in our laboratory [6], and the structure was confirmed by NMR and MASS. Purity (95%) was measured by HPLC analysis. RY10-4 was dissolved in dimethyl sulfoxide (DMSO) to make a 10 μM stock solution, and this was stored at -20ºC. The working dosage was freshly prepared in basal medium with a final DMSO concentration of less than 0. 1%. The antibody against β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the antibodies against AKT, p-AKT, Notch1, NICD, and HES1 were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody against Dll4 was purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA).",
"section_name": "Reagents",
"section_num": null
},
{
"section_content": "MDA-MB-231 and MCF-7 human breast cancer cells were obtained from American Type Culture Collection. SKBR3 and BT474 human breast cancer cells were kindly provided by Dr. Qin Yan (Department of Pathology, Yale School of Medicine). They were propagated in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. All cell lines were maintained at 37ºC in a humidified atmosphere of 5% CO 2.",
"section_name": "Cell culture",
"section_num": null
},
{
"section_content": "Exponentially growing cells (1 × 10 4 ) were plated in a 48-well plate and treated with various concentrations of RY10-4 dissolved in DMSO (giving a final DMSO concentration of ≤ 0. 1%) in media after 24 h growth. Incubation was carried out at 37ºC for 24 h. Controls received vehicle in DMSO at a concentration equal to that of RY10-4 treated cells. Media was removed, and cells were washed with phosphate buffered saline (PBS), air dried, stained with 0. 3 ml methylene blue (2. 5 g in 250 ml EtOH + 250 ml H 2 O), left at room temperature for 2 h, and then washed with water. To each well, 0. 5 ml 1% Sarkosyl was added, and plates were rotated at room temperature for 3 h. Transferring 150 μl to a 96-well plate allowed for determining the OD using a plate reader at 595 nm. Cell viability assays were performed with three independent experiments.",
"section_name": "Methylene blue cell proliferation assay",
"section_num": null
},
{
"section_content": "The effect of RY10-4 on breast cancer cell proliferation was also assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium (MTT)bromide assay. Exponentially growing cells (0. 5 × 10 4 ) were plated in 96-well plates and treated with concentrations of RY10-4 in media (giving a final DMSO concentration of ≤ 0. 1%) after 24 h of growth. Incubation was carried out at 37ºC for 24 h. Controls received DMSO vehicle at a concentration equal to that of drug-treated cells. MTT solution was added to each well (2. 5 mg/ ml) and incubated for 4 h. Supernatants were removed from the wells, and the reduced MTT bromide dye was solubilized in 200 μl/well DMSO. Absorbance at 570 nm was determined on a plate reader.",
"section_name": "MTT cell proliferation assay",
"section_num": null
},
{
"section_content": "Breast cancer cell lines were plated on 6-well plates at a density of 1 × 10 3 cells per well and treated with RY10-4 and/or DAPT. Media was changed after 24 h of incubation, and colonies were observed over seven days. Colonies were then stained with 0. 5% of methylene blue in 50% ethanol for 2 h. Quantification of colony formation was processed using the \"ColonyArea\" plugin on ImageJ [34].",
"section_name": "Colony-formation assay",
"section_num": null
},
{
"section_content": "Total RNA was extracted from cells using the RNeasy Mini kit (Qiagen, CA, USA) according to the manufacturer's protocol. Total cDNA was reversetranscribed from the total RNA with random hexamers using the MultiScribe™ Reverse Transcriptase Kit (Applied Biosystems, CA, USA) according to the manufacturer's recommendations. Analysis of transcript copy number relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an endogenous control, was carried out by quantitative real-time PCR (BIO-RAD iQ™5, USA) using iTaq™ SYBR Green. Gene expression was determined by normalizing to reference genes using the comparative C T method. A list of the oligonucleotide sequences used for qPCR can be found in Table 1.",
"section_name": "RNA isolation and qPCR",
"section_num": null
},
{
"section_content": "The CSL (CBF1/RBP-J) luciferase reporter, (a gift from Dr. Yungchi Cheng, Department of Pharmacology, Yale School of Medicine), which contains a firefly luciferase gene under the control of the multimerized CSL responsive element upstream of the minimal promoter, was transiently transfected into cells using the FuGene (Roche, San Francisco, USA) transfection reagent. The pRL plasmid, a vector that constitutively expresses the Renilla luciferase gene under the control of the cytomegalovirus promoter, was used as an internal control. Total DNA was kept constant by adding empty vector as needed. After transfection, cells were treated with RY10-4, DAPT, or si-Notch1 for 6 hours. All transfections were carried out in triplicate. The luciferase activity was measured with the luciferase assay kit (Promrga, USA) and the Tecan FARCyte luminometer (GE Healthcare, USA) according to the manufacturers' instructions.",
"section_name": "Luciferase reporter assays",
"section_num": null
},
{
"section_content": "Cells were transfected with siRNA using Oligofectamine (Life Technologies, USA), performed according to the supplier's instructions. One day prior to transfection, 2 × 10 5 cells per six-well plate were seeded without antibiotics, corresponding to a density of 40%-50% at the time of transfection. Cells were treated with siNotch1 or control, a scrambled sequence, at a concentration of 100 nM. After each treatment, cells were incubated at 37ºC for 4 h followed by addition of fresh culture media. Cells were harvested 24 h after transfection for protein analysis.",
"section_name": "In vitro transfection of siRNA",
"section_num": null
},
{
"section_content": "Cells were harvested and lysed on ice for 30 min in lysis buffer containing 50 mM Tris-Hcl, pH 8. 0, 150 mM NaCl, 20 mM EDTA, 50 mM NaF, 1% NP-40, and 0. 02% NaN 3 with protease inhibitor (1 mM phenylmethanesulfonyl fluoride (PMSF) and, 1 μg/ml aprotinin) to prevent proteolysis and dephosphorylation. After centrifugation at 16099 g for 10 min, the supernatant was harvested as the total cellular protein extract. Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Scientific, USA). The total cellular protein extracts were separated by SDS-PAGE and transferred to polyvinyldifluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 5% (w/v) nonfat dry milk in TBST (1 M Tris buffer saline, pH 7. 4, 5 M NaCl, and 0. 1% Tween-20) for 1 h at room temperature and incubated overnight at 4ºC with primary antibody. Blots were washed three times in TBST buffer, followed by incubation for 1 h at room temperature with the corresponding HRP-linked secondary antibodies. Specific proteins were visualized using enhanced chemiluminescence reagent (Thermo Scientific, USA).",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "Cells were incubated with RY10-4 and/or DAPT for 24 h. After treatment, cells were washed twice with PBS and once with binding buffer (Abcam, USA). After adding 5 μl of Annexin V-PE to the media, cells were incubated at room temperature for 15 min in the dark. Analysis was performed using a fluorescence microscope (Olympus IX71) with the filter set for rhodamine.",
"section_name": "Annexin V labeling",
"section_num": null
},
{
"section_content": "Six-week-old female nude mice (BALB/c) were inoculated subcutaneously at the right flank with 2 × 10 6 SKBR3 cells suspended in 0. 2 ml PBS (pH 7. 4). When tumors reached an average diameter of 3 mm (usually in seven days), the mice were randomized into 4 groups and administered DAPT, RY10-4, combination of DAPT and RY10-4, or vehicle. Each group consisted of five animals. RY10-4 was administered intraperitoneally (i. p. ) every other day at 200 mg/kg, and DAPT was administered i. p. every day at 100 mg/kg. The control group was given vehicle (Figure 6A ). Tumor size was measured using calipers, and tumor volumes (mm 3 ) were calculated according to a standard formula: width 2 × length/2. Upon termination of the experiment, the mice were sacrificed, and the tumors were excised for weighing.",
"section_name": "In vivo tumor xenograft study",
"section_num": null
},
{
"section_content": "After treatment over 14 days, the mice were sacrificed by euthanasia. The tissues were fixed overnight in 4% paraformaldehyde, dehydrated, and coated with wax. Tissue sections were sliced to 4 μm in thickness and dyed with the primary antibody. Results were captured by the Nikon TS100 microscope.",
"section_name": "Immunohistochemistry",
"section_num": null
},
{
"section_content": "The data represent mean ± SD from independent experiments. Statistical analysis was performed using the student's t test and the ANOVA post test. The level of significance was set at P < 0. 05.",
"section_name": "Statistics",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "We would like to sincerely thank professor Yung-Chi Cheng of Yale University for providing us with a good environment and facilities to perform this project. Also, we want to especially thank Dr. Qin Yan for giving us two kinds of human breast cancer cell lines to enable our study proceeds. Financial support for the research came from the National Nature Science Foundation of China (No. 81173093 ).",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
},
{
"section_content": "The authors declare no conflicts of interest.",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
}
] |
10.1186/s12885-021-07864-y
|
A novel scoring system integrating molecular abnormalities with IPSS-R can improve the risk stratification in patients with MDS
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>The treatment strategies for Myelodysplastic Syndromes (MDS) are usually based on the risk stratification system. However, few risk signatures which integrate the revised international prognostic scoring system (IPSS-R) with gene mutations can be easily applied in the real world.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>The training cohort of 63 MDS patients was conducted at Zhongda Hospital of Southeast University from January 2013 to April 2020. The validation cohort of 141 MDS patients was obtained from GSE129828. The mutation scoring system was based on the number of mutations and a unique favorable prognostic factor, which is <jats:italic>SF3B1</jats:italic> mutation. Univariate Cox, multivariate Cox, and LASSO regression analyses were used to determine the significant factors that influenced the overall survival. The receiver operating characteristic curve (ROC) was used to evaluate the efficiency of the prognostic model.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>A novel risk scoring system we named “mutation combined with revised international prognostic scoring system (MIPSS-R)” was developed based on the results derived from multivariate analysis which assigned points to the IPSS-R and the mutation scores according to their relative statistical weight. Based on the quintile of the new scores, patients were divided into five risk levels. The Kaplan-Meier curves showed the superiority of MIPSS-R in separating patients from different groups, comparing with IPSS-R both in the training cohort (<jats:italic>p</jats:italic> = 1.71e-08 vs. <jats:italic>p</jats:italic> = 1.363e-04) and validation cohort (<jats:italic>p</jats:italic> = 1.788e-04 vs. <jats:italic>p</jats:italic> = 2.757e-03). The area under the ROC of MIPSS-R was 0.79 in the training cohort and 0.62 in the validation cohort. The retrospective analysis of our house patients showed that the risk levels of 57.41% of patients would adjust according to MIPSS-R. After changing risk levels, 38.71% of patients would benefit from treatment strategies that MIPSS-R recommends.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>A mutation scoring system was conducted based on the number of mutations and a unique favorable prognostic factor. MIPSS-R, the novel integral risk stratification system was developed by integrating IPSS-R and the mutation scores, which is more effective on prognosis and treatment guidance for MDS patients.</jats:p> </jats:sec>
|
[
{
"section_content": "Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by ineffective and dysplastic hematopoiesis that causes cytopenia, which are also likely to progress to the development of acute myeloid leukemia (AML) [1, 2]. MDS is predominantly diagnosed among older adults, and more than half of the patients exceed the age of 75 [3, 4]. The treatment strategies are usually based on the risk stratifications like the revised International Prognostic Scoring System (IPSS-R), which consists with bone marrow (BM) cytogenetics, blast percentage, and peripheral blood (PB) cytopenia [5]. However, with the rapid development of high through-put technology like nextgeneration sequence (NGS), multiple mutations have been revealed as significant factors in MDS [6], and approximately 90% of MDS patients have at least one mutation [7]. One large-scale genomic research showed that TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1 mutations appeared in more than 10% of MDS cases, and many mutations were correlated to higher risk groups or high blast counts [6]. According to National Comprehensive Cancer Network (NCCN) guidelines, epigenetic mutations such as TET2, DNMT3A, ASXL1, IDH1/2, and EZH2 commonly occur in MDS; Splicing factor-related mutations such as SF3B1, SRSF2, U2AF1 and ZRSR2 are not specific mutations of MDS but occur more frequently in MDS than in other myeloid tumors. SF3B1 mutation predicts a good prognosis; SRSF2, RUNX1, U2AF1, ASXL1, and TP53 mutation predict high risks of progressing to AML [8]. It is well known that mutations have the prognostic effect in MDS [9, 10], but a perfect scoring system based on mutation or combined with IPSS-R has not yet appeared. \n\nBased on these concepts, we would like to build a novel prognostic system that integrated the mutations with IPSS-R. To address these issues and to expand the knowledge about predictive factors, the data of 63 patients from our clinical center was utilized as a training cohort, and the data of 141 patients from GSE129828 was utilized as a validation cohort. This research aims to provide physicians with practical information, support them in choosing the best treatment plan, and provide consultations for patients.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "A total of 63 de novo MDS patients were collected in the department of hematology, Zhongda Hospital from January 2013 to April 2020, which were conducted as the training cohort. The diagnostic criteria and subclassified standards were referenced to the World Health Organization (WHO) in 2008 [11]. All samples were collected with patient consent under protocols approved by institutional review boards and by the Declaration of Helsinki. The prognostic risk stratifications of all patients were according to the IPSS-R prognosis integral systems [5, 12]. \n\nMeanwhile, A total of 141 patients with treatmentnaive MDS from dataset GSE129828 [13] were composed to a validation cohort, which was similar to the training cohort and was obtained from the Gene Expression Omnibus (https://www. ncbi. nlm. nih. gov/geo/). Clinical data to determine the IPSS-R scores, mutations and French-American-British (FAB) classification were available at the time of sample collection.",
"section_name": "Patient cohorts",
"section_num": null
},
{
"section_content": "All patients in the training cohort submitted BM aspirates at the time of admission. Cytogenetic analysis was conducted by conventional G-banding technology and fluorescence in situ hybridization (FISH). Each sample with three or more abnormal genetic characteristic BM cells was considered a sample with abnormal clones after analyzing at least 20 metaphases. Taking the FISH examination, each probe analyzed at least 200 cells. When the proportion of abnormal signal cells of a sample exceed the threshold, the sample considered with cytogenetic abnormality. By using DNA extracted from each aspirate, the mutational analysis was taken with an ampliconbased, NGS panel targeting the entire coding regions of 31 genes frequently mutated in MDS (supplement Table 1 ). Only mutations that have been previously reported to be pathogenic either in the Catalogue of Somatic Mutations in Cancer (COSMIC) ID or other databases or in the literature were considered in the present study.",
"section_name": "Cytogenetic and molecular biology determination",
"section_num": null
},
{
"section_content": "The mutation risk stratification was constructed by the number of mutated genes and only one favorable prognostic mutated gene. Patients with no mutant or with only SF3B1 mutation classified into the low-risk; with one mutant except SF3B1 classified into intermediate-1-risk; with two to four mutants classified into intermediate-2-risk; with five or more mutants classified into high-risk. Patients in low, intermediate-1, intermediate-2, and high risk were assigned 0, 1, 2, and 3 points, respectively. The mutation combined with revised international prognostic scoring system (MIPSS-R) was developed based on the results derived from multivariate analysis which assigned points to the IPSS-R and the mutation scores according to their relative statistical weight. Based on the quintile of the MIPSS-R scores, patients were divided into very low-, low-, intermediate-, high-, and very high-risk groups, respectively.",
"section_name": "Mutation risk stratification and MIPSS-R",
"section_num": null
},
{
"section_content": "The overall survival (OS) was defined as the time in days from the date of MDS diagnosis to the date of last follow-up or death, regardless of causes. The univariate, multivariate Cox and Least absolute shrinkage and selection operator (LASSO) regression models, receiver operating characteristic curve (ROC) analyses, and Kaplan-Meier (K-M) survival curve with Log Rank analysis were performed using R studio (version 3. 6. 3). The univariate, multivariate Cox regression and K-M survival analyses were performed with the package of \"survival\" in R. The LASSO regression analysis was performed with R package of \"glmnet\" in R. The prediction ability of the model was assessed by the area under the curve (AUC) of ROC with the package of \"survivalROC\" in R. Quantitative data were exhibited as the mean ± standard deviation (SD). Mann-Whitney U test and Fisher exact test analyzed continuous variable and categorical variables respectively by using SPSS 26. 0 software. All statistical tests were bilateral, with a p-value < 0. 05 being statistically significant.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The follow-up deadline of the training cohort was April 20, 2020. 9 out of 63 patients were removed due to losing follow-up. In the validation cohort, 33 out of 141 patients were removed from the present study since lack of survival data. Finally, data of a total of 162 patients were analyzed, 54 patients in the training cohort and 108 patients in the validation cohort. The clinical characteristics for each patient were summarized in Table 1. The baseline characters including the age, gender, BM blasts proportion, and mutation risk stratifications had no differences between the two cohorts. Most of the patients were low-risk ones according to the IPSS-R category, 38. 9% of patients in the training cohort, and 28. 7% in the validation cohort (p = 0. 015). The most common subtypes in the training cohort were multilineage dysplasia (MLD, 42. 6%) based on 2008 WHO classification, and in the validation cohort were refractory anemia with excess of blast (RAEB, 35. 2%) based on FAB classification. \n\nIn terms of the mutation risk stratification, most of the patients were in the intermediate-2 risk group, both in the training cohort (46. 3%) and validation cohort (56. 5%, Table 1 ). The most common mutations were ASXL1, TET2, TP53, SRSF2, and SF3B1, accounting for 31. 5, 27. 8, 18. 5, 14. 8, and 14. 8%, respectively, in the training cohort; meanwhile, the most common mutations in the validation cohort were TET2, ASXL1, SF3B1, RUNX1, and SRSF2, making up 31. 5, 27. 8, 25, 17. 6, and 16. 7%, respectively. A total of 20 same mutated genes were detected in both two cohorts, and there was no differences between cohorts (Supplement Table 2 ). In terms of abnormal karyotypes, 5q-was the most common one both in the training cohort (20. 4%) and the validation cohort (9. 3%). Complex karyotype was defined as more than or equal to three abnormal karyotypes, constituting 9. 3% in the training cohort and 8. 3% in the validation cohort. The expression of karyotype was similar between the two cohorts except for the 20q-(p = 0. 007, Supplement Table 3 ).",
"section_name": "Baseline characteristics",
"section_num": null
},
{
"section_content": "For the training cohort, with a median follow up of 13. 5 months (range, 0. 39-88. 24 months), the median OS per IPSS-R scoring system was > 60, > 60, > 60, 11. 34, and 5. 92 months for very low-, low-, intermediate-, high-, and very high-risk, respectively, p = 5. 759e-06 (Fig. 1 a ). The median OS per mutation risk stratification was > 60, 38. 9, 11. 3, and 2. 7 months for low-, intermediate-1-, intermediate-2-, and high-risk, respectively, p = 6. 096e-04 (Fig. 1 b ). For the validation cohort, with a median follow up of 22. 2 months (range, 0. 66-139. 51 months), the median OS per IPSS-R scoring system was 25. 59, 20. 93, 16. 8, 9. 73, and 4. 7 months for very low-, low-, intermediate-, high-, and very high-risk, respectively, p = 2. 757e-03(Fig. 1 c ). The median OS per mutation risk stratification was 33, 37, 22, and 18. 2 months for low-, intermediate-1-, intermediate-2-, and high-risk, respectively, p = 4. 242e-03 (Fig. 1 d ). \n\nTo identify all independent factors for OS, we next performed univariate Cox proportional hazards regression analysis and LASSO regression analysis in the training cohort. Univariate analysis demonstrated that age, TP53 mutation, mutation risk stratifications, IPSS-R, progression to AML, + 8, -7/7q-, and complex karyotype were the prognostic factors (supplement Fig. 1 a ). LASSO regression analysis was performed to select factors, and -7/7q-, IPSS-R, and mutation risk stratification were retained according to the optimal lambda value [log(lambda. min) = -1. 64, supplement Fig. 1 b, c ]. Next, in multivariate analysis, we confirmed the IPSS-R (p < 0. 01) and mutation risk stratification (p < 0. 001) as significant predictors for OS (supplement Fig. 1 d ).",
"section_name": "Survival analysis",
"section_num": null
},
{
"section_content": "In the next step, we aimed for the development of a practical risk score based on the results derived from multivariate analysis. A novel risk scoring system, we named MIPSS-R was developed based on a linear combination of the mutation risk stratification score and the IPSS-R score multiplied by regression coefficients obtained from the multivariate analysis: MIPSS-R score = mutation score × 1. 047 + IPSS-R × 0. 641. Based on the quintile of the MIPSS-R scores (ranged from 1. 28 to 8. 59), patients were divided into very low-(1. 28-2. 24), Meanwhile, the AUC value of the MIPSS-R was 0. 790, which was higher than IPSS-R (0. 731) and mutation scoring system (0. 672) alone in the training cohort (Fig. 2 c ). MIPSS-R in the validation cohort had an equal AUC value to the IPSS-R (0. 620), but higher than the mutation scoring system (0. 555, Fig. 2 d ).",
"section_name": "Incorporating mutation risk stratification into IPSS-R",
"section_num": null
},
{
"section_content": "To highlight the clinical significance of the MIPSS-R, we compared the risk stratifications changes in the training cohort (supplement Table 4 ). 27. 78% (15/54) patients had a decreased risk, and 29. 63% (16/54) patients had an elevated risk. Patients #49 and #58 were classified as intermediate-risk and low-risk based on IPSS-R and received demethylation therapy but died shortly owing to toxicity thereafter. These two patients would benefit from supportive care or other non-intensive therapy",
"section_name": "The clinical significance of the MIPSS-R",
"section_num": null
},
{
"section_content": "MDS are a group of highly heterogeneous diseases which are commonly occurred in the elderly population, characterized by pancytopenia and a high risk of progressing to AML. Various factors including the BM blasts, pancytopenia, cytogenetic characteristics, and genetic mutations affect the prognosis of the disease [14]. The gold criteria for assessing conditions of MDS patients is IPSS-R which is the latest version of IPSS revised by the MDS Prognosis International Working Group in 2012 [5]. However, independent prognostic elements such as red blood cell transfusion dependence, genetic mutations are not included in the scoring system, especially the gene mutations that help to the accurate assessment [15]. The advance of modern technology has improved the genome-wide analysis of genetic mutations in MDS [6, 16]. Although the evolution of molecular technology has introduced new challenges, it is also leading to novel recognition of accurate diagnosis and therapy. One large scale molecular research analyzed 994 MDS patients and revealed the genomic landscape of the disease [6]. The most frequently mutated genes were TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1, that all accounted for more than 10% in these patients. Another whole-exome sequencing study of 699 patients revealed distinct patterns of clonal evolution in MDS [17]. The data showed that MDS patients with SF3B1 mutations were enriched in the low-risk group, but patients with GATA2, NRAS, KRAS, IDH2, TP53, RUNX1, STAG2, ASXL1, ZRSR2, and TET2 mutations were enriched in the high-risk group. Patients with FLT3, PTPN11, WT1, IDH1, IDH2, NPM1, and NRAS mutations were significantly correlated to the AML progression. Meanwhile, they also found that most of the patients have unique mutated patterns, leading to a great deal of heterogeneity [6, 17]. \n\nIn the past few years, some studies have integrated mutations with IPSS-R to improve the prognostic values for MDS patients. One of the researches by Bejar et al. described a mutation landscape of 439 MDS patients and screened out five mutations, TP53, EZH2, ETV6, RUNX1, and ASXL1, that could predict the poor overall survival of MDS patients independently [18]. Haferlach et al. utilized the predictors including age, gender, IPSS-R, and 14 mutations genes, building a novel prognostic model (model-1) and separating patients into four risk groups, which showed significantly different 3-year survival rate of 95. 2, 69. 3, 32. 8, and 5. 3%, respectively [6]. Comparing with another model built by the 14 mutations alone (Model-2), and with IPSS-R, model-1 was more superior. Nazha et al. incorporated mutated EZH2, SF3B1, and TP53 with IPSS-R and improved the predictive ability in MDS [19]. Notably, MDS patients enrolled in the study were serial samples with different time points during their disease courses. The new model classified patients into 4 risk groups with a median OS of 37. 4, 23. 2, 19. 9, and 12. 2 months, respectively. The new model also had a better C-index than IPSS-R. The results of the paired samples also confirmed the new model had a dynamic prognostic potential. Hou et al. built an integrated risk-stratification model consisting with the age, IPSS-R, and 5 mutations (CBL, IDH2, DNMT3A, ASXL1, and TP53) [20], diving patients into four risk groups, and the median OS of each group was 250. 7, 38. 4, 17, and 8. 9 months respectively. They also showed that the new model could be well applied not only in the FAB-defined MDS patients but also those defined by WHO. Recently, Naqvi et al. also developed a new prognostic system incorporating 27-item Adult Comorbidity Evaluation (ACE-27) and TP53 mutation with IPSS-R which improved outcome prediction in patients with MDS [7]. The C-index for the new model is 0. 822, and the survival curves between risk groups of the new model were more well-separated than those of IPSS-R risk groups. The study also highlighted that clonal hematopoiesis of indeterminate potential (CHIP) associated mutations were associated with a higher frequency of prior history of cardiovascular events and poor prognosis in patients with MDS. \n\nCertainly, the specific effects of some mutations are commonly accepted, besides the mutated genes aforementioned utilized in multivariable models, DNMT3A, U2AF1, SRSF2, CBL, PRPF8, SETBP1, and KRAS have also been reported the association with decreased OS [6, 18, [21] [22] [23]. Only mutated SF3B1 is correlated to favorable outcome [6, 24]. Dr. Bejar. argue that the mutation patterns of MDS are diverse and no two are the same [25]. Due to the heterogeneities of mutations in MDS patients, it is difficult to utilize the prognostic model with specific mutants to assess patients with mutants that with uncertain prognostic values. Hence, we constructed a more simple-to-use mutation scoring system which contained only one favorable factor and the number of mutations, assigning 0 to 3 points to patients respectively. Then, the novel prognostic scoring system, MIPSS-R, was constructed by the linear combination of the IPSS-R and the mutation scoring system. Although MIPSS-R would lose some specific information about mutated genes, it overcomes the heterogeneities of mutation patterns of MDS patients. The prognostic value was also verified in the validation cohorts. Moreover, retrospective analysis of our house patients showed that more than half of the patients would adjust the risk stratification based on the MIPSS-R. Through a comprehensive analysis of the treatment strategies and outcomes of these patients, we found that part of the patients may obtain better prognosis under the guidance of MIPSS-R. \n\nHowever, this study still has some limitations. First, our study was retrospective, so there may be some inherent biases. Secondly, the number of patients was limited and large-scale prospective researches are prospected. Third, the prognostic model needs to be verified in other cohorts, such as specific treatment strategies cohort and paired cohort.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In summary, by integrating IPSS-R and gene mutations, MIPSS-R, a novel risk stratification system for MDS patients has been developed. This system is more effective in prognosis and will be helpful to reduce treatmentrelated deaths, to recognize MDS patients with high-risk or AML progression risk earlier.",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Not Applicable.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This work is supported in part by The National Natural Science Foundation of China ( 81770172 ); Jiangsu Provincial Special Program of Medical Science ( BE2017747 ); Jiangsu Province \"333 \" project ( BRA2019103 ); The Fundamental Research Funds for the Central Universities ( 2242019K3DZ02 ); Milstein Medical Asian American Partnership (MMAAP) Foundation Research Project Award in Hematology (2017); Key Medical of Jiangsu Province ( ZDXKB2016020 ). The funders had no roles in study design, data collection, data analysis, and interpretation, or writing of the manuscript.",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Patients' data that before December 2018 support the findings of this study are available on request from the Beijing Hyster Technology Co., Ltd. Patients' data after December 2018 that support the findings of this study are available on request from the corresponding author. Data from GSE129828 was obtained from the Gene Expression Omnibus (https://www. ncbi. nlm. nih. gov/geo/).",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi. org/10. 1186/s12885-021-07864-y.",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "Abbreviations MDS: Myelodysplastic Syndromes; IPSS-R: Revised International Prognostic Scoring System; ROC: The receiver operating characteristic curve; AML: Acute myeloid leukemia; BM: Bone marrow; PB: Peripheral blood; NGS: Nextgeneration sequence; NCCN: National Comprehensive Cancer Network; WHO: World Health Organization; FISH: Fluorescence in situ hybridization; COSMIC: Catalogue of Somatic Mutations in Cancer; OS: Overall survival; LASSO: Least absolute shrinkage and selection operator; K-M: Kaplan-Meier; AUC: Area under the curve; SD: Standard deviation; MDS-SLD: MDS with single lineage dysplasia; MDS-MLD: MDS with multilineage dysplasia; MDS-RS: MDS with ring sideroblasts; MDS-5q-: MDS with isolated del 5q; MDS-EB: MDS with excess blasts; MDS-U: MDS, unclassified; BM: Bone marrow; ANC: Absolute neutrophil count; Hb: Hemoglobin; PLT: Platelet; MIPSS-R: The mutation combined with revised international prognostic scoring system Authors' contributions SG and JZ were involved in the conception and design of the study. JX and YT were involved in the collection and assembly of the data. SG and ZG were involved in data analysis and interpretation and were involved in the writing of the manuscript. All authors have read and approved the manuscript.",
"section_name": "Additional file 1.",
"section_num": null
},
{
"section_content": "The written informed consent was provided by all the patients by the Declaration of Helsinki before enrollment in the study. The Institutional Review Board of Zhongda Hospital Southeast University, Nanjing, China, approved the study with approval number: 2017ZDKYBA001. 0, 2017ZDKYBA002. 0, 2019ZDSYLL033-P02.",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Not Applicable.",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.1186/s12263-016-0537-z
|
Fatty acid extract from CLA-enriched egg yolks can mediate transcriptome reprogramming of MCF-7 cancer cells to prevent their growth and proliferation
|
Our previous study showed that fatty acids extract obtained from CLA-enriched egg yolks (EFA-CLA) suppressed the viability of MCF-7 cancer cell line more effectively than extract from non-enriched egg yolks (EFA). In this study, we analysed the effect of EFA-CLA and EFA on transcriptome profile of MCF-7 cells by applying the whole Human Genome Microarray technology.We found that EFA-CLA and EFA treated cells differentially regulated genes involved in cancer development and progression. EFA-CLA, compared to EFA, positively increased the mRNA expression of TSC2 and PTEN tumor suppressors as well as decreased the expression of NOTCH1, AGPS, GNA12, STAT3, UCP2, HIGD2A, HIF1A, PPKAR1A oncogenes.We show for the first time that EFA-CLA can regulate genes engaged in AKT/mTOR pathway and inhibiting cell cycle progression. The observed results are most likely achieved by the combined effect of both: incorporated CLA isomers and other fatty acids in eggs organically modified through hens' diet. Our results suggest that CLA-enriched eggs could be easily available food products with a potential of a cancer chemopreventive agent.
|
[
{
"section_content": "Conjugated linoleic acid (CLA) term includes several isomers of linoleic acid (18:2), naturally present in ruminant and dairy products, due to the activity of the rumen microflora [25, 30]. In numerous studies, CLA was shown to have several beneficial properties on human health. Researchers examined its effect on stimulating the immune system [1], reducing cancerogenesis [32, 52], atherogenesis [34], diabetes, and obesity [54]. However, most of the available literature was focused on the activity of isolated, pure substances. In addition, according to available data, the consumable quantities of naturally occurring CLA are relatively too low to effectively impact human health [27]. The recommended effective dose of CLA was estimated at least 1. 5-3. 5 g/day [13], while natural ruminant products contain between 1. 2 and 12. 5 mg per gram of fat [25, 30, 47, 78] and in poultry CLA concentration remains relatively low, at 0. 6 to 0. 9 mg per gram of fat [13]. Enhancing CLA concentration in food products such as eggs, chosen dairy products/yogurts, and meat could then become an alternative to synthetic CLA supplements. Indeed, studies have shown an easy incorporation of CLA into eggs of chickens by diet fortification [11, 65, 74] and that CLA-enriched eggs meet the requirements of functional food [24]. \n\nLittle is known about the effect of fatty acids (FA) from CLA-enriched food products on cancer cells [16, 46]. Our previous study showed that FA extracts obtained from CLA-enriched egg yolks (EFA-CLA) suppressed the viability of MCF-7 cancer cell line more effectively than the extracts from non-enriched egg yolks (EFA) [32]. To analyze the potential molecular mechanism, we decided to compare the effects of both extracts on MCF-7 cells transcriptome profile. \n\nThe whole-genome DNA microarray technology has become a very powerful tool to analyze global gene expression profiles, and in multiple studies, it has been shown to be an effective method for detecting genomic variation of closely related samples. Finally, we identified and analyzed differently expressed genes based on family, molecular functions, biological processes, cellular components, or pathways. As suggested in this article, relationships between studied genes require a confirmation at protein level; nevertheless, the microarray results are a valuable and multi-faceted source of information for other scientists and a foundation for further in vivo research [67].",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "The Animal Ethics Committee of the National Institute of Animal Production (Poland) approved all experiments involving animals (approval number: 851/2011). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. \n\nForty-eight Isa Brown laying hens (26 weeks old) were housed in a controlled room under 14/10 h light/dark cycle, given free access to water and commercial starter diet ('DJ' feed). After a 1-week adaptation period, an equal number of hens was randomly allocated to the control or experimental group for 4 months of the experiment. Diets (Additional file 1: Table S1 ) were calculated to provide 2700 kcal/kg and 17 % crude protein. The 0. 75 % dietary CLA (TONALIN FFA 80, BASF Company, Germany) concentration was based on previously determined formula [24] and contained 80 % CLA in 50:50 ratio for cis-9,trans-11 and trans-10,cis-12 isomers. Eggs were collected daily for the period of 10 weeks and stored at 4 °C. Yolks were separated from albumen, homogenized with rotary homogenizer, and frozen at -20 °C. Samples were then lyophilized (Martin Christ Model Alpha 1-4, Germany) and again stored at -20 °C. The total dry matter was determined by oven drying method [3] and the total fat content was determined by Soxhlet method (Soxtec Avanti's 2050 Auto Extraction Unit, Tecator Foss, Sweden) using petroleum ether as a solvent [23].",
"section_name": "Hens' and eggs' management",
"section_num": null
},
{
"section_content": "Lipids from control and CLA-enriched yolks were extracted by using modified Folch method [22]. One gram per liter of butylated hydroxytoluen (BHT) was used as an antioxidant. Briefly, after overnight incubation with chloroform/methanol (2:1) solution, samples were filtrated and mixed with 4 mL of 0. 88 % sodium chloride solution to obtain phase separation. Chloroform lipids layer was then carefully dried under nitrogen. Ten milligrams of each lipid extract was subjected to saponification (20 min, 60 °C) with 0. 5 M KOH/methanol followed by methylation with 14 % (v/v) BF3/methanol (15 min, 60 °C) and extraction with hexane. The obtained fatty acid methyl esters (FAME) were analyzed by GC/MS (Additional file 2: Table S2 ). The profile of EFA-CLA and EFA was expressed as percentage (%) of relative area, obtained by area normalization (FA peak area relative to chromatogram total area). For the treatment, lipid extracts were subjected to the basic hydrolysis (0. 5 M KOH, 60 °C, 15 min) and extracted with hexane. The free fatty acids were then dissolved in ethanol at the stock concentration 1 g/mL and stored under nitrogen in the temperature of -20 °C.",
"section_name": "Fatty acids extraction and GC/MS analysis",
"section_num": null
},
{
"section_content": "Human breast adenocarcinoma cell line MCF-7 (ATCC ® HTB22 TM ) was purchased from the American Type Culture Collection. Cells were cultured according to the manufacturer's procedure. Cells were seeded in culture plates (BD Biosciences) for 24 h. After that time, growing medium was replaced by a medium containing (a) fatty acid extract from CLA-enriched egg yolks (EFA-CLA) and (b) fatty acids extract from non-enriched egg yolks (EFA), both at the concentration of 0. 5 mg/mL. We used (c) cell cultures only in growth medium (empty control (EC)) and (d) cell cultures treated with only a solvent of fatty acids (ET-ethanol) at final concentration 0. 1 %, as a negative control (NC).",
"section_name": "Cell culture and treatment",
"section_num": null
},
{
"section_content": "Cell proliferation was determined with 5′-bromo-2′-deoxy-uridine (BrdU) Labeling and Detection Kit III (Roche), according to manufacturer's instruction.",
"section_name": "Cell proliferation",
"section_num": null
},
{
"section_content": "Whole Human Genome Microarrays containing about 50 000 probes (Agilent Technologies, USA) were used to establish the expression profile of each tested sample. Total RNA was isolated from cells using RNA isolation kit (A&A Biotechnology, Poland). RNA quantity was measured with NanoDrop (NanoDrop Technologies, USA). The analysis of its quality and integrity was performed with BioAnalyzer (Agilent, USA). Only samples with RNA integrity number (RIN) ≥8. 0 were included in the analysis which was performed using SurePrint G3 Human Gene Expression 8x60K v2 Microarray. Each slide contained eight microarrays representing about 50 000 probe sets. The Low Input Quick Amp Labeling Kit, two-color (Agilent, USA) was used to amplify and label target RNA to generate complementary RNA (cRNA) for oligo microarrays used in gene expression profiling. The experiment was performed using a common reference design, where the common reference was a pool of equal amounts of RNA from control cells. On each of two-color microarrays, 300 ng of cRNA from the pool (labeled Cy3) and 300 ng of cRNA (labeled Cy5) were hybridized. In total, 12 microarrays were run-three for each experimental group. Microarray hybridization was performed with the Gene Expression Hybridization Kit (Agilent Technologies, USA), according to the manufacturer's protocols. RNA Spike In Kit (Agilent Technologies, USA) was used as an internal control. Acquisition and analysis of hybridization intensities were performed using the Agilent DNA microarray scanner. Data were extracted and background was subtracted using standard procedures contained in the Agilent Feature Extraction (FE) Software version 10. 7. 3. 1. FE performs Lowess normalization. Samples underwent quality control and the results showed that each sample had a similar QC metric profile. The next step was filtering probe sets by flags to remove poor quality probes (absent flags). Microarray data were deposited at the Gene Expression Omnibus data repository under the number GSE65397 and followed MIAME requirements. To identify signaling pathways and gene functions, the microarray data were analyzed using Panther Classification System-an online database.",
"section_name": "Microarray analysis of gene expression profile",
"section_num": null
},
{
"section_content": "Reverse transcription was performed using 1 μg of total RNA isolated from the cells by using the Maxima first Strand cDNA Synthesis kit for RT-qPCR (Thermo Scientific). A quantitative verification of genes was performed using the CFX96 Touch™ Real-Time PCR Detection System instrument (Bio-Rad), utilizing the SYBR Green Precision Melt Supermix kit (Bio-Rad). Conditions of individual PCR reactions were optimized for given pair of oligonucleotide primers (Additional file 3: Table S3 ). Basic conditions were as follows: 95 °C for 10 min, 45 PCR cycles at 95 °C, 15 s; 59 °C, 15 s; 72 °C, 15 s, followed by melting curve analysis (65-97 °C with 0. 11 °C ramp rate and five acquisitions per 1 °C). Results were normalized using at least two reference genes (GAPDH, HPRT1, ACTB, or HSP90AB1) and were calculated using the 2 -ΔΔC T method [39].",
"section_name": "RT and real-time PCR analysis",
"section_num": null
},
{
"section_content": "Each treatment included three replicates and the experiment was repeated three times. Statistical analysis for microarrays was performed using Gene Spring 12. 6. 1 software (Agilent, USA). Statistical significance of the differences was evaluated using a one-way ANOVA and Tukey's HSD Post hoc test (p < 0. 05). A multiple testing correction was performed using Benjamini and Hochberg false discovery rate (FDR) <5 %. Other experiments were assessed by Student's t tests.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "There were no significant differences in egg production between hens as well as egg characteristics [33]. Fatty acid profile in CLA-enriched egg yolks was significantly affected by dietary CLA fortification (Fig. 1 ). Both CLA isomers were found incorporated into the yolk, and their concentration did not reflect their initial proportion in the experimental diets (Additional file 1: Table S1 ), with the preference for the cis-9,trans-11 isomer. Compared to the control, feeding with 0. 75 % of dietary CLA significantly increased (p < 0. 001) total SFA concentration at the expenses of MUFA (p < 0. 001) (Fig. 1 and [33] ).",
"section_name": "Bird performance and egg composition",
"section_num": null
},
{
"section_content": "EFA-CLA extract at a concentration of 0. 5 mg/mL suppressed MCF-7 cell proliferation more effectively than the extract from non-enriched egg yolks. Specifically, treatment with EFA-CLA reduced the cell proliferation by approximately 40 % compared to the negative control, while EFA reached 20 % (Fig. 2 ). Moreover, this effect was weaker for estrogen-negative MDA-MB-231 (Additional file 4: Figure S12 ) and not observed for a non-tumorigenic MCF-10A cell line (Fig. 3 ).",
"section_name": "Cell proliferation",
"section_num": null
},
{
"section_content": "The analysis was performed on 1589 transcripts, of which 160 were differently expressed between EFA-CLA and EFA studied groups (Fig. 4 and Additional file 5: Table S4 ). We omitted 21 genes, which could have been directly affected by the solvent (ET) leaving 139 genes (Additional file 6: Table S10 ). Further analysis showed 69 transcripts shared by all three comparison groups: EFA-CLA vs. EFA, NC vs. EFA-CLA, and NC vs. EFA and 36 transcripts shared by EFA-CLA vs. EFA and NC vs. EFA. We determined 34 transcripts unique only to EFA-CLA vs. EFA. Among those, 11 were uncharacterized in available databases. Finally, we identified 18 (underlined) that, according to the available data, can be linked to cancer development and/or progression and which are involved in important cellular processes including regulation of cell cycle, apoptosis, or cell metabolism (Table 1 ). For those, the differences in expression between EFA and NC were statistically insignificant (Additional file 7: Table S5 ).",
"section_name": "Effect of applied treatments on MCF-7 cell line transcriptome profile",
"section_num": null
},
{
"section_content": "To validate microarray data, we selected eight random genes from EFA-CLA vs. EFA comparison group: NOTCH1, HIGD2A, PPKAR1A, UPC2, NAP1L1, CAM-SAP2, PPP2R5E, and TSC2 (p < 0. 05, Table 2 ). Our analysis showed a significant decrease in the messenger RNA (mRNA) expression of NOTCH1, HIGD2A, PPKAR1A, and UCP2 and an increase in the expression of CAMSAP2, PPP2R5E, and TSC2 genes due to the EFA-CLA treatment. Interestingly, only RT-qPCR results for NAP1L1 exhibited changes in the opposite direction than the microarray.",
"section_name": "Real-time PCR",
"section_num": null
},
{
"section_content": "Next, we examined the Gene Ontology (GO) for EFA-CLA vs. EFA differently regulated genes, using Panther Classification System. Results obtained from analysis of the signaling pathways, biological processes, molecular functions, and protein classes are presented in supplementary material (Additional file 8: Table S6, Additional file 9: Table S7, Additional file 10: Table S8, and Additional file 11: Table S9 ).",
"section_name": "GO molecular complete analysis",
"section_num": null
},
{
"section_content": "In addition to signaling pathways listed in Additional file 8: Table S6, we aimed to study the connections between all the transcripts from Table 1, especially in terms of oncogenic pathways (Fig. 5 ) [79]. Our results showed that EFA-CLA treatment affected the downstream genes of the mammalian target of rapamycin (mTOR) signaling pathway. The increased mRNA expression of PTEN, PPP2R5E, and TSC2 and decreased expression of GNA12, UPC2, AGPS, ANAX5A, and HIF1A, together with observed reduced proliferation of MCF-7, suggest that EFA-CLA negatively regulates AKT/mTOR pathway.",
"section_name": "Effect of EFA-CLA on oncogenic pathways",
"section_num": null
},
{
"section_content": "Breast cancer is one of the most common malignancies among women [21] ; however, despite extensive research, the cellular processes that lead to carcinogenesis have not yet been fully explained. In the current research, we chose the estrogen receptorpositive (ER+) MCF-7 breast cancer cell line-the most studied cellular model of breast cancer [26, 59, 73]. One of the main reasons behind our choice is that the (ER+) breast cancers are the most frequently diagnosed breast cancer subtype. \n\nCLA is an extensively studied compound, and research findings showed a variety of possible beneficial effects of dietary CLA on human health. In addition, some of the molecular aspects of CLA mechanisms of action have been already described, and to our knowledge, Murphy et al. [50] applied microarray technique to show the effects of CLA isomers on the global gene expression using Caco-2 cells. However, most of published results are based on studying pure, isolated isomers which may not reflect the effect of a food product naturally enriched in CLA. Thus, our research would be the first to address the effects of the extract from CLA-enriched egg yolks on a breast cancer cell model in a wide spectrum of the whole human genome. \n\nOur previous experiments showed that EFA-CLA extract suppressed the viability of MCF-7 breast cancer cell line more effectively than extract from non-enriched egg yolks [32]. Our current study supports those findings not only for estrogen receptor-positive MCF-7 but also for estrogen receptor-negative MDA-MB-231; however, the EFA-CLA effect was most notable for MCF-7 (Fig. 2 and Additional file 4: Figure S12 ), which we proposed to be associated with affected mTOR signaling pathway [8]. In addition, we also performed experiments on commercially available and described as a non-tumorigenic MCF-10A cell line (ATCC). Interestingly, we did not observe a decreased proliferation in that cell line after treatment with tested fatty acids (Fig. 3 ). Some authors recommend caution when using MCF-10A cell line as non-transformed human breast epithelial cells in carcinogenesis research. They point out their potential for morphological and phenotypic transformation [56]. However, it should be noted that, to some extent, this could be due to the modification of the cell line microenvironment, including culture conditions, presence of serum, and used medium [53, 76]. \n\nIn the present manuscript, we discuss selected genes, which expression differs the most between cells treated with FA from CLA-enriched and non-enriched egg yolks (Table 1 ), specifically, in terms of their potential significance in the neoplastic process. Based on a scheme of the interactions between those genes (Fig. 5 ), we pointed the anti-proliferative and pro-apoptotic properties of EFA-CLA, specifically through the regulation of AKT/ mTOR signaling pathway. mTOR interacts with several Fig. 5 Potential interactions between differently regulated genes treated with EFA-CLA in MCF-7 cells (listed in Table 1 )\n\nproteins and forms two distinct complexes named mTOR complex 1 (mTORC1) and 2 (mTORC2) of which mTORC1 is currently better characterized. mTOR is a central controller of protein synthesis, cell growth, cell proliferation, and cell viability [37]. Several components of the PI3K/PTEN/AKT/mTOR pathway (Fig. 5 ) are frequently mutated in human cancers. \n\nMost notably, our results showed that treatment of MCF-7 cells with EFA-CLA (compared to EFA) increased the mRNA expression of known tumor suppressors, such as TSC2, PTEN, PPP2R5E, and LMCD1. TSC2 complex is a key upstream regulator of mTORC1, and it constitutes of two proteins, TSC1 and TSC2, that interacts with each other. Mutations in either of them result in the development of the tuberous sclerosis complex (TSC), characterized by the growth of benign tumors in multiple vital organs [29]. Reduced expression of TSC2 was determined in the invasive breast cancer compared to normal mammary epithelium [45]. Another mTOR pathway inhibitor, PTEN, was also found up-regulated. PTEN is a p53-regulated tumor suppressor, which transcription can be enhanced by p53 protein-acting as a transcription factor. Although our microarrays did not show statistically significant change in TP53 mRNA expression in the EFA-CLA cells vs. EFA group, additional Western blot analysis clearly showed an accumulation of p53 at the protein levels in the EFA-CLA treated cells (data not shown). PTEN is one of the most frequently mutated genes in various human cancers [9, 17, 70], including breast cancers, and is linked to aggressive tumors [64]. Information on PPP2R5E is limited, but it has been suggested as a potential negative regulator of PI3K/ AKT signaling (2016). It has been also found to act as a tumor suppressor in breast cancer [19] and gastric cancer cells [38]. \n\nDuring our analysis, we have also found other genes potentially associated with PI3K/AKT/mTOR pathway, which were down-regulated in cells: AGPS, ANXA5, STAT3, NOTCH1, PRKAR1A, and HIF1A (Table 1 ), after the treatment with EFA-CLA. Whether observed decrease in gene expression is the cause or the result of inhibition of AKT/mTOR pathway requires further study. Zhu et al. [77] showed that phosphorylation of AKT1 in glioma and hepatic carcinoma cell lines was reduced simultaneously with AGPS silencing, whereas Benjamin et al. [6] showed that silencing of AGPS in breast cancer (including MCF-7) and melanoma cells manifested in a life-time reduction of cancer cells viability, tumor growth, and invasiveness. Although we did not find a direct link between ANXA5 and AKT/mTOR pathway (Fig. 5 ), its up-regulation may be a predictive factor for tumor stage and clinical outcome of colorectal cancer [71]. ANXA5 has been also found in a group of proapoptotic genes [48]. These data may suggest that the expression of ANXA5 could be dependent on the expression profile of other superior genes being a part of anti-tumor response of cells. In our study, we show a significant down-regulation of STAT3 and NOTCH1 gene under the influence of EFA-CLA. Phosphorylated STAT3 is being observed in nearly 70 % of human cancers. Acting as an oncoprotein, it is constitutively activated in many primary human tumors, being activated by a number of different cytokines as well as oncoproteins, i. e., Src and Ras. NOTCH1 is associated with PI3K and PI3K-dependent activation of AKT1. It has been shown to play a role in growth, proliferation, and inhibition of apoptosis [10, 58]. It has been also reported that Hes1, NOTCH1's downstream target protein, negatively regulates PTEN expression [51]. A fly model of tumorigenesis induced by NOTCH1 showed a synergism of NOTCH1 signaling and PI3K/AKT pathway, suggesting that the interplay between these two signaling pathways was conserved during the evolution process. \n\nPRKAR1A has been found to be down-regulated in MCF-7 cells, due to the treatment with EFA-CLA. Information about PRKAR1A in available literature is ambiguous. Some studies have shown its up-regulation in many tumors, including breast cancer [7, 40, 41], suggesting its role in cell cycle regulation, growth, and/or proliferation. Other studies have pointed its tumor suppressing properties in osteosarcoma [49] and follicular thyroid cancer [55]. Due to EFA-CLA treatment, we determined a reduction in the mRNA expression of UCP2, which belongs to the family of mitochondrial carriers. Significant amount of studies is available on UCP2, but its functions are still under debate. It has been recently proposed to control routing of mitochondrial substrates [20, 69]. The overexpression of UCP2 has been shown in various tumors, including breast cancer [44]. Some data reveal that up-regulation of UCP2 may facilitate an increased chemoresistance as well as cancer adaptation to oxidative stress via mitochondrial suppression of reactive oxygen species (ROS) [4, 15, 18]. Sayeed et al. [60] have shown that UCP2 gene silencing rapidly led to the induction of apoptosis and differentiation in breast cancer cells, concurrent with reduced cell survival and proliferation. These results may be supported by numerous studies reporting evidences suggesting a correlation between oxidative stress and breast cancer, due to mitochondrial dysfunction [57]. Being the source of ROS, mitochondria are particularly exposed to potential oxidative DNA damage. Several studies have determined a higher rate of mitochondrial DNA (mtDNA) mutations in breast tumor tissue, specifically, they identified somatic mutations in the D-loop region as, probably, the major factor leading to decreased mtDNA level in breast tumors and indicating a poor prognosis [36]. Recent results have shown that a reduced number of mtDNA copy may be involved in cancer development and/or progression [66, 75] and mtDNA content might be potentially used as a tool to predict prognosis. Interestingly, in our unpublished studies on prostate cancer cells and melanoma, we determined a significant increase in the levels of mtDNA when treating with EFA-CLA extracts (compared to EFA). \n\nWe also determined a down-regulation of HIGD2A, a subunit of the cytochrome C oxidase (COX, complex IV). However, little is known about its mechanism of action. A study presented by An et al. [2] on HIGD1A, another member of HIG1 gene family, showed that expression of HIGD1A is directly dependent on binding of HIF-1α to HRE (hypoxia-response element) site at -32 bp in the HIG1D1A promoter. Transfecting RAW264. 7 cell line with HIGD1A under hypoxia condition promoted cell survival, whereas silencing the endogenous gene with siRNA resulted in hypoxia-induced apoptosis. The authors have proposed the inhibition of cytochrome C release and the reduction of caspases as a potential mechanism. They also obtained similar results for HIGD2A. It should be noted that similarity between HIG1 gene family members might have influenced obtained microarray results as probe specificity for gene isoforms is limited [35]. Interestingly, in our unpublished results on prostate cancer cells and melanoma, we determined a down-regulation of HIF-1α, after treatment with EFA-CLA. In our present study, we also observed down-regulation of HIF-1α in MCF-7 cells after EFA-CLA treatment, which is a positive signal confirming the anti-proliferative activity of EFA-CLA in the AKT/mTOR pathway (Fig. 5 ). HIF-1α is responsible for the activation of transcription of various genes, such as VEGF, EDN1, or CDKN1A, which are involved in cell cycle regulation, neovascularization, and metastasis [62]. Overexpression of HIF-1α correlates with an advanced tumor stage and poor survival [63, 68]. Majumder et al. [43] have demonstrated that the expansion of AKT-driven prostate epithelial cells requires mTOR-dependent survival signaling and activation of HIF-1α. \n\nFinally, our results showed a down-regulation of GNA12 mRNA expression after the treatment with EFA-CLA. Although we did not find a direct association with mTOR, GNA12 has been found to participate in oncogenesis and metastasis in pathological conditions [28]. Kim et al. [31] ), while studying breast cancer cells, proposed that GNA12 up-regulates the activity of matrix metalloproteinase (MMP)-2 via p53-dependent manner and promotes malignant phenotypic conversion of this cancer cells. A recent study supported those findings [12]. Their results also show that GNA12 stimulates the expression and activity of tumor promoting cytokines IL-6 and IL-8 and MMP-2 via binding and activation of NF-kB. \n\nAlthough in current manuscript we focused on differences between the CLA-enriched (CLA-EFA) and non-enriched egg (EFAs), attached data (database GSE65397) suggest that EFAs can change the gene expression profile as well; however, they are unable to suppress the cell proliferation as efficiently as CLA-EFA can. The analysis of MCF-7 transcriptomes revealed that some of the observed changes may not be solely caused by CLA isomers present in the pool of other fatty acids identified in the enriched egg yolk. As shown in Fig. 1, the incorporation of CLA was accompanied by significant changes in the general FA profile of egg yolk. The most notable was an increase in total SFA concentration at the expense of MUFA. Our calculations showed that the altered SFA/ MUFA ratio can affect the expression of some genes including HIGD2A and SMS (Additional file 12: Table S11 ) suggesting that the changed FA ratio in EFA-CLA extract could be responsible, at least in part, for the specific response of the cancer cells (Fig 2 ). It seems therefore that our functional products, obtained through the process of modification of hens' diet, may show specific features determined by both the presence of CLA and altered SFA/MUFA ratio. This may also suggest that CLA-enriched eggs cannot be simply replaced with a synthetic CLA supplements. \n\nAlthough available literature on effects of CLA on other FAs is limited, some authors have shown that treatment of cells with synthetic CLA increased the SFA/MUFA ratio in cell culture [14, 72]. As potential explanation, authors suggested that CLA could reduce the expression of SCD gene, which is responsible for conversion of SFA into MUFA. Interestingly, overexpression of SCD was associated with increased cancer cell proliferation, both in vitro for breast, prostate and lung cancer cells as well as in vivo [5, 14]. Our results showed a decreasing tendency for SCD mRNA for EFA-CLA vs. EFA treatment groups (Table 1 ); however, these results were statistically non-significant. Nevertheless, comparison with the negative control revealed a significant reduction in SCD expression for EFA-CLA at the level of transcription (Additional file 7: Table S5 ). It should be noticed, however, that the SCD mRNA levels does not necessarily correspond with this enzyme activity that has been shown by Choi et al. [14] for both MDA-MB-231 and MCF-7 cells treated with synthetic cis-9, trans-11 and trans-10, cis-12 CLA isomers. SCD has been also reported to be involved in mTOR pathway. Scaglia and Igal [61] have showed that the down-regulation of SCD reduces the activity of AKT in A549 cell line (SCD-ablated A549 cells). In addition, Luyimbazi et al. [42] have observed an increase in SCD protein expression when using activators of mTOR pathway in both MCF-7 (ER+) and MDA-MB-231 (ER-) cell lines, while the use of selective mTOR inhibitors showed an opposite effect. All these data may suggest that the observed decrease in MCF-7 proliferation in the presence of EFA-CLA could result from down-regulation of AKT/mTOR signaling pathway and reduced expression of SCD and other genes involved in mTOR pathway (database GSE65397). However, the role of CLA, other egg FAs, and the altered SFA/ MUFA ratio in mTOR-dependent down-regulation of cell proliferation needs further studies. \n\nAlthough some of our results may require to be confirmed at protein levels, the microarrays are a valuable and multi-faceted source of information and may explain the adequacy of further in vivo research, according to 3R principles (replacement, reduction, and refinement).",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In summary, our study presents the first evidence that the fatty acids extracts from CLA-enriched egg yolks (EFA-CLA) can affect transcriptome of MCF-7 cancer cells and inhibit their proliferation. We found this effect to be accompanied by changes in gene expression associated with down-regulation of AKT/mTOR signaling pathway. EFA-CLA increased expression of TSC2 and PTEN tumor suppressors as well as decreased the expression of oncogenes including NOTCH1, AGPS, GNA12, STAT3, UCP2, HIGD2A, HIF1A, and PPKAR1A. The observed results are most likely achieved by the combined effect of both incorporated CLA isomers and other fatty acids in eggs organically modified through hens' diet. It seems, therefore, that in contrast to synthetic CLA supplements, CLA-enriched eggs with an altered SFA/MUFA ratio could be easily available food products with a potential of a cancer chemopreventive agent. Although this concept needs further in vivo studies, it is clear that our microarray-derived results are a rich source of information on pathways in which fatty acids from CLA-enriched egg yolks can modify the response of the cancer cells at the level of transcription.",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This work was supported by the Polish National Science Center (grant number 2011/03/B/NZ9/01423 ) \"Conjugated linoleic acid (CLA)-induced transcriptional activation of PPAR-an investigation of molecular mechanisms of putative anticancer action of fatty acids of CLA-enriched egg yolks. \"",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Microarray data were deposited at the Gene Expression Omnibus data repository under the number GSE65397.",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "Additional file 1: S1. Composition of hens' experimental diets (%). (DOCX 12 kb) Additional file 2: S2. FAME analysis GC/MS conditions. (DOCX 12 kb) Additional file 3: S3. Nucleotide sequences of primers. ACTB, actin, beta; CAMSAP2, calmodulin regulated spectrin-associated protein family, member 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIGD2A, HIG1 hypoxia inducible domain family, member 2A; HPRT1, hypoxanthine phosphoribosyltransferase 1; HSP90AB1, heat shock protein 90 kDa alpha (cytosolic); NAP1L1, nucleosome assembly protein 1-like 1; NOTCH1, Notch homolog 1, translocation-associated; PPKAR1A, protein kinase, cAMPdependent, regulatory, type I, alpha; PPP2R5E,protein phosphatase 2, regulatory subunit B', epsilon isoform; TSC2, tuberous sclerosis 2; UCP2 uncoupling protein 2 (mitochondrial, proton carrier). Abbreviations AKT, protein kinase B; BF3, boron trifulouride; BHT, butylated hydroxytoluen; BrdU, 5′-bromo-2′-deoxy-uridine; CLA, conjugated linoleic acid; COX, cytochrome C oxidase; DNA, deoxyribonucleic acid; EC, empty control; EFA, fatty acids extract from non-enriched egg yolks; EFA-CLA, fatty acids extract from CLA-enriched egg yolks; FA, fatty acids; FAME, fatty acid methyl esters; FC, fold change; GC/MS, gas chrmoatography/mass spectrometry; GO, Gene Ontology; HRE, hypoxia-response element; KOH, potassium hydroxide; MCF-7, human breast adenocarcinoma cell line; mtDNA, mitochondrial DNA; mTOR, mammalian target of rapamycin; NC, negative control; QC, quality control; RNA, ribonucleic acid; ROS, reactive oxygen species; siRNA, small interfering RNA Authors' contributions AAK, AM, and PL made substantial contributions to the conception and design of experiments. DD, EP, MM, and AAK participated in performing the experiments. AAK and AM participated in the analysis and interpretation of data. AAK and PB participated in drafting the article. PL and AM participated in critically revising article for its important intellectual content. TL gave the final approval of the version to be submitted and any revised version. All authors read and approved the final manuscript.",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "The Animal Ethics Committee of the National Institute of Animal Production (Poland) approved all experiments involving animals (approval number: 851/ 2011). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.",
"section_name": "Ethics approval and consent to participate",
"section_num": null
}
] |
10.3390/curroncol30100643
|
Next-Generation Sequencing Analysis of Mutations in Circulating Tumor DNA from the Plasma of Patients with Head–Neck Cancer Undergoing Chemo-Radiotherapy Using a Pan-Cancer Cell-Free Assay
|
<jats:p>Using next-generation sequencing (NGS), we investigated DNA mutations in the plasma tumor cell-free circulating DNA (ctDNA) of 38 patients with inoperable squamous cell head neck cancer (SCHNC) before and after the completion of chemoradiotherapy (CRT). Baseline mutations of the TP53 were recorded in 10/38 (26.3%) and persisted in 4/10 patients after CRT. ΤP53 mutations were further detected post CRT in 7/38 additional patients with undetectable mutations at baseline (overall rate 44.7%). Furthermore, 4/38 patients exhibited baseline mutations of the EGFR, AR, FGFR3, and FBXW3, and four new gene mutations were detected after CRT (MTOR, EGFR3, ALK, and SF3B1). Τ4 stage was related with a significantly higher rate of mutations (TP53 and overall). Mutations were observed in 8/30 (26.6%) responders (complete/partial response) vs. in 6/8 (75%) of the rest of the patients (p = 0.03). Significant poorer LRFS was noted for patients with mutations detected before and after CRT (p = 0.02). Patients who had detectable mutations either before or after CRT had significantly worse DMFS (p = 0.04 overall, and p = 0.02 for TP53 mutations). It was concluded that assessment of mutations before and after the end of CRT is essential to characterize patients with a high risk of locoregional recurrence or metastatic progression.</jats:p>
|
[
{
"section_content": "During disease progression, cancer cells and especially stem cells acquire genetic mutations that define clinical aggressiveness, invasion, metastasis, and resistance to radiotherapy (RT) and chemotherapy [1, 2]. Such mutations can appear even during therapy, either as a result of direct DNA damage and failure to properly repair the DNA strand breaks or as an accumulation and prevalence of existing resistant cancer cell clones with specific mutations [3, 4]. \n\nThe profile of genetic mutations of a tumor can be assessed with next-generation sequencing (NGS) based on tissue biopsy material. Indeed, this has been established as a routine test to identify molecular fingerprints that can guide therapy with molecular inhibitors or monoclonal antibodies, e. g., therapies targeting epidermal growth factor receptor (EGFR) or other gene mutations [5]. Nevertheless, NGS can also be applied in cellfree DNA (cfDNA) extracted from the plasma or the saliva and other body fluids of patients. \n\nFragmented DNA released by cancer cells through vesicles and exosomes or fragments from dying cancer cells that enter the circulation (circulating tumor DNA-ctDNA) can be isolated from the blood and body fluids. Testing ctDNA for tumor mutations in liquid biopsies has emerged as a convenient and reliable method for tumor profiling. In fact, Parkh et al. suggested that analysis of a single-lesion tumor biopsy alone is less effective than ctDNA analysis in identifying tumor genetic heterogeneity and alterations associated with resistance to therapy [6]. Liquid biopsies, being non-invasive procedures, can be repeatedly obtained from patients without any discomfort during their therapy, conferring an important advantage over tissue biopsy analysis. \n\nSquamous cell head neck cancer (SCHNC) accounts for approximately 600,000 new cases annually, ranking 7th in prevalence among different cancer subtypes, with smoking and human papilloma virus (HPV) infections being major risk factors [7]. Combination of surgery and adjuvant RT or chemoradiotherapy (CRT) and definitive/radical RT or CRT for inoperable cases are the established treatment modalities of this malignancy, offering high curability rates. While the incidence of distant metastases is less than 30%, locoregional recurrence eventually occurs in more than 50% of patients with a locally advanced disease [8]. Specifically, the 2-year progression-free survival rates of locally advanced head and neck cancer patients treated with radical CRT range from 30% to 60% [9]. \n\nA number of studies investigating the cfDNA levels in the plasma or other body fluids of SCHNC patients before or after administration of RT or CRT have suggested that this method could potentially predict response to treatment and patient prognosis [10]. In a prospective trial, we quantitatively assessed the cfDNA concentration in the plasma of a cohort of patients with locally advanced SCHNC treated with CRT [11]. Increased levels were noted in 55% of patients and this was related to poorer response to therapy and worse prognosis. Beyond the quantity of cfDNA, gene mutation analysis could identify ctDNA that would ultimately prove to be of further prognostic and predictive relevance. In the current study, we report the analysis of DNA mutations in this cohort of patients. Detection of mutations was based on a panel of selected genes involved in the cell cycle, cell death pathways, cell signaling, and metabolism. These were assessed before and at the end of CRT, aiming to identify specific gene mutations involved in resistance to CRT and also assess an eventual prognostic role of persistent or newly emerging mutations after treatment.",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "As previously reported [11], patients with histologically diagnosed inoperable SCHNC were prospectively treated with RT combined with chemotherapy (cisplatin and/or cetuximab). Only patients with a conventional type of squamous cell cancer of the head-neck area, as identified by the 5th edition of the World Health Organization Classification of Head and Neck tumors [12], were included. Thirty-eight patients were included in the current analysis. No patient selection was performed (patients sequential in time). Inclusion criteria were good performance status (0-1), no previous chemotherapy or RT treatment, normal blood and biochemical tests, and absence of major heart, kidney, lung, autoimmune, hematological or psychiatric disease. Pregnant women were also excluded. Supplemental Table S1 presents details regarding patient and disease characteristics. The median follow-up was 15 months (2-36 months), while for patients alive at the time of last follow-up, this was 18 months (6-36 months).",
"section_name": "Patients",
"section_num": "2.1."
},
{
"section_content": "Patients were treated with image-guided RT (IGRT) and a Volumetric Modulated Arc Therapy (VMAT) technique as previously reported [11]. Briefly, a simultaneous integrated boost (SIB) technique was applied to deliver 22 fractions, 5 fractions per week, within 30 days. Areas receiving prophylactic irradiation (e. g., neck) were treated with 2. 15 Gy/fraction, while a daily booster dose of 0. 40-0. 55 Gy was administered to the primary tumor. The dose to enlarged nodes was increased using a daily booster dose of 0. 3-0. 4 Gy per fraction. \n\nThe SIB regimen has been widely applied in our department for the treatment of SCHNC, as this provides an equivalent dose delivered in 2 Gy fractions (EQD2) of 62-66 Gy, using the linear quadratic formula for tumor α/β = 4-10 Gy. As this dose is delivered with a 15-day acceleration, the time-corrected (T) EQD2-T (for a λ-value = 0. 4-0. 8 Gy/day) reaches an estimated biological dose of 68-78 Gy. This has been analyzed in detail in a previously reported study [11]. A recent radiobiological study by Shuryak et al. has suggested that optimized hypofractionated and accelerated RT in the range of the above-reported regimen can be better tolerated and is highly effective [13]. \n\nPatients received concurrent chemotherapy with intravenous administration of cisplatin at a dose of 35-40 mg/m 2 per week, or cetuximab at a dose of 250 mg/m 2 /week, or a combination of both, as previously reported [14]. Thirteen patients were treated with cisplatin, four patients received cetuximab and twenty-one patients were treated with both agents.",
"section_name": "Treatment Technique",
"section_num": "2.2."
},
{
"section_content": "A CT or an MRI scan was performed two months after RT completion to assess tumor response, and these were repeated six-monthly after that during the follow-up of patients. The WHO criteria [15] were applied to assess response to CRT as follows: complete response (CR) was defined as a 95-100% reduction in 2D dimensions of all detectable and measurable lesions. Partial (PR) and minimal response (MR) refer to 50-95% and 25-49% reduction in tumor dimensions (2D), respectively. An increase in tumor dimensions by more than 25% was defined as progressive disease. All other cases were considered to correspond to stable disease.",
"section_name": "Assessment of Response",
"section_num": "2.3."
},
{
"section_content": "Twelve ml of venous blood were collected in vacuum blood collection test tubes containing ethylenediaminetetraacetic acid (EDTA) vials. The first sampling was performed immediately before the administration of the first RT fraction and chemotherapy infusion. A second blood sample was obtained on the day of the last RT fraction. The technique of PBMC and plasma isolation and storage has been previously reported [11].",
"section_name": "Plasma Collection",
"section_num": "2.4."
},
{
"section_content": "Isolation of cell-free DNA was performed using the bead-based MagMAX™ Cell-free DNA extraction kit (catalog no. : A36716, Thermo Fisher Scientific, Waltham, MA, USA), specialized for high-quality isolation and specific enrichment of nucleic acids from liquid biopsies, as previously reported [11].",
"section_name": "Extraction and Quantification of Plasma cfDNA",
"section_num": "2.5."
},
{
"section_content": "NGS analysis was performed using the Oncomine Pan-cancer cell-free assay (Thermo Fisher Scientific, USA; https://www. thermofisher. com/order/catalog/product/A37664, accessed on 25 September 2023) according to the manufacturer's instructions. Library preparation was performed using cfDNA with concentrations ranging from 0. 5 ng to 4 ng. Quantification of the isolated libraries was again performed via the Qubit system. The input range of each library used ranged from 10 ng to 20 ng. Sample analysis was performed using the Ion 540™ Kit-Chef system (Thermo Fisher Scientific). Automated preparation of Ion 540™ chips was performed, with each chip having six patient samples with different barcodes, and finally, sequencing of the samples was performed through the next-generation sequencing system using the Ion S5™ system. High-depth sequencing of the samples was performed using the Ion S5 sequencer, while the analysis of the samples was performed using Torrent Suite™ Software v. 5. 12. 3 and Ion Reporter™ version 5. 20. 2 (Thermo Fisher Scientific), using the Homo sapiens reference genome (hg19) as a reference library, according to the manufacturer's instructions, for the analysis of point mutations, deletions, insertions, fusions and CNV's in a panel of genes occurring in various cancer types, including AKT1, ALK, AR, ARAF, BRAF, CHEK2, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ESR1, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MAP2K1, MAP2K2, MET, MTOR, NRAS, NTRK1, NTRK3, PDGFRA, PIK3CA, RAF1, RET, ROS1, SF3B1, SMAD4, SMO (Hotspot genes (SNVs) and short indels), ALK, BRAF, ERG, ETV1, FGFR1, FGFR2, FGFR3, MET, NTRK1, NTRK3, RET, ROS1 (Gene fusions), MET (exon 14 skipping), CCND1, CCND2, CCND3, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, MET, MYC (CNVs) APC, FBXW7, PTEN, TP53 (Tumor suppressor genes), with a limit of detection (LOD) of a 0. 1% allele frequency for SNVs and 1% for fusions. These genes are frequently mutated in multiple cancer types [16, 17], including head and neck cancer [18].",
"section_name": "NGS Analysis",
"section_num": "2.6."
},
{
"section_content": "We used the GraphPad Prism 7. 0 package for statistical analysis and graph presentation. The chi-square and Fisher's exact t-test were used to test associations between categorical variables, as appropriate. Kaplan-Meier locoregional relapse-free survival (LRFS), disease-specific overall survival (OS), and distant metastasis-free survival (DMFS) curves were plotted. For statistical significance, we considered a p-value < 0. 05.",
"section_name": "Statistical Analysis",
"section_num": "2.7."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "The quality control of the analyzed DNA samples from patients before and after CRT showed a QC-test-limit-of-detection LoD % range from 0. 1 to 1. 3 (median 0. 4) and from 0. 1 to 1. 2 (median 0. 5), respectively. Table 1 reports the genes, mutations, and molecular frequencies recorded in patients before and after CRT. \n\nThe baseline pre-CRT analysis showed a clear prevalence of mutations of the TP53, recorded in 10/38 (26. 3%) of patients. Single mutation was recorded in 7/10 patients, while multiple mutations of the gene were recorded in 3/10 patients (two mutations in two patients and three in one). After CRT, TP53 mutations were undetectable in 5/10 of these patients, while baseline mutations persisted in 4/10. In one (1/10) additional patient, disappearance of the pre-existing TP53 mutation was noted, while a new mutation became detectable. Reduction in the number of TP53 mutations was observed in patients who presented with multiple mutations at baseline: patient no. 22 had two mutations post CRT (at baseline, three mutations), and patient no. 35, with two mutations at baseline, had only one post CRT. Both mutations in patient no. 30 were undetectable post CRT. Of interest, mutations of the TP53 gene were further detected post CRT in 7/38 additional patients with undetectable mutations at baseline. In this way, the rate of TP53 mutation detection (before and/or after CRT) was 17/38 (44. 7%). Figure 1a shows the mutation rates of TP53 and changes after CRT, while Figure 1b shows the mutations of TP53 before, after CRT, and these persisted throughout therapy. Furthermore, 4/38 more patients exhibited mutations of EGFR, AR, FGFR3, and FBXW3 (one patient, respectively) before CRT. After CRT, the FGFR3 mutation remained detectable in the patient, while the rest were undetectable. Moreover, four additional mutations became detectable after CRT in four patients (one each) without pre-treatment detectable mutations. These concerned the MTOR, EGFR3, ALK, and SF3B1 genes, respectively (Table 2 ). We did not detect any CNVs or gene fusions in the current cohort of patients. Figure 1c shows the genes and mutation rates recorded before and after CRT. Appendix A reports the detected genes with mutations and their main biological functions in cancer biology. Analysis of TP53 mutations detected after CRT according to the chemotherapy regimen applied (cisplatin alone vs. cetuximab with or without cisplatin) did not reveal any statistically significant difference (mutation rate: 4/13 vs. 8/25 patients, respectively; p = 0. 99). \n\nFurthermore, 4/38 more patients exhibited mutations of EGFR, AR, FGFR3, and FBXW3 (one patient, respectively) before CRT. After CRT, the FGFR3 mutation remained detectable in the patient, while the rest were undetectable. Moreover, four additional mutations became detectable after CRT in four patients (one each) without pre-treatment detectable mutations. These concerned the MTOR, EGFR3, ALK, and SF3B1 genes, respectively (Table 2 ). We did not detect any CNVs or gene fusions in the current cohort of patients. Figure 1c shows the genes and mutation rates recorded before and after CRT. Appendix A reports the detected genes with mutations and their main biological functions in cancer biology. \n\nTable 2. Univariate tables from Kaplan-Meier loco-regional relapse-free survival, disease-specific overall survival and distant metastasis-free survival analysis according to the presence of overall and TP53 mutations. The grouping of cases was performed using 4 variables: i mutations detected before (B) chemo-radiotherapy (CRT), ii. mutations detected after (A) CRT, iii. mutations detected before and/or after CRT, and iv. mutations detected before and after CRT. Abbreviations: LRFS = locoregional relapse-free survival, OS = disease-specific overall survival, DMFS = distant metastasis-free survival.",
"section_name": "Gene Mutations",
"section_num": "3.1."
},
{
"section_content": "",
"section_name": "All Mutations",
"section_num": null
},
{
"section_content": "",
"section_name": "LRFS",
"section_num": null
},
{
"section_content": "Supplemental Table S2 reports the distribution of overall and TP53 mutations according to the age of patients and histopathological variables. For cases with the T4 stage, there was a significantly higher chance of detecting mutations before and after CRT (6/17 T4 patients vs. 1/21 T0-3 patients; p = 0. 03). No other association with age, T stage, N stage, or histopathological grade was noted. Regarding TP53 mutations, these prevailed in the T4 stage compared to other stages, reaching a maximum significance for patients who had mutations both before and after CRT (5/17 T4 patients vs. 0/21 T0-3 patients; p = 0. 007).",
"section_name": "Associations with Histopathological Variables and Patient Age",
"section_num": "3.2."
},
{
"section_content": "Supplemental Table S3 reports the distribution of overall and TP53 mutations (recorded before and after CRT) in patients according to the response obtained after CRT. The only statistically significant association concerned the analysis of overall mutations assessed after the end of CRT. Mutations were observed in 8/30 (26. 6%) responders (CR/PR) vs. in 6/8 (75%) of the rest of patients (p = 0. 03). This difference showed a statistical trend after analysis for TP53 mutations (p = 0. 08).",
"section_name": "Associations with Response to CRT",
"section_num": "3.3."
},
{
"section_content": "Table 2 and Figure 2 report the univariate and Kaplan-Meier LRFS analysis, according to the presence of overall and TP53 mutations. Significantly poorer LRFS was noted for patients with persistent detection of mutations (mutations detected before and after CRT) (p = 0. 02). A marginal association was observed for patients with detectable mutations after CRT (p = 0. 08). \n\nUnivariate tables from Kaplan-Meier loco-regional relapse-free survival, diseasespecific overall survival and distant metastasis-free survival analysis according to the presence of overall and TP53 mutations. The grouping of cases was performed using four variables: i. mutations detected before (B) CRT, ii. mutations detected after (A) CRT, iii. mutations detected before and/or after CRT, and iv. mutations detected before and after CRT. We found no association of mutations with the OS (Supplemental Figure S1 ). Analysis of DMFS showed that patients with mutations after CRT and patients with mutations after CRT had a marginally poorer outcome (p = 0. 11 and 0. 09, respectively), which reached significance for patients who had detectable mutations either before or after CRT (p = 0. 04) (Table 2 and Figure 3 ). None of the patients without mutations (before or after CRT) developed metastasis during their follow-up. Analysis of TP53 mutations showed that patients with mutation after CRT and patients with mutations either before or after CRT had a significant association with poor prognosis (p = 0. 05 and 0. 02, respectively). \n\nWe found no association of mutations with the OS (Supplemental Figure S1 ). Ana sis of DMFS showed that patients with mutations after CRT and patients with mutatio after CRT had a marginally poorer outcome (p = 0. 11 and 0. 09, respectively), wh reached significance for patients who had detectable mutations either before or after C (p = 0. 04) (Table 2 and Figure 3 ). None of the patients without mutations (before or af CRT) developed metastasis during their follow-up. Analysis of TP53 mutations show that patients with mutation after CRT and patients with mutations either before or af CRT had a significant association with poor prognosis (p = 0. 05 and 0. 02, respectively).",
"section_name": "Survival Aanalysis",
"section_num": "3.4."
},
{
"section_content": "Supplemental Tables S4 and S5 present the specific gene mutations recorded before and after CRT, respectively, in patients whose disease progressed or did not progress after CRT. Regarding TP53 mutations detected before CRT, p. R248W, p. V157F, p. Y220C, p. C238Y and p. C135S characterized patients who progressed after therapy. Regarding TP53 mutations detected after CRT, p. H179L, p. R213=, p. R248W, p. C238Y and of two newly detected mutations p. S241F, and p. V157F characterized patients who progressed after therapy. \n\nAmong other gene mutations, the p. R505C mutation of the FBXW7, the p. E894K mutation of the AR, and the p. F384L mutation of the FGFR3 genes detected before CRT were recorded in patients with disease progression. In addition, detection of the p. F384L mutation of the FGFR3 and the p. R2217W mutation of the mTOR after CRT were found in two patients, respectively, with disease progression.",
"section_name": "Specific Gene Mutations and Disease Progression",
"section_num": "3.5."
},
{
"section_content": "Gene mutations are frequently present in SCHNCs. These mutations concern genes involved in cell proliferation, survival, and death regulation pathways (e. g., p53 and EGFR signaling pathway), cellular differentiation (e. g., Wnt, NOTCH1, Hedgehog pathway), or regulation of the cell cycle (e. g., cyclins and related genes). TP53 mutations seem to have a dominant role in the biology of SCHNC [19]. Huang et al. reported that mutations of the TP53 were noted in tissue samples of 55% of SCHNCs, and this rate was similar in HPV-positive and -negative tumors [20]. Using NGS, TP53 mutations can also be detected in the plasma of SCHNC patients [21]. Economopoulou et al. reported a 32. 6% rate of TP53 mutations in a series of 45 SCHNC patients [22]. Furthermore, in an investigation detecting gene alterations in the ctDNA from the saliva of SCHNC patients, mutations were recorded in 76% of cases [23]. In the current study, we confirmed an evident prevalence of mutations of TP53 in SCHNCs, which concerned 26. 3% of patients examined at baseline. In addition, we identified multiple TP53 mutations in a minority of patients, while mutations of other genes, like EGFR, AR, FGFR3, and FBXW3, were noted in 10% of patients. The rate of TP53 mutations reported herein are similar to the one reported by Economopoulou et al. [22], but certainly lower than the 50-80% rates reported in studies on tissue samples [19, 20]. Of interest, Porter et al. and Galot et al. recorded ctDNA TP53 mutations in 68% and 50% of patients with head and neck cancer, respectively [24, 25]. However, in both studies, blood samples were drawn from patients with recurrent or metastatic disease, a parameter that could potentially explain the higher rates of TP53 mutations. \n\nIn this investigation, we also performed an analysis of gene mutations detected in the blood of SCHNC patients immediately after the end of CRT. In this way, we could identify the persistence, disappearance, or new mutations of genes after therapy. After CRT completion, TP53 mutations were undetectable in about half of patients with baseline detectable mutations. This may be a result of high intrinsic radiosensitivity and early elimination of cancer cell clones bearing these mutations during the course of CRT. Although TP53 mutations are involved in apoptosis inhibition and resistance to RT, this effect is not consistent as specific TP53 mutations have been linked with enhanced apoptotic tendency after irradiation [26]. Additional molecular pathways may also counteract p53-mediated radioresistance and sustain radiosensitivity [27]. For example, the FBXW7 gene, mutations of which were noted in one patient before CRT, has been shown to confer survival of cancer cells during RT by induction of p53 protein degradation and blockage of apoptosis [28]. \n\nPersistent detection of specific TP53 mutations, and, for one case, mutations of FGFR3, was noted in about 10% of patients after the end of CRT in our study. In addition, in 18% of the patients with undetectable TP53 mutations at baseline, new TP53 mutations could be detected post CRT. In this way, the total rate of TP53 mutations recorded in ctDNA was 44. 7%. Moreover, new mutations of other genes, undetectable at baseline, were also recorded in a minority of patients, and these concerned MTOR, EGFR3, ALK, and SF3B1. Emerging mutations in patients with esophageal cancer progressing after CRT have also been noticed in a study by Azad et al. [29]. Persistence of baseline mutations and the emergence of new detectable mutations could indicate radioresistance of the cancer cell compartment bearing these very gene mutations. Indeed, in the current study, patients with detectable mutations after CRT had a significantly lower tumor response rate. In this context, an interesting study in medulloblastoma suggested that the dominant clone at recurrence after RT emerges through selections of pre-existing minor clones [30], which may also apply to patients where new mutations were recorded after CRT. \n\nAs far as prognosis is concerned, patients with persistent detection of mutations after CRT (detectable mutations at baseline) had significantly worse LRFS. A marginal association was also noted for patients with mutations detected after CRT. Although we found no significant association with OS, patients with detectable mutations after CRT, or mutations before and/or after CRT had a significantly higher rate of development of distant metastases. This finding was noted after taking into account all gene mutations and when analysis concerned TP53 mutations only. A retrospective mutation analysis of the ctDNA of 75 patients with SCHNC (stages I-IV, stage IV 62. 7%) demonstrated that both overall ctDNA alterations and TP53 mutations significantly correlated with advanced tumor progression status and OS [31]. In addition, it has been reported that the presence of ctDNA mutations either before or before and after treatment with CRT was linked with decreased survival [22]. Taylor et al. published the results of a study in SCHNC patients treated with chemotherapy or immunotherapy, suggesting that, although baseline ctDNA abundance was not associated with OS, changes in the ctDNA variant allele frequency were predictive of progression-free survival [32]. Two additional studies, although performed in squamous cell esophageal cancer, showed a significant association of ctDNA mutations with prognosis. Wang et al. reported that detectable ctDNA alterations one or several months after RT were linked with inferior progression-free survival of patients, while a better prognosis was recorded for patients whose ctDNA disappeared one month after therapy [33]. Azad et al. also found an increased risk of disease progression in patients with squamous cell esophageal cancer when ctDNA mutations were recorded after CRT [29]. \n\nBeyond the well-known limitations related to the NGS procedure (quality of the isolated DNA, bioinformatic analysis variations, false negative results), other limitations of the study include the relatively low number of patients recruited in the prospective trial due to predefined funding and the high cost of NGS experiments. Moreover, although the study focused on SCHNC, this includes different primary tumor locations with eventual different pathogenesis, clinical behavior and prognosis. In addition, the HPV status was not studied in parallel with NGS. Longer follow-up could also have allowed the extraction of more robust conclusions. Nevertheless, the treatment was consistent for all patients and the inclusion of liquid biopsies after the end of therapy provided further insights of the biology behind the interplay of CRT with tumor biology.",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "Despite the aforementioned limitations, it is suggested that detection of TP53 and other gene mutations in the ctDNA from the plasma of patients with SCHNC treated with radical CRT can be achieved with NGS. Assessment of mutations before and after the end of CRT is, however, essential to characterize patients with high risk of locoregional recurrence or even metastatic progression. Persistent detection of mutations, pre-existing or new, appeared as the major identified parameter that predicted locoregional progression after CRT. Although TP53 mutations prevailed, detection of less frequently recorded mutations of other genes, like FGFR3, MTOR, EGFR3, ALK, and SF3B1, mutations after CRT seem also to contribute to the overall association of the mutational burden with disease progression. The genomic alterations post CRT described herein provide a platform for novel therapeutic approaches for SCHNC that test combined targeted therapies and CRT.",
"section_name": "Conclusions",
"section_num": "5."
},
{
"section_content": "The following supporting information can be downloaded at: https:// www. mdpi. com/article/10. 3390/curroncol30100643/s1, Figure S1 : Kaplan-Meier disease specific overall survival curves according to the existence of overall mutations and TP53 mutations detected before chemo-radiotherapy, after CRT, before and/or after CRT and, finally, before and after CRT; Table S1 : Patient and disease characteristics; Table S2 : Distribution of overall and TP53 mutations, according to the age of patients and histopathological variables; Table S3 : Distribution of overall and TP53 mutations; Table S4 : Distribution of specific gene mutations before CRT in patients according to the progression status (after CRT); Table S5 : Distribution of specific gene mutations at the end of CRT in patients according to the progression status (after CRT).",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
[
{
"section_content": "Data Availability Statement: All data are available in the files of the Department of Radiotherapy. and Oncology, Democritus University of Thrace. The data presented in this study are available on reasonable request from the corresponding author.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Institutional Review Board Statement:",
"section_num": null
},
{
"section_content": "Author Contributions: Conceptualization, I. K. and C. N. B. ; validation, M. I. K., C. N. B. and I. K. ; formal analysis, M. I. K., E. X., S. P. F., C. K., I. M. K. and N. K. ; investigation, M. I. K., C. N. B., E. X., S. P. F. and N. K. ; writing-original draft preparation, I. M. K., writing-review and editing, M. I. K., E. X., S. P. F., C. K., I. K., C. N. B. and N. K. ; supervision, M. I. K. and C. N. B. ; funding acquisition, M. I. K. and C. N. B. All authors have read and agreed to the published version of the manuscript. \n\nFunding: This research has been co-financed by the European Regional Development Fund of the European Union and Greek National Funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH-CREATE-INNOVATE (project code: T2EDK-03266, project acronym, and title: \"BIOKAKETRA-Identification of genomic and transcriptomic prognostic bio-signatures in head and neck cancer\").",
"section_name": "",
"section_num": ""
},
{
"section_content": "The study has been approved by the local Ethics and Research Committee (ES1 23-01-2019 and ES2 22-02-2019) and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Written informed consent to enter the trial was obtained from all patients before therapy.",
"section_name": "Institutional Review Board Statement:",
"section_num": null
},
{
"section_content": "Written informed consent to enter the trial was obtained from all patients before therapy. Patients' consent included permission to publish their clinical and laboratory data for research and educational purposes anonymously.",
"section_name": "Informed Consent Statement:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. \n\nAppendix A Table A1. Mutated genes and principal functions.",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "",
"section_name": "GENE FUNCTION",
"section_num": null
},
{
"section_content": "A tumor suppressor gene. Encodes the tumor protein p53, a crucial regulator of apoptotic response, and guardian of the genome integrity. It also regulates DNA repair proteins and can induce cell cycle arrest at the G1/S cell cycle phase. Also involved in cellular senescence.",
"section_name": "TP53",
"section_num": null
},
{
"section_content": "The Epidermal Growth Factor Receptor or ErbB-1 gene encodes a transmembrane receptor that is activated by specific ligands like EGF and TGF-α, initiating a cascade of signaling events involved in proliferation, metabolism and resistance to chemotherapy and radiotherapy. Amplification and mutations of the gene promote aberrant activation lading to carcinogenesis and tumor progression.",
"section_name": "EGFR/ErbB1",
"section_num": null
},
{
"section_content": "Androgen receptor gene encodes ARs, transcription factors that, following their binding to testosterone, enter the nuclei to activate several genes involved in tumor progression.",
"section_name": "AR",
"section_num": null
},
{
"section_content": "Encodes a member of the fibroblast growth factor receptor family, a membrane protein that binds to the fibroblast growth factors of the tumor stroma, promoting proliferation and differentiation. Mutations of the FGFR3 have been detected in bladder cancer and glioblastomas and are involved in cell proliferation and resistance to anti-cancer therapy.",
"section_name": "FGFR3",
"section_num": null
},
{
"section_content": "The F-box and WD repeat domain containing 7 gene encodes a member of the F-box protein family with critical tumor suppressor functions. It controls the degradation of several oncoproteins (c-myc, mcl-2, mTOR, jun, cycline E) through the proteasome pathway. Its mutations promote carcinogenesis and tumor growth.",
"section_name": "FBXW7",
"section_num": null
},
{
"section_content": "The mammalian target of rapamycin gene regulates cell proliferation, autophagy, apoptosis and metabolism pathways including glycolysis. Its mutations promote carcinogenesis.",
"section_name": "mTOR",
"section_num": null
},
{
"section_content": "Encodes a member of the EGFR family protein. Activating mutations lead to resistance to anti-cancer therapy.",
"section_name": "ErbB3",
"section_num": null
},
{
"section_content": "The anaplastic lymphoma kinase gene can be activated in a subgroup of solid tumors, driving cell growth and resistance to chemotherapy. Specific targeting drugs have been approved for the treatment of ALK-positive patients with lung cancer.",
"section_name": "ALK",
"section_num": null
},
{
"section_content": "It encodes subunit 1 of the splicing factor 3b protein complex. Mutations of the gene are linked with chronic lymphocytic leukemia, myelodysplastic syndromes, breast cancer, and orbital melanoma.",
"section_name": "SF3B1",
"section_num": null
}
] |
10.3390/jcm11082076
|
Old and New Drugs for Chronic Lymphocytic Leukemia: Lights and Shadows of Real-World Evidence
|
<jats:p>Several novel treatments for chronic lymphocytic leukemia (CLL) have been recently approved based on the results of randomized clinical trials. However, real-world evidence (RWE) is also requested before and after drug authorization in order to confirm safety and to provide data for health technology assessments. We conducted a scoping review of the available RWE for targeted treatments of CLL, namely ibrutinib, acalabrutinib, idelalisib, and venetoclax, as well as for chemoimmunotherapy (CIT). In particular, we searched studies published since 1 January 2010 and reported outcomes of the above treatments based on health databases, registries, or phase IV studies, including named-patient programs. We included both full papers and abstracts of studies presented at major meetings. Overall, 110 studies were selected and analyzed: 28,880 patients were treated with ibrutinib, 1424 with idelalisib, 751 with venetoclax, 496 with acalabrutinib, and 14,896 with CIT. Reported discontinuation rates were higher than in clinical trials, while effectiveness could not be indirectly compared with clinical trials since a detailed case mix, including cytogenetic risk factors, was partially available and propensity scores rarely applied. RWE on CLL can help to set realistic outcomes with novel treatments, however, real-world studies should be fostered, and available data shared.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is an indolent lymphoproliferative neoplasm harbored by 5. 6 in 10,000 inhabitants (https://seer. cancer. gov/statfacts/html/clyl. html accessed on 30 December 2021). Clinical practice guidelines formerly recommended frontline chemoimmunotherapy (CIT) for all the patients [1] [2] [3], but several novel treatments, along with several combinations, have been progressively approved in the last 5 years by FDA and EMA: ibrutinib, idelalisib, venetoclax, obinutuzumab, acalabrutinib. The current therapeutic alternatives are, therefore, more diverse than in the past and several concerns apply on patient selection to a personalized treatment sequence. Moreover, patient selection bias due to severe restrictions of patient eligibility to clinical trials is a major hurdle for evidence-based medicine and health technology assessment of novel drugs [4]. Real-world evidence (RWE) gathered either retrospectively or prospectively from electronic health records, medical claims, databases, registries, or patient-generated data can complement the information reported by experimental studies. The pivotal role of RWE is witnessed by the 21st Century Cures Act requiring the FDA to expand the role of RWE and by the European Medicines Agency (EMA) draft guideline on real-world studies (RWS) developed either in the pre-or post-marketing authorization phase of drugs and devices (Table 1 ). (EMA draft guideline on real world studies 24 September 2020). \n\nTable 1. Aims of real-world studies (RWS).",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "To describe the characteristics of the target population To record the incidence of disease outcomes in the clinical practice To identify the determinants of disease outcomes in the clinical practice To provide information on standards of care",
"section_name": "Purposes of RWS before Drug Authorization",
"section_num": null
},
{
"section_content": "To confirm safety and effectiveness in the target population (i. e., phase IV studies) To confirm safety in subpopulations (i. e., comorbid patients) To survey modified patterns of care and health-care resource consumption\n\nThe present scoping review aims at scrutinizing RWE for novel targeted treatments of CLL [5, 6], namely ibrutinib, acalabrutinib, idelalisib, and venetoclax, as well as CIT. In particular, we aimed at checking the quality of available registries and named-patient program (NPP)-based studies, patient selection bias (such as age or comorbidities), the quality of available clinical and non-clinical data, and the length of follow-up.",
"section_name": "Purposes of RWS after Drug Authorization",
"section_num": null
},
{
"section_content": "We searched EMBASE, the largest bibliographic database of medical literature, by applying the following mast query: 'chronic lymphocytic leukemia' AND (RWD OR 'real life' OR 'real world' OR EHR OR registry OR register OR registries OR 'phase IV' OR postmarketing OR population-based) AND (bendamustine OR fludarabine OR chlorambucil OR venetoclax OR ibrutinib OR idelalisib OR rituximab OR ofatumumab OR obinutuzumab OR acalabrutinib OR rituximab) AND (2010:py OR 2011:py OR 2012:py OR 2013:py OR 2014:py OR 2015:py OR 2016:py OR 2017:py OR 2018:py OR 2019:py OR 2020:py OR 2021:py) AND 'article'/it. The search was limited to English-language publications reported from 1 January 2010 to 31 December 2020. \n\nTwo independent reviewers excluded the inappropriate records and retrieved the data from the selected records. Unspecific reports were deleted. Studies were also excluded if targeted at etiology (familiar lymphoproliferative disorders and exposures), survival, and prognosis (molecular biomarkers and undesirable events). \n\nThe following information were retrieved from selected records: Geographic limits",
"section_name": "Methods",
"section_num": "2."
},
{
"section_content": "Overall, 433 records were retrieved (Figure 1 ) and showed that the overall trend of publications addressing RWE of CLL rapidly had increased in the last 10 years (Figure 2 ). The full reference list is reported in Appendix A. Overall, 102 studies were selected from the list and 8 studies were added from the authors, therefore, the search query proved to be quite sensitive. Five studies were retrieved for more than one treatment. The full list of selected RWS is included in Appendix A. Major study characteristics are reported in Table 1. Most of the studies were from single institutions and size was often lower than 500 patients. Patients were representative of the real-life CLL population according to age. However, collected clinical data were unfortunately sparse in most of the published studies, for example, comorbidities were rarely systematically recorded and response rates were not reported by most of the studies. Moreover, follow-ups were short for most of the studies involving new oral inhibitors. Only 1 phase IV study was reported by Moreno, C. et al. [7] ; the study reported the outcomes of 103 patients treated with ofatumumab monotherapy after a median of four prior lines. The study reported a 5-month progression-free survival (PFS) and 11-month overall survival (OS), thus, confirming the data of the published randomized trial comparing ibrutinib versus ofatumumab. Only 1 phase IV study was reported by Moreno, C. et al. [7] ; the study reported the outcomes of 103 patients treated with ofatumumab monotherapy after a median of four prior lines. The study reported a 5-month progression-free survival (PFS) and 11-month overall survival (OS), thus, confirming the data of the published randomized trial comparing ibrutinib versus ofatumumab.",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "Ibrutinib is an orally administered irreversible inhibitor of bruton tyrosine kinase (BTK), representing the first-in-class of a new family of targeted drugs. Ibrutinib covalently binds to cysteine-481 within the active site of BTK, blocking the signal transduction from the B-cell receptor (BCR) and, thus, impairing CLL cells survival and proliferation (reviewed in [8] ). Ibrutinib has changed the therapeutic approach for patients with CLL both in first and subsequent lines of therapy, due to impressive response and survival rates, and acceptable tolerability observed in large, randomized clinical trials [9] [10] [11] [12] [13]. \n\nMany RWS addressed ibrutinib. Overall, 58 nonduplicated reports enrolled more than 50 CLL patients treated with the drug, mostly in the last 10 years. Enrolled populations were potentially representative of the clinical practice: median age was 69 years and median time from diagnosis was 56 months. Unfortunately, only a few studies were powered enough to detect predictors of safety or effectiveness endpoints in a multivariate analysis, namely large registries in the U. S. or healthcare databases. Moreover, both naive and relapsed/refractory patients were usually included. Many pieces of information were missing from many studies: median doses or median treatment durations, response rates, survival rates, and TP53 mutational status. Several other flaws could be observed in the retrieved studies: limited outcomes were analyzed (i. e., safety or hospitalization rate) and there were no studies that applied propensity-adjusted analyses. Despite the above limitations, the studies provided high discontinuation rates in the short time horizon analyzed.",
"section_name": "Ibrutinib",
"section_num": "3.1."
},
{
"section_content": "Acalabrutinib is a next-generation irreversible BTK inhibitor that was developed to reduce ibrutinib-mediated adverse effects, being more selective and lacking the inhibition towards other kinases (reviewed in [14] ). Acalabrutinib has entered clinical practice based on data demonstrating its high efficacy and enhanced tolerability, in both the frontline and relapsed/refractory setting [15]. \n\nWe retrieved four RWS reporting 496 patients with CLL treated with acalabrutinib; only one RWS was fully reported. A variable preportion of the patients (27-100%) were pretreated with and usually intolerant to ibrutinib. The discontinuation rate reported by two studies ranged from 19% in the first 6 months from treatment initiation to 30% after a median of 19 months. Cardiovascular events occurred in 6% of the largest reported cohort, but led to treatment discontinuation only in half of the cases and corresponded to a rate of 21/1000PY, which was lower than that reported in ibrutinib-treated patients [16]. \n\nThe overall response rate (ORR) was consistently above 60% in two studies and complete response (CR) was lower than 10%. Survival was documented in two cohorts (median age 64 years); 75% of the patients survived free of progression at three years and 75% were alive after five years, despite a high comorbidity burden, namely mean Charlson comorbidity score 1. 4 and 67% of the patients had a prior cardiac history. Patients experiencing a major cardiovascular event reported lower survival rates, namely 50% at 5-year follow-up. \n\nLong-term outcomes such as secondary neoplasms were also investigated in patients exposed to acalabrutinib, however, the increased hazard ratio of 2. 2 reported in CLL patients treated with either ibrutinib or acalabrutinib was possibly related to the disease itself, rather than to the treatment. No difference in risk of secondary neoplasms was reported in a multivariate analysis between ibrutinib and acalabrutinib. \n\nA recent retrospective analysis of a large CLL cohort from a single U. S. center specifically investigated the bleeding outcomes [17], however, 85% of the analyzed patients had been enrolled into clinical trials, 18% had prior bleed history, and 51% were on concomitant antiplatelet or anticoagulant medications. Overall, 835 of the patients experienced at least one bleeding event while on acalabrutinib; 98 out of 289 individuals experienced a clinically relevant or major event, but only 6% of the patients had a major bleed and 3% were CTCAE grade 3-5 (2 CNS fatal hemorrhages). Definitive discontinuation of acalabrutinib was decided for 6 patients with clinically relevant/major bleeds, while it was only temporarily held in 44 individuals and concomitant drugs were discontinued in 24 cases. Surgery-or invasive procedure-related bleedings were reported in 28 out of 1263 cases. Concomitant medications and a prior bleeding history were major predictors of bleeding events.",
"section_name": "Acalabrutinib",
"section_num": "3.2."
},
{
"section_content": "Venetoclax is an oral BH3-mimetic drug designed to inhibit the function of the Bcl-2 protein, thus, inducing apoptosis in tumor cells (reviewed in [8] ). Venetoclax, alone or in combination with an anti-CD20 monoclonal antibody, has demonstrated efficacy for the treatment of patients with treatment-naïve or relapsed CLL, eventually allowing a fixed-duration treatment [18] [19] [20]. \n\nSeven studies reported RWE on venetoclax in CLL patients; one study reported the French national early-access program and the other studies were retrospective national (n = 1) or multicenter (n = 5) studies. Overall, 751 patients were enrolled into the above studies, most of which were not reported as full papers. Enrolled patients had received a median of from three to four treatment lines, 47% harbored TP53 mutation, and many showed high-risk features, such as complex karyotype (27-61%) or unmutated IGVH status (81-87%). Grade 3-4 adverse events ranged from 23% to 39% and were mainly due to hematologic toxicity. Discontinuation rate was reported only by two studies and was quite low (4-11%); median treatment duration ranged from 12 to 18 months in two other studies. Response rates were quite high: median ORR was 74% and median CR was 25%. Richter transformation occurred in 4-5% of the patients but was reported only by two studies and the median duration of follow-up was shorter than 20 months in the four studies reporting this piece of information. Median PFS and OS were not reached in any study. Multivariate analyses were performed by five studies: survival was predicted by response to therapy, TP53 mutation (in two out of three studies), BCR-inhibitor discontinuation, multiple lines of target therapies, complex karyotype, performance status, and IGVH mutational status.",
"section_name": "Venetoclax",
"section_num": "3.3."
},
{
"section_content": "Traditionally, CIT was the standard approach in both the frontline and the relapsed/ refractory setting of CLL. Common CIT regimens include fludarabine/cyclophosphamide/ rituximab (FCR), bendamustine/rituximab (BR), and chlorambucil/rituximab. Older patients or those with comorbidities were recommended to not receive FCR due to the high risk of neutropenic fever and infections, despite very high rates of response in most of the patients [1, 2]. More recently, obinutuzumab combination with chlorambucil has progressively replaced chlorambucil/rituximab for the longer median PFS and OS despite similar toxicity, as reported by the CLL14 trial [21, 22]. \n\nWe retrieved 428 studies assessing CIT in real life by health registries (n = 13), electronic record databases (n = 2), or retrospective data collections (n = 11). Most of the studies involved multiple centers (n = 15) and followed a median of 277 patients (IQR 174-817) for a median of 37 months (Tables 2 and 3 ). The median patient age was 70 years (IQR 64-71) and most of the studies (n = 18) selectively enrolled naive individuals, but clinical information was often incomplete (Table 4 ). In particular, Rai stage and TP53 status were reported by 64% and 75% of the studies, respectively, and comorbidity data were missing in 61% of the studies. Response rates were reported by only 13 studies: median ORR rate was 61% and complete response (CR) rate 6%. Discontinuation rate was reported by only five studies: median rate was 20%. Patient survival was described by 75% of the studies (Table 3 ): median PFS and OS among the studies was 42 and 74 months, respectively.",
"section_name": "Chemoimmunotherapy",
"section_num": "3.4."
},
{
"section_content": "Idelalisib is an orally administered, selective, reversible inhibitor of the δ isoform of the phosphatidylinositol-3-kinase (PI3Kδ). The inhibition of PI3K downstream pathways (i. e., Akt/mTOR) hampers cellular growth, proliferation, and survival (reviewed in [23] ). From the clinical standpoint, the efficacy of the combination of idelalisib and rituximab has been demonstrated in the setting of relapsed CLL [8]. \n\nOverall, 16 studies reported one or more outcomes of real-world cohorts treated with idelalisib; most of the reported patients were relapsed/refractory and more than one third harbored TP53 disruption. \n\nThe largest cohort was reported by Bird et al. [24] ; 294 Medicare patients were compared with 89 patients enrolled into clinical trials. Detailed comorbidity profiles of the patients were provided showing that in the two cohorts, 71% versus 30% of the patients reported cardiac comorbidities, respectively. Similarly, 36% versus 7% showed a Charlson comorbidity score of 5 or higher. Median treatment duration in the Medicare cohort was much shorter than in the trial cohort, namely 173 versus 473 days and on-treatment mortality was 9. 9% versus 4. 5%. Serious infections were not different in the two cohorts, however, fatal infections occurred at a rate of 18. 4/100PY in the Medicare population versus 9. 8/100PY in the trial population. RETRO-idel was the highest quality retrospective study selected; it enrolled 110 patients treated with idelalisib in UK or Ireland, and was fully published in 2021 [25]. The study reported high discontinuation rates both in naiive and relapsed/refractory patients, namely 64% and 44%, respectively, but high ORRs (88%). Overall, 46% of the registered deaths were attributed to progressive disease and OS was 56% after 3 years. Median time-to-next treatment after stopping idelalisib was 29 months. \n\nTwo studies were specifically designed to patients reporting autoimmune cytopenias before starting idelalisib or receiving such a therapy as a bridge to stem cell transplantation [26, 27]. Only one study reported secondary neoplasms in 12. 9% of the patients after a median of 21 months from start of idelalisib treatment; the rates were not statistically different to those reported in ibrutinib-treated patients [28].",
"section_name": "Idelalisib",
"section_num": "3.5."
},
{
"section_content": "CLL is the most frequent leukemia and a large set of modern therapeutic options are available ranging from CIT to oral targeted drugs. These therapeutic options have all reported high rates of responses but also a relevant rate of toxicity in the published clinical trials, which accurately selected candidate patients. We, therefore, aimed at reviewing the available RWE on such therapies in order to test the quality of RWE and to retrieve real-world information for safety and effectiveness. \n\nEMA specifically fostered the development of patient registries, namely data collection systems on an unselected group of people defined by a particular disease or condition, serving a predetermined scientific, clinical. and/or public health purpose. RWE is particularly relevant for validating safety, including cardiovascular events, and especially for reporting rare events, such as SPM, rare infections, or unexpected events. RWE is also necessary for completing drug effectiveness profiles, including rare events, such as Richter transformation and concurrent disorders. Furthermore, treatment outcomes according to heterogeneous adoption of supportive care are fundamental in order to forecast the overall drug safety in the real world and provide management recommendations [29]. \n\nThe present manuscript systematically reviewed 117 RWS published from 2010 to 2021 and reported 46,447 CLL patients treated with CIT, idelalisib, ibrutinib, venetoclax or acalabrutinib. Unfortunately, 77 studies had been reported only at meetings and only limited data were available. Most of the studies were multicenter retrospective analyses and most of them targeted only a subset of outcomes. A complete clinical dataset, including age, stage and duration of the disease, comorbidity, and biologic risk status was provided only by a few studies, in particular, stage and disease duration were missing in most of the studies, while TP53 status was available in 57% of the studies. Only 26% of the RWS reported treatment discontinuation rates and 40% registered response rates; the median response rates ranged from 61% for acalabrutinib to 79% for idelalisib, and CR rate from 6% for acalabrutinib to 30% for CIT. Survival was also poorly described, since median OS or PFS were usually not reached in the follow-up period, which was usually shorter than 2 years in RWS assessing oral target drugs. Of notice, some research networks devoted to CLL published further high-quality RWS both before [30, 31] and after the cutoff date of our review [32] [33] [34] [35] [36], thus, demonstrating the effort of the scientific community in ameliorating the RWS quality. \n\nThe present systematic review aims at fostering further efforts of the scientific community towards RWE, which may become a very useful tool for both researchers and third-party payers. Institutional databases are major prerequisites for RWE, however, further efforts should be aimed at registering response rates, time-to-next treatment, and comorbidities [37]. Finally, large datasets would allow fine analyses including artificial intelligence algorithms and data mining. Gilead srl and speaker fees from Amgen.",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "",
"section_name": "Appendix A",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Funding: This research received no external funding. \n\nInstitutional Review Board Statement: Not applicable. \n\nInformed Consent Statement: Not applicable.",
"section_name": "",
"section_num": ""
},
{
"section_content": "No conflict of interest to declare for A. V. M. M. received consultancy fees from",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "",
"section_name": "Author Names Title Source",
"section_num": null
},
{
"section_content": "Author Contributions: M. M., A. V. (Alessandra Vasile), M. C., A. C., G. M. R., A. V. (Andrea Visentin), L. S. and C. V. contributed equally to the literature review. M. M. performed literature search and wrote the draft manuscript. M. M., A. V. (Alessandra Vasile), M. C., A. C., G. M. R., A. V. (Andrea Visentin), L. S. and C. V. equally contributed to revise the manuscript. All authors have read and agreed to the published version of the manuscript.",
"section_name": "",
"section_num": ""
},
{
"section_content": "DaCosta Byfield S., Blauer-Peterson C., Dawson K., Masaquel A. \n\nWhat are the health care utilization and costs associated with patients newly initiating anti-cancer systemic therapy for chronic lymphocytic leukemia? Pula B., Iskierka-Jazdzewska E., Dlugosz-Danecka M., Szymczyk A., Hus M., Szeremet A., Drozd-Sokolowska J., Waszczuk-Gajda A., Zaucha J. M., Holojda J., Piszczek W., Steckiewicz P., Wojciechowska M., Osowiecki M., Knopinska-Posluszny W., Dudzinski M., Zawirska D., Subocz E., Halka J., Pluta A., Wichary R., Kumiega B., Budziszewska B. K., Jurczak W., Lech-Maranda E., Giannopoulos K., Robak T., Jamroziak K. \n\nLong-term efficacy of ibrutinib in relapsed or refractory chronic lymphocytic leukemia: Results of the polish adult leukemia study group observational study\n\nAnticancer Research (2020) 40:7 (4059-4066). Date of Publication: 1 Jul 2020 Puła B., Iskierka-Jazdzewska E., Długosz-Danecka M., Szymczyk A., Hus M., Szeremet A., Drozd-Sokołowska J., Waszczuk-Gajda A., Zaucha J. M., Hołojda J., Piszczek W., Steckiewicz P., Wojciechowska M., Osowiecki M., Knopi ńska-Posłuszny W., Dudzi ński M., Zawirska D., Subocz E., Hałka J., Pluta A., Wichary R., Kumiega B., Budziszewska B. K., Jurczak W., Lech-Mara ńda E., Giannopoulos K., Robak T., Jamroziak K. \n\nLong-term real-world clinical outcomes for ibrutinib monotherapy treatment in relapsed refractory chronic lymphocytic leukemia-observational study of the polish adult leukemia study group (PALG)\n\nHemaSphere (2020) 4 Supplement 1 (867-868). Date of Publication: 1 Jun 2020",
"section_name": "Author Names Title Source",
"section_num": null
},
{
"section_content": "Puła B., Iskierka-Jazdzewska E., Długosz-Danecka M., Szymczyk A., Hus M., Szeremet A., Rybka J., Drozd-Sokołowska J., Waszczuk-Gajda A., Zaucha J. M., Hołojda J., Piszczek W., Steckiewicz P., Wojciechowska M., Osowiecki M., Knopi ńska-Posłuszny W., Kopacz A., Dudzi ński M., Zawirska D., Piotrowska M., Subocz E., Hałka J., Kumiega B., Gil L., Szukalski L., Wichary R., Budziszewska B. K., Lech-Maranda E., Jurczak W., Giannopoulos K., Robak T., Warzocha K., Jamroziak K.",
"section_name": "Author Names Title Source",
"section_num": null
}
] |
10.3324/haematol.2009.010173
|
Differential diagnosis of cyclin D2+ mantle cell lymphoma based on fluorescence in situ hybridization and quantitative real-time-PCR
|
Mantle cell lymphoma is characterized by the t(11;14) chromosomal translocation, resulting in the overexpression of cyclin D1 (CycD1). Recently, cases of mantle cell lymphoma negative for cycD1 but positive for cycD2 or cycD3 were identified by gene expression profiling and confirmed by immunohistochemistry. We analyzed 4 cases of cycD2(+) mantle cell lymphoma with a translocation involving the CCND2 locus, and its differential diagnosis from 35 mature B-cell non-Hodgkin's lymphomas based on immunohistochemistry, quantitative RT-PCR and FISH analysis. Bona fide cycD2(+) mantle cell lymphoma carried translocations involving the CCND2 gene, and IGH and IGK loci were identified as partners. As a result of this translocation, cycD2 mRNA was highly over-expressed when compared with normal lymphoid tissue and other B-cell non-Hodgkin's lymphomas, including chronic lymphocytic leukemia, making this technique ideally suited to identify cycD2(+)mantle cell lymphoma. In contrast, positive immunostaining for cycD2 was found in most B-cell non-Hodgkin's lymphomas, and therefore, it is not specific for a diagnosis of cycD2(+)mantle cell lymphoma.
|
[
{
"section_content": "Mantle cell lymphoma (MCL) is a distinct subtype of aggressive B-cell non-Hodgkin's lymphoma (NHL) with specific clinical and pathological features that accounts for approximately 6% of all lymphomas. 1 The genetic hallmark of mantle cell lymphoma (MCL) is the t(11;14) (q13;q32) chromosomal translocation that juxtaposes the immunoglobulin heavy chain (IGH) gene on 14q32 to the CCND1 gene on 11q13 resulting in the overexpression of cyclin D1 (cycD1) mRNA and protein. 1 Recently, a gene expression profiling study of MCL identified a small subset of tumors negative for cycD1 mRNA expression but morphologically, immunophenotypically, and by global expression profile otherwise undistinguishable from conventional MCL. 2 Interestingly, these cases instead expressed cycD2 or cycD3 mRNA, suggesting that any of these cyclins can functionally substitute for cycD1 in MCL. \n\nAccordingly, cycD1 negative MCL cases lacked the t (11;14) translocation by fluorescence in situ hybridization (FISH) analysis, 2 and were negative for cycD1 protein expression by immunostains. 3 However, no evidence of chromosomal translocations involving the corresponding CCND2 and CCND3 gene loci were identified. 3 The controversy surrounding cycD1 negative MCL was ended with the demonstration of bona fide cases of cycD2 positive MCL secondary to gene translocations involving the CCND2 locus on chromosome 12p13 with either the IGK locus on chromosome 2p12 t(2;12) (p12;p13), 4, 5 or a t(12;14) (p13;q32) translocation juxtaposing the CCND2 gene next to the IGH locus. 6 he diagnosis of cycD1 negative MCL is challenging because some low-grade B-cell lymphomas, such as chronic lymphocytic leukemia (CLL), marginal zone lymphoma (MZL) and follicular lymphoma (FL), may mimic MCL both morphologically and immunophenotypically. Indeed, the dif- Mantle cell lymphoma is characterized by the t(11;14) chromosomal translocation, resulting in the overexpression of cyclin D1 (CycD1). Recently, cases of mantle cell lymphoma negative for cycD1 but positive for cycD2 or cycD3 were identified by gene expression profiling and confirmed by immunohistochemistry. We analyzed 4 cases of cycD2 + mantle cell lymphoma with a translocation involving the CCND2 locus, and its differential diagnosis from 35 mature B-cell non-Hodgkin's lymphomas based on immunohistochemistry, quantitative RT-PCR and FISH analysis. Bona fide cycD2 + mantle cell lymphoma carried translocations involving the CCND2 gene, and IGH and IGK loci were identified as partners. As a result of this translocation, cycD2 mRNA was highly over-expressed when compared with normal lymphoid tissue and other B-cell non-Hodgkin's lymphomas, including chronic lymphocytic leukemia, making this technique ideally suited to identify cycD2 + mantle cell lymphoma. In contrast, positive immunostaining for cycD2 was found in most B-cell non-Hodgkin's lymphomas, and therefore, it is not specific for a diagnosis of cycD2 + mantle cell lymphoma. ferential diagnosis is important and relevant for patient treatment and prognosis. Until now, the recognition of potential cycD1 negative MCL has been based on microarray analysis, 2,3 a technique which is not available in routine practice. Although IHC for cycD2 and cycD3 has been proposed as a surrogate marker for cycD1 negative MCL, 3 the need to develop a reliable and accessible technique which is useful in the differential diagnosis is of utmost importance. The aim of this study was to investigate means to differentiate 4 cases of cycD2 + MCL with a CCND2 translocation from low-grade Bcell NHL, based on IHC, quantitative RT-PCR and FISH analysis with special interest on CD5 + B-cell NHL, including CLL and a subset of MZL.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Design and Methods",
"section_num": null
},
{
"section_content": "Formalin-fixed and paraffin-embedded biopsies from 35 well-characterized B-cell lymphomas, including 12 CLL, 8 MZL (5 cases CD5 + ), 5 FL and 10 cycD1+ MCL were selected from the files of the Institute of Pathology, Technical University of Munich, Germany. All cases were classified according to the guidelines of the World Health Organization (WHO) Classification of Tumors of Hematopoietic and Lymphoid Tissues. 7 Four cases of cycD2 + MCL with a CCND2 translocation were collected from the University Hospital Schleswig-Holstein Campus Kiel, Germany, CHU Sart Tilman, Liege, Belgium, Cleveland Clinic, USA, and Technical University of Munich, Germany. Two of these cases have been the subject of previous publications. 4, 6 As controls, 9 cases of normal lymph nodes were used.",
"section_name": "Tissue samples",
"section_num": null
},
{
"section_content": "All cases were previously studied by paraffin section immunohistochemistry (IHC) to assess lymphoid immunophenotype. The expression of cyclin D1 (SP4 clone, LabVision Corporation) and cyclin D2 (rabbit polyclonal, Cell Signaling Technology) was investigated in paraffin-embedded sections. IHC was performed on an automated immunostainer (Ventana Medical Systems, Inc., Tuczon, AZ, USA) according to the com-pany´s protocol. 8",
"section_name": "Immunohistochemistry",
"section_num": null
},
{
"section_content": "Real-time quantitative RT-PCR analysis was performed using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). For the quantification of cycD2 we used the following sequences: 5'-CGCAAGCATGCTCAGACCTT-3', 5'- CLL case with cyclin D2 nuclear expression in a percentage of tumor cells. Note how some paraimmunoblasts reveal stronger cyclin D2 expression (F). Images were recorded using a Hitachi camera HW/C20 installed in a Zeiss Axioplan microscope with Intellicam software. FISH images were acquired with a 63x/1. 40 oil-immersion objective in a Zeiss Axioskop2 fluorescence microscope (Zeiss, Göttingen, Germany) equipped with the appropriate filter sets (AHF, Tübingen, Germany). Image processing was carried out with Zeiss computer software (AIM 3. 2). Maximum projection mode was used to produce extended focus image stacks. \n\nTGCGATCATCGACGGTGG-3', 5'-FAM-TGCCACC-GACTTTAAGTTTGCCATGT-TAMRA-3'. The sequences of cycD1, cycD3 and TBP (TATA box-binding protein), as housekeeping gene have already been described. 9, 10 The assay and analysis were performed as previously described. 11",
"section_name": "Real-time quantitative RT-PCR",
"section_num": null
},
{
"section_content": "Locus-specific interphase FISH was performed on paraffin-embedded tissue sections according to the manufacturer's instructions (Abbott/Vysis) with minor modifications. The t(11;14) was investigated using commercially available probes (LSI IGH/CCND1; Vysis, Downers Grove, IL) in all MCL and CD5+MZL. Translocations affecting the CCND2 (12p13) and IGK (2p12) loci were investigated using recently described probes. 3",
"section_name": "FISH analysis",
"section_num": null
},
{
"section_content": "The 4 cases of cycD1 negative MCL showed clinical, morphological and phenotypic characteristics of MCL. Cases 1 and 2 are 2 male patients aged 71 and 54 years, who presented with stage IV disease. These cases have been previously reported. 4, 6 Cases 3 and 4 are 2 novel cases that corresponded to an 82-year old female with involvement of the Waldeyer's ring and cervical lymph nodes (Case 3, Figure 1A-C ) and to a 59-year old male with stage IV disease. The lymph nodes in the 4 cases showed a nodular and diffuse growth pattern with a CD20 +, CD5 +, CD10 -, CD23 -(4/4), and p27-(3/3) phenotype, but lack of cycD1 expression. Instead, cycD2 was positive. Interphase FISH demonstrated an IGK-CCND2 fusion indicating the presence of a t(2;12) (p12;p13) translocation in Cases 1 and 3. A cytogenetically cryptic translocation t(12;14) (p13;q32) involving the IGH locus in chromosome 14q32 and leading to IGH-CCND2 juxtaposition was present in Case 2. 6 In Case 4, interphase FISH demonstrated a clear CCND2 break with normal IGK and IGH, indicating the probability of a novel translocation partner, in addition to the already described translocations with IGK and IGH. Unfortunately, hybridization with an IGL probe failed repeatedly. Immunohistochemical analysis was performed in 35 cases of small B-cell lymphomas for cycD1 and cycD2 proteins. Due to the difficulties in the differential diagnosis of CD5 + small Bcell lymphomas, MZL expressing CD5 were preferentially included in the study. CycD1, as expected, was positive only in the 10 MCL cases, all of which had an IGH-CCND1 juxtaposition indicating t (11;14). In contrast, cycD2 was positive in all normal lymph nodes and lymphomas analyzed. This finding is not completely unexpected since cycD2 is the main cyclin expressed in normal B cells. The percentage of positive cells and intensity of positivity varied from case to case; however, CLL cases showed the strongest reactivity among the lymphomas analyzed (Figure 1D-F ). This result clearly indicates that immunohistochemical detection of cycD2 is not helpful in the differential diagnosis of cycD1 negative MCL. On the contrary, reliance on cycD2 IHC may well lead to the overdiagnosis of cycD1 negative MCL in phenotypically and morphologically difficult cases, such as CLL and CD5 + Figure 2. Cyclin D2 mRNA expression in mature B-cell NHL. Quantitatitve RT-PCR analysis of cyclin D2 was performed relative to the TBP housekeeping gene and results are depicted as ratio of cyclin D2/TBP transcript numbers. The horizontal line indicates the cut-off value for altered cyclin D2 expression (cycD2/TBP ratio=15. 4), which corresponds to the mean value of cyclin D2 in normal lymph nodes (cycD2/TBP ratio=6. 8) plus three standard deviations (SD:2. 8). The difference in cyclin D2 expression between cyclin D2 + MCL and the remaining cases is statistically significant (p=0. 04). MZL. FISH analysis for the IGH-CCND1 fusion indicating t(11;14) and for chromosomal translocations affecting the CCND2 locus in 12p13 was negative in all the CD5 + MZL analyzed. Since the identification of cycD1 negative MCL was based primarily on gene expression profile, 2 it seemed logical to consider that quantitative analysis of cycD mRNA levels could be the appropriate method to diagnose cases of cycD1 negative MCL. Therefore, we investigated the levels of the three D-type cyclins in normal lymph nodes and the selected B-NHL cases. The findings are summarized in Table 1. Normal lymph nodes showed, in every case, preferential expression of cycD2 (cycD2/TBP ratio=6. 8) with lower expression levels of cycD1 and cycD3. Interestingly, MCL cases with t(11;14) translocation have very low expression of cycD2mRNA. Accordingly, we recently reported that in MCL the low levels of cycD2 are the consequence of downregulation through the abnormally high levels of cyclin D1. 12 In general, CycD2 mRNA levels were slightly increased in MZL and FL, moderately increased in CLL and strikingly increased in cycD2 + MCL (Figure 2 ). Although some CLL cases have up to 6 times the amount of cycD2 mRNA found in lymph nodes (mean cycD2/TBP 21 vs. 7, p<0. 001), the levels of expression were far below the cycD2 mRNA levels found in cycD2 + MCL with a translocation involving the CCND2 locus (cycD2/TBP 21 vs. 202, p=0. 004). These results indicate that quantitative RT-PCR and/or FISH are ideal methods to confirm the diagnosis of cycD2 + MCL. Accordingly, our previous studies concerning cycD1 mRNA expression in MCL 10 and multiple myeloma, 13, 14 showed that very high levels of cycD1 mRNA were always associated with translocation involving the CCND1 locus. Importantly, the cases reported of cycD2 + MCL without translocation need to be analyzed carefully concerning the amount of cycD2 mRNA expression, and other possible mechanisms of cycD2 deregulation. It is of note that cycD2 + MCL are extremely rare, and in order to avoid overdiagnosis it is mandatory to perform either quantitative analysis of cycD2 mRNA and/or FISH.",
"section_name": "Results and Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Authorship and Disclosures",
"section_num": null
},
{
"section_content": "LQ-M was the principal investigator and takes primary responsibility for the paper; LQ-M and FF designed research, coordinated the research, analyzed and interpreted data and drafted the manuscript; JS-H, IK, MK, and SG performed the laboratory work for this study; LdL, EH, RS, and WK, contributed with case material and discussed data. \n\nThe authors have no conflict of interests to declare.",
"section_name": "Authorship and Disclosures",
"section_num": null
}
] |
10.3324/haematol.2009.005561
|
Predictive value of 2-microglobulin ( 2-m) levels in chronic lymphocytic leukemia since Binet A stages
|
We read with interest the study by Rossi and coworkers, reporting CD49d expression as risk factor of treatment free survival (TFS) in Binet A CLL patients.[1][1] In this paper, a close association between CD49d and CD38, LDH and β2-m is also described. We would like to add further information about
|
[
{
"section_content": "We read with interest the study by Rossi and coworkers, reporting CD49d expression as risk factor of treatment free survival (TFS) in Binet A CLL patients. 1 In this paper, a close association between CD49d and CD38, LDH and β2-m is also described. We would like to add further information about the prognostic power of β2-m. It is generally believed that β2-m is released constitutively by CLL cells and that its level approximately correlates with tumor mass. 2 Based on these premises the predictive value of β2-m serum concentration could vary in the course of the disease and be relatively low in the early disease stages, when tumor mass is low, irrespective of the subsequent clinical outcome. Therefore, β2-m determination could exhibit a lower predictive power particularly at the early disease stages compared to the newer biological markers, such as IgVH gene status, ZAP-70 and CD38, which represent intrinsic cell features that can be determined since the earliest disease stages and never (IgVH) or rarely (ZAP-70 and CD38) change in the course of the disease. 3 n order to explore this issue, we have measured β2m value in 222 Binet stage A patients at diagnosis. IgVH gene status and CD38 expression were also determined in all cases studied. Unlike β2-m, which was measured at diagnosis, these markers were determined in the course of the disease when marker determinations became available. This approach, although irrelevant for the IgVH gene status, may introduce some, albeit minor, biases for CD38 for the reasons alluded to above. The median β2-m value was 2 mg/dL (range 0. 4-19). ROC analysis determined that the cut-off value capable of discriminating between patients whose disease progressed and required treatment from those with stable disease was 2. 4 mg/dL (AUC:0. 67, p=0. 005). Accordingly 149/222 patients (67%) were β2-m neg and 73/222 (33%) as β2-m pos. Overall, the results did not substantially change when arbitrary cut offs used by other authors [4] [5] [6] [7] were employed. \n\nThe patients' features are summarized in Table After a median follow-up of 3. 5 years, 55 of 222 Binet stage A (25%) required treatment. β2-m neg cases showed a significantly longer TFS than β2-m pos cases; in particular the projected median TFS was 5. 3 years for β2-m pos versus not reached for β2-m neg (Figure 1A ). TFS represented a reliable measure of disease progression since all centers agreed to follow NCI guidelines for treatment start. \n\nIn order to ascertain whether β2-m identifies a patient subset of those with good prognostic markers, we calculated TFS of both CD38<30% and IgVHmutated CLL cases grouped according to the β2-m expression. β2-m pos CD38<30% cases exhibited a TFS which was significantly lower than that of β2-m neg CD38<30% cases (3. 5years TFS probability: β2-m neg vs. β2-m pos 91% vs. 83%; p=0. 05). However, these differences were not seen in the IgVHmutated cases (3. 5-years TFS probability: β2m neg vs. β2-m pos 89% vs. 84%; p=ns). \n\nAt Cox univariate analysis, β2-m pos (HR:2. 3, p=0. 003), CD38>30% (HR:3. 9, p<0. 0001) and IgVHunmutated (HR:3. 2, p<0. 0001) showed a statistically significant impact on TFS. At Cox multivariate analysis, all the three markers maintained an independent prognostic impact (β2-m pos, HR:1. 8, p=0. 047; CD38>30%, HR:2. 0, p=0. 03; IgVHunmutated, HR:2. 7, p=0. 022). When a scoring system in which one point was assigned to each unfavorable prognostic marker was utilized, the risk of an early treatment was highest (Figure 1B ) in patients presenting all the three adverse prognostic markers. Cases with two, one or none of the unfavorable prognostic factors showed lower risk for an early treatment (Figure 1C ). \n\nCollectively, this study shows that β2-m levels represent valuable predictors in early CLL stages, when the neoplastic cell burden is low. This finding raises a number of questions regarding the mechanisms governing the β2-m levels. This molecule is constantly shedded 8 Table 1. Comparisons of clinical and laboratory features among chronic lymphocytic leukemia patients devised according to β2-m expression.",
"section_name": "Predictive value of β2-microglobulin (β2-m) levels in chronic lymphocytic leukemia since Binet A stages",
"section_num": null
},
{
"section_content": "β β2-m <2. by lymphocytes and it is expected that its levels steadily increase together with the progressive expansion of the leukemic clone suggesting a close correlation between stage (which measures tumor burden) and β2m levels. Although a correlation with disease stage likely exists, there was a substantial proportion of patients with high β2-m levels already at Binet A stage (low tumor burden). Possibly, CLL cells from these patients are more activated in vivo and shed more abundant β2m. Taken all the above into consideration, the data indicate that the role of β2-m as a prognostic tool should be re-evaluated possibly in prospective studies involving large patient cohorts. Massimo Gentile, 1 Giovanna Cutrona, 2 Antonino Neri, 3 Stefano Molica, 4 Manlio Ferrarini, 2, 5 and Fortunato Morabito",
"section_name": "All patients",
"section_num": null
},
{
"section_content": "Anti-doping control of erythropoietin (Epo) relies on the differentiation by isoelectric profile of natural endogenous hormone from the recombinant hormone used for doping. The first and second generations of recombinant Epo were detectable in urine.",
"section_name": "Detection of continuous erythropoietin receptor activator in blood and urine in anti-doping control",
"section_num": null
}
] |
[
{
"section_content": "Funding: supported from Associazione Italiana Ricerca sul Cancro (AIRC) (to FM and MF) and Fondazione 'Amelia Scorza' Onlus, Cosenza, Italy. Acknowledgments: we would like to acknowledge Dr. Vincenzo Callea, Prof Luca Baldini, Dr Ugo Consoli and Dr Serena Matis for their contribution and useful suggestions. We thank Laura Veroni and Brigida Gulino for precious secretarial assistance. Key words: β2-microglobulin, CD38, IgVH mutational status, CLL, prognosis. Correspondence: Fortunato Morabito, Unità Operativa Complessa di Ematologia, Dipartimento di Medicina Interna, Azienda Ospedaliera di Cosenza, Viale della Repubblica, 87100 Cosenza, Italy. Phone: international +39. 0984. 681329. Fax: international +39. 0984. 791751. E-mail: fortunato_morabito@tin. it Citation: Gentile M, Cutrona G, Neri A, Molica S, Ferrarini M, and Morabito F. Predictive value of B2-microglobulin (B2-m) levels in chronic lymphocytic leukemia since Binet A stages. Haematologica 2009; 94: 887-888. doi:10. 3324/haematol. 2009. 005561",
"section_name": "",
"section_num": ""
}
] |
10.3390/math8040564
|
Sensitivity Analysis of Mathematical Model to Study the Effect of T Cells Infusion in Treatment of CLL
|
<jats:p>In this paper, we considered a mathematical model concerned with the treatment of Chronic Lymphocytic Leukemia (CLL) taking into account the effect of superficially infused T cells in this particular type of tumor. The model is described thoroughly by the system of non-linear differential equations explaining the interaction of naïve, infected, cancer and immune cell population. The detailed sensitivity analysis with the application is the major part of this paper. The basic objective is to provide insight to how parameters’ behavior varies model results by elaborating the results obtained from the application of sensitivity analysis. The sensitivity of the model was evaluated not only theoretically, but also with the help of a numerical approach, producing graphs providing better imminent of results. We argue that the application of the sensitivity analysis method endows an insight into how and which parameters are of primary significance in controlling the spread of leukemia.</jats:p>
|
[
{
"section_content": "The inherited concept of irrelevance of mathematics and biology has been changed to a great extent, somewhat because of the realization of the scientific world [1] of the undeniable services of mathematics in helping to gain a better understanding of various biological phenomena occurring in nature on daily basis. Mathematicians feel pleased and proud of their being praised by other scientists due to the role of mathematics in biology. Mathematical modeling is a remarkable example of the healthy support provided by mathematicians for better understanding [2] [3] [4]. Even complex biological models can be better understood by computational and mathematical models [5] [6] [7]. \n\nMillions of people's lives are in danger due to cancer throughout the world. There are many definitions of cancer available in the literature but the simplest one is the out of control development and splitting up of dungeons. There are several types of tumor, among which one is known as Leukemia, usually known as blood cancer [8]. This disease starts from blood stem cells. These cells are assumed to grow either as lymphoid or myeloid branch cells. The scheme of growth of Lymphoid Stem Cells (LSC) is as follows: these grow into lymphocytes, a type of white corpuscle acting to boost the body immune sense, which helps encounter viruses and wipe out unusual cells, whereas Myeloid Stem Cells (MSC) breed as erythrocyte and thrombocytes. Erythrocytes award oxygen to all tissues and thrombocytes form clots to avoid hemorrhage [9, 10]. As the stem cells grow, these become blast cells, known to be undeveloped blood cells. In leukemia, there is an overproduction of these cells. These cells keep on multiplying without any bound and do not become mature ones in the long run. This multiplication of abnormal cells then stops not only the proper functioning of normal cells, but also the normal distribution of the remaining standard cells, which, in turn, gives rise to tumor, and such persons are diagnosed as Leukemic patients. \n\nThe explanation of the physiology of the Lymphatic System in body homeostasis is of prime importance for better understanding the causes of Chronic Lymphocytic Leukemia. The main components of the lymphatic system are immigration dendritic cells, a type of white blood cell called macrophages, lymph, lymphatic vessels, lymphoid tissues, lymphocytes, and phagocytes. Lymphatic system has two basic sections; one is known as peripheral and the second as central [11]. \n\nThe basic function of this system is to transfer lymph all over the body and also provide support to throw out waste and unnecessary material. The key function of the lymphatic system is to provide enhanced immunity to fight against cancer [12, 13]. \n\nA sufficiently large number of people have been diagnosed with leukemia tumors. According to very recent research, a total of 60,300 new cases were registered, including 35,030 males and 25,270 females in this total, and a number of deaths up to 24,370, including 14,270 males and 10,100 females. This research has been carried out by the United States, with results published under an article titled \"Estimated new cancer cases and deaths by sex, United States 2018\" [14, 15]. \n\nA variety of treatment options are accessible, that generally include chemotherapy, targeted therapy, radiation therapy, and stem cell transplant [16, 17]. Though all the methods mentioned above have been used to treat cancer for many years, each of these treatments have some side effects associated with them. To avoid these unwanted side effects, scientists found a new way of treating tumor, known as Adoptive Immunotherapy [18, 19]. This is a category of immunotherapy in which leukocytes are united with a nature-produced augmentation aspect to enhance their cancer fighting capability [20]. The sole purpose of this practice is to boost the immune response of that individual [21]. \n\nMathematical models are brought into service to estimate various, highly complex engineering, physical, environmental, social, economic and biological phenomena. Mathematical modeling plays a vital role as it adds to the ability to understand the true nature of the problem and also to predict system behavior that will, in turn, define the problem and its solution in a physical sense. Many syndromes have been studied in which the spread of an infection takes place from cell to cell. Many scientists and mathematicians have considered cancer treatment by immunotherapy, treating normal and cancer cells as competitors [22]. Many mathematical models have been developed and studied that show competition between tumors and immune system, considering the role of antibodies. \n\nThe model under study is a mathematical model consisting of four nonlinear differential equations that describe the change in the population of naïve, infected, cancerous and immune cells with respect to time, which studies the spread of leukemia with the consequence of outdoor engineered T cells' permutation in cancer patients. The model also considers blood transfusion, as it is much needed in the treatment of cancer patients [23]. \n\nSensitivity analysis is defined to be the study of how the ambiguity in the output of a model can be distributed to different sources of doubt in its input. It can alternatively be defined as the methodical exploration of model reaction to either (1) the perturbation of the model's quantitative cause (e. g., input and/or parameters), or (2) a distinction in the model's qualitative aspects (e. g., arrangement, connectivity). Model constraints with most influence on the model results are recognized through 'Sensitivity Analysis' [24, 25]. In this paper, we applied sensitivity analysis in a local sense (one input is varied by a small amount at a time while keeping the others fixed) to the mathematical model of chronic lymphocytic leukemia [26], with immunotherapy technique application to predict the behavior of all the populations and, in particular cancer, and immune cell population. These are of prime importance in learning how the change in the input parameters causes a change in the output of the model, and what kind of change it is.",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "To advance healing either by indicating the spaces in the model where improvements could be made or by optimizing the offered therapies is the key objective of mathematical modeling, which, in turn, encourages mathematicians to form improve novel therapies. The model under argument assumes the spread of leukemia in a blood-circulating system. Let x be the population of susceptible, y be the population of dysfunctional blood cells, c s is the population of leukemic and z is the population of immune cells [23]. The endemic model is proposed as follows:",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "Since we will be discussing the sensitivity of all of these parameters throughout, it is of prime importance to provide details of what a parameter means. Below is a list providing a description of the parameters. \n\nA : Employment rate of naive blood cells inflowing into circulatory blood from different sections as well as from blood transfusion; a 0 : Natural death rate of susceptible blood cells; β : Decay rate of naive cells killed upon contact with tumor cells and becoming dysfunctional; β 0 : Natural death rate of infected cells; k : Recruitment rate of cancer cells into blood system; k 0 : Normal death rate of malignant cells; k 1 : Loss of cancer cells due to encounter with immune cells; B : Rate of external intravenous re-infusion of T cells; b : Propagation rate of resistant cells in case of cancer setback; b 0 : Natural death rate of immune cells; b 1 : Loss rate of immune cells due to encounter with cancer cells. Now, we will discuss the method in the formal way. As we are going to apply a sensitivity analysis, before applying it to the mathematical model, we state some basic definition and produce the understanding of the sensitivity of a parameter in terms of mathematical equation. This method of computing sensitivity will be adopted throughout.",
"section_name": "Nomenclature",
"section_num": "2.1."
},
{
"section_content": "The procedure used to find out how self-determining variable values will influence a particular dependent variable under a specified set of hypotheses is defined as sensitivity analysis. It is also known as the what-if analysis. The basic principle of this analysis is \"change the model and observe the behavior\". The technique employed in this paper is local sensitivity analysis, derivative-based method. Local sensitivity is also known as one-factor-at-a-time (OFAT) technique and this involves (1) affecting one input variable, keeping others at their baseline values, (2) returning the variable to its nominal value, then repeating this for each of the other inputs in the same way. Now, we state the basic technique employed in this paper as follows. \n\nThe classical sensitivity of y with respect to p at p 0 is defined as\n\nwhere the sensitivity of naïve, infected, cancer and immune cells with respect to all parameters is given by y (p 0 ) of eth formal definition of (2). This will give us the effect of variation in parameters on the model output, i. e., y = y(p). \n\nWithout presenting the complete procedure opted, sensitivity equations of various parameters, after employing the sensitivity definition (2), are listed as follows.",
"section_name": "Sensitivity Analysis",
"section_num": "2.2."
},
{
"section_content": "This term ∂y ∂A provides much-needed sensitivity equations. We define\n\nEquation (3) shows the sensitivity expression with respect to parameter A.",
"section_name": "Sensitivity Analysis with Respect to Parameter A",
"section_num": "2.2.1."
},
{
"section_content": "This term ∂y ∂a 0 provides the much-needed sensitivity equations. We define\n\nEquation (4) shows the sensitivity expression with respect to parameter a 0.",
"section_name": "Sensitivity with Respect to Parameter a 0",
"section_num": "2.2.2."
},
{
"section_content": "This term ∂y ∂β provides the much-needed sensitivity equations. We define\n\nEquation (5) shows the sensitivity expression with respect to parameter β. provides the much-needed sensitivity equations. We define\n\nEquation (6) shows the sensitivity expression with respect to parameter β 0.",
"section_name": "Sensitivity with Respect to Parameter β",
"section_num": "2.2.3."
},
{
"section_content": "This term ∂y ∂k provides the much-needed sensitivity equations. We define\n\nEquation (7) shows the sensitivity expression with respect to parameter 'k'. provides the much-needed sensitivity equations. We define\n\nEquation (8) shows the sensitivity expression with respect to parameter 'k 0 '.",
"section_name": "Sensitivity with Respect to Parameter k",
"section_num": "2.2.5."
},
{
"section_content": "This term ∂y ∂k 1 provides the much-needed sensitivity equations. We define\n\nEquation (9) shows the sensitivity expression with respect to parameter 'k 1 '.",
"section_name": "Sensitivity with Respect to Parameter k 1",
"section_num": "2.2.7."
},
{
"section_content": "This term ∂y ∂B provides the much-needed sensitivity equations. We define\n\nEquation (10) shows the sensitivity expression with respect to parameter 'B'.",
"section_name": "Sensitivity with Respect to Parameter B",
"section_num": "2.2.8."
},
{
"section_content": "This term ∂y ∂b provides the much-needed sensitivity equations. We define\n\nEquation (11) shows the sensitivity expression with respect to parameter 'b'. provides the much-needed sensitivity equations. We define\n\nEquation (12) shows the sensitivity expression with respect to parameter 'b 0 '.",
"section_name": "Sensitivity with Respect to Parameter b",
"section_num": "2.2.9."
},
{
"section_content": "This term ∂y ∂b 1 provides the much-needed sensitivity equations. We define\n\nEquation (13) shows the sensitivity expression with respect to parameter 'b 1 '.",
"section_name": "Sensitivity Analysis w.r.ro Parameter b 1",
"section_num": "2.2.11."
},
{
"section_content": "Here, we justify our sensitivity equations numerically by opting the nominal values for the parameters under consideration for sensitivity. The chosen nominal values are as follows. \n\nWe assumed nominal values for the initial condition of the differential equations system. We carried out numerical simulations using MATLAB [27]. \n\nWith the help of MATLAB, we produced graphical results of the sensitivity equations that we obtained from the application of the definition of sensitivity. Since the results obtained were in analytical form, to facilitate the reader's understanding of their meaning, the parameter sensitivity results are drawn in the form of graphs.",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "As we increase the recruitment rate of naïve cells, it is observed in Figure 1 that the population of naïve and infected blood cells increases abruptly, and then abruptly decreases in both the populations and, with further increase, there is no change. This is because, with the increase in the recruitment rate of naïve cells, naïve cells increase in number then, because of their death rate and interaction with cancer cells, their population decreases. A similar pattern is followed by infected cells because of their death rate, and the availability of less susceptible cells that become infected in the long run. No change is observed in cancer and immune cells because there is no interaction of these populations with the rest.",
"section_name": "Parameter A",
"section_num": "3.1."
},
{
"section_content": "As we increase the value of 'a 0 ', which shows the natural death rate of naïve cells, an initially abrupt change is observed in the naïve and infected cells' population as seen in Figure 2. Both the populations decrease abruptly and, when this rate is further increased, a negligible decrease is observed in both populations and no change is observed, further increasing their natural death rate. The rest of the two populations remain unchanged, as is obvious from mathematical model as well as from the sensitivity equations.",
"section_name": "Parameter a 0",
"section_num": "3.2."
},
{
"section_content": "As seen in Figure 3, all the populations remain unchanged as we increase the decay rate of naïve cells. This is because, upon the interaction of these two populations, i. e., naïve and cancer cells, naïve cells are either killed or become dysfunctional and, since the dysfunctional cells become part of the infected cells' population, they further disappear because of their natural death rate. Because of this fact, there remains a balance in both populations, and hence no change is observed. The rest of the two populations also remains unchanged because of their lack of connection with the previous populations.",
"section_name": "Parameter β",
"section_num": "3.3."
},
{
"section_content": "Since 'β 0 ' represents the natural death rate and is associated with the infected cells, there is an abrupt decrease in the infected populace, as an increased number of infected cells becomes available as shown in Figure 4, and then, because of their natural death rate, the population lessens with a negligibly small change, and with a further increase at this rate, no change is observed under the study of dysfunctional cells.",
"section_name": "Parameter β 0",
"section_num": "3.4."
},
{
"section_content": "As far as the behavior of 'k' is concerned, we notice in Figure 5 that, as the value of 'k', i. e., the recruitment rate of cancer cells is increased, an abrupt increase in the number of infected and cancer cells is observed. However, as the value is increased further, then, because of the already high population of cancer cells, there is a saturation level for this population and, due to an increased death rate and high number of encounters with immune cell, there is no further increase in infected and cancer population, and behavior is somewhat stable in the long run. The other population remains invariant.",
"section_name": "Parameter k",
"section_num": "3.5."
},
{
"section_content": "It can be seen from Figure 6 that a noticeable decrease occurs in the population of infected and cancer cells as the natural death rate is increased. The decay of these populations is much faster because of the high population and, with the passage of time, there is a lower population and change occurs negligibly. Finally, a stage comes when there are no more infected and cancer cells.",
"section_name": "Parameter k 0",
"section_num": "3.6."
},
{
"section_content": "It can be seen in Figure 7 that there is an abrupt decrease in the number of infected and cancer cells. This noticeable change occurs with respect to the increase in the coefficient 'k 1 ', whereas, at the same time, there is abrupt increase in the number of immune cells. After a short time period, the numbers reach the steady state.",
"section_name": "Parameter k 1",
"section_num": "3.7."
},
{
"section_content": "When there is no external re-infusion of T cells, the number of available infected and cancer cells is higher and there are less immune cells. As we increase the external re-infusion of T cells into cancer patients, the amount of infected and cancer cell decreases and a relative increase in the amount of immune cells is observed as shown in Figure 8.",
"section_name": "Parameter B",
"section_num": "3.8."
},
{
"section_content": "Since 'b' is the proliferation rate of T cells due to cancer antigen-presenting cells in the blood of cancer relapse patients, this term behaves similarly to the external re-infusion and similar behavior is observed graphically in Figure 9. As we increase the death rate of immune cells, it is observed in Figure 10 that, initially, there is an abrupt decrease in the amount of immune cells and, due to this, an abrupt increase in infected and cancer cells. However, as we go on increasing its death rate then, due to the lack of immune cells present in the body because of the higher death rate and their being killed by cancer cells, the change in the population of immune cells is almost negligible and, finally, there comes a stage when there is an absence of immune cells, and hence no further change is observed in the population of immune cells as well as cancer and infected cells, because there is no interaction between cancer and immune cells. However, the population of susceptible cells remains unchanged.",
"section_name": "Parameter b",
"section_num": "3.9."
},
{
"section_content": "As 'b 1 ' is the loss rate of immune cells due to encounters with cancer cells, similar behavior is observed in Figure 11 as that observed in the case of 'b 0 '.",
"section_name": "Parameter b 1",
"section_num": "3.11."
},
{
"section_content": "In this paper, we considered a mathematical model developed for studying the effect of genetically engineered T cells on the spread of Leukemia. The model is directed by four non-linear ordinary differential equations. The model is investigated for sensitivity purposes analytically as well as numerically. \n\nAnalytically, we applied a very basic approach to compute the sensitivity of each parameter in a local sense by taking the partial derivative of all dependent variables with respect to the parameters whose sensitivity is to be determined, by varying only that parameter while keeping others fixed. Since we received a large amount of reliable information from the numerical approach, we applied a numerical approach using the sensitivity equations obtained from the analytical approach, and plotted a graph to provide visual insight, as discussed in [28]. \n\nThe sensitivity analysis justified the use of immunotherapy. All the parameters are sensitive, but some of them are identified as having negligible effect on the system. The most important parameter found in controlling leukemia is the transfusion of genetically engineered T cells, as this showed a noticeable decrease in infected and cancer cells, which was the key purpose of this paper. One of the edges of this parameter is that it produces a dormant memory, which, in turn, helps to fight leukemia in case of relapse. \n\nThe conclusions obtained after applying a sensitivity analysis from a numerical approach graphically suggest that immunotherapy with T cell infusion is better than other techniques available for treatment as it has less harmful effects to the body, and the dormant memory of immune cells to fight leukemia is extremely beneficial. \n\nLast but not least, sensitivity analysis provides insight into the operational principles of the system, providing an opportunity for mathematicians to improve it and help improve treatment.",
"section_name": "Conclusions",
"section_num": "4."
}
] |
[
{
"section_content": "Funding: This research received no external funding. \n\nAcknowledgments: Authors acknowledge the support given by their respective universities.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest.",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.7150/jca.30690
|
Effects of Notch Signaling Pathway in Cervical Cancer by Curcumin Mediated Photodynamic Therapy and Its Possible Mechanisms in Vitro and in Vivo
|
Curcumin, as a high effect and low toxicity anti-cancer drug and photosensitiser, has synergistic and complementary effects with photodynamic therapy (PDT). However, due to its unclear mechanism, PDT's application and efficacy were limited. Notch signaling pathway, which is highly correlates with carcinogenesis and development of cervical cancer, could be a potential therapeutic targets to improve the effectiveness of PDT. Therefore, in this study, we explored the effects of Notch signaling pathway in cervical cancer by curcumin mediated PDT with/without Notch receptor blocker (DAPT), and hope to elucidate its mechanism. Firstly, the effect on the proliferation of cervical cancer Me180 cells were detected with MTT assay, and apoptosis were detected with Annexin V-FITC/PI combined with flow cytometry. Secondly, after establishment of nude mice model, dividing the experimental animals into model group, curcumin PDT group, simple DAPT group, and curcumin-PDT+DAPT group, and analyzing tumor volume changes as well as HE staining in each group. mRNA and protein expression of gene Notch-1 and its downstream NF-κB and VEGF were observed with RT-PCR, immunohistochemical staining and Western-blot with/without inhibition of Notch signaling pathway by DAPT, both in vivo and in vitro experiments. We found both DAPT and curcumin-PDT can inhibit the proliferation and induce apoptosis of cervical cancer cell. The two have synergistic effect in vitro and in vivo. This effect can effectively block the conduction of Notch signaling pathway, which is associated with down-regulation of the expression of Notch1 and NF-κB. Notch signaling pathway could be one of the targets of curcumin-PDT photodynamic therapy.
|
[
{
"section_content": "Cervical cancer is the third most common cancer in women worldwide and the fourth leading cause of tumor-induced death. China is a country with high incidence of cervical cancer in the world. About 150,000 women are infected every year, accounting for one-third of new cases globally and the incidence rate is six times higher than that of developed countries. It has become one of the most serious threat to the life and health of women in this country [1, 2]. At present, the incidence of cervical cancer is moving to younger female [3]. Traditional treatment, like surgery and radio/chemo therapy, could cause serious damage to Ivyspring International Publisher ovarian and vaginal functions [4]. While photodynamic therapy (PDT), as its high possibilities to retain patients' organ and reproductive function, is a new and effective noninvasive therapy to cancers with bright prospect. It has been proven in treatment of some malignant tumor, such as nasopharyngeal carcinoma and basal cell carcinoma [5]. \n\nIn recent years, there has been an increasing number of reports on the clinical treatment of cervical cancer in PDT. Hillemanns used ALA to treat CIN, and the remission rate of CIN was between 50%-95% [6]. Choi made a bold exploratory study on the PDT treatment of early cervical cancer. 21 patients with early cervical cancer received intravenous photogem for PDT treatment. The complete remission rate was 95. 2%, and only one recurred [7]. \n\nCurcumin is a new type of photosensitizer with high efficiency, low toxicity and dual anticancer effect [8]. Most important of all, it has synergistic and complementary effects with PDT [9]. Our preliminary studies demonstrated that, PDT is an effective measure for cervical cancer treatment. However, due to its unclear mechanism, PDT's application and efficacy were limited. Notch signaling pathway, which is highly correlates with carcinogenesis and development of cervical cancer, could be a potential therapeutic targets to improve the effectiveness of PDT at the same time. \n\nOur study is aiming to, on both cellular and molecular levels, analyze the role of Notch and downstream genes, such as NF-κB, VEGF, before and after curcumin mediated PDT on cervical cancer cell, Me180; based on which, to further observe the influences of PDT on animal model of cervical cancer; identify the possible targets in PDT on cervical cancer by blocking Notch receptor. We hope our studies could provide new ideas and experimental evidences for clinical PDT on cervical cancer.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Curcumin, purchased from Aldrich and Sigma chemical (St. Louis, MO, USA), was dissolved in PBS, and its PH was adjusted to 7. 4 by addition of 5M of NaOH. The stock solutions of 50 mmol/L were made and kept in -20 o C before use. The Notch receptor blocker (N -[N-[3, 5-difluorobenzene acetyll-propionyl)] -(S) -phenylglycine tert-butyl, DAPT) was purchased from Calbiochem,USA. All of the other chemicals used were of the highest purity commercially available. The 445nm laser source was purchased from Beijing Laserwave Optoelectronics Tech Co, Ltd.",
"section_name": "Chemicals and Instruments",
"section_num": null
},
{
"section_content": "The Me180 cell line was purchased from Shanghai FuXiang company. The cells were routinely propagated in monolayer cultures in Dulbecco's modified Eagles' medium (Gibco-BRL, Paisley, Scotland), supplemented with 5% heat-inactivated fetal bovine serum, 0. 37% sodium bicarbonate, 30mM HEPES, and penicillin/streptomycin. The cells were cultured in a 5% CO2 incubator at 37 o C. The cells were serially subcultured normally and the cells at the logarithmic growth phase were chosen for experiments.",
"section_name": "Cell culture",
"section_num": null
},
{
"section_content": "The Me180 cell lines were inoculated into an 96-well at a volume of 100μl (5×10 4 cells/well) for stationary culture. Our preliminary orthogonal experimental research shows that curcumin mediated PDT induced apoptosis of cervical cancer cells with the best combination of curcumin concentration of 5µmol/L, laser dose of 100J /cm 2, irradiation time of 180s, incubation time of 24h. Adding different concentrations of DAPT 2 hours before PDT. The treatment factors were added and divided into control group, simple DAPT group, curcumin-PDT group and curcumin-PDT+DAPT group for routine MTT detection. MTT method was used to calculate interaction index (CDI) of the two drugs, including DAPT and curcumin-PDT. The synergistic effect of DAPT and curcumin-PDT was judged by combined index (CI). The formula is CI=AB / (A×B). A and B represent two different drugs respectively. AB was the T/C value of the combination of the two drugs, A and B were the T/C value of the single drug, and T/C value was the ratio of the drug group to the control group.",
"section_name": "Cell proliferation assay",
"section_num": null
},
{
"section_content": "Me180 cells were seeded in 6 Hole culture plates and cultured for 24 h, then culture medium replaced. At this time, DAPT (1µmol/L) and curcumin (2. 5µmol/L) were added and blank control was set up. After incubation for 6 hours, fresh culture medium was changed immediately after 180 seconds irradiated with 100 J/cm 2 laser. After 24 hours of continuous culture, the cells of four groups were collected and cleaned by PBS twice. Cell suspensions were centrifuged and resuspended in PBS to a concentration of 10 6 cells/ml. For flow cytometric analysis, cells were incubated with 5 μl of Annexin V-fluorescein isothiocyanate (Beijing baosai biotech co, ltd. ) and 10 μl of propidium iodide (Sigma chemical, USA) in the dark at room temperature for 10 min followed by fixation with 2% formaldehyde. The stained cells were analyzed for DNA content by fluorescence activated cell sorting (FACS) in a FACScan (Beckman Coulter Epics XL, USA). The forward-and side-scatter gates were set to exclude any dead cells from the analysis. At least 10,000 events were collected for each sample.",
"section_name": "Flow cytometry",
"section_num": null
},
{
"section_content": "Total mRNA was isolated from Me180 cell lines of 4 groups and cells using Trizol Reagent (QIAGEN's RNeasy® RNA extraction kit) according to manufacturer's instructions. After DNAse I treatment, the RNA was reverse transcribed and the cDNA was used for real-time PCR. Real-time PCR was performed with the following sets of primers:\n\nβ-actin: forward: 5'-TTGTTACAGGAAGTCCC TTGCC-3'; reverse: 5'-ATGCTATCACCTCCCCTGT GTG-3'. \n\nNotch1: forward: 5'-CGGAGTGTGTATGCCA AGAGT-3'; reverse: 5'-GGTTCTGGAGGGACCAA GA-3'. \n\n2 -ΔCt method was used to calculate the relative mRNA level of each group. The ΔCt values was calculated according to the formula: ΔCt target gene of PDT group = Ct target gene of PDT group -Ct β-actin of the same sample ;ΔCt target gene of control group = Ct target gene of control group -Ct β-actin of the same sample ; Values of ΔCt between different groups were compared.",
"section_name": "Real-time Reverse Transcription-PCR",
"section_num": null
},
{
"section_content": "Me180 cells of 4 groups were plated in 6-well plates at the density of 1×10 6 cells per well in 2 mL of theculture medium and cultured at 37 o C overnight under 5% CO 2. Cells were collected and lysed after 60 minutes, and total protein concentrations were determined with a Bio-Rad BCA® kit. Equal amounts of cell lysates were loaded onto 10% SDS gel and separated by electrophoresis. Separated proteins were then electro-transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). After being blocked with 1× Tris-buffered saline (TBS) containing 0. 1% Tween-20 and 5% bovine serum albumin (BSA), the membranes were incubated with primary antibodies at room temperature for 2 hours or at 4 o C O/N, then washed with 1×TBS containing 0. 1% Tween-20 and followed by treatment with the horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for another 1 hour. The targeted proteins were visualized using an enhanced chemiluminescence (ECL) plus system (Thermo Fisher Scientific, Waltham, MA). Primary antibodies against Notch1 were purchased from Abcam (Cambridge, UK). NF-κB, VEGF were purchased from Santa Cruz Biotechnology. β-Actin and the horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG were purchased from Cell Signaling Technology.",
"section_name": "Western blot",
"section_num": null
},
{
"section_content": "Female BALB/c nude mice were obtained from Vitalriver Laboratory Animal Technology Co. (Beijing, China) and maintained under specific pathogen-free conditions. The mice were 6-8 weeks old and weighed 20-25 g when the experiments started.",
"section_name": "Animals",
"section_num": null
},
{
"section_content": "About 5×10 6 cells ( 200 μL) were injected subcutaneously into the back of BALB/C nude mice to set up cervical cancer xenograft model. After successful establishment of the model, experimental animals are divided into four groups with 12 animals each (model group, the curcumin-PDT group, only DAPT group and curcumin-PDT with DAPT group). \n\n(A) In the model group, 200μL saline was injected around the tumor and then natural light was given. (B) In curcumin mediated PDT group without Notch blocker, curcumin solution of 150μL was injected locally around the tumor for 200μL, and PDT was performed 4 hours later at a dose of 100 J/cm 2. (C) In the single DAPT group, DAPT solution was injected intraperitoneally every other day from the first week before treatment with a dose of 100 mg/kg. (D) In curcumin-PDT therapy combined with Notch blocker group, One week before PDT, DAPT solution was injected intraperitoneally every other day from the first week before treatment with a dose of 100 mg/kg. Then on the day of treatment, curcumin solution of 150μL was injected locally around the tumor for 200μL. PDT was performed 4 hours later at a dose of 100 J/cm 2. \n\nEach group has 12 mice. After one-day treatment, 6 mice were killed to cut tumor tissue for follow-up experiments. The remaining 6 mice continued to record tumor volume and tumor size. And it was measured 2 times per week until 21 days after treatment.",
"section_name": "Animal model",
"section_num": null
},
{
"section_content": "The tumor volume was calculated and tumor growth curve was drawn. Tumor volume V=a×b 2 /2 (a is the longest diameter of tumor and b is the shortest ). Observe and record any adverse reactions that occur during the treatment, including weight loss, death, etc.",
"section_name": "Inhibition of tumor growth",
"section_num": null
},
{
"section_content": "After measuring the gross tumor size, the specimens were embedded in paraffin, cut into 5-μm slices, and mounted on slides. The tumor xenografts was stained with hematoxylin-eosin and examined under a light microscope.",
"section_name": "Histopathologic examination",
"section_num": null
},
{
"section_content": "After fixed in 4% paraformaldehyde for 1 day, the tumor was routinely dehydrated and embedded to prepare the pathological sections, which were stained to observe the changes of the expression of Notch, NF-κB, and VEGF in each group. Briefly, tissue sections were incubated overnight at 4°C with an anti-Notch1(Abcam Cambridge, UK), NF-κB, and VEGF monoclonal antibody (Santa Cruz Biotechnology). Then, the sections were incubated 40 min with biotinylated anti-rabbit secondary antibody, and another 40 min with the avidin-biotinylated peroxidase complex. The sections were washed with distilled water (10 min), treated with diaminobenzidine (DAB) to visualize positive staining. IPP6. 0 analysis software was used for image analysis to measure optical density (IOD) value. Based on the average of the model group, the percentage of the data divided by the model group is used to analyze the immunohistochemical results.",
"section_name": "Immunohistochemical staining",
"section_num": null
},
{
"section_content": "The experiments were carried out in triplicate and values were shown as the mean± standard deviation (SD). The single factor variance analysis was used between groups. All statistical tests with P<0. 05 were considered significant. SPSS 19. 0 software was used for statistical analysis.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "DAPT itself can inhibit the growth of cervical cancer Me180 cells. When the concentration of DAPT was 1μmol/L or above, it could significantly inhibit the growth of Me180 cells. After curcumin-PDT treatment, the Notch signaling pathway was blocked by DAPT, and the inhibition of cell proliferation was more significant (Fig. 1 ). The inhibition rate of 1μmol/L DAPT on Me180 cells was 15. 79%. However, that of curcumin -PDT combined with blocker DAPT was 77. 18%. As shown in Table 1, when curcumin -PDT was added to the DAPT group, the concentration of DAPT was greater than 1 μmol/L and CI<0. 8. Both have moderate synergistic effects.",
"section_name": "Effects of curcumin-PDT and DAPT on the survival rate of cervical cancer Me180 cells",
"section_num": null
},
{
"section_content": "Both curcumin-PDT and 1μmol/L DAPT can induce apoptosis in cervical cancer cell line Me180. The total mortality rate of the former is 43. 9% and the latter is 8. 33%. There were significant differences between early and late apoptosis rates. After curcumin-PDT plus DAPT, there was a synergistic interaction between curcumin-PDT and DAPT, and the difference was significant(P<0. 01). The total mortality rate is 68. 61% (Fig. 2 ).",
"section_name": "Curcumin -PDT and DAPT induces apoptosis in cervical cancer Me180 cells",
"section_num": null
},
{
"section_content": "Curcumin-PDT and DAPT both inhibited Notch1 mRNA expression in Me180 cervical cancer cells. The inhibition rates were 32. 33% and 39. 99% respectively (Table 2 ). Curcumin-PDT combined with DAPT could inhibit Notch1 most significantly. Curcumin-PDT combined with DAPT had synergistic effects. The inhibition rate was 79. 27%. Compared with the control group and curcumin-PDT, the differences between them were statistically significant (P < 0. 05 or P < 0. 01).",
"section_name": "Downregulation of Notch1 mRNA expression in vitro",
"section_num": null
},
{
"section_content": "",
"section_name": "Protein expression of Notch and downstream genes detected by western blot in vitro",
"section_num": null
},
{
"section_content": "with the control group, curcumin-PDT group significantly inhibited the expression of Notch1, NF-κB and VEGF-A. Compared with the control group, DAPT also inhibited the expression of Notch1, NF-κB and VEGF-A. When curcumin-PDT was blocked by DAPT, the expression of Notch1, NF-κB and VEGF-C was inhibited more significantly than that of the two alone (Fig. 3 ).",
"section_name": "Compared",
"section_num": null
},
{
"section_content": "We used Me180 cell xenograft nude mice to investigate PDT anticancer activity in vivo. Tumor volume in model group increased gradually. Tumor volume of curcumin-PDT group, DAPT group, curcumin-PDT+DAPT group remained unchanged or slightly decreased from 1 to 7 days after treatment (Fig. 4 ). There was significantly different from the model group (P < 0. 05). Then they were gradually increased after 14 days.",
"section_name": "Xenograft tumor growth inhibition by curcumin-PDT",
"section_num": null
},
{
"section_content": "The results of Hematoxylin-Eosin (HE) staining demonstrate that, there is no appreciable change in the control group. The tumor cells were nested in the model group. Cell components are relatively single and relatively large in size. The cell boundaries were not clear, ranging from round to oval. Chromatin is rough and mitotic figures are common. Occasionally necrosis occurs. However, compared with the model group, the cells in each treatment group arranged sparsely. The volume was reduced, the ratio of nucleus to plasma was reduced, part of the nucleus was pyknosis, vesicular nucleus and perinuclear halo were observed, and the mitotic image was less than the model group. Among them, the necrosis of curcumin-PDT with blocker DAPT was the most obvious (Fig. 5 ).",
"section_name": "Pathological morphology observation of xenografts after curcumin-PDT",
"section_num": null
},
{
"section_content": "Curcumin-PDT and DAPT both inhibited Notch1 mRNA expression in cervical cancer xenografts. The inhibition rates were 40. 54% and 42. 17% respectively (Table 3 ). Curcumin-PDT combined with DAPT could inhibit Notch1 most significantly. Curcumin-PDT combined with DAPT had synergistic effects. The inhibition rate was 79. 22%. Compared with the control group and curcumin-PDT, the differences between them were statistically significant (P < 0. 05 or P < 0. 01). \n\nTable 3. The inhibition rate of Notch1 mRNA in cervical cancer xenografts (̅ ± s, n = 3) Compared with the model group, * P <0. 05, ** P <0. 01, *** P <0. 001; Compared with curcumin-PDT group, Δ P <0. 05, ΔΔ P <0. 01, ΔΔΔ P <0. 001; Compared with DAPT group,\n\n• P <0. 05,",
"section_name": "Downregulation of Notch1 mRNA expression in vivo",
"section_num": null
},
{
"section_content": "The results of immunohistochemical staining showed that the expression of Notch-1, NF-κB and VEGF protein was down-regulated in all groups compared with the model group, and the difference was statistically significant (P<0. 05). Curcumin-PDT with DAPT group had the strongest inhibitory effect Compared with curcumin-PDT group and DAPT group, the difference was statistically significant (P < 0. 05). The inhibitory effect of DAPT on Notch-1 expression was slightly stronger than that of curcumin-PDT (P < 0. 05) (Fig. 6 ), but the inhibitory effect on the expression of NF-κB (Fig. 7 ) and VEGF-A (Fig. 8 ) was slightly weaker than that of curcumin-PDT (P < 0. 05). For Notch and its downstream genes and protein expression downregulation, it was verified by western blot further. There was no significant difference in the expression of NF-κB and VEGF-A between curcumin-PDT and DAPT alone group. In addition to the above differences, the remaining results were consistent with immunohistochemistry (Fig. 9 ).",
"section_name": "Immunohistochemical assessment and Western Blot analysis of the protein expression of Notch and its downstream genes in vivo",
"section_num": null
},
{
"section_content": "Cervical cancer is the second leading cause of death in women with gynecological malignancies [10, 11]. There are nearly 500,000 new cases and about 270,000 deaths worldwide every year [12]. In some developing countries, the incidence rate of cervical cancer ranks first [13]. However, a large number of studies and practices have shown that cervical cancer is the only type of malignant tumor whose morbidity and mortality can be reduced by medical intervention [14]. \n\nWith the development of Nano-tech, Biotech and Photology, and their fusion with Medical Science, photodynamic therapy become an increasingly valid alternative for cervical cancer treatment. It can treat early tumors and also effective in the treatment of advanced tumors [15]. Photodynamic therapy (PDT) is an FDA-approved anticancer modality that has been shown to enhance anti-tumor immunity. Numerous studies have shown that stimulation of the host immune system can result in the generation of anti-tumor immune responses capable of controlling metastatic tumor growth [16]. Studies have reported that refractory advanced lung cancer can be alleviated by photodynamic therapy (PDT) combined with chemotherapy [17]. Photodynamic therapy has achieved certain curative effect. This study confirmed the effectiveness of PDT in the treatment of cervical cancer in vitro and in vivo, which echos the results of previous reports done by other researchers. \n\nMogan, a genetic pioneer, first discovered Notch signaling protein in 1916, named it because a partially disabled mutant would create a notch at the edge of a fruit fly's wing [18]. A total of four Notch genes, 9q34, 1p13-p11, 19p13. 2-p13. 1 and 6p21. 3, were identified in mammals, encoding four Notch receptors (Notch1-4) [19]. However, the relationship between Notch and tumor is complex, not only promoting cancer but also inhibiting cancer in different circumstances [20]. It is reported that Notch is highly expressed in low-grade cervical tumors. In a more aggressive, high-grade cervical cancer, the expression is lower [21]. Daniel et al. found that the expression of activated Notch-1 was closely related to the severity of HPV-related early cervical epithelial injury [22]. Notch-1 signaling pathway is involved in the occurrence and development of cervical cancer [23]. Clinical impact of de-regulated Notch-1 and Notch-3 in the development and progression of HPV-associated different histological subtypes of precancerous and cancerous lesions of human uterine cervix was studied. The findings suggest that Notch-1 and Notch-3 may play an important role with synergistic effect of HPV in regulating development and proliferation of cervical cancer through the deregulation of Notch signalling [24]. The abnormal expression of Notch signaling pathway is related to the occurrence and progression of cervical cancer [25]. Therefore, to explore the mechanism of Notch in PDT treatment of cervical cancer and to fully understand the regulation of its downstream genes by Notch will help enriching the strategy of PDT treatment and expand its clinical application. \n\nWe studied the effects and mechanisms of Notch signaling pathway in cervical cancer by curcumin mediated PDT. In addition, we identify the possible targets in PDT on cervical cancer through blocking Notch receptor. γ-secretase inhibitor (DAPT) was chosen to block Notch signaling pathway. Our studies had shown that DAPT itself inhibited Notch-1 mRNA and protein expression in cervical cancer in vitro and in vivo. DAPT and curcumin PDT demostrates great synergistic effects. After curcumin-PDT was added into the blocking agent DAPT, the efficacy of PDT could be further improved. Activated expression of Notch-1 protein may be associated with carcinogenesis of normal cervical epithelium and may affect the development of cervical cancer. Therefore, we have good reasons to suspect that Notch-1 is one of the key targets for curcumin-PDT in the treatment of cervical cancer. It is suggested that the therapeutic effect of PDT could be improved by regulating the target of Notch-1 in future clinical practice. \n\nThe regulation of cell differentiation and apoptosis by Notch signaling pathway is very complex [26]. PDT treatment on signaling pathways with different photosensitizers and on different cell types also lead to different results [27]. Moreover, it increases the complexity of its mechanism of action, and generates various efficacy of PDT in different diseases. Based on the results of our studies, Notch signaling pathway is one of the key, however not the only, targets among the mechanism of PDT. We analyzed and concluded the complexity and diversity of the mechanism could be related to the following factors: (1) Notch itself is divided into four types. Although the members of Notch pathway are fixed, the ligands and target genes of Notch pathway have different subtypes, so their roles in different tumors are different [28]. (2) There are interactions between Notch and other signaling pathways, such as Ras and Wnt, which can also regulate cell's growth by activating Notch pathway [29]. (3) Notch is ubiquitous in many tissues and cells, and is highly conservative in biological evolution [30]. Many different stimuli can activate Notch pathway. Sharing of Notch signaling pathway is one of the reasons for its complex function. \n\nNotch pathway controls the activation of NF-κB pathway by activating CSL protein in the side effector cells [31]. An important downstream target gene of NF-κB pathway is VEGF, which is closely related to the invasion and metastasis of cervical cancer [32]. In this study, our observation proved that curcumin-PDT can block Notch signaling pathway and tune down the expression of key factors such as Notch-1 and NF-κB, thus inhibiting tumor growth. \n\nIn conclusion, this study successfully demonstrated that Curcumin-PDT combined with DAPT could inhibit the expression of Notch-1 and its downstream related proteins in cervical cancer in vitro and in vivo. We conclude that Notch-1 could be one of the targets of curcumin-PDT in the treatment of cervical cancer. Receptor blocker DAPT has synergistic effect on curcumin-PDT in the treatment of cervical cancer, which is mainly related to the down-regulation of Notch-1 and NF-κB expression.",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This work was supported by the National Nature Science Foundation of China (Foundation No. 81202966 )",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Competing Interests",
"section_num": null
},
{
"section_content": "The authors have declared that no competing interest exists.",
"section_name": "Competing Interests",
"section_num": null
}
] |
10.4322/acr.2021.278
|
De novo double-hit B-cell precursor leukemia/lymphoma - an unusual presentation as peritoneal lymphomatosis
|
Peritoneal lymphomatosis (PL) is a rare presentation of extranodal precursor leukemia/lymphoma.The presentation is often non-specific, leading to delayed diagnosis and treatment.In this case, though the preliminary diagnosis was established on ascitic fluid cytology, the disease progressed rapidly, leading to demise before initiating chemotherapy.Immunophenotyping and molecular studies, performed later, established a diagnosis of de novo B-cell precursor leukemia/ lymphoma with MYC, BCL2 rearrangements (Double-hit lymphoma).MYC, BCL2 rearrangements are rarely reported in precursor B-lymphoma/leukemia which carry dismal prognosis.In this report, we illustrate autopsy findings of PL in an elderly gentleman who presented with ascites for evaluation.
|
[
{
"section_content": "Peritoneal lymphomatosis (PL) is a rare presentation of extranodal Non-Hodgkin lymphoma (NHL). Since the clinical presentation of PL is non-specific, the diagnosis is often delayed. Peritoneal carcinomatosis accompanied by malignant ascites is relatively common; however, 'peritoneal lymphomatosis' is rarely reported in the literature. 1 Differentiation of PL from other morbid entities with similar imaging features such as tuberculous peritonitis, peritoneal carcinomatosis, and peritoneal mesothelioma is difficult without a histological diagnosis. 2 In the present genomic era, the appropriate immunohistochemical and molecular workup of lymphoma is indispensable for guiding therapy and prognostic information. The 2016 revised World Health Organization (WHO) classification of lymphoid neoplasms has included the category of high-grade B cell lymphomas (HGBLs) with combined MYC and BCL2 and/or BCL6 rearrangements termed as double-hit (DH) or triple-hit (TH) respectively. 3, 4 DH is usually reported commonly in diffuse large B-cell lymphoma (DLBCL), less frequently in follicular lymphomas and rarely in B-lymphoblastic lymphoma/ leukemia (B-LBL). 4, 5 There is limited information on cases of B-LBL with MYC and BCL2 rearrangement. In addition, there are cases of HGBL which can express Tdt rendering a diagnostic dilemma. Herein, we present autopsy findings of an elderly gentleman who presented with ascites for evaluation.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "A 55-year-old gentleman presented with abdominal distension to the gastroenterology service. The distension was insidious in onset, along with decreased urine output over the last 20 days. On examination, the patient had tense ascites with bilateral pitting pedal edema. There was no history of jaundice, hematemesis, melena, altered sensorium, fever, night sweats, or weight loss. Ultrasound revealed ascites with mild hepatomegaly and hydroureteronephrosis of the right kidney. Upper gastrointestinal endoscopy did not reveal any varices. Hepatotropic viruses' markers were negative. His renal function tests were deranged. Blood urea was 128 mg/dl (reference range [RR]; 15-40mg/dl) and serum creatinine was 2. 9 mg/dl (RR; 0. 6-1. 2mg/ dl). The liver function test on admission day was as follows: Total bilirubin: 0. 4 mg/dl (RR: 0. 2 -1. 2 mg/ dl), conjugated bilirubin 0. 2 mg/dl (RR: 0 -0. 3 mg/dl); AST: 37 (RR: 2-40 U/L) ALT: 32 (RR: 2-41 U/L) alkaline phosphatase: 62 IU/L (RR; 44-147 IU/L). However, terminally (3 days after admission), the AST/ALT/ ALP were 408/90/2042 IU, respectively. Abdominal ultrasonography did not reveal features of acute Budd-Chiari syndrome such as thickened walls, intraluminal echogenicity, and compressed hepatic veins or inferior vena cava. \n\nThe ascitic fluid analysis revealed a high serum ascites albumin gradient (SAAG) ascites (1. 2 g/dl). Initial ascitic tap showed sheets of polymorphs, and the subsequent cytology showed numerous intermediate size lymphoid cells with a high nuclear/cytoplasmic ratio. Immunocytochemistry showed lymphoid cells strongly positive for CD45, CD10, Tdt, and C-MYC. Focal positivity for CD20, CD19, CD79a was seen in these atypical lymphoid cells, and a working diagnosis of B-cell lymphoblastic lymphoma/leukemia was kept. Peripheral blood smear did not show blasts. CSF tap did not show atypical cell infiltration. CECT abdomen and PET-CT scan were deferred due to deranged renal function test. Differentials for ascites were considered based on the serum-ascites-albumin gradient (SAAG). Cutoff of >1. 1mg/dl is considered as high SAAG, which includes conditions like cirrhosis, heart failure, Budd-Chiari syndrome, sinusoidal obstruction syndrome. Low SAAG includes malignancy, infection, and pancreatitis, among others. The ultrasonography, endoscopy, and hepatic viral markers were negative for cirrhosis, varices, and viral hepatitis, respectively; enabling a rapid ascitic fluid malignant cytology analysis. It is interesting to note that despite being a case of PL, this case showed high SAAG ascites. \n\nSince the initial two ascitic fluid taps showed neutrophils, the patient was empirically started on IV cefoperazone-sulbactum, oral acyclovir, oral fluconazole and twice weekly cotrimoxazole. Low molecular heparin was given as deep venous thrombosis (DVT) prophylaxis. Fluconazole and cotrimoxazole were withheld later in view of worsening liver function tests (LFT), and acyclovir was also stopped in view of renal failure. Pre-emptive steroids and chemotherapy could not be started because of the poor general status of the patient. The patient's renal and liver function deteriorated, and he had a sudden cardiac arrest and could not be revived despite resuscitation efforts. A complete autopsy was performed after obtaining informed written consent from the patient's next of kin.",
"section_name": "CASE REPORT",
"section_num": null
},
{
"section_content": "The peritoneal cavity yielded 2 liters of strawcolored fluid. The mesentery and the omentum appeared thick and nodular, forming adhesions to the intestinal wall (Figure 1A ). Either side of the diaphragm showed similar greyish white deposits. The liver weighed 2300 g (RR;1330-2100g) and was diffusely enlarged. The porta hepatis showed a greyish white mass measuring 4x3x2cm infiltrating the hepatic parenchyma (Figure 1B ). This tumor also infiltrated the pancreas, stomach, the serosal surfaces of the small and large intestine and right kidney. A similar deposit was seen in the periadrenal fat. No significant lymph node enlargement was noted. The heart and brain were grossly and microscopically unremarkable. \n\nThe microscopy revealed relatively uniformappearing atypical lymphoid cells with blastoid morphology. These cells were infiltrating the peritoneum, porta hepatis, renal pelvis, serosa and mucosa of the gastrointestinal tract (Figure 1C ). The cells were intermediate-sized with round to convoluted nuclei with condensed nuclear chromatin, indistinct nucleoli, and scanty cytoplasm. These cells were positive for CD45, CD20, CD10, TdT, CD99 (dotlike), Bcl2 (>70%), and C-MYC (>40%) while they were negative for CD3, CD5, CD34, CD23, Bcl6, Mum1, Cyclin D1 and SOX11 (Figure 1D, 2A, 2B, 2C and 2D). \n\nImmunophenotypic diagnosis of B-lymphoblastic lymphoma/leukemia was considered. The fluorescence in-situ hybridization (FISH) was positive for BCL2 rearrangement (18q21) (Vysis LSI BCL2 Dual Color Break Apart Rearrangement Probe) (Figure 3A ) and CMYC-IGH fusion (IGH/MYC/CEP 8 Tri-Color Dual Fusion Probe) (Figure 3B ). However, FISH was negative for BCL6, BCR-ABL-1, KMT2A translocation, ETV6-RUNX1 translocation, and chromosome-21 amplification. Real-time PCR was negative for CRLF2 overexpression. Copy number abnormalities revealed homozygous deletions of Exon 2 & 4 of CDKN2A and Exon 2 of CDKNA2B (Figure 4 ). Bone marrow biopsy was normocellular with adequate representation of all marrow elements. There was no infiltration by lymphoma or involvement by leukemic process. The peripancreatic and peribiliary lymph nodes did not show the lymphomatous process. The autopsy did not reveal features of hepatic venous outflow obstruction. There was no evidence of pulmonary thromboembolism. There was focal subpleural infiltration through the diaphragm (direct extension) by lymphoma on the right side.",
"section_name": "AUTOPSY FINDINGS",
"section_num": null
},
{
"section_content": "Non-Hodgkin lymphoma presents primarily as an extranodal disease in 25-40% of cases. 6 It can affect organs and sites such as the biliary tree, liver, spleen, gastrointestinal tract, adrenals, peritoneal cavity, among others. Peritoneal surfaces can be involved by malignancies from all three cell-lineages: epithelial (carcinomatosis), mesenchymal (sarcomatosis), and lymphoid (lymphomatosis). Peritoneal lymphomatosis (PL) is defined as the \"intraperitoneal spread of lymphoma\". It can be seen either in association with a primary visceral site of involvement, or without any visceral involvement. 7, 8 The route of dissemination is postulated to be through visceral peritoneal surfaces, gastrocolic ligament, and transverse mesocolon. PL is an exceptional clinical scenario, which is frequently associated with primary gastrointestinal NHL (high grade) and is radiologically identical to peritoneal carcinomatosis. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma presenting as PL. Multiple patterns of PL include infiltrative mass, distinct nodular, and ascites. 8 In the present case, there was a diffusely infiltrative mass presenting with ascites. \n\nBased on the immunohistochemical and molecular workup, the present case was a de novo B-LBL with MYC, BCL2 rearrangements. Double-hit/triplehit lymphoma is characterized by MYC (8q24) rearrangement in combination with a BCL2 (18q21) and/ or a BCL6 (3q27) rearrangement. 9,10 Double-hit lymphomas (DHL) are rare and represent 4-8% of all diffuse large B-cell lymphomas. They tend to occur in extranodal sites. Four morphological variants have been described in DHL; DLBCL-like, features previously designated as B-cell lymphoma unclassifiable (BCL-U) in 2008 WHO classification, Burkitt lymphoma-like and lymphoblastic lymphoma-like. 4, 11 ere are few case reports of B-ALL with double hit genetics reported in literature. 12, 13 In most of those cases, the blasts were L3 type (French-American-British classification) and majority of them had acute leukemia like presentation. Few of the cases had a leukemic transformation from DLBCL. Conversely, on the other end of the spectrum, there can be acute lymphoblastic leukemia-like high grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement lacking Tdt and other immaturity markers. 14 Ok et al. 15 described a series of 13 cases of high-grade B-cell lymphomas with TdT expression grouped into three categories. These included cases of de novo high-grade B-cell lymphoma with Tdt expression as well as blastic transformation of a low grade B-cell lymphoma which acquired Tdt during relapse. Features that favoured HGBL over LBL were positivity for BCL6 and monotypic surface immunoglobulin. All the cases showed dismal outcome, despite appropriate therapy. \n\nIn the present case, copy number analysis revealed homozygous deletions of Exon 2 & 4 of CDKN2A and Exon 2 of CDKNA2B gene. These genes are involved in cell cycle regulation and homozygous deletion carries poor prognosis in ALL. 16 ere are two reported cases of doublehit lymphoma expressing Tdt and presenting as ascites. 17, 18 Unlike ours, one case was a transformation from follicular lymphoma, and the other had involvement of the pancreas primarily. Primary effusion lymphoma (PEL) driven by HHV-8 is another differential. PEL cells typically display a \"null\" lymphocyte phenotype, i. e., CD45 is expressed, but routine B-cell (including surface and cytoplasmic immunoglobulin, CD19, CD20, CD79a) and T-cell (CD3, CD4, CD8) markers are absent. Instead, various markers of lymphocyte activation (CD30, CD38, CD71, human leukocyte antigen DR) and plasma cell differentiation (CD138) are usually displayed. 19 Peritoneal lymphomatosis is treated non-surgically and often shows dramatic improvement with chemotherapy. Therefore, early and precise diagnosis is of utmost importance. However, PL with double-hit phenotype has a belligerent course and a poor outcome despite appropriate treatment. 20 uble-hit genetics (MYC, BCL2 rearrangement) have inferior outcome with standard chemotherapy. This case highlights that double hit genetics can be seen in B-precursor lymphoma/leukemia and a separate classification of this entity may be beneficial for prognostication and may facilitate further research and design the novel therapeutic approaches such as MYC and BCL2 inhibitors.",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "Autops Case Rep (São Paulo). 2021;11:e2021278 This work was carried out at the Post Graduate Institute of Medical Education and Research (PGIMER). Chandigarh, India.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "CONCLUSIONS",
"section_num": null
},
{
"section_content": "• There should be early institution of empirical treatment while awaiting diagnostic results as it has an aggressive course and poor outcome;\n\n• De novo double-hit B-cell precursor leukemia/lymphoma needs separate recognition for facilitating further research and targeted therapy. \n\nAuthors' contributions: Balamurugan Thirunavukkarasu was involved in data collection, literature review and drafting of the manuscript. Amanjit Bal contributed to drafting of the manuscript and supervised it. Balamurugan Thirunavukkarasu, Amanjit Bal and Prateek Bhatia were involved in the diagnosis on autopsy. Jayanta Samanta managed the patient clinically. All authors have read and approved the manuscript. \n\nEthics statement: Informed consent by the next of kin was retained by the institution where the autopsy was performed. \n\nConflict of interest: None. \n\nFinancial support: None.",
"section_name": "CONCLUSIONS",
"section_num": null
}
] |
10.1038/cddis.2014.391
|
Gefitinib targets ZAP-70-expressing chronic lymphocytic leukemia cells and inhibits B-cell receptor signaling
|
<jats:title>Abstract</jats:title><jats:p>Chronic lymphocytic leukemia (CLL) can be divided into groups based on biomarkers of poor prognosis. The expression of the tyrosine kinase ZAP-70 (member of the Syk tyrosine kinase family) in CLL cells is associated with shorter overall survival in CLL patients. Currently, there is a lack of targeted therapies for patients with ZAP-70 expression in CLL cells. The tyrosine kinase inhibitor gefitinib has been shown to be effective at induce apoptosis in acute myeloid leukemia through inhibition of Syk. In this study, we sought to test the efficacy of gefitinib in primary human ZAP-70+ CLL cells. We demonstrate that gefitinib preferentially induces cell death in ZAP-70-expressing CLL cells with a median IC<jats:sub>50</jats:sub> of 4.5 <jats:italic>μ</jats:italic>M. In addition, gefitinib decreases the viability of ZAP-70+ Jurkat T leukemia cells but fails to affect T cells from CLL patients. Western blot analysis shows gefitinib reduces both basal and B-cell receptor (BCR)-stimulated phosphorylation of Syk/ZAP-70, ERK, and Akt in ZAP-70+ CLL cells. Moreover, gefitinib inhibits the pro-survival response from BCR stimulation and decreases pro-survival proteins such as Mcl-1. Finally, ZAP-70 expression sensitizes Raji cells to gefitinib treatment. These results demonstrate that gefitinib specifically targets ZAP-70+ CLL cells and inhibits the BCR cell survival pathway leading to apoptosis. This represents the likelihood of tyrosine kinase inhibitors being effective targeted treatments for ZAP-70+ CLL cells.</jats:p>
|
[
{
"section_content": "The clinical course of chronic lymphocytic leukemia (CLL) is highly variable, and although some patients are treated at diagnosis, others may not require therapy for years. 1 Biomarkers can help stratify these patients into indolent and aggressive disease categories. The aggressiveness of CLL is dependent on whether the leukemia cells have (60% of CLL population) or lack (40% of CLL population) mutations of the immunoglobulin variable region of the heavy chain (IgV H ). Thus, patients with early-stage disease have a median survival of 8 years if they have unmutated IgV H (Un-IgV H ) and 24 years if they have mutated IgV H (Mu-IgV H ) disease. 2 A surrogate marker for IgV H mutational status is the expression of zeta-chain-associated protein 70 (ZAP-70); IgV H mutated CLL cells are frequently ZAP-70 negative, whereas IgV H unmutated cells are more typically ZAP-70 positive. 3 ZAP-70 staining in CLL is not an all-or-nothing phenomenon, and to maximize the correlation with IgV H mutational status, a ZAP-70-positive case is defined as ≥ 20% of the CLL cells staining for ZAP-70. Like IgV H status, overexpression of ZAP-70 in CLL cells is associated with aggressive disease; time to treatment is 2. 6 years for ZAP-70+ patients compared with 8 years for ZAP-70patients independent of Rai stage. 3 Thus, ZAP-70 is a rationale target for therapy in CLL. \n\nAlthough the clinical relevance of ZAP-70 in CLL is well known, its molecular function is less understood. ZAP-70 is a member of the Syk family of protein tyrosine kinases and is normally involved in signal transduction of the T-cell receptor in T cells. ZAP-70 overexpression in malignant B cells, such as CLL cells, enhances the B-cell receptor (BCR) pathway. This pathway is a key mechanism for cell survival in CLL. 4, 5 Upon activation of the BCR, tyrosine kinase Lyn phosphorylates and activates Syk, leading to activation of downstream signaling pathways and upregulation of anti-apoptotic proteins, such as Mcl-1. CLL cells with both Un-IgV H and high ZAP-70 expression show increased activation of proteins downstream of the BCR such as Akt, mitogen-activated protein kinase (MAPK), and NF-κB. 4, 6, 7 This suggests that alterations in the BCR signaling pathway through increased expression of the tyrosine kinase ZAP-70 are important in CLL disease progression. \n\nGefitinib is a tyrosine kinase inhibitor known for targeting the epidermal growth factor receptor (EGFR) and is used in the treatment of non-small-cell lung cancer and other cancers of epithelial origin. 8 The drug is well tolerated, with rash and diarrhea being the only dose-limiting toxicities. Importantly to leukemias, it is not myelosuppressive. 9 Apart from its effects on EGFR activity, gefitinib has shown activity against 420 other kinase targets, including Lyn and Syk. 10, 11 Gefitinib has been shown activity in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and acute lymphocytic leukemia (ALL), inducing both differentiation and cell death in vitro. 12 These effects are associated with inhibition of Syk phosphorylation. Thus, although gefitinib is used to treat lung cancer by inhibiting EGFR, it has potential utility in the treatment of CLL patients with high expression of Syk family members that include ZAP-70. \n\nIn this study we show that gefitinib selectively induces apoptosis in ZAP-70-expressing CLL cells, both when unstimulated and BCR activated. These effects are associated in both cases with a reduction in overall tyrosine phosphorylation and specific decreases in Lyn/Lck, Syk/ ZAP-70, ERK1/2, and Akt phosphorylation. These changes produce a decreased expression of Mcl-1 and blocked antiapoptotic signaling. Forced overexpression of ZAP-70 by lentiviral infection in the Raji B-cell line increases the sensitivity of the cells to gefitinib-induced apoptosis. However, normal T cells from CLL patients, which also express ZAP-70, are not affected by gefitinib. These results suggest that tyrosine kinase inhibitors such as gefitinib are a viable treatment option for ZAP-70+ CLL patients.",
"section_name": "",
"section_num": ""
},
{
"section_content": "Gefitinib targets ZAP-70+ CLL cells and leukemia cell lines. Given the efficacy of gefitinib in targeting Syk in AML, 11, 12 we investigated its efficacy in CLL cells expressing ZAP-70. CLL samples were defined as being ZAP-70+ if the number of positive cells was ≥ 20%. The concentration of drug required to reduce cell viability by 50% (IC 50 ) for gefitinib in ZAP-70+ and ZAP-70 -CLL cells was assessed using the MTT (3,3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay. The median gefitinb IC 50 for ZAP-70+ CLL cells was 4. 5 μM and 415 μM for ZAP-70cells that was statistically significant (Table 1 and Figure 1a ). Overall, 77% of ZAP-70+ patient cells and 30% of ZAP-70patients responded to gefitinib treatment as defined by the median IC 50 concentration. In addition, there did not appear to be cross-resistance to fludarabine in the ZAP-70+ samples, and most fludarabine-resistant cases were sensitive to gefitinib (Supplementary Table 1 ). \n\nInterestingly, another EGFR tyrosine kinase inhibitor, erlotinib, had no activity against the ZAP-70+ CLL cells. There was also no significant difference between the median IC 50 of fludarabine in ZAP-70+ and ZAP-70 -CLL cells (5. 4 μM compared with 7. 0 μM). There was also no significant difference in the median IC 50 values of gefitinib when all cases were stratified by IgV H (7. 0 μM for Un-IgV H compared with 8. 3 μM or Mu-IgV H ) or if ZAP-70+ cases were stratified by mutational status (4. 0 μM for ZAP-70+/Mu-IgV H compared with 6. 0 μM for ZAP-70+/Un-IgV H ). \n\nTo confirm the MTT results, primary CLL cells were treated in vitro with gefitinib and cell death was analyzed by flow cytometry after 24 h. Although the median IC 50 was 4. 5 μM by MTT assay 72 h post treatment, a higher dose of 10 μM was chosen for cell death experiments because of the shorter time frame of 24 h to detect cell death. Gefitinib treatment increased apoptosis in ZAP-70+ primary CLL cells as detected by an increase in annexin V-stained cells. After 24 h, the number of annexin V-positive CLL cells ranged from 25 to 85% (Figure 1b ). After 72 h, cell death was seen at doses as low as 1 μM and increased in a dose-dependent manner (Figure 1c ). Cell death was accompanied by increased cleavage of poly (ADP-ribose) polymerase (PARP) and caspase 3 and decreased Mcl-1 expression (Figure 1d and Supplementary Figure 1 ). \n\nTo test whether gefitinib could be give a synergistic apoptotic response with standard chemotherapeutic agents used to treat CLL, gefitinib was added alone or in combination with fludarabine. The combined effect of the two drugs failed to give a synergistic apoptotic response (Supplementary Figure 2a ). \n\nBased on standard definitions, ZAP-70+ CLL samples were defined as having ≥ 20% of their leukemia cells expressing ZAP-70, 3 but changing the threshold of positive cells did not influence the results. To determine whether gefitinib was targeting only the ZAP-70+ cells or was affecting all cell populations, the viability of both ZAP-70+ and ZAP-70cells was measured within the same sample. Gefitinib decreased the percentage of viable annexin V-ZAP-70+ CLL cells compared with dimethyl sulfoxide (DMSO)-treated controls in six different CLL samples (Supplementary Figure 2b ). This indicates gefitinib is selectively inducing apoptosis in ZAP-70+ CLL cells. This preference for ZAP-70+ CLL cells cannot be from another gefitinib target, as CLL cells do not express EGFR and do not differentially express receptor-interacting protein kinase 2 (RIP2) or cyclin G-associated kinase 1 (GAK1; Supplementary Figure 3 ). \n\nTo support the role that ZAP-70 expression plays in gefitinibinduced apoptosis, we examined four leukemia/lymphoma cell lines for their sensitivity to gefitinib treatment. The B cellderived NALM6, BJAB, and I-83 cell lines do not express ZAP-70, whereas the T cell-derived Jurkat cell line does express high levels of ZAP-70. Conversely, the B-cell lines expressed Syk, whereas Jurkat cells do not (Figure 1e ). Human epithelial kidney cell line HEK293 was used as a negative control for both Syk and ZAP-70 expression. Gefitinib treatment over an 18-h time course revealed that only Jurkat cells showed significant apoptosis as detected by annexin V staining assay (Figure 1f ). When the cell lines were treated with a range of gefitinib concentrations (0. 1 to 30 μM) only Jurkat cells showed a significant increase in apoptosis; there were 80% annexin V+ Jurkat cells but o40% annexin V+ B-cell lines after treatment of 30 μM gefitinib for 18-24 h (Figures 1f and g ). The Src tyrosine kinase inhibitor dasatinib did not show this bias toward Jurkat cells (Supplementary Figure 4 ), but this cannot rule out differential internalization of gefitinib in different cell lines. As gefitinib had a significant effect against the Jurkat leukemia T-cell line, we determined whether normal T cells were also sensitive to gefitinib. We isolated mononuclear cells from peripheral blood of ZAP-70+ patients with lymphocyte count o40 × 10 9 cells/l and treated the cells with 10 μM gefitinib. This dose was chosen to agree with gefitinib treatments on CLL cells. The T and B cells were kept at the ratio observed in the peripheral blood. The CLL cells showed apoptosis following treatment with gefitinib, whereas the T cells were resistant (Figures 2a and b ). Even after 72 h of treatment with 10 μM gefitinib, only ZAP-70+ CLL cells responded to treatment (Supplementary Figure 5 ), suggesting that gefitinib only targets malignant leukemia cells expressing ZAP-70. \n\nGefitinib inhibits basal and BCR signaling, preventing downstream ERK and Akt activation. In cell lines, gefitinib treatment reduced tyrosine phosphorylation of ZAP-70 in a dose-dependent manner in CD3-stimulated Jurkat T cells, but not Syk in BCR-stimulated BJAB B cells. There was no decrease in Lyn or Lck phosphorylation in BJAB or Jurkat cells, respectively (Figure 3 ). In primary CLL cells, we found gefitinib treatment reduced total cellular tyrosine phosphorylation, both from the basal level and from the BCR-stimulated level, in ZAP-70+ CLL cells over a range of gefitinib concentrations and incubation times (Figures 4a-c ). However, this was not observed in ZAP-70 -CLL cells. The decrease in overall cellular tyrosine phosphorylation was evident after 1 h, and decreased further after 24 h (Figures 4a-c ). Tyrosine phosphorylation did not decrease in ZAP-70 -CLL cells treated with increasing doses of gefitinib (Supplementary Figure 6a ). However, even at doses as low as 1 μM, gefitinib decreased tyrosine phosphorylation in ZAP-70+ CLL cells (Supplementary Figure 6c ). As a control, erlotinib failed to decrease tyrosine phosphorylation in primary CLL cells (data not shown). Unlike cell lines, there were specific decreases in both Syk/ZAP-70 and Lyn/Lck phosphorylation in ZAP-70+ CLL cells after 1 h of gefitinib treatment (Figure 4d ). Quantification of these decreases showed that Syk/ZAP-70 phosphorylation appeared to decrease slightly more than Lyn/Lck phosphorylation after gefitinib treatment (Figure 4e ). \n\nBecause of the fact that phospho-antibodies recognized both Syk and ZAP-70 phosphorylation, we performed immunoprecipitation using specific Syk and ZAP-70 antibodies and western blotting for phospho-tyrosine after BCR stimulation. Each approach showed that both Syk and ZAP-70 phosphorylation decreased following gefitinib treatment (Figure 4f and Supplementary Figure 6d ). Immunoprecipitation was also performed with phospho-tyrosine antibodies and then blotted first for ZAP-70 and then for Syk (data not shown). \n\nThe activity of gefitinib was compared with ibrutinib and dasatinib, the two tyrosine kinase inhibitors under clinical investigation in CLL. The downstream effect of gefitinib was the same as these other two drugs and all three prevented phosphorylation of Akt and ERK after BCR stimulation (Figures 4g and h ). Even when the primary CLL cells had high basal Akt phosphorylation, which did not increase with BCR stimulation, there was still an inhibition of phosphorylation with the tyrosine kinase inhibitors (data not shown). This inhibition was not observed with fludarabine that was used as a negative control. BCR stimulation alone served as a positive control. As signaling through the BCR promotes cell survival, we determined whether inhibition of this signaling pathway by gefitinib decreased survival. Primary CLL cells were treated with 10 μM gefitinib, BCR activated for 30 min by immobilized anti-IgM, and cell death quantitated by annexin-V staining after 24 h. We found that anti-IgM protected CLL cells from spontaneous apoptosis but failed to protect CLL cells from gefitinib treatment (Figure 4i ). \n\nForced overexpression of ZAP-70 increases sensitivity to gefitinib. To determine whether ZAP-70 plays a role in the susceptibility of a patient to gefitinib, we tested the response of the ZAP-70-negative lymphoma-derived B-cell line Raji transduced with GFP-expressing vector or ZAP-70-expressing vector. Expression of ZAP-70 was firmed by western blot (Figure 5a ) and flow cytometry and expression of GFP was confirmed by flow cytometry (Supplementary Figure 7 ). We found that gefitinib treatment lowered Syk/ZAP-70 phosphorylation but not Lyn phosphorylation in ZAP-70-expressing Raji cells (Figure 5b ). Western blot analysis showed constitutive phosphorylation of Syk/ ZAP-70 in Raji cells overexpressing ZAP-70 (Figure 5b ), which is congruent with previously published literature. 13 pon BCR activation, Syk/ZAP-70 binds to the tyrosine phosphorylated co-receptor CD79a. We found that there was no difference in Syk or ZAP-70 binding to CD79a after gefitinib treatment (Figure 5c ). This suggests that CD79a is still tyrosine phosphorylated after gefitinib treatment. \n\nWe next investigated the sensitivity of gefitinib in ZAP-70-Raji cells. Raji cells overexpressing ZAP-70 had increased sensitivity to gefitinib compared with cells with vector alone as measured by greater degradation of Mcl-1 (Figure 5d ), greater DNA fragmentation as measured by sub-G1 peak analysis (Figure 5e ), and greater annexin V and 7-amino-actinomycin D (7AAD) staining (Figure 5f ). These results were confirmed with different cell passages, and different cell death staining techniques (PI staining and annexin V staining). We further knocked down ZAP-70 in Raji cells and showed reduced Erk phosphorylation and Mcl-1 expression, indicating changes in downstream targets of gefitinib were due to ZAP-70 overexpression (Supplementary Figure 8 ). In addition, gefitinib treatment was compared with dasatinib and ibrutinib. Gefitinib had the greatest effect of all the drugs on Raji cells overexpressing ZAP-70, as compared with Raji cells with vector alone (Figure 5f ). Dasatinib had little effect on Raji cells, whether they overexpressed ZAP-70 or not, only increasing cell death by 3-10%. Ibrutinib had a greater effect on ZAP-70-overexpressing Raji cells, as compared with the non-ZAP-70-expressing cells, but there was less cell death than seen with gefitinib using this same drug dosage (20 μM). This increased sensitivity of the ZAP-70expressing Raji cells was not seen with fludarabine treatment, and this was used as a negative control (Figure 5f ). Unfortunately, the complementary experiments to treat Jurkat cells with ZAP-70 knockdown were not feasible because knockdown of ZAP-70 led to increased cell death compared with control siRNA (Supplementary Figure 9 ).",
"section_name": "Results",
"section_num": null
},
{
"section_content": "In this study the tyrosine kinase inhibitor gefitinib, originally used to inhibit EGFR kinase activation in lung cancer, is also cytotoxic to primary CLL cells that overexpress ZAP-70. When these cells undergo BCR activation, gefitinib can inhibit phosphorylation of Lyn/Lck, Syk/ZAP-70, ERK1/2, and Akt within 1 h. These results are similar to those seen with 5-10 μM gefitinib in AML and MDS cells, 11 where gefitinib functions through an EGFR-independent mechanism targeting Syk activation. Using the MTT assay, which we and others have shown to be predictive of clinical response to fludarabine and chlorambucil, 14 the median IC 50 of gefitinib was 4. 5 μM in ZAP-70+ CLL cells but 415 μM in ZAP-70cells. \n\nThere has been considerable interest in the evaluation of tyrosine kinase inhibitors for the treatment of CLL. Dasatinib is normally used to treat chronic myeloid leukemia and is a tyrosine kinase that inhibits the Src family member Abl. 15 However, it is also cytotoxic to CLL cells, including ZAP-70+ cells, and it has been suggested that dasatinib is targeting tyrosine kinase Lyn. 15 In addition, Syk has been targeted in CLL with the tyrosine kinase inhibitor, R406 (fostamatinib). The effect of R406 was greatest in cells with high levels of Syk that were Un-IgV H and expressed ZAP-70. 16 However, R406 had no effect on the phosphorylation of other tyrosine kinases, such as ZAP-70. 16 Recent evidence has indicated that these findings are clinically relevant as the pro-drug for R406, fostamatinib disodium (FosD), is clinically active in CLL patients. 17 Two novel Syk inhibitors, PRT318 and P505-15, have recently been shown to suppress CLL activation and migration in vitro. 18 Besides these tyrosine kinase inhibitors, the Bruton's tyrosine kinase inhibitor, ibrutinib, has shown potent activity in CLL, both in vitro and in vivo, 19, 20 and the multikinase inhibitor, sorafenib, has also been shown to induce apoptosis in CLL cells in vitro. 6 These inhibitors were active against both ZAP-70-and ZAP-70+ CLL cells, whereas gefitinib was the only tyrosine kinase inhibitor shown to selectively sensitize ZAP-70+ CLL cells to undergo apoptosis. The long-term toxicity and efficacy of these tyrosine kinase inhibitors in CLL is unknown and it will be years before they become FDA approved for use in this disease. In contrast, gefitinib is already approved by the FDA for the treatment of lung cancer and has been used for many years in the clinic. It has the potential advantage in CLL in being specifically cytotoxic to ZAP-70+ CLL cells, and not being myelo-or immuno-suppressive. \n\nGefitinib has recently been shown to accumulate in solid tumors. Haura et al. 21 found 22 μM in lung tumor, and McKillop et al. 22 found 16. 7 μM in breast tumor. These doses were 40 and 42 times higher than the concentration observed in the plasma, respectively. We predict that this accumulation of gefitinib at the cancer site would also occur in leukemia patients. Therefore, low plasma concentrations from patients with solid tumors may not be appropriate values to consider when testing doses of gefitinib on leukemic cells that reside in the blood and lymphoid tissues. In addition, in vitro experiments cannot recapitulate the dosing scheme that would be used in vivo. Gefitinib cytotoxic concentrations in ZAP-70+ CLL cells were similar to its concentrations that induce apoptosis in AML cells. In the future, we will focus on in vivo models testing gefitinib in various drug combinations for effectiveness. \n\nThe blood and lymphatic systems consist of distinct microenvironments that include blood, bone marrow, spleen, and lymph nodes. As cells traffic through these microenvironments, dynamic cell-cell interactions occur between mobile cells and tissue-resident cells. ZAP-70+ CLL cells tend to localize to the nodes and this is associated with more aggressive disease. 3 One of the most important signals from the microenvironment for cell survival is BCR activation. 5, 23, 24 pon activation of the BCR, the tyrosine kinase Lyn phosphorylates and activates Syk, leading to activation of downstream signaling pathways such as Akt, MAPK, and NF-κB, upregulation of anti-apoptotic proteins such as Mcl-1, and inactivation of pro-apoptotic protein BIM. These changes lead to increased cell survival. 24, 25 CLL cells with both Un-IgV H and high ZAP-70 expression show increased BCR signaling. 24, 25 his suggests that alterations in the BCR signaling pathway are important in CLL disease progression. In the present study, we showed that gefitinib blocked both ERK and Akt activation leading to a decrease in Mcl-1 expression and apoptosis. This mechanism of cell death may be common among the tyrosine kinase inhibitors. 26 The evidence that ZAP-70 expression sensitizes cells to gefitinib and that gefitinib targets the BCR pathway both indicate that this drug may have activity in the microenvironment. In particular, gefitinib may have an effect in the lymph node microenvironments where BCR signaling occurs 27 and ZAP-70 expression is upregulated. 28 It is important to note that the complexity of feedback loops and interactions of ZAP-70 in CLL cells are not clearly understood, making it difficult to definitively determine the precise action of gefitinib. This will be the focus of future investigations. \n\nDespite inefficient tyrosine kinase activity in CLL, 29 ZAP-70 still plays an important role in the overactivation of the BCR pathway. Although the kinase domain is not required for enhanced signaling, inhibition of its kinase activity may cause steric hindrance or prevent conformational changes of signaling complexes preventing downstream signaling events. \n\nOverall, gefitinib selectively targets CLL cells expressing ZAP-70. This indicates that tyrosine kinase inhibitors could be used to selectively treat patients with high ZAP-70-expressing CLL cells. As gefitinib is already in clinical use in lung cancer patients, and lacks suppression of the bone marrow or immune system, further studies are warranted to investigate the clinical activity of gefitinib in ZAP-70+ CLL patients.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Cell isolation and culture. Peripheral blood samples were collected from patients following informed consent in accordance with the Research Ethics Board at the University of Manitoba. Samples were mixed with RosetteSep (Stemcell Technologies, Vancouver, BC, Canada) if the lymphocyte count was o40 × 10 9 /l and then purified on a Ficoll-Paque gradient (GE Healthcare, Cleveland, OH, USA). Red blood cells (RBCs) were lysed with a RBC lysis buffer (eBioscience, San Diego, CA, USA). All blood samples were processed within 24 h after collection and used fresh. For experiments, the leukemia cells were grown in Hybridoma serum-free medium (SFM, Life Technologies, Carlsbad, CA, USA). \n\nBJAB (ATCC, Burlington, ON, Canada), NALM6 (DSMZ, Braunschweig, Germany), I83 (kind gift from Dr. Panasci, McGill University, Montreal, Canada), Jurkat (ATCC), and Raji+/ -ZAP-70 (kind gifts from Dr. Marshall, University of Manitoba, Winnipeg, MB, Canada) cell lines were all cultured in Hyclone RPMI with 10% Hyclone fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA). \n\nDrugs and stimuli. Gefitinib (LC Laboratories, Woburn, MA, USA), erlotinib (LC Laboratories), dasatinib (LC Laboratories), ibrutinib (SelleckBio, Houston, TX, USA), and fludarabine (Sigma, St. Louis, MO, USA) were all dissolved in DMSO (Thermo Fisher Scientific) and added. B cells were stimulated with 10 μg/ml biotinylated Fab 2 'IgM (Southern Biotech, Birmingham, AL, USA) and T cells were stimulated with 2 μg/ml LEAF-purified anti-CD3 (BioLegend, San Diego, CA, USA). For western blot experiments, 10 μg/ml soluble anti-IgM was added and cells were lysed within 1 h. For cell death analysis after 24 h, 0. 1 μg anti-IgM was immobilized in Hanks' buffered salt solution (HBSS, Life Technologies) in a 96-well Falcon plate (BD, Franklin Lakes, NJ, USA) overnight, and then washed before addition of CLL cells. \n\nCell viability assays. For MTT assays, 3 × 10 7 CLL cells in 3 ml of Hybridoma SFM were added to tubes (Sarstedt, Nümbrecht, Germany) and treated with 1, 2, 5, 10, or 15 μM of the drug for 24 h. Cells were washed in HBSS and seeded into 96-well plates for 3 days. On day 3, MTT (Sigma) was added to a final concentration of 0. 25 mg/ml. Plates were incubated for 5 h at 37 °C with 5% CO 2, and absorbance was measured at a wavelength of 540 nm. \n\nFor flow cytometry, samples were collected, washed with 1 × annexin V Binding Buffer (BD Biosciences, Franklin Lakes, NJ, USA), and then stained with 7AAD (BD) and annexin V-fluorescein isothiocyanate (FITC; BD) or annexin V-allophycocyanin (APC; BD). Transduced Raji cells were stained instead with propidium iodide (PI) or annexin V-APC (BD) because of the GFP vector. Samples were examined using a BD FACSCalibur. \n\nWestern blotting and immunoprecipitation. Cell lysates were collected at the indicated times in 1% NP-40 lysis buffer with complete protease inhibitor tablet (Roche, Basel, Switzerland), 1 mM phenylmethanesulfonylfluoride (PMSF), and 2 mM sodium orthovanadate (New England BioLabs, Ipswich, MA, USA). Protein levels were quantified with a Pierce BCA kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Samples were run on 8-10% polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) blocked in 5% BSA (Sigma) or milk in TBS-T as per the antibody manufacturer's suggestions. Primary antibodies included rabbit or mouse anti-ZAP-70 (Cell Signaling, Beverly, MA, USA), rabbit anti-Syk/ZAP-70-P (Cell Signaling), rabbit anti-Lyn (Cell Signaling), rabbit anti-Lyn-P (Epitomics, Burlingame, CA, USA), mouse anti-Lck (Cell Signaling), rabbit anti-Btk (Cell Signaling), rabbit anti-Btk-P (Cell Signaling), rabbit anti-ERK1/2 (Cell Signaling), rabbit or mouse anti-ERK1/2-P (Cell Signaling), rabbit anti-Akt (Cell Signaling), rabbit anti-Akt Ser-P (Cell Signaling), mouse anti-tyrosine-P (Millipore, Darmstadt, Germany), rabbit anti-Mcl-1 (Cell Signaling), rabbit anti-PARP (Cell Signaling), rabbit anti-cleaved caspase 3 (Cell Signaling), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma), rabbit anti-α-tubulin (Cell Signaling), and rabbit or mouse anti-βactin (Sigma). Secondary antibodies were goat anti-rabbit-HRP or anti-mouse-HRP (Bio-Rad). Detection of protein was with Pierce ECL or Pierce Supersignal Pico (Thermo Fisher Scientific) reagents. Co-immunoprecipitation was carried out with 500 μg of protein in 0. 2% CHAPS lysis buffer containing 150 mM NaCl, 20 mM Tris, 10% glycerol, 2 mM EDTA, 1 mM PMSF, 2 mM sodium orthovanadate, and complete protease inhibitor tablet (Roche). Lysates were incubated at 4 °C with primary antibody mouse anti-Syk (Abcam, Cambridge, UK) or mouse anti-ZAP-70 (Cell Signaling) on a rotator overnight. Pierce protein G plus agarose bead slurry (Thermo Fisher Scientific) was added in a final dilution of 1 : 10 for 2 h and the procedure for western blot was followed. Entire immunoprecipitate supernatant was loaded on to a gel lane. Immunoprecipitations were done in the same way, with the exception of using 1% NP-40 lysis buffer. \n\nFlow cytometry. For extracellular staining of T and B cells, peripheral mononuclear cells were stained with annexin V-FITC, anti-CD3-PE, 7AAD, and anti-CD19-APC (BD Biosciences). Cells gated on either CD19+ or CD3+ were then analyzed for expressions of annexin V and 7AAD. \n\nFor intracellular staining of ZAP-70 in cell death experiments, RosetteSep and ficoll-purified CLL cells were first surfaced stained with annexin V-APC and 7AAD, then fixed with solution A (Beckman Coulter, Brea, CA, USA) for 12 min at 37 °C, washed with PBS, permeabilized with solution B (Beckman Coulter) for 5 min at room temperature, and then stained with mouse anti-human ZAP-70-FITC (Beckman Coulter) for 15 min. Mouse IgG1-FITC (BD) was used as an isotype control. Unstained and single-stained controls were always included in all flow cytometry experiments. All sample data were acquired on BD FACSCalibur and analyzed using CellQuest Pro software (BD). CLL cell samples were considered ZAP-70+ if ≥ 20% of the cells stained positively. \n\nThe ZAP-70 status of each patient sample was determined by diagnostic flow cytometry. Whole blood was stained with anti-CD19, anti-CD5, anti-CD38, and anti-ZAP-70. The negative control is a normal blood sample that should not have CD19+CD5+ leukemic cells. The positive control is the autologous CD19-CD5+ ZAP-70+ T cells. \n\nTransduction of ZAP-70. Vectors encoding GFP pWPTS or vector encoding ZAP-70 pWPTS-ZAP-70 were packaged into lentivirus by co-transfection with pCMV-R8. 91 and pMD. G into HEK-293 T cells. 30, 31 All vectors were kindly provided by Dr. Ferenc Boldizsar (University of Pécs, Pécs, Hungary). Raji cells (originally from DSMZ) were transduced with lentiviral particles using a spin protocol as previously described. 32, 33 Expression of ZAP-70 was confirmed by western blot and flow cytometry. \n\nStatistical analysis. Graphs were created and statistics were performed using GraphPad Prism4 software (GraphPad Software Inc., San Diego, CA, USA). Unless otherwise noted, a paired or unpaired two-tailed t-test was performed according to the nature of data. Statistical significance was noted in the figures as *Po0. 05, **Po0. 01, or ***Po0. 001. Densitometry was calculated using ImageJ (Wayne Rasband; National Institute of Mental Health, Bethesda, MD, USA).",
"section_name": "Materials and Methods",
"section_num": null
}
] |
[
{
"section_content": "Acknowledgements. We thank the Manitoba CLL Tumor Bank for organization of patient samples, Michelle Brown for the mutational analysis of patient samples, and Dr. Aaron Marshall and Hongzhao Li for the transduced Raji cell lines. This research is supported by the CancerCare Manitoba Foundation operating grant and by CLL Global Research Foundation.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Conflict of Interest",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest.",
"section_name": "Conflict of Interest",
"section_num": null
},
{
"section_content": "RFD, WX, J-YY, and EN designed and performed experiments. RD and SBG wrote the paper. HL and AJM provided vital reagents. VB, JBJ, and SBG provided intellectual input and editorial advice.",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4. 0 International Licence. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons licence, users will need to obtain permission from the licence holder to reproduce the material. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4. 0 Supplementary Information accompanies this paper on Cell Death and Disease website (http://www. nature. com/cddis)",
"section_name": "Cell Death and",
"section_num": null
}
] |
10.1186/1471-2164-14-s7-s1
|
Automatic B cell lymphoma detection using flow cytometry data
|
Flow cytometry has been widely used for the diagnosis of various hematopoietic diseases. Although there have been advances in the number of biomarkers that can be analyzed simultaneously and technologies that enable fast performance, the diagnostic data are still interpreted by a manual gating strategy. The process is labor-intensive, time-consuming, and subject to human error.We used 80 sets of flow cytometry data from 44 healthy donors, 21 patients with chronic lymphocytic leukemia (CLL), and 15 patients with follicular lymphoma (FL). Approximately 15% of data from each group were used to build the profiles. Our approach was able to successfully identify 36/37 healthy donor cases, 18/18 CLL cases, and 12/13 FL cases.This proof-of-concept study demonstrated that an automated diagnosis of CLL and FL can be obtained by examining the cell capture rates of a test case using the computational method based on the multi-profile detection algorithm. The testing phase of our system is efficient and can facilitate diagnosis of B-lymphocyte neoplasms.
|
[
{
"section_content": "Flow cytometry (FC) involves conjugating fluorochromes to antibodies, allowing them to bind different cell biomarkers, and passing the stained cells through the path of a laser where the fluorochromes are excited and fluorescence emission is measured. Forward and side scatter of cells give information about the size and complexity of the cells. FC is a valuable tool in the diagnosis of lymphocytic neoplasms. Most of the current software supplied by the cytometer manufacturer provides a 2-parameter visual representation of the multi-dimensional data. Pathologists must manually select the areas that include the cells of interest and view these cells using two other attributes, a process known as gating. These areas of interest are not fixed due to instrument, operator, and sample differences. The pathologists use the clustering of the cells, the distribution and cell size of a cluster, and the relative location of the clusters to make the selection. The process is tedious, time-consuming, and subject to bias. Thus, there is an urgent need to develop a fast and unbiased diagnostic approach [1]. \n\nOur ultimate goal is to establish an automated process for clustering cells of interest to replace manual gating [2, 3]. Cell populations can be identified in an automated fashion (automated gating) by employing clustering algorithms. The most challenging aspect of the automated process is finding the best clustering algorithm for high-dimensional data sets [4] [5] [6] [7]. Many existing dimension-reduction approaches may cause useful information to be lost [8] [9] [10] [11] [12] [13]. There have been several attempts to use machine learning technique to automate the gating process [14] [15] [16] [17] [18] [19] [20]. The most commonly used approach is the k-mean algorithm [21], which assigns a cell to its nearest cluster. There are several versions of k-mean algorithms such as fuzzy k-mean, K-medoid, Gath Geva, and the Gustafson Kessel algorithm [22]. \n\nOther common approaches are hierarchical clustering [23] [24] [25] [26] and density-based clustering [27]. \n\nRecently, model-based clustering has been gaining popularity [28] [29] [30] [31], including use of the expectationmaximization (EM) algorithm [32]. However, most approaches only focus on the first stage of FC data analysis that identifies cell populations, some approaches are only semi-automatic [33], and some only target certain types of lymphocytic neoplasms [34, 35]. This paper proposes a novel 3-dimensional (3-D) 5-parameter model that detects multiple types of B-lymphocyte neoplasms. \n\nIn this proof-of-concept study, we will apply this methodology to differentiate between selected subtypes of B-lymphocyte neoplasms and identify biomarkers that contribute to the classification of certain subtypes, such as chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). Our goal is to develop software solutions to allow pathologists to quickly interpret the FC data without bias.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "The multi-profile lymphoma detection described in this article can detect whether the FC data of an individual matches the profile of a particular type of lymphoma or that of a healthy donor. The objectives of the computational detection system were: (1) minimum human intervention in the detection process, (2) ability to detect various types of B-lymphocyte neoplasms, (3) efficient computation complexity, and (4) reasonable detection rate with a low false-negative rate. \n\nOur system is a multi-anomaly detection system in which a healthy subject's profile and several anomaly profiles are used to determine the closest match. We will first describe a single profile detection system. The system must be trained with known data to gain knowledge about a healthy donor and a patient with a particular type of lymphoma. To achieve the goal of diagnosis, the system has to learn the profile of healthy donors through a training phase and match the profile to the test subject through a fitting (testing) phase. The profile building and fitting will be discussed later. The overall strategy of the testing is summarized in Figure 1. The FC data is categorized into three groups: normal donor, patients with CLL, and patients with FL. A \"profile building algorithm\" is used to capture the attributes of one individual group, and the profile is a collection of ellipsoids defined on the cytometry map of selected attributes. These ellipsoids are the area of the map where the cells concentrate. One can imagine the profile as a multi-dimensional ellipsoid which filters out some of the fringe outlier cells. A \"fitting algorithm\" is used to compare a test subject with the profile. Certain features of the profile and the test data have to be extracted for comparison. We needed a metric to measure the fitness of the profile and the test case, defined as the cell capture rate (CCR). This is the ratio of the number of cells captured inside the ellipsoid(s) to that of cells within the test clusters. How the CCR was calculated will be explained in a subsequent section. The CCR estimates how good a test subject matches the profile of healthy donors. Ideally, in a single-profile system, most of the test cells should fall within the ellipsoids defined in the normal profile, thus we can use the CCR to determine if the test case matches the normal profile. In a multi-profile detection system, a test subject then has a vector that includes three CCRs, one each for the normal, CLL, and FL profiles. We will use the vector to diagnose the outcome of the test subject.",
"section_name": "A multi-profile approach for lymphoma detection",
"section_num": null
},
{
"section_content": "Our system focused on characterizing B lymphocyte neoplasms to develop a model-based clustering approach to identify normal or abnormal B cell populations that share similar biological functions. There were two steps in the pre-processing: (1) removal of the doublets (Figure 2 ) and (2) selecting the lymphocytes (Figure 3 ). The algorithm for removing the doublets is relatively simple and can be found in [36]. Our system was designed to learn from the user's knowledge and to select and classify these cells automatically. There were five Cluster of Differentiation (CD) biomarkers that were used for data modelling: CD5 (labelled with PerCP), CD10 (APC), CD19 (APC-Cy7), kappa light chain (PE), and lambda light chain (FITC). In addition, CD45-AmCyan was used for lymphocyte gating. Normal B lymphocytes are positive for CD19, but not express (negative for) CD5 and CD10, and composed of nearly even populations of cells expressing kappa or lambda light chains. In CLL, the lymphoma cells are positive for CD19 and CD5, but negative for CD10. In contrast, FL cells are positive for CD19 and CD10, but negative for CD5. Importantly, cells of CLL and FL exhibit kappa or lambda light chain restriction (a feature of malignancy). The five lymphocytic biomarkers were combined into a 3-D image as shown in Figure 4. The x-axis represents kappa light chain and lambda light chain. The y-axis represents CD19, a pan-B cell marker. The z-axis is used for CD5 and CD10 expression. Since a B lymphocyte is either kappa light chain or lambda light chain positive, we subtracted the value of kappa by lambda to eliminate the background signal; likewise, the value of CD5 minus CD10 is presented in the z-axis. We use the notation of \"Lambda+\" and \"Kappa+\" to indicate the difference in expression between the two CD biomarkers. A cell with a Lambda+ value in Figure 4 means the cell is expressing Lambda, and lacking Kappa expression. \"CD5+\" and \"CD10+\" are used in the figure in a similar fashion. For y-axis, only one biomarker is used, and the cell expressed with CD19 is noted as \"CD19+\". By analyzing the digital FC data in this novel 3-D 5-parameter model, lymphoma cells can be easily distinguished from the normal cell population and further classified into three sub-types: (a) B cell lymphoma with immunophenotyping prolife CD5+, no CD10-expression, light chain restriction (expressing kappa or lambda alone): this profile could be seen in CLL, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), and CD5+ diffuse large B cell lymphoma (DLBCL), (b) B-cell lymphoma with immunophenoting prolife CD10+, no CD5-expression, light chain restriction: this profile is specific for FL, Burkitt lymphoma, and CD10+ DLBCL, and (c) B-cell lymphoma with immunophenotyping prolife no CD5-or CD10expression, light chain restriction: including MCL, DLBCL, and other non-classified B cell lymphomas. Notably, this model is expandable and can be used to analyze any type of B lymphocyte neoplasm. For proof-of-concept demonstration, the two most common B cell lymphomas, CLL and FL, were studied. We will include additional neoplasms as the data becomes available.",
"section_name": "A 3-D 5-parameter flow cytometry data model",
"section_num": null
},
{
"section_content": "After removing the doublets and selecting the lymphocytes by gating on CD45 and side scatter, the healthy donors' FC data was plotted using the 3-D 5-parameter model defined above (see Figure 5 ). Our objective was to identify all clusters so that the lymphocytes can be characterized by the clusters in the 3-D 5-parameter model. In Figure 5, a healthy donor's cells form three clusters: (1) pink cells: B lymphocytes expressing kappa light chain, (2) blue cells: B lymphocytes expressing lambda light chain, and (3) green cells: T lymphocytes. \n\nThe EM algorithm was used to cluster the cells because it gave better results in most of our cases. We used the EM algorithm to produce the final clustering result with means and covariances, and an ellipsoid was constructed with the means and covariances of clusters. The means gave us the center of the ellipsoid and the covariances gave us the orientation and the shape of the ellipsoid. The standard deviation dictated the size of the ellipsoid. Once the means, covariances, and standard deviation were determined, the three ellipsoids for healthy donors were constructed (Figure 6 ). In other words, the orientation, size, and location of the ellipsoid were considered as the profile of this cluster of healthy donors. \n\nThe details of the algorithm are listed below. Step 1: [Initialization] Given X, use the K-mean algorithm to find k clusters of X. The output of K-mean are: M (i), V (i) and W (i), the means, co-variance, and the weight of the k clusters. \n\nStep 2: [Clustering] Use the EM (expectation maximization) algorithm to compute a better clustering of × with initial values M = M (i), V = V (i), and W = W (i). \n\nStep 3: [Ellipsoid Construction] Construct k ellipsoids with Means M and Co-variance V and weight W. The ellipsoid should include all data points within m × std of the center of its cluster. \n\nThe process of building a cancer patient's profile (ellipsoids) was the same as that for the healthy donors except the training data were selected from patient cases. However, the modelling of the patient's profile was more complicated. Although most healthy donors show very similar representation in the 3-D 5-parameter model, CLL data are more diverse, most likely due to different stages and severity of disease. While healthy donors have two cell clusters for kappa and lambda light chains, CLL patients show only one cluster because lymphoma cells are restricted to either kappa or lambda light chains. Thus, in the profile-building algorithm, there are only 2 clusters (k = 2) instead of 3 clusters. For example, in Figure 7a, CLL patient 453 only showed one cluster of cells expressing kappa light chain, which is defined as kappa dominant; in Figure 7b, CLL patient 338 showed B cells that were lambda light chain dominant. It is very important to have a good CLL profile to test the subjects, and this can be cross validated by subsequent experiments. \n\nThe FL profile was built by a similar approach, but both clusters 1 and 2 cells are present in only the CD10 + side of the z-axis (CD5-CD10) since FL cells express only CD10 and lack CD5 expression. For example, in Figure 8a, FL patient 1444 only showed one cluster in the kappa light chain and is defined as kappa dominant. In Figure 8b, FL patient 1284 showed B lymphocytes that were lambda light chain dominant.",
"section_name": "Profile building",
"section_num": null
},
{
"section_content": "Once we had constructed the normal profile, a test case (Figure 9b ) was \"fitted\" to the profile (Figure 9a ). Our goal was to use these ellipsoids to capture cells of a test case, and count the numbers of captured cells inside an ellipsoid. After the means and co-variances were computed by the normal profile building algorithm, the three profiles were represented as ellipsoids (Figure 9a ). However, due to manual handling of the samples, environmental conditions, and the calibration of the instrument, the cell clusters and the ellipsoids may not align very well. An example of cells of a normal test case overlaid with the three-ellipsoid profile is shown in Figure 9c. Thus, a fitting algorithm was required to realign the cell clusters to match with the ellipsoids. \n\nThe ratio of the number of cells captured inside the ellipsoid(s) and that of cells of the test clusters is defined as the CCR of the profile on the test case. In other words, the ratio calculation requires two numbers: the number of B cells and the number of overall cells. The number of cells captured by a profile ellipsoid can be used as the numerator of CCR. For the denominator, there are three possibilities: all blood cells, all lymphocytes, or B cells. In the next two paragraphs, we shall describe how the CCR is computed. \n\nTo find out the B cells captured by an ellipsoid in the profile, it was necessary to partition the cells into clusters. However, most clustering algorithms are ineffective in dealing with clusters that are very close or intersecting each other. Thus, our first step was to use a hierarchical divisive clustering (\"top-down\") approach by separating the T cells from the rest of the test cells by using the value of CD19. The parameter k is defined as the number of clusters (k = 3 for normal profile and k = 2 for patient profile) and X[c,j] represents the observation data of c-th cell. In the first step, T cells were identified and assigned with a label k. The next step was to find the center of the T cells. This can be easily achieved by calculating the mean of cells with label k. Technical variation, such as different operators, machines, etc., may cause the data to shift. Thus, the third step of alignment is to fix the variation by moving the profile to \"fit\" the test data. We have tried several methods for alignment. In one approach for fitting the normal profile, we divided B cells into two clusters representing lambda light chain dominant and kappa light chain dominant and obtained the centers of the two clusters. Then we aligned the ellipsoids individually to the corresponding center. This approach fails to detect changes in the distance between clusters. In addition, the clustering algorithm used to separate two clusters that are very closely aligned was not very effective and this may result in misclassifications. In our current work, we adopted a hierarchical approach: we first found the center of the T cells in the test case, and then calculated the difference between center of T cells in the profile and test case. Finally, we aligned all ellipsoids by the difference. In our system, we used only the one or two ellipsoids that represent B cells and left out the ellipsoid that represents T cells since we are detecting B lymphocyte neoplasms. After aligning the ellipsoids to the center of the corresponding clusters, we obtained the numbers of the captured B cells, which is the numerator of the CCR. \n\nFor the denominator of the CCR, we tried out all the three possibilities mentioned above. If we use the total number of the blood cells as the denominator, the CCR is compressed to a small range thus it is difficult to distinguish healthy donors and patients. In a preliminary paper we reported [37], the B cell CCR is calculated by the number of B cells inside the ellipsoid divided by the total number of lymphocytes. \n\nThat approach gave us a higher CCR to compare since the denominator is smaller. Even though the CCR in [37] was able to distinguish the patients from healthy donors, the CCR for healthy donor using the normal profile is somewhat small (about 13% on average). In this paper, we decide to use a third approach by using the total number of B cells as the denominator. This approach gives us a much higher CCR for healthy donor compared with the normal profile (over 80% on average). \n\nThe detail of the fitting process is given below, and the final B cell CCR is defined as the ratio of the number of B cells inside the ellipsoid over the total number of B cells. Output: Cell capture rate of × against the profile P. Algorithm:\n\nStep 1: [Clustering of cells] This is achieved by a hierarchical divisive clustering approach to identify the Tcells with the CD19 first. Let cluster[c] be the cluster of cell c, thus cluster[c]=k for all cell c in the T-cell cluster. For the rest of the cells, use the K-mean algorithm on X[c,j] to find the remaining k-1 cluster(s). The B-cell clusters are numbered as cluster 1,. ., k-1. \n\nStep 2: [Finding the centers] For each cluster, find the center MC[c, i] (c = 1,. . ., k, i = 1,. . ., d) of the cluster by computing the mean of the cells in that cluster i. \n\nStep",
"section_name": "Profile-fitting algorithm",
"section_num": null
},
{
"section_content": "More formally, we defined the diagnosis decision process by finding the distance of the CCR vector to the axes. The algorithm is described in the equations below. Let D = {Normal, CLL, FL} be the set of all diagnoses, and CCR j be the CCR for each j in D. Compute the distance from the CCR vector of a test case to the corresponding axis as\n\nand\n\nThe algorithm calculates and selects the axis with the smallest distance to the CCR of the test case.",
"section_name": "Diagnosis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Single-profile testing means testing against only the normal healthy profile. We used 72 data sets from 36 healthy donors, 21 CLL patients, and 15 FL patients in our experiment. We constructed the normal profile with 12 randomly selected healthy donors and tested it with the remaining 60 cases. Our hypothesis was that the CCR computed in the fitting algorithm of the healthy cases would fit the normal model better than the patient cases. The result is shown in Figure 10. By comparing the CCRs, the CLL cases were distinguishable from the healthy subjects, but there was overlap between the healthy donors and FL patients. Thus, we were not able to identify all patient samples using only the normal profile. To improve these results, we used multi-profile testing, which combines the results of testing against all three profiles (normal, CLL, and FL).",
"section_name": "Single-profile testing",
"section_num": null
},
{
"section_content": "The hypothesis to be tested was that the healthy donors' cases would capture more cells using the healthy subject model, and patients' cases would capture more cells using the patient model. In Figure 11 we show a test case (healthy subject) against all three profiles, and as it is a healthy subject, the normal profile fits better. \n\nThe unresolved issue is how to select a suitable profile to best represent the healthy donors, and CLL and FL patients. Building normal profiles is easier since samples from healthy donors are generally consistent and less variable; building patient profiles is more difficult since patient samples can vary dramatically. As a first step we used cross validation to test our approach. Because of the differences noted previously, we used a 3-fold cross validation for building and fitting the normal profile, and leave-one-out cross validation for building and fitting the disease profile. We did not use the leave-oneout method for healthy profile testing for two reasons:\n\n(1) leave-one-out will create a lot more cases, and (2) healthy donor samples are more homogenous than CLL and FL cases. Thus we use a 3-fold validation technique for the testing against the normal profile. In this experiment, we used the same 72 data sets from 36 healthy donors, 21 CLL patients, and 15 FL patients (summarized in Table 1 ). The normal profile is built by merging 12 healthy donors (3-fold) and processed by our profilebuilding algorithm. Then 60 CCR normal (from testing the 24 remaining healthy donors, 21 CLL patients, and 15 FL patients) are obtained by processing our profile-fitting algorithm. Since it is 3-fold cross validation, we then have 180 CCRnormal (72 normal, 63 CLL, and 45 FL). The CLL profile is built by every CLL subject (leave-one-out) and processed by our profile-building algorithm. Then 72 CCR CLL (from testing 36 healthy donors, 20 remaining CLL patients, and 15 FL patients) obtained by processing our profile-fitting algorithm. Since it is leave-one-out cross validation, we then have 1491 CCR CLL (756 normal, 420 CLL, and 315 FL). Doing similar processes for FL patients, we then have 1065 CCRFL (540 normal, 315 CLL, and 210 FL). Averaging the CCR produces the results shown in Table 2. The average of the 108 CCR normal is 69. 9%, which means 69. 9% of B cells are inside the normal ellipsoid/ profile for healthy donors. In other words, 69. 9% of the B cells can be captured by our normal profile. The CLL profile has a 39. 5% capture rate for CLL cases, and the FL profile has a 65. 7% capture for FL cases. For each test case, we obtained three cell capture rates (CCR Normal, CCR CLL, and CCR FL ). By applying the diagnosis decision formula, our system decides which profiles fit better. Based on the cross validation, there are 54,810 test cases and the result is shown in Table 3. Our system can identify 80. 7% of healthy donors from all the healthy donors, 61. 1% of CLL patients among all the CLL patients, and 64. 5% of FL patients among all the FL patients. \n\nSince we adopted the leave-one-out approach for building for the CLL and FL profiles, some of the cancer patients fit the profile better than others. A more carefully selected profile is needed to improve the accuracy of the diagnosis, which is discussed in the next section.",
"section_name": "Multi-profile testing with cross validation",
"section_num": null
},
{
"section_content": "As mentioned previously, there is no need to pre-select healthy donors to build the normal profile since healthy donors' samples are fairly consistent in composition. To choose a better ellipsoid to represent the CLL, we used the distance between the center of cluster 3 to 1 (or 2) as our selecting criteria in Figure 7a and 7b. We selected approximately 15% of the CLL cases that have a closer value to the mean of the distance. For FL (Figure 8 ), we will perform the same process to pre-select 15% of FL data for our training cases. The CLL and FL profiles are built by merging the training cases. \n\nIn this experiment, we used 80 data sets from 44 healthy donors, 21 CLL patients, and 15 FL patients (see Table 4 ). We used the pre-selected 15% of data to build the profile, and tested the remaining 68 cases. In Figure 12, the average CCR shows a higher value than the average data listed in Table 2. For each test case, we obtained three CCRs. We plotted the test cases using the three CCRs and they clustered in three regions in 3-D space (Figure 13 ). Because the CCR of the matched profile gave a much higher value than the unmatched ones, most of the test cases were very close to the axis representing the corresponding profile. The final result is shown in Table 5. Our system successfully identified 36 out of 37 normal cases, 18 out of 18 CLL cases, and 12 out of 13 FL cases.",
"section_name": "Multi-profile testing with a data selection strategy for profile building",
"section_num": null
},
{
"section_content": "As a proof-of-concept study, we have demonstrated a multi-profile B lymphocyte neoplasm analysis methodology to automate the detection of certain types of B lymphocyte neoplasms by FC. A profiling method was described that characterized both the healthy donors and patients with different types of B-lymphocyte neoplasms. A CCR was defined to measure the fitness of a test case against the profile. We have demonstrated that one can obtain an automated diagnosis of CLL and FL by examining the CCRs of a test case against all three profiles. Although we only looked at FL and CLL in this study, this novel 3-D 5-parameter detection system should be capable of identifying other types of B The table shows the number of cases used to build profiles (7 cases for building normal profile, 3 cases for building CLL profile, and 2 cases for building FL profile). The remaining cases are used for testing purpose. \n\nlymphocyte neoplasms. Moreover, since the analysis is computational, it is possible to track FC data for monitoring disease progression of a lymphoma patient. Additionally, this 3-D 5-parameter detection system provides a novel way for pathologists to interpret FC data. Instead of manually gating on numerous 2-parameter plots, they can analyze 5-parameters in a 3-D image that can be rotated and viewed from various angles. This would allow them to see small clusters of cells that may be obscured in a 2-D image. In this way the 3-D 5-parameter detection system has the potential to improve a process that is labor-intensive, time-consuming, and subject to human error through automation and improved data interpretation. \n\nThis article is an expanded paper previously presented at the 2012 IEEE 2nd International Conference on Computational Advances in Bio and Medical Sciences (ICCABS) [37]. We expanded the preliminary result presented at the ICCABS conference and added the following new components. 1. Detail algorithms of our method: In the ICCABS paper, we only included the brief descriptions of building profiles and using the profile to test a new subject. In our current submission, we have included the detail steps of the Profile Building Algorithm and Fitting Algorithm. We presented a new overview of the methodology which gives a better explanation of the system, and we used box plots to compare the cell capture rate of using various profiles. This gives reader a better understanding of the distribution of the CCRs.",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This project was supported in part by NIH grants R01CA151955 (YZ) and R33CA173382 (YZ).",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. \n\nAuthors' contributions SH and CC conceived the idea of a computational model of processing the flow cytometry data. YZ proposed the 3-D 5-parameter data model and draft the manuscript. RD prepared the flow cytometry data collected at the Methodist Hospital under an approved IRB protocol and helped with the manuscript writing. MS and SH conceived and designed the experiment, analyzed the data and wrote the paper. All authors read and approved the final manuscript.",
"section_name": "Competing interests",
"section_num": null
}
] |
10.3390/cells9071600
|
Loss of ZC4H2 and RNF220 Inhibits Neural Stem Cell Proliferation and Promotes Neuronal Differentiation
|
<jats:p>The ubiquitin E3 ligase RNF220 and its co-factor ZC4H2 are required for multiple neural developmental processes through different targets, including spinal cord patterning and the development of the cerebellum and the locus coeruleus. Here, we explored the effects of loss of ZC4H2 and RNF220 on the proliferation and differentiation of neural stem cells (NSCs) derived from mouse embryonic cortex. We showed that loss of either ZC4H2 or RNF220 inhibits the proliferation and promotes the differentiation abilities of NSCs in vitro. RNA-Seq profiling revealed 132 and 433 differentially expressed genes in the ZC4H2−/− and RNF220−/− NSCs, compared to wild type (WT) NSCs, respectively. Specifically, Cend1, a key regulator of cell cycle exit and differentiation of neuronal precursors, was found to be upregulated in both ZC4H2−/− and RNF220−/− NSCs at the mRNA and protein levels. The targets of Cend1, such as CyclinD1, Notch1 and Hes1, were downregulated both in ZC4H2−/− and RNF220−/− NSCs, whereas p53 and p21 were elevated. ZC4H2−/− and RNF220−/− NSCs showed G0/G1 phase arrest compared to WT NSCs in cell cycle analysis. These results suggested that ZC4H2 and RNF220 are likely involved in the regulation of neural stem cell proliferation and differentiation through Cend1.</jats:p>
|
[
{
"section_content": "During mammalian embryonic development, the multipotent neural stem/precursor cells (NSCs/NPCs) undergo sequential differentiation to form the nervous system. The NSCs may self-renew, differentiate, or remain quiescent [1], depending on the intrinsic and external signaling environment. In the NSCs/NPCs, cell cycle exit and cell differentiation have to be coordinated to generate the appropriate number of different neurons. Some basic helix-loop-helix (bHLH) proneural factors have been shown to promote cell cycle exit and specific neuronal differentiation [2]. A number of studies have indicated that the changes in cell cycle length of neural stem/precursor cells can influence their cell fate and that lengthening of the G0/G1 phase is responsible for the shift from symmetric/proliferative pattern toward an asymmetric/neuron-generating pattern [3]. The lengthening of the G0/G1 phase might increase the levels of the proneural factors that are specifically expressed at this phase, thereby promoting neurogenesis [2]. \n\nIn humans, mutations in ZC4H2, a zinc-finger nuclear factor, have been reported to cause various clinical phenotypes, including arthrogryposis multiplex congenita, intellectual disability, epilepsy, spasticity, hypotonia, etc., which are now referred to as ZC4H2-associated rare disorders (ZARD) [4] [5] [6] [7] [8] [9]. Mechanistically, ZC4H2 has been suggested to be involved in neural development and dendritic spine density [4, 9]. Recently, we and other groups showed that ZC4H2 works as a co-factor to stabilize RNF220, an ubiquitin E3 ligase involved in many neural developmental processes [10, 11] via different mechanisms. During ventral spinal cord patterning, RNF220 and ZC4H2 are both required for proper development of the V1 and V2 interneurons, where they promote K63-linked polyubiquitination and nuclear exportation of Gli, thus limiting Shh signaling [11, 12]. During cerebellum development and medulloblastoma progression, RNF220 promotes Shh signaling epigenetically through targeting EED for degradation, overwriting its effects on Gli ubiquitination. During the development of the central noradrenergic neurons in the locus coeruleus, the RNF220/ZC4H2 complex targets Phox2a/2b for monoubiquitination, which is required for their full activities [13]. However, whether ZC4H2 and RNF220 are involved in neural stem cell proliferation and differentiation remains largely unknown. \n\nHere, we established mouse neural stem cells from wild type (WT), ZC4H2 -/-, and RNF220 -/- mouse cortex and analyzed their mRNA expression profiling. We found that ZC4H2 and RNF220 likely regulate NSC proliferation and differentiation through modulating the expression of Cend1, a key regulator of cell cycle exit and differentiation of neuronal precursors.",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "The ZC4H2 -/-and RNF220 -/-mice were used and genotyped as previously reported [11, 12]. NSCs were prepared from E14. 5 embryonic cortex using the methods as previously described [14], and the NSCs were cultured in a non-coated 6 cm plate with proliferation medium containing DMEM/F12 (Life Technologies, Carlsbad, CA, USA), N2 (Life Technologies), B-27 (Life Technologies), EGF (20 ng/mL; Peprotech, Rocky Hill, NJ, USA), bFGF (20ng/mL; Peprotech), Heparin (Stemcell, Vancouver, Canada), and 100× penicillin/streptomycin [14]. Neurospheres were allowed to form after 4-7 days of culture, which was considered passage 0. The size of neurospheres was assessed and the results shown are representative of multiple independent litters. The neurospheres were passaged 2-3 times using Accutase (Life Technologies) digestion [1, 15, 16]. For adhered culture, the NSCs were seeded in 6 cm plates coated with poly-L-ornithine and laminin.",
"section_name": "Isolation and Culture of NSCs",
"section_num": "2.1."
},
{
"section_content": "The NSCs were collected and total RNA was extracted using Trizol (TianGen, Beijing, China) reagent; the cDNA was synthesized using a PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan) according to the manufacturer's instructions. RT-qPCRs were performed with specific primers (Table 1 ) and the PCR Master Mix (Roche, Basel, Switerland) on QuanStudio 3 Applied Biosystems (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels were calculated using the 2 -∆∆Ct method on the basis of β-actin for normalization.",
"section_name": "Real-Time Quantitative PCR (RT-qPCR)",
"section_num": "2.2."
},
{
"section_content": "The NSCs plated in the chamber were fixed with 4% paraformaldehyde for 20 min, then washed three times with PBS. After having been permeabilized with 0. 5% Triton X-100 for 15 min and blocked with 5% normal goat serum for 1 h, the chambers were sequentially incubated with respective primary antibodies overnight at 4 • C, and then DAPI (4-6-diamidino-2-phenylindole), or DyLight 488-or DyLight 555-labeled secondary antibodies (1:400, Thermo Fisher) were added for 1. 5h at room temperature. Stained cells were visualized and imaged using a laser scanning confocal microscope (Olympus, Tokyo, Japan). Primary antibodies used in this study are as follows: rabbit anti-Ki67 (Abcam, Cambridge, USA, Cat# ab15580), mouse anti-Nestin (Abcam, Cat# ab6142), rabbit anti-β-tubulin III (Biolegend, San Diego, CA, USA, Cat# PRB-435P), mouse anti-MAP2 (Sigma-Aldrich, St. Louis, MO, USA, Cat# M9942), and goat anti-Sox2 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-17320). The plated cells in chambers were processed for EDU staining using the Click-iT EdU Cell Proliferation Kit according to the manufacturer's protocol. The numbers of TUJ1+, Ki67+, Edu+, and MAP2+ cells were quantified with ImageJ software.",
"section_name": "Immunofluorescence Staining",
"section_num": "2.3."
},
{
"section_content": "Approximately 8000-10,000 NSCs per well were seeded onto the laminin-and poly-L-orthenine-coated chambers containing proliferation medium for the first 12 h. Then, the cells were subjected to differentiation medium (50% Neural Basal, 50% DMEM-F12/Glutamax, 1× N2, 1× B27 without vitamin A, 0. 075% BSA, 0. 1 mM nonessen amino acids with the addition of 200 M ascorbic acid, 2 M db-cAMP, 20 ng/mL BDNF, 20 ng/mL GDNF, and 100× penicillin/streptomycin) [17]. The medium was changed every 2-3 days. Specific markers were analyzed after differentiation.",
"section_name": "Neural Stem Cell Differentiation",
"section_num": "2.4."
},
{
"section_content": "Harvested NSCs were washed twice in PBS and fixed in ice-cold 75% ethanol for 48 h. The cells were then washed twice with cold PBS and incubated in PBS mixed with 50 µg/mL propidium iodide (Sigma) and 20 µg/mL RNase A for 30 min at 37 • C. The samples were run on a flow cytometry (BD, LSR Fortessa, San Jose, CA, USA), and the data were analyzed using FlowJo VX software.",
"section_name": "FACS and Cell Cycle Analysis",
"section_num": "2.5."
},
{
"section_content": "The NSCs were lysed in RIPA (CWBIO, Beijing, China) lysis buffer, and the total proteins were fractionated by 8-15% SDS-PAGE (polyacrylamide gel electrophoresis). The membranes were blocked with 5% BSA. Before incubation with secondary antibody, membranes were incubated with the following primary antibodies: rabbit anti-RNF220 (Sigma-Aldrich, Cat# HPA027578), rabbit anti-ZC4H2 (Sigma-Aldrich, Cat# HPA049584), mouse anti-Sin3B (Santa Cruz, Cat# sc-13145), rabbit anti-Cend1 (Abcam, Cat# ab113076), rabbit anti-Neurogenin1 (Abcam, Cat# ab66498), mouse anti-p53 (Abcam, Cat# ab26), rabbit anti-Hes1 (Abcam, Cat# ab108937), rabbit anti-Notch1 (Abcam, Cat# ab52627), mouse anti-Six3 (Rockland, Cat# 35944), mouse anti-Neuritin (Santa Cruz, Cat# sc-365538), rabbit anti-Ascl1 (BD Pharmingen, Cat# 556604), rabbit anti-CyclinD1 (Cell Signaling Technology, Danvers, MA, USA, Cat# 2922S), mouse anti-CyclinB1 (Cell Signaling Technology, Cat# 4135S), rabbit anti-P21 (Abcam, Cat# ab109199), and mouse anti-α-tubulin (ProteinTech, Rosemont, IL, USA, Cat# 11224-1-AP). Band intensities were quantified using ImageJ software and normalized to α-tubulin.",
"section_name": "Western Blot Analysis",
"section_num": "2.6."
},
{
"section_content": "All cellular experiments were repeated at least three times. GraphPad 5. 0 software was used for statistical analysis. Comparisons were performed using the two-taileds Student's t-test. Probabilities of p < 0. 05 were considered significant (* p < 0. 05; ** p < 0. 01; *** p < 0. 001).",
"section_name": "Statistical Analysis",
"section_num": "2.7."
},
{
"section_content": "Sequencing libraries were generated and sequenced by BGI-Genomis on an Illumina platform, and 150 base-pair paired-end reads were generated. The raw sequencing reads were first processed by Trimmomatic (version 0. 38) software [15] to remove the adapter sequences and low-quality sequences using the following parameters \"LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36\". After reads filtering, the clean reads were aligned to the mouse reference genome GRCm38 (https: //www. ncbi. nlm. nih. gov/grc/mouse) using STAR (version 2. 6. 0c) [18]. Next, the aligned reads in bam format generated by STAR were subjected to featureCounts function of the Subread package (version 1. 5. 1) [19] to assign and count the uniquely mapped fragments to genes using the annotation file of GRCm38. We used the rlogTransformation function of R package DESeq2 [20] to normalize and scale the reads counts. Principle component analysis (PCA) and differential expression analysis were performed using the DESeq2 R package [20] on the basis of the count table. p-values were adjusted (p. adjust) by using the Benjamini and Hochberg (BH) method. Genes were identified as differentially expressed between different cells if the adjusted p-value < 0. 05. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using R package clusterProfiler [21] with the differentially expressed genes as input.",
"section_name": "RNA Sequencing and Processing of RNA-Seq Data",
"section_num": "2.8."
},
{
"section_content": "The RNA-sequencing data were submitted to the National Genomics Data Center (NGDC) under accession number CRA002818.",
"section_name": "Accession Numbers",
"section_num": "2.9."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "NSCs were isolated from E14. 5 mouse embryonic cortex of WT (wild type) or ZC4H2 -/- (ZC4H2-KO) littermates. The NSCs formed classical neurospheres in the uncoated dish and expressed the NSC markers Nestin and Sox2 (Supplementary Figure S1 ). We checked whether loss of ZC4H2 affected NSC proliferation. The neurospheres formed by WT NSCs were bigger than that of ZC4H2 -/- NSCs (Figure 1A-C ) under proliferation culture conditions, suggesting slower proliferation of the ZC4H2 -/-NSCs. Indeed, the ZC4H2 -/-NSCs showed lower EDU incorporation ratio and Ki67 positive ratio than WT NSCs (Figure 1D-I ). Under the differentiation condition, the NSCs were efficiently induced to differentiate into neurons, as indicated by the expression of the neuronal markers β-tubulin III (TUJ1) (Supplementary Figure S1 ). However, the proportion of differentiated MAP2-positive and TUJ1-positive cells increased in the ZC4H2 -/-group (Figure 1J-O ) relative to the WT NSCs. The expression levels of TUJ1 and MAP2 mRNA were upregulated after NSC differentiation in ZC4H2 -/-NSCs compared with WT NSCs (Figure 1R, S ). On the contrary, the stem cell markers (Nestin and Vimentin) had a lower expression level in the ZC4H2 -/-NSCs than in WT NSCs (Figure 1P, Q ). ZC4H2 has been suggested to stabilize the ubiquitin E3 ligase RNF220 during neural patterning [11]. To explore whether ZC4H2 works through RNF220 in NSCs, we established and characterized the RNF220 -/-NSCs. Like the ZC4H2 -/-NSCs, the RNF220 -/-NSCs also showed a lower proliferation rate and higher neural differentiation rate than WT NSCs (Figure 2A-L ). Similar changes were found for the expression levels of the stemness marker genes (Nestin and Vimentin) and the neuronal marker genes (TUJ1 and MAP2) under differentiation conditions in the RNF220 -/-NSCs (Figure 2M-P). \n\nTaken together, our results indicated that both ZC4H2 and RNF220 are required for proper neural stem cell proliferation and differentiation. ZC4H2 has been suggested to stabilize the ubiquitin E3 ligase RNF220 during neural patterning [11]. To explore whether ZC4H2 works through RNF220 in NSCs, we established and characterized the RNF220 -/-NSCs. Like the ZC4H2 -/-NSCs, the RNF220 -/-NSCs also showed a lower proliferation rate and higher neural differentiation rate than WT NSCs (Figure 2A-L ). Similar changes were found for the expression levels of the stemness marker genes (Nestin and Vimentin) and the neuronal marker genes (TUJ1 and MAP2) under differentiation conditions in the RNF220 -/-NSCs (Figure 2M-P ). \n\nTaken together, our results indicated that both ZC4H2 and RNF220 are required for proper neural stem cell proliferation and differentiation.",
"section_name": "ZC4H2 and RNF220 are Required for Proper NSC Proliferation and Differentiation",
"section_num": "3.1."
},
{
"section_content": "Through the RNA-Seq analysis, 132 genes (100 upregulated and 32 downregulated) were shown to be differently (p-adjust <0. 05) expressed in WT NSCs versus ZC4H2 -/-NSCs (Supplementary Figure S2A ). In GO analysis, these differentially expressed genes (DEGs) were enriched in the biological processes of axon functions, neural precursor cell proliferation, telencephalon development, and so on (Figure 3A ). These genes were indicated as being involved in different terms of CC (cellular component) and MF (molecular function) (Figure 3A ), such as component of synaptic membrane, extracellular matrix, and transmitter-gated channel activity (Figure 3A ). In KEGG pathway analysis, the most significantly enriched pathways included the PI3K-Akt signaling pathway, neuroactive ligand-receptor interaction, ECM-receptor interaction, and GABAergic and glutamatergic synapse (Figure 3B ). These data strongly suggested an active role of ZC4H2 in neural development or function. 3. 2. RNA Profiling of the ZC4H2 -/-and RNF220 -/-NSCs Through the RNA-Seq analysis, 132 genes (100 upregulated and 32 downregulated) were shown to be differently (p-adjust < 0. 05) expressed in WT NSCs versus ZC4H2 -/-NSCs (Supplementary Figure S2A ). In GO analysis, these differentially expressed genes (DEGs) were enriched in the biological processes of axon functions, neural precursor cell proliferation, telencephalon development, and so on (Figure 3A ). These genes were indicated as being involved in different terms of CC (cellular component) and MF (molecular function) (Figure 3A ), such as component of synaptic membrane, extracellular matrix, and transmitter-gated channel activity (Figure 3A ). In KEGG pathway analysis, the most significantly enriched pathways included the PI3K-Akt signaling pathway, neuroactive ligand-receptor interaction, ECM-receptor interaction, and GABAergic and glutamatergic synapse (Figure 3B ). These data strongly suggested an active role of ZC4H2 in neural development or function. \n\nSimilarly, RNA-Seq analysis of the RNF220 -/-and WT NSCs identified 433 DEGs (192 upregulated and 241 downregulated; p < 0. 05) (Supplementary Figure S2B ). In GO analysis, these genes were mostly enriched in the processes, including pattern specification, embryonic skeletal system development, cell fate commitment, extracellular matrix, transcription factor activity, and so on (Figure 4A ). In KEGG pathway analysis, the downregulated genes in RNF220 -/-NSCs were suggested as being involved in ECM-receptor interaction, focal adhesion, and calcium signaling pathways (Figure 4B ), whereas no clear enrichment was found among the upregulated genes. \n\nUnexpectedly, only 24 of the DEGs between RNF220 -/-and WT NSCs were found to be shared with those between ZC4H2 -/-and WT NSCs (Figure 5A, Table 2 ). Among them, Sp8, Nrn1, Cend1, Six3, SLC6A13, and Ngfr (Table 2 ) were reported to be actively involved in the regulation of NSC proliferation/differentiation. The limited overlapping of affected genes between NSCs loss of ZC4H2 and RNF220 was compatible with the speculation for RNF220-independent roles of ZC4H2 in NSCs.",
"section_name": "RNA Profiling of the ZC4H2 -/-and RNF220 -/-NSCs",
"section_num": "3.2."
},
{
"section_content": "In order to understand the underlying regulatory role of ZC4H2/RNF220, we focused on the group of potential common targets of ZC4H2 and RNF220. Among these DEGs that were identified by the above RNA-Seq, Cend1, Nrn1, and Six3 have been reported to promote neuronal development and neurogenesis [23] [24] [25] [26] [27]. We further confirmed the upregulation of Cend1, Nrn1, and Six3 by real-time PCR and Western blot (Figure 5B -H and Figure S3A, B ). The neuronal lineage-specific protein Cend1 has been shown to promote cell cycle exit and neuronal differentiation during neurogenesis through regulating the p53-CyclinD1 and Notch1 signaling pathways [23, 28, 29]. The protein and mRNA level of CyclinD1 was downregulated both in ZC4H2 -/-and RNF220 -/-NSCs, whereas those of p53 and p21 were elevated (Figure 6A,G-L, and Supplementary Figure S3C, D ). The expression of CyclinB1 remained unchanged (Figure 6B,M,N and Figure S3E-F ). We characterized the ZC4H2 -/-and RNF220 -/-NSC cell cycle transition by flow cytometry analysis, and found that loss of ZC4H2 or RNF220 increased the ratio of G0/G1 cell population and decreased the ratio of S cell population (Figure 6O -T), which is consistent with a lower EDU incorporation ratio in ZC4H2 -/-and RNF220 -/-NSCs (Figure 1D -F and Figure 2A-C ). In addition, the mRNA and protein levels of Notch1 and Hes1 were downregulated in both ZC4H2 -/-and RNF220 -/-NSCs (Figure 6A,C-F and Supplementary Figure S3C, D ). These data were consistent with the model wherein the RNF220/ZC4H2 complex likely regulates neurogenesis through the Cend1/p53-CyclinD1/Notch cascade.",
"section_name": "ZC4H2/RNF220 Regulate Cend1 Expression During NSC Proliferation",
"section_num": "3.3."
}
] |
[
{
"section_content": "Funding: This work was supported by grants from the Chinese National Science foundation ( 31671521 and 31871483 to B. M) and the Bureau of Frontier Sciences and Education of Chinese Academy of Sciences ( QYZDJ-SSW-SMC005 to Y. G. Y. ).",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "In this study, we provide direct evidence that ZC4H2 and RNF220 are likely involved in the regulation of neural stem cell proliferation and differentiation through Cend1. Loss of either ZC4H2 or RNF220 inhibits the proliferation and promotes the differentiation abilities of NSCs in vitro (Figures 1 and 2 ). Through RNA-Seq analyses, we identified Cend1 as a key molecule that is upregulated in both ZC4H2 -/-and RNF220 -/-NSCs (Figure 5B-D ). Cend1 is a key regulator of proliferation and differentiation of neural precursors, and overexpression of Cend1 promotes cell cycle exit at the G0⁄G1 transition point and differentiation of neuron [28]. Cend1 triggers p53 and its downstream effector p21, whereas it downregulates CyclinD1, resulting in withdrawal from the cell cycle at the G0/G1 transition. Indeed, the expression of p53 and p21 were upregulated and that of CyclinD1 downregulated in both ZC4H2 -/-and RNF220 -/-NSCs. In neuronal precursors, Cend1 has been reported to suppress Notch signaling and its expression is regulated by proneural genes such as Neurogenin1 (NEUROG1) and Ascl1 [23, 29, 30]. In NSCs, in terms of loss of ZC4H2 or RNF220, we did observe downregulation of the expression of Notch1 and its target Hes1. However, no clear changes were found for the expression of Neurogenin1 and Ascl1 in the NSCs (Figure 6B and Supplementary S3E,F), which suggested that other proneural genes might be involved in this process. \n\nHow RNF220/ZC4H2 act to regulate Cend1 expression remains unknown. ZC4H2 has been reported as a key regulator of RNF220 stability [11]. In the NSCs, loss of ZC4H2 did reduce RNF220 protein level (Figure 5B and Supplementary Figure S3A, B ), but not mRNA level (Figure 5I, J ). It is reasonable that RNF220/ZC4H2 regulate Cend1 expression through a common target. Although loss of RNF220 and ZC4H2 in NSCs both lead to elevated Shh signaling [11, 12], it is less likely to be responsible for the observed phenotype, since Gli signaling is well established as promoting cell proliferation. Another RNF220 target, Sin3B [31], is elevated only in RNF220 -/-NSCs, but not in ZC4H2 -/-NSCs (Figure 6B and Supplementary Figure S3E, F ). In cerebellar granule neuron progenitors and medulloblastoma cells, RNF220 targets EED for degradation and promotes Shh signaling epigenetically [32]. However, RNF220 does not interact with EED in NSCs [32]. Thus, the direct target of ZC4H2/RNF220 involved in Cend1 regulation remains to be explored. \n\nInterestingly, during rhesus monkey embryonic stem cell (rESC) neural differentiation [33], through analysis of the reported data [33], we found that ZC4H2 had similar expression dynamics of RNF220 from ESCs to early rosette neural stem cells (R-NSCP1) and late (R-NSCP6) passages (Supplementary Figure S4 ), suggesting that ZC4H2 and RNF220 might have closely related function in NSCs in early development. However, from R-NSCP6 to neural progenitor cell (NPC) differentiated stage, ZC4H2 and RNF220 have opposite expression dynamics (Supplementary Figure S4 ), and in our study, the DEGs identified in the ZC4H2 -/-and RNF220 -/-NSCs were largely non-overlapping, which might suggest RNF220-independent roles of ZC4H2 in NSCs. Indeed, the PI3K-Akt signaling pathway seems to be affected in the ZC4H2 -/-but not the RNF220 -/-NSCs (Figures 3 and 4 ). How ZC4H2 works in this context awaits further analysis. \n\nIn short, we characterized the role of ZC4H2/RNF220 in the proliferation and differentiation of NSCs, and identified Cend1 as a potential mediator for the signaling.",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "The following are available online at http://www. mdpi. com/2073-4409/9/7/1600/s1, Figure S1 : Isolation of NSCs from wild type E14. 5 mouse cortex. Figure S2 : Heat map analysis of the differentially expressed genes between WT and ZC4H2 -/-NSCs and WT and RNF220 -/-NSCs. Figure S3 : Quantification of Western blot data. Figure S4 : Re-analysis of the expression patterns of ZC4H2 and RNF220 from rhesus monkey embryonic stem cells (ESCs) to rosette neural stem cells (R-NSCs) at early (R-NSCP1) and late (R-NSCP6) passages, and neural progenitor cells (NPC). Supplementary Material 1: DEGs of RNA-Seq and rhesus monkey ESC-NPC FPKMs. Supplementary Material 2: GO annotations and KEGG enrichment.",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest.",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.1590/fst.90721
|
Role of oleanolic acid in relieving psoriasis and its underlying mechanism of action
|
Abstract The present study aimed to determine whether oleanolic acid (Ole) could be used to treat psoriasis and its related underlying mechanism of action via in vitro analysis. HaCaT cells were stimulated with IL-22 to established an in vitro psoriasis cell model. MTT, flow cytometry and TUNEL assays, respectively. Transmission electron microscopy was used to observe the cell ultrastructure. LC3B protein expression levels were analyzed using immunofluorescence, other protein expression levels were determined using western blotting. Cell viability was significantly increased, while the apoptotic rate was significantly decreased in the model group (P < 0.001). In addition, Notch1, Hes1, beclin 1 and LC3B protein expression levels were significantly downregulated, while P62 protein expression levels were significantly upregulated in the model group compared with the control group (P < 0.001). Supplementation of Ole, the increased levels of proliferation were significantly suppressed, while cell apoptosis was significantly increased (P < 0.05) in a dose-dependent manner, which was discovered to occur via Notch1 upregulation. Notably, the transfection with small interfering RNA-Notch1 significantly reversed the effect of Ole treatment (P < 0.001) and the levels of autophagy were also decreased. In conclusion, the findings of the current study suggested that Ole may relieve psoriasis via upregulating Notch1, which subsequently regulates cell autophagy.
|
[
{
"section_content": "Psoriasis is a common and recurring chronic inflammatory skin condition, and its prevalence among adults ranges from 0. 91 to 8. 5% in different Please clarify, are you referring to different countries and ethnic (Griffiths et al. 2017). However, the exact pathogenesis of psoriasis remains unclear. At present, studies have confirmed that the onset of psoriasis is closely associated with autoimmunity and inflammation, and the excessive proliferation of keratinocytes and parakeratosis are key factors underlying its pathogenesis (Perera et al. 2012). Autophagy is a process of self-phagocytosis in cells that occurs when cells are under stress conditions, such as those of nutrient deprivation or infection (Jo, et al. 2012). It was previously reported that oleanolic acid (Ole), which is an extensively used plant-derived triterpenoid, exerted effective antioxidant effects in cell model (Guo et al. 2020). In an in vitro antioxidant activity evaluation model, Ole was discovered to clear free radicals and inhibit reactive oxygen species (ROS) generation through chemical reactions, or by inhibiting lipid peroxidation or stimulating the cellular antioxidant defense mechanisms (Wang et al. 2020 ). In addition, Ole has been demonstrated to possess hepatoprotective, antiinflammatory, anticancer and other pharmacological properties (Zolnourian et al. 2019, Zhang et al. 2019a ). Moreover, Ole was discovered to exert therapeutic benefits in liver cancer (Zhou et al. 2020), diabetes-induced cell injury (Chen et al. 2019 ) and bladder cancer (Song et al. 2017 ) via regulation of autophagy. Kim et al. (Varshney and Saini, 2018) found autophagy was correlation with psoriasis. However, to the best of our knowledge, the effects of Ole in psoriasis remain unknown. \n\nPrevious studies have reported that the levels of autophagy in psoriasis skin lesions were lower compared with those in the normal skin in vitro study (Haruna, et al. 2008) ; blocking autophagy could significantly increase the secretion of inflammatory factors in human epidermal cells (Lee et al. 2011) ; and increasing the levels of autophagy in psoriasis skin lesions, mainly via regulation of hes family bHLH transcription factor 1 (Hes1), beclin 1, LC3B and P62 protein expression (Zhang et al. 2019b ), may represent a potential therapeutic strategy for psoriasis. However, the effects of Ole on the autophagy of human keratinocytes remain unclear. Therefore, the present study aimed to investigate the effect of Ole on the autophagy of HaCaT cells and to determine the related underlying mechanism of action.",
"section_name": "Introduction",
"section_num": "1"
},
{
"section_content": "Reagents and antibodies. Ole (cat. no. O5504; mass fraction, ≥ 97%), DMSO (cat. no. V900090) and MTT reagent (cat. no. V900888) were purchased from Sigma-Aldrich (Merck KGaA); the HaCaT cell line was purchased from the American Type Culture Collection; EDTA-free pancreatin, DMEM and FBS were purchased from Gibco (Thermo Fisher Scientific, Inc. ); the rabbit anti-LC3B",
"section_name": "Materials and methods",
"section_num": "2"
},
{
"section_content": "Yan LIU 1 * , Dong-Mei YAN 2, Li-Li DENG 1, Yan-Jun ZHU 1, Cai-Yun BIAN 1, Hui-Ru LV 3 a monoclonal antibody was obtained from Abcam, cat. ab239416, UK; the rabbit anti-P62 monoclonal antibody was purchased from Abcam, cat. ab91526, UK; the HRP-conjugated goat anti-rabbit secondary antibody was acquired from Santa Cruz Biotechnology, Inc. ; the anti-Notch1 (cat. no. ab52301), antibeclin 1 (cat. no. ab217179), anti-Hes1 (cat. no. ab119776) and anti-GAPDH (cat. no. ab8245) antibodies were obtained from Abcam; and the TUNEL Apoptosis Staining kit was purchased from Beyotime Institute of Biotechnology (cat. no. C1090). \n\nCell culture. HaCaT cells (which were authenticated via STR profiling) were cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Thermo Fish, Waltham, MA, USA), and maintained in an incubator with 5% CO 2 at 37 °C. Cells in the logarithmic growth phase were collected and used in subsequent experiments. \n\nEstablishment of the psoriasis cell model. A psoriasis cell model was established as previously described (Wu et al. 2020). Briefly, upon the confluence reaching 60-70%, HaCaT cells were cultured in serum-free DMEM for 12 h, then stimulated with 100 ng/mL IL-22 for 12 h to simulate the proliferation of the cells. Cell transfection. Cells in the logarithmic growth phase were seeded into a 6-well microplate at a density of 2x10 5 cells/ well. Upon reaching 60% confluence, cell transfection was performed in accordance with the manufacturer's instructions. Small interfering RNA (si)-negative control (NC) (sense, 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense, 5'-ACGUGACACGUUCGGAGAATT-3') and si-notch 1 receptor (Notch1; sense, 5'-UGGACAAGAUCGAUGGCUATT-3' and antisense, 5'-UAGCCAUCGAUCUUGUCCATT-3') were designed and synthesized by Nanjing KeyGen Biotech Co., Ltd (KG-D-19060715). Following transfection, the cells were further incubated in an incubator containing 5% CO 2 at 37 ˚C ready for use in subsequent experiments. The cell transfection efficiencies were shown in Supplementary Figure 1.",
"section_name": "Role of oleanolic acid in relieving psoriasis and its underlying mechanism of action",
"section_num": null
},
{
"section_content": "MTT assay. Cells in all groups were treated by difference methods for 48 then 20 μL MTT was added to each well for further incubation for 4 h. Following incubation, the medium was discarded and 100 μL DMSO was added to each well. After the mixture had been centrifuged as 8,000 x g at 4 °C for 10 min, the absorbance of each well was measured at a wavelength of 570 nm using an ELISA microplate reader. The assay was repeated three times to calculate the proliferative rate of the cells. \n\nFlow cytometric analysis of apoptosis. Cells in all groups were treated by difference methods for 48, then digested with EDTA-free pancreatin. The cells were washed with pre-cooled PBS buffer solution twice and centrifuged at 1,000 x g for 5 min to collect 1x10 5 cells. Subsequently, 5 μL Annexin V-FITC and PI were added to the cells and mixed, following which the cells were incubated in the dark at room temperature for 5-10 min. Apoptotic cells were analyzed within 1 h using a flow cytometer at an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The early apoptotic cells in the lower right quadrant and the late apoptotic cells in the upper right quadrant were counted as apoptotic cells. The apoptotic rate was calculated as the number of apoptotic cells/total number of cells. The analysis was repeated three times. \n\nTUNEL assay. Cells in all groups were treated accordingly for 48 h, then washed with PBS three times. The TUNEL assay was performed using a TUNEL Apoptosis staining kit, according to the manufacturer's protocol. After staining, the cells were sealed with anti-fluorescence quenching sealing solution containing DAPI and visualized using a fluorescence microscope in three highly magnified (x200) randomly selected fields of view, with each field containing ≤ 100 cells. The apoptotic rate of HaCaT cells was calculated using the following equation: Apoptotic rate (%) = FITC-positive cells/100 x100%. The average apoptotic rate was obtained from three fields. The assay was repeated in triplicate.",
"section_name": "Preparation of",
"section_num": null
},
{
"section_content": "Autophagosome detection was performed using TEM. Briefly, cells in each group were treated accordingly for 48 h and then washed with PBS thrice. Cells were subsequently collected and centrifuged at 1,200 x g for 5 min. The supernatant was discarded and cells were fixed with 4% glutaraldehyde overnight. Following incubation, the cells were rinsed, dehydrated and embedded, then cut into ultra-thin slices, which were observed under a transmission electron microscope and photographed. \n\nWestern blotting. Cells in each group were treated accordingly for 48 h and then washed with PBS thrice. Total protein was extracted from the cells using RIPA lysis buffer supplemented with a protease inhibitor. The cells were centrifuged at high speed to extract the supernatant and total protein was quantified using a BCA protein assay kit (cat. no. ab102536; Abcam). Loading buffer was subsequently mixed with the protein, and boiled, following which 50 μg protein/lane was separated via SDS-PAGE. The separated proteins were transferred to PVDF membranes and blocked with 3% BSA at room temperature for 2 h. The membranes were then incubated with the following primary antibodies overnight at 4 ˚C: Anti-Notch1 (1:500), anti-Hes1 (1:500), anti-beclin 1 (1:500), anti-P62 (1:500) and anti-GAPDH (1:500). Following the primary antibody incubation, the membranes were incubated with an HRP-conjugated antirabbit secondary antibody (1:2,000; cat. no. ab6728, Abcam) for 2 h. Protein bands were visualized using chemiluminescence. The experiment was repeated three times to obtain the average protein expression. \n\nDetection of the autophagy marker protein, LC3B, using immunofluorescence. Cells in each group were treated accordingly for 48 h, washed with PBS thrice, then fixed with 4% formaldehyde at room temperature for 10 min. Subsequently, the cells were washed with PBS twice and incubated with 0. 5% Triton X•100 at room temperature for 5 min. The cells were subsequently washed with PBS twice and incubated with 5% BSA at room temperature for 1 h. Then, the cells were incubated with an anti-LC3B antibody (1:200) for 1 h at room temperature; the primary antibody was replaced with PBS for the control group. Following the primary antibody incubation, the cells were washed with PBS twice and incubated with the corresponding fluorescent-conjugated secondary antibody (1:2,000; cat. no. ab6728, Abcam). The cells were washed with PBS, counterstained with DAPI, and further washed with PBS before being sliced and sealed with glycerinum. Stained cells were observed using a fluorescence microscope in five randomly selected fields of view (magnification, x200) to count the number of autophagosome-positive cells and calculate the percentage of positive cells. Cells with more than three high-density green fluorescent spots in the perinuclear and extranuclear regions were regarded as autophagosome-positive cells. The experiment was repeated three times to calculate the average value. \n\nStatistical analysis. Statistical analysis was performed using SPSS 22. 0 software (IBM Corp. ) and data are presented as the mean ± SD. Statistical differences between groups were determined using one-way ANOVA followed by Tukey post hoc test. P < 0. 05 was considered to indicate a statistically significant difference.",
"section_name": "Transmission electron microscopy (TEM).",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3"
},
{
"section_content": "Compared with the 0 μmol/L group, the proliferative rate of HaCaT cells in the 200. 0 μmol/L group was significantly inhibited with apoptosis rate significantly up-regulating (P < 0. 001; Figure 1A, C ), while no significant differences were observed among the 25. 0, 50. 0 and 100. 0 μmol/L groups in cell viability and apoptosis rate (all P > 0. 05; Figure 1A ). Thus, it could be inferred that, in a normal environment, Ole at concentrations of 25. 0, 50. 0 and 100. 0 μmol/L, was nontoxic to HaCaT cells. A psoriasis cell model was subsequently established by stimulating HaCaT cells with 100 ng/mL IL-22 for 12 h. Compared with the control group, the proliferative rate was significantly increased in the model group (P < 0. 001; Figure 1B ). After Ole intervention, compared with the model group, the proliferative rates in the Ole groups were significantly inhibited (all P < 0. 05; Figure 1B ), and significant differences were identified in the proliferative rates among the different Ole groups (all P < 0. 05; Figure 1B ). 25. 0, 50. 0 and 100. 0 μmol/L Ole was safe to HaCaT cell in normal treatment; in HaCaT cell psoriasis model, 25. 0, 50. 0 and 100. 0 μmol/L Ole had effects to depress HaCaT hyperproliferation.",
"section_name": "Effects of different concentrations of Ole on the viability of the HaCaT cell psoriasis model.",
"section_num": null
},
{
"section_content": "The results from the flow cytometry and TUNEL staining assays revealed that, compared with the control group, the apoptotic rate was significantly decreased in the model group (all P < 0. 001; Figure 2A and B ). Following Ole intervention, compared with the model group, the apoptotic rate was significantly increased in all Ole groups (all P < 0. 05; Figure 2A and B ). Moreover, significant differences were observed in the apoptotic rates among the different Ole groups (all P < 0. 05; Figure 2A and B ). \n\nIn HaCaT cell psoriasis model, 25. 0, 50. 0 and 100. 0 μmol/L Ole had effects to promote HaCaT cell apoptosis; and 100. 0 μmol/L Ole had best effects. \n\nOle promoted the autophagy of HaCaT cell psoriasis model. The formation of autophagosomes was visualized using TEM. Autophagosomes with a bilayer membrane and containing numerous organelles and folded proteins were observed in the control group, while no autophagosomes were observed in the model group. After Ole intervention, the number of autophagosomes in all the Ole groups was increased (Figure 3 ). In HaCaT cell psoriasis model, 25. 0, 50. 0 and 100. 0 μmol/L Ole had effects to increase autophagosome number; and 100. 0 μmol/L Ole had best effects. \n\nOle regulated the expression levels of Notch1, Hes1, Beclin1 and P62 proteins in HaCaT cell psoriasis model The results of the western blotting experiment revealed that, compared with the control group, the protein expression levels of Notch1, Hes1 and beclin 1 were significantly downregulated in the model group, while the protein expression levels of P62 were significantly upregulated (all P < 0. 001; Figure 4 ). After Ole intervention, compared with the model group, the protein expression levels of Notch1, Hes1 and beclin 1 were significantly upregulated in all Ole groups, while the protein expression levels of P62 were significantly downregulated (all P < 0. 001; Figure 4 ). Furthermore, there were significant differences observed in the expression levels of these proteins among the Ole groups (all P < 0. 05; Figure 4 ). 25. 0, 50. 0 and 100. 0 μmol/L Ole improved psoriasis cell model might be correlation with Notch1, Hes1, Beclin1 and P62 proteins changing; and 100. 0 μmol/L Ole had best effects.",
"section_name": "Ole promoted cell apoptosis in HaCaT cell psoriasis model.",
"section_num": null
},
{
"section_content": "HaCaT cell psoriasis model. The results of the cell immunofluorescence analysis showed that, compared with the control group, the protein expression levels of LC3B were significantly decreased in the model group (P < 0. 001; Figure 5 ). Compared with the model group, the protein expression levels of LC3B were significantly upregulated in all Ole groups (all P < 0. 05; Figure 5 ). Furthermore, the differences in the expression levels of LC3B protein among the Ole groups were statistically significant (all P < 0. 05; Figure 5 ). 25. 0, 50. 0 and 100. 0 μmol/L Ole improved psoriasis cell model might be correlation with autophagy; and 100. 0 μmol/L Ole had best effects.",
"section_name": "Ole increased the protein expression levels of LC3B in",
"section_num": null
},
{
"section_content": "HaCaT cell viability. No significant difference in the proliferative rate was identified between the model and model + si-NC groups (P > 0. 05; Figure 6 ), while the proliferative rate in the Ole groups was significantly decreased (all P < 0. 001; Figure 6 ). Following the transfection of si-Notch1 into cells, the proliferative rate in the Ole + si-Notch1 group was significantly increased compared with that in the Ole groups (all P < 0. 001; Figure 6 ). With Notch1 knockdown, Ole's treatment effects was disappear in HaCaT cell psoriasis model.",
"section_name": "Notch1 knockdown improved the Ole-induced inhibition of",
"section_num": null
},
{
"section_content": "The results of the flow cytometry and TUNEL staining assays revealed that, compared with the model group, the apoptotic rate in the model + si-NC group was not significantly altered (P > 0. 05; Figure 7A and B ). Conversely, compared with Model group, the apoptotic rates in the Ole groups were significantly increased (all P < 0. 001; Figure 7A and B ). In addition, following si-Notch1 transfection into cells, the apoptotic rate in the Ole + si-Notch1 group was significantly decreased compared with that in the Ole groups (all P < 0. 001; Figure 7A and B ). With Notch1 knockdown, Ole's increasing cell apoptosis effects was disappear in HaCaT cell psoriasis model.",
"section_name": "Notch1 knockdown depressed the Ole-induced increased in the apoptosis of HaCaT cell psoriasis model .",
"section_num": null
},
{
"section_content": "in HaCaT cell psoriasis model. Formation of autophagosomes was observed using TEM. The results demonstrated that the number of autophagosomes in the model and model + si-NC groups was decreased (Figure 8 ). Conversely, following Ole intervention, the number of autophagosomes was increased; however, after si-Notch1 was transfected into cells, the number of autophagosomes in the Ole + si-Notch1 group was decreased. With Notch1 knockdown, Ole induced autophagosome increasing was depressed in HaCaT cell psoriasis model. Expression levels of Notch1, Hes1, Beclin1 and P62 proteins in HaCaT cell psoriasis model. Compared with the model group, the protein expression levels of Notch1, Hes1, beclin 1 and P62 in the model + si-NC group were not significantly altered (all P > 0. 05; Figure 9 ). Compared with Model group, the protein expression levels of Notch1, Hes1 and beclin 1 were significantly upregulated in the Ole groups, while the protein expression levels of P62 were significantly downregulated (all P < 0. 001; Figure 9 ). Following the transfection of si-Notch1 into cells, compared with the Ole groups, the protein expression levels of Notch1, Hes1 and beclin 1 were significantly downregulated in the Ole + si-Notch1 group, while the protein expression levels of P62 were significantly upregulated (all P < 0. 001; Figure 9 ). With Notch1 knockdown, Ole induced Notch1, Hes1, Beclin1 and P62 proteins changing was recovery in HaCaT cell psoriasis model.",
"section_name": "Notch1 knockdown downregulated the formation of autophagosomes",
"section_num": null
},
{
"section_content": "The results of the immunofluorescence analysis revealed that, compared with the model group, the protein expression levels of LC3B in the model + si-NC group were not significantly altered (P > 0. 05; Figure 10 ). Compared with Model group, the protein expression levels of LC3B were significantly upregulated in the Ole groups (all P < 0. 001; Figure 10 ). Following si-Notch1 transfection into cells, compared with the Ole groups, the protein expression levels of LC3B were found to be significantly downregulated in the Ole + si-Notch1 group (P < 0. 001; Figure 10 ). With Notch1 knockdown, Ole induced LC3B proteins changing was recovery in HaCaT cell psoriasis model.",
"section_name": "Expression levels of LC3B protein in HaCaT cell psoriasis model",
"section_num": null
},
{
"section_content": "As a natural pentacyclic triterpenoid, Ole has been reported to exert antitumor and anti-inflammatory effects (Wang et al. 2019; Abdelmageed et al. 2017), and has been shown to be effective in the treatment of multiple types of tumor (Chu et al. 2017; Dong et al. 2020; Han et al. 2021). The results of the present study revealed that Ole intervention could effectively inhibit the abnormal proliferation of HaCaT cells induced by IL-22 stimulation and promote their apoptosis, which may subsequently inhibit the development of psoriasis. Autophagy is a protective mechanism that occurs in eukaryotic cells. It removes dysfunctional and denatured, damaged organelles, macromolecules and invading microorganisms by forming autophagic lysosomes, thereby maintaining cell homeostasis and renewal (White et al. 2010; Mizushima & Komatsu, 2011). LC3B is a characteristic marker of autophagy, and the expression levels of LC3B reflect the autophagy level (Satyavarapu et al. 2018). The findings of the current study demonstrated that, following Ole intervention, the number of autophagosomes was increased, the protein expression levels of autophagy marker proteins, Hes1 (Yao et al. 2015), beclin 1 (Maejima et al. 2016 ) and LC3B (Satyavarapu et al. 2018), were significantly upregulated, and the protein expression levels of P62 (Lamark et al. 2017 ) were significantly downregulated. The highly conserved Notch gene family was first discovered in Drosophila melanogaster by Morgan et al. in 1917, and comprises four transmembrane receptors, Notch1, Notch2, Notch3 and Notch4, which are involved in regulating the viability, proliferation and differentiation of cells and the development of organs (Nantie et al. 2014; Ebens & Maillard, 2013). Previous studies have reported that the upregulated expression of Notch1 promoted the autophagy of cells (Xu et al. 2019, Lee et al. 2020). Similarly, the results of the present study also showed that the protein expression levels of Notch1 were significantly increased Ole treated groups. Therefore, it was hypothesized that Ole may play an important role in reversing the IL-22-induced excessive proliferation of HaCaT cells by increasing autophagy via upregulation of Notch1. Furthermore, the therapeutic effect of Ole was reduced after the expression of Notch1 was knocked down by transfection with si-Notch1. These findings suggested The findings of the current study also revealed that treatment with 25, 50 or 100 μmol/L Ole was non-toxic to HaCaT cells in a normal environment, but could inhibit the IL-22-induced proliferation of HaCaT cells. Moreover, the inhibition of proliferation occurred in both a time-and dose-dependent manner. Treatment with 25, 50 or 100 μmol/L Ole could also significantly increase the apoptotic rate of HaCaT cells in a dose-dependent manner. Similarly, 25, 50 or 100 μmol/L Ole was discovered to induce the autophagy of HaCaT cells. Ole treatment also upregulated the protein expression levels of Hes1 and beclin 1, and downregulated those of P62. Immunofluorescence analysis further revealed that the number of LC3B-fluorescing accumulation points was increased and TEM analysis observed the presence of autophagosomes with bilayer membranes, proving that the autophagy levels of cells were increased, while degradation of P62 was increased. However, following the transfection of si-Notch1 into cells, the effects of Ole on the cells disappeared. \n\nThere were certain limitations to the present study. First, a positive control drug group was not used. Second, only in vitro cellular studies were performed, and the effects of Ole on an imiquimod-induced psoriasis animal model were not investigated. Finally, considering that mTOR serves an important role in autophagy, the present study did not determine the association between mTOR and Notch1 during Ole treatment, in our study, we just discussed autophagy (autophagosome changing) in Ole's affects to positively cell model, the lysosomes' effects had been unclear; meanwhile, there were no Model+si-Notch1 and Ole+si-NC groups in second part of our present research. Therefore, these points will be addressed in future studies, with the effects of Ole on animal models of psoriasis being prioritized. were stimulated with IL-22 and treated with 100. 0 μmol/L Ole. ### P < 0. 001 vs. model; @@@ P < 0. 001 vs. Ole. Ole, oleanolic acid; si, small interfering RNA; NC, negative control; Notch1, notch 1 receptor. Ole + si-Notch1, HaCaT cells transfected with si-Notch1 were stimulated with IL-22 and treated with 100. 0 μmol/L Ole. ### P < 0. 001 vs. model; @@@ P < 0. 001 vs. Ole. Ole, oleanolic acid; si, small interfering RNA; NC, negative control; Notch1, notch 1 receptor. \n\nOriginal Article",
"section_name": "Discussion",
"section_num": "4"
}
] |
[
{
"section_content": "",
"section_name": "Supplementary Material",
"section_num": null
},
{
"section_content": "Supplementary material accompanies this paper. Supplementary Figure 1. Notch1 mRNA expression in difference groups NC: HaCaT cells were treated with normal medium; si-NC: HaCaT cells were transfected with si-NC; si-Notch1: HaCaT cells were transfected with si-Notch1. ***: P < 0. 001, compared with NC group This material is available as part of the online article from https://www. scielo. br/j/IDSCIELO",
"section_name": "Supplementary Material",
"section_num": null
}
] |
10.1038/s41408-021-00556-7
|
Survival of patients with chronic lymphocytic leukemia before and after the introduction of chemoimmunotherapy in Germany
|
<jats:title>Abstract</jats:title><jats:p>Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults in western countries. Therapy is indicated in symptomatic and advanced stages and has changed fundamentally since 2010 when rituximab, an anti-CD20 antibody, has been approved for treatment of CLL. Until then therapy had been based on chemotherapy drugs. This study investigates whether survival in CLL patients improved at the population level after the introduction of combined chemoimmunotherapy. Data from the cancer registry North-Rhine Westphalia was used to calculate relative survival (RS) by applying period analyses. Age-standardized 5-year RS increased from 79% in 1998–2002 (75% in 2003–2007) to 81% in the calendar period 2008–2012 and 88% in 2013–2016 for men and continuously from 71% in 1998–2002 to 92% in 2013–2016 for women. In CLL patients aged 15–69 years 5-year RS increased from 83% to 90% for men and from 82% to 94% for women after adding an anti-CD20-antibody to chemotherapy while in the older age group of 70–79-year-old CLL patients an increase by 20 percentage points was observed. These findings show marked improvements in the survival of CLL patients at the population level subsequently to the approval of anti-CD 20 antibodies like rituximab, ofatumumab or obinutuzumab for CLL treatment.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is the most common leukemic disease in western countries. The age-standardized incidence rate is four to five cases per 100,000 person-years [1, 2] and about 5500 patients in Germany are newly diagnosed with CLL annually. Men are more often affected than women. CLL is a disease of the elderly with a median age at diagnosis of 70 years. While agestandardized rates remained constant over the last 15 years, the absolute number of cases increased [1]. Hence, healthcare needs will rise further during the next years. CLL is basically diagnosed after routine blood analyses. The diagnosis requires ≥5 × 10 9 /L clonal B-lymphocytes in the peripheral blood and immunophenotyping is mandatory to confirm the CLL with coexpression of CD5 /CD19, CD20, CD23 and low levels of CD20 and CD79b. Expression of either κ or λ immunoglobulin light chains confirms the clonality [3]. \n\nThe clinical presentation of CLL is diverse and varies between an indolent and a highly aggressive course. Phase 3 trials investigating treatment in early and asymptomatic stages, could not demonstrate an advantage for overall survival [4, 5]. Standard of care for these patients remains a watch-and-wait strategy. Antineoplastic treatment is indicated in advanced stages (Binet C), if symptoms occur (organomegaly, anemia, thrombocytopenia, B-symptoms (fatigue, fever, night sweat, weight loss), or with a lymphocyte doubling time of <6 month [3]. Therapy of CLL has changed fundamentally during the last decade. Until 2009 antineoplastic treatment was based on chemotherapeutic drugs, like chlorambucil or fluradabine as single-agents or fluradabine in combination with cyclophosphamide (FC). Results of the CLL8 study showed a marked improvement of progression-free and overall survival in patients treated with a combination of FC and rituximab, a monoclonal anti-CD20 antibody, in comparison to FC alone [6]. The CLL11 protocol [7] corroborated the notion that survival benefits could be obtained even for older and less-fit patients with marked comorbidities by adding an anti-CD20antibody (rituximab or obinutuzumab) to chemotherapy (with chlorambucil). The combined chemoimmunotherapy has then rapidly become a standard regime in first-line therapy of CLL patients [8] from its introduction until recently when novel noncytostatic, targeted agents have led to another paradigm shift in the treatment of CLL, and chemoimmunotherapy has been replaced as standard of care for many but so far not all patients with CLL. \n\nAlthough controlled clinical trials have shown an improvement in survival achieved by chemoimmunotherapy, firm evidence is still lacking at a population-based level. Until now, there are only two representative population-based studies from Denmark and Sweden that analyzed survival for CLL patients in relation to the introduction of chemoimmunotherapy [9, 10]. The aim of the work presented here was to investigate survival in CLL before and after the introduction of chemoimmunotherapy in a population of 2. 6 million people using data of the population-based cancer registry of North Rhine-Westphalia (LKR NRW).",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "The Cancer Registry of North Rhine-Westphalia (NRW) covers a population of 18 million people and is the largest cancer registry in Germany. Age-standardized incidence rates were calculated for the administrative district of Münster and for Germany for the years 2013-2016 using the old European Standard [11]. \n\nTo estimate cancer specific (net-)survival we calculated 5-year relative survival (RS). RS for a calendar period is defined as the ratio of the observed survival of cancer patients and the expected survival of the general population of the same age, sex and calendar period [12]. Survival time per patient was calculated by the difference between date of diagnosis and death or right censoring whatever came first. RS was calculated using the period approach [13]. Period analysis provides more up-to date survival estimates than the traditional cohort approach, since it exclusively reflects the survival experience of patients within a most recent calendar period, for which mortality follow-up is available. This is achieved by left truncation of observations at the beginning of this period, in addition to right censoring at its end [14]. Therefore, with the period approach changes in the prognosis of cancer patients can be detected timely. Expected survival was estimated by the Ederer II method [15, 16]. We used life tables of the administrative district of Münster stratified by age-, sex-and calendar year. RS was estimated for the calendar periods 1998-2002, 2003-2007, 2008-2012 and 2013-2016. Five-year RS was age-standardized according to the International Cancer Survival Standard (ICSS) [17]. We also computed age-specific RS for the age-groups 15-69 years and 70-79 years. All calculations were done with SAS, version 9. 4. RS was estimated by applying the period macro published by Brenner et al. [14].",
"section_name": "MATERIAL AND METHODS",
"section_num": null
},
{
"section_content": "During the years 1993 to 2016 in total 3175 patients (1805 men, 1370 women) were registered with CLL in the administrative district of Münster. The median age at diagnosis was 69 for men and 73 for women. After restricting the analyses to patients aged 15-79 years and exclusion of DCO-cases, 2327 cases (1412 men, 915 women) remained for relative survival analyses. The proportion of DCO-cases that had to be excluded was 7. 5% for men and 6. 1 % for women. The age-standardized incidence rate for 2013 to 2016 was 5. 0/100. 000 for men and 2. 7/100. 000 for women in the administrative district of Münster (Germany: 5. 1/100. 000 men, 2. 7/100. 000 women) (Table 1",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "We found marked improvements in relative survival at population level subsequently to fundamental changes in first-line therapy of patients diagnosed with CLL. For both, men and women, the 5-year age-standardized RS increased about 13 percentage points after the approval of anti-CD 20 antibodies like rituximab, ofatumumab or obinutuzumab for the treatment of CLL. Results of clinical studies have shown previously that CLL patients treated with chemoimmunotherapy achieved markedly longer progression-free survival, which also translated into prolonged overall survival [6, 7]. The population-based registry data shown in this analysis confirm these findings. \n\nTwo population-based studies from Denmark and Sweden also indicated improvements in the survival of CLL patients following the introduction of chemoimmunotherapy. However, both studies analysed data up to 2013 and hence the time period studied in which chemoimmunotherapy was available was short. Further Sylvan et al. [10] analysed survival by treatment group but did not differentiate these groups into chemotherapy only and combined chemoimmunotherapy except for therapy with fluradabine, cyclophosphamide and rituximab in combination for which the longest progressionfree and overall survival was observed. \n\nWith the selected method of relative survival, CLL-associated enhancements seem to be most probably associated with survival improvements shown in this work. Treatment for CLL probably is a main factor, because of the profound change of treatment options following the approval of monoclonal antibodies. Although rituximab was available prior to 2010, it was not approved for the treatment of CLL in Germany and therefore the effect on the survival of CLL patients in the earlier time periods is negligible. At this point, we cannot formally exclude that improvements in other measures such as supportive medication, or improved anti-infective therapies had a positive influence on survival. However, there were no marked changes in this patient management with regard to these health problems during the reported time period. \n\nUnfortunately, we cannot analyse the results according to the known prognostic factors in CLL, as this data is not reported to the registry. However, the distribution of factors such as deletion 17p, TP53 mutation, deletion 11q or an unmutated IGHV status is relatively constant over time, so that the influence of these factors on the present work can most likely be neglected. \n\nTwo points make the results particularly relevant and demand further transfer of knowledge between clinical and epidemiological research and routine care. First, the improved survival rates of patients with CLL in routine care show that the knowledge gained from clinical research and the advantages of newly approved drugs are also evident in routine care. The results presented here show a marked increase of the overall survival in the era with monoclonal anti-CD20 antibodies when compared to the era before, where these antibodies were not available for CLL therapy. It must therefore remain a goal to publish relevant study results without delay and to update guidelines for routine care as quickly as possible. \n\nOn the other hand, the work also results in a gain of knowledge for the registry strategy. In order to be able to do populationbased research accompanying clinical studies, it should be discussed to expand and specify the collected data sets. While data on therapies and on the course of the cancer have recently been added to cancer registration and will be available for future analyses, data on genetic prognostic factors should furthermore be included in the registry data sets. \n\nWe recognize that our analysis has some limitations. First, some of our survival estimates are based on small numbers and consequently survival is imprecisely estimated especially when we stratify for age. Second, our time trend analysis of survival assumes that the stage distribution of CLL remained constant over time. Unfortunately, data on stage (Binet) were too incomplete for a meaningful analysis of stage shifts over time. Early stage and asymptomatic CLL cases are usually diagnosed and treated in the ambulatory setting (including private practitioners) and reporting of incident CLL cases by private practitioners may be less complete. As a consequence, our report may underrepresent cases with early stages of CLL. However, the age-standardized incidence rate in the studied region is the same than that estimated for Germany and there is no indication that private practitioners' reporting to the cancer registry gradually increased over our study period; therefore, our time trend analysis of survival would not be substantially affected by an under-registration. Third, the high DCO among patients aged 80 years or more forced us to exclude this age group and therefore our results may not be generalizable to CLL cases aged 80 years or more. Fourth, as data on therapy were too incomplete for the study period, we could not directly study the influence of the introduction of chemoimmunotherapy on survival. Our interpretations are based on an ecologic approach, which has independent confirmatory value nevertheless. Fifth, we realize that the same type of analysis now needs to be done after the introduction of targeted agents such as BTK inhibitors or venetoclax to test for the effect of these drugs on overall survival. This may take a few years until the registry data have matured enough for this analysis. \n\nTaken together, our results suggest that chemoimmunotherapy with anti-CD20 antibodies has improved survival in CLL patients in North Rhine-Westphalia, Germany.",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Reporting summary",
"section_num": null
},
{
"section_content": "Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.",
"section_name": "Reporting summary",
"section_num": null
},
{
"section_content": "Prof. Hallek received research support by Roche, Gilead, Janssen, Celgene, Pharmacyclics, Abbvie, AstraZeneca and honoraria (speakers bureau and/or advisory board) by Roche, Gilead, Janssen, Celgene, Pharmacyclics, Abbvie, AstraZeneca. Dr. Fink received research support by Celgene, honoraria by Janssen and travel grants by AbbVie. The remaining authors declare no potential competing interests.",
"section_name": "COMPETING INTERESTS",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi. org/10. 1038/s41408-021-00556-7. \n\nCorrespondence and requests for materials should be addressed to Hiltraud Kajüter. \n\nReprints and permission information is available at http://www. nature. com/ reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "ADDITIONAL INFORMATION Supplementary information",
"section_num": null
}
] |
10.1186/s12885-024-12063-6
|
Integrated analysis of transcriptome and genome variations in pediatric T cell acute lymphoblastic leukemia: data from north Indian tertiary care center
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Introduction</jats:title> <jats:p>T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with poor prognosis and inferior outcome. Although multiple studies have been perform on genomics of T-ALL, data from Indian sub-continent is scarce.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>In the current study we aimed to identify the genetic variability of T-ALL in an Indian cohort of pediatric (age ≤ 12 years) T-ALL patients (<jats:italic>n</jats:italic> = 25) by whole transcriptome sequencing along with whole exome sequencing and correlated the findings with clinical characteristics and disease outcome.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>The median age was 7 years (range 3 -12 years). RNA sequencing revealed a definitive fusion event in 14 cases (56%) (including a novel fusions) with <jats:italic>STIL::TAL1</jats:italic> in 4 (16%), followed by <jats:italic>NUP21::ABL1, TCF7::SPI1, ETV6::HDAC8, LMO1::RIC3, DIAPH1::JAK2, SETD2::CCDC12</jats:italic> and <jats:italic>RCBTB2::LPAR6</jats:italic> in 1 (4%) case each. Significant aberrant expression was noted in <jats:italic>RAG1</jats:italic> (64%), <jats:italic>RAG2</jats:italic> (80%), <jats:italic>MYCN</jats:italic> (52%), <jats:italic>NKX3-1</jats:italic> (52%),<jats:italic> NKX3-2</jats:italic> (32%), <jats:italic>TLX3 </jats:italic>(28%), <jats:italic>LMO1</jats:italic> (20%) and <jats:italic>MYB</jats:italic> (16%) genes. WES data showed frequent mutations in <jats:italic>NOTCH1</jats:italic> (35%) followed by <jats:italic>WT1</jats:italic> (23%), <jats:italic>FBXW7</jats:italic> (12%), <jats:italic>KRAS</jats:italic> (12%), <jats:italic>PHF6</jats:italic> (12%) and <jats:italic>JAK3</jats:italic> (12%). Nearly 88.2% of cases showed a deletion of <jats:italic>CDKN2A/CDKN2B/MTAP</jats:italic> genes. Clinically significant association of a better EFS and OS (<jats:italic>p</jats:italic>=0.01) was noted with <jats:italic>RAG2</jats:italic> over-expression at a median follow up of 22 months, while a poor EFS (<jats:italic>p</jats:italic>=0.041) and high relapse rate (<jats:italic>p</jats:italic>=0.045) was observed with <jats:italic>MYB</jats:italic> over-expression.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>Overall, the present study demonstrates the frequencies of transcriptomic and genetic alterations from Indian cohort of pediatric T-ALL and is a salient addition to current genomics data sets available in T-ALL.</jats:p> </jats:sec>
|
[
{
"section_content": "T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive form of ALL and represents 15-20% of pediatric ALL cases [1]. Even with highly intensified therapy, 25% of T-ALL patients experience relapse and have lower post-relapse survival compared to the B-lineage ALL [1]. The genetic heterogeneity of the disease makes T-ALL risk stratification difficult and hence all cases are treated upfront as high-risk with intensified therapy regimen. The high dose multi-agent chemotherapy is often associated with severe toxicities and long-term side effects. Thus, improving understanding of T-ALL biology through the identification and characterization of carcinogenic lesions is essential for better prognostic classification and treatment of the disease. \n\nWith the advent of next-generation sequencing techniques, many genetic abnormalities have been found in T-ALL over the last few decades. Aberrant expression of genes such as LMO1, LMO2, TAL1, TLX1, TLX3, NKX2-1 and other transcription factors (TFs) have long been known [2]. Whole-exome sequencing (WES), and RNA sequencing (RNA-seq) have extended the list of genetic abnormalities in T-ALL [3, 4]. Besides aberrant expression that constitutes about 40-50% T-ALL [5], RNA seq data has expanded the fusion gene list (30-40%) in T-ALL. Such fusion transcripts can either generate an over-expressing protein as in the case of TAL1 as a result of STIL::TAL1 fusion [6, 7] or lead to over-expression of two truncated peptides such as SET&NUP214 in SET:: NUP214 fusion [8]. A number a novel fusion transcripts have been also identified in studies from different cohorts such as ZBTB16::ABL1, RCBTB2::LPAR6, DLEU2::SPRYD7, TRAC::SOX8 etc. [9, 10]. Further, in a holistic approach WES along with RNA seq has identified number of gene abnormalities in pathways regulating differentiation, proliferation, self-renewal, and survival of T-cell precursors. High mutation frequencies such as NOTCH1, JAK-STAT, PI3K-AKT or RAS-MAPK pathway genes have been noted in multiple studies although the frequency varies among cohorts and population being adult or pediatric [11, 12]. Moreover, copy number variations (CNVs), especially high frequency of CDKN2A/ CDKN2B deletions have been consistently shown across multiple studies [13] [14] [15]. \n\nThus, the complex interplay of gene fusions, sequence aberrations and transcriptional expression profiles needs to be increasingly investigated in different cohorts to further refine current models of T-cell leukemia and to identify potential new biomarkers and therapeutic targets. In the current study, RNA-seq and WES analyses were performed in a prospective cohort of pediatric T-ALL cases. A number of rare gene fusions, mutations, aberrant transcripts, and CNVs were identified. Overall, our results point to the need for further large-scale genomic studies to improve patient stratification and optimize treatment strategies for pediatric T-ALL, especially in relation to our distinctly ethnic sub-continental population.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Newly diagnosed pediatric T-ALL cases (age ≤12 years) confirmed on morphology and immunophenotype (flow cytometry) were enrolled for the study. Cases were classified immunophenotypically by flow cytometry into immature (pro T-and pre T-), cortical and mature T-ALL based on the EGIL criteria [16]. ETP-ALL was recognized based on the previously defined criteria [17]. Complete immunophenotype data in 3 patients was unavailable. Patients were considered as good prednisolone responder at day 8 if absolute blast counts (ABC) were < 1000/ul and poor prednisolone responder if ABC > 1000/ul. Day 8 ABC data was unavailable in6 cases. Patients were treated and followed up uniformly as per the ICiCLe treatment protocol (Clinical Trials Registry-India number, CTRI/2015/12/006,434) [18]. Written informed consent in agreement with the Declaration of Helsinki was taken from children and or guardians and the study was approved by the Institutional Ethics board.",
"section_name": "Patients and samples",
"section_num": null
},
{
"section_content": "RNA was extracted from PBMCs isolated from patient blood/bone marrow samples by the RNA blood kit (Qiagen Inc. ) as per manufacturer's protocol. NEBNext RNA Ultra II directional protocol was used to prepare the libraries for total RNA. Paired-end whole transcriptome sequencing was performed on the Illumina NovaSeq to generate 60 M, 2 × 150 bp reads/sample. The raw reads were filtered using Trimmomatic for quality scores and adapters. Filtered reads were aligned to Human genome (hg19) using splice aware aligner HISAT2 to quantify reads mapped to each transcript. Alignment percentage of reads were in the range of 91. 7-97. 5%. Total number of uniquely mapped reads were counted using feature counts. The uniquely mapped reads were then subjected to differential gene expression using Deseq2 (supplementary data-TALL Deseq2 data).",
"section_name": "RNA sequencing",
"section_num": null
},
{
"section_content": "The gene fusion studies of allsamples were carried out by detecting fusion events using two different prediction tools namely, FusionCatcher [19] and STARFusion [20]. The read alignment obtained from the above tools were considered for the event prediction. The tools provide both junction and spanning reads from the mapped bam file. The mapped reads from two different tools were considered as best hit. True fusions typically form from exon-exon fusion. The genomic coordinates were checked to ensure they were identical across tools with minimum distance between 5`gene and 3`gene. Manual review was applied to generate the final fusion gene list. Fusions were only considered for further analysis, if they were called by both the callers with at least 5 reads and were not detected in control samples. Novelty of fusions were checked on Mitelman Database of Chromosome Aberrations and Gene Fusions, and ChimerDB [21, 22]. \n\nNovel fusion validation: Novel fusion (ETV6::HDAC8) identified was validated by qRT-PCR using forward primer T",
"section_name": "Gene fusion analysis",
"section_num": null
},
{
"section_content": "DNA was extracted from PBMCs isolated from blood/ bone marrow samples using Qiagen DNA blood mini kit (Qiagen Inc. ). The libraries were prepared by standard protocol of Illumina platform. Paired-end sequencing (2 × 101 bp read length) was performed using the Illumina HiSeq 2000/2500 platform. Exome sequencing analysis was performed using Dragen server (Illumina Inc. ). The fastq files after demultiplexing were first aligned to reference genome and then the output Sam files were converted to bam. The bam file was then sorted followed by duplicate removal, realignment and re-calibration. Variant caller based on haplotypecaller of GATK was used to generate the variant call files (vcf ). VCF files were then uploaded on GeneYX tool [23] for variant identifications, annotations and subsequent reporting.",
"section_name": "Whole exome sequencing",
"section_num": null
},
{
"section_content": "Treatment outcome parameters analyzed included relapse free survival (RFS) or relapse rate (RR) -defined as time period from onset of therapy to disease relapse for those achieving complete remission with censoring at death in remission or last contact. Overall survival (OS)defined as time period from onset of therapy to death with censoring at last contact. Event free survival (EFS)defined as time period from onset of therapy to any event (relapse/death/abandonment of treatment against medical advice) with censoring at the time of event or last contact. Continuous variables were represented as mean/median (range) and categorical variables as ratio/ proportion. Chi-square test was performed for categorical variables between different clinical, hematological and treatment outcome parameters and genetic events. Survival curves (EFS, RR, OS) for overall cohort in relation to different genetic aberrations were calculated using Kaplan Meier curve and log-rank tests. A p-value of < 0. 05 was considered as significant. All statistical analysis was performed using SPSS v26. 0.",
"section_name": "Outcome assessment and statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Twenty five cases of newly diagnosed pediatric T-ALL were enrolled for the study. The median age of the patients at diagnosis was 7 years (range 3-12 years) with male to female ratio of 11. 5:1. The median WBC count was 184. 18 × 10 9 /L (range 45-785 × 10 9 /L). Mediastinal mass was observed in 12 (48%) and bulky disease was observed in 9 (36%) patients. Immunophenotypically, 45% (10/22) 1.",
"section_name": "Clinical characteristics of the patients",
"section_num": null
},
{
"section_content": "Based on RNA sequencing data we identified 14 cases (56%) with fusion genes. Eight different fusions were noted. The most common fusion event noted was STIL::TAL1 in 4 patients (16%). All 4 cases of STIL::TAL1 fusion were noted with either cortical or mature immunophenotype (3&1, respectively) as depicted in Fig. 1. The remaining 7 cases had fusions of NUP21::ABL1, TCF7::SPI1, ETV6::HDAC8, LMO1::RIC3, DIAPH1::JAK2, SETD2::CCDC12, and RCBTB2::LPAR6; one (4%) in each case. Among above fusion genes ETV6::HDAC8 has not been reported in literature earlier. Besides above fusion gene, 2 cases had KMT2A rearrangements and 1 case had MLL10 rearrangement. RNA sequencing data also revealed TCR re-arrangements in 10 (40%) cases, predominantly TRG@ in 8 cases (32%), 2 (8%) cases had TRA@ and 1 (4%) case had both TRA@ and TRB@ rearrangement.",
"section_name": "Overview of fusion transcripts",
"section_num": null
},
{
"section_content": "The well-known expression marker genes in T-ALL, such as TLX2/3, LMO1, RAG1/2, NKX3-1/2, MYB, MYCN were noted to be the most common over expressed genes in our cohort (Fig. 1 ). Five patients (20%) had over-expression of TLX2 while 7 patients (28%) had over-expression of TLX 3. LMO1 gene over-expression was noted in 4 cases (16%). RAG1 and RAG 2 gene overexpression cases were also high in number (n = 16 & 20, respectively). The newly identified NKX3-1/2 were over expressed in 13 (52%) and 8 (32%) cases, respectively. \n\nHigher number of MYCN overexpressed cases (n = 13, 52%) were noted than MYB gene over expressed cases (n = 4, 16%). WT1 gene was over expressed in 13 (52%) cases. Other notable genes were GATA3 (n = 7, 28%), RUNX1 (n = 12, 48%) and PIK3R3 (n = 4, 16%), however their fold change compared to control cases were lower than other mentioned genes.",
"section_name": "Gene expression analysis",
"section_num": null
},
{
"section_content": "T-ALL patients were screened for mutations by WES. Data was further re-analyzed for 60 genes previously known to be associated with T-cell leukemogenesis (supplementary Table 1 ). Eight out of 25 patients did not have sufficient DNA and therefore could not be processed for whole exome sequencing. Although focused analysis was done for 60 genes, only 23 genes showed missense or frameshift mutations predicted to result in amino acid change or change in protein length (Fig. 1 ). Seventeen cases that were analyzed for gene mutation had a median of 3 variants per case (range 1-6). The most commonly mutated gene was NOTCH1 with six cases (6/17, 35%) showing a pathogenic mutation. Five cases (5/6, 83%) showed single nucleotide variation (SNV), while the remaining one had frameshift insertion resulting in smaller predicted protein. Two cases had multiple mutations, SNV/s or frameshift mutation/s. Four cases (23%) showed mutation in WT1 gene. Three out of four cases harboring WT1 mutation was frameshift while one case had both SNV and a frameshift mutation. FBXW7, KRAS, PHF6 and JAK3 had variants in 3 (18%) cases each. While NRAS, PTEN, CDKN2A,FLT3 and IKZF1 genes were noted to have mutation in 2 (12%) cases each. MPL, ABL1, BCOR, CBL, CREBBP, KMT2A, KRAS, PDGFRA, SF3B1, SMC3, ATM and PAH genes harbored mutation in 1 (6%) case each. One case had overlapping mutation of KRAS and NRAS. All these genes had SNV except for CBL which had an insertion. The details of the variants, including the gene list and mutational frequency, are highlighted in supplementary Table 2.",
"section_name": "Whole exome sequencing variations",
"section_num": null
},
{
"section_content": "The most common CNV noted through WES data was deletion of 9p21. 3 locus that consists primarily of CDKN2A, CDKN2B and MTAP genes. In our study cohort 15/17 cases (88. 2%) revealed deletion of CDKN2A/CDKN2B/MTAP genes (Fig. 1 ). Seven out of 15 (47%) cases had heterozygous while the remaining 8 (53%) had homozygous deletion. Only one case each for CDKN2B and MTAP did not show deletion with the loss of CDKN2A gene.",
"section_name": "Copy number variation",
"section_num": null
},
{
"section_content": "Considering the cases with fusion gene aberration, two of the STIL::TAL1 fusion cases that had data available for DNA sequencing and they did not show any common gene mutations. While both the two cases with KMT2A rearrangement had either WT1 and PTEN gene mutations. The one case having NUP214::ABL1 fusion had NOTCH1 mutation. While MED12::IRF2BPL and RCBTB2::LPAR6 fusion cases were noted to have NRAS mutations. One case each of KMT2A rearrangement, TCF7::SP1, RCBTB2::LPAR6 fusion had KRAS gene mutation. DIAPH1::JAK2 fusion case had FBXW7, CDKN2A and CREBBP mutations. SETD2::CCDC12 case harboured BCOR mutation (Fig. 1 ). \n\nCorrelations among genetic alterations were analyzed in those sub-groups with ≥ 5 cases carrying positive genetic lesions. NOTCH1 mutations were examined for any correlation with deletion of CDKN2A and overexpression of TLX2, TLX3 and NKX3-1. NOTCH1 mutation was significantly associated with TLX3",
"section_name": "Association among subgroups of genetic alterations",
"section_num": null
},
{
"section_content": "Prognostic relevance among genetic alterations were analyzed for sub-groups with ≥ 4 cases carrying positive Although patients with both gene mutations showed slightly better overall survival (OS), this was not significant (supplementary Fig. 1A ). STIL::TAL1 fusion cases had better EFS and lower RR, however did not reach statistical significance (supplementary Fig. 1B ). Further, over-expression of TLX2/TLX3/LMO1/RAG1/2/NKX3-1/2/MYB/ MYCN for EFS, RR and OS revealed that patients with RAG2 over-expression showed better EFS (p = 0. 01) and OS (p = 0. 01). The OS of RAG2 over-expressed cases was 95% compared to 60% in those without over-expression (at 95% CI) for a median follow up 22 months (Fig. 2A ). \n\nOver-expression of MYB was noted to be associated with poor EFS (p = 0. 041) and RR (p = 0. 045). The EFS of MYB over-expressing cases were only 25% while the remaining patients were 71% (at 95% CI) as depicted in Fig. 2B. The remaining aberrantly expressed genes were not found to have any significant association with EFS, RR and OS in T-ALL patients.",
"section_name": "Prognostic analyses related to the genetic features of pediatric T-All patients",
"section_num": null
},
{
"section_content": "Studying genetic alterations in T-ALL is the way forward in improving patients' diagnosis and treatment. Being a genetically heterogeneous disease, multi-genomics approach needs to be applied across different cohorts to elucidate the genetics of T-ALL leukemogenesis. Different studies have used different approach to identify prognostic markers and targets of therapy in T-ALL such as whole transcriptomics studies, whole exome sequencing, targeted DNA sequencing etc. However, no data from Indian subcontinent has been reported till date from study comprising transcriptomic and genomic sequencing. In the current pilot study, we have applied whole RNA and DNA sequencing to unravel maximum genetic alterations both at genes as well transcripts level in Indian cohort of pediatric T-ALL patients. We found that each of the 25 cases of our T-ALL study population harboured at least one major genetic abnormality, including gene fusions, CNV, recurrent gene mutation and/or aberrant expression of genes that are key to leukemogenesis. 56% (14/25) of our T-ALL cases harboured fusion genes. As expected co-occurrence of fusion genes in the same case was not observed, suggesting their role as driver mutations. Further, fusions were noted to be coexisting with either point mutations or aberrant expression suggesting their possible cooperative effects. STIL::TAL1 fusion being the most common (16%) in the current study, have been reported earlier by us and others in Indian cohort, however the frequency ranged from 18 to 27% [24, 25]. Earlier reports were based mainly on multiplex ligation dependent probe amplification assay (MLPA) or RT-PCR. We are for the first time, studying the combined genetic heterogeneity in T-ALL cases from Indian cohort by RNA Seq. \n\nWe also noted a novel fusion of ETV6::HDAC8 in our cohort. An interesting study by Fisher MH et al. has shown that cytoplasmic localization of ETV6 due to inherited mutation leads to over-expression of HDAC3-regulated interferon response genes that predisposes to malignancy [26]. Fusions like TCF7::SPI1 and LMO1::RIC3, are also rarely reported. One case carrying TCF7::SPI1 fusion in a recent study cohort of 121 cases [27]. Interestingly we also noted one case having RCBTB2::LPAR6 fusion that has previously been reported in B-ALL and has been suggestive of partial loss of RB1 gene [9, 28]. \n\nIn the current study we have demonstrated a number of genes with aberrant expression profile in transcription factors and related genes. The highest expression showing gene is TLX3 which was noted in 30% of cases. Cryptic translocation of t(5;14) (q35;q32), have been shown to result over-expression of TLX3 expression in pediatric T-ALL cases. Earlier studies have noted 20-25% cases of T-ALL with TLX3 rearrangement/ over-expression [29, 30]. Further, this genetic aberration is also shown to be associated with NOTCH1 mutation and/or NUP214::ABL1 amplifications [29]. Interestingly, all 7 cases with over expressed TLX3 had NOTCH1/ FBWX7 mutation. Moreover, one cases that harboured NUP214::ABL1 fusion in our subjects, had the highest expression of TLX3. Over-expression of this case was noted with the fold change of > 7000 compared to controls. Further, over-expression of TLX3 was noted to be significantly associated with NOTCH1 mutations (p = 0. 04). However, when prognostic outcome was analyzed in such cases, the result was not significant. \n\nOther noteworthy genes having aberrant expression in our cohort are LMO1, RAG1, RAG2, NKX3-2, NKX3-1, MYB and MYCN. Among these, RAG2 and MYB over expression showed a correlation with outcome parameters. RAG2 over expresssion showed better EFS (p = 0. 01) and OS (p = 0. 01) in pediatric T-ALL patients. A recent study in cell lines and mouse model, has shown that RAG1 and RAG2 expression in both primary and transformed thymocytes is mediated by NOTCH1 dimerization. Since many earlier studies suggest better outcome of patients with NOTCH1 mutations [31, 32], further investigation of NOTCH1-RAG2 axis in T-ALL cells may provide indirect evidence of mechanism behind better prognosis. Over-expression of MYB was also noted to associated with poor EFS (p = 0. 041) and RR (p = 0. 045). However, the case number over-expressing MYB (n = 4) was too small to draw any definite conclusion and further study in larger cohort is needed to validate the association. \n\nMutational analysis of T-ALL cases revealed most frequent mutation in NOTCH1 gene as expected. Studies have shown the frequency of NOTCH1 mutation in T-ALL from 50 to 70% [33] [34] [35] while in Indian cohorts the frequency ranges from 40 to 50% [36, 37]. The lower frequency observed in current population could be due to the smaller cohort size. In our cohort the 6 cases that had NOTCH1 mutation only one had relapse but no significant association was noted with any outcome parameters. \n\nThe frequency of PHF6 mutation noted in our study population is 12% similar to other studies describing the range of 5-19% in pediatric patients [37] [38] [39]. Somatic mutations and deletions of PHF6 in pediatric T-ALL have been reported exclusively or predominantly in males [13, 40]. In our cohort one out of 3 cases bearing PHF6 mutation was female. Further, in the present study, 3 cases that had PHF6 mutation did not have relapse or any other event in the median follow up of 22 months. \n\nWT1 mutations were noted in 23% of current study cohort. Earlier studies have reported relatively lower frequency in WT1 gene [13]. In addition, we noted that all 3 cases having mutation in WT1 gene coincided with mutations/deletions of NOTCH1/FBXW7 or PHF6 genes. Similar observation has been reported in an earlier study suggesting that loss of function in WT1 gene may cooperate in disease pathogenicity of T-cell leukemia [13]. \n\nWe noted 5 cases with mutations occurring in NRAS or KRAS gene. Earlier studies have suggested NRAS mutation as an independent predictor of a poor outcome in ALL but a few other studies have shown favorable prognosis of RAS mutation in T-ALL [13]. In our cohorts 5 occurrence of NRAS-or KRAS mutation only one had relapse however, study on the larger cohort needs to be done before establishing any conclusion. Interestingly, FLT3 mutation was noted in 2 cases in our study population. As a target of therapy such mutations may contribute in personalized treatment of patients. \n\nCDKN2A/CDKN2B gene deletion has been most common genetic lesion in our population as reported previously by us and other groups in India [24, 25]. Similarly, in the current study population too, we noted 88% of cases (15/17) to have CDKN2A/ CDKN 2B deletion. Previous studies have shown that though it is the most common mutation in T-ALL with poor prognosis, it is suggested to be acquired during the course of leukaemia progression of T-ALL and is not a driver mutation of the cancer cells [14]. However more studies are needed to utilize this gene as prognostic marker or target of therapy in future. \n\nAlthough studies specially focused on pediatric T-ALL are scarce however, in the previous large studies based on pediatric T-ALL genomics and transcriptomic analysis, almost similar results have been reported. Masafumi et al., on analysis of 121 pediatric T-ALL patients reported similar results as our study where NOTCH1 and CDKN2A were the most frequently affected genes, they also reported USP7 gene in which we did not find any mutation. They also documented a SPI1 fusion and associated it with reduced overall survival, a finding we observed in our patient cohort too. However, we did not observe a statistically significant correlation, possibly attributable to our smaller sample size [27]. In a separate interesting study that analyzed both pediatric and adult samples, common fusions identified in the pediatric population included KMT2A, MLLT10, STIL-TAL1, and LMO2 fusions. Additionally, commonly mutated genes in this population were NOTCH1, KRAS, NRAS, and CDKN2A. These findings closely align with our results [41]. LEF1, WT1 and BCL11B copy number abnormalities were reported from the TARGET study of 2471 pediatric cancer patients whereas in our group we found CDKN2A, CDKN2B and MTAP harboring most copy number variations [42]. \n\nThus, despite the major limitation of small cohort size of the study, we present relevant mutations and aberrant expression profile in pediatric T-ALL from Indian cohort. As ethnicity has been shown to be involved the variations in T-ALL genomics, our study is an addition of current genomics data sets available in pediatric T-ALL. Further, it will be interesting in future to study the non-coding mutations, such as microRNAs and lncRNAs to add to the cancer-related gene regulatory network changes underlying leukemogenesis of T-ALL.",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi. org/10. 1186/s12885-024-12063-6.",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "",
"section_name": "Supplementary Material 1-T-ALL Deseq2 data",
"section_num": null
},
{
"section_content": "Author contributions M. S., P. S. and P. B. performed the experimental work, data analysis and in silico data analysis; R. T. assisted in experimental work; P. B., A. T. and S. S. enrolled patients and performed diagnosis of patients, clinical evaluation and clinical data analysis. M. S. conceptualized the study and wrote the manuscript with P. S. and P. B. All authors reviewed and edited the manuscript.",
"section_name": "Supplementary Material 2-T-ALL WES and outcome data",
"section_num": null
},
{
"section_content": "The study is funded by Institutional Intramural Research Grant (PGIMER, Chandigarh, India).",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "All the data from current manuscript has been uploaded as supplementary files.",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "The study was approved by Institutional Ethics Committee of Postgraduate Institute of Medical Education & Research, Chandigarh. Informed consent was taken from all the participants or parents.",
"section_name": "Declarations Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Not Applicable.",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The authors declare no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.1038/s41598-021-92412-8
|
Immunomodulatory effects of different intravenous immunoglobulin preparations in chronic lymphocytic leukemia
|
<jats:title>Abstract</jats:title><jats:p>Hypogammaglobulinemia is the most frequently observed immune defect in chronic lymphocytic leukemia (CLL). Although CLL patients usually have low serum levels of all isotypes (IgG, IgM and IgA), standard immunoglobulin (Ig) preparations for replacement therapy administrated to these patients contain more than 95% of IgG. Pentaglobin is an Ig preparation of intravenous application (IVIg) enriched with IgM and IgA (IVIgGMA), with the potential benefit to restore the Ig levels of all isotypes. Because IVIg preparations at high doses have well-documented anti-inflammatory and immunomodulatory effects, we aimed to evaluate the capacity of Pentaglobin and a standard IVIg preparation to affect leukemic and T cells from CLL patients. In contrast to standard IVIg, we found that IVIgGMA did not modify T cell activation and had a lower inhibitory effect on T cell proliferation. Regarding the activation of leukemic B cells through BCR, it was similarly reduced by both IVIgGMA and IVIgG. None of these IVIg preparations modified spontaneous apoptosis of T or leukemic B cells. However, the addition of IVIgGMA on in vitro cultures decreased the apoptosis of T cells induced by the BCL-2 inhibitor, venetoclax. Importantly, IVIgGMA did not impair venetoclax-induced apoptosis of leukemic B cells. Overall, our results add new data on the effects of different preparations of IVIg in CLL, and show that the IgM/IgA enriched preparation not only affects relevant mechanisms involved in CLL pathogenesis but also has a particular profile of immunomodulatory effects on T cells that deserves further investigation.</jats:p>
|
[
{
"section_content": "have been identified, for example: direct and indirect inhibition of T-cell activation 5, induction of anergy and impairment of BCR-and TLR-signalling on B cells 6, 7, and inhibition of the mononuclear phagocytic system 8, 9. \n\nThe immunomodulatory capacity of Ig preparations on CLL cells was not directly addressed until recently when Spaner, D. et al. showed that a SCIg preparation impaired BCR signaling, activation and cytokine secretion by CLL cells stimulated in vitro 10. Interestingly, in that report they found that patients receiving IgRT that increases IgG levels over 9 g/L showed evidence of disease control, suggesting that high doses of Ig may have anti-leukemic activity in CLL patients. \n\nBecause both, its particular isotype composition and the chemical treatments during manufacturing might affect the immunomodulatory capacity of an IVIg preparation, our aim was to explore in vitro the immunomodulatory capacity of Pentaglobin, an IVIg enriched in IgM/IgA (IVIgGMA) and Vigam, an IVIg preparation with more than 95% of IgG (IVIgG) in CLL. Given the capacity of IVIg to affect T cell compartment and the particular characteristics of T cells from CLL patients 11, we extended our analysis not only to leukemic B cells but also to T lymphocytes.",
"section_name": "",
"section_num": ""
},
{
"section_content": "The in vitro activation of T cells from CLL patients in response to TCR-stimulation is diminished by IVIgG but not IVIgGMA. Several reports have shown that IgG preparations decreased the activation of T cells from healthy subjects in vitro 5, 12, 13. In order to evaluate whether IVIgGMA and IVIgG differentially regulate the activation of T cells from CLL patients, PBMC were stimulated in vitro with immobilized anti-CD3 mAb for 24 h, in the presence of IVIgGMA, IVIgG or HSA at equimolar concentration as control. Because previous reports showed that the inhibitory effect of IgG is observed at high concentrations 5, 12, 13, we used both IVIg preparations at a final concentration of IgG of 10 mg/mL. We found that, as already reported for T cells from healthy donors, IVIgG impaired the up-regulation of the activation markers CD25, CD69 and PD-1, while IVIgGMA did not modify their expression (Fig. 1a-c ). When the effects of both preparations were compared, we observed that the up-regulation of CD25 and CD69 was significantly lower in the presence of IVIg than in the presence of IVIgGMA (Fig. 1a, b ), while no differences were found for PD-1 (Fig. 1c ). The inhibition on T cell-activation mediated by IVIgG was dose-dependent showing to be statistically significant at 10 and 1 mg/mL but not at lower doses as previously described for T cells from healthy donors 5 (see Supplementary Fig. S1 online ). Moreover, as shown in Fig. 1d, none of the IVIg preparations affected the viability of CD3 + cells of CLL patients.",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Then we evaluated the effect of the different IVIg preparations on T cell proliferation in response to TCR-stimulation and also in response to IL-15, a cytokine involved in homeostatic proliferation of memory T cells. To that aim CFSE-stained PBMC from CLL patients were cultured with immobilized anti-CD3 mAb or IL-15 in the presence of IVIgG, IVIgGMA or HSA. As shown in Fig. 2a, b we found that, contrary to what happened with early activation markers, both IVIg preparations were able to impair T cell proliferation when cells were stimulated through the TCR. This was observed both on CD4 + (Fig. 2a ) and CD8 + T cells (Fig. 2b ). Nevertheless, proliferation of T cells in response to TCR-stimulation was significantly lower in the presence of IVIgG than in the presence of IVIgGMA (Fig. 2a, b ). T cell proliferation in response to IL-15 was impaired in CD4 +, but not in CD8 + T cells, only by IVIgG preparation (Fig. 2c, d ). Again, the proliferation in response to IL-15 was lower in the presence of IVIgG than in the presence of IVIgGMA (Fig. 2c, d ). Same results were found when the proliferation of CD8 + T cells in response to IL-2 was evaluated (see Supplementary Fig. S2 online). \n\nAs shown in the Supplementary Fig. S3 online, the inhibitory effect of the IVIg preparations on the proliferation of T cells from CLL patients was due to a direct effect on this cell population, given that same results were obtained with purified T cells (> 95% T cells).",
"section_name": "The proliferation of T cells from CLL patients in response to TCR-stimulation or IL-15 is differentially affected by the two IVIg preparations.",
"section_num": null
},
{
"section_content": "As mentioned before, it was recently reported that a SCIg preparation impaired CLL cell activation when stimulated through the BCR in vitro 10. We asked if the IVIg preparations evaluated herein were also able to decrease leukemic B cell activation. To that aim, PBMC from CLL patients were activated with immobilized anti-IgM mAb to induce BCR crosslinking, in the presence of IVIgGMA, IVIgG or HSA, and after 24 h the expression of the activation markers CD69 and CD86 was assessed. As shown in Fig. 3a, b, both preparations decreased the upregulation of CD69 and CD86, without affecting CD19 + cell viability (Fig. 3c ). In this case, the inhibitory effect of both preparations was not statistically significant different. The inhibitory effect did not depend on the presence of accessory cells given that similar results were observed with purified leukemic B cells (see Supplementary Fig. S4 online ). Moreover, the inhibition on the up-regulation of the activation markers was accompanied with a decrease in the signaling pathway downstream the BCR as shown by a reduced phosphorylation of key molecules such as Syk, Btk and Erk 1/2 (Fig. 4 ). \n\nThe up-regulation of the activation markers CD69 and CD86 in response to BCR cross-linking was significantly reduced by IVIgGMA at 1 and 10 mg/mL or IVIgG at 10 mg/mL, while both preparations at 0. 1 mg/mL had no effect (see Supplementary Fig. S5 online ). Moreover, the inhibition exerted by both preparations at 10 mg/ mL on CLL cell activation seems not to be a general effect but rather specific to particular signalling pathways, given that the inhibition was not observed on CXCL12 or CpG-activated CLL cells (see Supplementary Fig. S6 online).",
"section_name": "Both preparations of IVIg impaired B cell activation in response to BCR crosslinking.",
"section_num": null
},
{
"section_content": "Although we observed that IVIg preparations did not affect the spontaneous apoptosis of leukemic cells (Fig. 1d, 3c ), we asked whether these preparations might affect the apoptosis induced by the BCL-2 inhibitor, venetoclax, currently employed in CLL treatment. To that aim PBMC from CLL patients were cultured with clinically relevant doses of venetoclax in the presence of IVIgGMA, IVIgG or HSA. We found that none of the IVIg preparations affect CLL cell apoptosis induced by venetoclax (Fig. 5a, b ). Given that we have previously reported that venetoclax induces the apoptosis of T cells from CLL patients 14, we also evaluated the effect of IVIg preparations on this cell population. Interestingly we found that IVIgGMA reduced T cell apoptosis induced by venetoclax while IVIgG did not (Fig. 5c, d ).",
"section_name": "IVIgGMA reduced T cell, but not CLL cell, apoptosis induced by venetoclax.",
"section_num": null
},
{
"section_content": "The potential benefit of IVIg preparations as an immune-modulator emerged when the infusion of high doses of IVIg in a patient with antibody deficiency and autoimmune thrombocytopenia results in the restoration of the platelets levels to its normal range 15. Since that initial observation, the use of Igs preparations in the treatment of autoimmune and inflammatory conditions has significantly increased. The study of the mechanisms behind this immunomodulatory effect has expanded as well, and several, not mutually excluding mechanisms have been proposed, including the inhibition of T and B cell activation 4, 16. In CLL, the immunomodulatory capacity of Ig preparations was first suggested in a study from Besa, et al. when they observed a decrease in leukemic cell counts in CLL patients treated with IVIg for recurrent infections and/or autoimmune complications 17. This observation was recently supported by the work of Spaner, et al. who showed an association between IgG levels above 8-9 mg/mL and a benign course of the disease 10. They also reported that a SCIg preparation has inhibitory effects on leukemic cells in vitro. \n\nHere we show that two IVIg preparations, one with mainly IgG, and the other enriched with IgA and IgM, interfere with the BCR signalling pathway, decreasing the phosphorylation of early signalling molecules downstream the BCR, as Syk and Btk, and the activation of leukemic cells in response to BCR-stimulation in vitro. Considering the central role of BCR-signalling on CLL pathogenesis, the capacity to inhibit leukemic cell activation adds an attractive immunomodulatory effect to IVIg preparations for CLL patients. The inhibitory effect of both IVIg preparations on BCR-activated leukemic cells did not depend on the presence of accessory cells, since the inhibitory effect was observed either using PBMC or purified leukemic cells from CLL patients. Although we do not know the mechanism by which these preparations impair BCR signalling on leukemic cells, we do know that CLL cells express both FcɣRIIb and CD22 18, which are receptors that negatively modulate BCR-mediated activation on B cells by interacting with IgG. In that regard, others have shown that blocking FcɣRIIb receptors on CLL cells abrogates IgG mediated inhibition of the activation through the BCR 10. Also, sialylated IgG present in these preparations might bind CD22 and diminish BCR activation as reported for normal B cells 19. \n\nWe also found that IVIgG impaired the activation of T cells from CLL patients in response to TCR-stimulation, and also in response to other two soluble factors, IL-2 and IL-15, a cytokine involved in homeostatic proliferation of memory T cells. Interestingly, we found that the IVIgGMA preparation had no effect on the upregulation of early activation markers on TCR-stimulated T cells and on the proliferation in response to IL15 or IL-2, while it was able to decrease the proliferation in response to TCR-stimulation. Remarkably IVIgGMA showed a significantly lower inhibitory effect on T cells compared to the IVIgG preparation. The IVIgGMA Pentaglobin, differs from other IVIg preparations, not only in its particular isotype composition (76% IgG, 12% IgM and 12% IgA), but also in its manufacturing process. In order to induce virus inactivation, this IVIgGMA is treated with β-propiolactone, a treatment that also modifies amino-acid residues in the Fc domain of the IgG affecting its binding capacity to monocytes through FcɣR and also its complement fixation capacity 20. Similar to what we have observed, others have reported that an anti-CMV hyperimmunoglobulin preparation treated with β-propiolactone was less effective in suppressing human T-cell activation in vitro compared to the same Ig preparation without this treatment. Thus, it is possible that β-propiolactone treatment is involved in the lower inhibitory capacity of the IVIgGMA preparation, although we cannot rule out the possibility that the different isotype composition also has consequences on its inhibitory capacity. \n\nWhen we evaluated the effect of IVIg preparations on the apoptosis induced by venetoclax, we observed that none of the two preparations affected CLL cell viability, while T cell apoptosis induced by venetoclax was significantly lower in the presence of IVIgGMA. Both, leukemic and T cells from CLL patients express high levels of the IgM receptor, the FcµR 21. Although this receptor was originally described as anti-apoptotic in T cells 22, convincing studies published later have demonstrated that the receptor has not an anti-apoptotic function per se 23. Thus the mechanism behind this interesting observation deserves further study. \n\nOur study has two main limitations. First, the fact that both IVIg preparations have differences in their isotype composition along with differences in the production process (mainly β-propiolactone treatment), does not allow us to provide a conclusive explanation for their different effects. Second, patients with unmutated IGVH genes are underrepresented in our cohort (see Supplementary Table S1 online). Considering that this group of patients obtain particular benefit from venetoclax 24, the observation that pentaglobin prevent T cell apoptosis induced by this drug should be validated with a larger cohort of unmutated patients. cells) were incubated with IVIgGMA, IVIgG (10 mg/mL of IgG) or HSA and anti-IgM (25 µg/mL) or the corresponding isotype control for 2 or 10 min. Then, cells were lysed and whole cell extracts were prepared as described in Material and Methods. Proteins were separated on a standard 10% SDS-PAGE and transferred to a PVDF membrane. Membranes were probed with primary antibodies for phospho-Syk (pSyk), phospho-Btk (pBtk), phospho-Erk1/2 (pErk) and β-actin, followed by the corresponding secondary antibody as described in Material and methods. Specific bands were visualized by enhanced chemiluminiscence (ECL) method. (a) Western blot analysis of pBtk, pSyk and pErk1/2 protein levels. β-actin was included as loading control. Cropped western blot images are shown, full-length blots are presented in Supplementary Fig. S7 CLL patient samples and cell separation procedures. The study was approved by the local ethics committee from Academia Nacional de Medicina, Buenos Aires, Argentina, according to the institutional guidelines (Approval number 15/20/CEIANM). Peripheral blood samples were obtained from CLL patients after signed informed consent. The study was conducted according to the principles of the Declaration of Helsinki. \n\nCLL was diagnosed according to standard clinical and laboratory criteria. At the time of the analysis patients were free from clinically relevant infectious complications and were either untreated or had not received treatment for a period of at least 6 months before investigation. Clinical characteristics of CLL patients included in the study are shown in Supplementary Table S1. \n\nPeripheral blood samples were obtained from CLL patients and peripheral blood mononuclear cells (PBMC) were isolated as previously described 14. \n\nT cells from CLL patients were purified by positive selection with the anti-CD3 Microbead isolation kit (purity obtained > 95%). Leukemic B cells from CLL patients were obtained by negative selection with the anti-B-CLL Microbead isolation kit (purity obtained > 98%). Magnetic separation was performed according to manufacturer's instructions.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "For T cell activation, PBMC (3 × 10 5 cells/150 µL RPMI 10% FCS) were pre-incubated for 30 min at 37 °C with IVIgGMA, IVIgG (0. 1-1-10 mg/mL of IgG) or HSA at equimolar concentration (control) and then cultured on a 96-well culture plate containing immobilized anti-CD3 mAbs (0. 5 µg/mL) or the corresponding isotype control for 24 h at 37 °C. Then, cells were stained with mAb for CD4, CD8, CD25, CD69 and PD1 and evaluated by flow cytometry as detailed in the Flow cytometry section. The presence of HSA did not affect T cell activation (not shown). \n\nT cell proliferation was evaluated using the CFSE dilution assay. PBMC or purified T cells, both from CLL patients (3 × 10 5 cells/150 µL RPMI 10% FCS) were labelled with CFSE (1 µM) and then pre-treated for 30 min at 37 °C with IVIgGMA, IVIgG (10 mg/mL of IgG) or HSA. Then, cells were cultured on a 96-well culture plate containing immobilized anti-CD3 mAbs (0. 5 µg/mL) or the corresponding isotype control or IL-15 (20 ng/ mL) or IL-2 (600 U/mL) for 5 days at 37 °C. Cells were then collected, stained with mAb for CD4 and CD8 and proliferation evaluated by flow cytometry. Percentage of proliferation was determinate as the % of T cells with low stain of CFSE. \n\nB cell cultures. PBMC or purified B-CLL cells (3 × 10 5 cells/150 µL RPMI 10% FCS) from CLL patients were pre-incubated with immobilized anti-IgM mAbs (25 µg/mL) or the corresponding isotype control for 30 min at 4 °C. Then, IVIgGMA, IVIgG (0. 1-1-10 mg/mL of IgG) or HSA was added, and cells were cultured for 24 h at 37 °C. The expression of CD19, CD86 and CD69 was evaluated by flow cytometry. The presence of HSA did not affect B cell activation (not shown). \n\nPBMC were pre-treated for 30 min at 37 °C with IVIgGMA, IVIgG or HSA and then CpG (1 µM) or CXCL12 (500 ng/mL) were added to cultures. After 24 h, B cell activation was evaluated by flow cytometry. \n\nVenetoclax-induced apoptosis cultures. PBMC from CLL patients (3 × 10 5 cells/150 µL RPMI 10% FCS) were pre-treated in a 96-well plate for 30 min at 37 °C with IVIgGMA, IVIgG (10 mg/mL of IgG) or HSA.",
"section_name": "T cell cultures.",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The authors would like to thank to María Tejeda and Romina Mariel Pagano from CONICET for their technical assistance, and Guillermo Atenza from Microsules Argentina for the support on the project and helpful discussions.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This work was supported by grants and fellowships from the Agencia Nacional de Promoción Científica y Tecnológica ( PICT 2017-2604 ), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Microsules Argentina.",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Statistical analysis.",
"section_num": null
},
{
"section_content": "www. nature. com/scientificreports/ Then, different doses of Venetoclax (0. 01-0. 1-1 µM) or DMSO (drug vehicle) were added to cultures. After 24 h cell viability was assessed by flow cytometric alterations of light scattering properties and confirmed by staining with CD19, CD3 and Annexin V. \n\nFlow cytometry. For surface staining, cells were incubated with the corresponding antibodies, anti-CD3, CD4, CD5, CD8, CD19, CD25, CD69 and CD86, for 30 min at 4 °C. The staining was performed in phosphatebuffered saline-0. 5% BSA. Cells were then washed and fixed with paraformaldehyde 1%. Gating of populations positive for any particular marker was based on fluorescence minus one (FMO) control of each activation marker. FMO was not modified by activation or IVIg treatment (data not shown). \n\nCell viability was determined by Annexin V staining performed on binding buffer. After cell surface staining, cells were washed once with binding buffer and then stained with Annexin V for 20 min at room temperature. Samples were then acquired in the flow cytometry. \n\nCell viability was also determined by flow cytometry by evaluating flow cytometric alteration of light scattering properties as previously described 14, 27. Briefly, apoptotic lymphocytes can be distinguished from viable lymphocytes by flow cytometry by their differences in cell morphology 28. Apoptotic cells show a reduction in cell size (lower FSC), and as a result of chromatin condensation, nucleus fragmentation and cytoplasmic protein cross-linking, in the late stages of the apoptosis process the scattering of light in SSC is decreases (lower SSC) 29. The analysis was performed evaluating the FSC-H vs SSC-H parameters, both on a linear scale. \n\nSamples were acquired with a BD FACSCalibur (BD Biosciences) and data were analyzed with FlowJo 10 software (FlowJo, USA). \n\nWestern blot. Purified B-CLL cells (2 × 10 6 cells) were activated with anti-IgM (25 µg/mL) or the corresponding isotype control at 37 °C, in the presence of IVIgGMA, IVIgG (10 mg/mL of IgG) or HSA. After 2 or 10 min, the reaction was stopped with cold saline solution. Whole-cell lysates were prepared using 75 µL of RIPA buffer containing protease inhibitors (Thermo Fischer Scientific, #78440). Lysates were vortex and incubated on ice twice, and then after centrifugation, supernatants were transferred to a new tube. 25 µL of loading buffer 4× containing β-mercaptoethanol was added and then samples were incubated for 5 min at 95 °C. 50 µL of protein extracts were separated on a standard 10% SDS-PAGE and transferred to PVDF membranes (GE HealthCare Science, #GE1060023). Membranes were then blocked with a PBST solution containing 5% non-fat dry milk for 2 h at room temperature. Then, PVDF membranes were cut to perform the incubation of the different sections of the same membrane with different primary antibodies, as previously reported 30. Membranes were incubated with primary antibodies over night at 4 °C as follows: membranes with molecular weight marker between 100 and 50 kDa were probed with anti-phospho-SYK and anti-phospho-BTK and membranes with molecular weight marker below 50 kDa were probed with anti-phospho-ERK1/2 and anti-β-actin. Membranes were then incubated with the corresponding secondary antibody, HRP-conjugated anti-rabbit or anti-mouse IgG mAb, for 1 h at room temperature. Specific bands were visualized by enhanced chemiluminiscence (ECL) method. The expression of β-actin was used as a loading control to normalize the protein levels detected in each lane of the same gel. The molecular weight marker was the Precision Plus Protein™ All Blue Prestained Protein Standards (10-250 kDa) from BioRad (#1610373). Densitometric measurements of specific bands were determinate by using ImageJ software (NIH).",
"section_name": "",
"section_num": ""
},
{
"section_content": "Statistical significance was determined using non-parametric tests: Wilcoxon matchedpairs signed rank test to compare between two paired groups and Friedman followed by the Dunn's post-test to compare three or more groups. Two-tailed tests were used and p < 0. 05 was considered statistically significant. The corresponding p value is indicated. Data were analysed using the GraphPad Prism software version 7.",
"section_name": "Statistical analysis.",
"section_num": null
},
{
"section_content": "",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The disclosures are the following: Bezares RF received compensation as speaker from Varifarma, Microsules, AstraZeneca and Abbvie. Borge M received a scientific research grant from Microsules and compensation as speaker from Bristol-Myers Squibb. The remaining authors declare no competing financial interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional information",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598-021-92412-8. \n\nCorrespondence and requests for materials should be addressed to M. B. \n\nReprints and permissions information is available at www. nature. com/reprints.",
"section_name": "Supplementary Information",
"section_num": null
}
] |
10.1186/s12890-019-0977-5
|
miR-34a in serum is involved in mild-to-moderate COPD in women exposed to biomass smoke
|
<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Chronic obstructive pulmonary disease (COPD) is characterized by persistent respiratory symptoms and airflow limitation that is due to airway and/or alveolar abnormalities. The main causes of COPD are Gene-environment interactions associated with tobacco smoking (COPD-TS) and biomass smoke (COPD-BS). It is well know that microRNAs (miRNAs) participate in the control of post-transcriptional regulation and are involved in COPD-TS; nevertheless, those miRNAS are participating in the COPD-BS are unidentified. Thus, we studied which miRNAs are involved in COPD-BS (GOLD stages I–II).</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>In the screening phase, the profile of the miRNAs was analyzed in serum samples (<jats:italic>n</jats:italic> = 3) by means of a PCR array. Subsequently, the miRNAs were validated with RT-qPCR (<jats:italic>n</jats:italic> = 25) in the corresponding study groups. Additionally, the serum concentration of Notch1 was measured comparing COPD-BS vs COPD-TS.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>miR-34a was down-regulated in COPD- BS vs COPD-TS. In the other study groups, three miRNAs were differentially expressed: miR-374a was down-regulated in COPD-BS vs C, miR-191-5p was up-regulated in COPD-BS vs H-BS, and miR-21-5p was down-regulated in COPD-TS compared to the C group. Moreover, the serum concentration of Notch1, one of the targets of miR-34a, was increased in COPD-BS compared to women with COPD-TS.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>This is the first study in patients with COPD due to biomass that demonstrates miRNA expression differences between patients. The observations support the concept that COPD by biomass has a different phenotype than COPD due to tobacco smoking, which could have important implications for the treatment of these diseases.</jats:p></jats:sec>
|
[
{
"section_content": "Chronic obstructive pulmonary disease (COPD) is a common, preventable and treatable disease, characterized by persistent respiratory symptoms and airflow limitation. COPD is caused by exposure to noxious particles or gases [1] ; tobacco smoke inhalation is a fundamental cause of COPD and affects both genders. \n\nBiomass smoke, such as that produced by wood combustion for cooking, is another risk factor that disproportionally affects women, particularly in low and middle-income countries [2]. \n\nCurrently, the COPD phenotype by biomass is considered different from that caused by tobacco smoke. Unlike COPD caused by tobacco, biomass COPD tends to remain in GOLD I and II stages [3] [4] [5] [6], and rarely progresses to emphysema [5]. Several hypotheses have been proposed to explain the plateau in the development of COPD by biomass. Among them, early airway remodeling is the most accepted explanation, since longitudinal studies have shown a different pattern in airway remodeling in these women. Still, the specific mechanisms that differentiate the phenotype of COPD by tobacco and biomass are largely unknown. \n\nBiomass COPD is usually characterized by chronic bronchitis, persistent cough and phlegm. Recently, the role of miRNAs in the pathophysiology of COPD has been explored, increasing our understanding of their role in the development of phenotypic heterogeneity of COPD. miR-NAs could help to the differences in COPD phenotypes; studies in tobacco COPD have reported differential expression of miR-20, miR-28-3p, miR-34c-5p, miR-100 and miR-7 in smokers, ex-smokers and non-smokers. \n\nThese miRNAs are involved in cancer detection, protein coding of inflammatory factors, macrophages and vascular inflammation regulators [7] [8] [9]. There are not reports regarding to determine the participation of miR-NAs in COPD by biomass. \n\nWe aimed to compare the expression of microRNAs in women with COPD due to biomass and tobacco smoke, as will as in control women, and to determine and quantify the target of miRNAs that are being differentially expressed by COPD phenotypes.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "A total of 125 women divided in five groups of 25 participants were recruited for the study. We included women with biomass COPD (COPD-BS), tobacco COPD (COPD-TS), smokers without COPD (H-TS), biomass exposed without COPD (H-BS), and healthy female controls (C), whom had not history of exposure to TS or BS, and with absence of any other respiratory or non-respiratory disease as controls (see Fig. 1 ). The diagnosis of COPD was established according to the history of smoking or exposure to BS and pulmonary function tests followed the recommendations of the American Thoracic Society and European Respiratory Society [1] and using standardized references for Mexican population [10, 11]. All women with COPD had I-II GOLD stages. \n\nDemographic, anthropometric and clinical data were collected including TS history (> 10 packs/year) and cumulative exposure to BS in hours/year by determining the average number of hours/day of exposure and the number of years of exposure; no patient with COPD was exposed to both factors. Wood was the only fuel used by women with COPD-BS, who came from rural and suburban, low-income regions of Mexico.",
"section_name": "Study population",
"section_num": null
},
{
"section_content": "Five mL of blood were collected in anticoagulant-free tubes (BD VACUTAINER, Becton, Franklin Lakes, NJ, USA), following the standard procedures at the INER, which included morning only bleeding with at least 8 h fasting. Samples were centrifuged at 5000 g X 15 min and room temperature, to obtain the serum, which was kept at -20 °C until their analysis.",
"section_name": "Blood samples",
"section_num": null
},
{
"section_content": "The extraction of the miRNAs was performed using the QIAGEN miRNeasy serum/plasma kit (Hilden, Germany) following the manufacturer's instructions. Aliquots of 200 μL of serum were transferred into 2 mL tubes; QIAzol lysis reagent, 3. 5 μL of spike (control), 1. 6 × 10 8 copies/μL, and 200 μL of chloroform were added and subsequently centrifuged at 12,000 g X 15 min at 4 °C. Aqueous phase was separated and 1. 5 volumes of 100% ethanol were added; later, an aliquot of 700 μL was passed through a 2 mL RNeasy spin MinElute column and centrifuged at 8000 g X 15 s. Then, 700 μL of Buffer RWT were added to the RNeasy spin MinElute column, which was centrifuged at 8000 g X 15 s followed by the addition of 500 μL of Buffer RPE. The resulting miRNA was eluted with 20 μL of RNase-free water by centrifugation at 10,000 g X 1 min. The miRNA was quantified, and integrity was assessed with the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).",
"section_name": "Isolation of serum microRNA",
"section_num": null
},
{
"section_content": "Serum miRNAs measurement was conducted in two stages: a screening stage to identify miRNAs differentially expressed in a small subsample of each participating group and a validation stage to confirm that such miRNAs were indeed different in all participants. In the screening stage we conducted a miRNAs-wide analysis, which included 96 miRNAs, using samples from three randomly selected patients from each study group. Quantitative real-time PCR (RT-qPCR) was used with the miScript miRNA PCR Array Human Serum/Plasma kit from QIA-GEN (Hilden, Germany) using the StepOnePlus™ Real-Time PCR System (Applied Biosystems-Real-Time PCR systems Foster City, California, USA). The data analysis was performed using software provided by the manufacturer (available at https://www. qiagen. com/ch/shop/ genes-and-pathways/data-analysis-center-overview-page/). Once miRNAs differentially expressed were identified, we implemented the validation stage in the remaining 25 participants of each group. The validation was performed by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), obtaining the cDNA of the miRNAs extracted with the RT kit and amplified withTaqMan Universal Master Mix II with the UNG kit, all from Applied Biosystems by Thermo Fisher Scientific (USA). Pre-designed commercial assays for each miRNA were obtained from Thermo Fisher Scientific: hsa-miR-150-5p (Assay ID 000473), hsa-miR-223-3p (Assay ID 0002295), hsa-miR-191-5p (Assay ID 002299), hsa-miR-374a-5p (Assay ID 000563), hsa-miR-21-5p (Assay ID 000397) and hsa-miR-34a-5p (Assay ID 000426). The expression level of each miRNA was evaluated using the comparative threshold cycle method (ΔΔCt) and normalized with a corresponding miRNA sequence from C. elegans as an exogenous normalizer in gene expression (spike-in cel-miR-39). The relative concentration of each miRNA was described by the equation ΔCt = (Ct miRNA-Ct spike). The cut-off value was set as the cycle ≤40 and it was considered that a gene was not detectable when the Ct was > 40 and the signal was under established limits [12, 13].",
"section_name": "RT-qPCR Array assay in serum",
"section_num": null
},
{
"section_content": "The serum concentration of the Notch1 protein, whose mRNA is the target of miR-34a, was performed using an ELISA kit (R & D Systems Human Notch1 DuoSet ELISA), following the manufacturer's instructions.",
"section_name": "Protein quantification",
"section_num": null
},
{
"section_content": "To obtain the sample size, the free software G Power (version 3. 1. 9. 2; Heinrich-Heine-Universität, Düsseldorf, Germany) was used. According to the results obtained in the screening phase where we found down-regulation of miR-34a, we calculated the sample size from 2 proportions to 30% between patients with COPD due to biomass and COPD due to tobacco. \n\nThe demographic and clinical characteristics of the study populations were expressed as mean ± SD. The statistical analysis was carried out by means of ANOVA Tukey's posthoc test to multiple comparisons and the differences, while comparison between two groups were determined by Student's t-test. The statistical analysis for qPCR array was perform with the Qiagen software (available at https://www. qiagen. com/us/shop/genes-and-pathways/data-analysis-center-overview-page/). RT-qPCR was analyzed by relative quantification (ΔΔCt method). The differential expression of a miRNA, and the Notch1 protein quantification was also evaluated by Student's t-test. The analyses were performed using the statistical package GraphPad version 6. 01 (Graph-Pad Software, Inc., La Jolla, CA, USA). P values less than 0. 05 were considered significant in all cases.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Table 1 shows the anthropometric, clinical, and physiological characteristics of the groups. FEV 1 % pred, and the FEV 1 /FVC ratio in both groups of women with COPD showed differences when compared with the H-BS, H-TS and C groups (P < 0. 01) with no difference between the groups with COPD. Women with COPD-TS, COPD-BS, H-TS and H-BS were shorter than the C (P < 0. 01). The average exposure to BS in the COPD-BS group was 361 ± 177 h/year, while in the COPD-TS group; there was an average cumulative tobacco consumption of 36 ± 23 packs/year.",
"section_name": "Patient characteristics",
"section_num": null
},
{
"section_content": "The analyses of expression of miRNAs were performed on samples from 3 women chosen in a simple random way in each study group. Six miRNAs were differentially expressed; 3 were up-regulation, miR-150-5p, miR-191-5p and miR-223-3p, in the COPD-BS group compared with the H-BS group, and the remaining 3 were downregulated, miR-374a-5p in the COPD-BS group compared with C, miR-21-5p in the COPD-TS group compared with C, and miR-34a-5p which was downregulated in the COPD-BS group compared with the COPD-TS group (Table 2 ).",
"section_name": "Differential expression of miRNAs in serum by PCR arrays",
"section_num": null
},
{
"section_content": "To validate the differentially expressed miRNAs obtained in the PCR matrices (n = 3), the cDNAs were obtained using the RT kit and TaqMan Universal Master Mix II with UNG (Applied Biosystems-Thermo Fisher Scientific). The six miRNAs validated by RT-qPCR (n = 25) were as follows: miRNA-34a-5p was down-regulated in the COPD-BS compared with the COPD-TS group (Fig. 2 ; P < 0. 001), miR-374a-5p was down-regulated in the COPD-BS compared with the controls (Fig. 3 ; P < 0. 001), miR-150-5p that was down-regulated in the PCR array analysis did not correspond with the study being decreased by RT-qPCR (Fig. 4a ; P < 0. 01). The same result was observed with miR-223-3p (Fig. 4b ; P < 0. 001), when comparing women from the COPD-BS group with those from the H-BS group, while miR-191-5p corresponded with the PCR result and was up-regulated in the COPD-BS group compared with the H-BS group (Fig. 4c ; P < 0. 01), and miR-21-5p was down-regulated in the COPD-TS group compared with the controls (Fig. 5 ; P < 0. 05).",
"section_name": "Validation of miRNAs by RT-qPCR",
"section_num": null
},
{
"section_content": "To interpret the possible biological relevance of the detected miRNAs in the pathogenesis of COPD, an analysis of the targets of miR-34a-5p was performed specifically because of the subexpression observed in the COPD-BS group compared with the COPD-TS group. \n\nThe target was searched in the updated database DIANA TOOLS, miRTarBase-bio. tools and TargetScan. The investigated focused on the participation of miR-34a in COPD-TS and other pulmonary diseases resulting in the Notch1 protein. The serum concentration of Notch1 was quantified by ELISA and was elevated in women in the COPD-BS group compared with women in the COPD-TS group (Fig. 6 ; P < 0. 001) and exhibited an inverse association with the expression of miR-34a-5p.",
"section_name": "Serum Notch1 concentration",
"section_num": null
},
{
"section_content": "We aimed to analyze the differential expression of miR-NAs across five groups of participants with and without COPD by tobacco and biomass exposure. The main finding was that miR-34a down-regulated was differentially expressed between COPD-TS and COPD-BS. We detected differences in 5 miRNAs, miR-374a miR-191-5p, miR-21-5p, miR-150, miR-223, yet, these differences were not statistically different between COPD-TS and COPD-BS. \n\nA differential expression of miR-34 in TS-COPD compared to healthy subjects has been reported; however, in the TS-COPD case miR-34 was up-regulated, with a consequent activation of p53. The expression of miR-34 has been also linked to the severity of TS-COPD, suggesting that miR-34a contributes to the pathogenesis of COPD, by activation in the HIF-1α pathway (hypoxia-inducible factor) [14]. Another study reported that miR-34a activation is induced by oxidative stress through PI3K (phosphoinositide-3-kinase) signaling, and it is implicated in aging responses to oxidative stress; thus, miR-34a could become a new therapeutic target and biomarker in COPD and age-related diseases driven by oxidative stress [15]. Contrary to the up-regulated of miR34a in COPD-TS, our results in COPD-BS are down-regulated, which probably gives us the preliminary basis for inferring that miR-34a could distinguish the genotypic characteristics of COPD-BS patients with respect to COPD-TS patients. \n\nTo understand one of the possible biological implications of the down-regulation of miR-34a in COPD-BS, one of its targets, the Notch1, was selected. miR-34a reduces the action of the Notch1 pathway, which plays an important role in the differentiation of the epithelium in the human airway. It has been observed that the addition of a Notch1 ligand, or the constitutive expression of its receptor, increases the number of mucosal cells containing MUC5AC and the number of secretory cells [16, 17]. Focusing on our findings, we can infer that patients with COPD by biomass, which have miR-34a down-regulated, do not supress the activation of Notch1 signaling, increasing the number of secretory cells, as has been shown in in vitro studies [17]. Our finding provides a potential explanation for the chronic bronchitis clinical expression of COPD-BS, likely mediated by the downregulation of miR-34a. This is relevant, because the regulation of Notch 1 could represent an important therapeutic target for these patients. \n\nAnother interesting finding of our study is the differential expression of three miRNAs between the study groups: miR-374a down-regulated in the group C vs COPD-BS, miR-191 up-regulated in the group H vs COPD-BS and miR-21 down-regulated in the group C vs COPD-TS. The miR-374a has been reported to be up-regulated in skeletal muscle in patients with COPD-TS, and associated with the development of extrapulmonary manifestations and co-morbidities in COPD-TS [18]. Base on the findings we suggest that it could be a good indicator of the comorbidities of COPD-BS patients, although more in-depth studies are needed to determine this possibility. miR-191, has been reported to be up-regulated in lung tissue and bronchoalveolar lavage (BAL) of mice exposed to TS, associated with the development of inflammatory cells in lung and lung parenchyma [19]. The results suggest that COPD due to tobacco and biomass could share the same pathway of inflammation; however, our results need to be evaluated with more studies. Another miRNA validated was miR-21, this miR in COPD-TS has been reported as upregulated in asymptomatic smokers [20]. Another study demonstrated that up-regulated of miR-21 in plasma and mononuclear cells of patients with COPD-TS may contribute to their pathogenesis and severity [21],\n\nsuggesting that high serum plasma levels of miR-21 may be a diagnostic and therapeutic indicator in COPD-TS [20], so that, our results were consistent with previous studies.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "The limitations of this study are related to its sample size. We used a small number of participants in the screening phase, to identify key miRNAs to be later validated in the total sample. This procedure will lead to the identification of miRNAs that are very different across groups but will fail to identify miRNAs that are more similar, which could cloud our understanding of partially expressed miRNAs. For this purpose, a larger sample size is needed. Additionally, only Notch1 was quantified, one of the many targets of miR-34a. Still, this limited analysis allowed us to begin to understand the relevance of miRNAs in COPD-BS, considering the great complexity of this disease. Coupled with this, characteristics such as the socio-economic level, the level of education, ethnic origin, genetic susceptibility and various other environmental factors, as well as the type and severity of exposure to BS or TS in the study groups could influence our results.",
"section_name": "Limitations of the study",
"section_num": null
},
{
"section_content": "This is the first study in patients with COPD due to biomass that demonstrates the genotypic difference between patients determined by miRNAs, supporting that COPD by biomass has a different genotype than COPD due to tobacco, which could have important implications for the treatment of these diseases.",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We are grateful to PhD. Eduardo Montes Martinez at Clinica of Asthma ), and to MSc. Christian Adolfo Trejo Jasso for his valuable advice and help in the development of RT-qPCR and ELISA techniques at National Institute of Respiratory Diseases Ismael Cosio Villegas (INER).",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This research was support by the Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico ; grant number: FOSISS; SALUD-2016-1-272301. The role of the funding of this study included the acquisition of reactive, consumables, laboratory equipment and all necessary to develop the research, CONACyT did not participate directly in the collection, analysis and interpretation of data, nor in the writing of the manuscript.",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Abbreviations BMI: Body mass index; BS: Biomass smoke exposure; C: Control healthy women; COPD-BS: COPD by biomass smoke exposure; COPD-TS: COPD by tobacco smoking; FEV 1 % pred: forced expiratory volume in the 1st sec (% predicted); FVC% pred: forced vital capacity (% predicted); GOLD: Global Initiative for Chronic Obstructive Lung Disease; H-BS: Women exposed to BS without COPD; H-TS: Smokers without COPD; miR: microRNA; miRNAs: MicroRNAs; RT-qPCR: Real-time reverse transcriptionase-PCR; TS: Tobacco smoking Authors' contributions YVT, VRL, OPB, CR, and MM made substantial contributions to conception and design. YVT, VRL, IBR, OPB, ARV, JPR, CR, and MM made acquisition of data. YVT, VRL, OPB, RFV, CR, and MM made analysis and interpretation of data. YVT, VRL, OPB, RFV, CR, and MM have been involved in drafting the manuscript. YVT, VRL, OPB, RFV, CR, and MM have been involved in revising it critically for important intellectual content. All authors have given final approval of the version to be published and agreed to be accountable for all aspects of the work.",
"section_name": "",
"section_num": ""
},
{
"section_content": "The Science, Bioethics and Biosafety Committees of the National Institute of Respiratory Diseases Ismael Cosío Villegas approved the study (INER) in Mexico City. All participants of the study were recruited at the COPD Clinic of INER, a signed informed consent form was obtained from all participants. The research was developed according with the Official Mexican Standard NOM-012-SSA3-2012, which establishes the criteria for the execution of research projects for human health. The protocol approved at INER was the B15 15.",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "",
"section_name": "Consent for publication Not applicable",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.1186/s12938-021-00973-6
|
The global burden and attributable risk factors of chronic lymphocytic leukemia in 204 countries and territories from 1990 to 2019: analysis based on the global burden of disease study 2019
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Chronic lymphocytic leukemia (CLL) is the most prevalent subtype of leukemia in Western countries, causing a substantial health burden on patients and society. Comprehensive evaluation of the epidemiological characteristics of CLL is warranted, especially in the current context of global population aging. The main objective of this study is evaluating the disease burden of CLL at global, regional, and national levels from 1990 to 2019. As secondary objectives, we studied the influence of demographic factors and performed risk factor analysis. We hope this study could provide evidence for the evaluation of the effectiveness of previous prevention strategies and the formulation of future global health policies.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>Based on data of CLL between 1990 to 2019 from the Global Burden of Disease (GBD) study 2019, we depicted the age, gender, and regional structure of the CLL burden population and described the impact of social development on the disease burden of CLL. The distribution and changing trends of attributable risk factors were also investigated. The global burden of CLL has increased dramatically. A high incidence has been achieved in males and elder people. Countries and territories with high social-demographic index (SDI) tended to have higher global burden than low-SDI region. Of risk factors, high body mass index and smoking were the major contributors for CLL-related mortality and disability adjusted life-years (DALYs).</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>In summary, the global CLL burden continues to rise over the past 30 years. The relocation of medical resource should be considered on a global scale.</jats:p> </jats:sec><jats:sec> <jats:title>Graphical Abstract</jats:title> </jats:sec>
|
[
{
"section_content": "",
"section_name": "Graphical Abstract",
"section_num": null
},
{
"section_content": "Chronic lymphocytic leukemia (CLL) represents a prevalent adult leukemia, which is characterized by abnormal accumulation of immunologically incompetent lymphocytes in blood, bone marrow, lymph nodes, and spleen. CLL accounts for 25-30% of all the leukemia in Western Countries [1], with over 100,000 incidence cases and over 40,000 death cases globally reported in 2019. Epidemiological studies found that the incidence of CLL rises exponentially with age and reaches a peak in elderly populations [2]. The incidence of CLL is approximately 2 times higher in males than that in females [3, 4]. Additionally, markable geographical imbalances were found in CLL-related incidence cases. While CLL is the most prevalent adult leukemia in Western countries, it is relatively rare in Asia, even in Asian immigrants moving to the Western hemisphere [4] [5] [6]. Another specific depiction of CLL epidemiology comes from the Arab world. It is reported that CLL incidence of the Jews in Israel is significantly higher than the Arabs in Israel and the Arabs in the surrounding Middle Eastern countries, which suggested that ethnic factors rather than geographical factors may be a more critical contributor [7]. \n\nDespite of promising results in emerging targeted medications including BCL-2 inhibitor venetoclax and Bruton tyrosine kinase (BTK) inhibitors represented by ibrutinib and zanubrutinib [8] [9] [10] [11], it cannot be neglected that the high-cost treatment and accompanied severe adverse events contributed to a heavy global burden to CLL patients. By far CLL's incidence and mortality are still increasing both in developing and developed countries, with a heterogeneous survival rate correlated with local medical conditions and economic settings [12]. But up to date, previous studies on CLL burden presented several limitations. Some studies only described the epidemiological profile of CLL. The lack of adequate indicators to reflect CLL's disease burden has weakened the intuitive understanding of the hazards of disease burden [13]. Besides, most studies focused on the disease burden of CLL in a localized region, within most underdeveloped areas ignored [14, 15]. In addition, significant differences of disease burden patterns were existed in heterogeneous types of leukemias. Previously, most studies using the Global Burden of Disease (GBD) platform focused on the entire leukemia category, leading to a neglect of the specific internal genetic differences existing across leukemia [16, 17]. Thus, there is no guarantee that the evidence offered by these studies could provide accurate guidance on targeting CLL burden management. In addition to the existence of the above-mentioned scientific gaps, we also considered that apart from genetic factors, social development and medical advance have also caused significant changes in the disease burden patterns of CLL. Therefore, an updated and comprehensive picture of global CLL-contributed disease burden is warranted for assessing the status of public health and formulating future policies of medical resource allocation. \n\nThe GBD study 2019 assessed epidemiologic data about 369 diseases across 204 countries and territories and provided an unprecedented opportunity to understand the trends in the global burden of CLL [18, 19]. In this study, we collected disability adjusted life-years (DALYs) from the GBD study 2019 to intuitively reflect the burden of disease and then calculated estimated annual percentage changes (EAPCs) to evaluate the disease burden changing trends. Based on the depict of overall trend of the disease burden caused by CLL from 1990 to 2019, we investigated the impact of age, gender, region and socio-demographic development level on the CLL burden. Further we performed a risk factor analysis on CLL-contributed disease burden to clarify the distribution and changing trend of risk factors. \n\nTo our best knowledge, this study is the first study to provide a comprehensive description of the epidemiology and global burden of CLL worldwide. Moreover, this study also extended the following hypotheses: (1) The higher the proportion of men in the population structure, the greater the disease burden of CLL; (2) The more serious the degree of aging, the greater the disease burden of CLL; (3) The higher the degree of social development, which could be measured by Social-demographic index (SDI), the greater the disease burden of CLL. Up to date, since the health care policies, epidemiology and disease burden trends of CLL have changed over time, understanding the updated CLLcontributed disease burden is critical to assess the effectiveness of previous prevention strategies. Besides, this study can also provide certain instructive evidence for the design of the follow-up health policies. This study described the age, gender, and regional structure of CLL-burdened population. Policymakers should consider the demographic structure of local CLL-burdened populations and social development level to adjust local medical care policies. In addition, the lifestyle and occupational environment of patients also have impact on their survival. Finally, knowledge of the attributable risk factors of CLL provides theoretical basis and specific directions for CLL prevention and management.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "",
"section_name": "CLL-related incidence, death, DALY burden and corresponding change trends",
"section_num": null
},
{
"section_content": "Globally, during the last 30 years, CLL-related incidence cases increased significantly from 40,537 in 1990 to 103,467 in 2019, with age-standardized incidence rate (ASIR) rising from 0. 76/100,000 persons in 1990 to 1. 34/100,000 persons in 2019 (EAPC: 1. 86, 95% CI: 1. 79-1. 92) (Table 1 ). Based on SDI-stratified regional analysis, the number of incidence cases and respective ASIR increased in all SDI categories between 1990 and 2019, with high-SDI quintiles exhibiting the highest ASIR in 2019. Of note, the most rapid increase was observed in middle-SDI quintiles. \n\nIn the geographical region levels, Western Europe, High-income North America, and Central Europe displayed the highest ASIR in 2019, while East Asia, Central Europe, and Andean Latin America showed rapidest growth. In the country or territory level, of 204 countries and territories, the USA, China, and India were the 3 countries with the highest incidence cases of CLL in 2019 (Additional file 1: Table S1 ) (Fig. 1a ). Croatia, Monaco, and Slovenia displayed the highest ASIR in 2019 (Additional file 1: Table S2 ) (Fig. 2a ).",
"section_name": "CLL-related incidence and its change trends",
"section_num": null
},
{
"section_content": "Global deaths cases of CLL had a prompt growth from 21,548 in 1990 to 44,613 in 2019, with age-standardized death rate (ASDR) rising from 0. 40/100,000 persons in 1990 to 0. 58/100,000 persons in 2019 (EAPC: 1. 17, 95% CI: 1. 07-1. 27) (Table 2 ). \n\nBased on SDI-stratified regional analysis, the number of death cases and corresponding ASDR increased in all SDI categories between 1990 and 2019, with high-SDI quintiles exhibiting the highest ASDR in 2019. Of note, the promptest death was observed in middle-SDI quintiles, consistent with incidence trends. \n\nIn the geographical region levels, the highest ASDR was found in Central Europe, Western Europe, and High-income North America in 2019. East Asia, Central Europe, and Andean Latin America showed the rapidest growth. In the country or territory level, India, the USA, and China were the 3 countries with the highest death cases of CLL in 2019 (Additional file 1: Table S3 ) (Fig. 1b ). Croatia, Latvia, and Lithuania displayed the highest ASDR in 2019 (Additional file 1: Table S4 ) (Fig. 2b ).",
"section_name": "CLL-related death and its change trends",
"section_num": null
},
{
"section_content": "Global DALY cases of CLL increased rapidly from 492,075 in 1990 to 948,464 in 2019, with age-standardized DALY rate rising from 9. 20/100,000 persons in 1990 to 12. 26/100,000 persons in 2019 (EAPC:0. 92, 95% CI: 0. 90-0. 94) (Table 3 ). \n\nCLL, chronic lymphocytic leukemia; ASIR, age-standardized incidence rate; EAPC, estimated annual percentage changes; SDI, social-demographic index According to SDI-stratified regional analysis, the number of DALY cases and corresponding age-standardized DALY rate increased in all SDI stratifications from 1990 to 2019. Among all SDI stratifications, high-SDI quintiles had the highest age-standardized DALY rate. Of note, the promptest DALY was observed in middle-SDI quintiles, consistent with incidence, also consistent with incidence trends. \n\nIn the geographical region levels, Central Europe, Western Europe, and Eastern Europe exhibited the highest age-standardized DALY rate in 2019, while East Asia, Central Europe, and Andean Latin America showed the rapidest growth. In the country or territory level, China, India, and the USA were the 3 countries burdened with the highest number of CLL DALY cases in 2019 (Additional file 1: Table S5 ) (Fig. 1c ). Latvia, Croatia, and Poland displayed the highest age-standardized DALY rate in 2019 (Additional file 1: Table S6 ) (Fig. 2c ).",
"section_name": "CLL-related DALY burden and its change trends",
"section_num": null
},
{
"section_content": "The incidence, death, and DALY burden differ by sex. Males were more likely to suffer from CLL than females (male: female in ASIR = 1. 13:1 in 1990, and 1. 14:1 in 2019) (Fig. 3a ). Whereas, the death gap between males and females continuously shrank and even reversed in the past 30 years (male: female in ASIR = 1. 05:1 in 1990, and 0. 98:1 in 2019) (Fig. 3b ). DALY burden of CLL was heavier in males than females (male: female in age-standardized DALY rate = 1. 12:1 in 1990, and 1. 04:1 in 2019), with an ongoing narrowing gap (Fig. 3c ). \n\nWe then evaluated CLL incidence and ASIR in 3 different age groups: 15-49 years, 50-69 years, and above 70 years in the globe and different regions based on SDI levels. The results revealed that most incidences occurred in the population with 50 years of age or older. Furthermore, in the high-SDI region, the incidence cases of patients above 70 years occupied the most proportion. While in low-SDI region, the incidence cases of patients aged 50-69 years accounted for the highest percentage (Fig. 4a ). In all age groups, patients aged above 70 years displayed the highest incidence rate, especially in the high-SDI region (Fig. 4b ).",
"section_name": "Sex and age patterns of CLL",
"section_num": null
},
{
"section_content": "We evaluated the relationship between ASIR of CLL in 1990 and corresponding EAPC and found that that the EAPC of ASIR was negatively correlated with ASIR (correlation coefficient = -0. 19, P = 0. 0058), indicating that the CLL incidence of countries and territories with low ASIR could be substantially underestimated (Fig. 5 ). Then, we investigated the correlation between SDI and EAPC values of ASIR, ASDR, and agestandardized DALY rate in 21 geographical regions across the globe. All age standardized ratios (ASRs) values displayed an apparent positive correlation with SDI (correlation coefficient of ASIR = 0. 70, of ASDR = 0. 68, of age-standardized DALY rate = 0. 67, all P values < 0. 0001) (Fig. 6a-c ), indicated that a heavier disease burden was more likely to be found in higher SDI regions.",
"section_name": "The correlation between SDI and CLL's disease burden",
"section_num": null
},
{
"section_content": "Based on GBD study 2019, four potential CLL-related mortality and DALY attributable risk factors including high body mass index, occupational exposure to benzene, occupational exposure to formaldehyde, and smoking were identified. Among these risk factors, smoking was the strongest risk factor to CLL-mediated death and DALY from 1990 to 2019 at a global scale (Fig. 7a-d ). Of note, compared with high-SDI areas, the proportion of CLL's disease burden attributable to high body mass index in low-SDI areas has a significant upward trend. In addition, although the percent of CLL deaths and DALYs attributed to occupational carcinogen-exposure only accounted for a very small proportion, a significantly higher risk of carcinogen exposure was found in low-SDI regions compared to high-SDI regions. \n\nCLL, chronic lymphocytic leukemia; DALY, disability adjusted life-year; EAPC, estimated annual percentage changes; SDI, social-demographic index",
"section_name": "CLL burden attributable risk factors",
"section_num": null
},
{
"section_content": "Our research focused on the current status and trends of the global burden of CLL based on the latest GBD study 2019 database. In this study, we collected the global incidence, mortality, DALY data attributable to CLL and evaluated the epidemiological trends from 1990 to 2019. The results revealed that global burden disease of CLL presented a constant growing trend during the past 30 years. Previous epidemiologic evidence has demonstrated that CLL predominately occurred in the elderly, with median diagnostic age above 70 years-old [2]. In our study, an age distribution of over 50 was found occupied the vast majority of CLL population, which is consistent with the previous reports. Aging affects the hematopoietic system. The aging of hematopoietic stem cells is accompanied by a series of biological changes including the accumulation of genomic damage, epigenetic changes, telomere shortening, and oxidative stress, which are closely associated to the occurrence of abnormal clonal hematopoiesis [20]. According to reports, in CLL, the ability to produce cloned B cells may have been acquired at the stage of hematopoietic stem cells [21]. Therefore, hematopoietic stem cells that have accumulated multiple key mutations accompanying aging are considered to play a central role in the occurrence of CLL. Recurrent Fig. 4 The incidence cases (A) and corresponding asir (B) of CLL across different age groups from 1990 to 2017 in the globe and various regions stratified by SDI. CLL, chronic lymphocytic leukemia; ASIR, age-standardized incidence rate; SDI, socio-demographic index mutated genes such as NOTCH1, MYD88, TP53, etc. have been identified in the occurrence of CLL [22]. Furthermore, based on the SDI-stratified regional analysis, we revealed that the majority of incidence cases were between 50 and 69 in regions with low SDI, while more than half of the incidence cases were over 70 in regions with high SDI. A generally expanding aging of the population in regions with high SDI may account for the differences between two regions. Besides, advanced ages imply a worse prognosis, accompanied with an increased disease burden due to bad health status and poor tolerance to chemotherapy toxicity [23]. Therefore, preferential attention should be paid to the rapid increase of CLL considering the current context of global population aging. \n\nIn the global context, the incidence and mortality of CLL in both genders displayed an increasing trend during the past 30 years, with males presenting a relatively larger proportion. Moreover, we found significantly divergent gender distribution of disease burden among different SDI regions. In regions with low SDI, we found that females account for the majority of incidence and mortality, which is in contrast to the global trends. Although several epidemiological studies in underdeveloped areas have reported similar gender ratio as our investigations, these studies are primarily limited to local place and contained insufficient sample size [24]. Even considering the poor health of local females due to unequal social status and the stronger tendency for females to seek medical assistance compared to males [25], the available evidence is not sufficient to fully explain the contrast incidence. Given the rapid increase in the incidence of CLL in these areas, it is necessary to conduct in-depth multi-center, large-sample CLL epidemiological investigations. Additionally, policies and strategies to improve the health status of women in these regions should be a priority. As for geographical variation factors, the results revealed that the incidence and mortality of CLL were higher in North America, Europe, and Australia, and lower in Asia, which is approximately consistent with previous studies recognized Caucasian race as risk factors [4]. Interestingly, when focused on the Middle East, we found that although CLL generally presented a low incidence in the Arab world, it is clearly manifested as a high burden disease pattern in Israel. Such situation could be explained by the main demographic composition of Israel which is dominated by Jews, and we speculated that other social environmental factors may also contribute to this difference. \n\nTo our knowledge, variations were observed in CLL burden across different SDI quintiles, with apparently heavier burden in regions with higher SDI. Notably, middle-SDI regions presented rapidly increasing incidence and mortality trends compared with high-or low-SDI regions. The possible explanation might be the huge imbalances of local healthcare environment and settings existing worldwide. Wide coverage of cancer screening and demographic characteristics of aging population are prevalent in high-SDI regions, contributing to a relatively stable disease burden growth trend although with high incident rates. In low-SDI regions, a long-standing lack of screening conditions, possible missed diagnosis, and incomplete case reports caused certain detection biases, leading to constant underestimation of the incidence and mortality. Besides, a catch-up development in annual increases of SDI was recorded in some developing countries [19]. In recent years, given that the improvement of basic medical conditions and the emphasis on early prevention in some middle-SDI regions, lost morbidity and mortality due to missed diagnosis and case underreporting are reducing gradually, thus exhibiting a trend of rapidly increasing disease burden [19]. Overall, these findings prompted us to rationally mobilize existing resources to accurately evaluate and reduce the CLL burden in less-developed areas, which required enhanced disease detection and early treatment management. \n\nWe also investigated the risk factors that affected CLL-related mortality and DALY. Smoking is the major contributor of the 4 risk factors across the world during the last 30 years. More than 60 compounds were identified as known carcinogens in tobacco smoke [26]. Although there is lack of definite association between tobacco use and CLL incidence up to date [27], various cohort studies have reported the association between smoking and the occurrence of myeloproliferative tumors [28, 29]. A striking variation in smoking rates was found among countries in a study investigating global trends for tobacco use from 1990 to 2010, with tobacco use in high-income countries effectively controlled, which may be partly attributed to the increase in awareness of smoking cessation and implementation of national tobacco control policies. Unfortunately, smoking prevalence is on the rise in low-income countries, suggesting that some interventions such as tobacco excise taxes and smoking cessation propaganda should be considered [30, 31]. \n\nHigh body mass index also behaves as an important risk factor for CLL, with a rapidly increasing trend especially in low-SDI regions. The difference in lifestyle between developed and underdeveloped regions may explain this difference in trends. In the past few decades, dependence on processed foods brought a high-sugar and fat diet across the world. Besides, mechanized production has replaced the original manual labor in most areas. These changes have accelerated a global obesity pandemic, which is even severer among low-and middle-income populations [32]. A meta-analysis on the correlation with obesity during adulthood and risk of lympho-hematopoietic cancers revealed that general adiposity in adulthood and early adulthood may increase the risk of CLL [33]. \n\nLater, several studies analyzing the impact of obesity on CLL patients indicated that a poorer baseline response to induction treatment, a lower complete remission rate, and a reduced progression-free survival time were observed in obese patients [34, 35]. According to these evidences, additional attention should be paid to advocate healthy diet and reasonable exercise in the public. \n\nBesides, the exposure risk of benzene and formaldehyde is significantly higher in low-SDI regions than in high-SDI regions. Inhalation is the predominant way for occupational exposure to benzene and formaldehyde. A study evaluating the genetic effects of long-term occupational exposure to formaldehyde showed that long-term exposure to formaldehyde caused higher frequencies of micronuclei in nasal cells and higher frequency of sister chromatid exchanges of peripheral lymphocytes [36]. A meta-analysis accessing the correlation between benzene exposure and leukemia showed that exposure to benzene at work increased the risk of AML and CLL in a dose-response pattern [37]. People at high risk of exposure to benzene and formaldehyde include workers in paint factory, shoe factory, furniture factory, and decorator. With globalization, a large number of manufacturing factories have moved to underdeveloped regions [38]. Meanwhile, strict control of carcinogenic occupations prevalent in developed countries has not been fully implemented in underdeveloped regions. Therefore, special attention should be paid to the risk of occupational exposure to carcinogens in these underdeveloped areas. \n\nSince there are limited available epidemiological studies on CLL in a global perspective, our research based on GBD study 2019 provides the latest global epidemiological distribution and trends on CLL for future research. However, several limitations are unavoidable in the study. As mentioned above, although GBD covers the data of disease burden in most countries and regions in the world, morbidity and mortality in underdeveloped regions may be underestimated due to missed diagnosis and lack of reliable disease information systems. This detection error is difficult to be completely corrected by the subsequent re-allocation algorithm of GBD. Secondly, GBD includes classifications of disease populations in geographical areas, but lacks ethnicity data to facilitate the analysis of genetic susceptibility. Thirdly, limited potential risk factors of CLL are included in the GBD, hampered further research on the distribution and trends of CLL risk factors.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In summary, the global burden of CLL has maintained a gradually increasing trend from 1990 to 2019. The disease tended to occur in males, the elderly populations, and people living in high-SDI regions. What cannot be ignored is the rapid growth of the disease burden in middle-SDI regions, which potentially indicated an underestimated incidence and mortality in underdeveloped countries. In addition, of attributable risk factors, smoking presented as the most contributed across the globe, with potential risk of carcinogen exposure containing a prominent issue in low-SDI regions which needs further investigation. Based on the evaluation of the increasing CLL global burden trends and the highly heterogeneous distribution pattern, policy-makers could assess the effectiveness of previous prevention strategies and rationally adjust the follow-up health policies to alleviate the growing burden.",
"section_name": "Conclusion",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "GBD study 2019 data resources were available online from the Global Health Data Exchange (GHDx) query tool (http:// ghdx. healt hdata. org/ gbd-resul ts-tool), containing a global collection of epidemiological data evaluating burden of disease worldwide with 369 diseases across 204 countries and territories [39]. The data for this study were collected from GBD study 2019, including the following epidemiological data:",
"section_name": "Data source",
"section_num": null
},
{
"section_content": "CLL-related incidences, deaths, DALYs, corresponding ASRs and the SDI are directly provided by the GBD study 2019. ASRs (including ASIRs, ASDRs, age-standardized DALY rates) could eliminate the interference of age structure and population size. DALY, refers to the total healthy life years lost due to disease from morbidity to death, is obtained by adding up the years lived with disability (YLDs) and the years of life lost (YLLs). SDI is an intuitive indicator which reflects the degree of social development [40]. The value of SDI which ranged from 0 to 1 were calculated through the comprehensive evaluation of fertility, education, and income to reflect the degree of social development. Countries and territories were then stratified into five levels (high SDI, high-middle SDI, middle SDI, low-middle SDI, and low SDI) according to SDI values obtained from GHDx. The attributable risk factors for CLL from the GBD study 2019 include: high body mass index, smoking, exposure to benzene, and exposure to formaldehyde. This study collected the above risk factors and presented the results through data visualization. \n\nTo assess trends in the CLL's disease burden, EAPC values (including EAPC based on ASIR, ASDR, and age-standardized DALY rate per 100,000 persons) were employed in this study. Taking the calendar year as the independent variable x while taking the natural logarithmic transformation of the ASR as the dependent variable y. A regression equation was fitted to the natural logarithm of the rates, i. e., y = α + βx, where x = year and y = ln (ASR). Further EAPC values were calculated according to the formula EAPC = 100* (e β -1). For the 95% confidence intervals obtained from the regression, if there is an overlap within 0 in the 95% confidence intervals, then the corresponding ASR is regarded stable. If the 95% confidence intervals fall entirely below 0, then the corresponding ASR is regarded declined, otherwise considered to be elevated [40]. \n\nTo assess whether the ASIR in low-incidence areas has been underestimated, we calculated the Pearson correlation coefficient between ASIR-based EAPC and ASIR in 204 countries and territories in 1990. Then, to analyze the correlation between the ASR trends and SDI, we calculated the Pearson correlation coefficient between the ASRs and SDI values of 21 geographical regions from 1990 to 2019. The Pearson correlation coefficient, ranged from -1 to 1, was used as a measure of linear correlation between two variables. The closer the absolute value of the correlation coefficient is to 1, the stronger the relevance [39]. \n\nAll data analyses in this study were performed with R software (R software, version 4. 0. 0). The R packages employed include: dplyr, stringr, Rcan, maps, and ggplot2. Dplyr and stringr were used for data collation. Rcan was used to calculate EAPC. Maps and ggplot2 were used for visualization and further correlation coefficient calculation. The map was used to present the current status of CLL burden worldwide. The histogram was used to show the changing trend of CLL-mediated disease burden. The scatter plot and regression curve were used for correlation analysis between ASRs and SDI. All tests were two-tailed, and a P value of less than 0. 05 was considered as statistically significant.",
"section_name": "Statistical analysis and data visualization",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Not applicable.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This work was supported in part by National Natural Science Foundation of China (No. 81800146 ); Key research and development program of Zhejiang Province, China (No. 2021C03123 ); Key international cooperation projects of the National Natural Science Foundation of China (No. 81820108004 ); Youth Natural Science Foundation of Zhejiang Province, China ( LQ18H080001 ).",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "The datasets generated and/or analyzed during the current study are available from the Global Health Data Exchange query tool (http:// ghdx. healt hdata. org/ gbd-resul ts-tool).",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "CLL: Chronic lymphocytic leukemia; GBD: Global Burden of Disease; SDI: Social-demographic index; DALY: Disability adjusted life-year; BTK: Bruton tyrosine kinase; EAPC: Estimated annual percentage change; ASIR: Age-standardized incidence rate; ASDR: Age-standardized death rate; ASR: Age-standardized rate; GHDx: Global Health Data Exchange; YLD: Year lived with disability; YLL: Year of life lost.",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12938-021-00973-6.",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "Authors' contributions YY, XL, FL contributed to the conception and drafting of the manuscript and figures. JJ and HW reviewed the manuscript and gave final approval of the version to be published. All authors read and approved the final manuscript.",
"section_name": "Additional file 1. Supplementary materials.",
"section_num": null
},
{
"section_content": "Ethics approval and consent to participate Not applicable.",
"section_name": "Declarations",
"section_num": null
},
{
"section_content": "Not applicable.",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. \n\n• fast, convenient online submission • thorough peer review by experienced researchers in your field",
"section_name": "Competing interests",
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},
{
"section_content": "• support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year",
"section_name": "• rapid publication on acceptance",
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{
"section_content": "At BMC, research is always in progress.",
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"section_name": "Publisher's Note",
"section_num": null
}
] |
10.1371/journal.pone.0233672
|
An exon skipping screen identifies antitumor drugs that are potent modulators of pre-mRNA splicing, suggesting new therapeutic applications
|
<jats:title>ABSTRACT</jats:title><jats:p>Agents that modulate pre-mRNA splicing are of interest in multiple therapeutic areas, including cancer. We report our recent screening results with the application of a cell-based Triple Exon Skipping Luciferase Reporter (TESLR) using a library that is composed of FDA approved drugs, clinical compounds, and mechanistically characterized tool compounds. Confirmatory assays showed that three clinical antitumor therapeutic candidates (milciclib, PF-3758309 and PF-030871) are potent splicing modulators and that these drugs are, in fact, nanomolar inhibitors of multiple kinases involved in the regulation the spliceosome. We also report the identification of new SF3B1 antagonists (sudemycinol C and E) and show that these antagonists can be used to develop a displacement assay for SF3B1 small molecule ligands. These results further supports the broad potential for the development of agents that target the spliceosome for the treatment of cancer and other diseases, as well as new avenues for chemotherapeutic discovery.</jats:p>
|
[
{
"section_content": "The use of targeted high-throughput screening (HTS) of recently available compound libraries composed of drugs, clinical compounds and advanced tool compounds offers the biomedical research community the opportunity to elucidate the mechanism of action (MOA), on-target specificity and potential for clinical repositioning of specific drugs, while at the same time developing a refined drug candidate profile for researchers in specific areas of drug discovery and drug development. The spliceosome is accountable for the post-transcriptional processing of pre-mRNA in the cells of metazoans by catalyzing the regulated exclusion of intervening sequences (introns) and the ligation of coding regions (exons) to produce mature mRNAs, and has recently emerged as a novel target in several therapeutic areas. [1] Small molecules that affect AS have been of interest for numerous therapeutic applications since they impact cellular function by modifying the abundance of different splicing isoforms that play a role in numerous disease states. [2] Given the important role that the spliceosome plays in the determination of cellular and organismal phenotypes it is not surprising that the function of the spliceosome is aberrant in most tumors. [3] Indeed, numerous genes are subject to splicing events that can be either oncogenic or serve to limit potential tumorigenesis, examples of this include BCL-X, VEGF-A, FAS, PKM or MDM2. [4] Additionally, numerous recurrent mutations occur in spliceosome regulatory components (including SF3B1, SRSF2, U2AF1 and others) in the myelodysplastic syndromes and other cancers. [5] These mutations result in a 'change in function' of the mutant spliceosome and a consequential change in the AS profile in the cells expressing these mutant proteins. [6] [7] [8] In parallel to these recent discoveries, there has been a proportional upsurge in interest in the potential application of several recently discovered small molecule modulators of pre-mRNA splicing to cancer chemotherapy. [9] [10] [11] This effort has resulted in Phase I clinical studies and advanced pre-clinical development, for a series of ligands of the SF3B1 spliceosomal protein. These innovative drugs include a derivative of the natural product pladienolide (E7107), [12] a synthetic analog of pladienolide [13, 14] (H3B-8800), [15] and sudemycin D6 (SD6) [16] a simplified synthetic analog of a natural product (FR-901,464). [17] SD6 is currently actively advancing through the 'investigational new drug' (IND) development process. Although the natural products which inspired these drugs were initially described as \"splicing inhibitors\", [12, 17] we now know that SF3B1 targeted agents act as potent modulators of AS through a change in 3' splice-site fidelity. [18] [19] [20] Tumor cells exposed to the splicing modulatory natural products (and analogs) display a profound change in AS, [19, 20] which shows similarities to the pharmacology that has been observed with kinase inhibitors that interfere with the regulatory phosphorylation of splicing factors. [10] Although the full range of molecular mechanisms responsible for the tumor selective toxicity of these agents remains to be fully elucidated, several mechanism types have been delineated. An early mechanism class to to be recognized is the sensitivity of tumor cells bearing spliceosomal mutations, for example chronic lymphocytic leukemia (CLL) cells bearing SF3B1 mutations, [21] and myelodysplastic syndrome (MDS) cells carrying U2AF1 mutations. [22] Additionally, it was found that tumors driven by MYC [23] or KRAS [24] are also sensitized to this class of drugs. More recently proposals have appeared for two additional general mechanisms that may account for the observed selective action of SF3B1 targeted agents in certain cancers, the first proposes that ~11% of all cancers have a partial copy of wild-type SF3B1 protein, which renders these tumors sensitive to SF3B1 targeted drugs; [25] another recent publication presents data which is consistent with the idea that certain tumors driven by BCL2A1, BCL2L2 and MCL1 are especially susceptible to SF3B1 targeted agents. [26] It is certainly possible that multiple mechanisms can account for the selective tumor toxicity that has been observed with these agents, which supports the concept that these agents have good potential for broad application in cancer chemotherapy. [9] Given these new insights into the relationships between carcinogenesis and spliceosome function we initiated a project aimed at the discovery of additional small molecules that target the spliceosome. This has been facilitated by our Triple-Exon Skipping Luciferase Reporter (TESLR) cell-based HTS assay, [27] which reports on a particular type of triple-exon skipping event in MDM2 pre-mRNA that we first observed in tumor cells treated with sudemycin analogs. [19] We applied this HTS assay to the unbiased screen of a collection of all FDA approved drugs, bioactive compound collections, and compounds in Phase I through Phase III clinical trials, in order to build on our previously reported pilot screening results with this assay, which identified two known cyclin-dependent kinases (CDK) inhibitors that were found to also inhibit several members of the cdc-like kinase (CLK) family. The hits from this pilot screen were then developed into SRI-30125, a selective inhibitor of CLKs 1, 2, and 4. [28] Importantly this work also showed that aminopurvalanol, [29] a commonly used tool compound that is often considered a \"selective CDK2 inhibitor\" is actually a multi-kinase inhibitor and a potent modulator of AS, which we showed acts through the inhibition of CLKs 1, 2, and 4. [28] This paper describes the results from our clinical compound focused HTS screen (see Fig 1), which led to the identification of three potent post-Phase I splicing modulatory drugs (1, 2, and 3: see Table 1 ), [30] [31] [32] that have not been previously recognized as splicing modulators. These investigational drugs were developed to target oncogenic kinases and were not previously reported to inhibit splicing regulatory kinases. All of these confirmed hits have been previously reported to display potent in vivo antitumor activity, which was proposed to be due to their activity to other kinases (CDK2, PAK4 or FAK, respectively), however we now report that they are also nanomolar inhibitors of subsets of the multiple kinases involved in the regulation of spliceosome activity. These observations also align with the previous report that another clinical antitumor agent CX-4945 (Silmitasertib) inhibits splicing regulatory kinases and modulates alternative splicing. [33] CX-4945 was developed as a CK2 inhibitor but was later recognized to be a modulator of alternative splicing following the initiation of clinical studies. [33] As discussed below, we propose that the splicing modulatory activity may positively contribute to the antitumor activity of all of these clinical drug candidates.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "SK-Mel-2-MDM2-Luc cells were cultured in MEM supplemented with 10% fetal bovine serum, 1 mM pyruvate, and 0. 1 mg/mL of the antibiotic G418; the cells were grown in a 37˚C incubator at 5% CO 2. [28] To perform the TESLR high-throughput screen, white 384 plates were seeded with 20 μL per well of growth medium without G418, at a density of 5,000 cells per well using a Multidrop peristaltic pump (Thermo Scientific). After 18 h at 37˚C in an incubator in 5% CO 2, cells were treated with 100 nL of compound using a pin tool attachment for a Janus MDT automated workstation (Perkin Elmer). SD6 at 5 μM was used as positive control for splicing modulation. After 5 h incubation at 37˚C with 5% CO2, an equal volume of One-Glow XL (Promega) was added using a Matrix WellMate dispenser (Thermo Scientific). Luminescence readings were taken immediately using an Envision multilabel reader (PerkinElmer). We screened 2035 compounds (seven 384-well plates) from SelleckChem at 10 μM in triplicate. The first 2 columns were reserved for the SD6 positive control, while the last two columns contained DMSO as a negative control. Z-scores were calculated from the DMSO-treated negative controls using the following equation: [X-μ(DMSO)]/σ(DMSO), where X = the luminescence intensity for a given well, and μ(DMSO) and σ(DMSO) are the average and standard deviation of the DMSO (calculated for each plate separately). Hits were determined as those wells having a median Z-score greater than 3. There were 12 hits that scored as positives by these criteria in both replicates, and these were repurchased, their purities confirmed by LCMS, and submitted to dose-response testing for confirmation of splicing modulation activity and to determine potency. All other experimental details are included in the Supporting Information section.",
"section_name": "High throughput screening methods",
"section_num": null
},
{
"section_content": "SK-MEL-2/MDM2-Luc stable cells were cultured in MEM medium with Earle's salts and Lglutamine containing 1 mM sodium pyruvate, 10% FCS and 10 mM Hepes and plated at a density of 10,000/well in 96-well plates and incubated overnight at 37˚C in 5% CO 2. The following day, cells were treated with serial dilutions of compounds for 4 hrs, ONE-Glo reagents (Promega) were added to measure the luciferase activity on an EnVision plate reader. SD6 and 0. 5% DMSO were used as positive and negative controls, respectively. Relative luminescent units were plotted against corresponding drug concentrations and fitted with a standard four parameter sigmoidal curve with GraphPad Prism.",
"section_name": "TESLR dose response",
"section_num": null
},
{
"section_content": "Enzymatic biochemical activities were evaluated in radiometric protein kinase assays (Eurofins, Dundee, Scotland). Kinases are incubated with buffer, substrate and [γ-33 P]-ATP (specific activity approx. 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 3% phosphoric acid solution. 10 μL of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting. All compounds are prepared to 50x final assay concentration in 100% DMSO. Positive control wells contain all components of the reaction with 2% DMSO. Blank wells contain all components of the reaction, with a reference inhibitor replacing the compound of interest. Each kinase is assigned a standard assay concentration of ATP within 20 μM of its apparent K m. Compounds were screened at 1 μM in duplicate or tested at 10 concentrations at half log dilution starting from 10 μM in singlicate. Dose response curves were fitted with four parameter logistic curve to obtain IC 50 values. The average coefficient of variation (CV) of the conducted assay. \n\nPercent inhibition was calculated by comparing to the positive control wells that contain all components of the reaction and 2% DMSO instead of compound (0% inhibition), as well as the blank wells that contain all components of the reaction, with a reference inhibitor (100% inhibition). Staurosporine is used as reference for all tested enzymes except for SRPK3 (reference inhibitor: K-252a. Nocardiopsis sp), NEK2 (reference inhibitor: 30% phosphoric acid), and CK2, CK2α2, MAPK1, MAPK2 (reference inhibitor: PKR Inhibitor). See Table 1 below for additional details.",
"section_name": "Biochemical kinase inhibition assays",
"section_num": null
},
{
"section_content": "RH-18 cells were cultured in RPMI 1640 medium containing 10% FCS. Cells are all maintained in humidified incubator with 5% CO2 at 37˚C. Rh18 cells were exposed to 0. 5% DMSO or SD6 10 μM, Cpd1 10 μM, Cpd2 5 μM or Cpd3 10 μM for 4 h. Total RNA was extracted and converted to cDNA. PCR was performed using NEB Q5 Master Mix and specific primers listed below (Table 2 ) according to the manufacturer's instruction and standard PCR protocols: 50 ng cDNA and 30 μl of the final reaction volume was used. PCR products were subjected to 3% agarose gel electrophoresis.",
"section_name": "Confirmation of splicing changes with RT-PCR",
"section_num": null
},
{
"section_content": "HCT116 cells were seeded at 3 million cells per 100 mm culture plate. The next day cells were treated with 5 μM or 10 μM of each compound for 4h and 8h. For harvesting, cells were washed 2X with ice cold PBS and then resuspended in 400 μl of nuclear isolation lysis buffer (150 mM NaCl, 10 mM Tris-HCl pH 7. 4, 1 mM EDTA pH8, 0. 5% NP-40, 1X Halt Protease and Phosphatase Inhibitor). Cells were scraped and transferred to a microfuge tube and spun at 2800 rpm for 5 minutes, 4C. The supernatant was removed and the nuclear pellet was resuspended in a nuclear extraction buffer (400 mM NaCl, 20 mM Hepes pH 7. 9, 1 mM EDTA pH 8, 1 mM DTT, 1X Halt Protease and Phosphatase Inhibitor). The pellet was snap frozen on dry ice and then thawed and spun at 12K rpm for 5 min, 4C. The supernatant was collected (nuclear extract) and protein concentrations determined using BCA Protein Assay Kit. Samples were run alongside molecular weight markers (Invitrogen SeeBlue™ Plus2 #LC5925) on 4-12% Bis-Tris protein gels (Invitrogen# NP0323BOX) using MOPS SDS running buffer (Invitrogen# NP0001). The proteins were transferred to PVDF membrane using a wet transfer system. The membranes were incubated for 30 minutes at room temperature in Odyssey blocking buffer (Li-Cor 927-40000), then incubated overnight at 4˚C with primary antibody in blocking buffer containing 0. 2% Tween-20 and 1h at 4˚C with secondary antibody in blocking buffer containing 0. 2% Tween-20 and 0. 02% SDS. After the secondary antibody incubation, membranes were washed 4X with PBS containing 0. 1% Tween-20 and 1X with PBS before imaging using the Li-Cor Odyssey imaging system. The primary antibodies used were as follows: SR Monoclonal Antibody (16H3) (Thermo Fisher #16H3E8), Phospho-SF3B1 (Thr313) (D8D8V) (Cell Signaling #25009), Anti-SF3B1 antibody [EPR11987(B)] (Abcam#170854), Anti-phosphoepitope SR protein (clone 1H4) (Millipore# MABE50). The secondary antibodies used are as follows: IRDye1 800CW Goat-anti-Mouse (Li-Cor 925-32210) and IRDye1 680RD Goat-anti-Rabbit (Li-Cor 926-68071).",
"section_name": "Western blot methods",
"section_num": null
},
{
"section_content": "SK-MEL-2/Luc-MDM2 stable cells were plated at a density of 20,000 cells/well in 96-well plates and incubated overnight at 37˚C in 5% CO 2. [27, 28] The following day, cells were",
"section_name": "Antagonist screen",
"section_num": null
},
{
"section_content": "Unless otherwise noted, all commercial reagents were obtained from commercially available sources and used without purification. Flash column chromatography was performed on a Biotage SP-1 chromatography system. TLC plates were visualized by exposure to ultraviolet light (254 nm). 1 H and 13 C spectra were recorded using 400 MHz, and 300 MHz, respectively, using CDCl 3, CD 3 OD, or DMSO-d 6 as a solvent. The chemical shifts are reported in parts per million (ppm) relative to residual solvent (for chloroform, δ 7. 24 ppm for 1 H NMR and δ 77. 02 ppm for 13 C NMR. For DMSO, δ 2. 47 ppm for 1 H NMR. For CD 3 OD, δ 49. 00 ppm for 13 C NMR). Coupling constants are reported in hertz (Hz). The following abbreviations are used to designate the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Mass spectra with electrospray ionization (ESI) were recorded on LCQ Fleet Ion Trap Mass Spectrometer (Thermo Scientific) coupled to the Finnigan Surveyor Plus HPLC System (Thermo Scientific). High-resolution mass spectra were recorded on LTQ-Orbitrap XL (Thermo Scientific) using static nanoelectrospray ionization in positive-ion profile mode at a nominal resolution setting of 100,000. Approximately 50 scans were averaged for each sample and the resulting Fourier-transformed frequency-domain spectrum was mass-assigned with calibration constants from an external calibration mixture. Experimental masses and isotope distributions were compared to theoretical values. All compounds reported are of at least 95% purity, as judged by HPLC (Waters XBridge C18, 250 mm X 4. 6 mm ID, 5 μm column; 10 μL injection; 10-100% MeCN/H 2 O + 0. 1% TFA gradient over 15 min; 1 mL/min flow; ESI; positive ion mode; UV detection at a wavelength of 310 or 340 nm).",
"section_name": "Chemistry",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "In order to expand on our initial pilot screen with the TESLR construct integrated into a stable cell-line, [28] we chose to screen the Selleckchem Bioactive Screening library (a collection of 2,035 small molecules), which includes FDA approved drugs, compounds that have entered clinical studies and validated tool compounds, see Fig 1. The TESLR construct is stably integrated into an engineered cell line and produces functional luciferase when three exons are skipped in an MDM2 based minigene construct. [28] This screen showed a low initial hit rate of 11 hits that had significant activity in replicates (see Fig 1 and Table 1 ), which is consistent with our previous experience with a pilot screen. [28]. The 11 initial hits were then tested in a dose-response format in the TESLR assay, which confirmed the activity of three compounds (see Fig 2). The three confirmed hits were milciclib [30] (compound 1, SRI-030867), PF-3758309 [31] (compound 2, SRI-030868) and PF-562271 [32] (compound 3, SRI-030871). These three actives were further confirmed by measuring MDM2 mRNA splicing modulation with RT-PCR using a previously reported gel-based assay that produced the expected MDM2 splicing alterations that are seen with SD6 treatment. Notably, the pattern of AS observed with compounds 1, 2, and 3 was not the same as that observed with SD6 (see Fig 3B ). [19] A dominant AS isoform at size of 1019 bp observed with SD6 treatment was not apparent in the gel with compound 1, 2, or 3 treatment, while an AS isoform of 785 bp was dominant with compound 1 and 2 treatment, while another AS isoform of lesser abundance (~950 bp) appeared with compound 1 and 2 treatment. A similar MDM2 AS isoform was previously reported following sudemycin C1, D1 and E treatment [19, 20]. The MDM2 AS pattern resulting from compound 3 was different than that observed with all other compounds, exhibiting the apperance of a dominant MDM2 transcript (the same size as the DMSO control) and an AS isoform of 785 bp, as shown in Fig 3B. To explore other AS splicing patterns caused by these compounds in additional genes, we chose RBM39 as a model, since it has previously been shown to undergo AS induced by SD6. [20] Interestingly, RBM39 is a member of the U2AF65 family of proteins that co-localizes in the nucleus with core spliceosome proteins and has been shown to play a role in both steroid hormone receptor-mediated transcription and alternative splicing. [34] Sudemycin induced splicing changes for RBM39 have been characterized and confirmed using RT-PCR. [20] Our results for compounds 1, 2 and 3 were consistent with the previously observed sudemycin induced splicing changes. Three differential RBM39 AS isoforms were identified with compound treatment, with a transcript at 673 bp showing the greatest abundance after treatment with compounds 1 and 2 (Fig 3C ). In general, we concluded that AS patterns induced by compounds 1 and 2 were the similar but distinct from the sudemycin induced changes and that these changes are slightly different from the AS patterns resulting from compound 3 treatment. It should be pointed out that these experiments suggest that genes showing AS changes within cells exposed to 1, 2, or 3 have the potential be used as pharmacodynamic biomarkers for these kinases, as we have previously shown for the SF3B1 targeted agent SD6. [27, 35] The three hits are all kinase inhibitors that show potent biochemical and cell-based inhibition of the phosphorylation of substrates All three hits have published discovery-related selectivity data from different small kinase panels, however none of these panels included members of the CMCG kinase family, which are known to be involved in AS regulation. [30] [31] [32]. Our previous pilot screening results [28] together with the fact that these three confirmed hits were known to be kinase inhibitors(see Fig 4 for the chemical structures of these three hits), led us to suspect that these compounds target splicing regulatory kinases. Therefore, we screened these compounds against a panel of kinases known to be involved in the regulation of AS. We then determined the inhibitory IC 50 s for any compound that showed > 50% inhibition at 1 μM for any enzyme in the panel. As shown in Table 3, the confirmed hits showed potent biochemical inhibition of splicing regulatory kinases and other members of the CMCG family, which has not previously been reported for any of these drugs. The relevance of the biochemical data to the cell-based activity of these compounds was demonstrated by measuring total and phosphorylated (phosphor) forms of SR proteins by immunoblots, since these phosphorylation events are known to affect AS (see Fig 4A and 4B ). Additionally, we investigated the effect of these drugs on SF3B1 phosphorylation (see Fig 4C and 4D ), using 0. 5% DMSO as a negative control, since a splicing modulatory natural product has been shown to reduce the phosphorylation of SF3B1. [36] Interestingly, compounds 1, and compound 2 reduced phosphorylation of both SR and SF3B1 proteins significantly in HCT116 and Rh18 cell lines, while treatment SD6 did not inhibit the phosphorylation of SR proteins but did inhibit the phosphorylation of SF3B1, which is expected based on the results with pladienolide B in JeKo-1 cells. [36] Compound 3 treatment inhibited the phosphorylation of SR proteins in both HCT116 and Rh18 cells, but to a lesser extent than compound 1 or 2. Compound 3 also inhibited the phosphorylation of SF3B1 protein in HCT116 cells, but not in Rh18 cells. SR proteins are known to be substrates for CLK and SRPK family members, [37] and DYRK1A has been reported to phosphorylate SF3B1 [38] ; however, the functional role of SF3B1 phosphorylation is not currently well understood. Based on the kinase inhibition profile of these compounds (Table 3 ) the reduction of SR protein phosphorylation may be attributed to inhibition of CLKs for compounds 1 and 2, and the modest inhibition of CLKs by compound 3 may explain the lack of reduction of phosphor-SR protein in cells exposed to this compound. The marked reduction in phosphor SF3B1 by compound 3 points to inhibition of a hypothetical SR kinase that was not included in the panel shown in Table 3. Alternately, it is possible that FAK plays an unrecognized role in the phosphorylation of SF3B1. These results also imply a possible role of SF3B1 phosphorylation in AS, which could be the subject of further experiments. \n\nFocused screening of selective tool compounds and kinase inhibitory drugs uncovered additional active compounds in the TESLR screen that inhibit a subset of splicing regulatory kinases DYRK, CLK, or CK but not SRPK 1 or 2\n\nGiven the potent and previously unrecognized splicing kinase inhibitory activity of these drugs against this kinase panel, we also decided to investigate the activity of other known splicing kinase inhibitory drugs and tool compounds that were not included in the initial TESLR screen. To this end we performed a dose-response screen with Mirk-IN-1 (a known DYRK1A inhibitor), [39] Sphinx31 (a selective SRPK1 inhibitor) [40] dinaciclib (a FDA designated orphan drug and a potent CDK 1, 2, 5, and 9 inhibitor), [41] SRPIN340 (SRPK 1 and 2 inhibitor), [42] SNS-032 (CDK 2,7 and 9 inhibitor), [43] flavopiridol (Alvocidib, a FDA designated orphan drug and a potent pan-CDK inhibitor), [44] palbociclib (a non-selective CDK/multikinase inhibitor that is a FDA designated orphan drug), [45] and silmitasertib (a CK2 inhibitor in Phase II). [33] Of these compounds we found that only Mirk-IN-1, palbociclib and silmitasertib were active (IC 50 s in the range of 10-300 μM) in the TESLR assay, while the other splicing kinase inhibitors showed IC 50 s >> 10 μM in inducing triple exon skipping (Fig 5). This shows that the type of alternate splicing detected by the TESLR assay is not induced by SPRK inhibitors and also indicates that the inhibition of the DYRKs, casein kinase 2 (CK2), or CLKs 1, 2, and/or 4 (and the resulting inhibition of phosphorylation of SR proteins or SF3B1) were the likely causes of the triple-exon skipping observed in the TESLR screen with compounds 1, 2, 3, and palbociclib, although the possibility that inhibition of FAK or other non-designated splicing kinases may also be involved, was not excluded by these studies. These results are completely consistent with recent findings from the chemoproteomic analysis of 243 kinase inhibitory drugs and tool compounds, which showed that drugs (e. g. palbociclib) and tool compounds that were touted as 'selective' actually potently inhibit numerous kinases, in addition to their designated targets. [45] Notably this comprehensive work clearly shows that palbociclib (trade name: Ibrance) has some affinity for CLK1 (IC 50 = 276 nM), [45] which presumably accounts for the modest splicing modulation observed with this approved antitumor drug.",
"section_name": "An unbiased screen of drugs and drug candidates identifies three investigation antitumor drugs that potently induce the same exon skipping event as the SF3B1 targeted antitumor drug SD6",
"section_num": null
},
{
"section_content": "In order to better understand the activity profile of TESLR hits we also decided to develop an improved assay for SF3B1 small molecule binding, since it is always possible that these hits could directly interact with SF3B1. We therefore decided to take advantage of the novel observation from the Jurica lab that 'inactive' analogs of natural product SF3B1 ligands can displace potent natural product splicing modulators in cell-free in vitro systems. [46] Since this report indicated that it was possible that our previously synthesized natural product analogs could be SF3B1 antagonists we screened a set of 14 of 'inactive' (minimal to no TESLR activity and noncytotoxic at the concentrations investigated) sudemycin and herboxidiene analogs in the TESLR assay in the presence of moderate concentrations of SD6. This assay identified two compounds that were able to potently antagonize the activity of SD6 as shown in Fig 6. Using this assay format, we were then able to show that sudemycinol C did not antagonize the TESLR activity of compounds 1, 2, or 3, which indicates that these hits do not bind to the same site on SF3B1 as SD6 (see Fig 7), which is consistent with our proposal that the inhibition of subsets of splicing regulatory kinases accounts for the splicing modulatory activity of these drugs. These results represent the first demonstration of SF3B1 antagonism in cells and the first HTS assay useful for the determination of binding to the splicing modulator binding site of SF3B1.",
"section_name": "Interrogating the SF3B1 small molecule binding site: The development of a competitive antagonist assay",
"section_num": null
},
{
"section_content": "One major goal of this work was the discovery of new splicing modulatory drug-like lead compounds that exhibit similar splicing changes to those observed with the SF3B1 targeted natural products and analogs. To accomplish this, we developed the TESLR screen that was designed based on a prevalent, unusual and characteristic modification of pre-mRNA splicing we observed in tumor cells treated with SF3B1 antitumor natural products and analogs. We are therefore pleased to identify three post-Phase I antitumor drugs that may exhibit a substantial component of their activity through the modulation of splicing in combination with the activity for their designated target. It is interesting to note that though all available drugs and available clinical compounds were included in the screen, only antitumor drugs were identified, though this could simply be due to the fact that large number of kinase inhibitors are under clinical investigation and many investigational antitumor drugs are kinase inhibitors that typically exhibit polypharmacology. [45] Naturally, these results suggest that the repurposing of these multitargeted drugs should be envisioned in light of this new clinically-relevant MOA information. These results also clearly highlight the need for careful scholarship and scrutiny when using 'selective' drugs as tool compounds for experiments in cell biology. [47] Fortunately, an authoritative publication of inhibitor binding data for 243 clinical kinase inhibitors with 220 human kinases makes this important kinase selectivity data readily available to the biomedical science community. [45] In order to evaluate the possible direct interaction of 1, 2, or 3 with the small molecule binding site of the SF3B subunit, we screened a small focused library of analogs of FR-901,464 and herboxidiene that show minimal activity in the TESLR assay, at the concentrations examined. Two of these compounds (sudemycinol C and E) were able to effectively reduce the TESLR activity of SD6 in a dose-dependent manner, presumably by competing with SD6 for the SF3B1 binding site without triggering the modulation of splicing detected by the TESLR assay. This observation is consistent with results from cell-free experiments, reported from the Jurica laboratory, which showed that binding to the small molecule SF3B1 site is necessary, but not sufficient, to induce splicing modulation and reported several different antagonists at this site. [46] Thus we discovered new simple antagonists and extended this type of assay into a format using living cells, which is useful for the high-throughput determination of SF3B1 small molecule binding. Using this screen, we were able to show that the activity seen with 1, 2, or 3 is not affected by the antagonist sudemycinol C, which is consistent with our hypothesis that these drugs exert their action through the inhibition of splicing modulatory kinases. \n\nIn summary, we report the novel characterization of three drugs as splicing modulators 1 (milciclib), 2 (PF-3758309), and 3 (PF-562271) that show potency on par with the SF3B1 targeted drug candidate SD6 in the TESLR screen, which reports on a triple-exon skipping event in MDM2 pre-mRNA that we first observed in tumor cells treated with sudemycin analogs. \n\n[19] We further demonstrated that in addition to modulation of splicing of the MDM2 gene these three drugs differentially induce AS in multiple genes that we examined, further supporting our hypothesis that the splicing modulatory activity is an important aspect of the antitumor activity of these three drugs. We also characterized the MOA of these drugs as the potent biochemical inhibition of a subset of the CMCG family of kinases including DYRK, CLK and CK. Additionally, we confirmed that these compounds show cell-based inhibition of the phosphorylation of SR proteins and SF3B1. Though the functional role of phosphorylation of SF3B1 is not currently fully understood, [38] our results suggest a possible regulatory role of this phosphorylation event on alternative splicing, and show that the SF3B1 protein appears to be a substrate for other kinases including the CLKs. \n\nWe also report the discovery of new potent SF3B1 antagonists (sudemycinol C and sudemycinol E) for the SF3B1-sudemycin binding site, and thereby confirm the previous report of the remarkable pharmacology of this critical spliceosome regulatory component. [46] We also show that these newly identified antagonists allow for a facile determination of the compounds that interact with the SF3B1 SD6 binding site, via a simple competition assay coupled to the TESLR splicing reporter screen. Using this new assay, we show that hit compound's splicing modulatory activity in TESLR is not mediated through binding to the SF3B1 small molecule binding site, which is consistent with our hypothesis that they act through their potent inhibition of different splicing factor kinases. Our results also highlight a previously unanticipated portrait of the receptor-like pharmacology of SF3B1 splicing protein that is beginning to emerge. \n\nThese new insights suggest novel opportunities for the future clinical repositioning of these agents for additional indications in oncology, since these drugs have already been evaluated through Phase I or Phase II clinical studies. This improved understanding of the MOA of these clinical agents can lead to the effective translation of basic research results into better informed future clinical studies with these drugs, with an increased likelihood of therapeutic success. Specifically, our work suggests that the pre-clinical efficacy of compounds 1, 2, and 3 should be examined for possible clinical repurposing in MDS, CLL and AML. Our work also adds new tools compounds, and further clarifies the activity of known splicing modulatory compounds for basic research in cell biology. These interconnected outcomes support a growing recognition of the substantial potential of modulators of pre-mRNA splicing in cancer therapy, and also suggest a profile for a new chemotherapeutic class of multi-targeted splicing kinase inhibitors with other potential clinical applications, which is of interest to scientists across a broad range of disciplines from drug discovery through clinical practice in oncology.",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "The authors wish to thank Karen Tenney of the Department of Chemistry and Biochemistry at UCSC for her contributions to the administration and organization of this project. We also gratefully thank Professor Melissa Jurica of the Department of Molecular, Cell & Developmental Biology at UCSC, for her very helpful critical review and edits to the draft manuscript and Dr. Philip M. Potter of St. Jude Children's Research Hospital for helpful discussion.",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This work was supported by the National Institute of Health (NIH https://www. nih. gov)/ National Cancer Institute (NCI https://www. cancer. gov) CA2147590 (to T. R. W. ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.",
"section_name": "",
"section_num": ""
},
{
"section_content": "Funding acquisition: Thomas R. Webb.",
"section_name": "",
"section_num": ""
},
{
"section_content": "All screening protocols and results are available at PubChem: https://pubchem. ncbi. nlm. nih. gov/.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Competing interests:",
"section_num": null
},
{
"section_content": "The authors have declared that no competing interests exist.",
"section_name": "Competing interests:",
"section_num": null
},
{
"section_content": "",
"section_name": "Supporting information",
"section_num": null
}
] |
10.1007/s12032-019-1300-2
|
Examination of clonal evolution in chronic lymphocytic leukemia
|
Chronic lymphocytic leukemia (CLL) is one of the most frequent lymphoproliferative diseases. CLL is characterized by unusual heterogeneity, which probably reflects its biological and genetic lack of homogeneity. Clonal chromosome aberrations belong to the most important prognostic and predictive factors in CLL. This research was aimed at observing clonal evolution in CLL at the chromosomal level, and assessing its clinical significance in relation to selected prognostic factors. The study involved 72 untreated patients with CLL. The preliminary investigations using cytogenetic banding analysis (CBA) and FISH were performed at the time of diagnosis, and again after about 24 months to observe clonal changes (clonal evolution). In addition, other parameters were evaluated, i.e., the expression of ZAP-70 kinase, CD38 antigen, and the mutation statuses of IGVH and NOTCH1 genes. Classic prognostic factors, i.e., categorized ZAP70 and CD38 expressions as well as mutations in IGVH and NOTCH1 genes did not influence the course of clonal evolution in the examined group of patients. Clonal evolution was detected in 45.8% of patients by means of CBA, and in 19.4% patients with FISH. Analysis of chromosomal aberrations in the examined group of patients showed that the incidence of 17p deletions and translocations in karyotypes has a negative impact on overall survival. CE was found to be a risk factor for the occurrence of disease progression (OR = 2.22). Our observations indicate that combined CBA and FISH are the most optimal techniques for monitoring clonal evolution in the course of CLL.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is one of the most common lymphoproliferative diseases that occur in elderly people in Europe and North America. It is a monoclonal disease, characterized by accumulation of small, morphologically mature, resting lymphocytes B in peripheral blood, bone marrow, lymph nodes, and spleen. Leukemic lymphocytes are characterized by a unique immunophenotype, which involves coexpressions of the surface antigens: CD19+, CD20+, CD5+ i, and CD23+; and poor expressions of the IgM and IgD immunoglobulins. CLL cells may show differentiated expressions of CD38, CD25, and CD79b antigens on the surface and ZAP-70 kinase in the cytoplasm [1] [2] [3] [4]. \n\nCLL belongs to the class of heterogenous diseases. There is a group of patients with benign, slow course of disease, which does not require treatment, and another comprising patients with rapid progression despite the use of aggressive therapy. There is also a group of patients with a moderate prognosis. Patients from these prognostic groups differ as to the mutation status of IGVH gene, the expression profile of the genes that encode the proteins ZAP70, LPL, and ADAM29, and expression of the CD38 antigen. In addition, mutations in the NOTCH1 gene were observed in patients with CLL. Mutations in the PEST domain of this gene are an unfavorable prognostic factor. There is also a high risk of CLL transformation in the Richter syndrome in patients with such a mutation [5]. \n\nGenetic alterations which include clonal chromosome aberrations also play an important role. \n\nCommonly used methods for detecting chromosome aberrations accompanying the expansion of a cell clone are the cytogenetic analysis of metaphase cells by banding (CBA) or the molecular techniques such as FISH used to metaphase chromosomes or interphase nuclei. \n\nThe studies using methods of classical cytogenetics have allowed karyotype lesions of a clonal character to be observed in 16-43% of patients with CLL [4, 6]. Unfortunately, in the study of clonal character, the use of methods of classical cytogenetics is associated with multiple restrictions, which are mainly due to the low mitotic index of leukemic cells in cultures in vitro. Thanks to the use of new mitogens in in vitro cultures, the efficiencies of the methods of classical cytogenetics and the assessments of karyotype in CLL patients have been increased. \n\nAt present, the reference method for detecting chromosomal changes is FISH. This technique enables an effective assessment of anomalies in more than 83% of CLL patients [7] [8] [9]. \n\nDetected genome alterations, including clonal chromosomal aberrations, are independent prognostic factors that enable identification of patients with CLL of various clinical course, time to first-line treatment, and overall survival. \n\nThe most frequently detected chromosomal aberrations in CLL by means of these research methods include deletions or translocations of chromosomes 13, 11, 14, 17, and 6 in regions 13q, 11q, 14q, 17p, and 6q, respectively, and trisomy of chromosome 12 [10]. \n\nThe phenomenon of clonal evolution (acquisition of chromosomal abnormalities during the course of the disease) that affects the clinical course of CLL is a subject of increasing attention. Our research focused on the observation of clonal evolution and its clinical significance in the course of CLL in relation to selected prognostic factors.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "The material for the research were peripheral blood lymphocytes from 72 individual subjects with chronic lymphocytic leukemia hospitalized over the years 2005-2015 at the Department and Clinic of Hematooncology and Bone Marrow Transplantation, Medical University, Lublin, Poland. \n\nInformed consent of patients for blood collection and positive opinion of the Bioethics Committee at the Medical University in Lublin (no. KE-0254/153/2010) were obtained. \n\nPatients with CLL were diagnosed according to standard morphological and immunophenotypic criteria [11]. The test group comprised 34 women and 38 men aged 36 to 86 years (on average 63. 22 ± 10. 42). The patients were at different stages of disease according to the classification by Rai et al. 20 patients (28%) was at Rai stage 0, the group of 13 patients (18%) at stage I, 29 patients (40. 2%) at stage II, 7 patients (9. 7%) at stage III while 3 people (4. 1%) at the fourth stage of disease progression. \n\nThe clinical and laboratory characteristics of the CLL patients are shown in Table 1. \n\nCytogenetic analyses (classical cytogenetic tests and FISH) were performed in all patients twice (baseline and follow-up) at the time of diagnosis and prior to possible start of treatment, and repeated after approx. 24 months. \n\nFor all the subjects, immunophenotyping was performed according to standard procedures using the FACSCalibur II flow cytometer from Becton-Dickinson, USA. The analysis of the results was obtained by the CellQuest Pro program (Becton-Dickinson); 10,000 cells for each sample were assessed after incubation with monoclonal mouse anti-human CD5-PE, CD19-PE-Cy5, CD23-FITC, CD38-FITC, and ZAP70 antibodies along with appropriate isotype controls (all from BD Bioscience). Antibodies were applied at 1 µg/100 µL of cell suspension (1 × 10 6 cells in 1% BSA/PBS), and samples were processed according to the manufacturer's instructions. The expression of the CD38 surface antigen and ZAP-70 has been categorised according to existing standards, i. e., high expression for CD38 (> 30%) and high expression for ZAP-70 (> 20%) [11]. Genomic DNA was extracted from 200 µL peripheral blood samples collected in EDTA with GeneMATRIX Quick Blood DNA Purification Kit (EURx, Poland), according to the manufacturer's manual. DNA quality and quantity were assessed by means of NanoDrop 200 spectrophotometer (ThermoScientific) and by agarose gel electrophoresis. For further investigation, samples containing nondegraded, high molecular weight DNA at the concentration of at least 100 ng/µL, with A260/A280 ratio between 1. 8 and 2. 0, were used. \n\nThe most common NOTCH1 mutations located in fragment of exon 34 were analyzed by direct sequencing. DNA was amplified with high fidelity Advantage HD polymerase (Clontech). The assessment of mutation state of the NOTCH1 gene was performed using the sequencing technique using an ABI Prism 3130 Genetic Analyzer (Applied Biosystems) [12]. All primers and conditions were designed at the Department of Cancer Genetics with the Cytogenetic Laboratory, Medical University of Lublin (sequences and detailed conditions are available on request). \n\nTesting of rearrangement and somatic hypermutation of the VDJ genes of the immunoglobulin heavy chain was performed based on the sequencing technique. The results of IGVH genes sequencing were compared with the germ line sequences, available in the Immunogenetics (IMGT) database, using the IMGT/V-QUEST software. The analysis identified the VDJ genes occurring in the rearrangement examined, the number and type of mutations present therein, and determined whether the rearrangement is of a functional nature. Sequences having identity of ≥ 98% with the germ sequence have been considered to be unmutated and those having < 98% have been deemed mutated.",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "The cultures were run in a standard manner in sterile Falcon (Sarstedt) vessels on the RPMI 1640 medium with l-glutamine with 15% inactivated fetal calf serum (FCS) vol/vol (Biomed), 100 U/mL penicillin G sodium salt, 100 µg/mL of streptomycin sulfate, and 0. 25 µg/mL of amphotericin B (Gibco, Invitrogen). The starting culture concentration was 2 × 10 6 cells/mL of the medium. The cultures were run at 37 °C in the atmosphere of 5% CO 2. \n\nTo obtain a large mitotic index, two versions of the culture were assumed. To both a suitable mix of stimulation factors has been added: in version A-PWM-2,5 µg/ mL (Pokeweed Mitogen, SIGMA Aldrich), TPA-10 ng/mL (12-O-tetradecanoylphorbol 13-acetate; Sigma Aldrich), calcium ionophore-7. 5 × 10 -7 mol/mL (4-bromo-calcium ionophore; Sigma Aldrich), oligoCpG DSP30-2 µmol/l (TibMolBiol), in version B-PWM-2. 5 µg/mL, TPA-10 ng/ mL, calcium ionophore 7. 5 × 10 -7 mol/mL, oligoCpG DSP30-2 µmol/, LPS-50 µg/mL (Escherichia coli lipopolysaccharide; Sigma Aldrich). \n\nThe mixtures of stimuli used have been adequately tested and selected for the type of the cell culture concerned [13]. \n\nThe cultures were run for 3 to 5 days and then terminated in a standard manner. The differential staining of metaphases was conducted in the cytogenetic preparations (G and R bands) and analyzed in a Nikon Upright Microscope eclipse Ni-U microscope with the magnification ×1000 under the immersion, lens 100× 1. 45 Plan Apoλ Oil. The chromosome analysis was carried out using the CytoVision computer system from Applied Imaging and Applied Spectral Imaging Case Data Manager Capture and Analysis V7. 1. Cytogenetic changes were described according to ISCN 2016 [14].",
"section_name": "Cytogenetic analyses",
"section_num": null
},
{
"section_content": "Selected chromosomal aberrations in interphase nuclei and metaphase chromosomes were assessed using the FISH method with the use of a fluorochrome-marked probe panel. The set of probes used for the study contained the following single probes: LSI D13S319 (13q14) labeled with Spec-trumOrange, LSI 13 (RB1) labeled with SpectrumOrange, LSI ATM (11q22. 3) labeled with SpectrumOrange, LSI p53 (17p13) SpectrumOrange, SPP 12 labeled with Spectrum-Green or SpectrumOrange, LSI MYB (6q23) labeled with SpectruAqua and the multi-coloured probes: LSI D13S319/ LSI 13q34/CEP12 marked with appropriate SpectrumOrange/SpectrumAqua/SpectrumGreen fluorochromes, LSI p53/LSI ATM probe marked with SpectrumOrange/Spec-trumGreen, LSI IGH/CCND1dual color, dual fusion translocation probe, LSI break apart IGH translocation probe firmy VYSIS-Abbott Molecular. We also used Kreatech 6q21/SE6 probes labeled with SpectrumOrange/SpectrumGreen, MYB deletion from Cytocell. For each of the probes, the cut-off point value was determined based on the tests carried out on the control group (average ± 3SD). It was respectively: for the probe for chromosome centromere 12-5%, for the RB1 gene probe-10%, for probes for del(13) (q14. 3) (D13S319), del(13) (q34) (LAMP1), del(11) (q22. 3) (ATM), del(17) (p13. 1) (TP53) it was 8%, and for probes for del(6) (q21-q23) it was 3%.",
"section_name": "FISH",
"section_num": null
},
{
"section_content": "The distribution of the features analyzed was assessed using the Shapiro-Wilk test. The following tests were used: t-Student, U Mann-Whitney, χ 2 (χ 2 Pearson, χ 2 Fisher). \n\nAccording to the adopted model of research, the odds ratio (OR) and the 95% confidence interval (95% CI) were calculated to estimate the association of important features for the clinical course of leukemia with clonal evolution. Survival was calculated using the Kaplan-Meier estimator. The effect of selected features on survival was assessed by the log-rank test and the F Cox test. The logistic regression model (GLZ Multiple Regression, Probit Model, Best subsets) was used to assess the links between the features under analysis and clonal evolution in the CLL process. The calculations were performed using the Statistica 12 software (StatSoft, Inc. ).",
"section_name": "Statistical analyses",
"section_num": null
},
{
"section_content": "Tests with the use of classical cytogenetics and FISH were performed in all the patients at the time of diagnosis and prior to treatment in patients requiring chemotherapy. In order to observe clonal changes (clonal evolution), the tests were repeated. The second test was assumed to be carried out after about 24 months. The time counted in the months between the tests was: median 25. 00 (range from 11 to 35 months, average 23. 52 ± 15. 00). The shorter time between the tests in some patients was associated with the occurrence of progression. Overall survival (OS) was measured from the diagnosis of CLL and from baseline analysis to the date of the last follow-up visit or death.",
"section_name": "Results",
"section_num": null
},
{
"section_content": "In the first test using classical cytogenetics in 33 patients (45. 83%), normal karyotypes were observed. In the remaining patients, detected clonal chromosome aberrations concerned the number and structure of chromosomes; Table 2. \n\nThe most commonly appearing chromosome aberration was deletion of the long arm of chromosome 13. It occurred in 14 patients (19. 44%), being the only observed chromosome aberration in 12 of those people (16. 67%). The deletion of chromosome 13 concerned a long-arm fragment with the breakpoints: del(13) (q14) or del(13) (q14q21). Moreover, chromosome 13 was, in two cases, involved in translocations. \n\nOther aberrations that occurred at high incidence included deletion of the long arm of chromosome 11 and trisomy of chromosome 12. \n\nThe deletion of the long arm of chromosome 11 was related to the regions of del(11) (q14) or del(11) (q22q23) and occurred in 5 patients (6. 94%). The loss of the entire chromosome 11 was observed in one case, and chromosome translocations were also observed in three patients, in which chromosome 11 was involved. \n\nTrisomy of chromosome 12 occurred in 5 patients (6. 94%).",
"section_name": "Clonal changes detected using classical cytogenetics technique",
"section_num": null
},
{
"section_content": "There were also changes in chromosome 6 in 4 patients (5. 55%). A deletion of a fragment of the long arm of chromosome 6 del(6) (q21) was detected in one patient, and the loss of the whole chromosome 6 was observed in the second patient. In addition, in two cases chromosome 6 was involved in the translocation. \n\nOther interesting changes detected in the karyotype of the subjects were changes related to chromosome 14. \n\nA complex karyotype was found in the test group in 9 patients (12. 5%) (occurrence of three or more chromosome aberrations). \n\nThe second cytogenetic test to analyse karyotypes of the subjects was performed to detect new clonal changes. The emergence of new chromosome aberrations in karyotypes indicates an occurrence of clonal evolution. \n\nDuring the second test, normal karyotype was observed in 17 patients (23. 61%). Clonal changes appeared in 19 (26. 38%) of the patients who had a normal karyotype in the previous test. However, in 15 people (20. 83%), the second test demonstrated the emergence of new clonal chromosome changes. \n\nA complex karyotype was observed in 15 patients (20. 80%). \n\nChromosomal aberrations were related to both the number and structure. The most common was the loss of chromosomes: 13, 14, 16, 17, but there was also trisomy of chromosomes 12, 18 and 21. The structural changes include additions, deletions, and translocations. \n\nAn analysis of the chromosome changes found during the first and second cytogenetic test has revealed the existence of clonal evolution in 33 patients (45. 8%). Clonal changes concern both the number and structure of chromosomes. A comparison of the share of individual chromosome aberrations in clonal evolution in CLL patients detected during the two tests is shown in Fig. 1.",
"section_name": "Changes in chromosome 17 included the loss of the whole chromosome in two cases (2.78%).",
"section_num": null
},
{
"section_content": "Owing to the use of a panel of probes to select chromosome areas and centromeric sequences for specific chromosomes, a broad spectrum of chromosome changes have been detected in the genome in the subjects; Table 2. \n\nIn the first test, the most frequently observed change was deletion of the long arm of chromosome 13. \n\nIn the group tested, in 50 subjects (69. 44%) a deletion was detected in the region 13q14. 3 in the locus D13S319. The deletion occurred as a single change in 25 subjects (34. 72%). In the case of 9 people (12. 50%), the deletion D13S319 was accompanied by a deletion in the RB1 gene (13q14. 2), while 13 patients (18%) had a deletion in D13S319. [20] 13q-13q-,17p-46,XX,del(13) (q14q21) [8] /46,XX [12] 46,XY,t(9;14) (p12;q32) [8] /46,XY [12] 13q-13q-,17p-45,XY,t(9;14) (p12;q32),-13,-16, +mar [6] /46,XY [15] 46,XY, del(13) (q14) [4] /46,XY [24] 13q-,17p-13q-,17p-45,XY,del(13) (q14),-20 [8] /46,XY [12] 46,XX [30] 13q-13q-,17p-46,XX [20] 46,XX [26] 13q biallel 13q biallel 46,XX,del(13) (q14) [11] /46,XX[9] 46,XY [19] 13q-13q-46,XY [24] 46,XY,del(13) (q14q21) [6] /46,XY [12] 13q-,17p-13q-,17p-46,XY,del(13) (q14q21) [ [28] 13q-,11q-13q-,17p-46,XY [24] 46,XY [27] 11q-11q-46,XY [24] 46,XX,del(13) (q12) [3] /46,XX [20] 13q-13q-46,XX,del (13) (q12) [6],46,XX [12] 46,XY [21] 13q-13q-46,XY [20] 46,XY [26] 17p-6q-,17p-46,XY [24] 46,XY [19] 13q-13q-46,XY,del(13) (q14) [6] /46,XY[14] 46,XX,del(13) (q14) [7] /46,XX [13] 13q-13q-,17p-46,XX,del(13) (q14) [8] /46,sl,del(17) (p12) [5] /46,XX [9] 46,XY [22] 13q-13q-46,XY [27] 46,XX,del(13) (q14) [4] /46,XX [15] 13q-13q-46,XX,del(13) (q14) [10] /46,XX [13] 46,XX,del(13) (q12q34) [3] /46,XX [15] 13q-,11q-13q-45,X,-X,del(13) (q12q34) [5] /46,XX [16] 46,XY,del(13) (q14) [8] /46,XY [12] 13q-13q-46,XY,t(6;13) (q15;q14) [2] /46,XY,del(13) (q14) [4] / 46,XY [12] 47,XY, +12 [3] /46,XY [19] +12 +12 47,XY, +12 [4] /46,XY [16] 46,XX,del(13) (q14q21) [5] /46,XX [14] 13q-, +12 13q-, +12 47,XX, +12,del(13) (q14q21)\n\n[cp6]/46,XX [13] 46,XY [25] 13q-,17p-13q-,17p-46,XY [24] 46,XX [21] 13q-13q-46,XX [23] 46,XY [27] 13q-13q biallelic 46,XY[25] 46,XX [22] 13q-13q-46,XX [20] 46,XX,del(13) (q14) [8] /46,XX [24] 13q-13q-46,XX,del(13) (q14) [4] /46,XX [18] 46,XX,del(13) (q14) [4] /46,XX [16] 13q-13q-47,XX,del(13) (q14), +mar [6] The second most frequently observed aberration was a deletion of the long arm of chromosome 11. A deletion of the long arm of chromosome 11 containing ATM (11q22. 3) was observed in 17 patients (23. 61%). \n\nAnother change that was detected with high frequency was the trisomy of chromosome 12. The presence of an additional copy of chromosome 12 was observed in 12 patients (16. 67%). In 8 patients (11. 11%), it was the only chromosomal aberration detected. \n\nIn the first test, a deletion in the short arm of chromosome 17 was detected in 11 patients (15. 27%). The most frequently occurring aberration accompanying a deletion of 17p was a deletion of 13q, and these changes were detected in 7 patients (9. 72%). \n\nAnother aberration was a deletion of the long arm of chromosome 6. Del(6) (q21) was observed in 4 patients (5. 56%). In the case of five patients (6. 94%) no genomic changes were observed at the level of resolution of the method. \n\nIn the second test (during follow-up observation), no chromosomal changes were observed in four patients (5. 55%), and a deletion of a fragment 13q was detected in 50 patients (69. 44%). In 22 patients (30. 55%), a del13q occurred as a single chromosomal aberration. In the whole group, in seven patients (9. 72%) the occurrence of monoand bi-allelic deletions were observed at the locus D13S319. The second most common aberration detected during the second test was a deletion of 17p13 which was found in 17 patients (23. 61%) and a deletion of 11q was observed in 15 patients (20. 83%). A deletion of 11q as a single aberration was observed in 3 patients. \n\nIn the next test, the trisomy of chromosome 12 as the only observed aberration was detected in 12 patients (16. 67%). \n\nThe loss of a fragment of the long arm of chromosome 6 was found in 6 patients (8. 33%). \n\nOwing to the use of the FISH technique, clonal evolution was detected in 14 patients (19. 44%). Clonal changes were associated with the emergence of a deletion of chromosome 17p in 6 patients, deletion of chromosome 6q in two patients, and changes in within chromosome 13: deletion of the RB1 gene in two patients and a bi-allelic deletion of the region 13q14. 2 (D13S319) in 7 patients. \n\nIn addition, 6 patients with clonal evolution (deletion of TP53) were in a high-risk group according to the FISH hierarchical risk category according to the Döhner's classification [10]. The comparison of chromosomal changes detected with the FISH technique during the 1st and 2nd tests is presented in Fig. 2. \n\nClonal evolution (CE) was detected in 33 (45. 8%) of patients after the application of classic cytogenetics techniques, and in 14 patients (19. 4%) after application of the FISH technique. \n\nA comparison of the number (changes) of particular chromosome aberrations detected by both techniques is shown in Table 3.",
"section_name": "Clonal changes detected using the FISH technique",
"section_num": null
},
{
"section_content": "The tests of mutation status of the IGVH genes performed using the sequencing method allowed us to detect changes in the sequence of examined genes in 12 patients (16. 7%), and in 60 (83. 3%) patients, no mutations were detected. \n\nThe performed cytogenetic tests allowed for the identification of occurrence of chromosomal aberrations in 11 patients with the mutation of IGVH. Only in the case of one patient with a mutation, no changes were detected after applying both research techniques. Analysis of the results obtained clearly indicates an increase in the number of chromosomal aberrations in patients without mutations in the IGVH genes. Studies on the mutational status of IGVH genes did not show a statistically significant correlation between the presence of mutations and Rai stage of the disease, age, and sex of patients and the analyzed clinical parameters. \n\nSequencing of a fragment of the NOTCH1 gene, allowed us to detect changes in the sequence of a fragment of exon 34 in 8 patients. All the subjects in whom the mutation was detected had the same change, i. e., a deletion of two nucleotides: cytosine and thymine at position 7544_7545 (7544-7545delCT, P2515fs). \n\nMutation in the NOTCH1 gene was statistically more frequent in men (p < 0. 05), but was not statistically significantly related to the age of patients with CLL. Having performed cytogenetic tests, it was found that the trisomy of chromosome 12 occurred statistically significantly more often in patients with a mutation in NOTCH1 (OR = 7. 00; p < 0. 05). Moreover, it was observed that a deletion of a fragment of the long arm of chromosome 13 del(13q) is more common in patients with no mutations in the NOTCH1 gene (OR = 2. 56). \n\nThe mutation of NOTCH1 was not related in a statistically significant way to clonal evolution evaluated in particular techniques.",
"section_name": "Clonal evolution and mutation status of the genes IGVH and NOTCH1",
"section_num": null
},
{
"section_content": "The expression of the CD38 surface antigen in the group of subjects with CLL was categorized in accordance with the applicable clinical standard, which allowed dividing the subjects into two groups, i. e., those with low expression (< 30%) and high expression (> 30%). In the test group, patients with high CD38 expression constituted 36. 11% while patients with no CD38 expression-63. 89%. The tests using cytogenetic techniques confirmed statistically significantly more frequent occurrence of a deletion of 13q14 (D13S319) in patients with low expression of the CD38 antigen (p < 0. 001). It was also found that high expression of CD38 is a risk factor for the occurrence of chromosome 12 trisomy (OR = 3. 02). The other clonal changes assessed by these methods were not statistically significantly related to CD38 expression. The categorized values of CD38 expression were not statistically significantly related to clonal evolution evaluated by specific techniques. \n\nIn the test group, ZAP-70 positive patients constituted 47. 22% while patients with no ZAP-70 expression-52. 78%. \n\nFISH tests have shown that the deletion of chromosome 11 del(11 q) is statistically more frequent in patients with high-expression of ZAP-70 (p < 0. 05). It was also found that high ZAP-70 values are a risk factor for the occurrence of a deletion of 6q22 (OR = 2. 40). The categorized values of ZAP-70 expression were not statistically significantly related to clonal evolution evaluated by specific techniques. \n\nWithin the group, 57 patients (79. 2%) were provided treatment due to the progressive nature of the disease. This group comprised 25 women and 32 men and the average age was 63. 60 years. \n\nThe time to treatment was 12. 54 months on average (0. 00-110 months). \n\nFurther observation allowed for the conclusion in 15 patients (26. 32%) complete remission, and 32 patients (56. 14%) had partial remission. Progression occurred in 10 patients (17. 54%). \n\nTesting of clonal changes with cytogenetic methods allowed us to observe statistically significantly more frequent occurrence of a deletion of 13q (p < 0. 01) in patients with complete remission. The other clones assessed using this technique were not significantly linked to the total remission in the subjects. \n\nHaving conducted statistical analyses, it was found that the complete remission was not related statistically with clonal evolution in the group of subjects covered by treatment. \n\nThere was no significant link between progression and subjects' sex, but progression occurred more statistically significantly in older patients (p < 0. 01). The analysis of clonal changes in the group of CLL patients treated with cytostatics has demonstrated statistically significantly more frequent occurrence of progression, in the case of the emergence of clones with a deletion of 17p (deletion of TP53) (p < 0. 05). The results obtained with the FISH technique have unambiguously indicated the deletion of 17p as a progression risk factor (OR = 4. 17). The other clones assessed by all the techniques were not significantly linked to the progression of the disease. The measured OR values for the deletion of 11q (OR = 4. 92) and 17p (OR = 4. 00) in classical cytogenetic tests indicate that these aberrations constitute a progression risk factor. \n\nThe statistical analyses carried out point to clonal evolution as a risk factor for the occurrence of progression (OR = 2. 22). \n\nThe average overall survival of the patients covered by the observation was 67. 40 Comparing the relationships between individual chromosomal aberrations detected using methods of classical cytogenetics and FISH, and the overall survival, a statistically significant relationship was found at the occurrence of 17p (del TP53) (p < 0. 0018) (Fig. 3 ), chromosomal translocations and the appearance of complex karyotypes during observation of clonal evolution (p < 0. 0423). \n\nClonal evolution assessed by methods of classical cytogenetics and the FISH method did not significantly affect the survival of patients with CLL.",
"section_name": "Correlations between clonal evolution and cytogenetics and clinical parameters",
"section_num": null
},
{
"section_content": "Genomic research on cancer cells that have been conducted in recent years have allowed noticing high heterogeneity and the presence of clonal populations [15, 16]. It was considered that probably clonal evolution in tumors takes place as a result of competition and interaction between genetically diversified cell clones [17]. The incidence, diversity, and evolutionary dynamics of clonal changes in CLL may contribute to the rate of progression of the disease and response to treatment [18]. In the observation of the process of clonal evolution in CLL essential is the slow growth of leukemia cells compared to other malignant tumors because it may take many months or years before a new cell clone fully replaces the previous clones [18, 19]. \n\nThe acquisition of chromosome anomalies during the course of the disease (clonal evolution -CE) was a rare phenomenon in CLL when using classical cytogenetic methods [20]. The detection of these changes has increased dramatically in recent years by the introduction of stimulating agents, such as DSP30/IL2, in cell cultures. [21]. The phenomenon of clonal evolution was more frequently observed with the use of a more sensitive method, namely the FISH technique [20, 22, 23]. \n\nThere are few reports in the literature on the subject, describing first attempts to compare these two methods in assessing clonal evolution [12, 24]. In our own studies, CE was more often detected by classical cytogenetics methods than by FISH (45. 8% vs. 19. 4%). The results obtained do not differ from the results obtained by other authors. Fegan et al. [25] observed CE with classical cytogenetics methods in 43% of patients with CLL [24]. Haferlach et al. observed clonal evolution detected with classical cytogenetic techniques in 30% of patients, and with the FISH technique in 11. 16% [26]. On the other hand, Wawrzyniak et al. detected CE in 39. 5% of patients with classical techniques and in 26. 3% of patients with the FISH technique [24]. \n\nOur research has shown that the majority of chromosome disorders acquired during CE, detected by classical cytogenetic tests, are structural aberrations including the following deletions: 13q, 17p, 11q, 6q, and translocations involving the chromosomes: 2, 3, 4, 6, 8, 9, 10, 11, 13, 14, and 22. Additional copies of chromosomes 12, 18, and 19 and marker chromosomes were also observed. On the other hand, the most frequently observed CE-related clonal changes detected using FISH are mono-and bi-allelic deletion of 13q14, deletion of 17p13, and deletion of 6q. \n\nIn our research, a comparison of both methods in the detection of so-called high-risk aberrations, i. e., deletions of TP53 or ATM detected with FISH and deletions of 11q22 or abnormalities of 17p detected using methods of classical cytogenetics have shown that FISH is more accurate and reliable in detecting high-risk changes. This aspect of testing is clinically significant due to the time and type of treatment being introduced. \n\nThe recent literature on the subject point to the value of classical cytogenetic studies in accurate determining the prognosis for CLL patients [27] [28] [29] [30]. \n\nRigolin et al. have demonstrated that classical cytogenetic tests are more effective than FISH in detecting complex karyotypes. The presence of complex karyotypes is of a prognostic nature and affects TFT, which was confirmed in clinical observations [28]. \n\nOur tests showed in 7 patients (9. 7%) the incidence of a complex karyotype during clonal evolution. The group of these patients was characterized by the early administration of treatment (shorter TFT), which is consistent with the observations of Rigolin et al. [28]. \n\nIt should be noted that in the course of the observation in the group of CLL patients there was no evidence of the acquisition of trisomy of chromosome 12, which correlates with reports in the literature [29, 30]. \n\nIn our research no relationship was found between clonal evolution and the mutation status in the IGVH genes. \n\nClonal evolution was observed in 8 patients with mutations in the IGVH genes. \n\nMulticenter research on clonal evolution and its relationship with the mutation status in the IGVH genes provide interesting results [26, 31]. The studies by Parikh et al. and their extensive literature review evaluating the prognostic relevance of mutation status of the IGVH genes and the studies using FISH to perform stratification of patients with newly diagnosed and/or previously untreated CLL indicate their reliable and coherent prognostic values [32]. Delgado et al. developed a prognostic system that depended on biomarkers for CLL patients based on the two most important prognostic parameters for this leukemia, i. e., the mutation status of IGVH genes and chromosomal aberration tests using the FISH technique. The researchers distinguished three clinical risk groups. The model developed by them can be extremely useful in clinical practice and used to stratify patients in clinical tests [33]. \n\nA correlation between the occurrence of mutations in the NOTCH1 gene and the presence of trisomy of chromosome 12 (p < 0. 05) was observed in the studies. \n\nMany research teams dealing with similar topics found the occurrence of mutations in the NOTCH1 gene in a small percentage of patients with CLL and these values ranged between 4. 6 and 12% [34] [35] [36]. Balatti et al. [37] observed an increase in the percentage of patients with a mutation in the NOTCH1 gene up to 42% in cases where the mutation was accompanied by trisomy of chromosome 12. There was also a high frequency of this mutation in patients where trisomy of chromosome 12 was accompanied by high expression of ZAP-70 and there was no mutation in the IGVH genes [5, 38]. \n\nThe occurrence of NOTCH1 mutations in CLL patients is considered an unfavourable prognostic factor. The mutation of NOTCH1 is very often accompanied by high expression of the CD38 antigen and the ZAP-70 kinase. It was also observed that in most patients with mutations in NOTCH1 there were no somatic mutations in IGVH, which was associated with adverse prognosis. In addition, attention was drawn to the lack of coexistence of mutations in NOTCH1 in patients' cells and the loss of the TP53 gene or changes in its sequence, the presence of which are an indicator of poor prognosis and shortened survival time [34, 39]. Our own studies did not show statistically significant correlations between clinical/laboratory parameters and the NOTCH1 mutation status in the group of CLL patients. Our studies did not show the presence of somatic mutations in genes for the immunoglobulin heavy chain variable region in patients with a mutation of NOTCH1. Multicentre observations point to significant correlations between the occurrence of mutations in the NOTCH1 gene and cytogenetic aberrations occurring in the course of CLL, especially trisomy of chromosome 12. Co-occurrence of this mutation and trisomy 12 in CLL leukemic cells is associated with an unfavorable prognosis and shortening of overall survival [35] [36] [37] [38] [39] [40] [41]. \n\nThe existence of correlation between the incidence of mutations in the NOTCH1 gene and the presence of trisomy of chromosome 12 was confirmed. This correlation has been confirmed in both research techniques. No relationship between mutations in the NOTCH1 gene and clonal evolution has been found in our research. \n\nParticular attention should be paid to the results of statistical analyses that indicated clonal evolution as a risk factor for the progression in CLL patients. The literature confirm the increased risk of progression in patients with clonal evolution [5, 42]. In our study, the analysis of clonal changes in the group of patients covered by treatment has demonstrated statistically significantly the incidence of progression, when the appearance of clones with a deletion of 17p was observed (deletion of TP53) (p < 0. 05). The emergence of deletions of 11q and 17p during clonal evolution was indicated as risk factors for progression. Further analyses indicated the phenomenon of clonal evolution as a risk factor for progression. \n\nIn our study, clonal evolution has not significantly affected the survival of the CLL patients during the periods under analysis. \n\nThe statistically significant lower OS rates were found in patients with a deletion of 17p (p < 0. 0018) and chromosomal translocations (p < 0. 0423) occurring alone or in complex karyotypes. Similar observations have been published by Wawrzyniak et al. who have not found significant differences in the length of OS in patients with or without clonal evolution [24]. Also Haferlach et al. made similar observations [26]. Lopez et al. in their study observed shorter TFS in patients who had acquired new chromosome abnormalities, but clonal evolution did not have a significant impact on their overall survival time [43]. \n\nHowever, studies conducted by Janssens et al. and Shanafelt et al. showed a relationship between clonal evolution and shortened survival time [29, 44]. \n\nFailure to note the effect of clonal evolution detected with tests of classical cytogenetics and FISH on the survival of CLL patients may result from the presence of other newly acquired aberrations (gene mutations) impossible to detect with these techniques. It is possible that these are mutations of such genes as TP53, ATM, BIRC3, SFB1, MYD88, POT1, CHD2, XPO1, FBXW7, DDX3X, EGR2, NFKBIE, or SETD2 [45]. \n\nThe results obtained showed that conventional karyotyping should be included in the observation of clonal evolution in addition to the FISH technique. These observations confirm the suggestions of many researchers that the assessment of risk of the clinical course of the disease and monitoring the treatment of patients with CLL indicate the need to include conventional karyotyping for screening these patients, similarly to those routinely performed in other hematological disorders [21, 28].",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The authors would like to thank M. Luterek, I. Pilecka, MSc, and M. Wojcierowska-Litwin, MSc for help in performed chromosome banding and genes sequencing.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "Author contributions DK, SP-M, and SZ designed the study, and EWS collected patients' data. DK wrote the manuscript, and AF revised and finally approved of the version to be submitted.",
"section_name": "",
"section_num": ""
},
{
"section_content": "Please contact with authors for any data.",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "Conflict of interest The authors declare no conflict of interest. Informed consent All patients provided informed written consent for all procedures in the study.",
"section_name": "Compliance with ethical standards",
"section_num": null
},
{
"section_content": "Open Access This article is distributed under the terms of the Creative Commons Attribution 4. 0 International License (http://creat iveco mmons. org/licen ses/by/4. 0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. \n\nPublisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Ethic approval",
"section_num": null
}
] |
10.3389/fphar.2021.772576
|
Danzhi Xiaoyao Powder Promotes Neuronal Regeneration by Downregulating Notch Signaling Pathway in the Treatment of Generalized Anxiety Disorder
|
<jats:p><jats:bold>Background:</jats:bold> Generalized anxiety disorder (GAD) is one of the most common types of anxiety disorders with unclear pathogenesis. Our team’s previous research found that extensive neuronal apoptosis and neuronal regeneration disorders occur in the hippocampus of GAD rats. Danzhi Xiaoyao (DZXYS) Powder can improve the anxiety behavior of rats, but its molecular mechanism is not well understood.</jats:p><jats:p><jats:bold>Objective:</jats:bold> This paper discusses whether the pathogenesis of GAD is related to the abnormal expression of Notch signal pathway, and whether the anti-anxiety effect of DZXYS promotes nerve regeneration in the hippocampus by regulating the Notch signaling pathway.</jats:p><jats:p><jats:bold>Methods:</jats:bold> The animal model of GAD was developed by the chronic restraint stress and uncertain empty bottle stimulation method. After the model was successfully established, the rats in the model preparation group were divided into the buspirone, DZXYS, DZXYS + DAPT, and model groups, and were administered the corresponding drug intervention. The changes in body weight and food intake of rats were continuously monitored throughout the process. The changes in anxiety behavior of rats were measured by open field experiment and elevated plus-maze test, and morphological changes and regeneration of neurons in the rat hippocampus were observed by HE staining and double immunofluorescence staining. Changes in the expression of key targets of the Notch signaling pathway in the hippocampus were monitored by real-time fluorescence quantitative PCR and western blotting.</jats:p><jats:p><jats:bold>Results:</jats:bold> In this study, we verified that the GAD model was stable and reliable, and found that the key targets of the Notch signaling pathway (Notch1, Hes1, Hes5, etc.) in the hippocampus of GAD rats were significantly upregulated, leading to the increased proliferation of neural stem cells in the hippocampus and increased differentiation into astrocytes, resulting in neuronal regeneration. DZXYS intervention in GAD rats can improve appetite, promote weight growth, and significantly reverse the anxiety behavior of GAD rats, which can inhibit the upregulation of key targets of the Notch signaling pathway, promote the differentiation of neural stem cells in the hippocampus into neurons, and inhibit their differentiation into astrocytes, thus alleviating anxiety behavior.</jats:p><jats:p><jats:bold>Conclusion:</jats:bold> The occurrence of GAD is closely related to the upregulation of the Notch signaling pathway, which hinders the regeneration of normal neurons in the hippocampus, while DZXYS can downregulate the Notch signaling pathway and promote neuronal regeneration in the hippocampus, thereby relieving anxiety behavior.</jats:p>
|
[
{
"section_content": "Generalized anxiety disorder (GAD) is a chronic anxiety disorder characterized by persistent significant tension, accompanied by autonomic nervous function excitement and over alertness. According to the World Health Organization (WHO) World Mental Health Survey, anxiety disorder is the most common cause of mental illness and disability worldwide, and generalized anxiety disorder (GAD) is one of the most common types of anxiety disorders (Demyttenaere et al., 2004). The global prevalence of anxiety disorder was 7. 3% (Stein et al., 2017). The lifetime prevalence of GAD ranged from 0. 8 to 5. 1% (Kessler, 2000; Hidalgo and Sheehan, 2012), seriously affecting the quality of life of patients. At the same time, GAD is a risk factor for many other diseases, such as alcohol dependence and drug dependence (benzodiazepine dependence) (Tyrer and Baldwin, 2006). Although GAD is common in clinics, it has not been studied as much detail in neurobiology as many other emotional disorders such as depression (Menard et al., 2016), and its pathogenesis remains to be unclear. At present, it is generally believed that its pathogenesis is closely related to neurotransmitter disorders, and neurotransmitter regulating drugs are often used as treatment, and their effects are not ideal and side effects may be obvious, such as drowsiness, dizziness, nausea, and addiction, etc. (Bandelow, 2020). \n\nTraditional Chinese medicine has been widely used in the treatment of emotional diseases for thousands of years. Danzhi Xiaoyao Powder is a classic Chinese prescription that has been used in the clinic. It originated from Xue Ji's Internal Medicine Abstract during the Ming Dynasty. It was modified by the \"Xiaoyao Powder. \" The entire prescription consisted of 10 g danpi, 10 g gardenia, 15 g bupleurum, 10 g Poria cocos, 15 g fried Atractylodes macrocephala, 10 g Angelica sinensis, 10 g white peony, 10 g licorice, 3 g mint, and 9 g ginger, which is widely used in clinical and experimental research on anxiety and depression disorder (Xu et al., 2020). Our team's previous research found extensive neuronal apoptosis and neuronal regeneration disorders in the hippocampus of GAD rats. DZXYS can improve the anxiety behavior of GAD rats, but its mechanism remains to be explored (Dong, 2015). \n\nIn recent years, some scholars have found that the occurrence of depression is related to the decline of adult hippocampal neurons, and stimulating hippocampal neurons is a new strategy for antidepressant treatment (Tanti and Belzung, 2013; Hill et al., 2015; Anacker et al., 2018). The hippocampus has become the focus of research on mental diseases because of its nerve regeneration function and unique regulation of emotion and cognition (Toda et al., 2019). In view of the similarities between depression and anxiety disorders in pathophysiological mechanisms and neuroanatomical changes (Hettema, 2008), we speculate that neuronal apoptosis and neuronal regeneration disorder in the hippocampus of GAD rats found in the previous team's research are also important pathological mechanisms leading to GAD. Hippocampal newborn neurons do not appear spontaneously; they are the result of proliferation and differentiation of neural stem cells in the granular cell layer (subgranular cells of the dentate gyrus, SGZ) of the hippocampal dentate gyrus (Aguilar-Arredondo et al., 2015; Zupanc et al., 2019). The maintenance of neural stem cell proliferation and its differentiation direction and timing are regulated by the Notch signaling pathway (Lutolf et al., 2002; Niessen and Karsan, 2007), which are expressed in the two neurogenic sites of the SGZ and the subventricular zone (SVZ) in the adult brain (Stump et al., 2002), and participate in adult neurogenesis. The activated Notch signaling pathway can maintain the cellular characteristics of NSCs, inhibit their fate of neuronal differentiation, and promote their differentiation into astrocytes (Guo et al., 2009; Snyder et al., 2012). Astrocytes are the type with the largest number of glial cells, and the ratio of their number to the number of neurons is 3: 1 in rodents and 1. 4:1 in the human central nervous system (Sofroniew and Vinters, 2010). Under normal conditions, astrocytes can regulate the concentration of ions inside and outside neurons, nutritional repair, synaptic transmission, and construct a neural tissue grid, forming the blood-brain barrier; however, excessive proliferation can form glial scars, secrete nerve axon regeneration inhibitors, inhibit neuron and nerve axon regeneration, and lead to nerve regeneration disorder (Pekny et al., 2007; Singh and Joshi, 2017). We speculate that the Notch signaling pathway plays an important role in the pathogenesis and treatment of GAD. Therefore, this manuscript discusses the role of Notch signaling pathway in the pathogenesis of GAD and whether the anti-anxiety effect of DZXYS is related to the regulation of Notch signaling pathway to promote nerve regeneration in the hippocampus and improve anxiety behavior (Ren and Zuo, 2012).",
"section_name": "INTRODUCTION",
"section_num": "1"
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": "2"
},
{
"section_content": "All the experiments were approved by the ethics review committee of Shandong University of Traditional Chinese Medicine (No. DWSY201703013) and carried out in accordance with \"the Guidelines for the Care and Use of Experimental Animals of the National Institutes of Health\". All the studies attempted to reduce the pain felt by the animals. Animals: Male Wistar rats (n 120, 6 weeks old, 200 ± 10 g) were purchased from Beijing Vital River Experimental Animal Technology Co., Ltd (Beijing, people's Republic of China; No. SCXK 2016-0006). During the standard 12 h light/dark cycle, the animals were kept at a room temperature of 21 ± 1 °C and relative humidity of 30-40%. After 1 week of adaptation, the rats were randomly divided into two groups according to their body weight: control group (n 24) and model preparation group (n 96). The control group did not have a model, a free diet, or drinking water.",
"section_name": "Animal Preparation",
"section_num": "2.1"
},
{
"section_content": "The GAD model was established in the model preparation group according to the uncertain empty bottle stimulation method and chronic restraint stress. The specific methods were as follows: first, 7 days of regular water feeding training, the rats were given a water bottle filled with water at 9:00-9:10 and 21:00-21:10 every day to drink for 10 min, and the rest of the time spent on removing the water bottle without water, so that the rats were in a thirsty state. After the rats were used to drinking water at two time points every day, from the 8th day to the 28th day, randomly select one time period to feed water to the water bottle for 10 min, and the other time period to stimulate the rats with empty water bottle, observe the rats' behavior of attacking the cage, looking around the water bottle and modification behavior, etc. At the same time, from 9:30 am to 15:30 pm every day, the rats were fixed with tubular rat fixator (Model: HL-DTS, Specification: 20*6 cm, Material: plexiglass, Heli Kechuang Technology Development Co., Ltd, Beijing) for 6 h for 21 days. On the 28th day, the elevated plus maze (EPM) and open field test (OFT) behavioral tests were performed after restraint, and the rats in the model preparation group were divided into DZXYS group, DZXYS + DAPT group, buspirone group, and model group with the time for rats to open arms in the EPM as the baseline, with 24 rats in each group. Different drug interventions were performed. During the drug intervention, the uncertain empty bottle stimulation and restraint methods were still carried out synchronously until the end of the drug intervention. From the beginning of the experiment to the 42nd day, the changes in body weight and food intake were measured every week (Figure 1 ).",
"section_name": "GAD Modeling Method",
"section_num": "2.2"
},
{
"section_content": "The traditional Chinese medicine compound used in the experiment was Danzhi Xiaoyao Powder Formula Granule (1,035,073, Yifang Pharmaceutical Co., Ltd., Guangzhou, China). The granule was dissolved in the distilled water at a concentration of 1. 05 g/ml. The drug concentration has been verified by a previous experiment conducted by the research group, and the curative effect is ideal (Dong Ning, 2015). The dose of Buspirone hydrochloride tablets (H19991024, Enhua Pharmaceutical Co., Ltd., Jiangsu, Beijing) were calculated to be 7 times the adult dosage (20 mg/60 kg/d), i. e., the dosage of rats was 2 mg/kg/d, which was dissolved in distilled water at a concentration of 0. 2 mg/ml (Garakani et al., 2020). The drug has no addiction and has good anti-anxiety effects and has been found to promote neuronal regeneration in the hippocampus as a 5-HT1A receptor agonist (Masayoshi et al., 2014). 5-Bromo-2deoxyuridine (BrdU Sigma Chemical Company, St. Louis, MO) was injected intraperitoneally into rats to label proliferating cells. BrdU solution was prepared from 0. 9% NS at a concentration of 15 mg/ml and a rat injection dose of 50 mg/kg/time. DAPT (r-secret inhibitor) (D7230, Solarbio, Beijing, China)is a Notch signaling pathway inhibitor, dissolve 50 mg DAPT in 1 ml DMSO before use, and then add 0. 9% NS to dilute it into a solution with a concentration of 4. 54 mg/ml, and the dosage of intraperitoneal injection was 20 mg/kg/d (Breunig et al., 2007; Zhang et al., 2019). \n\nIn this experiment, only Notch signaling pathway inhibitors were used without agonists, for two reasons. Firstly, by consulting the literature, we have learned that the activated Notch signaling pathway can maintain the cellular characteristics of NSCs and inhibit their fate of differentiation into neurons, so as to promote the differentiation into astrocytes (Guo et al., 2009; Snyder et al., 2012). We speculate that excessive upregulation of the Notch signaling pathway is an important reason for the excessive proliferation and differentiation of neural stem cells into astrocytes, resulting in the obstacle of neuronal regeneration, and the application of Notch signaling pathway inhibitors can often inhibit the excessive proliferation of neural stem cells and restore the normal differentiation of neural stem cells into neurons (Breunig et al., 2007). If the application of the signal pathway inhibitor can help Chinese medicine reverse anxiety behavior, it confirms our hypothesis, so as to avoid excessive sacrifice of experimental animals, which is also in line with our animal ethical practices. In addition, the commonly used and stable Notch signaling pathway agonists are Notch signal pathway ligands such as Jagged1 and Dll4 (Saffarzadeh et al., 2019). Another important topic of our team was to explore whether DZXYS can regulate Notch ligands. If such agonists are used, our research will be disturbed.",
"section_name": "Drug Preparation",
"section_num": "2.3"
},
{
"section_content": "From the 28th day after successful modeling, the rats in each group were given an oral stainless steel gavage needle 30 min before stimulation with an uncertain empty bottle of water every day. The DZXYS, DZXYS + DAPT and buspirone groups were given DZXYS solution and buspirone solution respectively according to the dosage of 1 ml/100 g body weight, and the blank group and model group were also given 0. 9% NS according to 1 ml/100 g body weight, lasting for 14 days. In addition, rats in the DZXYS + DAPT group were intraperitoneally injected with DAPT and the rats in each group were injected intraperitoneally with BrdU during the modeling period. The injection frequency was as follows: BrdU was injected intraperitoneally twice a day, 5 days before the start of modeling, and then twice a week. One day before death, the rats were injected every 8 h three times (Figure 2 ).",
"section_name": "Drug Administration",
"section_num": "2.4"
},
{
"section_content": "Open-field test: The OFT experiment was conducted on the 28th day after modeling, 7 days after intragastric administration (35th day), and 14 days after administration (42nd day). The animals were placed in a quiet test room illuminated by red light for 30 min in advance to adapt to the environment. The open field is a black square device (length*width*height is 100 cm*100 cm*50 cm), and the bottom of the field is divided into nine equal squares. Each rat was placed separately in the center of the site and allowed to explore freely for 6 min. A camera was installed directly above the open field and connected to a computer on the side. The XR super-maze tracking system records the total distance, speed, time, number of times entering the central area, number of times standing up, and the number of times the horizontal grids were crossed (Ennaceur et al., 2009). After each rat was tested, the site was disinfected with 75% ethanol to remove any residual odor. When the animals stayed around the open field for a significantly longer time than in the central area, it proved that the rats had anxiety tendencies. \n\nElevated Plus Maze: The experiment was conducted in a quiet room illuminated by red light. EPM is a polypropylene plastic cross-shaped device 76 cm above the ground and consists of two closing arms (50 × 10 cm), two open arms (50 × 10 cm), and a central platform (10 × 10 cm). We put the rat's head towards the closed arm on the central platform, and used the XR-super Maze tracking system to record the action track of rats within 6 min, including the frequency (OE) and time (OT) of entering the open arm, and the frequency (CE) and time (CT) of entering the closing arm (Biedermann et al., 2017). OE% and OT% were calculated as follows: OE% OE/(OE + CE) × 100% and OT% OT/(OT + CT) × 100%. If the OE% and OT% of rats were low, the anxiety behavior of rats was obvious.",
"section_name": "Animal Behavior Test",
"section_num": "2.5"
},
{
"section_content": "Pentobarbital (2%) was injected into the abdominal cavity of rats to induce deep anesthesia (40 mg/kg). Some rats were decapitated, and their brains were removed. The hippocampus in the brain tissue was quickly stripped and stored in liquid nitrogen for subsequent western blotting and RT qPCR detection. The remaining rats in each group were perfused with 0. 9% NS 500 ml and 4% paraformaldehyde 150 ml, the brain tissue was quickly removed and soaked in 4% paraformaldehyde, fixed for 24 h, and the area of the hippocampus was trimmed into small pieces with a thickness of 5 mm. The small pieces were dehydrated with gradient concentration of ethanol (75, 85, 95% for 2 h, 100% for 1 h), transparent in xylene (45 min), and soaked in paraffin (three times, 1 h each time). The tissues were embedded in an embedding machine to make paraffin blocks (Mohamed et al., 2017). Each paraffin block was cut into four 4 um pathological sections using a Leica paraffin slicer (Histocore Biocut Paraffin Slicer, Leica, United States).",
"section_name": "Sample Collection and Preparation",
"section_num": "2.6"
},
{
"section_content": "First, paraffin sections of brain tissue from each rat were taken, dewaxed and hydrated, washed with 0. 1 M PBS three times, then immersed in hematoxylin dye for 5 min, washed with tap water after removal, differentiated with 1% hydrochloric acid alcohol for several seconds, returned to blue with 0. 6% ammonia, and rinsed with running water for 5 s. They were dyed in eosin solution for 2 min and rinsed with running water. Finally, the slices were successively placed in gradient alcohol and xylene, dehydrated, transparent, and sealed with neutral gum (Feldman and Delia, 2014).",
"section_name": "HE Staining",
"section_num": "2.7"
},
{
"section_content": "Three paraffin sections were obtained from the brain tissue of each rat. Each section was double-stained with anti-BrdU/anti Nestin (anti-BrdU mouse mAb, GB12051, Servicebio; Anti-Nestin Mouse mAb, GB12137, Servicebio), anti-BrdU/anti NeuN (Anti-NeuN Mouse mAb, GB11138, Servicebio), and anti-BrdU/anti GFAP (anti-GFAP rabbit pAb, GB11096, Servicebio). The staining steps of immunofluorescence double staining are briefly described as follows: First, the sections were dewaxed and hydrated, repaired with EDTA antigen repair solution, and the sections were washed with 0. 1 M PBS, and then incubated in goat serum for 30 min. Each slice was then incubated with different pairs of primary antibodies at 4 °C overnight. The next day, after recovery to room temperature, they were washed with 0. 1 M PBS three times, and mixed secondary antibody (Alexa Fluor ® 488 labeled goat anti-rabbit IgG, GB25303, Servicebio; Cy3 labeled goat anti-rabbit IgG, GB21303, Servicebio), incubated in the dark at room temperature for 20 min, washed with 0. 1 M PBS for 3 times, Cy3-TSA was added dropwise and incubated at room temperature for 10 min, and the nuclei treated with 4′,6diamidino-2-phenylindole (DAPI; ThermoFisher Scientific, Waltham, MA, United States). Finally, two drops of fluorescence quencher were added to each slice to seal it and was observed under an upright fluorescence microscope (Leica SP8, Hamburg, Germany) (Mohan et al., 2008; Lightbody and Nicol, 2019).",
"section_name": "Double Immunofluorescence",
"section_num": "2.8"
},
{
"section_content": "Total RNA was extracted from 60 rat hippocampal tissues using Trizol (CWBIO, CW0580s) according to the manufacturer's instructions. A large amount of cDNA was obtained using a HiFiScript cDNA Synthesis Kit. In the ultrasound mixture realtime fluorescence quantitative PCR system, the detection of fluorescence quantitative PCR is efficient and sensitive. Using 2 -ΔΔCT, the relative expression of each gene was calculated by the CT method, and the expression of each protein was standardized by the GAPDH housekeeping gene (Derveaux et al., 2010). Gene-specific primers for quantitative RT-PCR included: GAPDH forward, 5-CCTTCCGTGTTCCTACCCC-3, GAPDH reverse, 5-GCCCAGGATGCCCTTTAGTG-3; Notch1 forward,5′-TGGATGCCGCTGACCTACG-3, Notch1 reverse, 5-TGGATGCCGCTGACCTACG-3; Hes1 forward, 5′-TTG AGCCAACTGAAAACACTGATT-3′, Hes1 reverse, 5-GTG CTTCACTGTCATTTCCAGAAT-3; Hes5 forward, 5-GAT GCTCAGTCCCAAGGAGAAAA-3, Hes5 reverse, 5-CCACGA GTAACCCTCGCTGTAGT-3'.",
"section_name": "Real Time-qPCR",
"section_num": "2.9"
},
{
"section_content": "Hippocampal samples were dissected on ice, homogenized in icecold lysis buffer, and the protein concentration of the supernatant was measured on a spectrophotometer. A loading buffer was added to the samples to boil them. Then, 50 ug protein was loaded into 10% Bis-Tris gel, and the strip was transferred to a polyvinylidene fluoride membrane (PVDF) and sealed with sealing solution. The primary antibodies Hes1(1:300) (#11988, CST, American), Hes5 (1:300) (22666-1-AP, Proteintech, China), and Notch1 (1:1000) (#36081, CST, United States) were incubated at 4 °C overnight. The secondary antibodies conjugated with horseradish peroxidase were added dropwise on the next day and visualized by enhanced chemiluminescence. The expression of each target gene was standardized using the GAPDH housekeeping gene (Pillai-Kastoori et al., 2020).",
"section_name": "Western Blotting",
"section_num": "2.10"
},
{
"section_content": "The data were analyzed using Graph Pad Prism 7. 00 software (Graph Pad Software, Inc, San Diego, California, United States). Outliers were eliminated by the statistical elution method (values that deviate from the mean ±2 times the standard deviation are excluded) (Nakagawa and Cuthill, 2009). The data were expressed as Mean ± SEM. In all group tests, whether using parametric analysis of variance or repeated measurement of parameters, the data before analysis of variance were subject to a normality test (Kolmogorov-Smirnov test) and variance homogeneity test (Levene test). If not, a nonparametric test was used. All data in this study were normal and homogeneous in terms of variance. The transcriptional expression levels of Notch1, Hes1, and Hes5 mRNA and the protein expression levels of Notch1, Hes1, and Hes5 in the hippocampus were analyzed by one-way ANOVA. Repeated measurement two-way ANOVA was used to analyze the food intake, body mass growth rate, total distance of exercise, distance to the central area, residence time in the central area, number of upright, and BrdU+/Nestin+, BrdU+/NeuN+, BrdU+/ GFAP + expression in the DG area of the hippocampus. For all analyses, if appropriate, post hoc comparisons were performed using the Bonferroni posthoc tests test. The significance level of ANOVA and post-test was set at p < 0. 05.",
"section_name": "Statistical Analysis",
"section_num": "2.11"
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": "3"
},
{
"section_content": "There are significant differences in food intake between groups (Figure 3A ). \n\nThe Bonferroni post hoc test showed that the model group was significantly lower than the control group (p < 0. 0001). There was no significant difference between the drug intervention groups and the model group after 7 days of drug intervention. After 14 days of DZXYS and DZXYS + DAPT intervention, the loss of appetite was reversed (p < 0. 0001 and p < 0. 0001, respectively). This shows that chronic stress stimulation affects the appetite of rats. The intervention of DZXYS or DZXYS + DAPT for a long time can improve the appetite of rats in the model group. Two-way ANOVA showed a drug treatment effect (F 4,52 90. 72, p < 0. 0001) and time effect (F 6,312 673. 5, p < 0. 0001). The growth rate of the body mass of the rats in each group was significantly different (Figure 3B ). On the 42nd day, after stress and drug intervention, there was a significant difference in body mass growth rate between the groups. The Bonferroni post hoc test showed that the model group was significantly lower than the control group (p < 0. 0001). After intervention with buspirone, DZXYS and DZXYS + DAPT, weight gain retardation was reversed on Day 35 and Day 42 respectively (p 0. 0103, p < 0. 0001, p < 0. 0001; p < 0. 0001, p < 0. 0001, p < 0. 0001). The results showed that stress had an impact on the normal physiological metabolism of rats, and the three drug interventions corrected the abnormal physiological metabolism of stressed rats to varying degrees. Two-way ANOVA showed significant drug treatment effect (F 4,52 1631, p < 0. 0001)And time effect (F 6,312 2,700, p < 0. 0001).",
"section_name": "DZXYS Increased Food Intake and Weight Gain in GAD Rats",
"section_num": "3.1"
},
{
"section_content": "In order to observe the effect of DZXYS on the improvement of anxiety behavior in GAD rats, we used the internationally widely used OFT and EPM behavioral testing methods to detect the changes in anxiety behavior in rats. As shown in Figure 4B, there was no significant difference in the total movement distance of rats in each group in the open field at each time node. Two-way ANOVA showed no significant treatment effect (F 4,52 1. 935, p 0. 1184). \n\nThere were significant differences among the groups in the central area exercise distance in the open field experiment (Figure 4C ). The Bonferroni post hoc test showed that the model group was significantly lower than the control group (p < 0. 0001), and buspirone, DZXYS, and DZXYS + DAPT were significantly higher than the model group after 7 days (Day 35) and 14 days (Day 42) (p 0. 0010, p 0. 0009, p < 0. 0001; p < 0. 0001, p < 0. 0001, p < 0. 0001, respectively). Two-way ANOVA showed that the movement distance of the central area had a significant processing effect (F 4,52 63. 28, p < 0. 0001) and time effect (F 2,104 147. 2, p < 0. 0001). The residence time in the central area of the rats in each group was significantly different between the groups (Figure 4D ). The Bonferroni post hoc test showed that the model group was significantly lower than the control group (p < 0. 0001), and buspirone, DZXYS, and DZXYS + DAPT were significantly higher than the model group on the 35th and 42nd days after intervention (p 0. 0377, p 0. 0115, p 0. 0006; p < 0. 0001, p < 0. 0001, p < 0. 0001, respectively). The treatment effect of residence time in the central area was as significant as the time effect (F 4,52 51. 7, p < 0. 0001) (F 2,104 51. 29, p < 0. 0001). The results showed that the model group rats had obvious anxiety-like behaviors. Buspirone, DZXYS, DZXYS, and DAPT alleviated anxiety-like behavior in stressed rats. \n\nThe number of upright times in the open field of rats in each group was significantly different between the groups (Figure 4E ). Bonferroni post hoc analysis showed that the model group was significantly lower than the control group (p < 0. 0001), and buspirone, DZXYS, DZXYS + DAPT were significantly higher than the model group after 7 days (Day 35) and 14 days (Day 42) (p 0. 0021, p 0. 0252, p < 0. 0001; p < 0. 0001, p < 0. 0001, p < 0. 0001, respectively). Two-way ANOVA showed that the number of upright rats had a significant treatment effect (F 4,52 60. 87, p < 0. 0001) and time effect (F 2,104 65. 22, p < 0. 0001). \n\nThe behavioral trajectories of rats in the different treatment groups in the EPM device are shown in Figure 4. In the EPM test, significant differences are observed in the percentage of time entering the open arm (OT%) (Figure 5B ) and the percentage of frequency entering the open arm entrance (OE%) (Figure 5C ). Bonferroni post-test results showed that the OT% and OE% values of the model group were significantly lower than those of the control group (p < 0. 0001, p < 0. 0001), including buspirone and DZXYS after DZXYS + DAPT intervention for 7 days (Day 35) and 14 days (Day 42), The OT% value was significantly higher than that in the model group (p < 0. 0001, p 0. 0046, p < 0. 0001; p < 0. 0001, p < 0. 0001, p < 0. 0001), and OE% was significantly higher than that in the model group (p 0. 0035,p 0. 0176, p 0. 0048; p < 0. 0001, p < 0. 0001, p < 0. 0001). \n\nTwo-way ANOVA showed that OT% had a significant treatment effect (F 4,52 80. 39, p < 0. 0001) and time effect (F 2,104 140, p < 0. 0001). The OE% value drug treatment effect was as significant as the time effect (F 4,52 85. 93, p < 0. 0001) (F 2,104 174. 6, p < 0. 0001).",
"section_name": "DZXYS Reverses Anxiety Behavior in GAD Rats",
"section_num": "3.2"
},
{
"section_content": "We found that after HE staining in the hippocampus of rats in each group (Figure 6 ), the pyramidal nucleus in the hippocampus of rats in the normal group was large and round, the morphology was intact, the cytoplasm was pink, the nucleus was blue, the chromatin was evenly distributed, the nucleolus was obvious, and the connection between cells was tight and arranged neatly, while in the model group, the number and layers of cells in the hippocampus decreased and were disorderly arranged. It can be seen that a large number of neurons have nuclear chromatin gathering on the lower edge of the nuclear membrane, nuclear pyknosis, deep staining, irregular morphology, cytoplasmic concentration, dark red, and even apoptotic bodies. Neurons lack blindness, the arrangement between cells is disordered, loose, and irregular, and there are gaps around the cells. This is especially true in the DG area of the hippocampus. After 7 days of drug intervention (Day 35), the morphology of neurons in the hippocampus of the buspirone, DZXYS, and DZXYS + DAPT groups was slightly improved compared with that of the model group, but the number of cell layers were still less than that of the control group. Some nuclear chromatin gathered under the nuclear membrane, along with nuclear pyknosis, deep staining, and irregular morphology. After 14 days of drug intervention (Day 42), the morphology of hippocampal neurons in the DZXYS + DAPT and buspirone groups was significantly improved compared to that in the model group. Most cells had a clear outline and were arranged closely. Only a small number of nuclei had slight pyknosis and deep staining of the cytoplasm, which was similar to that in the control group. The morphology of hippocampal neurons in the DZXYS group was further improved compared to that at 7 days, but the arrangement was loose, which was slightly worse than that in the DZXYS + DAPT and buspirone groups.",
"section_name": "DZXYS Improves the Morphology of Hippocampal DG Neurons in GAD Rats",
"section_num": "3.3"
},
{
"section_content": "To observe the proliferation of neural stem cells (NSCs), we labeled newly proliferating cells with BrdU (red) antibody and neural stem cells with nestin antibody (neural progenitor cellspecific marker; green) (Bernal and Arranz, 2018) in the dentate gyrus of rats. There were significant differences in the number of newborn NSCs in the DG area of the hippocampus between the groups (Figures 7A-C ). The Bonferroni post hoc test showed that the number of newborn NSCs in the model group was significantly higher than that in the control group (p < 0. 0001), and the number of buspirone, DZXYS, DZXYS + DAPT on the 35th and 42nd days after intervention was significantly lower than that in the model group (p 0. 0004, p 0. 0004, p < 0. 0001; p < 0. 0001, p < 0. 0001, p < 0. 0001, respectively). Two-way ANOVA showed that the number of neonatal NSCs in the DG area of rats had a significant treatment effect (F 4,20 52. 16, p < 0. 0001) and time effect (F 1,20 5. 488, p 0. 0296). \n\nTo observe the differentiation of newborn NSCs into neurons (Zupanc et al., 2019), we double-labeled with anti-BrdU (red) and NeuN (neuron specific protein; green) (Gusel'nikova and Korzhevskiy, 2015) antibodies. After 7 days (Day 35) and 14 days (Day 42) of drug intervention, the number of newborn neurons in the DG of the hippocampus in each group was significantly different between the groups (Figures 8A-C ). The Bonferroni post hoc test showed that the model group was significantly lower than the control group (p < 0. 0001), and the number of newborn neurons in buspirone, DZXYS, and DZXYS + DAPT was significantly higher than that in the model group (p < 0. 0001, p < 0. 0001, p < 0. 0001; p < 0. 0001, p < 0. 0001, p < 0. 0001, respectively). Two-way ANOVA showed that the number of newborn neurons in the DG area had a significant processing effect (F 4,20 50. 39, p < 0. 0001) and a significant time effect (F 1,20 11. 64, p 0. 0028). To observe the differentiation of neonatal NSCs into astrocytes in the rat hippocampus, we double-labeled them with antibodies against BrdU (red) and GFAP (a specific marker of astrocytes; green) (Baba et al., 1997). There was a significant difference in the number of new astrocytes in the DG area of the hippocampus between groups on the 35th and 42nd days after modeling (Figures 9A-C ). Bonferroni post hoc analysis showed that the number of new astrocytes in the model group was significantly higher than that in the control group (p < 0. 0001), and it was significantly lower than that in the model group after buspirone, DZXYS, and DZXYS + DAPT intervention for 7 days (Day 35) and 14 days (Day 42) (p < 0. 0001, p < 0. 0001, p < 0. 001; p < 0. 0001, p < 0. 0001, p < 0. 0001). Two-way ANOVA showed that the number of newborn neurons in the hippocampus had a significant processing effect (F 4,20 119. 2, p < 0. 0001) and a significant time effect (F 1,20 39. 06, p < 0. 0001).",
"section_name": "DZXYS Promotes Neurogenesis in DG Region of Hippocampus in GAD Rats",
"section_num": "3.4"
},
{
"section_content": "DZXYS upregulated the expression of Notch1, Hes1, and Hes5 in the hippocampus, and there was a significant difference in the expression of Notch1 mRNA in the hippocampus of rats in each group (F 7,16 29. 57, p < 0. 0001) (Figure 10A ). The Bonferroni post hoc test showed that the expression of Notch1 mRNA in the model group was significantly higher than that in the control group (1. 783 ± 0. 093 vs. 1. 000 ± 0. 058, p < 0. 0001), and decreased compared to the model group after 7 days of intervention with buspirone, DZXYS, and DZXYS + DAPT (1. 370 ± 0. 012 vs. 1. 783 ± 0. 093, p 0. 0019; 1. 380 ± 0. 047 vs. 1. 783 ± 0. 093, p 0. 0025; 1. 270 ± 0. 017 vs. 1. 783 ± 0. 093, p 0. 0002), and decreased significantly compared with that in the model group after 14 days of drug intervention (0. 970 ± 0. 017 vs. 1. 783 ± 0. 093, p < 0. 0001; 1. 010 ± 0. 067 vs. 1. 783 ± 0. 093, p < 0. 0001; 0. 900 ± 0. 069 vs. 1. 783 ± 0. 093, p < 0. 0001). \n\nThere was a significant difference in the expression of Hes1 mRNA in the hippocampus of rats in different treatment groups (F 7,16 24. 13, p < 0. 0001) (Figure 10B ). Bonferroni post test showed that the expression of Hes1 mRNA in the model group was significantly higher than that in the control group (1. 7 ± 0. 10 vs. 1. 000 ± 0. 115, p < 0. 0001), and decreased after 7 days of intervention with Buspirone, DZXYS, and DZXYS + DAPT (1. 317 ± 0. 017 vs. 1. 7 ± 0. 10, p 0. 0144; 1. 293 ± 0. 007 vs. 1. 7 ± 0. 10, p 0. 0083; 1. 28 ± 0. 042 vs. 1. 7 ± 0. 10, p 0. 0061). After 14 days of drug intervention (Day 42), it was significantly lower than that in the model group (0. 867 ± 0. 073 vs. 1. 7 ± 0. 10, p < 0. 0001; 0. 913 ± 0. 019 vs. 1. 7 ± 0. 10, p < 0. 0001; 0. 783 ± 0. 017 vs. 1. 7 ± 0. 10, p < 0. 0001). \n\nThere was a significant difference in the expression of Hes5 mRNA in the hippocampus of each group (F 7,16 23. 86, p < 0. 0001) (Figure 10C ). The Bonferroni post-test showed that the expression of Hes1 mRNA in the model group was significantly higher than that in the control group (1. 600 ± 0. 052 vs. 1. 000 ± 0. 115, p < 0. 0001), and decreased after 7 days of intervention with Buspirone, DZXYS, and DZXYS + DAPT (1. 18 ± 0. 02 vs 1. 600 ± 0. 052, p 0. 0023; 1. 3 ± 0. 058 vs. 1. 600 ± 0. 052, p 0. 0494; 1. 227 ± 0. 064 vs. 1. 600 ± 0. 052, p 0. 0073). After 14 days of drug intervention (Day 42), the expression was significantly lower than that in the model group (0. 820 ± 0. 035 vs. 1. 600 ± 0. 052, p < 0. 0001; 0. 913 ± 0. 019 vs. 1. 600 ± 0. 052, p < 0. 0001; 0. 783 ± 0. 017 vs. 1. 600 ± 0. 052, p < 0. 0001). \n\nThere was a significant difference in the amount of Notch1 protein in the hippocampus of rats in different treatment groups (F 7,16 207. 3, p < 0. 0001) (Figure 10D ). Bonferroni post hoc analysis showed that the amount of Notch1 protein in the model group was significantly higher than that in the control group (2. 828 ± 0. 037 vs. 1. 000 ± 0. 028, p < 0. 0001, p < 0. 0001). The amount of Notch1 protein in the model group was significantly lower than that in the model group on the 35th day of the experiment after the intervention with buspirone, DZXYS, and DZXYS + DAPT (1. 718 ± 0. 035 vs. 2. 828 ± 0. 037, p < 0. 0001; 1. 957 ± 0. 078 vs. 2. 828 ± 0. 037, p < 0. 0001; 1. 440 ± 0. 059 vs. 2. 828 ± 0. 037, p < 0. 0001). It continued to decline on the 42nd day of the intervention (1. 302 ± 0. 049 vs. 2. 828 ± 0. 037, p < 0. 0001; 1. 255 ± 0. 033 vs. 2. 828 ± 0. 037, p < 0. 0001; 0. 714 ± 0. 026 vs. 2. 828 ± 0. 037, p < 0. 0001). \n\nThere was a significant difference in the amount of Hes1 protein in the hippocampus of rats in different treatment groups (F 7,16 46. 57, p < 0. 0001) (Figure 10E ). Bonferroni's post hoc test showed that the amount of Hes1 protein in the model group was significantly higher than that in the control group (2. 633 ± 0. 033 vs. 0. 995 ± 0. 058, p < 0. 0001). After 7 days of buspirone, DZXYS, and DZXYS + DAPT intervention (Day 35), the amount of Hes1 protein in the model group was significantly lower than that in the model group, especially in the DZXYS + DAPT group (2. 067 ± 0. 067 vs. 2. 633 ± 0. 033, p 0. 02; 2. 067 ± 0. 033 vs. 2. 633 ± 0. 033, p 0. 02; 1. 643 ± 0. 054 vs. 2. 633 ± 0. 033, p < 0. 0001). Fourteen days after drug intervention (Day 42), the amount of Hes1 protein in the model group was significantly lower than that in the model group (1. 327 ± 0. 037 vs. 2. 633 ± 0. 033, p < 0. 0001; 1. 427 ± 0. 239 vs. 2. 633 ± 0. 033, p < 0. 0001; 0. 587 ± 0. 047 vs. 2. 633 ± 0. 033, p < 0. 0001). \n\nThere was a significant difference in the amount of Hes5 protein in the hippocampus of rats in each group (F 7,16 74. 51, p < 0. 0001) (Figure 10F ). Bonferroni post hoc analysis showed that the protein content of Hes1 in the model group was significantly higher than that in the control group (1. 751 ± 0. 055 vs. 1. 000 ± 0. 011, p < 0. 0001). After 7 days of intervention with buspirone, DZXYS, and DZXYS + DAPT (Day 35), the protein content was lower than that in the model group, and the most significant was the DZXYS + DAPT group (1. 542 ± 0. 059 vs. 1. 751 ± 0. 055, p 0. 0295; 1. 542 ± 0. 025 vs. 1. 751 ± 0. 055, p 0. 0304; 1. 260 ± 0. 020 vs. 1. 751 ± 0. 055, p < 0. 0001). Fourteen days after drug intervention (Day 42), the protein content of Hes1 in the model group was significantly lower than that in the model group (1. 063 ± 0. 037 vs. 1. 751 ± 0. 055, p < 0. 0001; 0. 995 ± 0. 039 vs. 1. 751 ± 0. 055, p < 0. 0001; 0. 882 ± 0. 025 vs. 1. 751 ± 0. 055, p < 0. 0001).",
"section_name": "DZXYS Downregulated the Expression of Notch1, Hes1, and Hes5 in the Hippocampus",
"section_num": "3.5"
},
{
"section_content": "The purpose of this study was to explore whether the pathogenesis of GAD is related to the abnormal expression of the Notch signaling pathway, and whether the anti-anxiety effect of Danzhi Xiaoyao Powder can promote nerve regeneration in the hippocampus by regulating the Notch signaling. In this animal experiment, we found that after the intervention of DZXYS and DZXYS + DAPT, the food intake and body weight of rats increased significantly compared with the model group; the anxiety-like behavior of the buspirone, DZXYS, and DZXYS + DAPT groups was significantly reversed after 14 days of drug intervention, especially DZXYS + DAPT; immunofluorescence double staining of the hippocampal DG area of rats in each group showed that there was more proliferation of neonatal NSCs in the DG area of rats in the model group, and a large number of them transformed into astrocytes, resulting in obstacles to neurons. After 14 days of treatment with the three groups of drugs, the number of neonatal NSCs in the DG area of rats gradually decreased, the number of neonatal neurons increased significantly, the number of neonatal astrocytes decreased, and the protein expression of key targets of the Notch signaling pathway (Notch1, Hes1, and Hes5) in the hippocampus was gradually lower than that in the model group, and the protein expression of DZXYS + DAPT group was the most significant. \n\nAt present, the etiology of GAD is unclear, but it is certain that environmental pressure is an important reason for its onset, repeated fluctuation, and deterioration (Wittchen et al., 2000). Based on this, this study used the uncertain empty bottle drinking water stimulation method (Lin Wenjuan, 2003; Zhang et al., 2020) and chronic restraint stress (Ma et al., 2019) preparation of the GAD rat model. After 21 days of chronic stress stimulation, we found that GAD rats showed significant changes compared with the control group, such as decreased body weight growth rate (Morris, 2019), reduced exploration behavior in the central area of the open field experiment (Deacon, 2013), and significantly reduced exploration time to the open arm in the elevated cross maze (Gasnas, 2021), and there were statistical differences, which proved that the physiological metabolism of the model group was disordered. Moreover, there was anxiety of decreased desire for exploration and fear and timidity, which was consistent with the behavioral performance of anxiety disorder rats prepared by Wenjuan Lin (Lin Wenjuan, 2003) and Shuichi Chiba (Chiba et al., 2012). In addition, during the modeling period, the rats in the model group showed characteristics of depressed expression, easy frightening, dark yellow hair color, loss of luster, curling up in corners, and loose stool (Zhang et al., 2020). It can be seen from the above that the GAD model prepared in our study is accurate and reliable, and the characterization is consistent with clinical symptoms (Luyten et al., 2011). \n\nObserving the changes of food intake of rats after drug intervention, we found that the food intake of rats in the DZXYS and DZXYS + DAPT groups increased significantly compared with the model group after 14 days of drug intervention; It was found that after 7 and 14 days of drug intervention, the body weight of rats in the buspirone, DZXYS and DZXYS + DAPT groups increased steadily compared with the model group, and had a time effect. DZXYS, the combination of DZXYS and DAPT had the most significant effect, indicating that the traditional Chinese medicine DZXYS, and the combination of DZXYS and DAPT can significantly improve appetite, restore normal physiological metabolism, and increase the body weight of rats, which is consistent with the animal experimental results of Yan et al. Yan et al. found that after the intervention of traditional Chinese medicine, the appetite and digestive ability of anxiety and depression rats can be significantly improved. It is speculated that this change is related to the change in the microbial population in the gastrointestinal tract, and its change is closely related to changes in brain metabolites (Qu et al., 2019). This phenomenon reflects the advantages of the multitarget treatment of traditional Chinese medicine. Through the classical open field experiment, the exploratory behavior and tension of rats in novel and unfamiliar environments were observed, and it was found that the overall activity of rats after modeling showed no significant change compared with the control group, It shows that the limb activity of rats is normal and can move freely (Ann-Katrin et al., 2019), but the activity distance, residence time, and standing times of the central area in the open field were significantly lower than those in the control group after 28 days of modeling. After 7 days (Day 35) and 14 days (Day 42) of drug intervention, the activity distance of the central area in the buspirone, DZXYS, and DZXYS + DAPT groups were higher than those in the model group; the residence time and standing times increased significantly, and the activity of the central area increased most significantly in the DZXYS + DAPT group. This is currently the gold standard for investigating the anxiety of animals at present (Biedermann et al., 2017). The values of OT% and OE% in the model group were significantly lower than those in the control group. After 14 days of drug intervention, the values of OT% and OE% in the buspirone, DZXYS, and DZXYS + DAPT groups were significantly and continuously increased, and the treatment effect and time effect were significant (Figure 5 ) (Taylor et al., 1985). The above behavioral experimental results show that DZXYS can increase the ability of exploration, reduce the tension, and improve the anxiety behavior of rats. This is consistent with the experimental results obtained by (Zhao et al., 2017). Among them, the antianxiety effect of DZXYS + DAPT was better than that of DZXYS alone. \n\n5-HT1A receptor agonists, such as buspirone and tandospirone, have been shown to promote the formation of neurons in the hippocampus of anxiety and depression rats and improve anxiety and depression. As shown in Figure 6, HE staining of rat hippocampus showed that the morphology of neurons in DG area of DZXYS group was slightly improved compared with that of the model group, most cells had clear outline and were closely arranged, only a small amount of nuclear pyknosis and deep staining of cytoplasm were seen, and the number and number of cell layers in DZXYS group were slightly lower than those in the control group (Kai et al., 2020). To understand the anti-anxiety role of DZXYS, we used double immunofluorescence staining (Donaldson, 2015) to observe neurogenesis in the DG area of the hippocampus of rats in each group at 7 days (Day 35) and 14 days (Day 42) after drug intervention. Proliferation of new neural stem cells (NSCs): Compared with the control group, the number of new NSCs in the GAD model group increased significantly. On the 35th day, the number of new NSCs in the DZXYS + DAPT group was lower than that in the model group. On the 42nd day, the number of new NSCs in the hippocampus of the buspirone, DZXYS, and DZXYS + DAPT groups decreased, especially in the DZXYS + DAPT group. This shows that compared with normal rats, neural stem cells in the DG area of the hippocampus of GAD rats had compensatory proliferation, while the proliferation of neural stem cells decreased or was successfully transformed into other neural cells after drug intervention. Proliferation of newborn neurons: Compared with the control group, the number of newborn neurons in the model group decreased significantly. After 7 and 14 days of drug intervention, the number of newborn neurons in the buspirone, DZXYS, and DZXYS + DAPT groups continued to increase significantly, with significant treatment effect and time effect; proliferation of new astrocytes: Compared with the control group, the number of new astrocytes in the DG area of the model group increased significantly. After 7 and 14 days of drug intervention, the number of new astrocytes in the hippocampus of the buspirone, DZXYS, and DZXYS + DAPT groups decreased significantly compared with the model group, and the time effect was significant, especially in the buspirone group which decreased most significantly. According to the above three fluorescent double-standard results, it can be seen that at all time points after the model was built by the model group, a large number of NSCs proliferated and mainly transformed into startype glial cells, and a small number of them were transformed into new neurons. After DZXYS intervention for 7 days, the proliferation of NSCs decreased, and the number of newly proliferated NSCs in the early stage was transformed into new neurons, The transformation to the new astrocytes decreased significantly and the trend was more obvious with time. We speculate that the neural stem cells in the hippocampus of the model group that received adverse stimulation were overproliferated and differentiated into astrocytes in large quantities, which could not be transformed into new neurons. Astrocytes are the most widely used glial cells in the brain. An appropriate number of astrocytes can maintain potassium homeostasis around neurons, regulate synaptic transmission, promote the supply of neuronal sugar, and have the ability to divide (Vasile et al., 2017). However, excessive proliferation of reactive astrocytes can form glial scars and secrete growth factors and interleukins, interferons, and other factors that inhibit nerve regeneration, inhibiting the regeneration of neurons and nerve axons (Rossi et al., 2007). Therefore, astrocyte activation plays a \"double-edged\" effect in the process of central nervous system injury and repair, and it is very important to ensure a steady state of astrocyte number (Lee et al., 2014; Pekny et al., 2016). Neurogenesis in the hippocampus is particularly important in cognition, emotion, and reproductive behavior, and dysfunctional neurogenesis leads to emotional and mental disorders, leading to the emergence of an anxious state. This is similar to Gisele's observation of neurons in the hippocampus of GAD rats (Dias et al., 2014). DZXYS, especially the combination of DZXYS and DAPT, dynamically reversed the excessive differentiation of neural stem cells into astrocytes, turned into newborn neurons, and improved anxiety behavior, which is similar to the mechanism of hippocampal neuron regeneration in the treatment of depression (Tanti and Belzung, 2013; Hill et al., 2015; Anacker et al., 2018). Therefore, we propose an important question as to how DZXYS dynamically reverses the differentiation direction of newborn neural stem cells at the molecular level. \n\nIt is generally recognized that Notch signaling plays an important role in maintaining the proliferation state of neural stem cells, regulating the timing of differentiation, and determining the fate of neural precursor cells (Lutolf et al., 2002; Niessen and Karsan, 2007). Previous studies have shown that the Notch signaling pathway is expressed in the SGZ and SVZ regions of the adult brain (Stump et al., 2002) and participates in adult neurogenesis. The Notch signaling pathway is composed of the Notch receptor, ligand, and CSL (CBF1-suppressor of hairless-lag-1) DNA-binding protein (Ehebauer et al., 2006). This pathway is initiated by the combination of the Notch receptor and its ligand, after which the Notch receptor releases the active Notch intracellular domain (NICD) into the cytoplasm, and the released NICD is transferred to the nucleus to directly regulate the functions of transcription factors CSL [CBF-1, Su(H), and LAG-1] and a series of downstream target genes, including Hes1 Hes5 and Hes related protein (HERP/HEY) genes (Louvi and Artavanis-Tsakonas, 2006). In this study, we observed fluctuations in Notch1, Hes1 and Hes5 mRNA transcription levels and protein expression in the rat hippocampus by PCR and western blotting. The transcription levels of Notch1, Hes1 and Hes5 mRNA in the model group were significantly higher than those in the control group. After 7 and 14 days of drug intervention, Notch1, Hes1 and the expression of Hes5 mRNA decreased gradually, with an obvious time effect. Among them, the transcriptional levels of Notch1, Hes1, and Hes5 mRNA in the hippocampus of rats in the DZXYS + DAPT group decreased significantly. The changes in the expression of the three proteins detected by western blotting were consistent with the PCR results. \n\nPrevious studies have shown that the activated Notch signaling pathway can maintain the cellular characteristics of NSCs and inhibit their differentiation into neurons, while promoting their differentiation into astrocytes (Guo et al., 2009; Snyder et al., 2012). The overexpression of Notch1 protein observed in this experiment will lead to a large number of transformations of newborn neural stem cells into astrocytes and neurogenesis disorder (Louvi and Artavanis-Tsakonas, 2006) as shown in Figure 11. Hes1 and Hes5, the main downstream targets of Notch signaling, are essential for regulating neurogenesis. They inhibit the transcription of precursor genes, leading to the inhibition of neuronal differentiation (Ohtsuka et al., 1999), the emergence of new astrocytes also depends on the activities of Notch downstream effectors Hes1 and Hes5 (Givogri et al., 2006). The transcriptional levels of Notch 1, Hes1, and Hes5 mRNA in the model group were significantly higher than those in the control group, which revealed that the deep-seated molecular mechanism of GAD was related to the activation of the Notch signaling pathway and the overexpression of key targets. DZXYS can downregulate the Notch signaling pathway, inhibit the continuous proliferation of neural stem cells, and promote their differentiation into neurons, thus improving the anxiety behavior of rats. DAPT in the DZXYS + DAPT group (c-secretase inhibitors) can inhibit the transmission of Notch 1 signaling, inhibit the activation of the Notch signaling pathway, reduce the excessive proliferation of neural stem cells, and promote their transformation to newborn neurons (Breunig et al., 2007). Therefore, the decrease in protein expression of the main target of the Notch signaling pathway in the hippocampus of rats in the combination group of DZXYS and DAPT was more significant than that in other drug intervention groups. The Notch signaling pathway was most effectively inhibited, the proliferation level of neural stem cells decreased, and then differentiated into a large number of newborn neurons. The anxiety behavior was most significantly improved, which also confirmed that inhibiting the overexpression of the Notch signaling pathway and promoting the regeneration of newborn neurons is the key to alleviating anxiety behavior. Many common active ingredients in DZXYS can regulate Notch signaling pathway and promote neuronal regeneration. It has been found that sodium ferulate combined with bone marrow stromal cells can down regulate Notch signaling pathway and promote neural regeneration after focal cerebral ischemia in rats (Zhao et al., 2013) ; It was also found that baicalin could down regulate the expression of basic helix-loop-helix (bHLH) protein downstream of Notch signaling pathway and induce the differentiation of human iPS cells into neurons (Morita et al., 2015). At present, mental diseases are less associated with the Notch signaling pathway, and there is a great controversy about how the Notch signaling pathway plays a therapeutic role in limited studies. For example, Guo et al. observed the Notch signaling pathway in the hippocampus of post-stroke depression rats and found that the Notch signaling pathway in the hippocampus of depression rats is inhibited, neuronal apoptosis and necrosis are increased, and there are few newborn neurons; after intervention with antidepressants, the Notch signaling pathway was activated, and the protein expression of key targets (especially Hes1 and Hes5) increased, which promoted the increase of newborn neurons and alleviated depressive behavior (Guo et al., 2009). We speculate that the differential expression of the Notch signaling pathway protein is related to the special pathological state of post-stroke brain tissue in this study. \n\nThe neuronal hypothesis has been the focus of international psychiatry research in the past 10 years, and it is also one of the most challenging research fields. For the treatment of depression, experimental and theoretical research on promoting hippocampal neuron regeneration and improving depressive symptoms has gradually matured. Whether it can effectively promote hippocampal neuron generation has become an important new target to determine the effectiveness of antidepressants. As a chronic mental disease (Fenton, 1996), generalized anxiety disorder (GAD) usually precedes depression. Some experts believe that the emergence of depression may indicate that compensatory activities cannot protect themselves from the chronic pressure imposed by GAD and eventually develop into depressive disorder (Kessler et al., 1999; Maron and Nutt, 2017). If GAD symptoms can be reversed in the early stage of the disease, it can not only greatly alleviate the great mental pain experienced by patients with GAD, but also avoid the occurrence of depression to a certain extent. DZXYS has been widely used in the clinical treatment of GAD and has a definite curative effect on the disease, but it has not received extensive international attention because of its unclear treatment mechanism (Xu et al., 2020). Through this study, we found that the deep-seated molecular mechanism of DZXYS promotes neuronal regeneration in the hippocampus to treat GAD by downregulating the Notch signaling pathway, which not only opens up new ideas for the development of more effective anti-anxiety drugs in the future, but also lays a foundation for further exploration of the effective components in the prescription. In addition, imaging tools can be developed to detect the level of adult hippocampal neurogenesis in patients with GAD, so that the degree of increased adult hippocampal neurogenesis can become a new biomarker for the efficacy of antianxiety drugs.",
"section_name": "DISCUSSION",
"section_num": "4"
},
{
"section_content": "This study shows that the excessive up regulation of Notch signaling pathway in the hippocampus of GAD rats leads to a large number of proliferation and differentiation of neural stem cells into astrocytes, and neuronal regeneration is impaired; DZXYS can play a positive role in neurogenesis by inhibiting the overexpression of Notch signal pathway in hippocampus, promoting neuronal regeneration in hippocampus and inhibiting the differentiation into astrocytes, and has a significant time effect. \n\nThere are still three deficiencies in the design of this subject. Firstly, the experimental model was prepared using the chronic mild stress paradigm. at the end of 21 days, we should not only use OFT and EPM to detect the formation of anxiety behavior in rats, but also use sucrose preference test to eliminate the depression phenotype (Hyeonwi et al., 2019), so as to eliminate the interference of depression phenotype rats to the experiment, In the future experimental design, we will supplement the sucrose preference experiment; Secondly, we only mechanically observed that DZXYS can downregulate the Notch signaling pathway, promote the increase of neuron number, but ignored the important part of neuronal synaptic plasticity, which is an important part of nerve regeneration. In the next experimental study, we need to focus on whether DZXYS can regulate NMDA receptor to a certain extent after intervening in Notch signal pathway in anxiety rats, promote neuronal synaptic plasticity without inducing its excitotoxic effects (Corlew et al., 2008; Moreau and Kullmann, 2013; Deutschenbaur et al., 2016). At the same time, in the next experiment, we will co locate Notch1 with BrdU, Nestin and NeuN by immunofluorescence in various regions of hippocampus, observe the specific expression changes of Notch1, and find the specific regions with elevated Notch1 level. At last, the abnormal expression of a single Notch signaling pathway is not sufficient to reveal the pathogenesis of generalized anxiety disorder. The therapeutic effect of traditional Chinese medicine is also multi-target and multichannel. We should seek other effective multi-signal transduction pathways and explore the cross connection and interaction between various pathways to further explore the pathogenesis of GAD and explore the deep treatment mechanism of traditional Chinese medicine.",
"section_name": "Summary and Limitations of Current Study",
"section_num": "4.1"
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "Thank Servicebio Technology Co., Ltd. for its help in the immunofluorescence experiment in this experiment. Thank Ran Xue for his guidance in the production of this picture.",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "FUNDING This work was supported by the National Natural Science Foundation of China (Nos. 81904108, 81974553 ), The Project of Shandong Traditional Chinese Medicine Science and Technology Development Plan (Nos. 2017-097, 2019-0240 ), the Natural Science Foundation of Shandong Province (Nos. ZR2020ZD17, ZR2019MH053 ), The Youth Scientific Research Innovation Team specializing in nerve regeneration mechanism based on SUI cognition of TCM and TCM neurological rehabilitation strategy, Shandong University of Traditional Chinese Medicine (University No. 54 ( 2020 )), and The Chinese Medicine and Brain Science Youth Scientific Research Innovation Team, Shandong University of Traditional Chinese Medicine (No. 22202101 ).",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "The animal study was reviewed and approved by the Ethics review committee of Shandong University of Traditional Chinese Medicine.",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "ND designed the subject, CL completed the manuscript, ZY conducted data sorting and statistical analysis, XW reviewed the data, ZL guided the animal experiment, LZ, XL, WG, JS, XF, and KY finished the experiment and SW supervised the animal experiment and revised the manuscript. All authors read and approved the final manuscript.",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. \n\nPublisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. \n\nCopyright © 2021 Liu, Ying, Li, Zhang, Li, Gong, Sun, Fan, Yang, Wang, Wei and Dong. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.",
"section_name": "Conflict of Interest:",
"section_num": null
}
] |
10.1101/2021.06.03.446738
|
The timing of differentiation and potency of CD8 effector function is set by RNA binding proteins
|
<jats:title>Abstract</jats:title><jats:p>CD8<jats:sup>+</jats:sup> T cell differentiation into effector cells is initiated early after antigen encounter by signals from the T cell antigen receptor and costimulatory molecules. The molecular mechanisms that establish the timing and rate of differentiation however are not defined. Here we show that the RNA binding proteins (RBP) ZFP36 and ZFP36L1 limit the rate of differentiation of activated naïve CD8<jats:sup>+</jats:sup> T cells and the potency of the resulting cytotoxic lymphocytes. The RBP function in an early and short temporal window to enforce dependency on costimulation via CD28 for full T cell activation and effector differentiation by directly binding mRNA of <jats:italic>NF-κB</jats:italic>, <jats:italic>Irf8</jats:italic> and <jats:italic>Notch1</jats:italic> transcription factors and cytokines, including <jats:italic>Il2</jats:italic>. Their absence in T cells, or the adoptive transfer of small numbers of CD8<jats:sup>+</jats:sup> T cells lacking the RBP, promotes resilience to influenza A virus infection without immunopathology. These findings highlight ZFP36 and ZFP36L1 as nodes for the integration of the early T cell activation signals controlling the speed and quality of the CD8<jats:sup>+</jats:sup> T cell response.</jats:p>
|
[
{
"section_content": "CD8 + T cells are instrumental for the clearance of pathogen infected or malignant cells and the provision of immune memory. \n\nUpon T cell receptor (TCR) sensing of peptide presented by MHC-I, naive CD8 + T cells exit quiescence and engage a differentiation program to form cytotoxic T-lymphocytes (CTL) accompanied by massive clonal expansion. High affinity antigen overrides inhibitory mechanisms which ensure the quiescent state of the CD8 + T cell and promotes growth and cell cycle entry 1, 2. Co-stimulatory signals, termed \"signal 2\", from CD28 are critical for lowering the activation threshold -especially when TCR stimulation is suboptimal 3. \n\nThe amount and duration of TCR stimulation correlates with the clonal expansion of antigen-specific cells 4. However, weak TCR signals are sufficient to induce a full differentiation program, albeit more slowly 5, 6. The cytotoxic effector differentiation program is installed early after activation with estimates ranging between 2and 48-hours after antigen stimulation 4, [7] [8] [9] [10]. Persistent exposure to IL-2, IL-12 and type-I interferon further shapes the response of CD8 + T cells by providing critical survival factors and promoting effector differentiation [11] [12] [13]. The convergence of these signals on the regulation of transcription and chromatin states has been clearly demonstrated [14] [15] [16]. By contrast, an understanding of the nature and influence of RNA binding proteins (RBP) on CD8 + T cell activation and differentiation is very limited 17. \n\nAmongst RBP, the ability of Roquin (Rc3h1) and Regnase1 (Rc3h12a) to limit CD8 + T cell responses has been linked to the repression of TCR signaling and cytokine production 18, 19. Their absence in T cells leads to T cell hyperactivation and autoimmune/inflammatory disease. The ZFP36 family of RBP bind AU-rich elements (AREs) present in the 3' untranslated region (3'UTR) of mRNAs and can effect different outcomes promoting RNA decay 19, suppressing translation 17, 20, 21 or directing localised translation 22 which are cell-context-specific 23. Of the three ZFP36 gene-family members expressed by CD8 + T cells, ZFP36L2 is present in naive and memory cells, while ZFP36 and ZFP36L1 are induced rapidly and transiently following TCR stimulation 20. Zfp36deficient mice show heightened immune responses and develop a severe autoimmune syndrome attributable to its function in myeloid cells 19 20. An enhanced CD8 response in Zfp36-deficient mice has been linked to the excessive production by myeloid cells of IL-27, of which the p28 subunit is a direct target of ZFP36 24. In quiescent memory CD8 + T cells the ZFP36-paralog ZFP36L2 suppresses the translation of cytokine mRNA 25, but no studies have yet investigated the biology of ZFP36L1 in T cells. The widespread expression of ZFP36-family members by haematopoietic and mesenchymal cells and the genetic redundancy between them has made it challenging to understand the contributions of these genes to T cell physiology. \n\nIn this study we show ZFP36 and ZFP36L1 function during an early temporal window after activation in CD8 + T cells to limit the tempo of effector differentiation and the functional capacity of differentiated effector cells. CTL deficient for both ZFP36 and ZFP36L1 show superior cell intrinsic cytotoxicity and confer greater protection against Influenza-A virus infection. A key function of ZFP36 and ZFP36L1 is to suppress T cell activation and enforce dependence upon costimulatory signals via CD28. \n\nThe RBP bind transcripts encoding subunits of the NF-ĸB pathway, Notch1, IRF8 and Il2 to regulate the abundance of their protein products early after T cell activation. Thus, the acquisition of the CD8 effector fate is under the dominant control of ZFP36 and ZFP36L1.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To examine the consequences of the absence of ZFP36 and ZFP36L1 in T cells on an in vivo response we infected control mice Zfp36 fl/fl /Zfp36l1 fl/fl (WT), Zfp36 fl/fl CD4 cre (ZFP36 KO),\n\nZfp36l1 fl/fl CD4 cre (ZFP36L1 KO) or Zfp36 fl/fl /Zfp36l1 fl/fl CD4 cre (dKO) mice intranasally with a sublethal dose of H1N1 Influenza A virus A/Puerto Rico/8/1934 (IAV/PR8). Following infection, using bodyweight as a surrogate for morbidity, we observe no significant difference between WT mice and mice with T cells deficient for ZFP36 or ZFP36L1 (Fig. 1a, b ). By contrast, dKO mice show significantly less weight loss when compared to WT mice (Fig. 1c ). \n\nMoreover, when challenged with a lethal dose of IAV/PR8 dKO mice show increased survival compared to WT mice (Fig. 1d ). We conclude that the absence of ZFP36 and ZFP36L1 in T cells does not promote immunopathology, but rather increases resilience following infection with a pathogenic virus. \n\nThe presence of viral RNA in lung tissues of dKO mice following IAV/PR8 infection is reduced throughout the infection and by day 10 we found no detectable viral RNA in five of ten infected dKO mice (Fig. 1e ). This is indicative of more efficient viral clearance when ZFP36 and ZFP36L1 are absent from T cells and may explain why the lungs of dKO mice contain reduced numbers and frequencies of CD8 + T cells specific for IAV nucleoprotein peptide NP(366-374) compared to WT mice by day 10 after infection (Fig. 1f, g ). We do not find differences in NP(366-374) specific CD8 + T cells at earlier timepoints suggesting that recruitment of antigen specific cells into the lung is not affected (Fig. 1f, g ). We also observed an increased frequency of GranzymeB (GzmB) positive total CD8 + T cells in the lungs of dKO mice compared to WT mice at four-and ten-days post-infection (Fig. 1h ). In addition, as shown by the increased GzmB staining intensity, dKO CD8 + T cells contain greater amounts of GzmB per cell (Fig. 1i ).",
"section_name": "ZFP36 and ZFP36L1 limit the anti-viral CD8 + T cell effector response",
"section_num": null
},
{
"section_content": "It has been previously shown that ZFP36 and ZFP36L1 are transiently expressed in naive CD8 + T cells after stimulation with anti CD3 and anti CD28 antibodies 20. We find that restimulation of in vitro differentiated CTL by antigen induces a similar transient expression of ZFP36 and ZFP36L1 with peak expression at 3-4h after stimulation (Supplementary Figure 3a, b ). Thus, to discriminate the function of the RBP during activation of naive CD8 + T cells from their role in limiting effector function in differentiated CTL, we activated OT-I cells in vitro with peptide in the presence of IL-2 and IL-12 for 24 hours and cultured them with IL-2 for seven days to generate CTL. We generated CTLs from OT-I-Zfp36 fl/fl /Zfp36l1 fl/fl CD4 cre mice where ZFP36 and ZFP36L1 are absent during initial T cell activation. We compared these with CTLs generated from OT-I-Rosa26L-stop_L-Cas9GFP-CD4 cre mice transduced at 24 hours by viruses expressing sgRNAs targeting Zfp36 and Zfp36l1. In the latter, both RBPs are present in OT-I cells during initial activation but absent in CTLs (Fig. 3a ). \n\nConsistent with this expectation the efficiency of knockout of ZFP36 and ZFP36L1 with CRISPR-Cas9 was very high as evident from Western blotting of CTL whole cell lysates (Supplementary Figure 3c-f ). The CTL generated from OT-I-Zfp36 fl/fl /Zfp36l1 fl/fl CD4 cre mice show increased cytotoxicity against SIINFEKL peptide-loaded EL4 cells compared to that of CTL derived from WT mice (Fig. 3b ; Supplementary Figure 4a ). The CTL numbers recovered after the 3 hours killing assay were the same for WT and dKO CTLs, suggesting a higher efficiency of killing on a per cell basis by the dKO CTL (Supplementary Figure 4b ). Moreover, dKO CTLs were also more efficient in killing target cells loaded with a lower affinity peptide SIIVFEKL (V4), highlighting their increased potency when triggered by weaker signals (Supplementary Figure 4c ). Notably, the CTL generated by Cas9-mediated knockout of Zfp36 and Zfp36l1 were not more potent than CTL generated following transduction with viruses expressing non-targeting sgRNAs (Fig. 3c ). Upon T cell stimulation the expression of Granzyme-B and TNF is increased when Zfp36 and Zfp36l1 were absent before T cell activation (Fig. 3d, e ). Notably, CTL produced increased TNF upon restimulation irrespective of the mode of genetic modification (Fig. 3f, g ) indicating that the RBP directly target and limit TNF in",
"section_name": "ZFP36 and ZFP36L1 determine the CD8 effector program early after T cell activation",
"section_num": null
},
{
"section_content": "To identify the direct targets of the ZFP36-family which act early in CD8+ T cells to regulate the CTL differentiation we employed an approach that was agnostic to whether this was mediated by and/or 18h post activation comprised the majority with 166 genes (81%) of which 67 transcripts are shared between both time points (Fig. 4a ). \n\nThe target mRNAs could be broadly clustered into five groups according to their expression dynamics following activation (Fig. 4b ; Supplementary Figure 5a, b ; Supplementary Data 1). \n\nThe group I cluster includes genes that are induced early and transiently following T cell activation and include transcription factors of the NF-κB pathway and genes involved in signalling and protein synthesis (Supplementary Figure 5c ). This group includes genes involved in metabolism (Slc7a1/5) and Kdm6b encoding a Histone-lysine specific demethylase. Group II comprises genes induced by six-hours and maintained high expression at 18 hours; it contains cytokines and chemokines",
"section_name": "Dynamic gene expression networks targeted by ZFP36 and ZFP36L1 early after CD8 + T cell activation",
"section_num": null
},
{
"section_content": "IL-2, which is an important cytokine for effector T cell differentiation, was identified as a direct target of ZFP36 and ZFP36L1 in the CLIP analysis (Supplementary Fig. 6a ). However, the regulation of cytokines by ZFP36 has been shown to be cell context dependent 23 and previous studies found that the absence of Zfp36 in CD8+ T cells did not affect IL-2 production in CD4 or CD8 + T cells 20, 24. In contrast, another study reported enhanced Il2 mRNA stability in mixed splenocyte and T cell populations from Zfp36 -/-mice 28. Therefore we sought to examine IL-2 production by dKO cells using a sensitive bispecific antibody-based capture method 29 which measured IL-2 secretion at the single cell level in the absence of a secretion inhibitor. Following peptide-stimulation the frequency of WT and dKO CD8 + T cells producing IL-2 peaked around six-hours following peptide-mediated T cell activation. \n\n(Fig. 5 a, b ). We found a substantial increase in the proportion dKO OT-I cells producing IL-2 compared to WT OT-I cells during the first 16 hours (Fig. 5a, b ). However, by 24 hours the frequency of IL-2 expressing cells is low and does not differ between dKO and WT cells. Therefore, the suppressive mechanisms that lead to the cessation of IL-2 production are intact in the absence of the RBP. \n\nIn contrast we find the same staining intensity for CD25 between WT and dKO OT-I cells during the first 16h after activation. \n\nHowever, expression of CD25 persists on dKO cells at 24h a time at which it shows diminished expression on WT cells (Fig. 5c ). For the WT cells the intensity of IL-2 staining showed little difference at any of the timepoints tested before 24 hours after activation. This is indicative of the all-or-none digital response to T cell stimulation. By contrast, at three and six hours after activation, OT-I cells lacking ZFP36 and ZFP36L1 stain more intensely than WT OT-I for IL-2 indicating that they produce more IL-2 per cell (Fig. 5d ). Thus, early after activation ZFP36 and ZFP36L1 limit the frequency of activated T cells producing IL-2 and the amount of IL-2 produced by the activated CD8 + T cell. \n\nTo investigate in detail how the RBPs regulate IL-2 expression we measured the Il2 mRNA and found increased amounts in dKO CD8 + T cells, especially three-and six-hours after stimulation with peptide (Fig. 5e ). To assess whether the increased abundance in Il2 mRNA is due to increased stability in the absence of the RBPs we activated naive OT-I WT and dKO cells for 3hours and treated them with Triptolide a global transcription inhibitor. The inhibition of transcription allowed us to assess the decay of Il2 (Fig. 5f ). The absence of both RBPs increased the stability of Il2 mRNA. To understand whether absence of the RBPs also alters the transcription of the Il2 gene, we measured the unprocessed Il2 pre-mRNA. Although this is not a direct measure of transcription, mRNA transcripts with retained introns are recently transcribed. \n\nHowever, we did not find differences in pre-mRNA transcripts between WT and dKO naive CD8 + T cells early after activation (1. 5 and 3h). Together these results suggest that IL2 mRNA is regulated directly by ZFP36 and ZFP36L1 (Fig. 5g ).",
"section_name": "ZFP36 and ZFP36L1 limit the magnitude of IL-2 production",
"section_num": null
},
{
"section_content": "The regulation of cytokines by ZFP36 and ZFP36L1 can influence other cells of the immune system and result in an altered inflammatory milieu which influences effector cell differentiation 12, 13, 16. Indeed, IL-2 has been proposed to promote effector differentiation 30. To test whether the increased production of IL-2 promotes rapid formation of SLEC, we co transferred 500 naive WT and 500 dKO OT-I cells, electroporated with a complex of Cas9 protein and a non-targeting guide RNA, into CD45. 1 recipients and infected them with attLm-OVA the following day. In another group of recipients, we co-transferred 500 naive WT OT-I cells together with 500 dKO OT-I, electroporated with Cas9 protein and a guide RNA targeting the Il2 gene. The dKO OT-I cells showed higher frequencies of KLRG1 + SLEC in blood of the recipients on day 5 post infection irrespective of their ability to produce autocrine IL2 (Supplementary Figure 6 b-e ). Thus, we conclude that dKO cells which are in the same inflammatory environment as WT cells still differentiate more into SLEC in the absence of autocrine IL-2.",
"section_name": "Increased autocrine IL2 production is not necessary for differentiation into SLEC",
"section_num": null
},
{
"section_content": "Multiple transcripts encoding components of the canonical and noncanonical NF-κB pathway, including Nfkb1, Nfkb2, Nfkbib and\n\nRel are induced at 6 hours post-activation and cluster in group I. \n\nThe NF-κB pathway is critical to integrate TCR signalling with CD28 costimulation and inflammatory cues. In concert with the NFAT/AP1 complex, NF-κB promotes the induction of IL-2 by T cells 3. Analysis of ZFP36-family interactions with RNA confirmed Nfkb1 (Supplementary Figure 6f ), Nfkb2 (Fig. 6a ) and Rel (Fig. 6b ) mRNA to be directly bound with accumulation of reads at AU-rich elements (AREs) in their 3`UTRs that are highly conserved between mammalian species (Fig. 6c, d : Supplementary Figure 6f ). Consistent with this interaction being consequential, we found that in CD8 + T cells from dKO mice the abundance of representative members of the non-canonical and canonical NF-κB pathways, NF-κB2 (p100) (Fig. 6e ) and cREL (Fig. 6f ) is greater compared to WT following activation with anti-CD3. While NF-κB2 (p100) abundance remained greater in the dKO than the WT over the whole course of T cell activation, increased cREL protein was observed in the dKO only at early time-points. \n\nThe NF-κB pathway has been shown to interact with other transcription factors including NOTCH1 to drive differentiation into CTLs by directly regulating the expression of the transcription factor Eomesodermin (EOMES) 31 and Granzyme-B 32. The Notch1 mRNA is bound by ZFP36 and ZFP36L1 according to T cell CLIP data (Supplementary Figure 7a ). This is consistent with an earlier biochemical demonstration of the interaction of ZFP36L1 with a 61 nucleotide sequence containing the Notch1 AREs 33. Although\n\nNotch1 is not among the mRNAs in the induced clusters, the expression of the NOTCH1 intracellular domain is increased in naïve dKO CD8 + T cells at three-, six-and 18-hours post stimulation compared to WT cells (Supplementary Figure 7b, c ). \n\nTCR induced EOMES expression is NF-κB and NOTCH-1 dependent and mediates the effector and memory differentiation program in CD8 + T cells by promoting cytokine and granzyme expression 31, 32, 34, 35. We found dKO naïve CD8 + T cells activated with plate bound anti-CD3 antibody showed greatly increased frequencies of Granzyme-B and EOMES positive cells compared to WT cells 72 hours after activation (Fig. 6g, h ). The increased expression of EOMES three days after activation was not due to relief of direct inhibitory effects of the ZFP36 -family on the Eomes mRNA as it was not identified as a target mRNA. EOMES becomes highly expressed during the later course of the immune response and not in naive T cells 31. This data highlights the more rapid acquisition of an effector state in dKO cells mediated via targets accelerating EOMES expression. Group II transcription factors IRF4 and IRF8 have also been shown to be important for CTL differentiation and function 36, 37. In particular Irf8 has been suggested to act independently of T-box transcription factors Tbet and Eomes to promote effector functions 36. Irf8 mRNA, which is rapidly induced upon T cell activation and continues to be highly expressed by 18h, is also bound by ZFP36 and ZFP36L1 in T cells (Supplementary Figure 7 d ). IRF8 protein is increased in dKO cells compared to WT cells, particularly so at later time points of stimulation (Supplementary Figure 7 e, f ). Taken together, these data suggest a collective of transcription factors known to form a network promoting T cell differentiation are regulated by ZFP36 and ZFP36L1.",
"section_name": "ZFP36 and ZFP36L1 directly limit the expression of NF-B, IRF8 and Notch1",
"section_num": null
},
{
"section_content": "T cell activation, proliferation and differentiation are intimately linked. A previous study suggested CD8 + T cells deficient for ZFP36 to proliferate more rapidly in response to TCR stimulation 20. \n\nMoreover, our CTL killing assays with low affinity peptide suggested a reduced activation threshold for dKO CTLs. To test whether naive isolated CD8 + T cells were more sensitive to TCR dependent activation we titrated the amount of plate-bound anti-CD3 and analyzed the proliferation of cell trace labeled dKO and WT cells after 72 hours. We found that dKO cells responded to lower doses of anti-CD3 with greater proliferation as compared to WT cells. Interestingly, upon stimulation with higher doses of anti-CD3 the dKO cells had a proliferative advantage as compared to their WT counterparts (Fig. 7a-c ). \n\nCostimulation by cytokines and cell surface ligand-receptorinteractions e. g., CD28 is critical for naive CD8 + T cell activation and subsequent differentiation. The dependency of T cells on CD28 is regulated by the NF-κB pathway 3 including NF-κB2 38. Also not only transcription of Il2 is dependent on CD28 costimulation via NF-κB 39 but CD28 also promotes the stabilization of the Il2 mRNA 40. This prompted us to test the co-stimulation dependence of dKO CD8 + T cells by the titration of anti-CD28 antibodies. \n\nFollowing stimulation with 5µg/ml plate bound anti-CD3, cell trace labelled dKO CD8 + T cells divide more than their WT counterparts (Fig. 7d ). The addition of anti-CD28 (clone 37. 51) increases the numbers of both, WT and dKO cells present after 72 hours culture, compared to cultures with anti-CD3 alone (Fig. 7d, e ). WT cells show a greater sensitivity to lower amounts of CD28 costimulation compared to the dKO CD8 + T cells. The latter are only mildly responsive to the inclusion of anti-CD28 as measured by the absolute cell numbers in the cultures after 72 hours with LogEC50 of 2. 2µg/ml for WT and 1. 2µg/ml dKO cells (Fig. 7e ). dKO cells As shown above stimulation of CD8 + T cells with anti-CD3 alone induced significantly greater amounts of both NF-κB2(p100) (Fig. 7h, i ) and IRF8 (Fig. 7j, k ) in dKO cells compared to WT. The addition of 5µg/ml plate bound anti-CD28 augmented the expression of both transcription factors, indicating they were responsive to co-stimulation and reduced the differences in NF-κB2 (Fig. 7h, i ) and IRF8 (Fig. 7j, k possible, but unexamined here, that the RBP also limit the rate of translation of Il2 mRNA. We speculated whether there is an early indirect transcriptional regulation of Il2 which would be partly accounted for by the RBP limiting expression of Nfkb2 and Rel subunits of NF-κB 38, 42, 43. However, we find that there is no increased transcription in the absence of the RBPs. It is worth noting that the NF-kB pathway integrates multiple costimulatory signals and acts on the transcription of different genes which are important to mediate the effects of costimulation in T cells independent of its regulation of IL2 44. This is consistent with our results where we find that enhanced differentiation of dKO OT-I cells into KLRG1 + effector cells is likely to be independent of This may as well be accompanied by the deregulation of target genes involved in anabolic metabolism and cell cycle processes as well as the restructuring of the chromatin landscape in the absence of RBP. We have not examined the latter experimentally, but we propose that ZFP36 and ZFP36L1 exert regulation across multiple cellular processes to limit T cell activation and differentiation. In this way, the RBP can coordinate the amounts of genes expressed within pathways and also integrate the activity of different pathways to regulate differentiation.",
"section_name": "ZFP36 and ZFP36L1 inhibit TCR mediated T cell activation and promote dependence on CD28",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Mice with single or combined floxed Zfp36 and Zfp36l1 alleles 33, 45, Rosa26 lsl Cas9-GFP 46, B6. Cg-Tg(CD4-cre)1Cwi mice 47 To measure antigen specific cytokine release single cell preparations from lungs were stimulated in the presence of 10μM NP(366-374) peptide and 1μg/ml Brefeldin A for 6 hours. Cells were stained for surface markers, fixed and permeabilized with the cytofix/cytoperm reagent (BD Biosciences, USA) and co-stained for intracellular cytokines and Granzyme-B.",
"section_name": "Mice",
"section_num": null
},
{
"section_content": "8-12-week old male and female mice were used for all experiments. \n\nBacteria were grown in BHI medium to an OD600 of 0. 1 before each experiment. Mice were infected with a sublethal dose of 5x10 6 CFU attenuated (ΔactA) listeria monocytogenes expressing OVA(257-264) 49 by intravenous administration. \n\nAdoptive transfer experiments: naive OT-I cells (if not otherwise stated)\n\nwere sorted by Flow Cytometry from spleens and LN of mice from a respective genotype and 1000 cells transferred intravenously on the day before infection. In all cotransfer experiments donor cells were mixed with 1*10 6 carrier splenocytes of the same genotype as the host. \n\nFor characterization of transferred OT-I cells after transfer mice were bled on indicated dates and spleens were collected at final points of analysis. \n\nCells were incubated with 10 -7 M N4 peptide for 3 hours in the presence of Brefeldin A (1µg/ml) in full cell culture medium. After surface staining, cells were fixed with 2% PFA for 20 min at 4°C and permeabilized with BD Perm/wash +1%FCS for 20 min at 4°C, before intracellular Cytokine staining.",
"section_name": "Infection with Listeria monocytogenes",
"section_num": null
},
{
"section_content": "For cell surface staining single cell suspensions from tissues or cultured cells were prepared in FACS buffer containing PBS 1% FCS +/-2mM EDTA (if not otherwise stated in the methods). All cells were blocked with Fcγ blocking antibody (24G2, BioXcell) and incubated with fixable cell viability dye eF780 (Thermo Fisher or BD) for 20min at 4°C.",
"section_name": "Flow Cytometry and monoclonal antibodies",
"section_num": null
},
{
"section_content": "Mouse lL-2 secretion assay was performed following manufacturer's instructions (130-090-491; Miltenyi Biotech). In brief; 5*10 5 /ml purified naive OT-I cells were activated with 10 -10 N4 peptide in cell culture medium for the indicated timepoints. Cells were washed twice and incubated with capture reagent for 1 h at 37°C while mixing the cells every 10 min. Cells were surface stained with antibody cocktail containing anti IL2-PE antibody.",
"section_name": "Cytokine assays",
"section_num": null
},
{
"section_content": "In vivo: Splenic leukocytes from naïve C57BL/6 mice (8-12 weeks old)\n\nwere pulsed for 1 hr with 10µg/ml influenza virus NP(366-374) peptide and were subsequently labeled for 10 min at 37 0 C with 5 μM CFSE (Invitrogen) (CFSE high ; loaded cells). Non-peptide loaded control cells were labelled with 0. 5μM CFSE low. The two populations of cells were then mixed together in a 1:1 ratio and 6x10 6 cells were transferred intravenously into mice that had previously been infected with influenza PR8 ten days before, or transferred into uninfected control mice. Mice were killed 1 hour later and the percentage of peptide loaded target cells and peptide non-loaded cells was quantified by flow cytometry. \n\nIn vitro: OT-I CTLs were cultured in the presence of N4 peptide -pulsed For CRISPR/Cas9-gene editing experiments 1*10",
"section_name": "Cytotoxicity assays",
"section_num": null
},
{
"section_content": "iCLIP data was mapped to GRCm38 mouse genome assembly using bowtie2. Barcoded adaptors of iCLIP sequenced reads were removed before mapping, crosslink sites were defined by the nucleotide preceding iCLIP cDNAs using Genialis iMaps web server:\n\nhttps://imaps. genialis. com/iclip.",
"section_name": "ZFP36L1 iCLIP mapping",
"section_num": null
},
{
"section_content": "HITS-CLIP datasets for ZFP36 in CD4 + T cells following 4h or 72h activation were obtained from GSE96074 20. This data, together with the iCLIP data, were processed using the Genialis iMaps web server (https://imaps. genialis. com/iclip). Data was deduplicated based on the random barcodes, trimmed with Cutadapt 50, and mapped to GRCm38 mouse genome assembly using STAR 51. Significant crosslink sites, defined by the nucleotide preceding iCLIP or HITS-CLIP cDNAs, were identified using the iCount pipeline (https://icount. readthedocs. io/en/latest/index. html). \n\nFor CLIP target gene identification, target transcripts were filtered for the presence of identical significant crosslink sites in their 3'UTRs in at least 3 out of 4 or 5 (for ZFP36) and 2 out of 3 (for ZFP36L1) biological replicates. \n\nUnique and common targets identified from the two datasets were then unified and taken as our final list of high confidence ZFP36/L1 target genes. \n\nRNA-seq data for naïve and in-vitro activated CD8 + T cells were obtained from GSE77857 27. Data were trimmed using Trim Galore (https://www. bioinformatics. babraham. ac. uk/projects/trim_galore), mapped to the GRCm38 mouse genome build using Hisat2 52, taking into account known splice sites from the Ensembl Mus_musculus. GRCm38. 90 annotation release. Raw read counts over mRNA features from the same annotation release were quantified using Seqmonk (https://www. bioinformatics. babraham. ac. uk/projects/seqmonk). \n\nDifferentially expressed genes at 6h and 18h after CD8 + T cells activation were identified using DESeq2 analysis with default parameters for each time point relative to 0h, and were selected for adj. pvalue <= 0. 05 and 1. 3 < log2 FC < -1. 3 (using 'normal' log2 fold change shrinkage). Genes with a lower baseMean expression than 30 normalized read counts were excluded from analysis. \n\nFor analysis of binding site conservation, a 70nt sequence around the identified CLIP binding site was analyzed for conservation among vertebrates using the UCSC genome browser. Downstream analysis of CLIP data was performed using R v4. 0. 4. conceptualisation, supervision, funding acquisition, writing review and editing. \n\nThe authors declare no conflict of interest.",
"section_name": "Data and statistical analysis",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "We thank the Babraham Institute Biological Support Unit, Flow Cytometry and Kirsty Bates, Anne Segonds-Pichon and the Bioinformatics Facilities for assistance; Elisa Monzon-Casanova, Adrian Liston, Sarah Ross and Arianne Richard for comments on the manuscript. We would like to thank Sarah Collison for formatting this manuscript version for BioRxiv, using a template generously provided by the Finkelstein Lab, Austin, TX (https://github. com/finkelsteinlab). This study was supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC) ( BBS/E/B/000C0407 ; BBS/E/B/000C0428 ; the BBSRC Core Capability Grant to the Babraham Institute ; and a Wellcome Investigator award ( 200823/Z/16/Z ) to M. T. FS was supported by European Molecular Biology Organization (EMBO) Long-Term Fellowship ( ALTF 880-2018 ). T. J. M. was supported by the BBSRC Cambridge doctoral training partnership. V. D'A. was supported by the Cambridge Trust.",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "All data generated and analyzed during this study are included in this article and its supplementary information, or have been made available in public repositories as follows: Sequencing data from ZFP36L1 iCLIP experiments performed in this manuscript is publicly available on: GEO: GSE176313 (http://www. ncbi. nlm. nih. gov/geo/query/acc. cgi?acc=GSE176313). Previously published datasets are available on GEO under the accessions GSE77857 (RNA-seq) (https://www. ncbi. nlm. nih. gov/geo/query/acc. cgi?acc=GSE77857) and GSE96074 (HITS-CLIP for ZFP36) (https://www-ncbi-nlm-nihgov. ezproxy. u-pec. fr/geo/query/acc. cgi?acc=GSE96074). Source Data is provided as a Source Data file for data sets presented in this study. Statistical analysis was performed with Graph Pad Prism 8. 1. 2 and R v4. 0. 4.",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "",
"section_name": "Author Contributions and Notes",
"section_num": null
}
] |
10.1186/s13075-023-03039-1
|
Notch signaling is activated in knee-innervating dorsal root ganglia in experimental models of osteoarthritis joint pain
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>We aimed to explore activation of the Notch signaling pathway in knee-innervating lumbar dorsal root ganglia (DRG) in the course of experimental osteoarthritis (OA) in mice, and its role in knee hyperalgesia.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>Cultured DRG cells were stimulated with the TLR4 agonist, lipopolysaccharide (LPS). Notch signaling in the cells was either inhibited with the γ-secretase inhibitor, DAPT, or with soluble Jagged1, or activated through immobilized Jagged1. CCL2 production was analyzed at mRNA and protein levels. In in vivo experiments, knee hyperalgesia was induced in naïve mice through intra-articular (IA) injection of LPS. The effect of inhibiting Notch signaling was examined by pre-injecting DAPT one hour before LPS. OA was induced through surgical destabilization of the medial meniscus (DMM) in male C57BL/6 mice. Gene expression in DRG was analyzed by qRT-PCR and RNAscope in situ hybridization. Activated Notch protein (NICD) expression in DRG was evaluated by ELISA and immunofluorescence staining. DAPT was injected IA 12 weeks <jats:italic>post</jats:italic> DMM to inhibit Notch signaling, followed by assessing knee hyperalgesia and CCL2 expression in the DRG.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>In DRG cell cultures, LPS increased NICD in neuronal cells. Inhibition of Notch signaling with either DAPT or soluble Jagged1 attenuated LPS-induced increases of <jats:italic>Ccl2</jats:italic> mRNA and CCL2 protein. Conversely, activating Notch signaling with immobilized Jagged1 enhanced these LPS effects. In vivo, IA injection of LPS increased expression of Notch genes and NICD in the DRG. Pre-injection of DAPT prior to LPS alleviated LPS-induced knee hyperalgesia, and decreased LPS-induced CCL2 expression in the DRG. Notch signaling genes were differentially expressed in the DRG from late-stage experimental OA. <jats:italic>Notch1</jats:italic>, <jats:italic>Hes1</jats:italic>, and NICD were increased in the neuronal cell bodies in DRG after DMM surgery. IA administration of DAPT alleviated knee hyperalgesia <jats:italic>post</jats:italic> DMM, and decreased CCL2 expression in the DRG.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>These findings suggest a synergistic effect of Notch signaling with TLR4 in promoting CCL2 production and mediating knee hyperalgesia. Notch signaling is activated in knee-innervating lumbar DRG in mice with experimental OA, and is involved in mediating knee hyperalgesia. The pathway may therefore be explored as a target for alleviating OA pain.</jats:p> </jats:sec>
|
[
{
"section_content": "Keywords Notch signaling, Dorsal root ganglia, Osteoarthritis, Pain, Mouse model",
"section_name": "",
"section_num": ""
},
{
"section_content": "Our incomplete understanding of the mechanisms underlying joint pain in osteoarthritis (OA) accounts for the general ineffectiveness of analgesics and hampers the development of new pharmacological treatments [1]. The first step in pain generation is nociception, a process whereby noxious stimuli activate nociceptors, specialized sensory neurons that innervate peripheral tissues [2]. These stimuli generate action potentials that carry the pain signal from the innervated tissues to the cell bodies in the dorsal root ganglia (DRG). From there, the signal is relayed to the dorsal horn of the spinal cord, where nociceptors synapse with neurons in central pain pathways, and then travels further up to the brainstem and higher brain regions, where it is consciously perceived as pain. \n\nThe pain signal can be modulated-often amplifiedat different stages along the pain pathway, including in the DRG, through neuronal crosstalk and interactions between neurons and non-neuronal cells [3]. All animal models of chronic pain are characterized by extensive changes in the DRG, including cellular, molecular, and biophysical changes. It can be expected that a detailed description of the precise nature of such DRG changes will deepen our understanding of mechanisms underlying pain and facilitate the identification of molecular targets. In neuropathic pain models, DRG changes have been extensively delineated [4, 5], and more recently, there has been increased characterization of the DRGs that contain the cell bodies of knee-innervating afferents in mouse models of OA [3, 6]. For example, in slowly progressive experimental OA induced by surgical destabilization of the medial meniscus (DMM) in the mouse knee, joint damage and the associated pain-related behaviors are accompanied by specific temporally regulated cellular and molecular changes in the lumbar L3-L5 DRG [3, 6]. \n\nA recent microarray analysis of the L3-L5 DRG at 4, 8, and 16 weeks after DMM revealed a strong regulation of innate neuro-immune pathways, especially in the later stages of the model, 8-16 weeks after DMM, when persistent pain is associated with severe joint damage [7]. Further analysis of these DRG microarray data revealed a clear regulation of genes encoding molecules in the Notch signaling pathway after DMM, also in the later stages of the model. Because this pathway has been extensively investigated in nervous system development [8], we decided to validate these findings, and explore if the Notch signaling pathway may play a role in knee joint pain. \n\nNotch signaling is a cell-to-cell signaling pathway that plays a major regulatory role in cell fate and differentiation, including in the nervous system [9]. Canonical Notch signaling is mediated by Notch receptors, which are transmembrane receptors that are activated by the Delta-like (Dll)/Jagged (Jag)-family ligands presented by neighboring cells. Following ligand-receptor interactions, the intracellular domain of Notch receptors (Notch intracellular domain or NICD) is released from the membrane by a sequence of proteolytic cleavage events mediated by ADAM10/17 (a disintegrin and metalloprotease 10/17), and subsequently the γ-secretase complex. The NICD then translocates to the nucleus and binds to the DNA binding transcription factor, RBPJ (recombination signal binding protein for immunoglobulin kappa J region), and Mastermind-family coactivators, facilitating the transcription of Notch downstream target genes such as HES (hairy and enhancer of split) family genes [10]. \n\nAn evolutionary conserved transcription factor cascade downstream of Notch signaling is necessary for both the maintenance of neural progenitor cell character and the progression of neurogenesis [11, 12]. Furthermore, links between Notch signaling and pain perception have been explored in rat models of peripheral neuropathic pain [13] [14] [15]. Therefore, we decided to investigate the Notch signaling pathway in experimental models of knee pain. Of interest, it has been reported that Notch signaling may regulate innate immunity and inflammation through a crosstalk with toll-like receptor (TLR) signaling [16]. Since we have previously reported that damage-associated molecular patterns (DAMPs) present in OA joints can excite nociceptors through neuronal TLR4 [17], and that the pro-algesic chemokine, CCL2, is released upon TLR activation and plays a key role in initiating and maintaining pain in the DMM model [17] [18] [19], we investigated the crosstalk between Notch signaling and TLR4 signaling in activating CCL2 production in the DRG and mediating knee hyperalgesia. We also explored whether Notch signaling is activated in the DRG of mice with experimental knee OA, and whether this activation contributes to knee hyperalgesia.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "All animal experiments were approved by the Institutional Animal Care and Use Committee at Rush University Medical Center. A total of 220 male C57BL/6 J mice were used for these experiments, including 101 naïve mice and 119 mice that underwent DMM or sham surgery. DMM or sham surgery was performed in the right knee of 10-week old mice under general anesthesia using isoflurane, as previously described [18, 20]. Briefly, the medial meniscotibial ligament (MMTL) was transected after medial parapatellar arthrotomy and dissection of the anterior fat pad. Sham surgery was through the same approach without MMTL transection.",
"section_name": "Animals and surgery",
"section_num": null
},
{
"section_content": "Bilateral L3-L5 DRG were collected from 10-week old male naïve C57BL/6 mice and pooled for enzymatic digestion using collagenase 4 and papain, as previously described [18]. A total of 36 mice were used. Dissociated DRG cells were plated on poly-L-lysine and laminincoated glass coverslips in multi-well plates, and cultured in F12 medium supplemented with 1 × N2 and 0. 5% fetal bovine serum. \n\nOn day 4, TLR4 signaling was stimulated by adding lipopolysaccharide (LPS) (Catalog# tlrl-3pelps, InvivoGen, San Diego, CA) at a concentration of 1 μg/mL. Notch signaling was inhibited either by a γ-secretase inhibitor, DAPT (N-[N-[3, 5-diflurophenylacetate]-l-alanyl]-(S)-phenylglycine t-butyl ester) (Catalog# 565784, Calbiochem, San Diego, CA), or by a soluble form of the Jagged1 (188-204) peptide (sJag1, CDDYYYGFGCNKFCRPR) (AnaSpec Inc., Fremont, CA). DAPT (25 μM) or sJag1 (40 μM) was added 1 h before LPS. Control groups were treated with vehicle (0. 1% dimethyl sulfoxide, DMSO) and scrambled Jagged1 (RCGPDCFDNYGRYKYC, AnaSpec), respectively. \n\nIn another set of cultures, Notch signaling was induced by using immobilized Jagged1 ligand as described [21]. Briefly, Corning ® BioCoat ™ Poly-D-Lysine/Laminin 4-well chamber slides (Corning, Bedford, MA) were coated overnight with 50 μg/mL of protein G (Invitrogen), and subsequently blocked with 1% bovine serum albumin (BSA, Invitrogen) for 2 h. The blocked plates were then incubated for 3 h with recombinant Jagged1-Fc chimera (R & D Systems, Minneapolis, MN) at a concentration of 1 μg/ mL. An equal amount of IgG-Fc fragment (Jackson Immu-noResearch, West Grove, PA) was incubated on the control plates. After washing 3 times with PBS, cells were immediately seeded onto the coated plates, and LPS (1 μg/mL) was added on day 4 of cell culture. \n\nIn both experiments, 24 h after LPS stimulation, RNA was extracted using RNeasy kit from cells in part of the wells for qPCR analysis of Ccl2 mRNA expression, while supernatants were collected for CCL2 protein measurement using Quantikine Mouse CCL2/JE/MCP-1 Immunoassay kit (R&D Systems Inc, Minneapolis, MN), normalized to total protein amount in the supernatants (N = 4-9 independent cultures per group). For the rest of the wells, coverslips were collected 24 h after LPS stimulation for detecting NICD expression (N = 5-9 culture wells per group). Cells were fixed with 100% ice-cold methanol for 10 min, followed by immunofluorescence (IF) staining for NICD.",
"section_name": "DRG cell cultures",
"section_num": null
},
{
"section_content": "Under isoflurane anesthesia, LPS (3 μg in 3 μL saline) was injected IA into the right knees of 12-week old naïve male mice, as described [22], and control mice were injected with saline only (N = 6 mice per group). Knee hyperalgesia was tested in a 24-h time-course after injection. Three additional independent batches of mice also received LPS or saline control injected IA. RNA was extracted from the ipsilateral L3-L5 DRG 4 h after LPS injection for qPCR analysis of Ccl2 and Notch signaling genes (batch 1, N = 6 mice per group). Proteins were extracted from the L3-L5 DRG 6 and 24 h after LPS injection for ELISA detection of CCL2 (batch 2, N = 4 or 5 mice per group), or 24 h after LPS injection for ELISA detection of NICD and CCL2 in the tissue lysates (batch 3, N = 4 mice per group). \n\nTo detect the effect of inhibition of Notch signaling on LPS-induced changes, DAPT (Catalog# S2215, Selleck Chemical, Houston, TX, 100 μg in 3 μL DMSO) was injected into the right knees of mice one hour prior to LPS injection, while the control group received 3 μL vehicle (N = 8 mice per group). Knee hyperalgesia was tested in a 24-h time-course after injection. Proteins were extracted from the ipsilateral DRG 24 h after LPS injection for measuring the levels of CCL2 and NICD in the DRG (N = 5 mice per group).",
"section_name": "Intra-articular (IA) injection of LPS",
"section_num": null
},
{
"section_content": "Knee hyperalgesia was measured using a Pressure Application Measurement (PAM) device (Ugo Basile, Varese, Italy), as previously described [22]. Briefly, mice were restrained by hand and the hind paw was lightly pinned with a finger in order to hold the knee in flexion at a similar angle for each mouse. With the knee in flexion, the PAM transducer was pressed against the medial side of the ipsilateral knee while the operator's thumb lightly held the lateral side of the knee. The PAM software guided the user to apply an increasing amount of force at a constant rate (30 g/s), up to a maximum of 450 g. If the mouse tried to withdraw its knee, the force at which this occurred was recorded. If the mouse did not try to withdraw, the maximum possible force of 450 g was assigned. Two measurements were taken per knee and the withdrawal force data were averaged. The operators (S. I. and J. L. ) were blinded to the treatment groups.",
"section_name": "Assessment of knee hyperalgesia",
"section_num": null
},
{
"section_content": "Ipsilateral L3-L5 DRG were collected 12 or 26 weeks after DMM or sham surgery (N = 6 mice per group at 12 weeks after surgery, and N = 3 mice per group at 26 weeks after surgery). Genes in the canonical Notch signaling pathway that were identified in the microarray study to be differentially expressed in DRG after DMM were selected for quantitative reverse transcription polymerase chain reaction (qRT-PCR), including Jag1, Adam17, Rbpj and Hes1 [7]. Ipsilateral L3-L5 DRG from each mouse were pooled and homogenized in Trizol (Invitrogen, Carlsbad, CA) on ice, vigorously mixed with chloroform (Sigma, St. Louis, MO), and centrifuged for 15 min at 12,000xg at 4 °C. RNA was extracted from the upper aqueous phase using RNeasy kit (Qiagen, Hilden, Germany). The ratios of A260/A280, which were assessed for the quality of RNA, ranged from 1. 86 to 2. 03, with an average of 1. 95. Reverse transcription was performed using RT 2 first strand kit (Qiagen). Quantification of mRNA was conducted on a Bio-Rad CFX96 machine using the Qiagen SYBR Green qPCR master mix and the RT 2 primer assays (Supp. Tab. 1). Gapdh was used as an internal control for normalization of target gene expression. The comparative 2 -ΔCT method was utilized for relative quantitation of gene levels of expression, presented as 2 -ΔCT(Gene of interest -Gapdh). \n\nTo localize the expression of Jag1, Notch1 and Hes1, RNA in situ hybridization (ISH) was performed in 4% paraformaldehyde (PFA)-fixed L4 DRG frozen sections from mice 26 weeks after DMM or sham surgery (N = 3 mice per group) using RNAscope ™ Multiplex Fluorescent v2 kit and RNAscope ™ probes (Advanced Cell Diagnostics, Newark, CA), according to the manufacturer's instructions. Hybridization signals were detected using Opal fluorophores (Akoya Biosciences, Marlborough, MA). Slides were mounted with ProLong Gold Antifade Mountant (Life Technologies, Eugene, OR) and viewed with an Olympus FV10i confocal laser scanning microscope (Tokyo, Japan). \n\nFor quantification of mRNA expression in neurons, H-score (ranged from 0 to 400) was calculated as sum of each (bin number x percentage of cell per bin) according to the 0-4 five-bin scoring system recommended by the manufacturer [23]. Results are presented as an average of the findings from two of the authors (J. L. and L. W. ), who were blinded to sample information.",
"section_name": "Notch signaling gene expression in DRG",
"section_num": null
},
{
"section_content": "Protein levels of NICD, the active form of Notch protein, in L3-L5 DRG from mice 12 or 26 weeks after DMM or sham surgery (N = 4-7 mice per group) were assessed using PathScan Cleaved Notch1 Sandwich ELISA Kit (Cell Signaling Technology, Danvers, MA) following the manufacturer's instructions. Proteins were extracted from DRG using 1 × Cell Lysis Buffer (Catalog# 9803, Cell Signaling Technology). NICD levels in DRG cell lysates are presented as absorbance at 450 nm normalized to total protein, which was measured using the BCA (bicinchoninic acid) assay (Thermo Fisher, Rockford, IL). \n\nTo detect NICD in DRG, IF analysis was performed on L4 DRG from mice 12 or 26 weeks after DMM or sham surgery (N = 3 mice per group). Mice were perfused transcardially with PBS followed by 4% PFA. The spinal column was dissected and postfixed in 4% PFA overnight followed by cryopreservation in 30% sucrose in PBS. L4 DRGs were embedded with OCT compound (Fisher Healthcare), frozen with dry ice, and cut into 12-μm sections. Sections were stained overnight at 4 °C with an antibody against active Notch (NICD) (Abcam Cat# ab52627, RRID:AB_881725; 1:200), followed by an Alexa Fluor 633-conjugated secondary antibody (Invitrogen, 1:1000) for 1 h at room temperature. The slides were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Fluorescent signals were quantified as mean fluorescence intensity (MFI) using ImageJ [24]. For each DRG, 2 or 3 sections were evaluated and averaged.",
"section_name": "Active Notch protein, NICD, in DRG",
"section_num": null
},
{
"section_content": "To detect the effect of inhibition of Notch signaling on DMM-induced OA pain, DAPT (100 μg in 5 μL DMSO), was injected IA into the right knees, 12 weeks after DMM. DAPT was administered IA, aiming to investigate the DRG that directly innervate the knee joints. IA injection also limits the systemic effects of Notch signaling inhibition and other γ-secretase substrates inhibition by DAPT. Knee hyperalgesia was tested in a 24-h timecourse after injection (N = 6 mice per group). \n\nIn another experiment, mice were injected IA with DAPT 12 weeks after DMM or sham surgery. RNA was extracted from the ipsilateral L3-L5 DRG 6 h after injection for Ccl2 qPCR analysis (N = 6 mice per group), or proteins were extracted from the L3-L5 DRG 24 h after injection for CCL2 and NICD ELISA analysis (N = 7 or 5 mice per group).",
"section_name": "IA injection of DAPT in DMM joints",
"section_num": null
},
{
"section_content": "Results are presented as mean ± SEM. Statistical analyses were performed to compare two groups using unpaired two-tailed Student's t-tests at a significance level of p < 0. 05. Time-course knee hyperalgesia measurements were analyzed by two-way repeated measures ANOVA with Šidák post hoc test at a significance level of p < 0. 05, and area under the curve (AUC) over the time course was analyzed using unpaired two-tailed Student's t-tests.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We have previously reported that TLR4 stimulation with LPS results in increased production of the proalgesic chemokine, CCL2, in cultured DRG cells [17]. We used the same approach to start exploring the potential link between Notch signaling and TLR activation in DRG cells by testing whether LPS activates Notch signaling in cultured DRG cells, and whether LPS-induced CCL2 expression is dependent on activation of Notch signaling. \n\nFirst, L3-L5 DRG cells from naïve 10-week old male C57BL/6 mice were isolated, cultured, and stimulated with LPS (1 μg/mL) for 24 h. This led to increased NICD expression in DRG cells (representative images in Fig. 1A ), with a higher MFI (88. 07 ± 6. 59) compared to the controls (62. 61 ± 7. 17) (p = 0. 023, N = 7 or 8 cultures per treatment group) (Fig. 1B ). Consistent with previous findings [17], LPS stimulation increased Ccl2 mRNA expression in these cell cultures, as well as release of CCL2 protein into the culture supernatants, compared to controls treated with vehicle (Fig. 1C-D ). \n\nNext, to test the effect of inhibition of Notch signaling on TLR4 stimulation, we blocked Notch signaling with the γ-secretase inhibitor, DAPT, or with soluble Jagged1. Pretreatment of the cells with DAPT (25 μM) attenuated the LPS-induced increase of NICD, Ccl2 mRNA, and CCL2 protein (p = 0. 013, p = 0. 013, and p = 0. 011, respectively, N = 4-8 independent cultures per treatment group) (Fig. 1B-D ). Similarly, blocking Notch ligandreceptor interaction with soluble Jagged1 (40 μM) also suppressed LPS-induced increases of NICD, Ccl2 mRNA, and CCL2 protein when compared to scrambled peptide control (p = 0. 005, p = 0. 036, and p = 0. 015, respectively, N = 4-9 cultures per group) (Fig. 1E-G ). \n\nConversely, we used immobilized Jagged1 to activate Notch signaling in DRG cultures. The activation was confirmed by the increase of NICD, as shown in representative cultures in Fig. 2A and quantified and CCL2 protein, when compared to cells incubated with immobilized IgG-Fc (p = 0. 042 and p = 0. 014, respectively, N = 6 cultures per group) (Fig. 2C-D ). \n\nTaken together, these in vitro findings suggest that LPS stimulation activates the Notch signaling pathway in cultured DRG cells, and Notch signaling is involved in CCL2 synthesis induced by LPS.",
"section_name": "LPS stimulation increases NICD in DRG cultures, and induces CCL2 expression in a partly Notch-dependent manner",
"section_num": null
},
{
"section_content": "In order to assess whether the in vitro findings in DRG cultures can be translated in vivo, we injected LPS intra-articularly into the knee joint cavity of naïve mice, and monitored the effect on Notch signaling in knee-innervating DRGs, as well as on knee hyperalgesia. LPS (3 μg in 3 μl saline) injection into the right knee joint of 12-week old naïve male C57BL/6 mice caused transient knee hyperalgesia, which peaked after 4 h and returned to baseline 24 h after injection (Fig. 3A ) (twoway ANOVA test: p < 0. 05 vs. saline control; post hoc test: p < 0. 001, p < 0. 0001, and p = 0. 005 at 2, 4, and 6 h after injection, respectively. N = 6 mice per group). \n\nWe then determined whether upregulation of Notch signaling genes in the DRG was observed in this model of transient knee joint pain. To this end, LPS or vehicle (N = 10 mice per group) was injected IA, and ipsilateral L3-L5 DRG were collected and pooled 4 h later for mRNA analysis (N = 6 mice per group), or 24 h after injection for We found increased mRNA expression of Adam17, Rbpj, and Hes1 in DRG 4 h after LPS administration compared to saline (p = 0. 017, 0. 012, and 0. 046, respectively), while Jag1 and Notch1 genes were unchanged (Fig. 3B ). Quantification of NICD protein levels by ELISA in the lysates revealed that NICD levels were increased in LPS-injected mice compared to vehicle controls (p = 0. 025) (Fig. 3C ). \n\nWe have previously shown that CCL2 is upregulated in the DRG that innervate painful knees, in the DMM model [18] as well as in knee hyperalgesia induced by TLR2 stimulation [22]. Hence, we measured CCL2 expression in the L3-L5 DRG after intra-articular LPS injection. We found that LPS-induced hyperalgesia was accompanied by increased CCL2 expression in the DRG: Ccl2 mRNA was upregulated 4 h after LPS injection, the time point when knee hyperalgesia peaked (Fig. 3D ), and CCL2 protein levels were increased both 6 and 24 h after injection (Supp. Figure 1 and Fig. 3E ). \n\nThese results show that IA injection of LPS models transient knee pain, and this is associated with activation of the Notch signaling pathway. Furthermore, knee pain and Notch signaling are accompanied by upregulation of the pro-algesic chemokine, CCL2, in kneeinnervating DRG.",
"section_name": "IA injection of LPS induces knee hyperalgesia and activates Notch signaling in DRG",
"section_num": null
},
{
"section_content": "Since IA administration of LPS in the knee was associated with knee hyperalgesia and Notch signaling activation in the lumbar DRG, we evaluated whether blocking Notch signaling through DAPT had an effect on LPSinduced knee hyperalgesia. We injected DAPT (100 μg) into the knees one hour prior to LPS administration, and monitored knee hyperalgesia over a 24-h time-course, as before. We found that pretreatment with DAPT alleviated LPS-induced hyperalgesia, with significant reductions 2 and 4 h after LPS injection (p = 0. 0002 and 0. 037, respectively, N = 8 mice per group) (Fig. 4A ). In order to confirm that this effect was mediated through inhibition of the Notch signaling pathway, we showed that pre-injection of DAPT also decreased the protein levels of NICD compared to vehicle control (p = 0. 031, N = 5 mice per group) (Fig. 4B ), and this was accompanied by decreased CCL2 protein in the ipsilateral DRG collected 24 h after LPS injection, compared to vehicle (p = 0. 002, N = 5 mice per group) (Fig. 4C ).",
"section_name": "Local administration of DAPT reduces LPS-induced knee hyperalgesia and CCL2 expression in DRG",
"section_num": null
},
{
"section_content": "The results described above suggest that the activation of the Notch signaling pathway in knee-innervating DRG has a functional role in knee pain through TLR activation and subsequent production of the pro-algesic chemokine, CCL2. Therefore, we aimed to broaden these findings to a model of experimental OA, induced by DMM surgery.",
"section_name": "Notch signaling in lumbar DRG after DMM, and its role in knee hyperalgesia",
"section_num": null
},
{
"section_content": "First, we explored whether genes in the canonical Notch signaling pathway are upregulated in a surgical model of OA induced by DMM-as previously suggested by our microarray studies, which revealed that genes in the canonical Notch signaling pathway are differentially regulated in the DRG from mice with latestage OA after DMM compared to sham-operated controls [7], including the Notch ligand, Jag1, the S2 Notch cleavage enzyme, Adam17, the Notch transcription moderator, Rbpj, and the Notch target gene, Hes1. Here, we sought to validate these findings using qRT-PCR to assess gene expression of these Notch signaling components in the ipsilateral L3-L5 DRG after DMM or sham surgery. Twelve weeks after DMM vs. sham surgery, DRG mRNA levels were increased for Jag1 (p = 0. 034), Adam17 (p = 0. 019), Rbpj (p = 0. 029), and Hes1 (p = 0. 049) (Fig. 5A-D ). These genes were still upregulated 26 weeks after DMM surgery (p = 0. 016, p = 0. 049, p = 0. 040, and p = 0. 011, respectively) (Fig. 5A-D ), confirming the findings from the microarray study. \n\nWe then used RNAscope to detect in situ expression of Notch signaling components (Jag1, Hes1, and Notch1) in the ipsilateral L4 DRG, 26 weeks after surgery. Neurons were identified by DAPI staining and phase contrast images, which is comparable to the method of identification of neurons with IF staining of a neuronal marker, PGP9. 5 (Supp. Figure 2 ). Jag1 was expressed in DRG neurons both after sham and DMM surgery, and the H-scores revealed no difference in expression level (319. 96 ± 2. 50 after sham vs. 329. 25 ± 3. 58 after DMM) (Fig. 6A ). Hes1 was also detected in DRG neurons, and increased 26 weeks after DMM compared to sham surgery, with H-scores of 238. 9 ± 4. 6 after sham vs. 263. 5 ± 2. 9 after DMM (p = 0. 010) (Fig. 6B ). Finally, RNAscope detected neuronal Notch1 expression, which increased 26 weeks after DMM, with an H-score of 337. 3 ± 20. 5 vs. 268. 9 ± 12. 6 after sham surgery (p = 0. 047) (Fig. 6C ). \n\nSince RNAscope ISH suggested neuronal expression of these Notch signaling components, we examined neuronal expression of additional Notch pathway genes in a recently completed single cell RNA sequencing (scRNAseq) analysis of L3-L5 DRGs of male naïve mice at 18 weeks of age [25]. The results showed that Notch ligand Dll3, Notch transcriptional binding complex genes (Rbpj, Maml3), target gene (Hey1), and γ-secretase complex component genes (Ncstn, Aph1a, Psen1, Psen2) are predominantly expressed in nociceptors (Supp. Figure 3A-B ). qRT-PCR confirmed the upregulation of many of these genes (Dll3, Rbpj, Maml3, and Aph1a) in DRG 26 weeks after DMM (Supp. Figure 3C-J ).",
"section_name": "Expression of genes in the Notch signaling pathway in the DRG after DMM vs. sham surgery",
"section_num": null
},
{
"section_content": "Having established that the Notch signaling pathway is regulated in DRG neurons after DMM surgery, we sought to assess whether the upregulation of these genes is accompanied by the release of the Notch intracellular domain, NICD, which is generated when γ-secretase cleaves the Notch receptor [10]. Here, we confirmed the presence of NICD by measuring NICD protein levels by ELISA in the tissue lysates of ipsilateral L3-L5 DRG. We found an increase 12 and 26 weeks after DMM, compared to sham-operated mice (12 weeks: p = 0. 034, N = 4 sham and 5 DMM; 26 weeks: p = 0. 026, N = 7 mice per group) (Fig. 7A ). \n\nTo confirm that NICD is generated after DMM, we performed an independent experiment, assessing IF staining on cryo-sections of the ipsilateral L4 DRG, 12 and 26 weeks after surgery. Increased NICD staining was shown in the neuronal cell bodies after DMM compared to sham-operated controls, with an MFI of 27. 87 ± 1. 09 after sham vs. 38. 66 ± 4. 43 after DMM 12 weeks after surgery (p = 0. 077, N = 3 mice per group) (Fig. 7B ), and an MFI of 54. 17 ± 10. 89 26 weeks after sham surgery vs. 100. 08 ± 4. 51 after DMM (p = 0. 018, N = 3 mice per group) (Fig. 7C ).",
"section_name": "Active Notch protein is increased in DRG neurons after DMM",
"section_num": null
},
{
"section_content": "Finally, we evaluated whether blocking Notch signaling in the DMM model through IA injection of DAPT had an effect on knee hyperalgesia. Twelve weeks after DMM, mice showed robust hyperalgesia at the operated knee, manifest as a lower withdrawal threshold, as we have described before [26] (Fig. 8A ). At that time point, we assessed the effect of IA injection of. Each dot represents one mouse DAPT in a 24-h time course. Compared to vehicle, IA administered DAPT (100 μg) attenuated knee hyperalgesia, as indicated by a higher withdrawal threshold (two-way ANOVA test, p = 0. 336 vs. vehicle; AUC for 0 to 6 h, t-test, p = 0. 010 vs. vehicle. N = 6 mice per group) (Fig. 8A ). Knee hyperalgesia returned to pre-injection levels by 24 h after injection of DAPT (Fig. 8A ). \n\nIpsilateral L3-L5 DRG were collected from another group of mice with the same treatment 6 h after injection for qRT-PCR. Ccl2 mRNA expression was increased in DMM mice compared to sham group after injection with vehicle (p = 0. 047, N = 6 mice per group) (Fig. 8B ), and these increases were attenuated when the DMM-operated knees were injected with DAPT (p = 0. 046, N = 6 mice per group) (Fig. 8B ). L3-L5 DRG were also collected and lysed from mice 24 h after DAPT injection for CCL2 and NICD detection in the lysates using ELISA. Compared to vehicle, DAPT injection had a trend to decrease both CCL2 protein (p = 0. 021, N = 7 mice per group) and NICD protein (p = 0. 078, N = 5 mice per group) in DRG (Fig. 8C-D ).",
"section_name": "IA administration of DAPT reduces knee hyperalgesia after DMM surgery, and suppresses CCL2 expression in the DRG",
"section_num": null
},
{
"section_content": "Our studies revealed that the TLR4 agonist LPS increased activated Notch protein in cultured DRG neurons from naïve mice. Activation of Notch signaling also occurred following LPS treatment in vivo, together with increased expression of the chemokine CCL2, which we have demonstrated occurs during the establishment of painful OA [18]. In DRG cell cultures, inhibition of Notch signaling led to decreased LPS-induced CCL2 production, and activation of Notch signaling led to increased LPS-induced CCL2 synthesis. Similarly, in vivo, preinjection of the γ-secretase inhibitor DAPT, resulted in attenuated LPS-induced knee hyperalgesia and decreased LPS-induced NICD and CCL2 in the lumbar DRG. In the DMM model of OA, qRT-PCR detected increased expression of several genes in the Notch signaling pathway in the L3-L5 DRG-including Jag1, Adam17, Rbpj, and Hes1-confirming our published microarray studies [7]. In situ hybridization analysis of the DRG showed that Notch1 and Hes1 were highly expressed in the neuronal cell bodies after DMM, and ELISA and IF staining showed that NICD was increased in DRG neurons. IA Crosstalk between Notch and TLR signaling pathways has been reported in several studies, most of which were focused on macrophages and monocytes under inflammatory conditions [16]. LPS may activate Notch signaling indirectly through upregulating the expression of Notch ligands [27] and receptors [28, 29], or directly through inducing expression of Notch target genes [30]. Conversely, Notch signaling amplifies TLR-mediated proinflammatory responses by increasing NF-κB activity [29] and enhancing expression of pro-inflammatory cytokines [28] [29] [30]. In a murine model of atherosclerosis, blockade of Notch signaling reduced atherogenesis, associated with diminished CCL2 expression and suppressed NF-κB activation in atherosclerotic lesions [31]. \n\nNotch signaling plays a fundamental role in neurogenesis and neuroplasticity [32]. Activation of Notch signaling is involved in the development of neuropathic pain [13, 14]. Interaction between Notch1 and TLR4 signaling pathways in DRG neurons has been reported in diabetic rats with neuropathic pain, where inhibition of either Notch1 or TLR4 signaling attenuated mechanical allodynia and thermal hyperalgesia [33]. In the current study, we showed that an interaction between Notch and TLR4 signaling also exists in the peripheral nervous system in a mouse model of OA. TLR4 signaling in response to DAMPs generated in the osteoarthritic joint may normally activate Notch signaling in lumbar DRG. Activated Notch signaling in sensory afferents may synergize with TLR4 signaling in promoting synthesis of pro-algesic mediators such as CCL2, and thereby mediate the development of knee hyperalgesia. TLR4 stimulation by LPS clearly activates Notch signaling, as is evident from the Of interest, a recently published comparative transcriptome profile analysis revealed conserved enrichment of genes in human and mouse DRG [34]. After reviewing their database of L2 lumbar DRG from male and female human donors with and without neuropathic pain [34, 35], we found that several Notch signaling pathway genes are expressed in human DRG with a TPM (transcripts per million) over 10. Compared to donors with no pain, the Notch target gene HES1 showed a 1. 86-fold increase in male and a 1. 65-fold increase in female donors with pain; Notch ligand JAG1 a 2. 66-fold increase in female donors with pain; and Notch moderator RBPJ a 2. 25-fold increase in females with pain (Supp. Figure 4 ). These data further support that Notch signaling activation in DRG may be associated with pain. \n\nNeurons located in L3-L5 lumbar DRG innervate the knee joint, but it is not clear from our experiments whether the neurons with increased NICD are those that innervate the knee or other structures. Furthermore, the DRG not only contain the cell bodies of sensory neurons, but also a wide variety of other cell types such as satellite glial cells and immune cells. Given the fact that Notch signaling normally proceeds through cell-to-cell interactions it is quite possible that its signaling influence in this case may not be cell autonomous. Further studies are required to answer this question. \n\nOne limitation of our microarray [7] and qRT-PCR data is that they reflect gene expression by all cells in the DRG. Nevertheless, our in situ hybridization results suggest that certain genes such as Notch1 may be preferentially enhanced in neurons even though the global expression as seen through qPCR did not change. Furthermore, scRNA-seq is a powerful tool for studying cellular diversity in the DRG and our laboratory and other groups have confirmed gene expression profiles of some Notch signaling molecules by scRNAseq analysis of DRG cells from naïve mice [25, 36]. The results revealed that Notch signaling genes are expressed in peptidergic and non-peptidergic nociceptive neurons, including genes encoding Notch ligands, receptors, γ-secretase complex components, transcriptional binding complex components, and downstream targets. \n\nTaken together, our studies revealed activation of Notch signaling in the DRGs that innervate the knee, and suggest a role for the pathway in mediating knee hyperalgesia in OA. These findings add to the existing literature implicating the Notch signaling pathway in OA pathogenesis. It has been reported that the pathway is highly activated in joint tissues, both human and murine, in the course of post-traumatic OA [37, 38]. While physiological Notch signaling is required for long-term maintenance of articular cartilage, enhanced Notch signaling leads to progressive OA pathology in a mouse surgical model [39]. Conditional inactivation of Notch signaling in mouse chondrocytes caused resistance to OA development, and inhibition of Notch signaling by IA injecting DAPT for 10 weeks prevented OA development in mice [37]. Combining our findings on the role of Notch signaling in mediating hyperalgesia in OA mice, it could be expected that modulating Notch signaling may not only alleviate OA pain, but also ameliorate the progression of OA. Notch signaling may therefore be explored as a target for controling both OA pain and progression of joint damage. Large-scale temporal gene expression profiling revealed that Notch activation by overexpression of NICD in chondrocytes not only decreased expression of chondrogenic markers and increased expression of inflammatory factors, but also increased the expression of the gene encoding the neurotrophin, nerve growth factor (NGF) [39]. Since structural changes in joint innervation, including NGF-induced sprouting, have been reported in OA joints and may be related to joint pain [40] [41] [42], upregulation of NGF through Notch activation in chondrocytes may constitute another way in which the Notch pathway contributes to OA pain.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "The presented findings suggest that peripheral neuronal Notch signaling contributes to joint pain in murine experimental OA. A model is emerging whereby TLR4 stimulation in the OA joint activates Notch signaling in lumbar DRG. Synergy between Notch signaling and TLR4 signaling promotes joint pain through increased synthesis of the pro-algesic chemokine, CCL2. The Notch signaling pathway in the peripheral nervous system therefore merits deeper exploration as a target for alleviating OA pain.",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Not applicable.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "Rush University Cohn fellowship (to LW); National Institutes of Health (NIAMS) ( R01AR064251, R01AR060364, P30AR079206 to AMM; R01AR077019 to REM).",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "All data generated or analyzed during this study are included in this published article.",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "A disintegrin and metalloprotease CCL2 Chemokine C-C motif chemokine ligand 2 DAMP Damage-associated molecular pattern DAPT N-[N- (3, 5",
"section_name": "ADAM",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13075-023-03039-1. \n\nAdditional file 1: Supp. Tab. 1. List of PCR primers and RNAscope probes used in the qRT-PCR and RISH. Supp. Fig. 1. Protein levels of CCL2 in DRG tissue lysates 6 and 24 h after IA injection of LPS or vehicle (N =4 or 5 mice per group). CCL2 levels are presented as CCL2 protein normalized to total protein (pg CCL2/mg total protein). Supp. Fig. 2. Identification of DRG neurons using phase contrast micrographs and DAPI staining, compared to the method using a neuronal marker PGP9. 5 IF staining. Sections were stained overnight at 4°C with an antibody against PGP9. 5 (Sigma-Aldrich Cat# SAB4503057, RRID:AB_10761291; 1:200), followed by an Alexa Fluor 488-conjugated secondary antibody (Invitrogen, 1:1000) for 1 h at room temperature. Scale bars, 50 µm. Supp. Fig. Gene expression was presented as 2 -ΔCT(Gene of interest -Gapdh). Each dot represents one mouse. Supp. Fig. 4. Notch signaling genes (JAG1, NOTCH1, RBPJ, and HES1) in human DRG. Raw data of TPM (transcripts per million) were obtained from comparative transcriptome profile analysis of L2 lumbar DRG of human donors [34, 35]. Gene expression was compared between donors without pain and those with neuropathic pain using unpaired 2-tailed Student's t-test. Totally 15 males (4 \"no pain\" and 11 \"pain\") and 12 females (4 \"no pain\" and 8 \"pain\") were analyzed. \n\nAuthors' contributions L. W. contributed to the design and interpretation of the experiments, performed cell culture, qPCR, RISH, IF, ELISA, IA injection experiments, and wrote the manuscript. S. I. performed surgeries, IA injection, and knee hyperalgesia experiments. J. L. performed surgeries, knee hyperalgesia experiments, and quantification of the images. R. E. M. contributed to the design and interpretation of the experiments. A-M. M. contributed to the design and interpretation of the experiments and was a major contributor in writing the manuscript. The authors read and approved the final manuscript.",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "All animal experiments were approved by the Institutional Animal Care and Use Committee at Rush University Medical Center.",
"section_name": "Declarations Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Not applicable.",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. \n\n• fast, convenient online submission\n\n• thorough peer review by experienced researchers in your field\n\n• rapid publication on acceptance\n\n• support for research data, including large and complex data types\n\n• gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "At BMC, research is always in progress.",
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{
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"section_name": "Learn more biomedcentral.com/submissions",
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"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.18632/oncotarget.24927
|
Assessment of micro RNAs expression in leukemic cells as prognostic markers in chronic lymphocytic leukemia: micro RNAs can predict survival in a course of the disease
|
Numerous genetic alterations predicting prognosis and clinical outcome are revealed recently in chronic lymphocytic leukemia (CLL). Among them the deregulated expression of micro RNAs that can induce tumor growth, or act as tumor suppressors seem to be of great importance. This study aimed to analyze the possible role of chosen micro RNAs as markers of prognosis in patients with CLL. We assessed the expression of miR-21, miR-34a, miR-181a, miR-199a/b and miR-221 in previously separated leukemic cells with the use of qRQ-PCR technique at the moment of diagnosis. The results were then analyzed in regards to presence of prognostic factors, clinical data and the end points like progression free survival (PFS), time to progression (TP) and overall survival time (OS). We detected significant correlations between expression of the analyzed micro RNAs and CLL prognostic markers particularly as far as miR-221 and miR-181a were concerned. The subsequent analysis revealed that high expression of miR-34a and miR-181a as well as low miR-21 expression indicated longer TTP, while miR-221 was predictor of OS. The obtained results prove the role of micro RNAs as CLL prognostic markers, particularly as factors predicting survival in a course of the disease.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL), one of the most frequently diagnosed leukemias, is characterized by the accumulation of leukemic CD19+/CD5+/CD23+ B cells in the blood, bone marrow, lymph nodes and spleen [1, 2]. Clinical course and prognosis of this type of leukemia are highly variable. Some patients with benign disease never require therapy and die because of causes other than leukemia. In others the treatment is started soon after diagnosis, because of the aggressiveness of the disease [3, 4]. Numerous factors are used to predict prognosis and clinical outcome of CLL patients like the mutational status of immunoglobulin heavy chain genes (IgVH)), ZAP-70 and CD38 expression as indicators for IgVH mutations as well as gene and genomic abnormalities [5, 6, 7]. \n\nLots of mechanisms involved in leukemic transformation of CLL are reported. The B cell receptor signaling plays an important pathogenic role because of BCR-dependent survival of leukemic lymphocytes [8, 9]. Pro-proliferative signals are also mediated from microenvironment composed of macrophages, T cells, or stromal follicular dendritic cells. This microenvironment produces various essential proteins like chemokines and cytokines that by interacting with leukemic cells may",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "induce their survival [7, 10, 11]. Additionally, numerous genetic alterations are revealed recently in CLL. These are single-nucleotide polymorphisms, chromosomal alterations and alterations in non-coding RNA, like micro RNA (miR) [7, 9, 12]. Micro RNAs belong to epigenetic regulators that modulate gene expression and cellular signaling pathways. Micro RNAs may be deregulated in human cancers, some of them induce tumor growth, while others act as tumor suppressors [13, 14]. They expression can be used to predict prognosis and clinical response to treatment in cancer patients [13, 15]. CLL was the first proliferative disorder that was reported to be connected with alterations in micro RNAs. In particular, miR-15a and mirR-16-1 both target BCL2 and MCL1 expression are dysfunctional in about 60% of patients with CLL [7, 16, 17]. Such abnormalities lead to the resistance of B lymphocytes towards apoptosis. Attention has also focused on other micro RNAs in CLL patients that are dysregulated and may be overexpressed or show low level. Thus extensive research are currently conducting to find the pattern of micro RNA expression in CLL patients which could be used as prognostic factor in everyday clinical practice. \n\nThe presented article aimed to analyze the possible role of chosen micro RNAs as a markers of prognosis in patients with CLL. The expression of miR-21, miR-34a, miR-181a, miR-199a/b and miR-221 in previously separated CD19+ leukemic cells was assessed with use of qRQ-PCR technique at the moment of diagnosis. The obtained results were then analyzed in regards to presence of prognostic factors, clinical data and the end points like progression free survival (PFS), time to progression (TP) and overall survival time (OS).",
"section_name": "Research Paper",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "Our analysis revealed an expression of all analyzed micro RNAs in leukemic cells except of miR-199a/b, so this micro RNA was not further assessed. Micro RNAs expression presented as mean ± standard deviation are shown in Table 1. According to the level of particular micro RNA expression the patients were divided into groups of low and high expression, respectively (see Table 1 ). The cut-off point was established as the mean expression of micro RNA in the study group.",
"section_name": "Assessment of micro RNAs expression in leukemic cells",
"section_num": null
},
{
"section_content": "We detected statistically significant negative correlation between expression of miR-221 and both leukocytosis and lymphocytosis. Other micro RNAs were not in correlation with parameters of complete blood count. The expression of both miR -181a and miR-221 were in adverse significant correlation with serum level of β2-mikroglobulin. These micro RNAs were also in negative correlation with expression of CD38 on leukemic cells, however such a relationship was not found as far as expression of ZAP-70 was concerned. Additionally, expression of miR-221 was detected to be different in regard to cytogenetic risk. In a group of standardcytogenetic risk the expression was higher in comparison to high-risk group. The results are shown in Figure 1. We did not detect any correlation between expression of miR-21, miR-34a, miR-181a, miR-221 and age and sex of the studied patients. There was no relationship between micro RNA expression and clinical stadium of CLL, either.",
"section_name": "Assessment of micro RNAs expression in leukemic cells in relation to CLL prognostic markers",
"section_num": null
},
{
"section_content": "The group of studied patients was assessed in regards to first line chemotherapy regimens as well as chemotherapy outcome. Only the subjects (20 persons) who required therapy because of the disease progression were enrolled into this part of study. The following schemes of chemotherapy were used: chlorambucil + prednisone (9 persons), fludarabine + cyclophosphamide (7 persons), fludarabine + cyclophosphamide + rituximab (2 persons), bendamustine (1 person), cyclofosfamide + vinkristin + doksorubicin + prednisone (1 person). The patients were classified as responders (those who obtain complete or partial remission) and non-responders (those who have stable disease or progression). Statistical analysis indicated that expression of analyzed micro RNAs was not a predictor of chemotherapy outcome, however the variability of regimens used and a low number of patients in each group might influence the results.",
"section_name": "Assessment of influence of microRNA expression on overall response rate",
"section_num": null
},
{
"section_content": "The study group was analyzed with reference to time to leukemia progression. In the moment of analysis 20 out of 40 enrolled patients were diagnosed because of leukemia progression. The others were in stable phase of the disease. The patients with progression detected were divided into 2 groups: the first one with moment of progression which occurred within 1 year after diagnosis (n = 14; 1. 93 ± 3. 97 months ) and the second one with time to progression longer than 1 year after beginning of leukemia (n = 6; 33. 50 ± 16. 29 months). Analysis of micro RNAs expression in the above groups revealed the statistically significant difference in expression of miR-34a. Similarly, Kaplan-Meier analysis of progression probability showed statistically significant difference between a group of patients with high versus low expression of miR-34a. These results are shown in Figure 2. \n\nAs far as expression of miR-221, miR-21, miR-181a were concerned no statistically significant differences were detected. However, the multivariate assessment based on linear regression model assessing influence of all studied micro RNAs on TTP revealed statistical significance (F = 7. 79; p < 0,001; ∆R 2 = 0. 59). High expression of miR-34a (β = 0. 67) and miR-181a (β = 0. 49) as well as low miR-21 expression (β = -0. 46) significantly influence TTP. The data are presented in Figure 3.",
"section_name": "Analysis of microRNA expression in regard to time to progression",
"section_num": null
},
{
"section_content": "The analysis of PFS was performed in the group of subjects who have completed first-line therapy (20 persons). They were divided into two groups: the first one of patients who progressed after the first line of treatment within one year (n = 12; M ± SD = 2. 0 ± 3. 232 months) and the second one who progressed in the period longer than one year (n = 8; M ± SD = 27. 0 ± 11. 402 months). Analysis of microRNA in the above groups revealed statistically significant difference only in case of miRNA-221 expression (0. 016 ± 0. 028 versus 0. 031 ± 0. 021, p = 0. 045). Analysis based on linear regression in regards to all studied micro RNAs showed no statistical significance (F = 0. 23; p < 0. 92; ∆R 2 = -0. 24). Assessment with Kaplan-Meier test showed no statistically significant difference in predicting PFS between groups with high and low micro RNA expression, respectively.",
"section_name": "Assessment of influence of microRNA expression on progression free survival",
"section_num": null
},
{
"section_content": "The length of patient observation was between 1 and 106 months (59. 90 ± 33. 88). Up to the moment of analysis 45. 2% of patients (n = 19) died. All subjects were divided into two groups: the first one -patients who survived less than 5 years (n = 16; 21. 87 ± 17. 23 months ) and the second one -those who survived more than 5 years (n = 24; 84. 24 ± 12. 08 months). Analysis of micro RNAs in the above groups revealed statistically significant difference in miRNA-221 expression. The linear regression assessment did not revealed statistical significance (F = 1. 31; p < 0. 29; ∆R 2 = 0. 03). Analysis with Kaplan-Meier test showed that the probability of survival was higher in the group of patients with elevated level of miR-221 expression in comparison to the group with lower miR-221 level, however the difference was not statistically significant (Figure 4 ).",
"section_name": "Assessment of microRNA expression as predictor of overall survival",
"section_num": null
},
{
"section_content": "Until the moment of investigation 19 persons of the study group died. With a use of linear regression analysis we assessed correlation between miR-21, miR-34a, miR-181a and miR-221 expression and risk of death. However no statistically significant differences were found.",
"section_name": "Influence of micro RNAs expression on patients morbidity",
"section_num": null
},
{
"section_content": "Micro RNAs seem to be very informative biomarkers of clinical value that are easy-to-assessed both in cells and in body fluids [18, 19]. The role of particular micro RNAs expression in CLL patients was investigated and reported, however the one micro RNAs profile for this disease was not precisely established yet. Thus we tried to assess the role of the expression of following micro RNAs: miR-21, miR-34a, miR-181a, miR-199a/b and miR-221 as prognostic factors in CLL in relation to course of the disease and survival of patients. \n\nFirstly, the correlation of micro RNAs expression and the established CLL prognostic factors was analyzed. We detected that low expression of miR-221 was observed together with higher leucocytes and lymphocytes concentration that may indirectly indicates the higher rate of leukemic cells proliferation. In the group of subjects with high cytogenetic risk (del 17p or del11q) miR-221 expression was significantly lower than in those with standard cytogenetic risk. Additionally, we detected negative correlation between miR-221 expression and (10) According to level of particular micro RNA expression the patients were divided into groups of low and high expression, respectively. The cut-off point was assumed to be the mean expression of a micro RNA in the study group. NA-not applicable, n-number of patients. β-2-mikroglobulin level. It can be concluded that a low expression of this micro RNA results in more progressive course of leukemia and is correlated with the presence of high-risk-cytogenetic profile. In case of miR-181a the correlation with CD38 expression was detected. In patients with low miR-181 level the expression of CD38 was significantly higher. Similarly, in patients with higher β-2-mikroglobulin concentration the expression of miR- 181a was significantly lower. There were no detectable difference between expression of other micro RNAs and prognostic factors. Expression of miR199a/b was not detected at any of studied patients indicating that this miRNA cannot be consider as prognostic factor in CLL. \n\nThe literature data on micro RNA expression in course of CLL are incomplete and sometimes confused. Rodrigez et al. [20] showed that miR-221 is significantly lower in patients with del 17p, del11q and del13q in comparison to other patients. Contrary, Callin et al. [21] indicated the high expression of this micro RNA in cases with poor cytogenetic risk and aggressive course of leukemia. MiR-199a is widely reported to be changed in tumors like ovarian cancer, thyroid gland cancer and breast cancer [22] [23] [24] [25]. Tropan et al. [26] assessed expression of this micro RNA in patients with DLBCL and indicted differences between cases with central nervous system infiltration and those without such manifestation. Pallash et al. [27] showed low expression of miR-199b in course of CLL, similarly to our results. Visone et al. [28] analyzed some micro RNAs expression in CLL patients of different cytogenetic risk groups. They indicated that in newly diagnosed subjects the presence of del 17p, high ZAP-70 expression and unmutated IgVH correlated with low expression of miR-29b, miR-29c, miR-223 and miR181 which indicated the shorter TTP. However, overexpression of miR-181a co-existing with trisomy 12 significantly downgraded prognosis [28]. The role of miR-34a expression in CLL course was previously examined as well. Its low expression was proved to be in correlation with del 17p and/or TP53 mutation. Additionally, abnormal miR-34a expression influenced expression of genes involving in CLL pathogenesis TCL1, BCL2, MCL1 as well as cykline D1 and p21 [29]. Similarly, miR-181a expression was demonstrated as regulator of TCL1 oncogene. In the advanced and aggressive stadium of leukemia miR-181a expression was significantly decreased [30]. Rossi et al. [31] indicated that in CLL patients with del 17p the overexpression of miR-21, miR-155, miR-15atogether with low miR-34a and miR-181b expression are characteristic. Additionally, the authors proved the correlation between the level of TP53 abnormalities and miR-34a and miR-155 expression [31]. \n\nOur analysis of micro RNAs expression in regards to end points in CLL patients revealed that high expression of miR-34a and miR-21 as well as low miR-181a expression significantly influenced TTP, while miR-221 and miR-181a indicated OS. Some literature data showed correlations of microRNA expression and the end points in hematologic disorders. Marcuccii et al. [32] indicated in group of patients with acute myeloid leukemia, that high miR-181a expression is connected with higher rate of complete remission and longer OS [32]. In DLBCL patients low level of miR-181a expression correlated with shorter OS [33]. Zhu et al. [34] assessing CLL patients showed correlation between low miR-181a expression and survival time. Analysis of miR-21 expression showed the correlation of its high level and poor prognosis in liver cancer, colon cancer and osteosarcoma [35] [36] [37]. There is also evidence on the correlation between high miR-21 expression and both OS and PFS in DLBCL patients [38, 39], however different results indicating that high miR-21 expression was in correlation with poor prognosis in this group of patients were also reported [40]. In regards to miR-221 expression there are results showing the correlation between its overexpression and shorter OS in liver cancer and non-small cell lung cancer [41, 42]. There is no evidence on miR-221 expression in CLL, however the correlation between its level and poor cytogenetic profile was reported. Thus, it may be presumed that this micro RNA overexpression will be observed together with shorter OS and poorer prognosis [20, 21]. The group of studied patients was assessed in regards to chemotherapy outcome, however no significant correlations were detected. Scientific literature data indicated that expression of specific micro RNAs may help to predict the chemotherapy outcome in CLL patients. Moussay et al. [43] showed the correlation of high miR-29, low miR-181 and low miR-221 expression with good response to fludarabine-based therapy. Similarly, Zhu et al. [44] proved the connection of fludarabine sensitivity and low miR-181 expression. Contrary, in patients with resistance to fludarabine the mir-21 overexpression and low expression of miR-34a were reported [45, 46]. In our study the variability of regimens used and a low number of patients in each group might influence the results. Thus further assessment in the bigger group of patients will be continued. \n\nTo summarize, based on our results and the presented literature data we can conclude that micro RNAs may be the prognostic factors in the course of the disease and the predictors for end points in CLL patients. Expression of miR-21, miR-34a and miR-181a may be useful in predicting TTP, while expression of miR-221 may indicate OS. Thus further studies are required in this field of CLL biology to prove the importance of micro RNAs expression in clinical practice.",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Forty newly diagnosed CLL patients, previously not treated, were enrolled into the study. The diagnosis of CLL was based on clinical examination and morphological and immunological criteria. The clinical characteristics are shown in Table 2. The local Bioethics Committee granted permission to conduct the research and patients were asked to sign the informed consents. Samples of peripheral blood were collected in syringes with the anticoagulant edetate (Sarstedt, Germany).",
"section_name": "Research material",
"section_num": null
},
{
"section_content": "Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation (Biocoll AG Biochrom, Germany). After washing with phosphatebuffered saline, the number and viability of cells were assessed with trypan blue staining. Viability below 95% was a disqualifying criterion for further study. PBMC were subsequently subjected to the procedure of leukemic cells separation. The magnetic activated cell sorting method was used according to manufacturer's instruction (MACS Cell Separation, Germany). PBMC were firstly incubated with magnetic beads coated with monoclonal antibody (MoAb) anti-CD19 in MACS buffer and then subjected to washing procedures to obtain the cell pellet for application to the MACS column. In this way the CD19+ leukemic cells were obtained, their viability was assessed and they were used for further procedures.",
"section_name": "Isolation of B-cells from peripheral blood mononuclear cells with MACS method",
"section_num": null
},
{
"section_content": "Total RNA was isolated from leukemic cells according to the modified method of Chomczynski and Sacchi [47]. The mirVana Isolation Kit (Ambion, USA) for obtaining RNA was used. RNA concentration and integrity was determined by spectrophotometer. Isolated RNA was stored at -20° C until further procedures.",
"section_name": "RNA extraction",
"section_num": null
},
{
"section_content": "Assessment of microRNA expression was performed using the TaqMan ® Small RNA Assays Kit (Applied Biosystems, USA). Initially, total cellular RNA was reverse-transcribed with specific primers using TaqMan ® MicroRNA Reverse Transcription Kit according to the manufacturer's protocol on Applied Biosystems 7500 Fast Real Time PCR Systems. The obtained complementary DNA (cDNA) was used for further procedures. The quantitative analysis of microRNA expression was done with quantitative reverse transcriptase real-time PCR, (qRT-PCR) method. Following probes were used: hsa-miR-21-5p, hsa-miR-34a-5p, hsa-miR-181a-5p, hsa-miR-199a-5p, hsa-miR-221-3p and hsa-miR-16 as endogenous control. A positive reaction was detected by accumulation of a fluorescent signal. The cycle threshold (Ct) was defined as the number of cycles required for the fluorescent signal to cross the threshold and exceeds background level. Ct levels were inversely proportional to the amount of microRNA in the sample. The expression of each micro RNA was normalized with the endogenous control. The comparative ΔΔCq method was then applied for data analysis, and fold changes were next calculated using 2 -ΔΔCq [48]. All PCR reactions were run in duplicate.",
"section_name": "MicroRNA quantification",
"section_num": null
},
{
"section_content": "Sample of PBMC cells were stained with the MoAbs anti-CD19 PE-Cy7, CD5 APC or CD3 PE (BD Pharmingen). After membrane staining, the cells were fixed by 1% paraformaldehyde solution. Next, anti-ZAP-70 antibody (Biomol Research Laboratories, USA) that was labeled by the ZenonTM Alexa Fluor ® 488 Mouse IgG2a Labeling Kit (Molecular Probes, USA) was added to the sample tubes. The samples were incubated for 30 min, washed, and examined by flow cytometry method. When ZAP-70 expression was detected in ≥ 20% of leukemic cells, the subject was considered positive for ZAP-70. To assess CD38 expression, PBMC were stained with anti-CD38 FITC, anti-CD19 PE, anti-CD5 CyChrome MoAbs, or IgG1 isotypic control (BD Pharmingen) for flow cytometry analysis. Patients were considered CD38 positive when expression was found in at least 20% of CLL cells.",
"section_name": "Analysis of ZAP-70 and CD38 expression",
"section_num": null
},
{
"section_content": "Fluorescence in situ hybridization (FISH) method was used to determine cytogenetic abnormalities in leukemic cells. The locus-specific probes 17p13. 1 (LSI TP53), 11q22. 3 (LSI ATM), 13q14. 3 (D13S319), 13q34, and the chromosome 12 centromere (Abott Diagnostics, USA) were used. Procedures were performed according to the manufacturer's protocol. Probes were denatured at 73°C for 5 min and then applied to the designated areas of the slides. Following an overnight hybridization the slides were stained with DAPI. The analysis was performed using a BX51 fluorescence microscope (Olympus, USA), and CytoVision image analysis system. At least 200 nuclei were assessed for each probe, and the border value for positive result was 20%.",
"section_name": "Fluorescence in situ hybrydization",
"section_num": null
},
{
"section_content": "In the group of subjects enrolled into the study the following clinical data were analyzed: time from diagnosis to starting of the therapy (time to progression -TTP), chemotherapy regimens ordered and chemotherapy outcome (overall response rate -ORR), probability of progression free survival (PFS) and probability of overall survival (OS). The criteria of therapy response proposed by WG-IWCLL in 2008 [49] based on WG-NCI criteria from 1996 [50] were used. The complete response required the absence of symptoms and organomegaly, normal complete cell counts of peripheral blood and less than 30% of lymphocytes in bone marrow for at least 2 months. When size of the lymph nodes, spleen and liver, together with the peripheral blood data, were at least 50% better than pre-treatment values, the partial response was achieved. Other patients were considered non-responders.",
"section_name": "Assessment of therapy outcome and survival time",
"section_num": null
},
{
"section_content": "Statistical analysis was performed with STATISTICA 12. 0 software for Windows. The results were shown as median or mean values with standard deviation. The Mann-Whitney and Wilcoxon tests were used for groups comparison. The Kaplan-Meier method was employed to calculate the survival analysis. Multivariate analysis of independent clinical factors for survival was tested by linear regression. Value of p < 0. 05 was considered to be statistically significant.",
"section_name": "Statistical analysis",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "This study was funded by the research grant of Medical University of Lublin [ DS176 ].",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "CLL: chronic lymphocytic leukemia; TTP: time to progression; PFS: progression free survival; OS: overall survival time; miR: micro RNA; MoAb: monoclonal antibody; IgVH: immunoglobulin heavy chain genes; PBMC: peripheral blood mononuclear cells; qRT-PCR: quantitative reverse transcriptase real-time PCR; cDNA: complementary DNA; Ct: cycle threshold; MACS: magnetic activated cell sorting; FISH: fluorescence in situ hybridization.",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "ASz was responsible for the data collection and analysis, interpreted the results, contributed to manuscript writing. SCh, AM, DSz contributed to the data collection, analysis and interpretation. MH contributed to the interpretation of the data and critical revision of the manuscript for important intellectual content. MP designed the study, contributed to the data collection and analysis and wrote the manuscript.",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "There were no conflicts of interest.",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
}
] |
10.3390/toxins16090394
|
Proteome and Phosphoproteome Profiling Reveal the Toxic Mechanism of Clostridium perfringens Epsilon Toxin in MDCK Cells
|
<jats:p>Epsilon toxin (ETX), a potential agent of biological and toxic warfare, causes the death of many ruminants and threatens human health. It is crucial to understand the toxic mechanism of such a highly lethal and rapid course toxin. In this study, we detected the effects of ETX on the proteome and phosphoproteome of MDCK cells after 10 min and 30 min. A total of 44 differentially expressed proteins (DEPs) and 588 differentially phosphorylated proteins (DPPs) were screened in the 10 min group, while 73 DEPs and 489 DPPs were screened in the 30 min group. ETX-induced proteins and phosphorylated proteins were mainly located in the nucleus, cytoplasm, and mitochondria, and their enrichment pathways were related to transcription and translation, virus infection, and intercellular junction. Meanwhile, the protein–protein interaction network screened out several hub proteins, including SRSF1/2/6/7/11, SF3B1/2, NOP14/56, ANLN, GTPBP4, THOC2, and RRP1B. Almost all of these proteins were present in the spliceosome pathway, indicating that the spliceosome pathway is involved in ETX-induced cell death. Next, we used RNAi lentiviruses and inhibitors of several key proteins to verify whether these proteins play a critical role. The results confirmed that SRSF1, SF3B2, and THOC2 were the key proteins involved in the cytotoxic effect of ETX. In addition, we found that the common upstream kinase of these key proteins was SRPK1, and a reduction in the level of SRPK1 could also reduce ETX-induced cell death. This result was consistent with the phosphorylated proteomics analysis. In summary, our study demonstrated that ETX induces phosphorylation of SRSF1, SF3B2, THOC2, and SRPK1 proteins on the spliceosome pathway, which inhibits normal splicing of mRNA and leads to cell death.</jats:p>
|
[
{
"section_content": "Epsilon toxin (ε-toxin, ETX), produced by Clostridium perfringens types B and D [1], is the causative agent of enterotoxaemia in animals [2, 3] and is also considered to be related to multiple sclerosis in humans, causing human health threats and economic losses to the global livestock industry [4, 5]. Due to its high toxicity, ETX has been classified as a potential bioterrorism or bio-warfare agent by the Centers for Disease Control and Prevention of the United States. ETX has been listed as one of the potential biological warfare agents by the Centers for Disease Control and Prevention of the United States and is also listed as a category B bioterrorism threat [6]. In in vivo challenge experiments in mice, it was found that the median lethal dose of ETX to mice was 70-110 ng/kg [7, 8]. \n\nETX is composed of 311 amino acids and has a molecular weight of 32. 7 kDa. At first, it is simply an inactive proETX that can be activated by extracellular serine-type enzymes [9]. Specifically, these enzymes hydrolyze to remove N-terminal and C-terminal peptides, allowing ETX to be converted to an activated form with toxic effects. Studies have shown that ETX can cause Madin Darby canine kidney (MDCK) cells to swell and rupture, eventually leading to cell death [10]. In recent years, an increasing number of cells have been found to be susceptible to ETX including the Caucasian renal leiomyoblastoma (G-402) cell line [11], the human kidney cell line ACHN [12], the murine renal cortical collecting duct principal cell line mpkCCDcl4 [13], and human erythrocytes [14]. However, MDCK cells are still the cells most used in ETX studies because they are the most sensitive [15]. \n\nThe exact mechanisms of action of ETX on target cells are barely understood. Previous studies have shown that ETX can directly form pores in the target cell membrane and is a typical pore-forming toxin [10, [16] [17] [18]. The formation of pores causes the exchange of materials between the inside and outside of the cell to be disrupted, which leads to the death of the cell [12]. However, some recent studies have found that ETX may also exert toxic effects through other pathways of action [12, 14, 19, 20]. For example, Wioland has reported that ETX can cause oligodendrocyte demyelination [20]. Gao demonstrated that ETX can cause hemolysis in human erythrocytes by activating P2 receptors on the surface of erythrocytes [14]. Interestingly, P2 × 7 and P2Y13 inhibitors inhibited hemolysis but not ETX space formation [14]. In addition, it has been suggested that oxidation of nucleotide-sensitive ICln chloride channels contributes to ETX-induced hemolysis [21]. These studies together indicate that ETX leads to cell death mode not only through the formation of membrane pores but also through a variety of other effective pathways. \n\nMost of the previous studies on ETX were on the pore-forming mechanism of ETX itself and on various mutants. To our knowledge, this is the first joint proteomic and phosphorylation proteomic study of the effects of ETX on MDCK cells. In this study, we profiled cell responses to ETX intoxication by performing quantitative proteomics and phospho-proteomics using MDCK cells to find clues about the pathogenesis of ETX.",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "2."
},
{
"section_content": "To determine the point times of ETX effects on MDCK cells, immunoblotting was performed. The result is shown in Figure 1A ; after being exposed to ETX for 10 min, the phosphorylated proteins were reduced. However, at 30 min, the number of phosphorylated proteins was increased. Therefore, we chose the 10 min and 30 min intervals for the following experiments. The flow chart of quantitative proteome and phosphorylated proteome is shown in Figure 1B.",
"section_name": "Pre-Assessment of Phosphorylation on MDCK Cells and Workflow",
"section_num": "2.1."
},
{
"section_content": "By TMT (Tandem Mass Tags) labeling, affinity enrichment, and mass spectromet 6086 proteins were identified, of which 5397 proteins contained quantitative informat (Table S1 ). We have identified 9989 phosphorylation sites, respectively, in 3199 of the p teins, of which 4916 loci with quantitative information correspond to 1688 proteins (Ta S2). This indicates that the larger the protein molecular weight, the smaller the covera (Figure 2A ). First, the accuracy of the mass spectrometer was normal after measurem (Figure 2B ) because most of the spectra have a first-order mass error within 10 ppm. T is also consistent with the law of tryptic hydrolysis because they are mostly present i certain number of amino acids (about 7-20). The length of the peptide meets the requi ments of mass spectrometry identification (Figure 2C ). We calculated and counted number of phosphorylation sites on proteins one by one. The protein with only one ph phorylation site accounted for 41. 8% of all phosphorylated proteins. The SRRM2 prot had 222 phosphorylation sites and was the protein with the most phosphorylation si (Figure 2D ). The PCA (principal component analysis) diagrams of quantitative proteom and quantitative phospho-proteomics show that each treatment group (MDCK ce treated with ETX for 0, 10, and 30 min) was clustered within the group and significan separated between the groups. It indicated that the biological repeatability within ea treatment group was good; meanwhile, there were obvious differences between grou and it was valuable to mine differential proteins and phosphorylated proteins (Figu 2E,F).",
"section_name": "Screening of Differentially Expressed Proteins and Differentially Phosphorylated Proteins",
"section_num": "2.2."
},
{
"section_content": "By TMT (Tandem Mass Tags) labeling, affinity enrichment, and mass spectrometry, 6086 proteins were identified, of which 5397 proteins contained quantitative information (Table S1 ). We have identified 9989 phosphorylation sites, respectively, in 3199 of the proteins, of which 4916 loci with quantitative information correspond to 1688 proteins (Table S2 ). This indicates that the larger the protein molecular weight, the smaller the coverage (Figure 2A ). First, the accuracy of the mass spectrometer was normal after measurement (Figure 2B ) because most of the spectra have a first-order mass error within 10 ppm. This is also consistent with the law of tryptic hydrolysis because they are mostly present in a certain number of amino acids (about 7-20). The length of the peptide meets the requirements of mass spectrometry identification (Figure 2C ). We calculated and counted the number of phosphorylation sites on proteins one by one. The protein with only one phosphorylation site accounted for 41. 8% of all phosphorylated proteins. The SRRM2 protein had 222 phosphorylation sites and was the protein with the most phosphorylation sites (Figure 2D ). The PCA (principal component analysis) diagrams of quantitative proteomics and quantitative phospho-proteomics show that each treatment group (MDCK cells treated with ETX for 0, 10, and 30 min) was clustered within the group and significantly separated between the groups. It indicated that the biological repeatability within each treatment group was good; meanwhile, there were obvious differences between groups, and it was valuable to mine differential proteins and phosphorylated proteins (Figure 2E, F ). Compared with the untreated group, a total of 44 DEPs were screened out in the 10 min ETX treatment group, including 9 up-regulated proteins and 35 down-regulated proteins (Figure 3A ). A total of 588 DPPs were screened out, including 398 proteins that were up-regulated and 190 proteins that were down-regulated (Figure 3B ). Compared with the untreated group, a total of 73 DEPs were screened in the 30 min ETX treatment group, including 36 up-regulated proteins and 37 down-regulated proteins (Figure 3A ), and a total of 489 DPPs were screened, including 313 up-regulated proteins and 176 down-regulated proteins (Figure 3B ). All up-and down-regulated DPPs and DEPs are listed (Table S3 ). There were 17 DEPs (Table 1 ) and 357 differential phosphorylation sites in the intersection of the 10 min group and the 30 min group (the phosphorylation sites up-and down-regulated in the top five are shown in Table 2 ). From the quantitative heat map of Compared with the untreated group, a total of 44 DEPs were screened out in the 10 min ETX treatment group, including 9 up-regulated proteins and 35 down-regulated proteins (Figure 3A ). A total of 588 DPPs were screened out, including 398 proteins that were up-regulated and 190 proteins that were down-regulated (Figure 3B ). Compared with the untreated group, a total of 73 DEPs were screened in the 30 min ETX treatment group, including 36 up-regulated proteins and 37 down-regulated proteins (Figure 3A ), and a total of 489 DPPs were screened, including 313 up-regulated proteins and 176 downregulated proteins (Figure 3B ). All up-and down-regulated DPPs and DEPs are listed (Table S3 ). There were 17 DEPs (Table 1 ) and 357 differential phosphorylation sites in the intersection of the 10 min group and the 30 min group (the phosphorylation sites up-and down-regulated in the top five are shown in Table 2 ). From the quantitative heat map of each treatment time, it can be seen that different clustered proteins had different protein expression and modification patterns. For example, some proteins and phosphorylated proteins were continuously up-regulated with the increase of treatment time, while others were continuously down-regulated (Figure 3C, D ). \n\nToxins 2024, 16, x FOR PEER REVIEW 5 of 21\n\neach treatment time, it can be seen that different clustered proteins had different protein expression and modification patterns. For example, some proteins and phosphorylated proteins were continuously up-regulated with the increase of treatment time, while others were continuously down-regulated (Figure 3C, D ).",
"section_name": "Screening of Differentially Expressed Proteins and Differentially Phosphorylated Proteins",
"section_num": "2.2."
},
{
"section_content": "We counted the percentage of subcellular localization of DEPs and DPPs. Eighty percent of the DEPs in the 10 min and 30 min groups were localized in the nucleus, mitochondria, and cytoplasm (Figure S1A ). Eighty percent of the DPPs in the two groups were located in the nucleus and cytoplasm, and the proportion of mitochondria decreased (Figure S1B ). \n\nIn order to explore the pathway involved in the toxicity of ETX, gene set enrichment analysis (GSEA) of KEGG was performed on proteomic and phosphorylated proteomic data. The five enriched pathways with the most significant up-regulated trends in the 10 min group of proteomic data were \"thermogenesis\", \"GABAergic synapse\", \"amyotrophic lateral sclerosis\", \"oxidative phosphorylation\", and \"mineral absorption. \" The two pathways with the most significant down-regulated trends were \"coronavirus disease\" and \"ribosome biogenesis in eukaryotes. \" The five enriched pathways with the most significant up-regulated trends in the 30 min group of proteomic data were \"carbon metabolism\", \"Glycolysis/Gluconeogenesis\", \"endocrine and other factor-regulated calcium reabsorption\", \"fatty acid degradation\", and \"endocytosis\". The three pathways with the most significant down-regulated trends were \"ribosome biogenesis in eukaryotes\", \"ribosome\", and \"RNA transport\" (Figure 4A ).",
"section_name": "Subcellular Localization and KEGG Enrichment Analysis",
"section_num": "2.3."
},
{
"section_content": "We used Motif-X to obtain the sequence characteristics around ETX-related phosphorylation sites and calculated the frequency of amino acid residues on both sides of typical phosphorylation sites (Ser/Thr sites). Taking threonine as the phosphorylation site, the frequency of Asp, Glu, Lys, Pro, and Arg in the surrounding amino acid residues was high, and the motif of Pro and Asp was highly enriched at the position of + 1 (Figure 5A, The five enriched pathways with the most significant up-regulated trends in the 10 min group of phosphorylated proteomic data were \"T cell receptor signaling pathway\", \"Yersinia infection\", \"Fc gamma R-mediated phagocytosis\", \"long-term potentiation\", and \"renal cell carcinoma. \" The five pathways with the most significant down-regulated trends were \"measles\", \"non-alcoholic fatty liver disease\", \"glycerophospholipid metabolism\", \"Huntington's disease\", and \"necroptosis. \" The five enriched pathways with the most significant up-regulated trends in the 30 min group of phosphorylated proteomic data were \"Herpes simplex virus 1 infection\", \"spliceosome\", \"ribosome\", \"coronavirus disease\", and \"lysine degradation. \" The five pathways with the most significant down-regulated trends were \"non-alcoholic fatty liver disease\", \"dopaminergic synapse\", \"necroptosis\", \"Parkinson disease\", and \"prion disease\" (Figure 4B ). The specific data are shown in Table S4.",
"section_name": "Motif Analysis of Phosphorylation Sites",
"section_num": "2.4."
},
{
"section_content": "We used Motif-X to obtain the sequence characteristics around ETX-related phosphorylation sites and calculated the frequency of amino acid residues on both sides of typical phosphorylation sites (Ser/Thr sites). Taking threonine as the phosphorylation site, the frequency of Asp, Glu, Lys, Pro, and Arg in the surrounding amino acid residues was high, and the motif of Pro and Asp was highly enriched at the position of + 1 (Figure 5A, Table S5 ). With serine as the phosphorylation site, the frequency of Lys, Pro, and Arg in the surrounding amino acid residues was high. At the same time, the motif of Pro was highly enriched at the position of + 1 (Figure 4B, Table S5 ).",
"section_name": "Motif Analysis of Phosphorylation Sites",
"section_num": "2.4."
},
{
"section_content": "In order to more comprehensively identify the toxic mechanism of ETX, we in grated the DEPs and DPPs of the 10 min group and the 30 min group into GO, KEG enrichment analysis, and PPI network construction. \n\nThere were 565 DEPs and DPPs in the 10 min group and 491 DEPs and DPPs in 30 min group (Figure 6A ). The two groups of DEPs and DPPs were enriched by GO a KEGG, respectively, and the co-enriched GO pathways were visualized. The most portant biological process (BP) in the two groups of cells is \"RNA processing\", the m important cellular component (CC) is \"ribonucleoprotein complex\", and the most portant molecular function (MF) is \"mRNA binding\" (Figure 6B ). The top three co-sign",
"section_name": "Integration Analysis of Differentially Expressed Proteins and Differentially Phosphorylate Proteins",
"section_num": "2.5."
},
{
"section_content": "In order to more comprehensively identify the toxic mechanism of ETX, we integrated the DEPs and DPPs of the 10 min group and the 30 min group into GO, KEGG enrichment analysis, and PPI network construction. \n\nToxins 2024, 16, 394 9 of 19 There were 565 DEPs and DPPs in the 10 min group and 491 DEPs and DPPs in the 30 min group (Figure 6A ). The two groups of DEPs and DPPs were enriched by GO and KEGG, respectively, and the co-enriched GO pathways were visualized. The most important biological process (BP) in the two groups of cells is \"RNA processing\", the most important cellular component (CC) is \"ribonucleoprotein complex\", and the most important molecular function (MF) is \"mRNA binding\" (Figure 6B ). The top three co-significant pathways were \"spliceosome\", \"focal adhesion\", and \"tight junction\" (Figure 6C ). There were 296 DEPs and DPPs in the intersection of the 10 min group and the 30 min group. The PPI (protein-protein interaction) network of intersection proteins was constructed, and the extent of each protein in the network was calculated using Cytoscape (version 3. 8. 2) software's Cytotumor plug-in to screen the top 20 hub proteins (Table S6 ). Using the MCODE plug-in, two sub-network clusters were selected from the complex network. Cluster1 had 26 points and 140 edges and contained the 12 proteins of the top 20 hub proteins ranked by degree in the total networks; its main biological processes are RNA processing and splicesome. Cluster2 had 10 points and 36 edges, including 1 protein (ANLN) of the top 20 proteins ranked by degree in the total network. The biological process it mainly participates in is related to the cell cycle (Figure 6D ).",
"section_name": "Integration Analysis of Differentially Expressed Proteins and Differentially Phosphorylated Proteins",
"section_num": "2.5."
},
{
"section_content": "After analyzing the 13 homologous key proteins of the KEGG pathway one by one in the cluster, 9 proteins could be found corresponding to the pathway (Table 3 ). To demonstrate whether these proteins are key to determining that ETX induced the death of MDCK cells, we conducted experiments to verify it. We found that these nine proteins were involved in three pathways, most of which were concentrated in the SR protein complex of the spliceosome pathway. Therefore, we selected SRPK1, a common upstream protein of this pathway, for verification. We used an inhibitor, SPHINX31, that has been shown to be effective in inhibiting the phosphorylation of serine/arginine-rich splicing factor 1 (SRSF1) and is also a selective inhibitor of serine/arginine-rich protein kinase 1 (SRPK1) [22, 23]. MDCK cells were treated with different concentrations of SPHINX31 for 12, 24, 36, and 48 h before being exposed to ETX (8. 93 nM), and it was found that the survival rate of MDCK cells was enhanced in a concentration-dependent manner (Figure 7A-D ). In addition, SRPIN340 is also an ATPcompeting SRPK inhibitor and has been shown to effectively inhibit SRPK1 expression. Similarly, different concentrations of SRPIN340 were applied to MDCK cells, and it can be seen that SRPIN340 could effectively inhibit the death of MDCK cells at low concentrations but had certain cytotoxicity when the concentration was greater than 80 µM (Figure 7E-H ). The results suggest that SRSF1 and SRPK1 are involved in ETX-induced toxicity on MDCK cells. \n\nconcentrations of SPHINX31 for 12, 24, 36, and 48 h before being exposed to ETX (8. 9 nM), and it was found that the survival rate of MDCK cells was enhanced in a concentra tion-dependent manner (Figure 7A-D ). In addition, SRPIN340 is also an ATP-competin SRPK inhibitor and has been shown to effectively inhibit SRPK1 expression. Similarly different concentrations of SRPIN340 were applied to MDCK cells, and it can be seen tha SRPIN340 could effectively inhibit the death of MDCK cells at low concentrations but ha certain cytotoxicity when the concentration was greater than 80 µM (Figure 7E-H ). Th results suggest that SRSF1 and SRPK1 are involved in ETX-induced toxicity on MDCK cells.",
"section_name": "Inhibitors Reduced Toxicity of ETX on MDCK Cells",
"section_num": "2.6."
},
{
"section_content": "In addition, to further identify the central proteins of MDCK that caused death by ETX, we selected six key proteins on the three key pathways obtained, NOP56, SF3B1, SF3B2, THOC2, SRSF1 and SRPK1, and designed lentivirus to knock down the expression levels of these proteins in MDCK cells, respectively (information on customized lentiviruses is provided in Table S7 ). The results expressed that ETX could lead to the increase of SRPK1 in MDCK cells, and the use of the four lentiviruses could reduce the expression of SRPK1 in MDCK cells (Figure 8A ). In addition, the levels of these six key proteins in MDCK cells were also knocked down by corresponding lentiviruses (Figure 8B ). The results of the cell cytotoxicity assay showed that the specific knockdown of SRSF1 and SRPK1 by lentivirus could effectively inhibit the cell death of MDCK cells (Figure 8C ), which was consistent with the results of previous inhibitors. However, NOP56 does not play a role in cell death, and decreased SF3B1 expression can promote the death of MDCK cells (Figure 8D, E ). \n\nAdditionally, to further confirm our conclusion, we selected two key proteins in the spliceosome pathway, SF3B2 and THOC2, and constructed lentiviruses to knock down their expression levels in MDCK cells. The results showed that SF3B2 could inhibit the toxicity of ETX to MDCK cells, while THOC2 could inhibit cell death at ETX = 1. 79 nM, but the inhibitory effect was not significant at high concentrations of ETX (Figure 8F ). The negative control of the lentivirus vector used had no effect on cell viability (Figure 8G ). These results support our findings in the inhibitor experiments above that SRSF1, SRPK1, and their spliceosome pathways play critical roles in ETX-induced cytotoxicity in MDCK cells (Figure 7 ).",
"section_name": "SRSF1 and SRPK1 Inhibit the Toxicity of ETX to MDCK Cells",
"section_num": "2.7."
},
{
"section_content": "ETX is highly toxic and can cause disease and even death in a variety of animals [24]. Because of this, it is considered a potential agent of biological and toxin warfare. After ETX poisoning, the course of the disease develops rapidly, and the mortality rate is high [25]. Therefore, it is necessary to reveal the pathogenesis of ETX, search for key signaling pathways and molecules, and strive to provide help and therapeutic targets for subsequent therapeutic research. \n\nIn the enriched ETX-related pathways, there were many transcription-and translation-related words, such as \"RNA transport\", \"ribosome\", \"splicesome\", \"RNA processing\", \"mRNA metabolic process\", etc., and most of the DEPs and DPPs were located in the nucleus, cytoplasm, and mitochondria. At the same time, the core sub-network in the PPI network was also enriched in \"RNA processing\", \"RNA spreading\", and other processes, indicating that the toxicity of ETX may be closely related to interfering with the transcription and translation of host cells. In order to verify the role of these hub proteins in ETX poisoning, we systematically summarized the pathways where these proteins were located, and they were found to be almost concentrated in three pathways: Herpes simplex virus 1 infection and spliceosome and ribosome biogenesis in eukaryotes. What is more interesting is that six of these proteins (SF3B1/2, SRSF1/2/6, and THOC2) exist in the \"spliceosome\" pathway. The spliceosome, which removes introns from genes and con-",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "ETX is highly toxic and can cause disease and even death in a variety of animals [24]. Because of this, it is considered a potential agent of biological and toxin warfare. After ETX poisoning, the course of the disease develops rapidly, and the mortality rate is high [25]. Therefore, it is necessary to reveal the pathogenesis of ETX, search for key signaling pathways and molecules, and strive to provide help and therapeutic targets for subsequent therapeutic research. \n\nIn the enriched ETX-related pathways, there were many transcription-and translationrelated words, such as \"RNA transport\", \"ribosome\", \"splicesome\", \"RNA processing\", \"mRNA metabolic process\", etc., and most of the DEPs and DPPs were located in the nucleus, cytoplasm, and mitochondria. At the same time, the core sub-network in the PPI network was also enriched in \"RNA processing\", \"RNA spreading\", and other processes, indicating that the toxicity of ETX may be closely related to interfering with the transcription and translation of host cells. In order to verify the role of these hub proteins in ETX poisoning, we systematically summarized the pathways where these proteins were located, and they were found to be almost concentrated in three pathways: Herpes simplex virus 1 infection and spliceosome and ribosome biogenesis in eukaryotes. What is more interesting is that six of these proteins (SF3B1/2, SRSF1/2/6, and THOC2) exist in the \"spliceosome\" pathway. The spliceosome, which removes introns from genes and connects exons together to synthesize pre-mRNA, plays a crucial role in gene expression [26, 27]. \n\nAbnormal RNA splicing causes a variety of diseases [28]. Interestingly, SF3B1/2 and SRSF1/2/6 are both present in the SR protein complex of this pathway; SR proteins promote the recruitment of spliceosome components through protein-protein interactions to promote early spliceosome assembly, and these functions are regulated by reversible phosphorylation [29]. Therefore, we suspect that these proteins may be the key proteins that determine ETX poisoning. Studies have proved that high expression of splicing factor SRSF1 can promote the occurrence and development of lung cancer [30], breast cancer [31], pancreatitis [32], and other diseases and cancers, indicating that SRSF1 is a key protein in the regulation of cancer cell expression. Studies have shown that the abnormal expression of the SRSF2 gene is strongly correlated with acute myeloid leukemia (AML) [33], liver cancer [22, 34], lung cancer [35], and other diseases. Recent studies have found that HSV-1 (herpes simplex virus-1) infection can up-regulate SRSF2 levels, and SRSF2 can negatively regulate HSV-1 transcription [36]. Therefore, SRSF2 is expected to be a therapeutic target for a variety of diseases. Multiple alternative splicing of SRSF6 also provides therapeutic targets for diseases such as rectal cancer [37], gastric cancer [38], and alcoholic liver disease [39]. SF3B1 is an important component of the spliceosome. Reduced expression of this gene can reduce the incidence of many human cancers and can also slow the progression of cancer [40] [41] [42]. But we have only found a finished inhibitor of the SRSF1 protein, SPHINX31, which can inhibit the phosphorylation of SRSF1, and no specific antibodies against them have been found. \n\nWe then went further up the pathway and found that the common upstream protein of these proteins was SRPK1. SRPK1 is a protein that can be involved in mRNA processing, including, of course, the alternative splicing mentioned earlier [43]. One of the unique features of SRPK1 is that it phosphorylates proteins containing the SR domain. Studies have shown that the high expression of SRPK1/2 is the cause of enhanced phosphorylation and nuclear translocation of SRSF1 [44]. Therefore, we have reason to believe that SRPK1 plays a key role in mediating ETX-induced MDCK cell poisoning. Interestingly, we found that SRPIN340 is an ATP-competitive SRPK inhibitor that inhibits SRPK1 activity. Therefore, in our results, we found that the reduction of SRSF1 and SRPK1 activity can effectively inhibit the death of MDCK cells induced by ETX. Lentivirus knockdowns of the two proteins produced the same results, and the increase in cell survival was more pronounced in the high-concentration ETX group. \n\nBased on the above results, we suspect that when ETX acts on MDCK cells, SRPK1specific kinase phosphorylates multiple proteins in the SR protein complex, thereby inhibiting the normal splicing of mRNA in the cells and preventing the synthesis of correct proteins, resulting in cell death. As has been reported in many studies, our study also demonstrates that SRPK1 does affect the normal life cycle of cells. However, it plays an important part in cancer treatment and diagnosis [45], whether it helps in the treatment of diseases caused by ETX is unknown, and this is a question that we must continue to address. This also suggests that ETX-induced disease and injury are not as simple as the formation of pores in the membrane but have more complex damage mechanisms, and our findings are an important starting point. \n\nUnfortunately, of the 13 proteins we screened, most currently have no finished inhibitors and antibodies, and several proteins are located in unknown pathways, making our research and validation extremely difficult. Further exploration of these unknown proteins is essential. The research on the pathogenesis of ETX needs to be ongoing. The above differential proteins can be used as potential therapeutic sites for ETX intoxication.",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "Taken together, we used quantitative proteomics and quantitative phospho-proteomics techniques to screen ETX pathogenic-related proteins, phosphorylated proteins, and some important pathways in the MDCK cell line. We identified the key proteins SRPK1, SF3B2, THOC2, and SRSF1 in ETX-induced cell death and verified that their high expression can lead to abnormal splicing of MDCK cells, leading to cell death. To our knowledge, this is the first joint proteomic and phosphorylation proteomic study of ETX on MDCK cells, which provides data support for clarifying the pathogenic mechanism, screening toxic biomarkers, and looking for therapeutic targets of ETX intoxication.",
"section_name": "Conclusions",
"section_num": "4."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "5."
},
{
"section_content": "SRPK1 Polyclonal Antibody was purchased from Immunoway (Plano, TX, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) antibody was purchased from Solarbio (Beijing, China). SPHINX31 (HY117661) and SRPIN340 (HY13949) were purchased from Med Chem Express Corporation (MCE, Romulus, MI, USA). SRSF1, SRPK1, SF3B1, and NOP56 four RNAi-custom lentiviruses were built by GENE Corporation (Shanghai, China). RIPA Lysis Buffer abs929 (strong) was purchased from Absin (Shanghai, China). \n\nRecombinant toxins, GST-labeled ETX, were expressed and purified according to the basic procedures previously performed in the laboratory [14, 46].",
"section_name": "Recombinant Toxin and Reagents",
"section_num": "5.1."
},
{
"section_content": "MDCK cells were cultured in DMEM complete medium (Dulbecco's Modified Eagle Medium, Gibco, Carlsbad, CA, USA), and the conditions of the incubator were set at 37 • C with 5% CO 2.",
"section_name": "Cell Culture",
"section_num": "5.2."
},
{
"section_content": "To assess the effects of ETX (0. 9 nM) on MDCK cells, 1 × 10 5 /mL cells were plated in 150 mm plates for 24 h. Subsequently, the cells were incubated with ETX (0. 9 nM) in DMEM for different durations (0, 0. 5, 5, 10, 15, 20, 25, and 30 min) at 37 • C. After incubation, cells were washed three times with PBS. According to the manufacturer's instructions (absin, Shanghai, China, abs9229), 25 µL of PMSF at a concentration of 100mM was added to 225 µL of RIPA lysate to make the final concentration of PMSF 1 mM. After mixing evenly, 250 µL of the mixture was added to each well, and the cells were lysed for 1 min. After which the cells attached to the bottom of the plate were scraped off with a scraper and collected into a centrifuge tube; this procedure was performed on ice. The lysates were then centrifuged at 14,000× g for 5 min at 4 • C. The supernatants obtained by centrifugation were collected for subsequent experiments. Samples were mixed with loading buffer and added to a 4-20% polyacrylamide SDS-PAGE gel for electrophoresis, after which they were transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder for approximately 3 h at room temperature by gentle shaking and washed three times using PBST. Then, the membranes were incubated with anti-SRPK1 polyclonal overnight at 4 • C and washed three times using PBST. The membranes were incubated with goat anti-rabbit IgG secondary antibody for 1 h. Finally, the color development solution was added, and the results were analyzed and imaged using an AE-1000 cool CCD image analyzer. \n\nThe Western blotting results were quantified using image J software (version number: Java 1. 8. 0-112).",
"section_name": "Western Blotting",
"section_num": "5.3."
},
{
"section_content": "MDCK cells treated with ETX for 10 min and 30 min were collected, then lysate (8 M urea, 1% protease inhibitor) was added and crushed using ultrasonic treatment on ice for 10 s. The crushed liquid was centrifuged at 4 • C, 14,000× g, for 10 min. The supernatant was collected, and the protein concentration was determined.",
"section_name": "Protein Extraction",
"section_num": "5.4."
},
{
"section_content": "The sample pretreatment before digestion was as follows [47] : The protein was reduced with 5 mM dithiothreitol for 30 min and then alkylated with 11 mM iodoacetamide under dark conditions for 15 min. Finally, the urea concentration of the sample was diluted to less than 2 M by adding 100 mM NH 4 HCO 3. Then, the protein solution was digested twice, first at a 1:50 ratio of trypsin to protein overnight, followed by a 1:100 ratio of trypsin to protein for 4 h. Finally, the peptides were desalted by Strata X SPE column.",
"section_name": "Trypsin Digestion",
"section_num": "5.5."
},
{
"section_content": "Tryptic peptides were first dissolved in 0. 5 M TEAB. Each channel of peptide was labeled with their respective TMT reagent (based on manufacturer's protocol, Thermo Scientific, Rockford, IL, USA, cat. 90406) and incubated for 2 h at room temperature. Five microliters of each sample were pooled, desalted, and analyzed by MS to check labeling efficiency. After labeling efficiency check, samples were quenched by adding 5% hydroxylamine. The pooled samples were then desalted with Strata X SPE column (Phenomenex) and dried by vacuum centrifugation.",
"section_name": "TMT Labeling",
"section_num": "5.6."
},
{
"section_content": "Separation was performed using a high pH reversed-phase HPLC on Agilent 300Extend C18 column (5 µm particles, 4. 6 mm ID, 250 mm long). The 8-32% acetonitrile gradient of PH9. 0 separated 60 fractions. The resulting fractions were recombined and dried, resulting in 18.",
"section_name": "HPLC Fractionation",
"section_num": "5.7."
},
{
"section_content": "The tryptic peptides were dissolved in solvent A (0. 1% formic acid, 2% acetonitrile/in water), directly loaded onto a homemade reversed-phase analytical column (25 cm length, 75/100 µm i. d. ). Peptides were separated with a gradient from 8% to 25% solvent B (0. 1% formic acid in 90% acetonitrile) over 40 min, 25% to 35% in 14 min, and climbing to 80% in 3 min, then holding at 80% for the last 3 min, all at a constant flowrate of 700 nL/min on an EASY-nLC 1000 UPLC system (Thermo Fisher Scientific, Waltham, MA, USA). \n\nThe separated peptides were analyzed in Q ExactiveTM (Thermo Fisher Scientific, Waltham, MA, USA) with a nano-electrospray ion source. The electrospray voltage applied was 2. 1 kV. The full MS scan resolution was set to 70,000 for a scan range of 350-1800 m/z. Up to 10 most abundant precursors were then selected for further MS/MS analyses with 15 s dynamic exclusion. The HCD fragmentation was performed at a normalized collision energy (NCE) of 28%. The fragments were detected in the Orbitrap at a resolution of 35,000. Fixed first mass was set as 100 m/z. Automatic gain control (AGC) target was set at 5E4, with an intensity threshold of 5E3 and a maximum injection time of 200 ms.",
"section_name": "LC-MS/MS Analysis",
"section_num": "5.8."
},
{
"section_content": "The resulting MS/MS data were processed using MaxQuant search engine (v. 1. 5. 2. 8). Tandem mass spectra were searched against the canis SwissProt database (25,492 entries) concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set as 0. 02 Da. Carbamidomethyl on Cys was specified as fixed modification. Acetylation on protein N-terminal, oxidation on Met, and deamidation (NQ) were specified as variable modifications. TMT-10plex quantification was performed. FDR was adjusted to <1% and minimum score for peptides was set to >40.",
"section_name": "Database Search",
"section_num": "5.9."
},
{
"section_content": "Enrichment was performed using IMAC kit (ThermoFisher Scientific, Rockford, IL, USA, Cat. A32992) as follows. Peptide mixtures were first incubated with IMAC microspheres suspension with vibration in loading buffer (50% acetonitrile/0. 5% acetic acid). To remove the non-specifically adsorbed peptides, the IMAC microspheres were washed with 50% acetonitrile/0. 5% acetic acid and 30% acetonitrile/0. 1% trifluoroacetic acid, sequentially. To elute the enriched phosphopeptides, the elution buffer containing 10% NH 4 OH was added, and the enriched phosphopeptides were eluted with vibration. The supernatant containing phosphopeptides was collected and lyophilized for LC-MS/MS analysis.",
"section_name": "Bio-Material-Based PTM Enrichment (For Phosphorylation)",
"section_num": "5.10."
},
{
"section_content": "First, the anti-library and the contaminated library were removed. Then, the data were first laterally homogenized (replacing 0 values with NaN and dividing the intensity values of each protein in all samples by the mean of the intensity values of all proteins in all samples) and then longitudinally normalized (dividing each column by the median of that column). Finally, unique peptides were grouped (peptide segments belonging to the same protein are grouped together), and the median of quantitative values in each group of peptides was used as the quantitative value of the protein.",
"section_name": "Standardization of Data",
"section_num": "5.11."
},
{
"section_content": "We performed principal component analysis (PCA) and visualization using the \"Fac-toMineR\" package and \"factoextra\" package of R software (version 4. 0. 3). To screen for DEPs and DPPs, we used an independent sample t-test. We then applied the Benjamin and Hochberg method to correct the p-value for multiple testing and to obtain the false discovery rate (FDR) value. The screening criteria were a fold change greater than 1. 2 or less than 0. 83, and an FDR less than 0. 05. We used Wolfpsort (https://wolfpsort. hgc. jp/) to predict the subcellular structural localization of these proteins. GO and KEGG enrichment analyses were performed using the \"clusterProfiler\" package of R software (version R-3. 5. 1). Motif analysis was conducted using soft motif X. We imported the protein list into the STRING (accessed on 12 July 2021) (https://www. string-db. org/) to export the relationship pairs and visualize the protein-protein interaction (PPI) network using Cytoscape software (version 3. 8. 2). We calculated the degree of each protein in the network using the CytoHubba. A higher degree value indicates a more central position for the protein in the network. We used the MCODE plug-in to filter the core sub-network, with a K-Core set to 2.",
"section_name": "Bioinformatics Analysis",
"section_num": "5.12."
},
{
"section_content": "In this experiment, we used two inhibitors, SPHINX31 and SRPIN340. MDCK cells were grown in 96-well plates at a density of 5 × 10 4 cells/mL for 16 h. The cells were then washed two or three times with PBS, and different concentrations of SPHINX31 and SRPIN340 (ranging from 2. 5 to 160 µM, diluted by double volume) were added to the wells. The control group received only medium, and three replicate wells were set for each concentration. The cells were incubated at 37 • C for 12, 24, 36, or 48 h. After the incubation period, 5. 37 nM GST-ETX was added to the corresponding well and incubated in the cell incubator for 1 h. To estimate cell survival, 100 µL DMEM complete medium and 20 µL MTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium inner salt) were added to the pore and absorbance at 492 nm was measured. As a positive control, cells were treated with toxin only (no inhibitor), and as a negative control, cells were treated with DMEM without the toxin and inhibitor.",
"section_name": "Cytotoxicity Assay",
"section_num": "5.13."
},
{
"section_content": "Six RNAi constructs were used to specifically knock down the expression levels of NOP56, SF3B1, SF3B2, THOC2, SRSF1, and SRPK1 proteins using lentivirus. MDCK cells were seeded in 96-well plates at 5 × 10 3 cells per well and cultured for 16 h. The cells were then gently washed two or three times with PBS. The lentivirus was diluted to a working concentration of 5 × 10 7 TU/mL (MOI = 100), and the lentiviral infection system (86 µL DMEM, 10 µL lentivirus, 4 µL 25 × HiTransG P) was added to the cells which were then incubated in the cell incubator for approximately 16 h. The culture was continued with DMEM culture medium and infected for about 48 h. After that, the cells were treated with different concentrations of diluted GST-ETX (0, 1. 79, 3. 58, 5. 37, 7. 16, 8. 95 nM) with three replicate wells for each concentration, and incubated in the cell incubator for 1 h. Cell",
"section_name": "Lentivirus-Mediated RNA Interference Assay",
"section_num": "5.14."
}
] |
[
{
"section_content": "",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "",
"section_name": "RT-qRCR",
"section_num": "5.15."
},
{
"section_content": "survival was estimated by adding 100 µL DMEM complete medium and 20 µL MTS to the wells and measuring absorbance at 492 nm. Lentivirus and toxin were not added as negative controls, while only toxin cells were added as positive control. \n\nWestern blotting was performed to determine if the lentivirus reduced protein levels. In simple terms, after infecting MDCK cells with lentivirus, total protein was extracted using RIPA lysate, separated by 4-20% SDS-PAGE at 130 V for 1 h, and transferred to a PVDF membrane. The membrane was then gently blocked with 5% skim milk at room temperature for 3 h, probed with SRPK1 rabbit antibody (1:1000, Immunoway YT4422, Plano, TX, USA), incubated overnight at 4 • C, and washed three times using PBST. The membranes were incubated with goat anti-rabbit IgG secondary antibody for 1 h. Finally, the color development solution was added, and the results were analyzed and imaged using an AE-1000 cool CCD image analyzer.",
"section_name": "",
"section_num": ""
},
{
"section_content": "The knockdown efficiency of lentivirus was verified by RT-qPCR. After MDCK cells were infected with lentivirus, the cells were collected. Total RNA in cells was extracted by Trizol and then quantified using Qubit 4 Fluorometer (Invitrogen, Carlsbad, CA, USA). After treatment with EVO M-MLV One Step RT-qPCR Kit (SYBR) (Accurate Biotechnology, Code NO. AG11732, Hunan, China), the detection was carried out on the QuantStudio TM 7 Pro Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Relative gene expression was calculated by the 2 -∆∆Ct method [48]. Primer information is shown in Table S8.",
"section_name": "RT-qRCR",
"section_num": "5.15."
},
{
"section_content": "Statistical analysis was performed with GraphPadPrism9. 0 software. The data were presented as the mean ± standard deviation (SD) of multiple independent experiments. Cell survival was logit transformed, and then the differences between groups were analyzed by independent sample one-way ANOVA. We prescribed that the significance level was p < 0. 05, and the difference was considered to be statistically significant.",
"section_name": "Statistical Analysis",
"section_num": "5.16."
},
{
"section_content": "The following supporting information can be downloaded at: https: //www. mdpi. com/article/10. 3390/toxins16090394/s1, Figure S1 : Subcellular localization statistics; Table S1 : Differential protein information; Table S2 : Phosphorylation sites information; Table S3 : All up-and down-regulated DPPs and DEPs' information; Table S4 : All DPPs' information; Table S5 : Motif-X analysis of significantly regulated phosphopeptides; Table S6 : Cytohubba results Top 20 degree. Table S7 : RNAi-customized-lentivirus information; Table S8 : Primer information for RT-qPCR.",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "",
"section_name": "Author",
"section_num": null
},
{
"section_content": "The authors declare no conflicts of interest.",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.1186/s12935-017-0496-5
|
A novel Notch1 missense mutation (C1133Y) in the Abruptex domain exhibits enhanced proliferation and invasion in oral squamous cell carcinoma
|
Notch1 has been regarded as a fundamental regulator in tissue differentiation and stem cell properties. Recently, Notch1 mutations have been reported intensively both in solid tumors and in hematopoietic malignancies. However, little is known about the biological effect and the clinical implication of these reported mutations. Previously, we discovered several missense mutations in the Notch1 receptor in a Chinese population with oral squamous cell carcinoma (OSCC).We selected a 'hotspot' mutation in the Abruptex domain (C1133Y). The expression of Notch1 was determined by western blot and real-time qPCR in OSCC cell lines transfected with pcDNA3.1-Notch1WT, pcDNA3.1-Notch1C1133Y, or pcDNA3.1 empty vector. CCK-8 assays were used to assess cell proliferation. Flow cytometry and western blot were used to confirm the alteration of cell cycle after transfection. Transwell assays and the detection of Epithelial-to-mesenchymal transition (EMT) markers were used to determine the invasive ability. The effects of Notch1 C1133Y mutation were analyzed by Immunofluorescence staining and the expression of EGFR-PI3K/AKT signaling.We demonstrated that Notch1C1133Y mutation inactivated the canonical Notch1 signaling. We identified an oncogenic phenotype of this mutation by promoting cell proliferation, invasion and by inducing EMT in OSCC cell lines. We found that the Notch1C1133Y mutation exhibited a decreased S1-cleavage due to the impaired transport of Notch1 protein from the endoplasmic reticulum (ER) to the Golgi complex, which was consistent with the observation of the failure of the Notch1C1133Y mutated receptor to present at the cell surface. Importantly, the mutated Notch1 activated the EGFR-PI3K/AKT signaling pathway, which has been confirmed as an overwhelming modulator in OSCC.Taken together, our findings revealed for the first time a novel Notch1 mutation that enhances proliferation and invasion in OSCC cell lines. The Notch1 C1133Y mutation impairs the processing of notch1 protein and the critical links between the mutated Notch1 and the activated EGFR-PI3K/AKT signaling pathway.
|
[
{
"section_content": "Oral squamous cell carcinoma (OSCC) is a locally aggressive epithelial neoplasm, having a propensity of lymph-node metastasis and a poor prognosis [1] [2] [3]. Although the etiology and mechanisms of OSCC malignant progression, such as tumor proliferation, invasion, metastasis and stem cell properties, are intricate and poorly understood, it has been clearly elucidated that both genetic and environmental elements play a role in OSCC progression [1, 2, 4]. \n\nNotch is a highly conserved and fundamental signaling system that mediates cell-cell interactions during animal development through highly context-dependent and celltype-dependent effects on cell growth, fate determination and survival. Aberrations in Notch signaling or components of the signaling system underlie various human diseases including carcinogenesis [5] [6] [7]. Recent whole exome sequencing of OSCC showed substantial high rates of somatic mutations, including Notch1, in Caucasian populations, revealing a common genetic cause for malignancy [8, 9]. A multitude of mutations in Notch1 has been reported in cutaneous and lung squamous cell carcinoma in Caucasian populations, in OSCC in Japanese populations, and in OSCC in Chinese populations [10] [11] [12]. Although many Notch1 mutations have been discovered by sequencing in numerous malignancies, subsequent functional studies are lacking. As a result, the oncogenic role of specific Notch1 mutations in tumor progression is still speculative and requires further verification. \n\nThe human Notch1 receptor is synthesized as a single 300 kD polypeptide in the endoplasmic reticulum (ER), and it is cleaved by a furin convertase during posttranslation in the Golgi complex into 120 and 180 kD fragments (S1 cleavage). The two fragments are then presented on the cell surface as a functional heterodimer. Multiple ligands (Jagged and Delta) can interact with the extracellular domain of the Notch1 receptor. Two additional cleavage reactions (S2 and S3) are then triggered, liberating the Notch intracellular domain (NICD). The intracellular domain then translocates into the nucleus where it interacts with the CSL DNA-binding protein (CBF1) to activate downstream target genes, particularly the members of the Hairy/Enhancer of Split family (HES) and Hairy/Enhancer-of-Split related with YRPW motif (HEY) [7, 13, 14]. \n\nEpithelial-to-mesenchymal transition (EMT) has been well-elucidated and associated with tumor invasion, metastasis and tumor survival, and EMT is frequently observed in OSCC [15] [16] [17]. EMT is a crucial early event in tumor progression and is characterized by the downregulation of epithelial markers (e. g., Beta-catenin and E-cadherin) and the up-regulation of mesenchymal markers (e. g., N-cadherin and Vimentin) [18]. The EMT process endows epithelial cells with mesenchymal cell properties, reduces intercellular adhesion, and increases the capacity for invasion [19]. In addition, Notch1 plays as a fundamental regulator in the induction as well as the maintenance of EMT and tumor progression [20, 21]. \n\nEpithelial growth factor receptor (EGFR) is composed of an extracellular ligand binding domain, transmembrane segment, and cytoplasmic domain with tyrosine kinase activity [22]. The binding of a ligand to the EGFR triggers EGFR autophosphorylation and downstream signaling transduction cascades, including MAPK, STAT3, and PI3K/AKT pathways. This process is of high biological and clinical significance because increased EGFR levels have been observed in ∼ 90% of the OSCC and believed to be an early event in OSCC pathogenesis [23]. Crosstalk between Notch1 and EGFR signaling in cell proliferation and cancer expression has been observed in genomics and can be either synergistic or antagonistic depending on the different contexts [24] [25] [26]. Notch1-dependent regulation of EGFR has been described in several types of cancers. \n\nIn our previous study, we examined Notch1 mutational status in OSCC in Chinese patients and observed a mutation rate of 43% in the patient population with a poorer clinical outcome [12]. We also identified the spectrum of Notch1 mutations. One of the common domains with mutations is the Abruptex domain (amino acids 907-1143) in the EGF-like repeats (EGF-like repeats 24-29), which has also been observed by other researchers [8, 9]. The Abruptex domain contains the most mutations (13 or 31%), including three nonsense mutations and a hotspot mutation (C1133Y). Because the Abruptex domain plays a role in suppressing cis-inhibition of Notch1 signaling, mutations in this region are thought to be gain-offunction [27, 28]. \n\nIn this study, we established full-length wild-type Notch1 (Notch1 WT ) and the Abruptex domain hotspot mutant Notch1 (Notch1 C1133Y ) vectors and discovered the functional effects of the mutation on Notch1 protein maturation and transportation. Results showed that compared with OSCC cell lines transfected with Notch1 WT, cells transfected with Notch1 C1133Y exhibited increased cell proliferative and invasive property. We also discovered an EMT-like phenotype in cells transfected with Notch1 C1133Y. Importantly, the Notch1 C1133Y activated the EGFR-PI3K/ AKT signaling pathway, which has been confirmed as an overwhelming modulator during oral carcinogenesis.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "CAL27 cell line was purchased from the American Type Culture Collection (ATCC), and the HN4, HN6, and HN13 cell lines were obtained from the Shanghai Ninth People's Hospital (Shanghai, China). All cells were cultured in Ham's F12 medium and Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin/streptomycin (Invitrogen) in humidified incubators at 37 °C in an atmosphere of 5% CO 2. The wild-type Notch1 or mutant Notch1 vectors containing full-length wild-type Notch1 (Notch1 WT ) or mutant Notch1 (Notch1 C1133Y ) cDNA inserted into pcDNA3. 1 were synthesized and constructed by Generay Biotech (Shanghai, China). For transfection, cells (5 × 10 5 cells per well in 6-well plates) were cultured to 50% confluence in complete growth medium, and the medium was then replaced with serum-free medium for 12-16 h. The purified pcDNA3. 1, pcDNA3. 1-Notch1 WT and pcDNA3. 1-Notch1 C1133Y plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Forty-eight hours after transfection, the medium was supplemented with 400 μg/ml G418 (Invitrogen). Two weeks later, successful transfection was confirmed by evaluating Notch1 mRNA by real-time qPCR (> 20-fold less than controls) and by western blot analysis using anti-Notch1 antibodies (see below).",
"section_name": "Cell culture, plasmid construction, and transfection experiments",
"section_num": null
},
{
"section_content": "Total protein was lysed using lysis buffer (Beyotime, China) containing phosphatase inhibitor and protease inhibitor cocktails. Coomassie Brilliant Blue was utilized to quantify the protein lysates, and bovine serum albumin (BSA) was used as the standard. All proteins (10 μg) were separated using SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore), which were then blocked with 5% BSA at room temperature for 2 h and hybridized with primary antibodies (diluted 1:1000) specific for Notch1 (clone D1E11), cleaved-NICD (clone D3B8), CDK2, CDK4, cyclin D1, cyclin D3, P21, P27, EGFR, p-EGFR, AKT, p-AKT, PI3K, p-Stat5, p-Shc, p-Gab1 (purchased from CST); HES-1 (purchased from Abcam); Beta-actin, Betacatenin, E-cadherin, Vimentin, N-cadherin, HES-2 and SNAI1, SNAI2 (purchased from Bioworld) overnight at 4 °C followed by incubation with anti-goat IgG HRP-conjugated secondary antibodies (Zhongshan Goldenbridge Bio) for 1 h at room temperature. Immunoreactive bands were detected using an Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized using the ImageQuantLAS4000 mini imaging system (General Electric). Protein expression levels were counted as gray values relative to Beta-actin according to the analyses of the bands using ImageJ software. Three independent experiments were analyzed for quantification.",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "According to the manufacturer's protocol, total RNA was extracted from cells using TRIzol reagent (Invitrogen) and was converted to cDNA using 5 × PrimeScript RT Master Mix (TaKaRa) at 37 °C for 15 min and 85 °C for 5 s. Primer sequences were obtained from the Primer Bank and were used in quantitative real-time PCR (RT-qPCR) with a 7900HT Real-Time PCR System (Applied Biosystems). The delta delta Ct method for quantitation of relative gene expression was used to determine the mean expression of each target gene normalized to the geometric mean of GAPDH. All primers were designed and synthesized to target the specific sequences of the genes as follows:\n\nNotch1: F: 5′-AGCAAGTTCTGAGAGCCAGG-3′ R: 5′-TAACAGGCAGGTGATGCTGG-3′ GAPDH: F: 5′-GAAGGTGAAGGTCGGAGTC-3′ R: 5′-GAGATGGTGATGGGATTTC-3′ HES-1: F: 5′-TCAACACGACACCGGATAAAC-3′ R: 5′-GCCGCGAGCTATCTTTCTTCA-3′ HES-2: F: 5′-CCAACTGCTCGAAGCTAGAGA-3′ R: 5′-AGCGCACGGTCATTTCCAG-3′ SNAI1: F: 5′-TCGGAAGCCTAACTACAGCGA-3′ R: 5′-AGATGAGCATTGGCAGCGAG-3′ SNAI2: F: 5′-TGTGACAAGGAATATGTGAGCC-3′ R: 5′-TGAGCCCTCAGATTTGACCTG-3′",
"section_name": "Real-time qPCR",
"section_num": null
},
{
"section_content": "For cell-cycle analysis, stable cells were harvested and washed in phosphate-buffered saline (PBS) and fixed in 75% ice-cold ethanol for 30 min at 4 °C. Cells were then washed twice in PBS, stained with propidium iodide (50 μg/ml) in the presence of 50 μg/ml RNase A (Sigma-Aldrich) and incubated for 1 h at room temperature. The cell-cycle analysis was performed on a FACSCalibur flow cytometer (BD Biosciences) and CellQuest Pro software (BD Biosciences). Flow cytometric analysis of apoptotic cells was performed by staining the cells using the Annexin V Apoptosis Detection Kit (BD Pharmingen) according to the manufacturer's protocol. The percentages of cells in specific cell-cycle stages in Notch1 C1133Y cells were compared with those in Notch1 WT cells.",
"section_name": "Flow cytometry",
"section_num": null
},
{
"section_content": "According to the manufacturer's instructions, Transwell chambers (8 μm pore size; Millipore) were used to detect cell invasive ability. A total of 3 × 10 4 cells were seeded into the upper chamber of each insert and incubated at 37 °C for 24 h. Similar inserts coated with Matrigel (BD Biosciences) were used to determine invasive potential in cell invasion assays. Chambers were fixed in 4% paraformaldehyde for 30 min and then dyed with crystal violet substrate. The noninvaded cells on the upper chamber surface were removed, and the invaded cells on the surface were subsequently viewed and counted by a microscopy (ZEISS, Germany).",
"section_name": "Transwell invasion assays",
"section_num": null
},
{
"section_content": "Cells were seeded into 96-well microplates at a density of 2 × 10 3 cells per well and incubated in fresh medium containing 10% CCK-8 reaction solution. After incubation for 1 h, the absorbance was measured on a microplate spectrophotometer (Multiskan MK3, Thermo) at a wave length of 450 nm according to the manufacturer's instructions. Five independent experiments were performed. The growth curves were illustrated using Graphpad Prism 6 software.",
"section_name": "CCK-8 proliferation assays",
"section_num": null
},
{
"section_content": "Statistical analysis was performed using the SPSS statistical package (version 18. 0). The results of quantitative data were expressed as the mean ± SD and evaluated using the Student's t-test. For Notch1 protein localization quantification, 100 cells from each cell line were counted and the values were calculated from three independent experiments. Pearson's Chi square test was used to verify the difference. All experiments were performed at least three times. All analyses were two-sided, and p < 0. 05 was considered statistically significant (*p < 0. 05, **p < 0. 01, and ***p < 0. 001).",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Although missense mutations in Notch1 have previously been identified in OSCC patients in various populations [8, 10], no functional analysis of any Notch1 alleles associated with OSCC has been elucidated. \n\nHere, we selected the C1133Y hotspot mutation and assessed its association with Notch1 function in OSCC cell lines with varying levels of endogenous Notch1 expression. Western blot analysis revealed that the level of endogenous Notch1 differed across cell lines. HN4 and HN13 cells demonstrated relatively higher Notch1 expression, while CAL27 cells showed moderate expression. Nearly undetectable Notch1 expression was observed in HN6 cells (Fig. 1a ). To establish specific Notch1 expression cell lines, HN6 and CAL27 cells were transfected with pcDNA3. 1-Notch1 WT, pcDNA3. 1-Notch1 C1133Y, or pcDNA3. 1 empty vector. Forty-eight hours later, transiently transfected HN6-/ CAL27-Notch1 C1133Y/WT or -pcDNA3. 1 cell lines were obtained and verified by real-time qPCR using common primers targeting Notch1 mRNA not covering the mutant nucleotides (Fig. 1b ). \n\nWe first examined Notch1 signaling pathway activation. Because HES family genes have been reported to be the regular target genes activated by Notch1 signaling [13], we performed real-time qPCR to selectively test the status of HES1-2 genes in HN6 and CAL27 cells after transfection. In HN6 cells, wild-type Notch1 induced downstream target genes up-regulated, compared to the control cells. When compared to the cells transfected with wild-type Notch1, cells transfected with the Notch1 C1133Y mutation induced HES1-2 mRNA down-regulated (Fig. 1c ). This trend was similar in Notch1 C1133Y -transfected CAL27 cells with HES1-2 mRNA being down-regulated, though wild-type Notch1 in CAL27 cells did not activate distinct downstream target genes (Fig. 1d ). Reduced HES-1 and HES-2 protein levels were also verified by western blot analysis (Fig. 1e ). To verify the Notch1 signaling status, cleaved-NICD, which is the key component of Notch1 signaling and serves as a marker of 'canonical Notch1 pathway' activation [6], was also evaluated by western blot analysis. As anticipated, the expression level of cleaved-NICD in Notch1 C1133Y cells was much lower than that in Notch1 WT or pCDNA3. 1 in HN6 and CAL27 cells (Fig. 1e ). Taken together, these results strongly suggest that the C1133Y mutation in Notch1 inactivates the Notch1 signaling pathway.",
"section_name": "Notch1 C1133Y mutation inactivates the Notch1 signaling pathway",
"section_num": null
},
{
"section_content": "To test the effect of the Notch1 C1133Y mutation on cell proliferation, CCK-8 assays were utilized. As shown in Fig. 2a-c, the CCK-8 growth curves indicated that the Notch1 C1133Y -transfected cells proliferated more frequently compared with the Notch1 WT -transfected cells. Moreover, we performed cell-cycle analysis by flow cytometry. The results demonstrated that Notch1 WT -transfected cells presented The overexpression of wild-type Notch1 attenuated cell growth in HN6, but it enhanced cell growth in CAL27 and HN13, compared with controls. However, Notch1 C1133Y mutation accelerated cell growth in all the tested cell lines, compared with Notch1 WT -transfected cells. d The cell-cycle analysis by flow cytometry revealed a less G1 phase arrest in Notch1 C1133Y -transfected cells. e Cell-cycle-specific proteins were analyzed by western blot analysis in HN6 and HN13. Notch1 C1133Y transfection resulted in the up-regulation of CDKs (2 and 4) and cyclins (D1 and D3), whereas the expression of P27 and P21 was decreased, suggesting an expedited cell-cycle induced by Notch1 C1133Y transfection. EGFR-PI3K/AKT signaling activities were evaluated by western blot analysis. Except that EGFR and AKT levels remained unchanged, wild-type Notch1 decreased the expression levels of p-EGFR, p-Stat5, p-Shc, and p-Gab1, while the Notch1 C1133Y mutation reversed the trend in all the tested cell lines. Notch1 C1133Y mutation increased p-AKT and PI3K levels in all the tested cell lines, though the p-AKT and PI3K expressions were down-regulated in HN6 or up-regulated in CAL27 and HN13 induced by wild-type Notch1 transfection a significantly higher percentage of cells in the G1 phases (81% in HN6 cells or 88% in HN13 cells) than the Notch1 C1133Y -transfected cells (68% in HN6 cells or 52% in HN13 cells). Meanwhile, Notch1 WT -transfected cells showed a lower percentage of cells in the S phase (10% in HN6 cells or 7% in HN13 cells) than the Notch1 C1133Y -transfected cells (23% in HN6 cells or 36% in HN13 cells) (Fig. 2d ). These results demonstrated a less G1 phase arrest induced by Notch1 C1133Y transfection, compared to Notch1 WT -transfected cells. In addition, changes in the expression of cell-cycle-specific proteins were also analyzed by western blot analysis [29]. As expected, Notch1 C1133Y transfection resulted in the up-regulation of cyclin-dependent kinases (CDKs) (2 and 4) and cyclins (D1 and D3), whereas the expression of P21 and P27 was decreased (Fig. 2e ). All these results suggest that the Notch1 C1133Y mutation expedites the proliferative ability by accelerating the cell-cycle. \n\nCell apoptosis using cleaved Caspase-3 and Flow cytometry was also tested in this study (Additional file 1: Figure S1A-B ). In HN6 and HN13 transfected cells, no apparent discrepancy has been discovered between wildtype Notch1 and C1133Y-mutant Notch1.",
"section_name": "Notch1 C1133Y mutation accelerates cell proliferation",
"section_num": null
},
{
"section_content": "Notch has been reported to promote epithelial-tomesenchymal transition (EMT) during tumor progression [30]. During the process of EMT, cells acquire more mesenchymal cell properties, exhibiting higher invasive ability. To determine if Notch1 C1133Y mutation also effects invasiveness in OSCC cells, we performed Transwell invasion assays. As shown in Fig. 3a, b, Notch1 WT -transfected cells demonstrated higher invasive ability in CAL27 and HN13 cells but lower invasive ability in HN6 cells compared with controls. In addition, our results revealed that Notch1 C1133Y -transfected cells exhibited even higher invasive ability in all three cell lines compared with Notch1 WT -transfected cells. \n\nNotch1 promotes EMT during cardiac development, involving down-regulation of epithelial markers (E-cadherin) and up-regulation of mesenchymal markers (Snail, Vimentin, and c-Myc) [31] [32] [33]. Notch1 mutations acting through a similar mechanism may also be involved in the EMT process of OSCC cells. Therefore, we utilized western blot analysis to assess the protein expression levels of EMT-specific markers. As expected, transfection of Notch1 WT induced upregulation of mesenchymal markers (N-cadherin and Vimentin) in CAL27 cells and down-regulation of these markers in HN6 cells, whereas the expression of the epithelial marker Beta-catenin was decreased in CAL27 cells or increased in HN6 cells (Fig. 3c-e ) compared with controls. There was no difference in E-cadherin expression between the two groups in both cell lines. An increased expression of mesenchymal markers (N-cadherin and Vimentin) and a decreased expression of the epithelial marker Beta-catenin were observed in Notch1 C1133Y -transfected CAL27 and HN6 cells compared with Notch1 WT -transfected cells, thus verifying its enhanced status of EMT induced by the C1133Y mutation. \n\nNotch1 can also enhance EMT during tumor progression via crosstalk with several regulators and growth factors relevant to EMT [20, 33, 34]. The Snail, Zeb, and cMET transcription factors are representative EMT regulators, and they have been previously implicated as inducers of EMT in many types of malignancies [17, 34]. To further elucidate the potential molecular mechanisms of EMT induced by the C1133Y mutation, we analyzed the gene expression levels of the classic EMT-inducers, Zeb (Zeb1 and Zeb2) and Snail (SNAI1 and SNAI2), by real-time qPCR. As shown in Fig. 3f, in both HN6 and CAL27 cells, an increase in SNAI1/2 gene expression was observed in Notch1 WT -transfected cells, while the expression of Zeb1 and Zeb2 was not significantly different in Notch1 WT -transfected cells compared with controls (data not shown). Astonishingly, decreased expression of SNAI1/2 was observed in Notch1 C1133Y -transfected cells, which was not consistent with the EMT phenotype. Altered SNAI1 and SNAI2 protein expression was also verified by western blot analysis (Fig. 3g, h ). Taken together, these data suggested that Notch1 C1133Y mutation induces cell invasion and EMT in HNSCC cell lines but down-regulates SNAI1/2 expression.",
"section_name": "Notch1 C1133Y mutation enhances cell invasion and regulates EMT markers",
"section_num": null
},
{
"section_content": "The data in Fig. 1 demonstrated a decreased cleaved-NICD expression after transfection of Notch1 C1133Y compared to Notch1 WT. One possible explanation for this could be that the C1133Y mutation induces functionally inert S3-cleavage, releasing less Notch1 intracellular domain. Other explanations lie in all the conditions that produce less structurally or functionally mature Notch1 protein. As S1-cleavage in Golgi complex has been described as required for canonical ligand-dependent Notch1 signaling prior to its presentation to the cell surface, the 120 kD band represents Notch1 protein that has undergone S1-or S2-/S3-cleavage, while the 300 kD band is expected to be the full-length Notch1 protein that has not undergone S1-cleavage [35]. We performed western blot analysis to evaluate the Notch1 protein expression pattern using the Notch1 primary antibody that detects endogenous Notch1 protein and recognize both full-length (300 kD) Notch1 and its cleaved intracellular region (120 kD). In HN6 cells, which express low levels of endogenous Notch1, Notch1 protein was detected mostly in the 300 kD form after transfection with Notch1 C1133Y. In Notch1 WT -transfected HN6 cells, nearly all the Notch1 protein was in the 120 kD form. For cells (CAL27 and HN13) expressing relatively moderate or high levels of Notch1, both the 120 and 300 kD forms of Notch1 protein were detected after transfection with Notch1 C1133Y, while the 120 kD form and a small amount of the 300 kD form were presented in Notch1 WT and pcDNA cells (Fig. 4a ). These data strongly imply that the C1133Y mutation causes reduced S1-cleavage. \n\nOnly S1-cleaved Notch1 receptors can be sent to the cell surface [36]. Thus the observed reduced S1-cleavage caused by the Notch1 C1133Y mutation may be attributed to either retention of Notch1 protein in the endoplasmic reticulum (ER) and failure of transporting to the Golgi complex or the functional impairment of S1-cleavage in the Golgi complex. To test this hypothesis, we performed immunofluorescence (Notch1-FITC) to visualize the intracellular localization of Notch1 protein [37, 38]. In HN6 cells, Notch1 protein in Notch1 WT -transfected cells was localized in the cytoplasm and on the cell surface (Fig. 4B a, d ). In contrast, Notch1 protein in C1133Y-mutated cells was mostly detected in the cytoplasm (Fig. 4B a′, d′). Quantification of Notch1 expression localization showed an obvious difference between the two groups (Fig. 4C ). The data indicated that 80% of Notch1 WT -transfected cells and 87% of Notch1 C1133Y -transfected cells exhibited cytoplasmic expression of Notch1, but that only 17% of Notch1 C1133Y -transfected cells exhibited cell surface expression compared with 76% of Notch1 WT -transfected cells exhibiting cell surface expression. Furthermore, we used a Golgi-specific marker (GM130-CY3) and an endoplasmic reticulum-specific marker (Calnexin-CY3) with immunofluorescence to define Notch1 protein localization. Consistent with the Notch1-FITC results, 90% of Notch1 WT -transfected cells (Fig. 4B d, e, f ) and 86% of Notch1 C1133Y -transfected cells (Fig. 4B d′, e′, f′) showed overlap between Notch1-FITC and the ER marker, while 76% of Notch1 WT -transfected cells (Fig. 4B a, b, c ) and only 12% of Notch1 C1133Y -transfected cells (Fig. 4B a′, b′, c′) showed overlapped staining between Notch1-FITC and the Golgi marker (p < 0. 01, Fig. 4D ). These findings strongly suggest that transport of Notch1 protein in Notch1 C1133Y -transfected cells from the ER to Golgi is impaired, leading to less protein transportation to the Golgi for S1-cleavage and ultimately less protein on the cell surface.",
"section_name": "Notch1 C1133Y mutation causes reduced S1-cleavage of the Notch1 receptor and accumulation of receptor protein in the endoplasmic reticulum",
"section_num": null
},
{
"section_content": "Overexpression of EGFR, which has been reported in up to 30% of solid tumors including 90% of OSCC [39, 40], generally correlates with a poor prognosis and promotes tumor proliferation. Notch1 has also been revealed to have an interplay with EGFR [26, 41, 42]. Thus, we investigated EGFR signaling activities using EGF receptor pathway-specific antibodies (including EGFR, p-EGFR, AKT, p-AKT, PI3K, p-Stat5, p-Shc, and p-Gab1) by western blot analysis. In all three cell lines, EGFR and AKT levels remained unchanged. The expression levels of p-EGFR, p-Stat5, p-Shc, and p-Gab1 decreased after cells were transfected with Notch1 WT. An opposite pattern was observed after the Notch1 C1133Y mutation was introduced (Fig. 5a ). Moreover, Notch1 WT transfection induced down-regulated p-AKT and PI3K in HN6 cells or up-regulated p-AKT and PI3K in CAL27 and HN13 cells. p-AKT and PI3K were increased in all three cell lines after cells were transfected with Notch1 C1133Y, compared with the Notch1 WT transfection (Fig. 5a ). Figure 5b showed the grey-scale analysis of the alteration of EGFR pathway in panel A.",
"section_name": "Notch1 C1133Y mutation activates the EGFR-PI3K/AKT signaling pathway",
"section_num": null
},
{
"section_content": "Recently, deep genome sequencing of cancers has produced a substantial amount of data involving Notch signaling in human malignancies, including OSCC. Since then, a substantial number of functional mutations have been discovered. In our previous study, we sequenced the entire coding region of the Notch1 gene in 51 OSCC tissues from a Chinese population and discovered that 13 (31%) mutations (including three nonsense mutations and a C1133Y hotspot mutation in three tumors) in the Abruptex region (Fig. 6a ). The mammalian Notch extracellular domain has 36 EGF repeats, and six of which (repeats 24-29) are detected by a series of Notch missense mutations in Drosophila, (See figure on previous page. ) Fig. 3 Notch1 C1133Y mutation enhances cell invasion and regulates EMT markers. a Images of Transwell invasion assays (left) and the quantification of the cells (right). Each data point represents the mean ± SD of data from 3 independent trials. b Quantitive analysis of a. c Western blot analysis was used to assess the expression of epithelial (E-cadherin and β-catenin) and mesenchymal (N-cadherin and Vimentin) markers in Notch1 C1133Y -or Notch1 WT -transfected cell lines (HN6 and CAL27). d, e The greyscale analysis of panel C. f Real-time qPCR was used to detect the expression of classic EMT-inducer Snail (SNAI1, SNAI2) mRNA in Notch1 C1133Y -or Notch1 WT -transfected cell lines (HN6 and CAL27). g Western blot analysis was performed to assess the expression of Snail (SNAI1, SNAI2) protein after transfections. h The greyscale analysis of panel G the called Abruptex domain [43]. These mutations result in amino-acid substitutions in the Abruptex domain and induce phenotypes opposite to those characteristics of decreased Notch expression during Drosophila development [44, 45]. For example, in sensory organ formation and vein differentiation, the Abruptex domain alleles are associated with the loss of these structures, whereas Notch1 loss-of-functional alleles cause the formation of extra sensory organs and vein tissue. Jose et al. [28] has determined that Abruptex alleles identify a domain in the Notch protein that mediates the interactions among Notch, its ligands and Fringe that result in suppression of Notch activity, suggesting that the Abruptex domain mediates interactions between Notch and other proteins that inhibit Notch signaling activity. Zifei P et al. [27] utilize protein-binding assays and determines that in addition to binding to the ligand-binding region (EGF repeats [11] [12], Delta can also bind to EGF repeats 22-27 of Notch, which overlap the Abruptex domain. As a result, the Abruptex domain competes with Delta for binding to the ligand-binding domain. Thus, the Abruptex domain may acts as a negative regulator of Notch signaling Fig. 4 Notch1 C1133Y mutation causes reduced S1-cleavage of the Notch1 receptor and accumulation of receptor protein in the endoplasmic reticulum. A Notch1 protein expression pattern was evaluated by western blot using a Notch1 primary antibody which could detect endogenous Notch1 protein and recognize both full-length Notch1 (FL, 300 kD, S1-uncleaved premature form) and its cleaved transmembrane/intracellular region (NTM, 120 kD, S1-cleaved and S2/S3-cleaved form). B The subcellular location of Notch1 receptors in HN6 cells was assessed by immunofluorescence. The Notch1-FITC staining revealed that Notch1 protein in Notch1 WT -transfected cells was localized in the cytoplasm as well as on the cell surface (a, d), while Notch1 protein in C1133Y-mutated cells was only localized in the cytoplasm (a′, d′). Costaining of Notch1 with the Golgi-marker GM130 (b, b′) demonstrated overlapped staining in Notch1 WT -transfected cells (c), but did not showed overlap in Notch1 C1133Y -transfected cells (c′). Costaining of Notch1 with ER-marker Calnexin (e, e′) showed strong overlapped staining in both Notch1 WT and Notch1 C1133Y -transfected cells (f, f′). Scale bars are 10 μm. C The localization of Notch1 in cytoplasm or on cell surface was assessed in 100 cells, and the percent of cells was shown. D Overlapped staining of Notch1 with Golgi-marker GM130, or Notch1 with ER-marker Calnexin was counted. Percentages of localization were calculated from three independent experiments activation, and any missense mutation in this region may reverse the antagonistic effects on Notch signaling activation. \n\nTo verify the activation of Notch1 pathway, we first tested downstream signaling using western blot and real time-qPCR in cells transfected with pcDNA3. 1-Notch1 WT, pcDNA3. 1-Notch1 C1133Y, or pcDNA3. 1 empty vector. Results showed Notch1C1133Y mutation inactivated Notch1 pathway. Further, CCK-8 and Transwell assays were performed in the experiment. Compared with cells transfected with Notch1 WT, cells with Notch1 C1133Y showed enhanced proliferative and invasive ability. \n\nTo detect the molecular mechanisms that may underlie the loss-of-function in Notch1 signaling through the C1133Y mutation, we examined Notch1 protein expression and localization. Notch1 C1133Y -mutant cells exhibited both reduced S1-cleavage and cell surface receptor level. Our findings further revealed that S1-uncleaved immature Notch1 protein localized to the ER in majority of Notch1 C1133Y -mutant cells, which contrasted with the usual Notch1 protein localization in Golgi complex and on the cell surface. These data may explain why the estimated gain-of-function mutation in Abruptex domain observed in transient cells adversely inactivated the Notch1 signaling in stable cells: the unexpected inactivation of Notch1 ligand-induced signaling was due to the retention or misfolding of Notch1 protein in the ER, which would lead to reduced transportation of full-length Notch1 protein from the ER to the Golgi complex for presumed S1-cleavage and ultimately presence on the cell surface, on which way the Notch1 signaling pathway was inactivated. Previous evidence has suggested that missense mutations in EGF repeats, not in the Abruptex domain, can cause Notch1 protein retention or misfolding. For example, a similar study [37] has found that a Notch1 A683T mutation (in EGF repeats 18) in left ventricular outflow tract defect patients causes deficient Notch1 protein localization induced by receptor retention in the ER. All these evidences hint that Notch1 C1133Y mutation leads to an ' Abruptex-specific' loss-of-function of Notch1 signaling. \n\nUntil now, there has been no functional analysis of Abruptex domain mutations in pathological diseases, The NICD translocates to the nucleus where it binds to the DNA-binding protein CSL and was recognized by the transcriptional coactivator Mastermind (MAM). The triprotein complex recruits additional coactivators (Co-A) to activate target genes. In this study, we find that the Notch1 signaling has an inhibitory effect on EGFR activation. When Notch1 C1133Y mutation occurs, Notch1 protein is arrested in endoplasmic reticulum and is unable to be transported to Golgi complex for S1-cleavage, thus the canonical Notch1 signaling activation is disrupted. The PI3K/AKT signaling is activated by Notch1 protein arrest in endoplasmic reticulum induced by Notch1 C1133Y mutation. Moreover, the loss of inhibitory effect by Notch1 loss-of-function mutation can also induces EGFR phosphorylation, thus activating PI3K/AKT signaling. NECD Notch1 extracellular domain, NICD Notch1 intracellular domain such as carcinoma. In this study, we selected the C1133Y Abruptex domain mutation and examined its functional effects on Notch1 signaling in OSCC. We found that Notch1 C1133Y transiently activated the Notch1 signaling pathway in OSCC cell lines compared with Notch1 WT -transfected cells, after which Notch1 C1133Y induced Notch1 signaling inactivated in stable cell lines. These data gave rise to evidence that C1133Y in Abruptex domain could be an ' Abruptex-specific' mutation (as mentioned above) as expected. \n\nWe detected the oncogenic phenotype alterations and found that over-expression of wild-type Notch1 had diverse effects on cell proliferation. Notch1 enhanced cell proliferation in CAL27 and HN13 cells, and it compromised cell proliferation in HN6 cells. We speculate that the interplay between the Notch1 and EGFR signaling pathways as well as the endogenous Notch1 level were responsible for these differences. Several studies have focused on the complicated cross-talk among Notch1, EGFR and their common downstream PI3K/AKT signaling, and they have revealed that the repressive interplay between EGFR and Notch1 could act as compensatory effectors towards the downstream targets, such as the AKT/PI3K pathway [26, 41, 42]. Specifically, communication between the Notch and EGFR pathways in cancer cells enables the cells to compensate for the loss of one pathway with the increase in the other. In HN6 cells, which had negligible endogenous Notch1 expression and a substantial amount of EGFR expression (data not shown), transfection of wild-type Notch1 did not overtly affect Notch1 pathway activation as distinct Notch1 over-expression did not evidently alter its downstream target genes. In HN13 cells, Notch1 signaling was drastically activated by wild-type Notch1 transfection, predicting the Notch1-dominant status in HN13, as well as the EGFR-dominant status in HN6. Because PI3K/AKT signaling is primarily responsible for cell proliferation and invasion in malignancies [46] [47] [48], in the EGFR-dominant HN6 cell line, inhibition of EGFR-AKT induced by Notch1 WT transfection induced an attenuated proliferative and invasive capacity, while in the Notch1-dominant HN13 cells, the activated Notch1 pathway mainly contributed to the enhanced cell invasion. The activated AKT pathway induced by Notch1 WT transfection also played a role in cell proliferation and invasion. Another contradiction in this study lies in the fact that in CAL27 or HN13 cells, Notch1 WT led to enhanced cell proliferation and invasion, while Notch1 C1133Y loss-of-function mutation still induced enhanced proliferative and invasive ability. We speculated that this inconsistence was mainly attributed to the role of the AKT pathway. Notch1-mediated activation of AKT signaling has been poorly reported. Several studies have indicated that AKT signaling could be activated by the activated form of Notch1 (NICD) [49] [50] [51] and is CSL-independent [52]. In this work, however, we found that AKT signaling could be activated by the 300 kD form of retarded Notch1 protein induced by Notch1 C1133Y mutation. These data indicate that AKT could be activated by Notch1 post-translationally and does not require ligand-dependent membrane cleavage of Notch1. Nevertheless, it remains unknown how 300 kD form of Notch1 in ER affects cytoplasmic signaling of PI3K-AKT. It is doubtful if the C1133Y mutation-induced AKT activation is attributed to Notch1 retention in cytoplasm directly or to the loss of inhibitory effect on EGFR phosphorylation. It's still unclear whether the opposite effect on oncogenic phenotypes induced by C1133Y lossof-function mutation is AKT signaling-dependent, thus requiring further investigation. \n\nAnother implication of this work is the explicit positive regulation of the Snail family induced by the Notch1 signaling pathway, which is closely related to EMT in HNSCC [53]. In this study, wild-type Notch1 up-regulated Snail expression in both HN6 and CAL27 cells although Notch1 WT -transfected HN6 cells exhibited a reverse EMT phenotype. Loss-of-function induced by the Notch1 C1133Y mutation decreased Snail expression, which was opposite to its gain-of-function manner, but not consistent with the EMT phenotype caused by Notch1 C1133Y mutation. The Snail superfamily of zinc-finger transcription factors is reported to be involved in the acquisition of invasive properties during tumour progression [54]. It has also been reported that Notch1 signaling pathway can promote metastasis mediated by Snail/Slug [55] and inhibits the expression of E-cadherin resulting in inhibition of expression of beta-catenin and destabilization of adherens junctions [56]. In Notch1 WT transfected CAL27 cells, up-regulation of mesenchymal markers (N-cadherin and Vimentin) and down-regulation of the epithelial marker Beta-catenin was discovered, which was consistent with the Transwell assays. The expression of Snail also coordinated with the alteration of mesenchymal markers, which verified its role in tumor invasion. It can be assumed that Notch1 could induce EMT through Snail activation. However, in Notch1 C1133Y transfected cells, although EMT phenotype was demonstrated in all three cell lines, the snail expression was less of which in the Notch1 WT cells. Such results implicated that in contrast with Notch1, the Notch1 C1133Y mutation-induced EMT was not attributed to the Snail family, thus indicating a complex role for Notch1 mutation in the EMT process. Further experiments would be implemented to uncover the EMT mechanism in this novel Notch1 mutation. \n\nThe findings in this work (summarized and illustrated in Fig. 6b ) found evidence for the first time that mutation in Notch1 Abruptex domain can have an inactivated effect on Notch1 signaling pathway and promotive effects on cell oncogenic phenotypes. They also consolidate our anticipation that a wide spectrum of OSCCs is genetically relevant and that a single genetic alteration could underlie oncogenic phenotypic outcomes. Our findings support the suggestion that OSCCs share a common genetic cause and depend upon crosstalk with other regulatory pathways.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Taken together, our data reveal that the Notch1 C1133Y mutation enhances proliferative and invasive ability in OSCC cell lines. This novel Notch1 mutation impairs the processing of notch1 protein, and activates the EGFR-PI3K/AKT signaling.",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Not applicable.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This project was supported by the National Natural Science Foundation of China ( 81402236 ), National Natural Science Foundation of China ( 81772887 ), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, 2014-37 ), Jiangsu Provincial Medical Innovation Team ( CXTDA2017036 ), Natural Science Foundation of Jiangsu Province of China ( BK20171488 ) and Jiangsu Provincial Medical Youth Talent ( QNRC2016854 ).",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "All original data are available upon request.",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "OSCC: oral squamous cell carcinoma; EMT: epithelial-to-mesenchymal transition; ER: endoplasmic reticulum; NICD: Notch intracellular domain; CBF1: CSL DNA-binding protein; HES: Hairy/Enhancer of Split family; HEY: Hairy/ Enhancer-of-Split related with YRPW motif; FBS: fetal bovine serum; BSA: bovine serum albumin; PVDF: polyvinylidene fluoride; EGFR: epithelial growth factor receptor; CDKs: cyclin-dependent kinases.",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "YZ and ZW performed the experimental study and data collection. YZ, XD and WZ analyzed and interpreted the data. YZ, ZW, GL and LL wrote and reviewed the manuscript. JC, HW, WG revised the manuscript and gave the material support. YW and XS conceived and supervised the whole project. All authors read and approved the final manuscript. Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, Brisbane, QLD 4006, Australia.",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "",
"section_name": "Author details",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "No parts of this manuscript are being considered for publication elsewhere.",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "Additional file 1: Figure S1. Cell apoptosis was analyzed in HN6 and HN13 transfected cells. (A) Cleaved Caspase-3 was utilized to detect the cell apoptosis in HN6 and HN13 cells. (B) Flow cytometry was used to determine the early and late stages apoptotic cells.",
"section_name": "Additional file",
"section_num": null
},
{
"section_content": "Not applicable.",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.1186/1755-8166-6-5
|
Atypical rearrangement involving 3′-IGH@ and a breakpoint at least 400 Kb upstream of an intact MYC in a CLL patient with an apparently balanced t(8;14)(q24.1;q32) and negative MYC expression
|
<jats:title>Abstract</jats:title> <jats:p>The t(8;14)(q24.1;q32), the cytogenetic hallmark of Burkitt’s lymphoma, is also found, but rarely, in cases of chronic lymphocytic leukemia (CLL). Such translocation typically results in a <jats:italic>MYC</jats:italic>-<jats:italic>IGH@</jats:italic> fusion subsequently deregulating and overexpressing <jats:italic>MYC</jats:italic> on der 14q32. In CLL, atypical rearrangements resulting in its gain or loss, within or outside of <jats:italic>IGH@</jats:italic> or <jats:italic>MYC</jats:italic> locus, have been reported, but their clinical significance remains uncertain. Herein, we report a 67 year-old male with complex cytogenetic findings of apparently balanced t(8;14) and unreported complex rearrangements of <jats:italic>IGH@</jats:italic> and <jats:italic>MYC</jats:italic> loci. His clinical, morphological and immunophenotypic features were consistent with the diagnosis of CLL.</jats:p> <jats:p>Interphase FISH studies revealed deletions of 11q22.3 and 13q14.3, and an extra copy of <jats:italic>IGH@,</jats:italic> indicative of rearrangement. Karyotype analysis showed an apparently balanced t(8;14)(q24.1;q32). Sequential GPG-metaphase FISH studies revealed abnormal signal patterns: rearrangement of IGH break apart probe with the 5’-IGH@ on derivative 8q24.1 and the 3’-IGH@ retained on der 14q; absence of MYC break apart-specific signal on der 8q; and, the presence of unsplit 5’-<jats:italic>MYC</jats:italic>-3’ break apart probe signals on der 14q. The breakpoint on 8q24.1 was found to be at least 400 Kb upstream of 5’ of <jats:italic>MYC</jats:italic>. In addition, FISH studies revealed two abnormal clones; one with 13q14.3 deletion, and the other, with concurrent 11q deletion and atypical rearrangements. Chromosome microarray analysis (CMA) detected a 7.1 Mb deletion on 11q22.3-q23.3 including <jats:italic>ATM</jats:italic>, a finding consistent with FISH results. While no significant copy number gain or loss observed on chromosomes 8, 12 and 13, a 455 Kb microdeletion of uncertain clinical significance was detected on 14q32.33. Immunohistochemistry showed co-expression of CD19, CD5, and CD23, positive ZAP-70 expression and absence of <jats:italic>MYC</jats:italic> expression. Overall findings reveal an apparently balanced t(8;14) and atypical complex rearrangements involving 3’-<jats:italic>IGH@</jats:italic> and a breakpoint at least 400 Kb upstream of <jats:italic>MYC</jats:italic>, resulting in the relocation of the intact 5’-<jats:italic>MYC</jats:italic>-3’ from der 8q, and apposition to 3’-<jats:italic>IGH@</jats:italic> at der 14q. This case report provides unique and additional cytogenetic data that may be of clinical significance in such a rare finding in CLL. It also highlights the utility of conventional and sequential metaphase FISH in understanding complex chromosome anomalies and their association with other clinical findings in patients with CLL. To the best of our knowledge, this is the first CLL reported case with such an atypical rearrangement in a patient with a negative <jats:italic>MYC</jats:italic> expression.</jats:p>
|
[
{
"section_content": "Chronic lymphocyctic leukemia (CLL) is the most common leukemia in the elderly with clinical presentation of lymphocytosis, bone marrow involvement, lymphadenopathy, hepatosplenomegaly, complex cytogenetics and heterogeneous clinical course [1]. Immunophenotypically, aberrant expression of CD5, CD20, CD22, CD23, CD38, CD43 and CD79 is diagnostic or prognostic of B-cells in CLL [2]. Common cytogenetic anomalies include deletion of 13q14. 3 (most frequent) and/or 13q34, deletion of 11q, deletion of 17p, trisomy 12 and IGH@ rearrangement [3]. \n\nWhile t(8;14) (q24. 1;q32), the cytogenetic hallmark of Burkitt's lymphoma, is a primary genetic event found in about 70-80% of cases, it is usually a rare secondary anomaly in other B-cell disorders including CLL (about 0. 2% to <1%) [4] [5] [6] [7] [8], lymphoblastic leukemia, DLBCL, and in other lymphoma transforming into a more aggressive disease [9]. In the latter, t(8;14) usually confers favorable prognosis, while a more aggressive phenotype and poor outcome are manifested when it is a part of a complex chromosome complement [5, 10]. \n\nIn a typical t(8;14) (q24;q32) translocation, the MYC at 8q24. 1 locus is spatiotemporally juxtaposed with the 3'-IGH@ locus on derivative 14q32 [11] [12] [13] [14] [15]. The IGH transcription factory, about 2. 5 Mb in size [12], localizes the regulatory elements for MYC deregulation and variable regions that promote translocation [13]. The IGH@ locus, is a hotspot for recombination and mutation of immunoglobulin genes during B-cell maturation, processes that usually promote translocations with oncogenic potential [11]. Whereas the breakpoint on chromosome 14 is within the IGH@ locus, usually located within the μ-gene, either within or adjacent to the variable (V), joining (J), diversity (D, or switch (S) regions, but other heavy-chain regions are occasionally involved [9]. While about 80% of translocations in Burkitt's lymphoma is typical and involve MYC and IGH@ (IG heavy chain) [16], others are involved in variant partnership with other IG chain loci; kappa light chain (IGK) at 2p12, or lambda light chain (IGL) at 22q11. 2 [16] [17] [18]. MYC is also involved with IGH in DLBCL [18], TCR alpha/delta in T-acute lymphoblastic leukemia/lymphoma, and IG kappa and lambda chains in plasma cell myeloma [18, 19]. \n\nMYC is a proto-oncogene that encodes for a transcription factor that regulates cell cycle progression, growth, differentiation, apoptosis, survival and biosynthesis [4, 6, 20]. It activates or represses transcription factories of other genes (about 10%), transcription factors, and chromatin modifying and remodeling complexes [20]. Rearrangements involving MYC drive cells into lymphomagenesis often through its deregulation and overexpression [5, 11, 12, 21, 22]. The oncogenic potential of MYC rearrangements is implicated not only in the initiation of lymphomagenesis but also in its transformation and progression of low-grade lymphomas into a more advanced disease and an unfavorable outcome [5, 17, 18, [21] [22] [23]. These findings suggest that the level of deregulated MYC expression of different stages of aberrant cellular maturation and differentiation may influence the neoplastic phenotype [9]. At 8q24. 1 locus, translocation breakpoints are located within or surrounding the MYC: regulatory region within MYC, from exon 1 to intron 1, (Class I and most common); transcription factor binding-site at or adjacent to 5'-MYC (Class II); and long-range regions up to 100-300 Kb or more upstream from an intact 5'-MYC-3' (Class III) [15, 16, 20]. It has been suggested that aberrant MYC expression is influenced by breakpoint location, mutation within the translocated region, deletion of regulatory elements, or transcription at cryptic sites other than the usual P1 or P2 initiation start site (promoter shift) [15, 20, 24]. Increased transcriptional activity is observed in breakpoints within exon 1 and intron 1 (Class I) than when it occurs within the most common breakpoint, 5' from MYC exon 1 (Class II) [15]. Longrange cis-acting enhancers regulate MYC expression through chromatin looping bringing the enhancers in close proximity to MYC [25, 26], or through increased distal enhancer activity utilizing preexisting loops [27]. Multiple genetic variants and SNPs, located in 1. 5 Mb \"gene desert\" regions 1, 2 and 3, up to 600 Kb upstream of MYC, are associated with increased susceptibility to prostate, colorectal, bladder, breast cancer, or chronic lymphocytic leukemia [26] [27] [28]. Although reporter expression studies revealed that long-range enhancers and other regulatory elements regulate MYC transcription, the clinical significance of MYC rearrangements upstream of MYC remain unclear and a subject of burgeoning field of investigation [4]. \n\nTo date, there are only very few reported cases of CLL with apparently balanced t(8;14) and atypical rearrangements [6, 8], none of which exhibits abnormal FISH signal patterns similar to what we detected in our patient. These abnormal patterns include: cryptic deletion on 8q24. 1 including MYC [6, 8], gain of an extra copy of MYC (+MYC) [4, 5, 29], or deletion of IGH@, usually 5' [3, 4, 6, 30, 31]. The prognosis for these cases is also variable, from indolent to transformed into a more aggressive course. \n\nHere we report a CLL case with complex cytogenetic findings of deletions of 11q and 13q, in addition to the apparently balanced t (8;14). We also present an undocumented atypical complex rearrangements involving 3'-IGH@ and at least 400 Kb upstream of 5'-MYC, unreported complex atypical rearrangements of IGH@ and MYC loci that did not result in IGH-MYC fusion and no subsequent MYC expression.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "Our patient is a 67 year-old Hispanic male with a medical past history of an end-stage kidney disease of uncertain etiology. His white blood cell count (WBC) was elevated and measured at 23. 15 × 10 3 per μL. Peripheral blood smear showed marked lymphocytosis with numerous atypical lymphoid cells including prolymphocytes, smudge cells, normocytic normochromic anemia and thrombocytopenia. The lymphoid-gated population constituted 87% of total cells, and consisted of 2% T cells, 70% B cells, and <2% NK cells (Figure 1A ). Flow cytometry showed co-expression of B-cell antigen (CD19) with CD5, CD23, CD20, and ZAP70 expression. These results were suggestive of CLL (Figure 1B ).",
"section_name": "Clinical report",
"section_num": null
},
{
"section_content": "A complete chromosome analysis was not possible due to low mitotic index. G-banded karyotype analysis of available metaphase cells revealed an abnormal male karyotype with an apparently balanced t(8;14) (q24. 1;q32) seen in 50% (6/12) of total cells examined (Figure 2 ). \n\nInterphase FISH studies did not reveal IGH@-CCND1 rearrangement, but instead, an extra copy of IGH@-specific signal in 30. 3% (91/300) of nuclei examined (data not shown). In addition, deletions of the 13q14. 3 (D13S319) (Figure 3A ) and 11q22. 3 (ATM) (Figure 3B ) in 8% (24/300) and 78% (294/300) of cells were also observed, respectively. Neither deletion of 17p13. 1 (TP53) nor trisomy 12 was detected (data not shown). Sequential GPG-metaphase FISH studies were performed on the same chromosome metaphase spread to determine the clonality of the structural abnormalities seen in our patient. Results showed that two different clones exist in the peripheral blood of our patient: one with deletion 13q14. 3 (seen only in interphase nuclei in our study), and another with concurrent deletion 11q (Figure 3C ) and t(8;14) (Figure 4D ). \n\nFurther sequential FISH studies on 10 metaphase nuclei using the IGH@ break apart probe showed splitting or rearrangement (1Y1G1R), with the 5'-IGH@ (green) translocated on chromosome 8q24. 1 and the 3'-IGH@ (red) retained on 14q (Figure 4A, B ) in all cells examined. The IGH@-MYC fusion (Figure 4C ) and MYC break apart (Figure 4D ) probes revealed atypical abnormal signal patterns in all 10 cells examined on derivative 8q24. 1: one green (5'-IGH@) and no red (deletion at least 400 Kb upstream of 5'-MYC-3'); and, on derivative 14q32: one yellow (relocation of 5'-MYC-3' and its flanking regions adjacent to 3'-IGH@). The estimated location of the translocation breakpoint upstream of 5'-MYC was determined by in silico mapping (Figure 5 ) by determining the base pair coordinates in the UCSC Genome Browser (hg19) of the STS markers mapped upstream of 5'-MYC-3' (Abbott Vysis FISH probes website). We based our calculations on the Spectrum Orange of the MYC break apart probe, the farthest probe from 5' of MYC (as compared to the MYC probe in the IGH-MYC fusion probe). The estimated distance of the translocation breakpoint (STS marker WI-1302) from 5'of MYC is at least 400 Kb (bp 128,354,420-128,747,680). This interval includes two RefSeq genes: POU5F1B (POU class 5 homeobox 1B), an intronless gene that encodes for a transcription factor (1. 6 Mb; bp 128,427,857-128,429,441) and a gene with no known function, LOC727677 (38. 8 Kb; bp 128,455,595-128,494,384). It also includes the SNPs implicated in several cancer types, rs1447295 (Region 1), rs16901979 (Region 2) and rs6983267 (Region 3) [26] and CLL SNP rs2456449 [28]. \n\nSNP CMA refined the 11q22. copy number loss at chromosome 11q22. 3-q23. 3, arr 11q22. 3q23. 3(107,888,769-115,016,307)x1 (data not shown). It deleted 62 RefSeq genes including ATM (ataxia telangiectasia mutated), a gene that encodes for a cell cycle checkpoint phosphorylating kinase that functions for regulating proteins for tumor suppression, checkpoint, DNA repair and maintenance of genome stability [32]. In addition, a 455 Kb heterozygous copy number loss on 14q32. 33 was also detected; arr 14q32. 33(106,530,533-106,985,955)x1, deleting two gene fragments or non-protein coding genes of no known function, LINC00226 and LINC00221 (data not shown) [32]. CMA did not detect a microdeletion within or surrounding the MYC locus despite its removal from der 8q24. 1 locus. This suggests that there was no net gain or loss despite the unbalanced rearrangements detected by FISH. In a lesser extent, a 61 Kb gain on 8q24. 12 was detected, but found to be unreportable with further in silico investigations. There were no clinically relevant gains or losses detected on chromosomes 12, 13 and 17. \n\nAccording to the ISCN [33], the overall findings from karyotype, FISH and CMA can be described as: 46,XY,t(8;14) (q24. 1;q32). ish der(8)t(8;14) (q24. 1;q32)del (8) (q24. 1q24. 1) (MYC-,5'IGH@+),der( 14 As mentioned above, deletion 13q14. 3 and deletion 11q22. 3 with t(8;14), detected by interphase and sequential metaphase FISH studies, are found as two different abnormal clones, indicative of mosaicism. CMA failed to detect gains or losses on 13q, since it only accounts for 8% of the total cell population, a number way below the detection limit (10-30%) of either SNP or BAC microarrays [30]. \n\nImmunohistochemistry studies using specific MYC antibodies did not detect any staining in our patient's sample, suggestive of absence of MYC activation (Figure 6A ). A strong positive staining for MYC was detected for the positive control sample (Figure 6B ).",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Our patient's clinical, morphological and immunophenotypic features are consistent with the diagnosis of CLL. Although complex cytogenetic findings including t(8;14) usually confers poor prognosis in CLL, a consistent genotype and phenotype correlation remains an unresolved issue. Our patient's case exhibits an unreported rearrangement involving IGH@ and MYC loci with absence of MYC expression. \n\nIn our patient, the FISH signal patterns detected are unique from those previously reported in CLL cases with atypical rearrangements and an apparently balanced t (8;14). These include a cryptic deletion on 8q24. 1 including MYC [6, 8], gain of an extra copy of MYC (+MYC) [4, 5, 29], or deletion of IGH@, usually 5' [3, 4, 6, 30, 31]. Although a deletion of the MYC-specific signal on der 8q24. 1 locus was also observed in our patient using IGH-MYC fusion probe (1Y2G1R), it is not identical to the reported deletion by Reddy et al. in 2006 [6,8]. The deletion reported on here did not show splitting of signals and no concomitant deletion of a 1. 6 Mb segment including the MYC locus. Instead, it showed two unsplit MYC probes (yellow) on the normal chromosome 8 and on der 14q32. We interpreted these findings as an atypical rearrangement never reported elsewhere, with the 5'-MYC-3' removed from the 8q24. 1 locus at a breakpoint at least 400 Kb upstream of its 5' region. We also showed that this deleted region is relocated to the 14q32 locus and apposed to the 3'-IGH@ locus. Neither gain of MYC nor deletion of the 5'-IGH@ locus was observed by FISH or CMA in our case. We have exhaustively searched the available literature and did not find any cases similar to the signal patterns reported on here. \n\nTo the best of our knowledge, expression levels of MYC and its correlation to disease progression have not been established in CLL with t(8;14), with or without MYC translocations [4, 6, 7]. MYC expression is generally at low levels in CLL [23], and similar in groups with either bad or good prognosis Increased expression even without MYC rearrangement has also been described in CLL with malignant Richter transformation and other higher risk cases for CLL progression [10]. Although, high levels of MYC are expressed as a result of the t(8;14) and its variant translocations in Burkitt's lymphoma and in some other B-cell malignancies including DLBCL and plasma cell myeloma, these translocations may not necessarily lead to increased expression of MYC in CLL [4, 6, 7]. These variable findings of MYC expression are most likely dependent on specific disruptions of regulatory regions, or characteristic genomic translocation breakpoints either at the MYC or IGH@ locus. The typical MYC-IGH fusion at der 14q32 expresses the MYC -deregulatting product, while the reciprocal IGH-MYC fusion at 8q24. 1 locus is transcriptionally silent [14, 35]. Despite the typical juxtaposition, overall MYC expression in some CLL cases remains within the normal range [20], or overexpressed through processes other than translocations [9]. It has been reported that the location of the genomic breakpoint influences MYC expression, with highest level when involving Class I breakpoints [15, 24]. The absence of Myc expression in our patient is most likely due to the atypical MYC-IGH fusion on der 14q32, with a Class III breakpoint (at least 400 Kb upstream of MYC) [16]. \n\nThe previously reported \"gene desert\" region upstream of MYC extends up to about 629 Kb [26] and includes genes and SNPs. Genome-wide association studies (GWAS) have shown the gene POU5F1B and several genetic variants or SNPs (Regions R1, R2, R3) (Figure 5 ) that are risk factors for various cancers including CLL exist in this region [26] [27] [28] [36] [37] [38]. The strongest evidence for risk or genetic susceptibility in CLL or monoclonal B-cell lymphocytosis is rs2456449 (8q24. 21) [28, 38]. In our patient, the breakpoint that we suggested (at least 400 Kb) is within this interval and includes POU5F1B, and SNPs R1 and R2. POU5F1B is one of the two RefSeq genes within the breakpoint on 8q24. 1 and 5'-MYC, is the most adjacent. Although it is not yet well studied, few reports described it as a pseudogene or a gene that encodes for a weak transcription factor that may play a critical role in stem cell pluripotency, eye development and carcinogenesis [36, 37, 39]. At the present time, there are no reports of a specific fusion involving 5'-POU5F1B The IGH@ break apart probe reveals splitting of signals (1Y1G1R) indicative of rearrangement, with 5'-IGH@ relocated to der 8q and 3'-IGH@ retained on der 14q. C: The IGH-MYC fusion probe shows 1Y2G1R2A, fusion of MYC-IGH der 14q32 (yellow), and deletion of MYC on der 8q24. 1 (green, no red). It also shows normal signal patterns for the other chromosome 8 (aqua for centromere and red for MYC), and chromosome 14 (green). D: The MYC break apart probe detects 2Y signals, one on normal chromosome 8 and the other is removed from der 8q24. 1 and relocated to der 14q32. These findings suggest that there is neither splitting of signals, or a deletion in between the red and green signals. \n\nand 3'-regulatory region of IGH@. It is possible that the breakpoint in our patient is further upstream, however, the paucity of available cells made it impossible for further characterization. In Figure 5, we extended the suggested breakpoint further upstream, from ~400 Kb to ~600 Kb, to include the farthest reported cancer-associated SNP (Region 2: rs16901979) and CLL SNP (rs2456449). To date, the genotype phenotype correlation underlying these associations remains unclear. However, it has been suggested by reported expression studies that MYC expression is influenced by such SNPs variants by altering its transcription regulation and amplification [40]. Despite such plethora of reports, replication of these findings and elucidation of its physiologic function and clinical significance remain an area of thorough investigation. Further in vivo and in vitro functional studies are needed to show consistent association of risk allele status and MYC expression levels. \n\nOn the other hand, transcription at the IGH@ locus is controlled by enhancers elements spread out as wide as 2. 5 Mb of the locus [12], and it contains regulatory elements necessary not only for MYC activation but also the promotion of translocation [13]. CMA detected a 455 Kb copy number loss on chromosome band 14q32. 2, not detected by FISH since the probe used was outside of this region. It is still a possibility, that the deletion in our patient may have removed some of the regulatory elements within this interval somehow affecting the regulation of Myc expression. No regulatory elements or high conservation data was seen in the UCSC Genome Browser. This microdeletion has been reported in other CMA studies of CLL patients using BAC-based array CGH, with some of the cases exhibiting the same findings as ours, i. e. with no IGH@ deletion by FISH [30]. It is still unclear whether this microdeletion is a The farthest probe upstream of 5' of MYC is the Spectrum Orange of the MYC break apart probe (upper panel) (adopted from Abbott Vysis website for FISH probes). We plotted the base coordinates for WI-1203 STS marker and the 5' of MYC (see inlet) into the UCSC Genome Browser (hg19) and determine the distance, RefSeq genes (POUF51B and LOC727677), STS markers and SNPs within this interval. The translocation breakpoint (with lightning icon) is centromeric of WI-1203 and about ~400 Kb upstream of 5'-MYC, a Class III breakpoint. We extended the breakpoint further upstream to show ~629 Kb region containing SNPs in different regions of the interval (R1, R2, R3 and CLL) that confer susceptibility risks for cancer [26, 28]. polymorphic feature of this locus and represents a region of frequent mutation and recombination, or it exhibits some susceptibility risks for CLL [3, 24, 31, 41]. \n\nAbout 20% of patients with CLL show ATM deletion, an anomaly also seen in almost all cancer, and is usually associated with an adverse outcome [1, 4, 31]. The collaboration of ATM and MYC in normal cell proliferation via an ATM-dependent pathway is well established. When deleted, ATM loses its protective checkpoint function leading to MYC-induced oncogenesis [4, 42]. This indicates that MYC alone is not capable of transforming lymphoid cells into neoplasia [4]. The ATM deletion and removal and relocation of MYC observed in our patient may explain the lymphomagenesis, but not necessarily the absence of Myc expression. \n\nGiven the limitations of this case report, we suggest that comprehensive retrospective studies in CLL patients should be performed to characterize the suggested ~400 Kb breakpoint and the region further upstream by sequential metaphase BAC FISH mapping since CMA does not detect the removal and relocation of an intact MYC locus. It is also possible that the absence of Myc expression is a false negative result given the specificity of immunostaining which is below 100%, and about 17% of cases may be overlooked for MYC rearrangements using this technique [43]. A more accurate quantitative approach such as RT-qPCR is recommended. Since variability in MYC breakpoints could still result in similar MYC expression [44], possibly due to flexible DNA looping [43, 45], reporter expression studies are needed to better understand the clinical impact and significance of long distance deregulation in in loci with atypical MYC rearrangement. \n\nThis paper presents an unreported atypical rearrangement involving the IGH@ and MYC loci detected by FISH, adding to the burgeoning cytogenetic data on CLL patients with atypical t (8;14). It also highlights the Class III translocation breakpoint upstream of MYC, including the cancer and CLL-associated SNPs within the interval. This report also provides important and promising findings for further studies correlating Myc expression with a specific type of genomic translocation breakpoint or copy number variants in CLL and in other B-cell disorders. Lastly, overall findings in our report highlight the utility of karyotype analysis, interphase and sequential metaphase FISH studies, CMA, and other molecular tools in approaching the diagnosis and prognosis of CLL in a more comprehensive manner.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Conventional GPG-banded chromosomal analysis was performed on peripheral blood lymphocytes that were cultured for 48 and 72 hours with and without pokeweed mitogen stimulation, following standard cytogenetics protocols. The karyotypes were described according to the ISCN 2009 nomenclature [33]. \n\nInitial FISH studies were performed on interphase cells using CLL panel probes (Abbott Molecular, Des Plaines, Illinois) specific for centromere 12, IGH@ break apart or IGH@-CCND1 fusion, and chromosome loci 13q14. 3 (D13S319)/13q34, 11q22. 3 (ATM) and 17p13. 1 (TP53). Sequential GPG-metaphase FISH studies were performed using IGH-MYC fusion (with centromere 8-specific probe), and break apart probes for the IGH@ and MYC loci. \n\nChromosome microarray analysis was performed on DNA sample from 48-hr culture of peripheral blood lymphocytes. DNA was extracted from Carnoy's fixed pellet cells Qiagen DNA extraction kit (Valencia, CA). DNA concentration and quality was checked using Nanodrop (Life Technologies, Carlsbad, CA) and gel electrophoresis, respectively. Whole genome chromosome SNP microarray was performed to assess for imbalances (i. e. gain or losses) in the genomic DNA sample tested. The assay compared the patient's DNA to a reference set from 380 normal controls (284 HapMap and 96 Affymetrix reference), using the Genome-Wide SNP Array CytoScan HD. This array platform contains 2. 6 million markers for Copy Number Variant detection (Affymetrix, Inc. ), which 750,000 are genotype SNPs and 1. 9 million are non-polymorphic probes, for the whole genome coverage. The analysis was performed using the chromosome analysis suite (ChAS), version CytoB-N1. 2. 2. 271(r4615). Oligonucleotide probe information is based on the 37 build of the Human Genome (UCSC Genome Browser, http://genome. ucsc. edu/cgi-bin/ hgGateway, hg19, February 2009). \n\nFFPE sections (4 μm thick) were stained for Myc using rabbit monoclonal human anti-Myc antibody (catalog #1472-1, Epitomics, Inc., Burlingame, CA, USA). Heat-induced epitope retrieval was accomplished by using ER1 for 20 min. Endogenous peroxidase was blocked using hydrogen peroxide. The slide was incubated in the primary antibody Myc for 30 min, followed by incubation in a post-primary 3,3-diaminobenzidine for 10 min, polymer 3,3-diaminobenzidine for 10 min, and chromogen 3,3-diaminobenzidine for 10 min. Subsequently, slide was incubated with post-primary alkaline phosphatase for 20 min, polymer alkaline phosphatase for 30 min, and fast red for 20 min. The nuclei were counterstained with hematoxylin and the slide was then dehydrated, cleared in xylene, and coverslipped. Appropriate positive controls were used.",
"section_name": "Materials and methods",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgement",
"section_num": null
},
{
"section_content": "We would like to thank the assistance of the following UCLA Cytogenetics Lab Staff : Lynn Yang for her technical assistance and her willingness to help and make this project possible. We would also like to acknowledge Ingrid Jaramillo, Gloria Lan, Pinky Bolire, Karen Park and Yun Lei and other FISH technologists in making this project possible. We also like to thank Dr. Kingshuk Das and the staff of UCLA Molecular Pathology Lab for assisting us in the DNA extraction. We are also grateful to Affymetrix for performing CMA in our patient's DNA sample.",
"section_name": "Acknowledgement",
"section_num": null
},
{
"section_content": "",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "The authors declare they have no competing interests. \n\nAuthors' contributions IA performed metaphase studies; gathered data for karyotype, interphase and metaphase FISH studies; analyzed, interpreted and wrote the cytogenetic report; analyzed and interpreted chromosome microarray data; and did necessary revisions in the manuscript as requested by the reviewers. PHB analyzed the molecular data, correlated clinical findings, and wrote the initial draft of the paper. BS provided the flow cytometry and immunohistochemistry data. SK reviewed the microarray data and paper draft. CT analyzed and reviewed all the data and drafted the paper. All authors read and approved the final manuscript.",
"section_name": "Competing interests",
"section_num": null
}
] |
10.1007/s10067-015-3014-y
|
Education for patients with rheumatoid arthritis in Latin America and the Caribbean
|
La educación del paciente es muy recomendable en la artritis reumatoide (AR) para apoyar el manejo del paciente. El desafío es cumplir con las recomendaciones para brindar educación sanitaria a los pacientes con AR en los países de América Latina y el Caribe (ALC) teniendo en cuenta factores como el analfabetismo en la salud de los pacientes, la falta de reumatólogos y la falta de recursos, incluido el acceso a medicamentos antirreumáticos modificadores de la enfermedad (FARME). Dado que el material educativo existente en los idiomas regionales no está fácilmente disponible y es inadecuado, proponemos desarrollar un programa educativo basado en la web que cumpla con los requisitos de la mayoría de los pacientes con AR en los países de ALC con énfasis en el uso correcto y seguro del metotrexato.
|
[
{
"section_content": "Rheumatoid arthritis (RA) is a chronic inflammatory disease of the joints affecting 0. 5-1 % of the adult population. RA produces pain, fatigue, and work incapacity, is potentially disabling, and shortens life expectancy. Much progress has been made in RA treatment in the last decades with methotrexate the cornerstone antirheumatic drug [1, 2]. Patient education is highly recommended in RA to support patient management. Guidelines for RA management say \"Education for patients with RA should be provided since first medical encounter\" [3]. Or recommend information to patients in the overarching principle \"Treatment of RA patients must be based on a shared decision between the patient and the rheumatologist. Shared decision-making includes the need to inform the patient of the risks of RA and the benefits of reaching the targeted disease activity states, as well as the pros and cons of respective therapies. It also means two-way communication and joint or shared decision-making on the therapeutic target and management plan as well as support for the patient to develop personal preferences\" [4]. Guidelines are endorsed by the Pan American League of Associations of Rheumatology (PANLAR) and all national rheumatology-affiliated societies and \"Grupo Latino Americano de estudio De Artritis Reumatoide\" (GLADAR) [5, 6]. Therefore, structured patient education should be available to all people with RA at initial diagnosis and on an ongoing basis, based on a formal, regular assessment of needs as the National Institute for Health and Care Excellence of the UK advocates (https://www. nice. org. uk/ guidance/qs33). Implementing these recommendations and providing patient education in clinical practice in Latin America and the Caribbean is a challenge. Some aspects that should be discussed in particular in the audio-visual material are listed here:\n\n1. Adverse effects of medications in contrast to benefits. Inequities in health care are a reality in Latin America and the Caribbean (LAC). There are many problems to face regarding the actual standard of care of patients with RA in LAC, including few rheumatologists and very few allied health professionals working in RA [6]. Moreover, rheumatology is not part of the undergraduate curricula in many medical schools (results of a 2014 PANLAR survey, unpublished communication). For the majority of clinical rheumatologists, the workload is heavy, time assigned per visit is short, and waiting lists are long [5, 6]. Methotrexate in oral form is the disease-modifying antirheumatic drug (DMARD) available in most centers due to both low costs and its effectiveness in at least 50 to 75 % of patients. However, this may also depend on whether treatment was initiated within \"the window of opportunity\" whereas other DMARDs or biologics are difficult to access due to their expensive costs [7]. Understandably, in these working conditions, to fully apply treat to target strategies [4] is not possible. Health education for patients with RA and caregivers is not a priority, although rheumatologists would agree that it is necessary. \n\nTherefore, it is relevant to develop a LAC healtheducational program to help doctors in busy clinical practices to provide education to RA patients, taking into account difficulties attributable to health system organizations and working conditions, poverty, and other socioeconomic factors. Particularly, when a recent study in early RA patients from the GLADAR cohort showed that low/middle low socioeconomic status (SES) is associated with more active disease and worse functional capacity [8]. It is possible that a health education program based on this reality could positively impact the outcome of RA or health-related quality of life. Thus, patients would be able to make optimal and safe use of methotrexate [9] and other medications and therapies and adopt healthier conduct. In our opinion, patients from low/middle low SES, illiterate or semi-illiterate patients, patients living in rural or suburban conditions, or manual laborers who are at risk of losing their jobs are in most need of health education if they suffer from RA and possibly would benefit more. In LAC, the most social and economic support for RA patients is provided by the family, as pensions for disability, sick leave, or anticipated retirement are low and under the requirements for a severe disease, and most patients find little or no social support from the state or their health system coverage. Every effort to provide health educational material to all LAC patients with RA and their families must be done. Especially when considering that there are few rheumatologists and difficulties in accessing medical visits and free medications across LAC countries.",
"section_name": "",
"section_num": ""
},
{
"section_content": "Health education is a planned learning experience that influences the knowledge of the patient about his/her illness aiming to modify health habits/conduct in order to be able to collaborate in their therapy [10] [11] [12]. It utilizes a combination of learning methods, counseling, and techniques to modify conduct. It is an interactive process to help the individual to participate in their self-care and to make optimal use of all health resources. The relevance of patient education safety programs has been put forward by the Joint Commission on Accreditation of Healthcare Organizations (JCAHO) (http://www. jointcommission. org/speakup. aspx). As mentioned by Fox [13], the JCAHO requires patient education standards as a condition of accreditation, including requirements related to understandability and readability, ease of access to materials, and consideration of language differences and patient abilities. However, it is not clear what type of educational interventions is most effective in improving health status for patients with chronic diseases [14]. Education strategies can vary from the provision of information only to the use of cognitivebehavioral strategies. Formats include verbal, written, audiovisual, and interactive computer-based educational programs [13]. The objectives of patient education in RA are to improve patient outcomes as well as to obtain the best of the affordable therapies available. From 31 education trials in RA included in a meta-analysis, significant benefits of patient education at first follow-up are modest (5-12 %). The most important benefit was observed in functional disability; additionally, behavioral programs versus controls had better results than information only or counseling [15]. Verbal information given by physicians on how to take prescriptions or drug side effects is not understood, not recalled, or not remembered incorrectly affecting patient adherence [16] even though it is the most common format used in hospitals/clinics. Therefore, printed material and multimedia are likely better options. \n\nPatient education is made up of knowledge, abilities and conducts, and psychological support. In RA, focus should be on obtaining and maintaining remission, prevention of relapses, and avoiding deformities and damage. If there is disability due to RA, how to adapt to this condition should be covered. Patients can learn the significance of adherence to therapies, attending regular controls, recognize possible complications on time, manage depression and anxiety symptoms, control co-morbidities, and avoid health risk conducts. Patients should be encouraged to maintain adequate body mass index and exercise, to follow vaccine programs, and to avoid infections. Patients should also adopt joint protection strategies [17]. Lack of awareness of health conducts directly affects patient's response to treatment and outcomes or obtaining an acceptable health-related quality of life (http:// www. uptodate. com/contents/arthritis-beyond-the-basics). Providing effective health education for RA patients has many difficulties. A recent study has shown that RA educational needs varies in relation to gender, personal characteristics, disease activity, and disease duration indicating that education be targeted more effectively. Correlations between educational needs and disease activity and function could enable identification of 'intervention points', which can be ideal opportunities for effective patient education [18]. \n\nA challenge often faced by LAC physicians when they approach patients with RA under their care is that they have health illiteracy or do not read, which both jeopardize patient c o m p l i a n c e [ 1 9, 2 0 ] (h t t p : / / w w w. h e a l t h. g o v / communication/). In LAC countries, patients often follow complementary and alternative medicine [21, 22] or magical cures that may greatly modify the therapeutic plan the rheumatologists consider more appropriate. An educational program should deal with doubts and help people with RA to take informed decisions considering risks and benefits [19]. When patients ask for information about RA, answers should be given in simple terms. However, this should not preclude offering the correct information particularly on methotrexate safe and correct use [19]. A respectful approximation of patient's beliefs identifying the preoccupations, expectations, emotions, and interests of patients would allow the \"coincidence of agendas of both patient and doctor\" [23, 24] that would permit choosing the individualized health educational material. Therefore, we propose including courses on patient education as well physician-patient communication skills at a professional level of competence at PANLAR meetings as part of the continuing medical education in rheumatology. \n\nThere are suitable programs that could be applied to RA patients in groups such as the Chronic Disease Self-Management Program of the Stanford University [11] given in six workshops to 10-12 participants. People with RA that attended this workshop did not show any benefit in biomedical or lifestyle outcomes at 3 years, although there were sustained improvements in some illness beliefs. However, in a short period, the benefit was evident [12, 15, 25]. In 2006, training of people from ten Latin American countries in Panama was started but the effort did not continue and workshops were not repeated. \n\nA review of web pages in Spanish shows sites providing information for patients and lay people on RA. The Medical Library of National Institute of Health from the US (http:// www. nlm. nih. gov/medlineplus) and the American College of Rheumatology provide information for Spanish-speaking people with RA living in the USA explaining drug mechanisms of actions and adverse effects using commercial names in the USA. The National Institute of Arthritis and Musculoskeletal and Skin diseases web page offers little information (http://www. niams. nih. gov) whereas Wikipedia (http://es. wikipedia. org) offers an up-to-date text appropriate for those with biomedical knowledge. The Sociedad Española de Reumatología (www. ser. es) has developed a brochure with detailed explanations on pathogenesis, clinical features, and therapy; however, it is difficult for LAC patients to identify the drug names. Pharmaceutical companies publish educational material for patients that lack individualization and need revisions to assure the absence of bias. Common to all pages is the use of a minimum visual support, lack of audio, and no interactive web page in Spanish or Portuguese. In summary, there is a need for web-based material for patients adapted to the specific needs of LAC patients with RA and the diverse reality in LAC countries.",
"section_name": "Education measures for RA",
"section_num": null
},
{
"section_content": "The challenge is to adhere to the recommendations for providing health education to RA patients in LAC taking into account the previously explained difficulties and lack of resources. As existent material is not easily available and inadequate, we propose developing an educational program that would fulfill the requirements of most patients with RA across LAC countries. The proposal will need support from PANL AR, resources, and funding, but costs and details are not within the scope of this manuscript. We will briefly review the program we conjecture our patients would benefit from. \n\nOur aim is that the RA patient educational material developed by PANLAR be offered to doctors and patients in a variety of formats the patient can choose from. The design will require short interventions with minimal contents explained clearly that could be given in each visit by the attending physician or under his/her direction and if possible, by health personnel and trained RA patients. This should be reinforced afterwards or at home. \n\nThe need to be scientifically valid is very importantly, and thus, every statement of the content will be based on evidencebased medicine or medical expert opinions when there is no evidence. As evidence changes, the program will be updated and reviewed annually. \n\nWe propose to develop a written and audio-visual material on a website based on plain local languages (in Spanish, including indigenous languages and Portuguese) that could be printed on one page in a letter case easily read (size 12), with headings and titles identifying the contents in a sequence that could be followed as needed. This material will be published online on the PANLAR educational website. As many patients, especially the elderly, do not have media access, they could be given printed material at the clinic visit, according to their needs. Videos should be posted on Youtube with frequently asked questions from a patient with an appointed doctor of about 5 min each. Rheumatologists, patients, linguist specialists, nurses, occupational therapists, physiotherapists, physiatrists, psychiatrists, psychologists, and education and multimedia experts will participate in the development of the material. \n\nAdditionally, patients will be directed to official ACR websites in Spanish and from the \"Sociedad Española de Reumatología\" and to EDUCAR, a website of PANLAR dedicated to continuing medical education in RA. Patient questions should be directed to the program by doctors or patients themselves to receive an expert response. A list of health providers containing rheumatology centers fulfilling local quality standards and qualified staff will be accessible from the web page of the educational site. A diffusion campaign to doctors and patient associations through email from PANLAR will be done. We are aware that the site should be found easily and be on the first two pages of Google.",
"section_name": "Educational program for LAC RA patients: a proposal for a solution",
"section_num": null
},
{
"section_content": "Knowledge. Basic articular anatomy notions, inflammation notions, DMARDs mechanisms of action, and adverse effects with emphasis on the correct and safe use of methotrexate. What rheumatologists assess in a clinic visit such as articular indices and registries. Qualified/accredited health providers locally and at a national level. A list of available resources for RA and patient associations in LAC will also be included. \n\nAbilities and conducts. Regular low-impact exercise combined with rest periods. Adaptation measures at work and in daily life. Promote adherence to therapy. Following vaccination schedule according to local regulations. Eating a healthy diet. How to proceed if an adverse effect appears. \n\nPsychological support including self-help and treatment for anxiety and depression when needed. Communication strategies, support from family, and available social agencies. The table lists some aspects that should be discussed in particular in the audio-visual material.",
"section_name": "Content of the education program",
"section_num": null
},
{
"section_content": "A survey will be sent out at 3 and 12 months to patient associations about readability, acceptance, and recall of the material and to rheumatologists about problems, questions, and suggestions. Also, a web page visit counts. This evaluation and revision will proceed annually. \n\nIn conclusion, there is an unmet need for patient health education in rheumatology in LAC, including patients with RA and their families. To reach more people with RA, information should be in various formats; written and audio-visual based on the web with an emphasis on the correct and safe use of methotrexate.",
"section_name": "Evaluation of the education program",
"section_num": null
}
] |
[] |
10.3389/fonc.2022.841630
|
Impact of Low-Burden TP53 Mutations in the Management of CLL
|
<jats:p>In chronic lymphocytic leukemia (CLL), <jats:italic>TP53</jats:italic> abnormalities are associated with reduced survival and resistance to chemoimmunotherapy (CIT). The recommended threshold to clinically report <jats:italic>TP53</jats:italic> mutations is a matter of debate given that next-generation sequencing technologies can detect mutations with a limit of detection of approximately 1% with high confidence. However, the clinical impact of low-burden <jats:italic>TP53</jats:italic> mutations with a variant allele frequency (VAF) of less than 10% remains unclear. Longitudinal analysis before and after fludarabine based on NGS sequencing demonstrated that low-burden <jats:italic>TP53</jats:italic> mutations were present before the onset of treatment and expanded at relapse to become the predominant clone. Most studies evaluating the prognostic or predictive impact of low-burden <jats:italic>TP53</jats:italic> mutations in untreated patients show that low-burden <jats:italic>TP53</jats:italic> mutations have the same unfavorable prognostic impact as clonal defects. Moreover, studies designed to assess the predictive impact of low-burden <jats:italic>TP53</jats:italic> mutations showed that <jats:italic>TP53</jats:italic> mutations, irrespective of mutation burden, have an inferior impact on overall survival for CIT-treated patients. As low-burden and high-burden <jats:italic>TP53</jats:italic> mutations have comparable clinical impacts, redefining the VAF threshold may have important implications for the clinical management of CLL.</jats:p>
|
[
{
"section_content": "The heterogeneous clinical course of chronic lymphocytic leukemia has highlighted the need to define prognostic and predictive markers to improve the management of patients (1). On one hand, prognostic markers reflect the underlying biology and natural history of CLL and are informative for untreated patients or those requiring treatment (2, 3). On the other hand, predictive markers provide information on the likely benefits or contraindications of a given treatment. TP53 abnormalities, namely, both deletion of the 17p chromosome and mutations at TP53 loci, are one of the gold standards of high risk in CLL because these abnormalities indicate both an adverse prognosis and predict chemoresistance (4, 5). In the past decade, the therapeutic landscape of CLL has considerably improved, offering the possibility for patients with TP53 defects to benefit from targeted therapy with BcR pathway or bcl2 inhibitors (6) (7) (8). Although the first-line treatment strategy may differ among countries, assessment of TP53 status has become essential, as it serves as a contraindication for the use of chemoimmunotherapy (CIT) (9). Hence, in daily clinical practice, the use of TP53 status as a predictive marker is mandatory for treatment decisions before the addition of each new line of treatment (10, 11). \n\nThe implementation of NGS sequencing technologies with high sensitivity has facilitated the detection of TP53 mutations with the possibility of detecting variants with allelic fractions (VAFs) below the current conventional threshold of 10% published by the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) in 2018 (12), above which TP53 mutations should be clinically reported. Nevertheless, the clinical and biological relevance of these minor clones is debated. \n\nThe definition of minor clones and their biological and clinical significance have been discussed in numerous studies. However, contradictory results are often reported that might be in part attributed to different cohort compositions and variable low-burden threshold definitions. To clarify the clinical role of low-burden TP53 mutations in CLL, the prognostic and predictive impact of TP53 mutations were analyzed in different cohorts. The results and conclusions are discussed in this review.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Del(17p) associated with TP53 mutations is the most common abnormality affecting the TP53 gene in CLL, accounting for approximately two-thirds of cases. The remaining cases either exclusively harbor TP53 gene mutation(s) or rarely a 17p deletion. Moreover, TP53 mutation can be accompanied by the mutation of the second allele or a copy number neutral loss of heterozygosity (13). \n\nHistorically, TP53 abnormalities were first analyzed by conventional karyotyping combined with Fluorescence In Situ Hybridization (FISH), which allowed the detection of cells carrying a deletion of chromosome 17p13. 1 (TP53) with a sensitivity of >5% positive cells (14). Despite a relatively good sensitivity of detection, cytogenetic techniques failed to detect approximately 30-40% of patients carrying only mutations in the gene. Later, TP53 mutation screening relied on Sanger sequencing covering exons 4 to 9 of the gene with a sensitivity of approximately 10-20%. Hence, combining FISH analysis and sequencing substantially improved the detection of TP53 aberrations. The advent of NGS technologies next provided the opportunity to reduce the threshold of detection of TP53 mutations and to deeply examine the clonal heterogeneity of CLL. In a retrospective analysis of newly diagnosed patient samples, NGS sequencing could detect low-burden TP53 mutations previously identified as unmutated by Sanger sequencing due to their low abundance in the tumor cell population (15). Altogether, Sanger sequencing led to misclassification of approximately 6% of newly diagnosed and untreated patients harboring low-burden TP53 mutations with a VAF ranging from 0. 3 to 11% (15) (16) (17) (18) (19). Of note, a fraction of patients harbored low-burden mutations associated with high-burden mutations, revealing the intratumoral heterogeneity of these mutations and the complexity of the TP53 clonal architecture. \n\nThe definition of minor clones often relies on the VAF threshold used to detect mutant alleles by Sanger sequencing, which is typically approximately 10-12%. This conventional threshold corresponds to the current recommendations published by ERIC in 2018, above which TP53 mutations should be clinically reported. Mutations with VAFs below the threshold are considered low allele frequency, whereas VAFs above the threshold are of a high allele frequency. This recommendation is still currently applied due to technical difficulties in detecting low-burden mutations. However, with the wide generalization and feasibility of NGS sequencing on a routine basis, the threshold to report TP53 mutations and hence to define minor clones is debated. \n\nIndeed, below this arbitrary threshold of 10%, a wide range of TP53 variants can be detected by NGS sequencing with high confidence until reaching a limit of detection as low as 0. 3% VAF (corresponding to three mutant alleles in a background of 1,000 wild-type alleles) while respecting specific procedures and quality criteria. First, CLL lymphocyte population purity greater than 80% reduces the possibility of dilution in nontumoral DNA that could underestimate a very low-burden mutation. Second, sufficient DNA corresponding to >6,000 diploid genomes and a third high target read depth is required to detect a very lowburden mutation with VAF<1% (20). Finally, robust bioinformatic workflows were developed to call true variants distinguished from background error noise. However, despite the very high confidence of TP53 variant detection by NGS sequencing, the limit of detection of these ultrasensitive technologies needs to be evaluated to distinguish true TP53 variants from background sequencing noise to avoid misdiagnosing TP53 unmutated patients as mutated. The sequencing background depends on sequencing technologies and library preparation, which differ in capture and ampliconbased processes (21, 22).",
"section_name": "WHAT IS A LOW-BURDEN TP53 MUTATION OR MINOR CLONE?",
"section_num": null
},
{
"section_content": "While TP53 abnormalities account for approximately 10% of naïve-treatment patients, these abnormalities are found in greater than 40% of patients with fludarabine-refractory CLL, which highlights the phenomenon of clonal evolution of TP53 mutation induced by chemotherapy (13). Despite the current recommendations that consider <10% of minor clones to be of uncertain significance, accumulating evidence based on longitudinal studies argues for the clinical relevance to report TP53 minor clones (15, 18, 20, (23) (24) (25). NGS sequencing of serial samples before and after treatment has allowed characterization of the dynamics of the minor clones under treatment and demonstrated their biological and clinical relevance. \n\nLongitudinal retrospective studies based on NGS sequencing of fludarabine relapsed/refractory TP53 mutated patient samples showed that low-burden TP53 mutations were detected early in the disease course and before the onset of chemotherapy. These pre-treatment samples were initially screened using Sanger sequencing, and mutations were missed due to the lack of sensitivity of the technique. Interestingly, longitudinal analysis indicated that the acquisition of TP53 mutations clearly preceded karyotype evolution, which highlights the genetic instability related to the presence of a TP53 mutation and its likely role in the development of a complex karyotype (24). It is widely accepted that chemotherapy plays a key role in driving the selection of clones carrying TP53 mutations (26). Fludarabine is a purine analog that inhibits DNA synthesis in tumor cells. In the case of defects in the TP53 pathway, CLL cells lose their capacity to stop cell division and to trigger apoptosis in response to chemotherapy. As a result, the mutation induces a fitness effect by conferring a growth and survival advantage to the low-burden TP53 mutation, which expands under the selection pressure of chemotherapy (27). The fact that a given low-burden TP53 variant detected at the time of treatment initiation is found at relapse after a fludarabine-based regimen clearly demonstrates that these minor clones are not sequencing artifacts and highlights the need to redefine this threshold for optimal clinical practice. \n\nFinally, relative stability in the TP53 variant allele frequency is observed in some patients as long as they are not treated with chemotherapy. This notion is particularly true for IGHVmutated patients, which have a more indolent disease course and can show the persistence of the mutated clone for years (28) (29) (30). On the other hand, given the natural clonal evolution of the disease with time, TP53 minor clones can also be acquired during the disease course, independent of any pressure of selection induced by chemotherapy. This finding justifies early and iterative screening for TP53 abnormalities during follow-up and before each new line of treatment with a sensitive sequencing technique.",
"section_name": "CLONAL EVOLUTION OF LOW-BURDEN TP53 MUTATION AFTER CHEMOTHERAPY",
"section_num": null
},
{
"section_content": "Given that TP53-mutated patients can benefit from targeted therapies with improved remission duration, there is a need to evaluate the impact of these therapies on the evolution of the TP53-mutated clone. Data on the clonal evolution of low-burden TP53 mutations upon targeted treatment are limited (23, 31). Malcikova et al. showed that upon the use of BcR or bcl2 inhibitors as a second line of treatment, the percentage of VAF in the residual lymphocytosis remains stable, which reflects the efficacy of these treatments on the mutated clones (23). Indeed, BcR and bcl2 inhibitors target the BcR signaling pathway and apoptosis, respectively, and therefore overcome the p53 pathway. However, the persistence of TP53-mutated clones after treatment shows the failure to eradicate the disease (32). In some progressive patients treated with targeted therapies, the major TP53 mutated clone becomes minor. However, in these cases, mutations that confer resistance to ibrutinib (i. e., BTK mutation) or Venetoclax (i. e., BCL2 mutations) are frequently found. In another longitudinal study including treatment-naïve and relapsed/refractory patients treated with BcR inhibitors, the dynamics of TP53 mutated clones were complex. Most of the TP53 mutations decreased or were undetectable, but one-third remained stable with no differences noted between low-or high-VAF clones. A small proportion of TP53 mutations increased. After a prolonged follow-up of greater than 2 years, the overall stability of low-burden TP53 mutations was noted, supporting the notion of the lack of specific positive selection of TP53 mutations under conditions of ibrutinib treatment (31). Nevertheless, all these observations need to be confirmed in a cohort of patients treated with novel agents in the frontline setting. To date, this has not been explored within clinical studies, and data are preliminary, especially for bcl2 inhibitors.",
"section_name": "IMPACT OF TARGETED AGENTS ON LOW-BURDEN TP53 MUTATIONS",
"section_num": null
},
{
"section_content": "In most studies focusing on the clinical impact of TP53 minor clones, an arbitrary threshold of 10-12% VAF was chosen to define patients with low-or high-burden TP53-mutated clones. Most studies conducted in untreated patients (15, 18, 20) showed that low-burden TP53 mutations significantly reduced the OS compared to cases with unmutated TP53 genes. Moreover, the impact on OS was the same for patients harboring minor clones or high-burden TP53 mutations (Table 1 ). The clinical consequence of TP53 mutations was similar when patients with low VAF were stratified into subclasses <1%, between 1% and 5% or 5% and 10%. Shorter OS was also confirmed when separately considering patients with single or multiple mutations classified as high VAF or low VAF (15, 20). \n\nThe presence of del(17p) and/or TP53 mutations are parameters of the CLL-International Prognostic Index (CLL-IPI), which combines five parameters (age, clinical stage, TP53, IGHV mutational status, serum b2-microglobulin) to predict survival and time-to-first-treatment (TTFT) in CLL patients. However, the value of the VAF threshold used to consider TP53 mutated considerably impacted this score. Indeed, revisited CLL-IPI combining both high-and low-VAF TP53 mutations significantly better discriminated high-risk patients than standard CLL-IPI, which exclusively considered high-VAF TP53 mutations (20, 23, 35). Therefore, minor clones should be considered to refine prognostication models. \n\nMost studies evaluating the predictive impact of TP53 mutations showed significantly reduced survival in CIT-treated patients harboring either low-or high-burden TP53 mutations (15, 20, 23, 33). Clonal expansion is likely the main factor contributing to the inferior survival of CIT-treated patients with low-burden TP53 mutations, as demonstrated by longitudinal studies comparing pre-and post-treatment samples showing that the mutation burden consistently increases at relapse (18, 20, 23). Furthermore, the risk of TP53 mutation expansion beyond the current threshold of 10% in the first relapse was significantly higher for patients carrying mutations with VAF >1% than for those with VAF <1% (23). Additionally, very low clonal abundance cell populations (as low as 0. 3%) are clinically relevant, as they are resistant to CIT, are positively selected and may become the dominant leukemic population at the time of relapse. Blakemore et al. 's (34) LRF CLL4 clinical trial could not demonstrate inferior survival associated with cases harboring <12% VAF TP53 mutations but rather an intermediate-risk group, revealing heterogeneity among studies based on the patients included, the duration of follow-up, and the thresholds used. \n\nTherefore, these observations strengthen the need to redefine the clinically relevant threshold of VAF, which better discriminates TP53-mutated patients who will benefit from a targeted therapy (15, 26, (36) (37) (38). \n\nThe literature on the impact of TP53 minor clones on targeted therapies is less abundant. One study (23) showed that in a cohort of relapsed/refractory patients entering treatment with BcR and bcl2 inhibitors, OS in response to targeted treatment in TP53-mutated patients did not significantly differ from that of TP53 wild type patients irrespective of VAF.",
"section_name": "LOW-AND HIGH-BURDEN TP53 MUTATIONS HAVE THE SAME UNFAVORABLE PROGNOSTIC IMPACT",
"section_num": null
},
{
"section_content": "The main focus of this review was to demonstrate that lowburden TP53 mutations have an impact on CLL survival. This review analyzing different retrospective and prospective CLL cohorts highlights the need to detect mutations with highly sensitive NGS technology in a routine setting due to the clonal expansion of minor clones after CIT. NGS sequencing technology can detect low-burden TP53 mutations that are as low as 0. 3% over the background noise using specific bioinformatics pipelines. The clinical relevance of these lowburden mutations is evaluated as prognostic or predictive markers, and most of the studies identified that cases bearing low-burden TP53 mutations (VAF <10%) experienced shorter OS similarly to cases with high-burden TP53 mutations (VAF >10%) compared to patients harboring wild type TP53. These concordant observations highlight the need to redefine the threshold used to identify TP53-mutated cases, as these findings may have important implications in the setting of CLL treatment. \n\nLow-VAF mutations showed the same molecular characteristics and distribution as high-VAF mutations, confirming that they are The overall survival (OS) in subgroups of patients with TP53 wild type, low-burden, or high-burden TP53 mutations is indicated in months, or the 5 years OS rate* is reported. P value corresponds to a comparison of OS of TP53 low-burden mutated patients vs TP53 wild-type patients. NR, not reached; NS, not significant. \n\nnot sequencing artifacts. Moreover, the pathogenicity of these mutations was confirmed using different databases (IARC TP53, UMD database) (39, 40). Accordingly, in longitudinal studies, sequential samples from CIT-treated patients showed that minor clones were positively selected and became dominant at relapse, confirming that these low-burden mutations that initially occur in a minority of cells are true mutations that expand under selective pressure (26). \n\nFocusing on studies designed to assess overall survival (OS) between cases harboring the wild-type TP53 gene versus cases with low-burden TP53 variant (15, 18, 20, 35), the frequencies of TP53 mutation ranged from 10. 6 to 27. 5%, of which 26. 8 to 45. 2% cases exclusively harbored low-burden TP53 mutations depending on the threshold used to discriminate between lowand high-VAF TP53 mutations. Blakemore et al. failed to demonstrate a clinical impact of low-burden TP53 mutations but identified an intermediate-risk group. These findings were probably due to the choice of an arbitrary threshold of 12% for discriminating low-and high-burden TP53 mutations and a minimum VAF >1% (34). \n\nThe impact of TP53 mutations on OS also depended on the composition of the cohort with different proportions of patients carrying mutated IGHV or 17p deletion or variable times to diagnosis. Indeed, newly diagnosed patients often harbor mutated IGHV, and TP53 abnormalities may not have a negative impact on the indolent disease course (23, (28) (29) (30) 35). These observations suggest that TP53 mutation testing should be performed exclusively before treatment. Conversely, Brieghel et al. demonstrated that neither high nor low burden TP53 mutations at the time of CLL diagnosis influenced OS independently (35). Surprisingly, patients with 17p deletion had an inferior outcome, and only the subgroup of patients with high-burden TP53 mutations and unmutated IGHV demonstrated an inferior OS. This discrepancy may be explained by the composition of the cohort and the more indolent nature of the disease for the patients included. The frequency of 17p deletion was only 2. 4%, whereas TP53 mutations without 17p deletions were more frequent (10. 7%). Furthermore, the proportion of newly diagnosed TP53-mutated patients with unmutated IGHV genes was low (32%) as compared to 57% (18) and 35. 5% (15). \n\nGiven that NGS technology can detect low-burden TP53 mutations at levels as low as 0. 3%, should this limit of detection be used as a threshold to identified patients with TP53 mutations? One study further stratified patients based on a 5% VAF threshold and observed shortened survival only for mutations with 5-10% VAF but not for mutations with 1-5% VAF. Interestingly, the subgroup carrying mutations with <1% VAF showed significantly shortened OS. In addition, the risk of a rapid expansion of the clone to greater than 10% in the first relapse after CIT treatment was higher for patients carrying mutations with >1% VAF than for those with <1% VAF (23). These results suggest that a >1% VAF threshold could be clinically relevant. \n\nFurther standardization (41) and bioinformatics development (42) may be necessary to identify the background noise at each position of the TP53 gene to validate very low-burden mutations (as low as 0. 3%). \n\nHence, there is a need to harmonize the methodologies used to detect minor clones and minimal requirements for the standardized assessment of such clones. An ERIC (European research initiative on CLL http://www. ericll. org/) multicenter study on the prognostic and predictive impact of low-burden TP53 mutations is in progress with three phases: 1) compare results among laboratories performing NGS analysis of TP53 mutations in CLL with a detection limit of ≤1% VAF, 2) assess the prognostic and predictive impact of low-VAF TP53 variants in patients entering first-line treatment, and 3) re-evaluate the cut-off for reporting of TP53 variants in CLL and, if needed, to update recommendations on minor TP53 variant detection, validation, and reporting. Forty-one laboratories participated in the 1st phase of the study and analyzed the same samples with low-VAF TP53 mutations. The collected results show that the 2% VAF cut-off could be reproducibly applied for the planned multicenter study on the clinical significance of low-VAF TP53 variants (43). The collection of clinical and biological data from a consecutive cohort of patients, namely, both wild-type and mutated TP53 CLL entering 1st-line therapy, is currently in progress to re-evaluate the cut-off for reporting TP53 variants.",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. \n\nPublisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. \n\nCopyright © 2022 Lazarian, Cymbalista and Baran-Marszak. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.",
"section_name": "Conflict of Interest:",
"section_num": null
}
] |
10.1371/journal.pone.0119723
|
Epidermal Growth Factor Receptor Inhibition Reduces Angiogenesis via Hypoxia-Inducible Factor-1α and Notch1 in Head Neck Squamous Cell Carcinoma
|
Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC). The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR) inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α) was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO) mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.
|
[
{
"section_content": "Head and neck squamous cell carcinoma (HNSCC) ranks as the sixth most frequent cancer worldwide with approximately 500,000 new cases per year worldwide [1]. Previous studies have established that risk factors, such as alcohol drinking, smoking, and human papilloma virus infection, contribute to the development of this fatal disease [2]. However, the five-year survival rate of HNSCC patients remains relatively unchanged at 40% to 50% during the past three decades [3]. Advanced-stage HNSCC patients have poor prognosis and often need both chemotherapy and radiotherapy [4]. However, only 30% of advanced-stage HNSCC patients survive for more than 5 years. Important factors that contribute to this scenario include the relative hypoxic and angiogenic conditions of high tumor burden in HNSCC. These conditions promote the stemness of cancer stem cells with both local and distant metastatic potentials [5]. \n\nEmerging basic, preclinical, and clinical findings indicated that epidermal growth factor receptor (EGFR)-mediated aberrant signaling transduction is crucial in HNSCC tumorigenesis and progression [6]. EGFR has been observed in 70% to 100% of all HNSCC lesions [7]. The high phosphorylation status of EGFR is frequently correlated with poor prognosis [8]. Activated EGF/EGFR pathway may promote cell proliferation, differentiation, angiogenesis, and antiapoptosis in HNSCC tumorigenesis and progression through the phosphoinositide-3-kinase (PI3K)/Akt, ras/raf/extracellular regulated protein (Erk), and signal transducer and activator of transcription pathways [9, 10]. Cetuximab is a chimeric IgG1 monoclonal antibody that is currently licensed for the treatment of HNSCC patients [11, 12]. This drug is used alone or in combination with chemotherapy as the first and second lines of treatment for advanced-stages patients [13]. Hypoxia-inducible factor-1α (HIF-1α) is a principal molecular mediator for tumor angiogenesis, and Notch pathway dysregulation is a leading genetic instability in HNSCC [14] [15] [16]. Previous reports suggested that the interaction between HIF-1α and Notch1 can influence tumor angiogenesis [17]. However, the mechanism by which the interaction between EGFR and HIF-1α or Notch1 in HNSCC regulates angiogenesis and tumorigenesis has yet to be elucidated. \n\nIn our previous studies, we established that Tgfbr1 and Pten conditional knock out (2cKO) mice demonstrate spontaneous fast HNSCC tumorigenesis with 100% penetration [18]. HNSCC mice are highly angiogenic as compared with Pten knock out HNSCC mice [19]. The present study shows that the overexpression and high phosphorylation of EGFR are crucial for the tumorigenesis of transgenic mouse models with combined Tgfbr1 and Pten loss. Furthermore, the cetuximab-induced inhibition of EGFR repressed tumor burden in xenograft HNSCC models. Chemopreventive treatment with cetuximab delays HNSCC onset in Tgfbr1/ Pten 2cKO mice and reduced HIF-1α-and Notch1-mediated angiogenesis. EGFR overexpression was correlated with HIF-1α and micro vessel density (MVD) in HNSCC clinical specimens. Thus, HIF-1α-and Notch1-mediated angiogenesis may be important for EGFR activation and may partially contribute to EGFR inhibitor sensitivity.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "All chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless indicated. Antibodies against EGFR, p-EGFR Tyr1068, HIF-1α, and Notch1, Notch1 intracellular domain (NICD), Hes1, VEGF, Histone H3 were obtained from Cell Signaling Technologies (Danvers, MA, USA), CD31 were obtained from BD Pharmingen (NJ, USA). Cetuximab was purchased from Merck (Darmstadt, Germany). N-[N-(3,5-difluorophenacetyl-l-alanyl)]-Sphenylglycine t-butyl ester (DAPT, γ-secretase inhibitor which inhibited cleavage of Notch1) was obtained from Sigma-Aldrich (St. Louis, MO, USA).",
"section_name": "Chemicals and reagents",
"section_num": null
},
{
"section_content": "The CAL27 cell line was purchased from ATCC and cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% FBS as previous described [20], in a humidified atmosphere of 95% air, 5% CO 2 at 37°C. CAL27 cells were serum-deprived for 12h and then treated with or without cetuximab (10 μg/ml) or DAPT (20 μM) in for indicated time (12h) in Anoxomat chambers (Mart Microbiology, Lichtenvoorde, the Netherlands) with appropriate oxygen concentrations for hypoxia (1% O 2 ) or normoxia (21% O 2 ). The cells were washed by phosphate buffer solution (PBS) two times and continue grow in serum-deprived endothelial basic medium (EBM, Lonza, Walkersville, MD, USA) medium for another 24h, and the cleared supernatants were collected as conditional medium (CM) and stored at -80°C. Pooled human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and cultured as previous described [19]. In vitro wound healing assay and Boyden chamber transwell migration assay and tube formation assay of HUVECs were performed as previous described [19] with detail in Supplementary Material and Methods in S1 File.",
"section_name": "Cell culture, conditional medium collection and in vitro migration assay",
"section_num": null
},
{
"section_content": "RNA interference were performed as previous described [20]. Briefly, CAL 27 cells were seeded in 6cm culture dishes and allowed to grown to 80% confluence, transfected with TGFBR1 siRNA or/and PTEN siRNA with Hiperfect transfection reagent (Qiagen) according to the manufacturer's instruction. The knock down efficiency with at least 84% decrease of TGFBR1 or PTEN protein at a indicated time (24h) were confirmed by western blot as previous described [20]. The expression of EGFR, p-EGFR Tyr1068 after the transfection was confirmed by Western blots.",
"section_name": "RNA interference",
"section_num": null
},
{
"section_content": "Immunofluorescence were performed as previous described [19] and detail described in Supplementary Material and Methods in S1 File. Cells immunofluorescence was photographed by microscopy (CLSM-310, Zeiss, Germany).",
"section_name": "Cell immunofluorescence and confocal microscopy",
"section_num": null
},
{
"section_content": "The nuclear/cytosolic fractionation of CAL27 cells was extracted using a Nuclear-Cytosol Extraction Kit (Applygen Technologies, Beijing, China) according to the manufacturer's instructions. Briefly, CAL27 cells treated with or without cetuximab were collected by centrifugation and resuspended in cytosol extraction buffer A. After incubation on ice for 10 min, the cells were mixed with cytosol extraction buffer B and further incubated on ice for 1 min. The lysates were separated by centrifugation, and the supernatant (cytosol extract) was collected and transferred into a new tube. The pellet was washed with cytosol extraction buffer A, and resuspended in cold nuclear extraction buffer. After incubation at 4°C for 30 min with constant rotation, the suspension was centrifuged at 12,000 g at 4°C for 5 min to collect the nuclear extract in the supernatant fraction. The nuclear and cytoplasmic extracts were subjected to Western blots analysis.",
"section_name": "Nuclear/cytosolic fractionation",
"section_num": null
},
{
"section_content": "All animal studies include nude mice and transgenic mice were approved and supervised by Animal Care and Use Committee of Wuhan University and conducted in accordance with the NIH guidelines for the Care and Use of Laboratory Animals. Female athymic nude mice (18-20 g; 6-8weeks of age) were obtained from the Experimental Animal Center of Wuhan University in pressurized ventilated cage according to institutional regulations. Mice were housed in appropriate sterile filter-capped cages and with an inverse 12 h day-12 h night cycle. Lights were turned on at 8:30 am at 22 ± 1°C and 55 ± 5% humidity in the Experimental Animal Center of Wuhan University. All cages contained wood shavings, bedding and a cardboard tube for environmental enrichment. Animals fed and watered ad libitum. \n\nFor heterotopic xenograft, nude mice were injected subcutaneously with CAL27 cells (4×10 6 in 0. 2 ml of serum-free medium) in the flank when cells exponentially grow. After tumors were established, the mice were divided into two groups randomly, which were received cetuximab (10 mg/kg i. p. twice per week; n = 5) or normal saline (vehicle, 100ul i. p. 2/week; n = 5) infusion for 3 weeks. Tumor growth was determined by measuring the size of the tumors 3 times per week. The formula (width 2 ×length)/2 was used to determine tumor volumes. All mice were monitored daily for abnormal behavior, e. g., inability to eat or drink, unable to run away when touched, no response to stimuli. There was no mice which was euthanized before the experimental endpoint. The maximum tumour sizes reached to 1. 2 cm during the course of this assay. The mice were euthanized using CO 2 and the tumors were harvested for the following immunohistochemical analysis and western blots analysis.",
"section_name": "Establishment and cetuximab treatment of CAL27 heterotopic xenograft tumors model in nude mice",
"section_num": null
},
{
"section_content": "The squamous epithelial tissue specific and time inducible combined Tgfbr1/Pten knockout mice (Tgfbr1/Pten 2cKO, K14-Cre ERtam ; Tgfbr1 flox/flox ; Pten flox/flox ) were maintained as previously described [18, 21]. The Tgfbr1/Pten 2cKO mice and their vehicles (Tgfbr1 flox/flox ; Pten flox/flox ) were from the same litter with mixed genetic background of C57BL/6; FVBN; CD1;129. Five day consequent tamoxifen oral gavage need to applied to knock out Tgfbr1/Pten in oral epithelial and head neck skin. The tamoxifen application procedure has been previously described [18, 21]. Only 4-to 8-week-old male and female Tgfbr1/Pten 2cKO mice were included in this study. For in chemopreventive assay, 2 weeks after the last dose of oral tamoxifen application of the Tgfbr1/Pten 2cKO mice were randomized into a vehicle group (100ul PBS. i. p. n = 5 mice) or a cetuximab group (10 mg/kg i. p. twice per week, n = 6 mice), based on our pilot study on the tumorigenesis and survival of 2cKO mice. All mice were monitored daily for abnormal behavior, e. g., inability to eat or drink, unable to run away when touched, no response to stimuli. There was no mice which was euthanized before the experimental endpoint. The maximum tumour sizes reached to 1. 0 cm during the course of this assay. At the end of studies, mice were euthanized using CO 2, tissues were harvest for histology immunohistochemical analysis and western blots analysis. .",
"section_name": "Chemopreventive study on Tgfbr1/Pten combined conditional knockout (2cKO) mice",
"section_num": null
},
{
"section_content": "For mouse phospho-RTK detection, we collected tissue of Tgfbr1/Pten 2cKO mouse tongue (n = 5), Tgfbr1/Pten 2cKO mouse tongue squamous cell carcinoma (n = 5), and their vehicles (Tgfbr1 flox/flox /Pten flox/flox tongue; n = 5) 6 weeks after the last oral tamoxifen dose. Antibody array was purchased from R&D system (proteome profiler mouse phospho-RTK array kit, ARY014). This array can detect the relative phosphorylation of 39 RTKs. Briefly, bovine serum albumin blocked the membrane containing immobilized phospho-RTK on a rocking platform at room temperature for 1 h. The membrane was then incubated with lysates of Tgfbr1/Pten 2cKO mouse tongue (n = 5), Tgfbr1/Pten 2cKO mouse tongue squamous cell carcinoma (n = 5), and their vehicles (Tgfbr1 flox/flox /Pten flox/flox tongue; n = 5) with Detection Antibody Cocktail overnight at 2°C to 8°C on a rocking platform. The membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Pierce Chemical, Rockford, IL) and then with chemiluminescent detection reagent. The membrane was scanned, and pixel density was presented by quantifying the mean spot densities from two experiments. For western blot, we collected tissue of Tgfbr1/Pten 2cKO mouse tongue (n = 2), Tgfbr1/Pten 2cKO mouse tongue squamous cell carcinoma (n = 5), and their vehicles (Tgfbr1 flox/flox /Pten flox/flox tongue; n = 2).",
"section_name": "Mouse phospho-Receptor Tyrosine Kinase (RTK) detection",
"section_num": null
},
{
"section_content": "HN803 tissue arrays which contain 10 cases of normal tongue mucosa, 4 cases of lymph node metastasis and 57 confirmed cases of HNSCC were obtained from Biomax US (Rockville, MD, USA). The tissue array clinical data, including pathological classification and TNM classification were also provided by Biomax.",
"section_name": "Human HNSCC tissues array",
"section_num": null
},
{
"section_content": "Antibodies against EGFR (1:50), p-EGFR Tyr1068 (1:200), HIF-1α, and Notch1, Hes1 (1:400) were stained in sections of xenograft samples and EGFR (1:50), HIF-1α, and Hes1 (1:400) were stained in sections of Tgfbr1/Pten 2cKO tongue SCC samples by immunohistochemistry. The methods and processes were described as previously reported [20]. CD31 were stained in both xenograft and Tgfbr1/Pten 2cKO tongue SCC samples by frozen section immunohistochemistry. All slices were scanned using an Aperio ScanScope CS scanner with background substrate for each slice, and quantified using Aperio Quantification software (Version 9. 1) for membrane, nuclear, or pixel quantification. Four random areas of interest were selected either in the epithelial or the cancerous area for scanning and quantification. Histoscore of membrane and nuclear staining was calculated as a percentage of different positive cells using the formula (3+)×3+(2+)×2+(1+)×1. Histoscore of pixel quantification was calculated as total intensity/ total cell number. The threshold for scanning of different positive cells was set according to the standard vehicles provided by Aperio.",
"section_name": "Histology, immunohistochemistry and scoring system",
"section_num": null
},
{
"section_content": "Western blot were performed as previously described [22] with detail in Supplementary Material and Methods in S1 File.",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "Graph Pad Prism version 5. 00 for Windows (Graph-Pad Software Inc) was used for data analyses. Student t tests were performed to analyze the differences between two groups. Two-way ANOVA analysis was used for analyzing differences between animal treatment results. Twotailed Pearson statistics were performed to correlate expression of EGFR with CD31, HIF-1α after confirmation of the sample with Gaussian distribution. All value was exhibited as Mean values ± SEM. P<0. 05 were considered statistically significant.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Tyrosine kinase dysregulation, overexpression and high activation are common phenomena in different cancers, including HNSCC. To examine the possible tyrosine kinase overexpression in the Tgfbr1/Pten 2cKO mouse model of HNSCC, we used a high-throughput antibody array with 39 RTKs to test the RTK expression of Tgfbr1/Pten 2cKO mouse tongue SCC in comparison with those of Tgfbr1/Pten 2cKO mouse tongue and Tgfbr1 flox/flox /Pten flox/flox tongue. Results revealed that the tyrosine kinases of EGFR, ErbB2, macrophage-stimulating protein receptor (MSPR) and platelet-derived growth factor receptor alpha (PDGFα) were highly expressed in Tgfbr1/Pten 2cKO mouse tongue SCC (Fig. 1A and 1B ). Particularly, EGFR overexpression seemed to be the predominant molecular event in mouse tongue SCC (Fig. 1A and 1B ). To confirm antibody array results, we used immunohistochemistry to directly observe the expression of EGFR in Tgfbr1/Pten 2cKO mouse tongue SCC. As shown in Fig. 1C, EGFR was almost negative in Tgfbr1 flox/flox /Pten flox/flox mucosa. The staining of EGFR in Tgfbr1/Pten 2cKO mouse HNSCC was even evidently stronger than that in in Tgfbr1/Pten 2cKO mouse mucosa (Fig. 1C ). The results from western blots analysis (Fig. 1D ) also validated this finding. More importantly, the activation of EGFR, p-EGFR Tyr1068 was much higher in Tgfbr1/Pten 2cKO mouse tongue SCC than that in the vehicle. Given that the mouse model was generated by conditionally knocking out Tgfbr1/Pten, we hypothesized that the expression levels of either EGFR or p-EGFR Tyr1068 increased after the knock down of TGFBR1 and PTEN, or both of them in vitro. The expression and activation of EGFR increased when the tongue cancer cells CAL27 were transfected with TGFBR1 and/or PTEN in siRNA (Fig. 1E ). These results strongly indicate that tyrosine kinase dysregulation, particularly EGFR, is an important molecular event in the Tgfbr1/Pten 2cKO mouse model of HNSCC carcinogenesis and the deletion of Tgfbr1 or Pten increased the expression and phosphorylation of total EGFR.",
"section_name": "High EGFR expression in the Tgfbr1/Pten 2cKO mouse model of HNSCC",
"section_num": null
},
{
"section_content": "We treated heterotopic xenograft tumors derived from CAL27 cells with cetuximab to further identify the possible function of EGFR in HNSCC development. The mice received the treatment at 21 d post implantation and were euthanized for Western blot and immunohistochemical analyses on day 42. Cetuximab significantly delayed tumor growth (Fig. 2A and 2B ). Fig. 2C showed the growth curves in tumors treated with cetuximab or vehicle, The mice administered with cetuximab showed partial tumor regression after 8 d of treatment., The cetuximab-treated mice showed significant tumor inhibition after 12 d of treatment (P < 0. 01) compared with the vehicle-treated group. We harvested and weighted the tumor at end point of experiment and results revealed cetuximab possessed antitumor activity because the tumor in the vehicle-treated group had significantly higher weight than those in the cetuximabtreated group (Fig. 2D ). The indicated dose of cetuximab exerted no significant toxicity to the mice because the mice weight between cetuximab-and vehicle-treated groups showed no significant difference (Fig. 2E ). These results demonstrated that EGFR blockade effectively prevented tumor growth.",
"section_name": "Cetuximab treatment of CAL27 heterotopic xenograft tumors",
"section_num": null
},
{
"section_content": "We performed a chemopreventive study on Tgfbr1/Pten 2cKO mice to determine whether or not an increase in EGFR was an early event in HNSCC tumorigenesis. We induced the onset of HNSCC tumor in Tgfbr1/Pten 2cKO mice as previously described [20]. The induction and drug administration strategies were shown Fig. 3A. Two weeks after the last tamoxifen oral gavages, the mice were treated with EGFR inhibitor or vehicle for 2 weeks. Cetuximab significantly (P < 0. 001, n = 6) delayed tumorigenesis in external head and neck (Fig. 3B with quantification in Fig. 3D ) and oral tongue tumors (Fig. 3C ) in Tgfbr1/Pten 2cKO mice as compared with the vehicle group (n = 5). No significant weight loss was observed, indicating that cetuximab exerted no significant toxicity to these immuno-sufficient mice (Fig. 3E ). These data indicated that EGFR blockade by cetuximab delayed the onset of HNSCC in 2cKO mice.",
"section_name": "Targeting EGFR by cetuximab delays HNSCC onset in Tgfbr1/Pten 2cKO mice",
"section_num": null
},
{
"section_content": "Digital pathology was performed to explore whether or not EGFR inhibition influences angiogenesis in 2cKO mice. Immunohistochemical staining showed that cetuximab downregulated EGFR, p-EGFR, and MVD in the xenograft tissues of CAL27 cells. Quantification of histoscore by using Aperio digital pathology validated the observation results. (S1A and S1B Fig. ). The inhibition of EGFR expression and phosphorylation was also confirmed by Western blot (S1C Fig. ). We collected the conditioned medium (CM) after pretreating CAL27 cells with cetuximab. We performed an in vitro migration assay to further confirm the function of cetuximab in angiogenesis in vitro. As shown in Fig. 4A, the CM from cetuximab-pretreated CAL27 cells reduced HUVEC migration as compared with the vehicle medium. Similar results were obtained in the Boyden transwell migration assay and tube formation assay under both hypoxic and normoxic culture conditions (Fig. 4B and 4C ). The findings exhibited that CM significantly decreased HUVEC migration and tube formation after cetuximab pretreatment under both normoxic and hypoxic conditions when compared with the negative vehicle (Fig. 4D ). Hypoxic culture conditions increased HUVEC migration as compared with normoxic culture conditions. The protein expression of HIF-1α and VEGFA were validated by western blots. Cetuximab reduced HIF-1α expression in normoxia and down-regulated VEGFA even in hypoxic condition (Fig. 4E ). We further confirmed that 24 h of treatment with 10 μg/ml cetuximab reduced HIF-1α nuclear translocation in CAL27 cells under hypoxic culture conditions (Fig. 4F ). To further detect the expression of HIF-1α, the protein levels expression of HIF-1α in the cytoplasmic and nuclear extracts were examined. As shown in Fig. 4G, cetuximab reduced expression HIF-1α in the nucleus in a concentration-dependent manner as compared with those in cells treated with vehicle. Aligned with this observation, cetuximab significantly inhibited HNSCC angiogenesis, and reduced HIF-1α nuclear translocation may be involved in this phenomenon.",
"section_name": "Cetuximab inhibits tumor-induced angiogenesis in vitro and in vivo",
"section_num": null
},
{
"section_content": "To further confirm whether Notch1 signaling pathway was involved in the preventive effect of cetuximab on tumor-induced angiogenesis, endothelial function assays were performed in the presence of DAPT, a widely used inhibitor for Notch1. As shown in Fig. 5A to 5C, the CM from cetuximab-or DAPT-pretreated CAL27 cells reduced HUVEC migration as compared with the vehicle medium using wound healing assays. HUVECs migration even were further inhibited when treated with the CM from Cetuximab combined with DAPT. Similar results were obtained in the Boyden transwell migration assays and tube formation assays (Fig. 5A to 5C). We next detected the expression of NICD, a cleaved fragment that transduced activated signals of Notch1, and VEGFA by western blots (Fig. 5D ). The results showed that the DAPT or cetuximab reduced the expression of NICD as well as VEGFA. More, cetuximab further reduced the expression of VEGFA even in the presence of DAPT, may suggesting other downstream molecule moderated VEGFA either. To explore the interaction between HIF-1α and Notch1, protein levels of HIF-1α and NICD were tested by western blots. And we found hypoxia up-regulated the activation of Notch1 consistent with the up-regulation of HIF-1α, while DAPT showed no effect on HIF-1α in hypoxia (Fig. 5F ), suggesting HIF-1α might play as upstream of Notch1 at least in CAL27 cell lines.",
"section_name": "Notch1 signaling pathway is involved in cetuximab-reduced angiogenesis in vitro",
"section_num": null
},
{
"section_content": "We next evaluated the immunoreactivity of EGFR to HIF-1α and CD31 in human tissue array to further assess the correlation of EGFR with HIF-1α and MVD in human HNSCC. Of 54 cases, 48 presented positive membrane staining in almost all epithelial tumor areas of HNSCC tissue; only 10% of the mucosa core showed staining, and this staining was limited in the basal layer (Fig. 6A ). Hypoxia is a common phenomenon in HNSCC. Intense HIF-1α nuclear staining was observed in a large proportion of tumor cells, suggesting hypoxia is a common phenomenon in HNSCC. The. staining of HIF-1α was considerably strong in invasive cancer. Most human HNSCC lesions were also highly angiogenic, as reflected by the strong staining of the vascular endothelial marker CD31 (Fig. 6A ). EGFR expression positively correlated with high expression levels of HIF-1α (P = 0. 0001, r = 0. 4192) and CD31 (P < 0. 0001, r = 0. 4296) (Fig. 6B ; statistic including normal mucosa and HNSCC, n = 71). These results further confirmed that increased EGFR expression was significantly associated with hypoxia and angiogenesis in HNSCC",
"section_name": "Increased EGFR expression is related to HIF-1α and MVD in human HNSCC tissue",
"section_num": null
},
{
"section_content": "We also examined the correlation of EGFR with Notch, another putative angiogenic molecule. Immunohistochemical staining showed that cetuximab treatment significantly reduced HIF-1α, Notch1, and Hes1 (putative downstream target of Notch1) (S2A and S2B Fig. Similar results were observed in 2cKO mouse HNSCC tissues, which are angiogenic and mimic human HNSCC in histological and molecule-expression patterns. Compared with the vehicle group (n = 7 from 5 mice), the residual cetuximab-treated HNSCC (n = 9 from 6 mice) showed downregulated HIF-1α, Hes1, EGFR, and CD31 expression (P < 0. 001, Fig. 7A with quantification in Fig. 7B ). The inhibition of EGFR expression and activation, HIF-1α, Hes1, VEGFA were also confirmed by western blots (Fig. 7C ). These data further demonstrated that cetuximab downregulated tumor-induced angiogenesis in the 2cKO mouse model of HNSCC by inhibiting the HIF-1α and Notch1 pathways.",
"section_name": "Cetuximab inhibits tumor-induced angiogenesis by downregulating HIF-1α and Notch1",
"section_num": null
},
{
"section_content": "Understanding the molecular mechanisms underlying HNSCC initiation and tumor evolution is important to delay tumor progression. Among the signaling events in HNSCC, the persistent overexpression and activation of EGFR have emerged as putative drug targets for HNSCC treatment in preclinical and clinical investigations [23] [24] [25]. EGFR inhibitors, including cetuximab and lapatinab, can dramatically reduce tumor burden in HNSCC animal models [26] or patients [11] In the present study, the EGFR pathway is frequently activated in Tgfbr1/Pten 2cKO mice. EGFR overexpression may be related with Tgfbr1 and Pten downregulation. We assessed EGFR inhibition and angiogenesis in xenograft and transgenic mouse models of HNSCC. Results showed that EGFR inhibition with cetuximab can reduce tumor growth and angiogenesis in HNSCC. \n\nStroma and immune cells serve important functions in tumor angiogenesis [27]. Thus, the implantation of human HNSCC cells in immunodeficient mice may not completely reflect the clinical situation and may not accurately evaluate the efficacy of the drug on HNSCC angiogenesis [28]. Tgfbr1/Pten 2cKO mice are characterized by 100% penetrance; in addition, they mimic human HNSCC with similar morphology and molecular alteration. Therefore, we analyzed the effect of EGFR on angiogenesis using this mouse model. Results showed that EGFR inhibitors at clinically relevant doses can reduce the regulation of HIF-1α and Notch1 in this tumor type with limited side effects. This phenomenon resulted in reduced angiogenesis and tumor shrinkage. \n\nIn previous studies, we proved that the angiogenesis in 2cKO mouse HNSCC is related to HIF-1α activation by miR-135b [19]. Herein, the blockade of EGFR in this experiment rapidly decreased HIF-1α, a hypoxic biomarker frequently observed in advanced-stage HNSCC [29]. This effect likely involves the impact of cetuximab on angiogenesis by reducing HIF-1α nuclear translocation and/or reducing migration and chemoattractants, such as vascular endothelial growth factor A (VEGFA), for endothelial cells. This phenomenon prevents angiogenic signaling. The Notch signaling pathway is involved in the regulation of stem cell and neuronal cell death [30, 31]. However, recent evidence has shown that the Notch signaling pathway serves an important function during blood vessel formation and remodeling [32]. The Notch signaling pathway is involved in endothelial cell biology; it influences the budding of endothelial tip cells during angiogenesis initiation [33]. Notch1 was confirmed to be regulated by HIF-1α in a culture cell system [34]. Notch blockade can abolish the tumor resistance of glioblastoma to VEGF inhibitors [35, 36]. Blocking both Dll4/Notch and VEGF pathways synergistically inhibits tumor growth, which indicates the potential application of Notch inhibitors as new adjuvant chemotherapy reagents [37]. Dll4/Notch transcription was activated by Erk and PI3K signaling pathways, which were also downstream of canonical EGFR transduction [38]. Notch1 downregulation also reduced VEGF expression [39]. Thus, we hypothesized that cetuximab can decrease VEGF production and reduce HNSCC tumor angiogenesis by inhibiting the Notch signaling pathway. The present results showed that cetuximab inhibited the Notch1 signaling pathway by decreasing Notch1, Hes1, and VEGF expression in both nude mouse xenograft and 2cKO mouse models. Although these possibilities remain to be proven, the present findings support a unique anti-angiogenic function of cetuximab. That is, cetuximab can exert its antitumor activity by decreasing primary tumor growth and size, reducing HIF-1α instability, preventing endothelial cell initiation and migration, and downregulating VEGFA. These phenomena lead to the prevention of HNSCC angiogenesis. \n\nHigh HIF-1α expression in HNSCC tissue is an important factor that predicts poor prognosis and resistance to chemotherapy and/or radiotherapy. The clinical application of EGFR as a molecular target of HNSCC therapy is a revolutionary event. However, the radiosensitization mechanism of cetuximab, a new adjuvant chemo-radiotherapy of HNSCC, still warrants further investigation. The emerging preclinical and clinical information about the promising beneficial angiogenetic effects of cetuximab on HNSCCs and our present findings on the capacity of cetuximab to downregulate Notch1 and HIF-1α signaling benefit HNSCC therapy. We can envision that the present study and prior reports may provide a rationale for the future clinical evaluation of cetuximab in an adjuvant setting, as a part of a molecular-targeted strategy after definitive treatment.",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "We thank Dr. Ashok B. Kulkarni of LCDB, National Institute of Dental and Craniofacial Research, NIH, USA for gift Tgfbr1/Pten 2cKO mice. We thank EssayStar for its linguistic assistance during the preparation of this manuscript.",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This work was funded by National Natural Science Foundation of China ( 81072203, 81272963 ) to Z. -J. Sun, ( 81371106 ) to L. Zhang, ( 81272946 ) to W. -F. Zhang, and ( 81170977 ) to Y. -F. Zhao. Z. -J. Sun is supported by program for new century excellent talents in university ( NCET-13-0439 ), ministry of education of China. Support also came from National Natural Science Foundation of China ( 81472528 ) to Z. -J. Sun. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.",
"section_name": "",
"section_num": ""
},
{
"section_content": "All relevant data are within the paper and its Supporting Information files.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Supporting Information",
"section_num": null
}
] |
10.1371/journal.pone.0247717
|
Subcutaneous immunoglobulins replacement therapy in secondary antibody deficiencies: Real life evidence as compared to primary antibody deficiencies
|
<jats:p>Secondary antibody deficiencies (SAD) may require immunoglobulin replacement therapy (IgRT). While the intravenous route (IVIG) is broadly considered effective in SAD, the use of subcutaneous immunoglobulins (SCIG) is mainly adopted from the experience in primary antibody deficiencies (PAD), where SCIG have been shown to perform as effective as IVIG. However, evidence-based data on SCIG administration in SAD patients are still insufficient. Herein we retrospectively evaluated the efficacy and safety profile of SCIG treatment in 131 SAD patients as compared to a group of 102 PAD patients. We found SCIG being equally effective in reducing annual infectious rate both in SAD and PAD patients. However, SAD patients required lower SCIG dosage and lower IgG through level to achieve similar biological effect in terms of infection burden, at the steady state. SAD patients also showed better correlation between SCIG dose and serum IgG achieved value. Furthermore, within SAD, SCIG were found to work irrespective of the underlying disease. Especially in Non-Hodgkin Lymphoma patients, whose indication to IgRT is still not included in all guidelines and for whom evidence-based data are still lacking, SCIG were as effective as in Chronic Lymphocytic Leukemia or Multiple Myeloma patients, and SCIG discontinuation, without evidence of B cell recovery, led to IgG decline and relapsed infections. Finally, treatment tolerance in SAD patients was comparable to the PAD cohort. Globally, our data suggest that SCIG, as already appreciated in PAD, represent a valuable option in SAD patients, independent on the disease leading to antibody deficiency.</jats:p>
|
[
{
"section_content": "The purpose of this core SmPC is to provide applicants and regulators with harmonised guidance on the information to be included in the summary of product characteristics (SmPC) for a human normal immunoglobulin for intravenous administration (IVIg). This guideline should be read in conjunction with the Guideline on the clinical investigation of human normal immunoglobulin for intravenous administration (IVIg) (EMA/CHMP/BPWP/94033/2007 rev. 2). For guidance on the clinical investigation of subcutaneous immunoglobulin products refer to CHMP/BPWP/410415/2011 Rev 1and the coreSPC CPMP/BPWG/143744/2011 Rev. 1. \n\nThe Quality Review of Documents (QRD) product information template with explanatory notes (′QRD annotated template') 1 and the convention to be followed for QRD templates 2 provide general guidance on format and text and should be read in conjunction with the core SmPC and the Guideline on summary of product characteristics 3. \n\nIt is necessary to provide information for health professionals on posology and method of administration at the end of the package leaflet. See the QRD annotated template for further guidance on how to present such information. \n\nThis core SmPC has been prepared on the basis of SmPCs of authorised medicinal products, and taking into account the published scientific literature. Any marketing authorisation application or variation of a marketing authorisation for <a human normal immunoglobulin> should be accompanied by the required data particulars, documents, literature and/or justification for the application to be valid. \n\nIn addition, for the content of sections 4.",
"section_name": "Introduction (background)",
"section_num": "1."
},
{
"section_content": "This core SmPC covers human normal immunoglobulin for intravenous administration defined by the European Pharmacopoeia monograph 0918. It does not apply to products intentionally prepared to contain fragments or chemically modified IgG. The maximum IgA content is {x} micrograms/ml.",
"section_name": "Scope",
"section_num": "2."
},
{
"section_content": "Produced from the plasma of human donors.",
"section_name": "Legal basis",
"section_num": "3."
},
{
"section_content": "For a full list of excipients, see section 6. 1.",
"section_name": "<Excipient(s) with known effect:>",
"section_num": null
},
{
"section_content": "[Product specific]",
"section_name": "PHARMACEUTICAL FORM",
"section_num": "3."
},
{
"section_content": "",
"section_name": "CLINICAL PARTICULARS",
"section_num": "4."
},
{
"section_content": "Therapeutic indications [Age ranges given in this section may require modification if there are any safety issues for the excipients used for a particular product e. g. sorbitol risk for babies and young children with hereditary fructose intolerance. ] Replacement therapy in adults, and children and adolescents (0-18 years) in:\n\n• Primary immunodeficiency syndromes (PID) with impaired antibody production * PSAF= failure to mount at least a 2-fold rise in IgG antibody titre to pneumococcal polysaccharide and polypeptide antigen vaccines Immunomodulation in adults, and children and adolescents (0-18 years) in:\n\n• Primary immune thrombocytopenia (ITP), in patients at high risk of bleeding or prior to surgery to correct the platelet count • Guillain Barré syndrome • Kawasaki disease (in conjunction with acetylsalicylic acid; see 4. 2) • Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) • Multifocal motor neuropathy (MMN)",
"section_name": "4.1",
"section_num": null
},
{
"section_content": "Replacement therapy should be initiated and monitored under the supervision of a physician experienced in the treatment of immunodeficiency.",
"section_name": "Posology and method of administration",
"section_num": "4.2"
},
{
"section_content": "The dose and dose regimen is dependent on the indication. \n\nThe dose may need to be individualised for each patient dependent on the clinical response. Dose based on bodyweight may require adjustment in underweight or overweight patients. [Product specific specify recommendation]. \n\nThe following dose regimens are given as a guideline.",
"section_name": "Posology",
"section_num": null
},
{
"section_content": "The dose regimen should achieve a trough level of IgG (measured before the next infusion) of at least 6 g/ L or within the normal reference range for the population age. Three to six months are required after the initiation of therapy for equilibration (steady-state IgG levels) to occur. The recommended starting dose is 0. 4-0. 8 g/kg given once followed by at least 0. 2g/kg given every three to four weeks. \n\nThe dose required to achieve a trough level of IgG of 6 g/L is of the order of 0. 2-0. 8 g/kg/month. The dosage interval when steady state has been reached varies from 3-4 weeks. \n\nIgG trough levels should be measured and assessed in conjunction with the incidence of infection. To reduce the rate of bacterial infections, it may be necessary to increase the dosage and aim for higher trough levels.",
"section_name": "Replacement therapy in primary immunodeficiency syndromes",
"section_num": null
},
{
"section_content": "The recommended dose is 0. 2-0. 4 g/kg every three to four weeks. \n\nIgG trough levels should be measured and assessed in conjunction with the incidence of infection. Dose should be adjusted as necessary to achieve optimal protection against infections, an increase may be necessary in patients with persisting infection; a dose decrease can be considered when the patient remains infection free.",
"section_name": "Secondary immunodeficiencies (as defined in 4.1.)",
"section_num": null
},
{
"section_content": "There are two alternative treatment schedules:\n\n• 0. 8-1g/kg given on day one; this dose may be repeated once within 3 days. \n\n• 0. 4 g/kg given daily for two to five days. The treatment can be repeated if relapse occurs. \n\nGuillain Barré syndrome 0. 4 g/kg/day over 5 days (possible repeat of dosing in case of relapse). Kawasaki Disease 2. 0 g/kg should be administered as a single dose. Patients should receive concomitant treatment with acetylsalicylic acid.",
"section_name": "Primary immune thrombocytopenia",
"section_num": null
},
{
"section_content": "Starting dose: 2 g/kg divided over 2 -5 consecutive days Maintenance doses: 1 g/kg over 1-2 consecutive days every 3 weeks. \n\nThe treatment effect should be evaluated after each cycle; if no treatment effect is seen after 6 months, the treatment should be discontinued. \n\nIf the treatment is effective long term treatment should be subject to the physicians discretion based upon the patient response and maintenance response. The dosing and intervals may have to be adapted according to the individual course of the disease.",
"section_name": "Chronic inflammatory demyelinating polyneuropathy (CIDP)",
"section_num": null
},
{
"section_content": "Starting dose: 2 g/kg given over 2-5 consecutive days. Maintenance dose: 1 g/kg every 2 to 4 weeks or 2 g/kg every 4 to 8 weeks. \n\nThe treatment effect should be evaluated after each cycle; if no treatment effect is seen after 6 months, the treatment should be discontinued. \n\nIf the treatment is effective long term treatment should be subject to the physicians discretion based upon the patient response and maintenance response. The dosing and intervals may have to be adapted according to the individual course of the disease.",
"section_name": "Multifocal Motor Neuropathy (MMN)",
"section_num": null
},
{
"section_content": "The posology in children and adolescents (0-18 years) is not different to that of adults as the posology for each indication is given by body weight and adjusted to the clinical outcome of the above mentioned conditions.",
"section_name": "Paediatric population",
"section_num": null
},
{
"section_content": "No evidence is available to require a dose adjustment.",
"section_name": "Hepatic impairment",
"section_num": null
},
{
"section_content": "No dose adjustment unless clinically warranted, see section 4. 4.",
"section_name": "Renal impairment",
"section_num": null
},
{
"section_content": "No dose adjustment unless clinically warranted, see section4. 4.",
"section_name": "Elderly",
"section_num": null
},
{
"section_content": "For intravenous use. \n\nHuman normal immunoglobulin should be infused intravenously at an initial rate of {indicate product specific rate} ml/kg/hr for {indicate product specific infusion time} hr. See section 4. 4. In case of adverse reaction, either the rate of administration must be reduced or the infusion stopped. If well tolerated, the rate of administration may gradually be increased to a maximum of {indicate product specific increased rate} ml/kg/hr.",
"section_name": "Method of administration",
"section_num": null
},
{
"section_content": "Hypersensitivity to the active substance (human immunoglobulins) or to any of the excipients (see sections 4. 4 and 6. 1). [Product specific contraindications]. \n\nPatients with selective IgA deficiency who developed antibodies to IgA, as administering an IgAcontaining product can result in anaphylaxis.",
"section_name": "Contraindications",
"section_num": "4.3"
},
{
"section_content": "In order to improve the traceability of biological medicinal products, the name and the batch number of the administered product should be clearly recorded.",
"section_name": "Special warnings and precautions for use [In addition to the text below, include any additional product specific precautions and warnings (e.g. those relating to excipients present in the product).] Traceability",
"section_num": "4.4"
},
{
"section_content": "Potential complications can often be avoided by ensuring that patients:\n\n• are not sensitive to human normal immunoglobulin by initially injecting the product slowly ({specify the product specific rate} ml/kg/min) • are carefully monitored for any symptoms throughout the infusion period. In particular, patients naive to human normal immunoglobulin, patients switched from an alternative IVIg product or when there has been a long interval since the previous infusion should be monitored at the hospital during the first infusion and for the first hour after the first infusion, in order to detect potential adverse signs. All other patients should be observed for at least 20 minutes after administration. \n\nIn all patients, IVIg administration requires:\n\n• adequate hydration prior to the initiation of the infusion of IVIg • monitoring of urine output • monitoring of serum creatinine levels • avoidance of concomitant use of loop diuretics (see 4. 5). \n\nIn case of adverse reaction, either the rate of administration must be reduced or the infusion stopped. The treatment required depends on the nature and severity of the adverse reaction.",
"section_name": "Precautions for use",
"section_num": null
},
{
"section_content": "Certain adverse reactions (e. g. headache, flushing, chills, myalgia, wheezing, tachycardia, lower back pain, nausea, and hypotension) may be related to the rate of infusion. The recommended infusion rate given under section 4. 2 must be closely followed. Patients must be closely monitored and carefully observed for any symptoms throughout the infusion period. \n\nAdverse reactions may occur more frequently • in patients who receive human normal immunoglobulin for the first time or, in rare cases, when the human normal immunoglobulin product is switched or when there has been a long interval since the previous infusion • in patients with an untreated infection or underlying chronic inflammation Hypersensitivity Hypersensitivity reactions are rare.",
"section_name": "Infusion reaction",
"section_num": null
},
{
"section_content": "• with undetectable IgA who have anti-IgA antibodies • who had tolerated previous treatment with human normal immunoglobulin\n\nIn case of shock, standard medical treatment for shock should be implemented.",
"section_name": "Anaphylaxis can develop in patients",
"section_num": null
},
{
"section_content": "There is clinical evidence of an association between IVIg administration and thromboembolic events such as myocardial infarction, cerebral vascular accident (including stroke), pulmonary embolism and deep vein thromboses which is assumed to be related to a relative increase in blood viscosity through the high influx of immunoglobulin in at-risk patients. Caution should be exercised in prescribing and infusing IVIg in obese patients and in patients with pre-existing risk factors for thrombotic events (such as advanced age, hypertension, diabetes mellitus and a history of vascular disease or thrombotic episodes, patients with acquired or inherited thrombophilic disorders, patients with prolonged periods of immobilisation, severely hypovolaemic patients, patients with diseases which increase blood viscosity). \n\nIn patients at risk for thromboembolic adverse reactions, IVIg products should be administered at the minimum rate of infusion and dose practicable.",
"section_name": "Thromboembolism",
"section_num": null
},
{
"section_content": "Cases of acute renal failure have been reported in patients receiving IVIg therapy. In most cases, risk factors have been identified, such as pre-existing renal insufficiency, diabetes mellitus, hypovolaemia, overweight, concomitant nephrotoxic medicinal products or age over 65. \n\nRenal parameters should be assessed prior to infusion of IVIG, particularly in patients judged to have a potential increased risk for developing acute renal failure, and again at appropriate intervals. In patients at risk for acute renal failure, IVIg products should be administered at the minimum rate of infusion and dose practicable. In case of renal impairment, IVIg discontinuation should be considered. \n\nWhile reports of renal dysfunction and acute renal failure have been associated with the use of many of the licensed IVIg products containing various excipients such as sucrose, glucose and maltose, those containing sucrose as a stabiliser accounted for a disproportionate share of the total number. In patients at risk, the use of IVIg products that do not contain these excipients may be considered. <{(Invented) name} contains <sucrose><maltose><glucose>. (See excipients above)> <{(Invented) name} does not contain sucrose, maltose or glucose. > Aseptic meningitis syndrome (AMS)\n\nAseptic meningitis syndrome has been reported to occur in association with IVIg treatment. The syndrome usually begins within several hours to 2 days following IVIg treatment. Cerebrospinal fluid studies are frequently positive with pleocytosis up to several thousand cells per mm 3, predominantly from the granulocytic series, and elevated protein levels up to several hundred mg/dl. AMS may occur more frequently in association with high-dose (2 g/kg) IVIg treatment. \n\nPatients exhibiting such signs and symptoms should receive a thorough neurological examination, including CSF studies, to rule out other causes of meningitis. \n\nDiscontinuation of IVIg treatment has resulted in remission of AMS within several days without sequelae.",
"section_name": "Acute renal failure",
"section_num": null
},
{
"section_content": "IVIg products can contain blood group antibodies which may act as haemolysins and induce in vivo coating of red blood cells with immunoglobulin, causing a positive direct antiglobulin reaction (Coombs' test) and, rarely, haemolysis. Haemolytic anaemia can develop subsequent to IVIg therapy due to enhanced red blood cells (RBC) sequestration. IVIg recipients should be monitored for clinical signs and symptoms of haemolysis. (See section 4. 8. ). \n\n<Neutropenia/Leukopenia> A transient decrease in neutrophil count and/or episodes of neutropenia, sometimes severe, have been reported after treatment with IVIgs. This typically occurs within hours or days after IVIg administration and resolves spontaneously within 7 to 14 days. \n\nTransfusion related acute lung injury (TRALI)\n\nIn patients receiving IVIg, there have been some reports of acute non-cardiogenic pulmonary oedema [Transfusion Related Acute Lung Injury (TRALI)]. TRALI is characterised by severe hypoxia, dyspnoea, tachypnoea, cyanosis, fever and hypotension. Symptoms of TRALI typically develop during or within 6 hours of a transfusion, often within 1-2 hours. Therefore, IVIg recipients must be monitored for and IVIg infusion must be immediately stopped in case of pulmonary adverse reactions. TRALI is a potentially lifethreatening condition requiring immediate intensive-care-unit management.",
"section_name": "Haemolytic anaemia",
"section_num": null
},
{
"section_content": "After the administration of immunoglobulin the transitory rise of the various passively transferred antibodies in the patient's blood may result in misleading positive results in serological testing. \n\nPassive transmission of antibodies to erythrocyte antigens, e. g. A, B, D may interfere with some serological tests for red cell antibodies for example the direct antiglobulin test (DAT, direct Coombs' test).",
"section_name": "Interference with serological testing",
"section_num": null
},
{
"section_content": "Live attenuated virus vaccines Immunoglobulin administration may impair for a period of at least 6 weeks and up to 3 months the efficacy of live attenuated virus vaccines such as measles, rubella, mumps and varicella. After administration of this medicinal product, an interval of 3 months should elapse before vaccination with live attenuated virus vaccines. In the case of measles, this impairment may persist for up to 1 year. Therefore patients receiving measles vaccine should have their antibody status checked.",
"section_name": "Interactions with other medicinal products and other forms of interaction",
"section_num": "4.5"
},
{
"section_content": "Paediatric population\n\n<The listed interactions apply both to adults and children. >",
"section_name": "Loop diuretics Avoidance of concomitant use of loop diuretics",
"section_num": null
},
{
"section_content": "",
"section_name": "Fertility, pregnancy and lactation",
"section_num": "4.6"
},
{
"section_content": "The safety of this medicinal product for use in human pregnancy has not been established in controlled clinical trials and therefore should only be given with caution to pregnant women and breast-feeding mothers. IVIg products have been shown to cross the placenta, increasingly during the third trimester.",
"section_name": "Pregnancy",
"section_num": null
},
{
"section_content": "According to the sequence:",
"section_name": "Class (SOC)",
"section_num": null
},
{
"section_content": "Overdose may lead to fluid overload and hyperviscosity, particularly in patients at risk, including elderly patients or patients with cardiac or renal impairment (see section 4. 4. ).",
"section_name": "Overdose",
"section_num": "4.9"
},
{
"section_content": "",
"section_name": "PHARMACOLOGICAL PROPERTIES",
"section_num": "5."
},
{
"section_content": "Pharmacotherapeutic group: immune sera and immunoglobulins: immunoglobulins, normal human, for intravascular administration, ATC code: J06BA02 Human normal immunoglobulin contains mainly immunoglobulin G (IgG) with a broad spectrum of antibodies against infectious agents. \n\nHuman normal immunoglobulin contains the IgG antibodies present in the normal population. It is usually prepared from pooled plasma from not fewer than 1000 donations. It has a distribution of immunoglobulin G subclasses closely proportional to that in native human plasma. Adequate doses of this medicinal product may restore abnormally low immunoglobulin G levels to the normal range. \n\nThe mechanism of action in indications other than replacement therapy is not fully elucidated. \n\n[Product specific: Clinical study results can be briefly summarised here]\n\nPaediatric population\n\n[Product specific: The text should be in line with the Paediatric Regulation and the SmPC guideline. In case of a full waiver or any deferral, include the standard statement in the SmPC guideline. ]",
"section_name": "Pharmacodynamic properties",
"section_num": "5.1"
},
{
"section_content": "",
"section_name": "Pharmacokinetic properties",
"section_num": "5.2"
}
] |
[
{
"section_content": "EMA/CHMP/BPWP/94038/2007 replaced guideline on core SmPC for human normal immunoglobulin for intravenous administration (IVIg) with reference number CPMP/BPWP/859/95.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Breast-feeding",
"section_num": null
},
{
"section_content": "Clinical experience with immunoglobulins suggests that no harmful effects on the course of pregnancy, or on the foetus and the neonate are expected.",
"section_name": "",
"section_num": ""
},
{
"section_content": "Immunoglobulins are excreted into human milk. No negative effects on the breastfed newborns/infants are anticipated Fertility Clinical experience with immunoglobulins suggests that no harmful effects on fertility are to be expected. \n\n[Any relevant product specific information should be added. ]",
"section_name": "Breast-feeding",
"section_num": null
},
{
"section_content": "Effects on ability to drive and use machines <{Invented name} has <no or negligible influence> <minor influence> <moderate influence> <major influence> on the ability to drive and use machines. > [describe effects where applicable. ]",
"section_name": "4.7",
"section_num": null
},
{
"section_content": "",
"section_name": "Undesirable effects",
"section_num": "4.8"
},
{
"section_content": "Adverse reactions caused by human normal immunoglobulins (in decreasing frequency) encompass (see also Section 4. 4):\n\n• chills, headache, dizziness, fever, vomiting, allergic reactions, nausea, arthralgia, low blood pressure and moderate low back pain • reversible haemolytic reactions; especially in those patients with blood groups A, B, and AB and (rarely) haemolytic anaemia requiring transfusion • (rarely) a sudden fall in blood pressure and, in isolated cases, anaphylactic shock, even when the patient has shown no hypersensitivity to previous administration • (rarely) transient cutaneous reactions (including cutaneous lupus erythematosus -frequency unknown) • (very rarely) thromboembolic reactions such as myocardial infarction, stroke, pulmonary embolism, deep vein thromboses • cases of reversible aseptic meningitis • cases of increased serum creatinine level and/or occurrence of acute renal failure • cases of Transfusion Related Acute Lung Injury (TRALI) Tabulated list of adverse reactions The table presented below is according to the MedDRA system organ classification (SOC and Preferred Term Level). \n\nFrequencies have been evaluated according to the following convention: very common (≥1/10); common (≥1/100 to <1/10); uncommon (≥1/1,000 to <1/100); rare (≥1/10,000 to <1/1,000); very rare (<1/10,000), not known (cannot be estimated from the available data). <Within each frequency grouping, adverse reactions are presented in order of decreasing seriousness. > Source of the safety database (e. g. from clinical trials, post-authorisation safety studies and/or spontaneous reporting)",
"section_name": "Summary of the safety profile",
"section_num": null
},
{
"section_content": "Human normal immunoglobulin is immediately and completely bioavailable in the recipient's circulation after intravenous administration. It is distributed relatively rapidly between plasma and extravascular fluid, after approximately 3-5 days equilibrium is reached between the intra-and extravascular compartments. \n\nHuman normal immunoglobulin has a half-life of about {insert product specific half-life} days. This halflife may vary from patient to patient, in particular in primary immunodeficiency. \n\nIgG and IgG-complexes are broken down in cells of the reticuloendothelial system. \n\nPaediatric population",
"section_name": "MedDRA System Organ Adverse Frequency Frequency",
"section_num": null
},
{
"section_content": "In the absence of compatibility studies, this medicinal product must not be mixed with other medicinal products, nor with any other IVIg products. \n\n[Product specific]",
"section_name": "Incompatibilities",
"section_num": "6.2"
},
{
"section_content": "[Product specific: reference should be made to the SmPC guideline for stability at different temporary storage conditions. ]",
"section_name": "Shelf-life",
"section_num": "6.3"
},
{
"section_content": "Special precautions for storage",
"section_name": "6.4",
"section_num": null
}
] |
10.1007/s12185-022-03414-9
|
ASXL1 mutations with serum EPO levels predict poor response to darbepoetin alfa in lower-risk MDS: W-JHS MDS01 trial
|
<jats:title>Abstract</jats:title><jats:p>Darbepoetin alfa (DA) is used to treat anemia in lower-risk (IPSS low or int-1) myelodysplastic syndromes (MDS). However, whether mutations can predict the effectiveness of DA has not been examined. The present study aimed to determine predictive gene mutations. The primary endpoint was a correlation between the presence of highly frequent (≥ 10%) mutations and hematological improvement-erythroid according to IWG criteria 2006 by DA (240 μg/week) until week 16. The study included 79 patients (age 29–90, median 77.0 years; 52 [65.8%] male). Frequently (≥ 10%) mutated genes were <jats:italic>SF3B1</jats:italic> (24 cases, 30.4%), <jats:italic>TET2</jats:italic> (20, 25.3%), <jats:italic>SRSF2</jats:italic> (10, 12.7%), <jats:italic>ASXL1</jats:italic> (9, 11.4%), and <jats:italic>DNMT3A</jats:italic> (8, 10.1%). Overall response rate to DA was 70.9%. Multivariable analysis including baseline erythropoietin levels and red blood cell transfusion volumes as variables revealed that erythropoietin levels and mutations of <jats:italic>ASXL1</jats:italic> gene were significantly associated with worse response (odds ratio 0.146, 95% confidence interval 0.042–0.503; <jats:italic>p</jats:italic> = 0.0023, odds ratio 0.175, 95% confidence interval 0.033–0.928; <jats:italic>p</jats:italic> = 0.0406, respectively). This study indicated that anemic patients who have higher erythropoietin levels and harbor <jats:italic>ASXL1</jats:italic> gene mutations may respond poorly to DA. Alternative strategies are needed for the treatment of anemia in this population. Trial registration number and date of registration: UMIN000022185 and 09/05/2016.</jats:p>
|
[
{
"section_content": "Myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized by ineffective hematopoiesis, and occasionally progress to acute myelogenous leukemia (AML) [1, 2]. The pathogenesis of MDS is thought to be a multistep process involving two or more genetic alterations that cause clonal proliferation of an abnormal stem cell [3, 4]. Our understanding of the molecular pathogenesis of MDS has improved in recent years, mainly through the identification of major mutational targets [5] [6] [7] [8] [9]. The majority of patients with lower-risk MDS of International Prognostic Scoring System (IPSS) [10] low or intermediate-1 risk have symptoms of anemia due to ineffective erythropoiesis [11], and need therapeutic intervention, including red blood cell (RBC) transfusion. Although RBC transfusions can temporarily reduce anemic symptoms, frequent transfusions usually lead to iron overload, which is associated with reduced survival and lower quality of life [12]. \n\nOn the other hand, the development of erythropoiesis in the fetal liver and adult bone marrow is regulated by the hormone erythropoietin (EPO) [13]. Therefore, recombinant human EPO (rHuEPO) and other erythropoiesisstimulating agents (ESAs) are used for the treatment of MDS-related anemia [14]. In Japan, darbepoetin alfa (DA), which is a re-engineered form of EPO, has been approved for clinical practice. Around 50% of the patients respond to EPO ± granulocyte-colony stimulating factors (G-CSF), and the median duration of response is 2 years [15, 16]. DA has also been reported to show an overall response rate of about 60% when employed for the treatment of anemia in lower-risk MDS [17]. \n\nPrediction of response to ESAs in anemic MDS patients is often based on clinical biomarkers such as volume of RBC transfusion, serum EPO levels, ferritin, and IPSS/ IPSS-R [18], and the use of Nordic score combined with serum EPO levels and transfusion volume has also been proposed [19]. However, the relationship between molecular pathogenesis of MDS and responsiveness to DA has not yet been well studied. Here we report the results of the West Japan Hematology Study Group (W-JHS) MDS01 trial, to determine gene mutations that predict the effectiveness of DA in treating anemia of lower-risk (low or int-1 in IPSS [10] risk category) MDS.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Patients with lower-risk MDS (low or int-1 in IPSS [10] category) were registered in the W-JHS MDS01 study (UMIN000022185) between February 2016 and May 2019 at 36 institutions in Japan. Other eligibility criteria were as follows: newly diagnosed patients with definite MDS based on diagnostic criteria of FAB classification [20] and the 4th edition of World Health Organization (WHO) classification [21] ; patients having anemia associated with MDS, clinically eligible for DA treatment and of age 16 years or older. Patients with present or past medical record of myocardial infarction, pulmonary infarction, cerebral infarction or similar disorders or risk of thromboembolism, uncontrollable hypertension, prior treatment with DA or other formulations of EPO, severe (requiring hospital care or judged by investigators) or uncontrollable complication, and those judged inappropriate for study participation due to complication of mental disease or psychiatric symptom and cognitive disorder were excluded. This study is registered at the University Hospital Medical Information Network Clinical Trials Registry on 09/05/2016, with ID No. 000022185.",
"section_name": "Patient eligibility",
"section_num": null
},
{
"section_content": "DA at a dose of 240 µg per body was administered once weekly for 16 weeks. Analysis was performed to confirm whether the presence of a specific gene mutation with a frequency of ≥ 10% affects the efficacy of DA. Peripheral blood sample of the subjects was collected before administration of DA, and genomic DNA was extracted. The presence of gene mutations was then analyzed with a panel of 376 genes in a previous report [6] using a nextgeneration sequencing method. In brief, 376 known target genes in MDS were examined for mutations in 79 patients from the cohort, using massively parallel sequencing (Illumina, Inc., San Diego, CA, USA) of SureSelect (Agilent Technologies Inc., Santa Clara, CA, USA)-captured target sequences. All sequencing data were analyzed using our in-house pipeline [22], through which highly probable oncogenic mutations were called by eliminating sequencing/mapping errors using an empirical Bayesian approach [23] and known/possible SNPs based on the available databases.",
"section_name": "Procedure",
"section_num": null
},
{
"section_content": "The primary endpoint was a correlation between highly frequent (≥ 10%) gene mutations and hematological improvement-erythroid (HI-E) according to the International Working Group (IWG) criteria 2006 [24] at week 16 after the initiation of DA treatment. Secondary endpoints were major and minor responses to DA at week 16 after the initiation of treatment in blood transfusion-dependent subjects, HI-E in blood transfusion-independent and dependent subjects, variety and frequency of gene mutations observed in all subjects, the correlation between highly frequent gene mutations and interval to achievement of the first HI-E according to the IWG criteria 2006 [24], and mortality (overall survival, OS) and progression to AML (progression-free survival, PFS) from 16 weeks to 1 year after the initiation of treatment. \n\nThe IWG criteria 2006 (HI-E) were used, defined as either hemoglobin increased by 1. 5 g/dL or more compared to pre-treatment (< 11. 0 g/dL) or RBC transfusion volume/8 weeks decreased by more than 4 units in RBC transfusion-dependent subjects. In Japan, one unit of red blood cell preparation is produced from 200 ml of whole blood. As for transfusion-dependent subjects, the major response was defined as no need for RBC transfusion (withdrawal from RBC transfusion dependence) for more than 56 consecutive days, and increase in the highest Hb concentration during the withdrawal period by at least 1. 0 g/dL compared to the baseline Hb concentration, while the minor response was defined as ≥ 50% decrease in RBC transfusion volume over 56 consecutive days compared to baseline transfusion volume. For AML progression, progression to AML or death without progression were stated as events, and the subjects without confirmed progression to AML were censored at the date of the last known survival. For overall survival (OS), death from any cause was considered an event, and for the survival cases, the study was terminated at the date of the last known survival.",
"section_name": "Assessment of response",
"section_num": null
},
{
"section_content": "All adverse events (AEs) were recorded in subjects who received DA at least once from the first administration to day 29 of the last cycle, and classified according to the Common Terminology Criteria for Adverse Events Version 4. 0 [25]. For the subjects who dropped out before completion of the study, AEs were monitored for two weeks after the last administration.",
"section_name": "Assessment of safety",
"section_num": null
},
{
"section_content": "Statistical significance of EPO levels before the treatment between non-responders and responders was evaluated by the Wilcoxon rank-sum test. Correlation between numbers of gene mutations and response to DA was analyzed by the Cochran-Armitage trend test and the chi-squared test. \n\nOdds ratios between the gene mutations and the outcomes were estimated using univariate and multivariable logistic regression models. The multivariable analysis was adjusted for baseline EPO levels (low: < 100, high: ≥ 100 mIU/mL) and RBC transfusion volumes (low: < 1 unit, high: ≥ 1 unit/ month) as the explanatory variables. \n\nThe survival curves were estimated using Kaplan-Meier methods, and the confidence intervals for the median survival time (MST) and the annual survival rate were calculated using Brookmeyer and Crowley's method and Greenwood's formula, respectively. In all analyses, p < 0. 05 (two-sided) was considered statistically significant. Statistical analysis was performed using SAS Ver. 9. 4.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "This study was conducted in compliance with the Act on the Protection of Personal Information (Act No. 57 of May 30, 2003), the Declaration of Helsinki (October 2013, translated by the Japanese Medical Association in the revised version of Fortaleza), the Clinical Research Act (Act No. 16 of 2017), the Ordinance for Enforcement of the Clinical Research Act (Ordinance No. 17 of the Ministry of Health, Labour and Welfare of 2018), and the Ethical Guidelines for Human Genome/Gene Analysis Research (February 28, 2017). \n\nThe protocol and an explanatory document regarding the protocol provided to patients were approved by the Ethics Review Committee of each participating institution. Prior to subject enrollment, the content of the study was explained to the patients using the explanatory document, and written informed consent was obtained from all participants. If a patient was under 20 years of age, written informed consent of the patient and his or her guardian was obtained.",
"section_name": "Ethics",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "A total of 85 patients underwent enrollment screening. Of these, 79 subjects were included in the full analysis set (FAS), after excluding 4 ineligible patients and 2 patients who withdrew their consent before the start of protocol treatment. The median (range) follow-up for FAS was 374 (44-1094) days. Baseline characteristics of the 79 subjects are shown in Table 1. Median (range) age was 77. 0 (29-90) years; 52 males (65. 8%) and 27 females (34. 2%) were included in the study. Median (range) Hb level was 8. 1 g/dL (4. 3-11. 8 g/dL) in FAS, and 7. 8 g/dL (4. 3-10. 2 g/ dL) and 8. 1 g/dL (4. 9-11. 8 g/dL) in transfusion-dependent and non-transfusion-dependent subjects, respectively. The number of transfusion-dependent cases was 15 (19. 0%).",
"section_name": "Subjects",
"section_num": null
},
{
"section_content": "Rate of overall response (achievement of HI-E according to the IWG criteria 2006) was 70. 9% (60. 0% in transfusiondependent cases, and 73. 4% in non-transfusion-dependent cases) (Table 2 ). Major/minor responses were observed in 46. 7% and 60. 0% of the RBC transfusion-dependent subjects (n = 15). When compared levels of EPO before the treatment between non-responders (n = 23) and responders (n = 56) by the IWG criteria 2006, the levels were significantly lower in responders than non-responders (p = 0. 008) (Fig. 1 ). The We analyzed 376 genes for mutations on 79 samples. The results are shown in Fig. 2. The highly frequent (10% or more) gene mutations included those in SF3B1 (24 cases, 30. 4%), TET2 (20 cases, 25. 3%), SRSF2 (10 cases, 12. 7%), ASXL1 (9 cases, 11. 4%), and DNMT3A (8 cases, 10. 1%). As reported previously, RNA splicing-and epigenetics-regulating genes were major targets for mutations in this study. \n\nAt first, we studied the rate of HI-E for each number of gene mutations. The rates were 63. 9% (23 cases/36 cases), 88. 9% (8/9), 75. 0% (9/12), 66. 7% (6/9), 87. 5% (7/8), 50. 0% (1/2), 100. 0% (1/1), 100. 0% (1/1) and 0% (0/1) in 0, 1, 2, 3, 4, 5, 6, 7 and 8 mutations, respectively. The Cochran-Armitage trend test indicated that there was no correlation between numbers of gene mutations and response to DA (p = 0. 7084). When compared the response between the presence or absence of gene mutations, and, the presence of > 2 or ≤ 2 gene mutations, we still could not notice any differences (p = 0. 210 and 0. 823, respectively). \n\nWe next evaluated the relationship between the presence of highly frequent (10% or more) mutated genes and the rate of HI-E by univariate analysis in FAS (Table 3 ). The univariate logistic regression analysis showed no significant association between these mutations with a frequency of ≥ 10% and therapeutic efficacy of DA. The same results were obtained when the analysis was limited to RBC non-transfusion-dependent subjects (n = 64). \n\nIn the multivariable analysis including baseline EPO levels and RBC transfusion volumes as variables, mutation of ASXL1 gene as well as baseline EPO levels was identified to independently predict poor response to DA with statistical significance (odds ratio 0. 180, 95% CI 0. 035-0. 928, p = 0. 040 for ASXL1 mutation, odds ratio 0. 146, 95% confidence interval 0. 042-0. 503; p = 0. 0023 for EPO levels) (Table 4 ). Transfusion volumes were not detected as predictive factors, possibly because that major part of our cohort was transfusion-independent. Response rates in subjects with low EPO (< 100 mIU/mL) + ASXL1 mutation(-) (n = 35), low EPO + ASXL1 mutation(+) (n = 5), high EPO (≥ 100 mIU/mL) + ASXL1 mutation(-), (n = 35) and high EPO + ASXL1 mutation(+) (n = 4) were 88. 6, 80. 0, 60. 0 and 0%, respectively (Table 5 ). The result of chi-squared test showed that the four groups were significantly different in terms of response (p = 0. 0006).",
"section_name": "Outcomes",
"section_num": null
},
{
"section_content": "Of the 79 subjects who were included in FAS, 56 subjects achieved the HI-E according to the IWG criteria 2006, and the median time to achievement (95% CI) was 7. 1 weeks (6. 1-10. 1 weeks). After adjustment for the baseline EPO levels (cut-off, 100 mIU/mL), although none of the highly frequent gene mutations had a significant association with the time to achievement of the first HI-E according to the IWG criteria 2006, mutation of ASXL1 gene showed a tendency of later achievement: (MST of mutation(+)/mutation(-), not reached/6. 7 weeks, p = 0. 1649).",
"section_name": "Correlation between highly frequent gene mutations and interval to the achievement of the first HI-E",
"section_num": null
},
{
"section_content": "Progression to AML was observed in 24 subjects from week 0 to year 1, and in 10 subjects from week 16 to year 1. A total of 23 subjects died between week 0 and year 1, and 9 subjects died between week 16 and year 1. \n\nPFS and OS at from week 16 to year 1 after the initiation of treatment are shown on the 58 subjects who continued treatment without progression to AML until week 16 in Fig. 3 A and B. PFS (95% CI) at year 1 was 81. 7% (68. 6-89. 7%) with MST (95% CI) of 37. 7 months (29. 5 months-not reached), and OS at year 1 was 83. 5% (70. 7-91. 1%) with MST (95% CI) not reached (30. 9 months-not reached).",
"section_name": "PFS and OS",
"section_num": null
},
{
"section_content": "Grade 3/4 adverse events observed during the study period were anemia in 33 subjects (41. 8%), neutrophil count decreased in 24 (30. 4%), platelet count decreased in 18 (23. 4%), white blood cell count decreased in 16 (20. 3%), lymphocyte count decreased in 15 (19. 0%), hyperglycemia in 3 (5. 9%), hypoalbuminemia in 2 (3. 1%), and aspartate aminotransferase increased in 1 (1. 3%).",
"section_name": "Safety",
"section_num": null
},
{
"section_content": "In this study focusing on DA-eligible lower-risk MDS patients, gene mutations frequently observed in MDS were found in SF3B1 (30. 4%), TET2 (25. 3%), SRSF2 (12. 7%),\n\nASXL1 (11. 4%), and DNMT3A (10. 1%), which is consistent with a previous report [6]. The response rate of DA up to 16 weeks was 70. 9% based on the IWG criteria (60. 0% in transfusion-dependent cases, and 73. 4% in non-transfusion-dependent cases). EPO levels before the treatment were significantly lower in the responders than non-responders. \n\nRegarding the association between gene mutations at a frequency of 10% or more and the response rate of DA, the univariate analysis showed no significant association between them. Multivariable analysis that included serum EPO levels and RBC transfusion volumes revealed that the presence of ASXL1 gene mutations and higher EPO levels (≥ 100 mIU/ mL) were independent factor that predicts for poor response to DA. In a previous meta-analysis, it had been shown that the serum EPO level < 100 IU/L is a biomarker for the effectiveness of DA [26]. Similarly, in our study, DA therapy was effective in subjects with low EPO levels (< 100 mIU/ mL), irrespective of the absence (response rate, 88. 6%) or presence (response rate, 80. 0%) of ASXL1 mutations. However, it is interesting to note that about 60% of the subjects with serum EPO levels ≥ 100mIU/mL and without ASXL1 mutations responded to DA therapy in our cohort. Even in patients with higher levels of EPO, there may be a chance to recover from anemia by DA therapy if they do not possess ASXL1 mutations. One-year PFS and OS rates was 81. 7% (95% CI 68. 6-89. 7%) and 83. 5% (70. 7-91. 1%), respectively. Considering that our cohort included 5 subjects with RAEB-1 according to the WHO classification, the PFS and OS were not worse than expected. DA is an ESA in which sustainable serum concentration has been obtained by substituting 5 amino acids in EPO [27, 28]. Like EPO, DA binds to erythropoietin receptors and thereby promotes erythropoiesis in early and late erythroid progenitor cells in the bone marrow [27, 29]. When injected subcutaneously once a week, sufficient serum levels are maintained for the treatment of anemia [28]. Nine studies performed in patients with MDS reported response rates to DA according to the IWG 2000 criteria with a range of 38-72. 5% within 12-24 weeks [26]. Similarly, in the present study, overall response rate to DA at week 16 was 70. 9% according to the IWG criteria 2006. There was a tendency that non-transfusion-dependent subjects had a better response to DA than transfusion-dependent subjects, with ORR of 73. 4 and 60. 0%, respectively. The earlier commencement of DA could enable avoidance of transfusion dependency in anemic patients with lower-risk MDS. \n\nKosmider et al. have reported that having > 2 somatic mutations was associated with lower HI-E in the ESA treatments for lower-risk MDS [30]. This result was opposite to ours. In our study, there was no co-relation between numbers of gene mutations and response to DA (p = 0. 7084). Even though we divided patients into two groups by the mutation numbers of > 2 or ≤ 2 like in their study, no differences in the response were observed (p = 0. 823). Although the sample size was the same (n = 79) and response rates were similar (70. 9 and 64. 5% according to the IWG 2006 criteria in our and their studies), these two studies had quite different designs. Our study was prospective, while Kosmider et al. 's study retrospective. Patients in our cohort were consistently treated with DA at a dose of 240 μg/week, while those in their cohort with EPO or DA at various doses with or without G-CSF. We analyzed 326 genes for mutations, but they only 37 genes. All these may have caused different conclusions. They also claimed that individual mutations of the frequently mutated genes had no significant impact on HI-E by the univariate analysis, which was the same result as ours. Importantly, however, by using multivariable analysis that included serum EPO level and RBC transfusion volumes as variables, we showed that the presence of ASXL1 mutations besides EPO level was significantly associated with poor response to DA among the highly frequent (> 10%) gene mutations observed in this study such as those in SF3B1, TET2, SRSF2, ASXL1, and DNMT3A. Because MDS are heterogenous not only in morphology but also in molecular pathogenesis, it was difficult for less frequent gene mutations to evaluate their predictive value on the DA treatment. Another important result of this study was that ASXL1 gene mutations were also correlated with possible prolonged interval to the achievement of the first HI-E. ASXL1 mutations are known genetic factors that predict unfavorable clinical courses [31], with a high rate of progression to AML [32]. Considering a balance between cost and benefit, DA perhaps should not be applied to patients with lower-risk MDS showing a higher level of EPO and carrying ASXL1 mutations. \n\nASXL1 is an epigenetics-regulating gene that supports the functions of polycomb complex PRC1 and represses the expression of oncogenes and other genes through methylation of K4 in histone H3 [33]. It has also been shown that mutant ASXL1 disrupts the function of PRC1 and causes derepression of expression in target genes [34], which possibly leads to the development of myeloid malignancies, including MDS [35]. The candidate target genes for the derepression include HOXA9 and MIR125A [36]. While HOXA9 is a known oncogene in hematopoietic tumors of the myeloid lineage [37], MIR125A is suggested to impair hematopoietic cell differentiation [38]. On the other hand, Shi et al. reported that ASXL1 loss impairs erythroid development and hinders erythroid differentiation [39], indicating that ineffective erythropoiesis of MDS may occur as a result of ASXL1 mutation. Although the precise mechanism in a regard to poor response to DA could not be identified for ASXL1-mutated subjects, the mutation could induce refractoriness to DA. From another point of view, Raimbault et al. reported that the low expressions of CD117/c-KIT + in lower-risk MDS erythroid precursors was correlated with ESA failure [40]. It would be interesting to analyze the association between ASXL1 gene mutations and expression levels of CD117/c-KIT + in a future study. \n\nThis prospective study provided the first evidence in the ESA therapy that the existence of some specific gene mutations (ASXL1 mutations) may be associated with response to specific treatments (DA). In our opinion, even though patients with lower-risk MDS show higher EPO levels, DA would be effective if they do not carry ASXL1 mutations. However, when patients have both the predictive factors of poor response, namely, higher levels of EPO and ASXL1 mutations, alternative therapies would be recommended as the first-line therapy for anemia. One of such candidates may be luspatercept which is a recombinant fusion protein binding TGF-β superfamily ligands and is effective in patients with increased ring sideroblasts and/or SF3B1 mutations [41]. Even in cytokine and other supportive therapies for anemia, molecular stratification needs to be established to determine their application in the near future.",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "Acknowledgements This study was funded by Kyowa Kirin Co., Ltd., and was supported by the Ministry of Education, Culture, Sports, Science and Technology under Grant number hp160219 and hp200138 and by AMED under Grand number JP20cm0106501h0005 to SO. We would like to thank all of the participated patients and their families. We are indebted to the physicians, all other co-medical staff and Independent Data Monitoring Committee ( Shuji Nakano, Naohito Fujishima and Kenichi Yoshimura ) who contributed to this study. We also thank the stuffs at the Clinical Research Support Center Kyushu (CReS Kyushu) for their excellent collection and management of data, secretarial assistance, and any other support.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Declarations",
"section_num": null
},
{
"section_content": "Author contributions Contribution: MI, HK, HS, YMo, YMa, KT, TM, TH, and KM designed the study; MI, HK, HS, YMo, YMa, KT, TM, TH, and HH collected clinical data; YN, YT, SM, and SO performed sample preparation and sequencing; MI, HK, HS, YMo, YMa, KT, TM, TH, YN, HH, IM, SO, KA, YK, and KM analyzed data; JK performed statistical analysis; MI, HK, HS, YMo, YMa, KT, TM, TH, YN, YT, SM, JK, YN, SO, and KM wrote the manuscript; and all of the authors reviewed and approved the final manuscript.",
"section_name": "",
"section_num": ""
},
{
"section_content": "Conflict of interest YN (Otsuka Pharmaceutical Co., Ltd. ), HK (Nip-Open Access This article is licensed under a Creative Commons Attribution 4. 0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. \n\nPublisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Declarations",
"section_num": null
}
] |
10.1038/s41598-024-59882-y
|
Targeting the ZMIZ1-Notch1 signaling axis for the treatment of tongue squamous cell carcinoma
|
<jats:title>Abstract</jats:title><jats:p>Zinc finger MIZ-type containing 1 (ZMIZ1) is a transcriptional coactivator related to the protein inhibitors of activated STATs (PIAS) family. Mounting evidence suggests that ZMIZ1 plays a crucial role in the occurrence and development of cancers. The function of ZMIZ1 in tongue squamous cell carcinoma (TSCC) and the mechanisms underpinning its role in this disease have not been fully clarified. We performed qualitative ZMIZ1 protein expression analyses using immunohistochemistry in 20 patient-derived, paraffin-embedded TSCC tissue sections. We used RNAi to knock down ZMIZ1 expression in the CAL-27 TSCC cell line and quantified the impact of ZMIZ1 knock down on proliferation, migration and apoptosis via CCK-8, scratch assay and flow cytometry, respectively. We used qRT-PCR and western blotting to investigate the role of ZMIZ1 in this cell line. Finally, we established a model of lung metastasis in nude mice to replicate the in vitro results. ZMIZ1 protein was significantly more abundant in TSCC case tissue samples. ZMIZ1 knockdown reduced the invasion and metastases of TSCC tumor cells and promoted apoptosis. ZMIZ1 knockdown was associated with the down-regulation of Notch signaling pathway related factors Jagged1 and Notch1, and invasion and metastasis related factors MKP-1, SSBP2 and MMP7 in vitro and in vivo, at the mRNA level. In vitro and in vivo data suggest that knock down of ZMIZ1 may inhibit TSCC invasion and metastasis by modulating Notch signaling. ZMIZ1 inhibition may therefore represent a new therapeutic target for TSCC.</jats:p>
|
[
{
"section_content": "www. nature. com/scientificreports/ Notch1 exhibited elevated expression and promotes TSCC invasion and metastasis by modulating the expression of matrix metalloproteinases (MMPs) and epithelial-mesenchymal transition 14. Moreover, Notch1 expression can be used as a primary biomarker to aid early diagnosis of TSCC as well as providing prognostic value 15. \n\nZMIZ1 (Zinc finger MIZ-type containing 1), also known as ZIMP10 or RAI17, is a transcriptional coactivator associated with the protein inhibitors of activated STATs (PIAS) family. ZMIZ1 expression is elevated in various tumors and is linked to clinical characteristics such as age of onset, progression, malignancy, prognosis, and chemotherapy resistance 16, 17. ZMIZ1 was reported to regulate Notch1 expression, targeting the Hes1 and Myc genes to modulate precursor T-cell development 18. Furthermore, ZMIZ1 is involved in transcriptional activation of Notch1 target genes 17. Through direct interaction with Notch1, ZMIZ1 was commonly co-expressed with activated Notch1 in T-cell acute lymphoblastic leukemia (T-ALL) 18. Knockout of ZMIZ1 inhibited the occurrence of Notch1-induced leukemia, and it was proposed that targeting ZMIZ1 could treat lymphocytic leukemia 19, 20. Together, these studies suggest that ZMIZ1 might act as a direct transcriptional co-factor for Notch1, and disrupting the Notch1-ZMIZ1 interaction may impact upon tumorigenesis, proliferation and metastasis in various cancers. \n\nIn this study, we aimed to explore the ZMIZ1-Notch1 axis as a potential therapeutic strategy in TSCC. We clarified the role of ZMIZ1 in the occurrence and development of TSCC. We knocked down ZMIZ1 in TSCC tumor cells and investigated the impact on TSCC invasion and metastasis in vitro and in vivo. Then, we detected the expression of invasion and metastasis related factors to explore the molecular mechanisms linking ZMIZ1 and Notch1, include mitogen-activated protein kinase phosphatase-1 (MKP-1), single-stranded DNA binding protein 2 (SSBP2) and matrix metalloproteinase 7 (MMP7).",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Twenty specimens of human TSCC embedded in paraffin were used. The specimens were collected from the first and second hospitals of Lanzhou University (Gansu, China). The basic clinical information of TSCC patients is shown in Supplementary Table 1. The informed consent forms were duly signed by and collected from all patients. This study was approved by the Medical Ethics Committee of Lanzhou University School of Stomatology (Gansu, China). And all authors confirmed that methods used were carried out in accordance with relevant guidelines and regulations.",
"section_name": "Patients and clinical specimens",
"section_num": null
},
{
"section_content": "LV-ZMIZ1-RNAi was designed by Shanghai Genechem Co., LTD. (Shanghai, China). Antibodies against ZMIZ1, Jagged1, Notch1, MKP-1, SSBP2 and MMP7 were procured from Abcam (MA, USA). Antibodies against GAPDH were purchased from ImmunoWay (Texas, USA). Secondary antibody was purchased from Wuhan Boster Company (Wuhan, China). All other reagents and compounds were of analytical grade and purchased from local chemical suppliers in China.",
"section_name": "Reagents",
"section_num": null
},
{
"section_content": "Human TSCC cell line CAL-27 was purchased from ATCC (https:// www. atcc. org/ produ cts/ all/ CRL-2095. aspx). STR analysis was performed in this cell line to ensure the authentication of human cell lines, and was cultured in a humidified CO 2 incubator at 37 °C. Cells were maintained in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 100 U/mL streptomycin. According to the manufacturer's protocol, LV-ZMIZ1-RNAi was used for cell transfection. CAL-27 cells were seeded into a six-well plate (3-5 × 10 4 cells per well), and cultured in 2 mL complete medium until cells were 60% to 80% confluent. Then, the medium was changed to 1. 8 mL complete medium and 200 μL LV-ZMIZ1-RNAi complex (containing 2 μl polybrene), and the transfection was confirmed under fluorescence microscope after 72 h selection with puromycin.",
"section_name": "Cell culture and transfection",
"section_num": null
},
{
"section_content": "According to the reagent instructions, total RNA in cells and tissues was extracted using TRIzol reagent (Invitrogen). RNA was reverse-transcribed into cDNA using Takara Reverse Transcription Kit (Takara Bio Inc. ), and qRT-PCR was performed using SYBR Premix Ex Taq II (Takara Bio Inc. ). The relative gene expression level was calculated according to the 2 -ΔΔCT method, and the relative expression level of mRNA was standardized using GAPDH. Table 1 listed all primers and related sequences used in the experiment.",
"section_name": "RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)",
"section_num": null
},
{
"section_content": "TSCC cell proliferation was detected by CCK-8 analysis. TSCC cells were inoculated into 96-well plates at 2000 cells/well, and 10 µL CCK-8 solution was added to each well after 24, 48, 72, 96 and 120 h as per the manufacturer's instructions (Dojindo China CO., Ltd, Shanghai, China). The absorbance value was measured at 450 nm after culturing at 37 °C for 2 h.",
"section_name": "CCK-8 assay",
"section_num": null
},
{
"section_content": "CAL-27 cells were inoculated in a six-well plate with DMEM containing 10% FBS until confluent. The cells were scraped with a sterile 200 μL pipette head to form an artificial wound. Three random images in each field were obtained with inverted microscope at 0, 24, 48, and 72 h after injury to assess wound healing. The average migration rate of cells of each group was calculated using ImageJ software.",
"section_name": "Wound healing assay",
"section_num": null
},
{
"section_content": "CAL-27 cells were cultured for 48 h following transfection, and whole protein was extracted using RIPA lysis buffer (Thermo Fisher Scientific) and quantified with the BCA kit (Beyotime Biotechnology). The sample (10 μg) was collected, separated by 10% SDS-PAGE, and transferred to PVDF membrane. Then, under gentle agitation, the membrane was blocked at 37 °C for 2 h. After that, primary antibody was added to incubate at 4 °C overnight, and secondary antibody was added to incubate at 37 °C for 2 h. Chemiluminescence was detected by ultra-sensitive chemiluminescence fluid (Millipore), and then processed and analyzed by a chemiluminescence imaging system. GAPDH was used as an internal reference protein. The antibodies and concentrations used in this experiment were as follows: ZMIZ1 (1:500; Abcam); Jagged1 (1:1000; Abcam); Notch1 (1:1000; Abcam); MKP-1 (1:1000; Abcam); SSBP2 (1:1000; Abcam); MMP7 (1:1000; Abcam); GAPDH (1:5000; ImmunoWay) and HRP-conjugated secondary antibody (1:10,000; Boster). The band density was analyzed using ImageJ software. All fold changes of band density were normalized to the control group.",
"section_name": "Western blots",
"section_num": null
},
{
"section_content": "For experimental purposes, all nude mice experiments were conducted in accordance with the \"Guidelines for Animal Care and Use\" (NIH), the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines, and were approved by the Medical Ethics Committee of Lanzhou University School of Stomatology. Male thymus free BALB/C nude mice (nu/nu) aged 4-6 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice were raised in a laminar flow cabinet free of specific pathogens, and had free access to food and high-pressure water with a dark/light cycle every 12 h. Nude mice were randomly divided into two groups (NC control group, LV-ZMIZ1 group). For the LV-ZMIZ1 group, 200 μL of 1 × 10 7 /mL CAL-27 cell suspension was injected through the tail vein (n = 8 animals in each group). Food, water consumption and body weight were measured every three days. At the 8th week after tail vein injection, mice were sacrificed, lung tissues were dissected and photographed, lung histopathological sections were used for hematoxylin-eosin staining (HE) staining and immunohistochemistry (IHC), and the numbers of lung tumor metastases were counted.",
"section_name": "In vivo metastasis assay",
"section_num": null
},
{
"section_content": "Lung samples were extracted from control and LV-ZMIZ1 lung metastasis models for HE staining. The lung tissue was fixed in 10% formalin for 24 h, embedded in paraffin, cut to 4 μm thickness, stained with HE, dehydrated, transparent, dry, and sealed with neutral gum. Each section was observed under the microscope (Leica Microsystems).",
"section_name": "Hematoxylin-eosin (HE) staining",
"section_num": null
},
{
"section_content": "In brief, the sections from the specimens of human TSCC and the lung samples of nude mice were dewaxed and dehydrated. Endogenous peroxidase activity was blocked with 3% methanol hydrogen peroxide solution for 20 min, and non-specific immune-reactivity was eliminated by 10% normal goat serum for 30 min. Then, the tissue sections were incubated with the primary antibody. Antibodies were anti-ZMIZ1 (1:100, Abcam), anti-Jagged1 (1:200, Abcam), anti-Notch1 (1:200, Abcam), anti-MKP-1 (1:200, Abcam), anti-SSBP2 (1:200, Abcam), anti-MMP7 (1:200, Abcam). For the negative control, the primary antibody was replaced by PBS. The specimens were washed 3 times with PBS, and incubated with the goat anti-rabbit secondary antibody (ZSGB-Bio) labeled with peroxidase polymer at room temperature until positive brown staining appeared. Samples were then incubated with diaminobenzidine, counterstained with hematoxylin, dehydrated and covered with glass slides. Sections were observed under the microscope. IHC was quantitatively analyzed using Image J software, with average optical density (AOD) serving as the intensity of staining. The \"Integrated density\" and \"Area\" tools were used. AOD = Integrated density/Area. The detailed information has been added in our article.",
"section_name": "Immunohistochemistry (IHC)",
"section_num": null
},
{
"section_content": "All the experiments were repeated at least three times. The data are expressed as mean ± standard deviation (SD). Statistical comparisons were performed by student T test or one-way ANOVA as implemented in the GraphPad Prism software (version 6. 0, GraphPad). P values < 0. 05 was considered statistically significant.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We first aimed to characterize ZMIZ1 protein expression in samples derived from TSCC patients. The protein abundance levels of ZMIZ1 were assessed through IHC in 20 paraffin-embedded TSCC tissue sections. ZMIZ1 demonstrated positive expression in both the cell membrane and cytoplasm. ZMIZ1 protein expression appeared to be abundant within the TSCC tumor tissue, but weakly expressed in adjacent non-tumor tissue (Fig. 1A and Fig. S1 ). \n\nHaving confirmed expression of ZMIZ1 protein in TSCC samples, we investigated the specific role of ZMIZ1 in TSCC. We established CAL-27 cell lines transfected with LV-ZMIZ1-RNAi (experimental group) and LV-Ctrl-RNAi (control group). Lentiviral vectors were used to construct LV-ZMIZ1-RNAi and LV-Ctrl-RNAi, then transfected into CAL-27 cells. An immunofluorescence assay confirmed that the efficiency of lentivirus transfection into CAL-27 cells exceeded 80% (Fig. 1B ). The expression of ZMIZ1 was then detected by qRT-PCR and western blotting. ZMIZ1 mRNA and protein levels were significantly decreased (mRNA knockdown to 56%, P < 0. 01; protein knockdown to 22%, P < 0. 01) in the LV-ZMIZ1-RNAi samples compared to controls (Fig. 1C-E ).",
"section_name": "ZMIZ1 expression is elevated in TSCC tissue",
"section_num": null
},
{
"section_content": "Having established a ZMIZ1-knockdown model in CAL-27 cells, we aimed to verify the role of ZMIZ1 in proliferation, apoptosis, and migration in CAL-27 cells. The CCK-8 assay was used to assess the proliferation ability of CAL-27 cells after ZMIZ1 was knocked down. The proliferation of the ZIMZ1 knockdown group was significantly inhibited compared with the control group (Fig. 2A ; P < 0. 05). \n\nThe proportion of apoptotic cells in each group was measured by flow cytometry (Annexin V-APC single staining), and the peak value of apoptosis and the percentage of apoptotic cells were obtained. The proportion of apoptotic cells was increased in the ZMIZ1 knockdown cells, linking ZMIZ1 to apoptosis in these cells (P < 0. 05; Fig. 2B, C ). \n\nTo investigate the role of ZMIZ1 in migration in CAL-27 cells we performed a wound healing assay. The results suggested that the scratch healing rate of ZMIZ1 knockdown cells was significantly lower than the control cells at 48 h (P < 0. 05), and was more even pronounced at 72 h. This indicated that the migration ability of CAL-27 cells was inhibited after ZMIZ1 was knocked down (Fig. 2D, E ).",
"section_name": "Knockdown of ZMIZ1 inhibits proliferation and migration, and promotes apoptosis in CAL-27 cells",
"section_num": null
},
{
"section_content": "Having shown that ZMIZ1 knockdown impaired the proliferation and migration of CAL-27 cells, and increased apoptosis, we investigated the mechanisms governing this behavior. Studies have shown that ZMIZ1 is an active cofactor of Notch1, and that ZMIZ1 may promote the invasion and metastasis of tumors by regulating the Notch1 signaling pathway 19, 20. We therefore investigated the expression of Notch1 signaling pathway proteins, and various markers of invasion and metastasis by IHC analysis of TSCC patient tissue sections. Our results indicated that expression of Notch1 protein and its ligand Jagged1 were significantly higher in tumor tissue compared to normal surrounding tissue (Fig. 3A and Fig. S2 ). Expression of invasion and metastasis-related proteins MKP-1, MMP-7, and SSBP2 were also significantly increased in TSCC tumor tissues, consistent with a propensity of tumor cells to metastasize (Fig. 3A and Fig. S2 ). \n\nHaving confirmed the involvement of Notch1 and metastatic factors in the patient TSCC samples, we aimed to elucidate the specific molecular mechanisms underlying the involvement of ZMIZ1 in TSCC. We employed qRT-PCR and western blot analyses to assess the expression levels of Notch1, Jagged1, MKP-1, SSBP2, and MMP7 following ZMIZ1 knockdown in the CAL-27 cell line model. The results confirmed that mRNA and protein expression levels of Notch1, Jagged1, MKP-1, SSBP2, and MMP7 were significantly decreased in the ZMIZ1 knockdown cells (Fig. 3B-D ). These results suggest that that ZMIZ1 knockdown can reduce the invasion and migration potential of TSCC tumor cells by modulation of the Notch1 signaling pathway and several invasion and metastasis markers.",
"section_name": "ZMIZ1 regulates Notch1 signalling to control CAL-27 cell invasion, metastasis and apoptosis",
"section_num": null
},
{
"section_content": "Following the in vitro experiments which demonstrated that the proliferation and migration of CAL-27 cells were inhibited by ZMIZ1 knockdown, we investigated the role of ZMIZ1 in TSCC metastasis in vivo. CAL-27 cells were transfected with LV-Ctrl-RNAi or LV-ZMIZ1-RNAi and were injected into the tail veins of nude mice to establish a lung metastasis model. We found no significant difference in the body weight of the mice between those inoculated with LV-Ctrl-RNAi and those with LV-ZMIZ1-RNAi (P > 0. 05; Fig. 4A ). After 50 days, the nude mice were sacrificed and lung tissues were isolated to observe metastases under the microscope. Counting, photography and HE staining were performed. The results revealed that there were fewer lung metastases in the mice inoculated with LV-ZMIZ1-RNAi CAL-27 cells compared to the control group, and the volume of metastases was larger in the control group (P < 0. 05; Fig. 4B ). Microscopic observation of lung metastatic tumors showed tightly arranged tumor cells with large and deep nuclei, infiltrated nuclear membranes, and distinct nucleoli in the control group. In contrast, tumors from mice with the LV-ZMIZ-RNAi CAL-27 cells displayed diffuse arrangements, appearing in streaks and clusters, accompanied by bleeding and necrosis in metastatic lesions (thickness = 4 μm, P < 0. 05; Fig. 4C, D ). Expression levels of Jagged1, MKP-1, MMP7, Notch1, SSBP2, and ZMIZ1 in tumor tissues of LV-ZMIZ1-RNAi CAL-27 mice were decreased compared to the control group (P < 0. 05; Fig. 4E-H and Fig. S3 ). These results suggest that targeted inhibition of the ZMIZ1-Notch1 signaling pathway may reduce the expression of invasion and metastasis-related factors (MKP-1, SSBP2, and MMP7), thereby suppressing the invasion and metastasis potential of TSCC tumor cells.",
"section_name": "ZMIZ1 regulates invasion and metastasis through the Notch1 signaling pathway in TSCC in vivo",
"section_num": null
},
{
"section_content": "TSCC is an oral and maxillofacial squamous cell carcinoma with high morbidity and mortality rates. Due to its location of origin, its prognosis and survival rate are poor. At present, gene-targeted therapy has become a treatment option due to its unique advantages 21. However, it has not been utilized in the treatment of TSCC. In this study, we found that ZMIZ1 and Notch1 expression of human TSCC were significantly elevated. Knocking down ZMIZ1 in CAL-27 cells markedly reduced Notch1 expression, diminished cell proliferation and promoted apoptosis. Hence, we speculated that ZMIZ1 gene may play a regulatory role in the occurrence, development, invasion and metastasis of TSCC, and this function may be achieved by regulating the Notch1 signaling pathway. Notch1, the first Notch receptor discovered in mammals, exhibits heightened expression in various tumors, closely associated with increased cancer stem cells, tumor invasion, metastasis, and drug resistance 22. In head and neck squamous cell carcinoma, Notch1 is the second most commonly mutated gene after TP53, where inactivating mutations exert tumor-suppressive effects, and activating mutations or upregulation of expression contribute to carcinogenesis 23. The expression of Notch1 is reportedly up-regulated in oral squamous cell carcinoma, correlating with T-stage and clinical staging 13. Additionally, membranous Notch1 expression serves as a robust independent prognostic factor for improved outcomes in head and neck squamous cell carcinoma 24. Our investigations show that Notch1 expression was elevated in TSCC patient tumor tissues compared to non-tumor tissues, which may be driving carcinogenesis. Consistent with our findings, Notch1 expression was upregulated in tongue cancer tissues, and inhibition of Notch1 reduced proliferation, invasion, and migration of tongue cancer cells 25. \n\nTargeting of Notch1 as a therapeutic strategy has undergone extensive investigation, utilizing various methods for its inhibition in cancer treatment, including Notch1 monoclonal antibodies, Notch1 siRNA, and γ-secretase inhibitors 26. In adenoid cystic carcinoma (ACC), mutations in the Notch1 gene which impair function correlate with higher metastatic rates and shorter survival periods. Treatment with the Notch1 monoclonal antibody bronticuzumab could delay ACC progression and contribute to partial reduction of tumor volume 27, 28. siRNA targeting Notch1 significantly reduces Notch1 expression, thereby inhibiting tumor cell proliferation and migration, inducing cell apoptosis, and exhibiting anticancer effects 29, 30. Among these approaches, research has focused on γ-secretase inhibitors (GSIs), which exert promising anticancer effects when used alone or in combination with other anticancer drugs 31. However, anti-tumor strategies targeting Notch1 all have side effects to varying degrees. Therefore, a balanced approach is necessary where there is a continual need to explore safer approaches for targeted Notch1 inhibition in anticancer strategies. \n\nPrevious research has confirmed that ZMIZ1 may be a direct and selective co-factor of Notch1 19, 32. ZMIZ1 collaborates with Notch1 to activate c-Myc transcription and promotes M2 polarization in Kupffer cells, thereby enhancing hepatocellular carcinoma transformation 33. However, knockdown of ZMIZ1 in Kupffer cells inhibited M2 polarization, which consequently restrained hepatocellular carcinoma progression. Similarly, in leukemia, the interaction between ZMIZ1 and Notch1 enhances C-MYC transcription and activity, facilitating leukemia initiation and progression, while ZMIZ1 inhibition attenuates leukemia progression 20. Crucially, ZMIZ1 plays a minor role in intestinal homeostasis or bone marrow suppression. Hence, targeting ZMIZ1 can avoid the associated toxicities related to direct Notch1 targeting 19. \n\nOur study demonstrated that knocking down ZMIZ1 significantly suppressed the expression of Notch1 and its ligand Jagged1, confirming the correlation between ZMIZ1 and Notch1. Simultaneously, the suppression of ZMIZ1 expression inhibited the proliferation and migration of CAL-27 cells while promoting apoptosis. These findings indicated that ZMIZ1 may be a potential therapeutic target for the treatment of TSCC. \n\nOur investigation further explored the specific mechanisms underpinning the ZMIZ1 influence on the invasion and metastasis of TSCC. Studies have shown that the expression of MMP7 was increased in tumor patients and was related to the depth of tumor invasion, size and number of lymph node metastases. MMP7 plays an important role in the malignant biological behavior of tumors 34. Therefore, MMP7 can be used as a related effector of invasion and metastasis. Similarly, MKP-1 is overexpressed in several cancer types, associated with cancer progression, and may be a potential prognostic marker 35. In T-ALL cell lines, cells with MKP-1 deficiency exhibited compromised tumorigenicity, which was regulated by Notch3 36. SSBP2 is widely studied as a tumor suppressor, and downregulation of SSBP2 expression inhibited tumor cell growth and increased susceptibility of malignant tumors 37, 38. However, some studies have indicated that overexpression of SSBP2 can exert oncogenic effects which correlated with adverse clinical outcomes in hepatocellular carcinoma and glioblastoma 39, 40. Our research observed increased expression of MMP-7, MKP-1, and SSBP2 in TSCC tissue, suggesting that the invasion and metastasis of TSCC may be related to the expression of these genes. Furthermore, our results found that knockdown of ZMIZ1 correlated with decreased expression of MMP-7, MKP-1, and SSBP2, indicating that ZMIZ1 may influence the invasion and metastasis of TSCC by regulating the expression of MMP-7, MKP-1, and SSBP2. \n\nIn summary, ZMIZ1 knockdown significantly inhibits the proliferation and migration of CAL-27 cells. This mechanism potentially involves ZMIZ1 as an oncogene that activates the Notch1 signaling pathway, thereby promoting the invasion and migration of TSCC. Thus, ZMIZ1 may be a potential treatment target for TSCC.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In conclusion, our investigation about the role of TSCC sheds light on its significance as a potential therapeutic target. Through a series of experiments involving ZMIZ1 knockdown, we observed a substantial inhibition in the proliferation and migration of CAL-27 cells. This suggests a pivotal role for ZMIZ1 as an oncogenic driver, possibly operating by activating the Notch1 signaling pathway, thereby facilitating TSCC invasion and migration. Additionally, our findings highlighted the association of ZMIZ1 with key factors involved in tumor progression, such as MMP7, MKP-1, and SSBP2, underscoring its multifaceted impact on TSCC biology. The identification of ZMIZ1 as a probable regulator of these invasive and metastatic factors emphasizes its potential as a promising therapeutic target for TSCC. The molecular mechanism of ZMIZ1 influencing the progression of TSCC needs to be further studied to provide new ideas for the targeted therapy of TSCC.",
"section_name": "Conclusion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This study was supported by the National Natural Science Foundation of China ( 81773942 ), the Scientific and Technological Foundation of Gansu Province ( 21JR1RA112, 20JR10FA670 ), and the School/Hospital of Stomatology Lanzhou University (lzukqky-2022-p04, lzukqky-2022-t09).",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "The full data used to support the findings of this study are available from the corresponding author upon request.",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "Y. P., Y. S. and Y. W. conceived and designed the study, performed statistical analyses, interpreted the data, and drafted the manuscript. J. L. partly contributed to the revision of articles and the supplementation of data during the revision stage. P. Q., and S. G. performed some experiments. W. Z. drafted the manuscript and contributed to the language polishing of manuscript. J. W. and J. C. contributed to conception, study design, and interpretation of the data, and drafted the manuscript. All authors critically revised the manuscript, gave final approval, and agreed to be accountable for all aspects of this study.",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional information",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s41598-024-59882-y. \n\nCorrespondence and requests for materials should be addressed to J. C. or J. W. \n\nReprints and permissions information is available at www. nature. com/reprints. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Supplementary Information",
"section_num": null
}
] |
10.1038/s41375-023-01845-9
|
Clinical impact of TP53 disruption in chronic lymphocytic leukemia patients treated with ibrutinib: a campus CLL study
|
NA
|
[
{
"section_content": "TO THE EDITOR Disruption of the TP53 gene, either by deletion at chromosome 17p13. 1 (del17p) or mutations, is the most important prognostic/ predictive biomarker in chronic lymphocytic leukemia (CLL), also in the context of the novel target therapies including ibrutinib [1] [2] [3] [4]. Although TP53 deletion and mutations mostly co-occur and are considered as equal prognosticators, the prognostic value of isolated or concomitant mutations and deletions remains unclear [2, 3]. Here we applied an ultra-deep next-generation sequencing (NGS) approach in CLL patients treated with ibrutinib, to investigate the clinical impact of TP53 mutations and del17p, either concomitant or isolated, or in relation to their disruption burden. \n\nThis study, generated in the framework of an institutional Italian multicenter working group on CLL (\"Campus CLL\"), is a retrospective/multicenter analysis of 229 CLL patients treated with ibrutinib in the current clinical practice. All cases have been either referred to a single institution for molecular and cytogenetic analyses (February 2014-February 2021), or retrospectively referred by delivering frozen cell samples taken prior to starting ibrutinib treatment. Clinical outcome data were updated as of October 2021. Eighty patients, included in a previous study [3], are presented here with an updated median follow-up (24. 7 months). As a stringent criterion, only patients assayed for TP53 mutation and 17p deletion in the same blood sample taken within 6 months prior to the start of ibrutinib were included. Median follow-up from ibrutinib treatment was 36. 3 months (95% CI 29. 5-41. 5 months); 51 patients were treatment naïve (TN) and, 178 refractory/relapsed (RR). In accordance with the ERIC recommendations for TP53 disruption [5], mutation analyses were always carried out on samples containing >80% tumor cells; when lower than the 80% cutoff, CD19 positive CLL cells were purified by cell sorting. Briefly, analysis of TP53 mutations was performed with an amplicon-based strategy, covering exons 2-11, as previously reported [4]. A minimum coverage of 2,000X was obtained for each sequence in 100% of the analyzed positions, with a limit of detection of 0. 3% VAF; TP53 mutated cases with less than 2% VAF were all confirmed by a second independent NGS run starting from DNA [4]. Moreover, selected low-VAF TP53 mutations were verified by a different experimental approach (digital droplet PCR, ddPCR). BTK and PLCG2 mutations related to ibrutinib resistance were studied by NGS. Interphase FISH was performed to detect del17p and 11q22. 3 deletion (del11q) [4]. Further methodological details are provided in Supplementary Information. The clinical and biological baseline characteristics of patients [6] are detailed in Supplementry Table S1. All statistical analyses were performed by using standard methods. Overall survival (OS) and progression free survival (PFS) were computed from date of ibrutinib treatment to date of death or progression/ suspension (events), respectively, or last follow-up (censoring). Molecular studies were blinded to the study end points. \n\nAmong 229 patients, 68 died and 57 progressed after median follow-up of 15. 6 months (95% CI 11. 9-20. 5 months) and 24 months (95% CI 16. 0-32. 7 months) from ibrutinib starting, respectively. As in previous reports [7] [8] [9], Rai stage, the number of previous treatments (0/1 versus >1), anemia and abnormal LDH values were found to associate with shorter PFS and/or OS by univariable analyses (Table 1 and Supplementry Fig. S1 ). CLL bearing del17p (n = 74; Supplementry Table S1 ) showed inferior OS and PFS compared to non-del17p cases (Fig. 1A and Supplementry Table S2 ), as previously reported [10]. Consistently, del17p was independent predictor in multivariable models for OS/ PFS (P = 0. 0209, OS; P = 0. 0057, PFS; Model 1 Supplementry Table S2 ). At baseline, before ibrutinib treatment, we identified a total of 296 TP53 mutations in 126 patients (median mutations per patient: 1; range of mutations/patient: 1-11; Supplementry Table S3 ). The relative high proportion of cases (126/229, 55%) with TP53 mutations can be explained by the use of an ultra-deep NGS strategy allows the detection of very small mutated clone (see also Supplementry Table S4 and Supplementry Fig. S2 for ddPCR validation of selected mutations) [4, 5]. By classifying TP53-mutated patients according to the VAF of the most prevalent TP53 mutation, VAF range for TP53-mutated cases was 0. 53-95. 24% (Supplementry Table S3 ). As in the chemo-immuno therapy setting [4], also in the ibrutinib setting, patients bearing TP53 mutations with low (<10%) and high (≥10%) VAF had shorter OS than TP53wt cases, either kept separate (Fig. 1B ), or when low-VAF and high-VAF cases were combined (Supplementry Fig. S3 ). These results suggest that even low burden TP53 alterations confer a negative impact on outcomes, widening previous findings [11]. Accordingly, TP53 mutations were associated with shorter OS/PFS intervals in univariable analyses (Supplementry Table S2 ), as well as in an OS multivariable model (P = 0. 0217; Model 2, Supplementry Table S2 ). Here, we expanded to low-VAF TP53-mutated patients previous observations on the clinical impact of TP53 disruption upon ibrutinib, as they emerged in the context of clinical trials [7], or in real-life [3, 6, 8], where TP53 disrupted patients were identified according to the current standard criteria (i. e. VAF ≥ 10%). \n\nThe combination of del17p with TP53 mutation data identified 95 cases without any TP53 aberrations (non-del17p/non-TP53mut), 8 del17p only cases, 60 TP53-mutated only cases (28 low-VAF), and 66 cases bearing both del17p deletion and TP53 mutations (7 low-VAF). Only patients with concomitant TP53 mutations and del17p showed significantly shorter OS/PFS intervals compared to non-del17p/non-TP53mut cases, while no difference in OS/PFS was found in patients presenting single aberration (Fig. 1C, D ). The simultaneous presence of TP53 mutations and del17p confirmed its detrimental clinical impact by univariable analysis and remained independent predictor for short OS/PFS by multivariable analyses together with the number of previous lines of therapy and anemia; consistently, these variables were the most frequently selected by internal bootstrap validation (Table 1 ). Given the low number of patients of some subgroups (e. g. 8 del17p alone cases), these results need to be confirmed in larger cohorts. \n\nAt variance from chemo-immunotherapy where the presence of a single TP53 mutation, even with a low-VAF, is associated with a worse outcome [4, 12], in the ibrutinib setting only cases presenting a more complex disruption of the TP53 function, due to the concomitant presence of mutations and deletions, fail to have the best benefit from therapy. Our results are in keeping with recent findings suggesting that only double-hit aberrations (i. e. more than one TP53 mutation or TP53 mutation and del17p) are independently associated with a shorter outcome in ibrutinib-treated patients, single-hit aberrations (a single TP53 mutation or del17p only) having an outcome comparable to that of TP53wt patients [2]. Differently from Brieghel et al. [2], however, in our cohort, TP53 mutated patients with more than one mutations but without del17p failed to experience a significantly worse prognosis respect to patients without any aberrations (data not shown). In the present series, 52/66 cases concomitantly bearing del17p and TP53 mutations (79%) bore TP53 mutations and/or 17p deletion in most of the neoplastic clone (Supplementry Table S3 ). We could, therefore, speculate that the genetic instability fostered by such a massive TP53 disruption might eventually lead to the development of more complex genetic lesions, known to correlate with dismal outcomes in the ibrutinib setting [11, 13]. Our finding may help to explain previous reports of ibrutinib-treated CLL in which TP53 mutations failed to have a prognostic impact [12], and in which the simultaneous presence of TP53 mutations and deletion was not investigated. \n\nThe evolution of TP53 mutated clones was assessed in 38 patients by longitudinal NGS analysis of paired samples collected at pre-treatment (median time, -0. 9 month; range -6. 0-0. 0) and during (non-relapsed cases; n = 22) or after (relapsed cases; n = 16) ibrutinib treatment (median time interval, 31. 8 months, range 3. 0-76. 9). For relapsed cases, the second time point was collected in close proximity of progression (median time, -0. 7 months, range -3. 0-1. 0 months). No significant differences were observed between relapsed and non-relapsed cases in relation to the timing of the second sampling (P = 0. 74). Of a total of 127 TP53 mutations, 92 were present before and 106 after treatment; among these, 21 mutations (median VAF, 1. 7%, range 0. 4-52. 3%) disappeared during the course of treatment, while 35 were newly identified (median VAF, 1. 0%, range 0. 4-95. 2%; Supplementry Table S5 ). Among relapsed cases, 15/16 showed either a prominent expansion (i. e. a VAF increase greater than 20%) or stability (i. e. VAF variations within the range of 20% VAF variation) over time of the TP53 mutated clone(s) (Fig. 1E ). Conversely, in the context of non-relapsed patients, 3 cases presented a VAF increase of the TP53 mutated clones, 13 remained stable, and 6 showed a VAF reduction (Fig. 1F ). These data support the idea of a general stability of TP53 subclones under ibrutinib [14], although a positive selection of TP53 mutations over time was slightly overrepresented in relapsed cases (P = 0. 04, χ 2 test), suggesting the occurrence of other genetic events complementing the clonal advantage due to TP53 disruption [11, 14, 15]. Considering the 127 TP53 mutations identified across the different time-points, 8 mutations were shared by ≥3 cases (Supplementry Table S5 ). Among them, G245S and R175H were found expanded (>20% VAF Fig. 1 Clinical impact of TP53 aberrations in ibrutinib-treated CLL. A Kaplan-Meier curves comparing OS probabilities of 155 non-del17p cases (green line), 74 cases with del17p (black line). B Kaplan-Meier curves comparing OS probabilities of 103 TP53wt cases (green line), 91 cases with high-VAF TP53 mutations (TP53mut_highVAF), i. e., ≥10. 0% of VAF (black line), and 35 cases with low-VAF TP53 mutations (TP53mut_lowVAF), i. e., <10. 0% of VAF (red line). C Kaplan-Meier curves comparing OS probabilities of 95 cases lacking del17p and TP53 mutations (non-del17p/non-TP53mut, green line), 8 cases with del17p only (del17p/non-TP53mut, black line), 60 cases with TP53 mutations only (non-del17p/TP53mut, red line), and 66 cases with concomitant TP53 mutation and del17p (del17p/TP53mut, blue line). D Kaplan-Meier curves comparing PFS probabilities of 95 cases lacking del17p and TP53 mutations (non-del17p/non-TP53mut, green line), 8 cases with del17p only (del17p/non-TP53mut, black line), 60 cases with TP53 mutations only (non-del17p/TP53mut, red line), and 66 cases with a concomitant TP53 mutation and del17p (del17p/TP53mut, blue line). In Kaplan-Meier curves, cases with more than one mutation are classified according to the mutation with the highest VAF (see Supplementry Table S3 ). The number of patients in each group is reported; P values refer to the logrank test. E Clonal evolution of TP53 mutations in longitudinal samples relapsed under ibrutinib treatment (relapsed cases). Graph reports results for 16 CLL patients (65 TP53 mutations) longitudinally studied at two different time-points, the 1 st (x-axis) collected at ibrutinib start and the 2 nd (y-axis) collected after ibrutinib interruption for relapse; overall, VAF values are referred to 65 TP53 mutations, as measured at the two time-points. F Clonal evolution of TP53 mutations in longitudinal samples during ibrutinib treatment (non-relapsed cases). Graph reports results for 22 CLL patients (62 TP53 mutations) longitudinally studied at two different time-points, the 1 st (x-axis) collected at ibrutinib start and the 2 nd (y-axis) collected during ibrutinib treatment; overall, VAF values are referred to 62 TP53 mutations, as measured at the two timepoints. The red color denotes a mutation with a VAF increase greater than 20%. The green color denotes a mutation with a VAF decrease greater than 20%. The dotted parallel lines denote the 20% interval on either side. increase) in 3/4 and 2/3 cases, respectively (Supplementry Table S5 ), suggesting their possible role in ibrutinib resistance. BTK and PLCG2 mutations, were retrieved in 3/7 relapsed cases presenting a positive selection for TP53 mutations at the relapse time (Supplementry S5). Overall, BTK and PLCG2 mutations were discovered in 9/16 (56%) relapsed cases versus 3/22 (14%) patients under ibrutinib treatment (P = 0. 006, χ 2 test; Supplementry Table S5 ). \n\nIn conclusion, here we provided evidence that only the copresence of TP53 deletion and mutations, the latter even with a low-VAF representation, and not the single aberrations have a negative prognostic impact in CLL patients under ibrutinib treatment. In practice, this finding points toward the need of a complete assessment of TP53 aberrations to be performed in all CLL patients prior to start ibrutinib treatment. A lower threshold for reporting TP53 mutations (e. g. VAF < 10%) must be evaluated in prospective clinical trial cohorts before it can be accepted as standard for routine practice. Moreover, low-VAF TP53 mutations should be always confirmed by orthogonal assays (e. g. ddPCR) or by repetition [4].",
"section_name": "",
"section_num": ""
}
] |
[
{
"section_content": "",
"section_name": "DATA AVAILABILITY",
"section_num": null
},
{
"section_content": "The data that support the findings of this study are available from the corresponding author upon request.",
"section_name": "DATA AVAILABILITY",
"section_num": null
},
{
"section_content": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "RB, designed the study, interpreted data, and wrote the manuscript; FMR, FV, TB, AZ, RP, ET, FP, MD, GF, performed and interpreted molecular studies, and contributed to data interpretation; FV, JP, and RB generated the bioinformatics pipeline of analysis, and performed statistical analyses; PB, RM, GR, LL, JO, AC, RL, MP, MIDP, AC, MG, FM, AT, FZ, RF, FDR, GDP collected clinical data and contributed to data interpretation; VG designed the study, interpreted data, and wrote the manuscript. All the Authors agreed on the final form of the manuscript with the only exclusion of GDP (deceased).",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The present study is supported in part by: Progetto Ricerca Finalizzata",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "The authors declare no competing interests.",
"section_name": "COMPETING INTERESTS",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi. org/10. 1038/s41375-023-01845-9. \n\nCorrespondence and requests for materials should be addressed to Riccardo Bomben or Valter Gattei. \n\nReprints and permission information is available at http://www. nature. com/ reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "ADDITIONAL INFORMATION Supplementary information",
"section_num": null
}
] |
10.1371/journal.pone.0039725
|
NOTCH1 Signaling Promotes Human T-Cell Acute Lymphoblastic Leukemia Initiating Cell Regeneration in Supportive Niches
|
Leukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated.We established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34(+) cells from NOTCH1(Mutated) T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1(Wild-type) CD34(+) cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1(Mutated) CD34(+) fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1(Mutated) T-ALL LIC-engrafted mice and resulted in depletion of CD34(+)CD2(+)CD7(+) cells that harbor serial transplantation capacity.These results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies.
|
[
{
"section_content": "Seminal research suggests that leukemia relapse occurs because standard chemotherapy fails to eradicate self-renewing leukemia initiating cells (LIC) [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]. While human myeloid leukemia xenograft studies demonstrate that LIC reside at the apex of a cellular hierarchy and are capable of serially transplanting leukemia [1] [2] [3] 6], cellular subpopulations within diagnostic precursor B cell acute lymphoblastic leukemia (ALL) samples demonstrate greater functional and genetic heterogeneity [16, 17]. Recently, DNA copy number alteration (CNA) profiling coupled with xenograft analysis suggested that patients with BCR-ABL1 ALL harboring a predominant clone at diagnosis have increased rates of early relapse thereby linking LIC clonal dominance with a poorer prognosis [18]. \n\nIn another leukemia subtype that is prone to early relapse [19], pediatric T cell acute lymphoblastic leukemia (T-ALL), serially transplantable LIC were found to be enriched in CD34 + CD4 2 and CD34 + CD7 2 fractions of newly diagnosed patient samples [12]. However, these results were obtained after suspension culture-mediated expansion prior to transplantation potentially leading to changes in LIC functional capacity. More recently, a CD7 + CD1a 2 glucocorticoid resistant LIC population, capable of engrafting leukemia in NOD/SCID IL2Rcnull (NSG) mice, was identified in primarily adult T-ALL without an in vitro expansion step [10]. While the LIC population was found to be an essential driver of therapeutic resistance and relapse, the NOTCH1 mutational status of the LIC population was not established; the cell surface phenotype changed during the prolonged engraftment period and niche dependence of LIC maintenance, which could ultimately contribute to relapse, was not elucidated. The high propensity for T-ALL relapse underscores the need for LIC characterization based on functional molecular drivers of survival and self-renewal and spatiotemporal tracking of niche dependence in bioluminescent serial xenotransplantation models. Together these compelling studies provided the impetus for investigating the potential LIC propagating capacity of NOTCH1 mutations, implicated in T-ALL therapeutic resistance [10] and sensitivity to targeted NOTCH1 inhibition within selective niches. \n\nWhile T-ALL represents only 25% of adult and 15% of pediatric ALL cases, they share an increased risk of early systemic and isolated central nervous system relapse often in the setting of mutational NOTCH1 signaling pathway activation [20]. A recent series of studies showed that NOTCH activation is associated with improved early therapeutic response (reviewed in [21] ). However, this early benefit translates into improved overall survival only in some series, most probably as a result of differences in therapy, and suggests that NOTCH-targeted therapies might represent promising therapeutic strategies. During normal hematopoiesis, NOTCH1 regulates cell fate decisions, proliferation and survival following ligand binding, which triggers a conformational change in the negative regulatory region (NRR) of the extracellular domain, enabling juxtamembrane ADAM protease cleavage [22, 23]. Subsequently, c-secretase complex mediated intramembrane proteolysis releases an intracellular domain of NOTCH1 (ICN1), which translocates to the nucleus and activates transcription of NOTCH target genes [22, 24]. In T-ALL, somatic activating mutations in the NOTCH1 heterodimerization domain (HD) or PEST domain or alternatively loss-of-function mutations in FBXW7, a NOTCH1 E3 ubiquitin ligase, increase release or stability of ICN1. This, in turn, leads to transcriptional activation of genes that promote proliferation and survival such as MYC and HES1 [22, 24]. \n\nDespite a plethora of reports describing mechanisms of NOTCH1 activation in T-ALL, the cell type and context specific role of NOTCH1 activation in the maintenance of therapeutically resistant self-renewing human LIC has not been established. Thus, we sought to examine (1) whether molecularly characterized LIC can be identified among specific hematopoietic subpopulations in pediatric T-ALL without preceding in vitro culture, (2) the role of NOTCH1 activation in LIC propagation, and (3) whether LIC have an intrinsic predilection for specific hematopoietic niches. For these purposes, lentiviral luciferase-transduced CD34-enriched (CD34 + ) and CD34-depleted (CD34 -) cells from molecularly characterized samples were transplanted into neonatal RAG2 2/2 c c 2/2 mice that permit high levels of human hematopoietic engraftment [6, 7]. In this study, the CD34 + fraction of pediatric NOTCH1 Mutated T-ALL samples had enhanced survival and self-renewal potential, characteristic of LIC, compared with their CD34 + NOTCH1 wild-type (NOTCH1 WT ) counterparts. These NOTCH1 Mutated LIC were uniquely susceptible to targeted inhibition with a therapeutic human NOTCH1 monoclonal antibody selective for the NRR (hN1 mAb), while normal hematopoietic progenitors were spared thereby highlighting the cell type and context specific effects of NOTCH signaling [13, [25] [26] [27] [28] [29] [30] and the importance of oncogenic addiction to NOTCH1 signaling in T-ALL LIC maintenance.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Molecular characterization of CD34 + cells from 12 T-ALL patient samples was performed by targeted exon sequencing analysis and focused on genes commonly mutated in T-ALL, including NOTCH1, PTEN, PIK3R1 and FBXW7 (Table 1 ). Selective NOTCH1 DNA sequencing revealed activating mutations in six of eleven newly diagnosed pediatric T-ALL samples and in one relapsed young adult T-ALL sample (Table 1 ). In addition, CD34 + T-ALL cells derived from these 12 samples were further sequenced to identify PI3K, PTEN and FBXW7 pathway mutations common to pediatric T-ALL. Some cases harbored mutations in PTEN (patients 01, 05, 06, 11) or PIK3R1 (patient 05) genes (Table 1 ) [31] [32] [33] [34] [35]. These data demonstrate that mutations in NOTCH1 and other genes capable of promoting LIC survival co-exist in the CD34 + fraction of T-ALL samples.",
"section_name": "T-ALL Molecular Characterization",
"section_num": null
},
{
"section_content": "To determine if lentiviral luciferase-transduced CD34 + and CD34 2 cells from NOTCH1 Mutated and NOTCH1 WT pediatric T-ALL samples differed in their capacity to propagate disease, quantitative non-invasive bioluminescent imaging was performed within 10 weeks of intrahepatic transplantation of neonatal RAG2 2/2 c c 2/2 mice (Figures 1A and B ). Mice transplanted with CD34 + enriched NOTCH1 Mutated T-ALL cells (patients 03, 05, 08, 11) demonstrated significantly greater leukemic engraftment than mice transplanted with CD34 2 cells (Figures 1B and C ; n = 79 mice, P = 0. 0005, Student's t-test). Conversely, both CD34 + and CD34 2 fractions from NOTCH1 WT T-ALL samples (patients 04, 07, 09, 10) exhibited equivalent engraftment capacity in primary transplant recipients (Figure 1C ; n = 76 mice). Hence, CD34 + cells from NOTCH1 Mutated samples gave rise to higher levels of bioluminescent engraftment in primary transplant recipients than their CD34 2 counterparts, indicative of LIC enrichment in the CD34 + fraction in NOTCH1 Mutated but not NOTCH1 WT samples. \n\nThe predilection of NOTCH1 Mutated T-ALL LIC for specific hematopoietic niches was determined in primary and serial transplants. Primary human NOTCH1 Mutated T-ALL CD34 + LIC engraftment was typified by thymic (Figure 1D ) and splenic (Figure 1D ) enlargement as well as pale marrow due to replacement by leukemic cells (Figure 1D ). Further analysis revealed that thymic (P,0. 01; Student's t test) and splenic (P,0. 001, Student's t-test) weights were significantly greater in both primary (1u) and secondary (2u) NOTCH1 Mutated T-ALL LIC transplant recipients than in no transplant control mice (Figures S1A and B ). Moreover, FACS analysis revealed robust CD34 + cell engraftment in marrow, spleen and thymus of primary and secondary NOTCH1 Mutated T-ALL LIC transplanted mice (Figures 1E and F ). NOTCH1-driven LIC have enhanced survival and self-renewal potential in supportive hematopoietic microenvironments.",
"section_name": "NOTCH1 Mutated T-ALL LIC are Serially Transplantable",
"section_num": null
},
{
"section_content": "The relative leukemic regenerative potential of NOTCH1 Mutated (Patients 03, 05, 08, 11), and NOTCH1 WT (Patients 04, 07, 09, 10) samples was determined in serial transplantation studies. FACS analysis of cells from bone marrow, spleen and thymus showed that while the levels of thymic engraftment were equivalent, NOTCH1 Mutated T-ALL LIC gave rise to a significantly higher CD34 + leukemic burden in the marrow and spleen of primary transplant recipients than NOTCH1 WT T-ALL samples (Figures 2A and B ; ***, P,0. 001; Student's t test). Hence, we sought to determine whether (1) selective NOTCH1 inhibition could reduce LIC burden, (2) NOTCH1 Mutated T-ALL LIC survival is dependent on activated NOTCH1 receptor signaling, and (3) selective NOTCH1 inhibition could spare NOTCH1 WT or normal cord blood CD34 + progenitors in engrafted mice. For these purposes, NOTCH1 Mutated T-ALL LIC-engrafted mice were treated with a selective NOTCH1-NRR/Fc mAb (hN1 mAb) that specifically inhibits NOTCH1 receptor signaling (Figures S2A and B ). \n\nAnimals were treated with hN1 mAb (10 mg/kg) or a control mouse IgG1 mAb every 4 days for 3 weeks, and over this time period both antibodies were well-tolerated in treated animals 1 ), were selected using immunomagnetic beads from T-ALL blood or bone marrow, transduced with lentiviral luciferase and transplanted (50000 cells/ mouse) intrahepatically into RAG2 2/2 c c 2/2 mice within 48 hours of birth. Engraftment was monitored over 10 weeks via non-invasive bioluminescent imaging system (IVIS 200, Caliper LifeSciences). After 10 weeks, mice were sacrificed and secondary transplants were performed with 50000 immunomagnetically-purified human CD34 + or CD45 + cells from primary CD34 + and CD34 (Figure S2C ). As anticipated, treatment with the hN1 mAb had no detectable toxicity (e. g. intestinal) or deleterious effects on survival in mice, as this antibody does not bind to endogenous murine NOTCH1 and is expected to only target activity of human NOTCH1 in transplanted human cells. Following hN1 mAb treatment of NOTCH1 Mutated T-ALL LIC-transplanted mice, FACS analysis revealed a significant reduction in leukemic CD34 + cell burden in both the marrow and spleen of hN1 mAb-treated mice (Figure 2C ; **, P = 0. 003 and Figure 2D, ***, P = 0. 001, respectively, Wilcoxon test). Levels of CD34 + cell burden in the thymus were similar in both groups (Figure 2E ), which is likely a result of relatively lower engraftment rates in this hematopoietic organ (Figure 2B ). Notably, LIC from one T-ALL NOTCH1 Mutated patient sample (patient 11), with a PTEN frame-shift mutation, retained sensitivity to hN1 inhibition (Figure 2 ). While survival of LIC from a sample (patient 05) with both PTEN and PIK3R1 mutations was not significantly inhibited in the bone marrow, LIC burden was significantly reduced in the spleen by hN1 mAb treatment (Figure 2C and D ), highlighting the influence of additional mutations and microenvironmental context in responses to selective NOTCH1 inhibitory strategies. Although engraftment rates of normal human cord blood progenitors were low, the survival of normal CD34 + hematopoietic progenitors was not significantly reduced by targeted NOTCH1 inhibition (Figures S3A and B ). These results suggest a greater functional dependence of NOTCH1 Mutated T-ALL LIC on NOTCH1 signaling in selective hematopoietic niches compared to NOTCH1 WT progenitors and normal hematopoietic stem cells (HSC). \n\nFollowing hN1 mAb treatment, immunohistochemical analyses revealed a marked increase in levels of activated caspase 3, and a concomitant reduction in levels of NOTCH1 in NOTCH1 Mutated (patient 08) T-ALL LIC-engrafted bone marrow compared with control IgG1 mAb-treated control bone marrow (Figures 3A and B ; **, P = 0. 005; *, P,0. 05, Student's t test). To assess whether hN1 mAb treatment could inhibit the generation of transcriptionally active NOTCH1 (intracellular NOTCH1, ICN1), which may be involved in promoting therapeutic resistance through induction of self-renewal, ICN1 immunohistochemical analysis was performed on bone marrow derived from the NOTCH1 Mutated LIC-engrafted mice after treatment with hN1 mAb or IgG1 control mAb. Treatment with the hN1 mAb was associated with a reduction in bone marrow ICN1 levels (Figure 3C ). These data corroborate that the antibody's mechanism of action involves both interference with ligand binding (Figure S2B ) and reduced human NOTCH1 cleavage and ICN1 generation (Figure 3C ). \n\nStrikingly, qRT-PCR analysis demonstrated a significant reduction in NOTCH1 expression in a subset of NOTCH1 Mutated T-ALL samples (patients 08, 11) after treatment with hN1 mAb (Figure 3D, **, P,0. 01; *, P,0. 05). One NOTCH1 Mutated sample (patient 05) showed only a trend towards reduction in NOTCH1 expression after hN1 mAb treatment. However, this patient harbored multiple additional mutations in other pathways (Table 1 ) that could contribute to resistance of the LIC population. Of note, one patient sample (patient 02) did not harbor a NOTCH1 mutation, as determined by DNA sequencing (Table 1 ), but exhibited increased NOTCH1 transcript levels (NOTCH1 High ) compared to cord blood progenitors (Figure S1C ), as well as increased bone marrow serial transplantation potential (Figure S4 ). Similar to the NOTCH1 Mutated samples, the CD34 + population of cells in the NOTCH1 High sample was reduced following hN1 mAb treatment compared to control IgG. \n\nAlternative NOTCH1 inhibitory strategies also recapitulated the effects of antibody-mediated NOTCH1 pathway downregulation, as demonstrated by in vitro lentiviral NOTCH1 shRNA knock down experiments (Figure S5 ). NOTCH1-shRNA treatment of CD34 + cells derived from NOTCH1 High (patient 02) or NOTCH1 Mutated (patient 05) samples resulted in reduced expression levels of NOTCH1 mRNA (Figure S5A ) and downstream target genes (c-MYC) (Figure S5B ). hN1 mAb Treatment Inhibits NOTCH1-Driven LIC Selfrenewal Following hN1 mAb treatment, FACS analysis revealed a reduction in both the populations of CD45 + CD34 + cells (Figure 4A, upper panel) and immature T cells identified by CD34 + CD2 + immunoreactivity (Figure 4A, lower panel) in NOTCH1 Mutated LIC (Patient 11) engrafted mouse bone marrows, as well as a reduction of the CD34 + NOTCH1 + cell population in bone marrow and spleen (Figure 4B ). To determine whether hN1 mAb treatment inhibited NOTCH1-driven LIC self-renewal, human CD34 + cells selected from the bone marrows of hN1 mAb (n = 4) and IgG1 mAb (n = 3) treated mice were serially transplanted into untreated secondary (2u) recipients. After 12 weeks, FACS analysis showed that mice transplanted with human CD34 + T-ALL cells obtained from control IgG1 mAb-treated mice exhibited higher CD45 + CD34 + leukemic burden compared to mice transplanted with CD34 + cells obtained from hN1 mAb-treated mice (Figure 4C ). Conversely, hN1 mAb-treated human T-ALL LIC Enrichment of LIC in the CD45 + CD34 + CD2 + CD7 +",
"section_name": "hN1 mAb Treatment Reduces NOTCH1 Mutated T-ALL LIC Survival",
"section_num": null
},
{
"section_content": "Recent studies have highlighted the importance of CD7 expression in discriminating the LIC population in T-ALL [10, 36]. In this context, we hypothesized that early T cell markers such as CD7 and CD2 might be enriched, and these populations might be serially transplantable, in NOTCH1-driven T-ALL LIC xenografted mice. While overall engraftment was similar between NOTCH1 High and NOTCH1 Mutated transplanted samples (Figure 5A (patients 04, 09, 10) T-ALL samples and cord blood (Figure 5B ). Following serial transplantation of NOTCH1-activated LIC, FACS analyses revealed that the CD45 + CD34 + CD2 + CD7 + population harbored serial leukemic transplantation potential at limiting doses (Table 2 and Figures 6A-C ). To test our hypothesis that the CD2 + CD7 + subset of the CD34 + human progenitor population identifies a LIC-enriched population in NOTCH1 Mutated T-ALL samples, CD34 + CD2 + CD7 + Lin -cells from NOTCH1 Mutated T-ALL samples (Patients 05 and 11) were FACS Aria purified and serial transplantations were performed. Serial transplantation of 1 500 CD34 + CD2 + CD7 + Lin 2 cells sorted from a NOTCH1 Mutated T-ALL (Patient 05) sample resulted in marked thymic enlargement, splenomegaly and pale marrows indicative of robust leukemic engraftment (Table 2 and Figure 6A ). Tertiary transplant experiments revealed that the human CD45 + CD34 + CD2 + CD7 + population propagated leukemia and seeded hematopoietic niches, which was demonstrative of LIC self-renewal capacity (Figures 6B and C ). As further evidence that this model recapitulates features of the human disease, infiltration of human CD45 + cells was detected in the brains of mice that received 3u transplants of the enriched LIC population (CD34 + CD2 + CD7 + ) from patient 11 (Figure S6 ). \n\nWe then sought to determine whether this LIC-enriched population was sensitive to hN1 antibody treatment. Remarkably, compared to control mAb-treated animals transplanted with the bulk CD34 + population, in hN1 mAb-treated T-ALL LIC engrafted mice, there was a significant reduction in the CD34 + CD2 + CD7 + population, but not the CD34 + CD2 + CD7 2 population (Figure 5C ). Taken together, in NOTCH1 Mutated T-ALL samples, a CD45 + CD34 + CD2 + CD7 + population is enriched for LIC that demonstrate serial leukemic transplantation capacity, and these cells are selectively targeted and depleted by hN1 antibody therapy.",
"section_name": "Population and Depletion Following hN1 Antibody Treatment",
"section_num": null
},
{
"section_content": "Cumulative reports reveal the protean nature of NOTCH signaling in the maintenance of normal and malignant hematopoiesis [13, [24] [25] [26] [27] 37, 38]. While Notch2 signaling regulates regeneration of mouse long-term HSC, ligand-driven NOTCH1 activation induces human hematopoietic progenitor expansion and differentiation [28, 39]. Ligand binding to the NOTCH1 extracellular domain activates ADAM family metalloprotease and c-secretase complex-mediated cleavage and intracellular release of the NOTCH1 intracellular domain (ICN1). Subsequently, nuclear translocation of ICN1 followed by engagement of transcriptional activators such as CBF1/Su(H)/Lag2 (CSL) and mastermind-like (MAML) sets the stage for NOTCH1 target gene transcription. Conversely, activation of NOTCH1 signaling through gain-offunction mutations in NOTCH1, first described in T-ALL [24], or loss-of-function mutations in NOTCH1 regulators, such as FBXW7 and NUMB, has been linked to therapeutic recalcitrance of hematologic malignancies [40, 41]. Chronic antagonism of both NOTCH1 and NOTCH2 processing with small molecule inhibitors of the c-secretase complex has been associated with loss of intestinal crypt progenitor cells, thereby providing the impetus for development of selective NOTCH1 inhibitors [42]. Recent pre-clinical studies demonstrate that inhibition of NOTCH1 using synthetic stapled peptides or monoclonal antibody-mediated strategies effectively decreases T-ALL cell line growth [22, 23]. However, the consequences of selective NOTCH1 inhibition for normal hematopoietic progenitor and patientderived T-ALL LIC survival and self-renewal have been unclear. \n\nIn this study, CD34 + cells from 6 of 12 T-ALL samples harbored NOTCH1 activating mutations. In these patients, NOTCH1 Mutated CD34 + LIC had greater engraftment and serial transplantation potential than their CD34 2 counterparts. Conversely, both CD34 + and CD34 2 subpopulations from NOTCH1 WT samples harbored roughly equivalent bioluminescent engraftment potential, albeit at lower levels than NOTCH1 Mutated LIC and with lower serial transplantation capacity. With the exception of one sample (patient 02) that harbored high NOTCH1 transcript levels in the absence of identifiable NOTCH1 mutations, bioluminescent imaging and FACS analyses of leukemic engraftment suggest that phenotypic markers other than CD34 will be needed to identify LIC in the NOTCH1 WT samples. In contrast to experiments with NOTCH1 WT and normal cord blood CD34 + samples, NOTCH1 Mutated LIC survival was significantly impaired by selective hN1 mAb-mediated inhibition, concomitant with reductions in ICN1 and NOTCH1 mRNA expression and protein levels. Furthermore, serial transplantation potential was also reduced by hN1 mAb treatment of mice transplanted with NOTCH1activated T-ALL samples. Thus, NOTCH1 Mutated CD34 + cells from these pediatric T-ALL patients constitute the apex of a leukemic hierarchy. \n\nNotably, patient samples with NOTCH1 activation, conferred either by mutation or elevated WT NOTCH1 expression levels, show enrichment of a subset of the CD34 + human progenitor cell population distinguished by co-expression of CD2 and CD7. Seminal studies reveal that CD7 expression enriches for a therapeutically recalcitrant LIC population [10, 36]. Our analyses of the serial transplantation capacity of the CD34 + CD2 + CD7 + population reveal that this population is maintained over multiple generations of T-ALL LIC transplantation, and these cells harbor robust leukemic initiating potential in medullary and extramedullary reservoirs of resistance. In experiments aimed at elucidating the fate of these cells in mice treated with hN1 mAb, we observed a significant reduction in this population compared to animals that received control IgG1 antibody. Taken together, these data further refine the markers that identify LIC in NOTCH1 Mutated T-ALL samples, and demonstrate that the CD34 + CD2 + CD7 + population is sensitive to and depleted following hN1 mAb treatment. While in the present studies, our analyses of the refined LIC marker were focused on the NOTCH1 Mutated samples, additional markers, or activation of other receptor-mediated signaling pathways such as insulin-like growth factor 1 receptor [43], may also be informative to determine the leukemic potential of LIC in non-NOTCH1 Mutated T-ALL patients. \n\nWhile mutations in tumor suppressor genes co-exist in some samples, NOTCH1 Mutated T-ALL LIC appear to be oncogenically addicted to NOTCH1 activation, rendering them uniquely susceptible to inhibition with a NOTCH1-targeted mAb, hN1. In contrast, hN1 mAb treatment did not significantly impair the survival of normal human hematopoietic progenitor cells. This favorable therapeutic index may be explained, at least in part, by mouse models of hematopoiesis, which demonstrate that Notch2, rather than Notch1, regulates mouse HSC regeneration [30]. In summary, characterization of LIC based on functional molecular drivers provides a useful paradigm for identification and selective elimination of malignant stem cells. Moreover, these findings provide a compelling rationale for clinical evaluation of hN1 mAb therapy in clinical trials aimed at eliminating self-renewing LIC that promote therapeutic resistance and relapse in T-ALL and potentially in other NOTCH1-driven malignancies.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Dr. Jamieson is the PI on an existing Institutional Review Board (IRB) approval for tissue banking, entitled ''Protocol 070678: Permission to Collect Blood and/or Bone Specimens and/or Tumor Samples and/or Saliva from Patients with Hematology Problems for Research (Adult). '' Approval was obtained from the UCSD Human Research Protections Program. Consent is always written, and clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The Human Research Protections Program office assists researchers in complying with federal, state and University policies regarding experimentation involving human subjects, and oversees the review and conduct of research conducted by federally registered IRBs. This study was carried out in strict accordance with the recommendations of the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego. The protocol was approved by the Committee under Animal Use Protocol Number S06015. All efforts were made to minimize suffering.",
"section_name": "Ethics Statement",
"section_num": null
},
{
"section_content": "",
"section_name": "Subjects and Samples",
"section_num": null
},
{
"section_content": "Methods required for establishment of bioluminescent LIC models using lentivirus-luciferase transduced primary patient samples in neonatal RAG2 2/2 c c 2/2 mice were described previously [6]. All animal experiments were approved by the Animal Experimental Committee of the University of California San Diego and were performed according to NIH recommendations for animal use.",
"section_name": "Bioluminescent Humanized T-ALL LIC Models",
"section_num": null
},
{
"section_content": "Immunophenotypic analysis was performed on all samples (FACSAria II system, BD Biosciences, Franklin Lakes, NJ). Human CD34 + cells were purified from T-ALL peripheral blood using a CD34 MicroBead Kit (Miltenyi Biotec, Auburn, CA) and CD34 + cell purity was assessed by FACS. For FACS sorting, mouse IgG1s conjugated to PE, FITC, or APC were used as isotype controls (BD Biosciences). Human CD45, CD34, CD38, CD2, and CD7 expression was assessed using anti-CD45-V450, anti-CD34-APC, anti-CD38-PE-Cy7, anti-CD2-FITC and anti-CD7-PE, respectively, together with a lineage cocktail including PE-Cy5. 5-conjugated antibodies against human CD4, CD8, CD14, CD19 and CD56. All antibodies for cell sorting FACS analysis were obtained from BD Biosciences. Propidium iodide (PI) was used to exclude dead cells and at least 10000 events were acquired for each sample.",
"section_name": "Human CD34 Initiating Cell Isolation and Immunophenotypic Analysis",
"section_num": null
},
{
"section_content": "Approximately 50000 human CD34 + and CD34 2 cells derived from both NOTCH1 Mutated and NOTCH1 WT samples (defined by DNA sequencing) were selected from T-ALL blood or bone marrow with the aid of immunomagnetic beads or FACSAria, and transduced with lentiviral luciferase which effectively transduced an average of 15-25% of cells, as measured by FACS (data not shown). Cells were transplanted intrahepatically into RAG2 2/2 c c 2/2 mice within 48 hours of birth [6]. For NOTCH1 shRNA knock down experiments, 100000 CD34 + cells selected from the T-ALL patient samples and cord blood (All Cells, Emeryville, CA) were transduced with lentiviral vectors expressing shRNA targeting human NOTCH1 (Dharmacon, Lafayette, CO).",
"section_name": "Lentiviral Transduction and Transplantation",
"section_num": null
},
{
"section_content": "Humanized LIC and normal hematopoietic progenitor mouse models were established by intrahepatic transplantation of neonatal RAG2 2/2 c c 2/2 mice with lentiviral luciferase-transduced CD34 + progenitor cells from 12 T-ALL samples or normal cord blood samples. Engraftment was monitored with the aid of an IVIS 200 system (Caliper Life Sciences, Inc. ). LIC mouse models were dosed starting at 6 weeks of age with either NOTCH1 mAb or IgG1 mAb control (both provided by Pfizer, Inc., La Jolla, CA) at 10 mg/kg intraperitoneally every 4 days for an average of 6 doses. To test whether hN1 mAb treatment could eliminate LIC in humanized T-ALL mouse models, mice were sacrificed one day after the last dose. At this endpoint (approximately 10 weeks of age) the majority of animals were sacrificed before the disease was evident. Bone marrow, spleen, thymus and liver were collected and processed for FACS analysis of human CD45, CD34, CD2, CD7 and NOTCH1 expression. NOTCH1 FACS analysis was performed using the same mAb as was used for treatment. Immunohistochemistry was performed using antibodies against NOTCH1 (1:50 dilution, Pfizer, Inc. ), CD45 (1:100 dilution, Cell Signaling, Danvers, MA), activated caspase 3 (1:50 dilution, Abcam, Cambridge, MA), as well as NOTCH1 intracellular domain (1:50 dilution, ICN1, Epitomics, Burlingame, CA) to examine expression levels in bone marrow sections following treatment with either control IgG1 mAb or hN1 mAb.",
"section_name": "Establishment, Treatment and Analysis of LIC Mouse Model",
"section_num": null
},
{
"section_content": "All statistical tests were performed for two sided p values. Continuous variables for each comparison group were assessed for distribution through univariate statistics. If the assumption of normal distribution could be supported, then the Student's t-test was performed for comparison of two samples with assessment of equality of variance with an F statistic. If the assumption of normal distribution was not supported, nonparametric testing was performed with the two sample Wilcoxon test using the t approximation for samples with N of less than 20. sorted from a NOTCH1 Mutated T-ALL (Patient 11) revealed the persistence of an expanded human CD45 + CD34 + CD2 + (including CD7 + and CD7 2 ) population in the transplanted mouse hematopoietic organs (bone marrow, spleen, thymus and liver). doi:10. 1371/journal. pone. 0039725. g006",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "Figure S6 Human cell infiltration in the tertiary CD34 + CD2 + CD7 + cells transplanted mouse brain. 30 000 CD34 + CD38 + CD2 + CD7 + Lin 2 cells sorted from T-ALL patient 11 with the aid of FACS were intrahepatically transplanted into neonatal RAG2 2/2 g c 2/2 mice, and serial transplantation were done by 50 000 mouse BM cells. Tertiary transplanted mouse brain was fixed in 4% PFA and 30% sucrose overnight, separately, and then embedded in OCT for section with the thickness of 16 mm. Mouse brain sections were stained with antihuman CD45 antibody, mounted with prolong gold (Invitrogen). Images were taken under the Fluoview FVi10 confocal microscope (Olympus).",
"section_name": "Supporting Information",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "We are grateful to Dennis Young of the University of California at San Diego Moores Cancer Center FACS facility for his expert assistance with FACS Aria analysis and sorting; Drs Mitchell B. Diccianni, Edward Kavalerchik, and Edward D. Ball for providing T-ALL patient samples and Ming Qiu and Qinghai Peng of the Oncology Research Unit at Pfizer Inc. for their technical assistance with hN1 antibody characterization. We thank Kimberly Wilson for excellent administrative support. The antibody utilized in this study was provided by Pfizer.",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This work was supported by the Ratner Family Foundation, the Leichtag Family Foundation, and Moores Cancer Center Donor Funds. Dr. Gutierrez 's work is supported by the NIH (grant 1K08CA133103 ), the William Lawrence Foundation, and the American Society of Hematology-Amos Medical Faculty Development program. Dr. Look 's work was funded by the NIH (grant 5P01CA68484 ). Dr. Jamieson 's work was supported by the California Institute for Regenerative Medicine (CIRM) Scientific Excellence through Exploration and Development (SEED) program, a CIRM New Faculty Award, and CIRM Disease Team and CIRM Early Translational ll grants. A portion of this research study was funded by a sponsored research agreement with Pfizer ( Pfizer/28-1076 ), and the antibody utilized in this study was provided by Pfizer. Authors Li, Gibson, and Wei were employees of Pfizer during the study, and these authors provided reagents for use in this study and contributed to data collection and analysis for antibody validation experiments.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Author Contributions",
"section_num": null
}
] |
10.1016/j.hoc.2013.01.006
|
Ibrutinib (PCI-32765) in Chronic Lymphocytic Leukemia
|
B-cell receptor (BCR) signaling is essential for chronic lymphocytic leukemia (CLL) cell survival. Many kinases in the BCR signaling pathway are being studied as potential therapeutic targets. Ibrutinib (PCI-32765) is a novel first-in-class selective inhibitor of Bruton tyrosine kinase. Preclinical evidence suggests that ibrutinib inhibits CLL cell survival and proliferation and affects CLL cell migration and homing. Early clinical data in patients with CLL and non-Hodgkin lymphoma is encouraging. It is likely that ibrutinib and other drugs targeting the BCR pathway will become an integral component of CLL therapy.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world with approximately 16,060 men and women expected to be diagnosed with CLL in year 2012 in the United States. 1 Most patients with CLL do not need treatment at diagnosis; however, the majority of patients will need CLL-directed therapy in their lifetime. Chemoimmunotherapy is the current standard of care for patients with CLL needing treatment. 2 One of the commonly employed regimens is FCR (fludarabine, cyclophosphamide, rituximab). Tam and colleagues reported long-term follow-up of the FCR regimen for frontline treatment of CLL with a complete remission (CR) rate 72% and median progression-free survival (PFS) of 80 months. 3 Despite these impressive results, certain subgroups of patients treated with chemoimmunotherapy have less than optimal outcomes. ] [7] [8] [9] [10] [11] These treatment strategies offer options after relapse post chemoimmunotherapy but outcomes are still less than satisfactory with approximately 4500 patients expected to die from CLL in the United States in the year 2012. 2 This underscores the need to develop better therapeutics for patients with CLL. 6] [17] Binding of a ligand to the membrane immunoglobulin leads to recruitment and phosphorylation of spleen tyrosine kinase (Syk) and Src family kinases (Lyn), which in turn recruit and phosphorylate many kinases and adapter proteins including Bruton tyrosine kinase (BTK). BTK is a nonreceptor tyrosine kinase of the Tec kinase family and plays a crucial role in BCR signaling. 18, 19 BTK is expressed in non-T cell hematopoietic cell lineages. 20, 21 The BTK gene is located on chromosome Xq21. 33-q22 and mutations in this gene result in X-linked agammaglobulinemia, a condition characterized by marked reduction in mature B-cells, severe hypogammaglobulinemia, and increased susceptibility to infections. 22 BTK activates downstream molecules such as nuclear-factor-kappa B and MEK/ERK, which are involved in many cellular processes including proliferation, survival, differentiation, apoptosis, and metabolism. Gene expression profiling has shown that BCR signaling is the most expressed signaling pathway in patients with CLL. 14 BCR signaling is enhanced in patients with poor prognostic markers such as ZAP-70 overexpression and those with unmutated immunoglobulin heavy chain gene (IGHV) rearrangement. 23, 24 Activated BCR signaling has also been shown to be required for cell survival in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). 25 Thus, multiple lines of data point to the crucial role of BCR signaling in CLL and other B-cell lymphoid malignancies. Many kinases in the BCR signaling pathway are now being pursued as therapeutic targets in CLL including Lyn, 26 Syk, [27] [28] [29] PI3K, [30] [31] [32] and BTK. 16, 17 rutinib (formerly PCI-32765, Pharmacyclics, Sunnyvale, California) (Figure 1 ) is an oral, selective and irreversible inhibitor of BTK and is the focus of this paper. Ibrutinib was initially developed by Celera Genomics (now Quest Diagnostics, Madison, New Jersey) and acquired by Pharmacyclics in 2006. Ibrutinib forms a specific bond with the cysteine-481 of BTK. 33 It leads to highly potent BTK inhibition with an IC50 0. 5nM (Table 1 ). 34 Ibrutinib is orally administered with daily dosing and has no cytotoxic effect on T-cells. 35",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Honigberg and colleagues showed that in a B-cell lymphoma cell line (DOHH2), ibrutinib irreversibly inhibited autophosphorylation of BTK (IC 50 11nM) and phosphorylation of downstream kinases such as ERK. 34 Phosphorylation of upstream kinases such as SYK was not affected. They also showed that ibrutinib blocked the transcriptional upregulation of Bcell activation genes in primary cultures of human peripheral B-cells. 34 In a mouse model for lupus nephritis, ibrutinib reduced proteinuria, lowered anti-dsDNA antibody levels and improved glomerular function, indicating the potential role for ibrutinib in autoimmune diseases. 34 Clinical activity of ibrutinib with once daily oral dosing was also seen in naturally occurring B-cell lymphoma in dogs. 34 rman and colleagues reported that BTK mRNA expression was significantly higher in CLL (CD19+) cells compared to normal B-cells. 35 They also noted that baseline BTK protein expression was highly variable in the CLL cells and protein expression did not correlate with known prognostic markers such as age, cytogenetics, IGHV status and ZAP-70 expression. Treatment of CLL cells with ibrutinib induced apoptosis in a dose-and time-dependent manner which was independent of baseline cytogenetics, IGHV mutational status or baseline BTK protein expression but dependent on caspase-pathway activation. 35 brutinib also induced apoptosis in normal B cells, but this was significantly less than that seen in CLL cells, indicating that CLL cells are more sensitive to ibrutinib than normal B cells. Ibrutinib treatment of CLL cells inhibited downstream signaling pathways including ERK1/2 phosphorylation, CD40L induced AKT phosphorylation and CD40L induced NF-kB DNA binding. 35 nader and colleagues evaluated the role of the tissue microenvironment of CLL cells and its effect on treatment with ibrutinib. 36 They reported that ibrutinib treatment significantly inhibited CLL cell migration and survival in a nurselike cell (NLC) coculture assay. In this model, ibrutinib treatment significantly decreased the levels of CCL3 and CCL4 and inhibited chemotaxis towards CXCL12 and CXCL13. In an adoptive transfer TCL1 mouse model, ibrutinib treatment was reported to delay CLL disease progression. 36 Overall the preclinical data suggests that ibrutinib is a selective, irreversible BTK inhibitor with effect on both CLL cell survival/proliferation and CLL cell migration/homing. 15",
"section_name": "PRECLINICAL STUDIES",
"section_num": null
},
{
"section_content": "Clinical studies with ibrutinib have only been published in abstract form thus far. Ibrutinib was evaluated in a phase I study in CLL and lymphoma [small lymphocytic lymphoma (SLL), follicular lymphoma, mantle cell lymphoma (MCL), DLBCL, marginal zone lymphoma, Waldenstrom macroglobulinemia] patients with a 28-day on / 7-day off schedule in 5 dose-cohorts (1. 25-12. 5 mg/kg orally daily) and once daily continuous dose in 2 dosecohorts (8. 3 mg/kg and 560-mg fixed dose). 37 Fifty-six patients with relapsed/refractory disease [median 3 prior regimens (range 1-10)] were enrolled. No dose-limiting toxicity was observed. MTD was not reached. Of the 50 evaluable patients, 30 (60%) patients achieved an objective response rate (ORR) [23% CR, 77% partial response (PR)]. Responses were seen in all NHL subtypes and irrespective of the dose levels. A unique pattern of response was noted, with a transient lymphocytosis lasting a few months. Transient lymphocytosis was also noted by Ponader and colleagues in an adoptive transfer TCL1 mouse model after treatment with ibrutinib. 36 This is postulated to be due to an initial compartment shift of CLL cells from lymphatic tissues into the peripheral blood. \n\nIn a phase IB/II study (PCYC-1102), patients with relapsed/refractory CLL and older adults (≥65 years) with untreated CLL were treated with 2 fixed doses of ibrutinib (420mg daily and 840mg daily). 38 Ibrutinib was given orally once daily for 28-day cycles until disease progression. Patient enrollment occurred from May 2010 to July 2011. Sixty-one patients were enrolled in the relapsed/refractory cohort (420mg cohort n=27, 840mg cohort n=34). The median age was 64 years (range, 40-81). The median number of prior therapies for the 420mg cohort was 3 (2-10) and for the 840mg cohort was 5 (1-12). High-risk molecular features were present in the majority of the patients [unmutated IGHV:79%; del(17p):36%; del(11q):39%]. The median follow-up for the 420mg cohort was 12. 6 months and for the 840mg cohort was 9. 3 months. Seventy-five percent of the patients were still on the study at the time of last follow-up. Treatment was well tolerated with the majority of adverse-events being grade I/II (diarrhea, fatigue, and nausea). Six patients needed dose reduction (2 in the 420mg cohort, 4 in the 840mg cohort). Grade 3/4 hematologic toxicity (neutropenia, anemia, thrombocytopenia), irrespective of attribution, was seen in 8%, 7%, 7% (420mg cohort) and 21%, 12%, 9% (840mg cohort), respectively. ORR was noted to be 67% (63% PR, 4% CR) in the 420mg cohort and 68% (all PR) in the 840mg cohort. An additional 22% (420mg cohort) and 24% (840mg cohort) of patients achieved nodal PR (>50% reduction in aggregate lymph node size) with residual lymphocytosis. Maximum change in tumor burden is shown in figure 2. Importantly, clinical responses were independent of the high-risk molecular features. Seventy-four percent of the patients with unmutated IGHV, 65% with del(17p), and 73% with del(11q) responded. Most clinical responses were nodal responses in the first 4-5 months of therapy which then improved to PR/CR with continued treatment over the next few months. Estimated 18-month PFS was 87. 7% in the 420 mg cohort. 39 A transient lymphocytosis typically peaking within the first 2 months of treatment, followed by gradual resolution over the next 6-8 months was noted (Figure 3 ). \n\nIn the update of the data presented at the European Hematology Association meeting in June 2012, O'Brien and colleagues reported the outcomes of 31 treatment naïve older patients (420mg, n=26; 840mg, n=5). 39 Enrollment in the 840mg cohort was terminated after similar results were noted between the 420mg and 840mg cohort in the relapsed/refractory cohort. Median age was 71 years (range, 65-84) with 75% of patients being older than 70 years. Forty-three percent patients had unmutated IGHV and 6% had del(17p). The majority of the adverse events (AE) were mild (grade I-II) and included diarrhea, nausea, and fatigue. Grade III non-hematological AE potentially related to the drug was seen in 6 (19%) patients (diarrhea 4 patients, hyponatremia 2 patients, hemorrhagic enterocolitis 1 patient). No grade 4 non-hematological toxicity was seen. Grade ≥3 hematological toxicity was seen in 4 (12%) patients and included 2 patients each with anemia and thrombocytopenia. Neutropenia was not observed. In the 420mg cohort, only 4 of the 26 patients have discontinued therapy, and only one patient for disease progression. With a median follow-up of 14. 4 months on the 420 mg cohort, 81% achieved a response (69% partial response, 12% complete response) by IWCLL criteria. An additional 12% of patients achieved a nodal response. Fifty percent of patients with baseline thrombocytopenia or anemia noted sustained improvement in blood counts. The responses were independent of high-risk features. Ninety-two percent of patients with unmutated IGHV responded. There were 2 patients with del(17p) and both responded. The estimated 15-month median PFS for the 420 mg cohort was 96%. \n\nGiven the impressive single-agent activity of ibrutinib in patients with CLL, trials exploring combinations of ibrutinib with either monoclonal antibodies (rituximab or ofatumumab), or with chemotherapy (bendamustine or fludarabine/cyclophosphamide/rituximab) have been initiated. Some of these trials have been reported in abstract form. Preliminary data has been reported for ibrutinib in combination with ofatumumab (PCYC-1109-CA trial). 40 Patients with relapsed/refractory CLL following ≥2 prior therapies, including a purine-nucleoside analog, were treated with ibrutinib 420 mg daily with addition of ofatumumab from cycle 2 onwards. Twenty-four patients with CLL/ pro-lymphocytic leukemia (PLL) and three with Richter's transformation were treated. The median age was 66 (range 51-85). High-risk cytogenetics were seen in the majority patients [10 patients with del(17p) and 9 patients with del(11q)]. The majority of AE were grade 1-2. All CLL/PLL patients and 2 of the 3 patients with Richter's transformation achieved PR. \n\nBrown and colleagues reported preliminary data on the combination of ibrutinib with bendamustine/rituximab (PCYC-1108-CA) in relapsed/refractory patients with CLL. 41 hirty patients were enrolled with a median age of 62 years (range 41-82). The median number of prior therapies was 2 (range 1-4). Twenty-three percent had deletion 17p and 43% had deletion 11q. No added toxicity was observed with the addition of ibrutinib. With a median follow-up of 8. 1 months, 23 of the 30 patients were still on study, with only 2 patients coming off-protocol for disease progression. The ORR was 93% (13% CR, 80% PR) which is higher than the 59% seen with bendamustine-rituximab in historical controls. As with single-agent ibrutinib, responses were independent of high-risk genetic and molecular features. \n\nIbrutinib has also been evaluated in other lymphoid malignancies including MCL, DLBCL, Waldenstrom macroglobulinemia, and multiple myeloma. In the preliminary results of a phase II study (PCYC-1104), Wang and colleagues reported outcomes of 48 relapsed/ refractory patients with MCL (29 bortezomib-naïve; 19 bortezomib-exposed) who were treated with single-agent ibrutinib. 42 Ibrutinib was administered orally at 560mg daily until disease progression. The median age was 67 years (range, 62-72). Therapy was well tolerated with most frequently reported adverse events being grade 1 or 2 diarrhea, fatigue, and nausea (similar to the CLL study). The ORR was 67% (16 of the 24 evaluable patients). Responses were seen in both bortezomib-naïve and bortezomib-exposed cohorts. \n\nMany ongoing/planned trials are exploring the role of ibrutinib in hematologic malignancies. Some examples include ibrutinib as a single agent in Waldenstrom macroglobulinemia and multiple myeloma, ibrutinib with rituximab in CLL, ibrutinib with bendamustine/rituximab in relapsed DLBCL/MCL, ibrutinib with R-CHOP chemotherapy in newly-diagnosed DLBCL. A phase III randomized, open-label registration trial of ibrutinib versus ofatumumab in patients with relapsed or refractory CLL (RESONATE trial) has been initiated. 43 The primary end-point of this trial is PFS with key secondary endpoints being overall response rate, overall survival, and quality of life measures. Another planned phase III study includes a randomized study of bendamustine/rituximab plus ibrutinib versus bendamustine/rituximab plus placebo in relapsed or refractory patients with CLL/SLL.",
"section_name": "CLINICAL STUDIES",
"section_num": null
},
{
"section_content": "It is clear from the preclinical and preliminary clinical data (as stated above) that BTK inhibitors (along with other BCR-signaling pathway inhibitors) are going to revolutionize the treatment of patients with CLL. Besides ibrutinib, there are other BTK inhibitors in clinical development such as AVL-292 (Avila Therapeutics, now part of Celgene Corporation, Summit, New Jersey) and ONO-WG-307 (Ono Pharmaceutical, Osaka, Japan). In the coming few years, there will be a barrage of preclinical and clinical data with these drugs. Thus far the clinical responses with ibrutinib have been impressive with manageable toxicities. It is likely that ibrutinib and other drugs targeting the BCR pathway will become an integral component of CLL and NHL therapy.",
"section_name": "SUMMARY",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "KEY POINTS",
"section_num": null
},
{
"section_content": "• B-cell receptor (BCR) signaling plays a crucial role in pathogenesis of chronic lymphocytic leukemia (CLL). \n\n• Many kinases in the BCR signaling pathway are being explored as therapeutic targets such as Lyn, Syk, PI3 kinase and Bruton tyrosine kinase (BTK). \n\n• Ibrutinib (PCI-32765) is a selective, irreversible and oral inhibitor of BTK. \n\n• Preclinical data suggests that ibrutinib affects both CLL cell survival/ proliferation as well as CLL cell migration/homing. \n\n• Preliminary clinical data in patients with CLL is very encouraging with 67% response rate in the 420mg dose cohort in the relapsed/refractory CLL setting. \n\n• Combination of ibrutinib with monoclonal antibodies and chemoimmunotherapy has also shown favorable early results. \n\n• BCR inhibitors will likely become an important component of CLL therapeutics in the near future.",
"section_name": "KEY POINTS",
"section_num": null
}
] |
10.3324/haematol.2011.049155
|
The cytotoxicity of anti-CD22 immunotoxin is enhanced by bryostatin 1 in B-cell lymphomas through CD22 upregulation and PKC- II depletion
|
In spite of potent first-line therapies for chronic lymphocytic leukemia, treatment remains palliative and all patients frequently relapse. Treatment options for these patients are more limited. BL22 is a recombinant protein composed of the variable region of a monoclonal antibody that binds to CD22 and of PE38, a truncated Pseudomonas exotoxin. BL22 is a very potent drug already used in patients with hairy cell leukemia, whereas in chronic lymphocytic leukemia its cytotoxicity is limited by a lower expression of CD22. Here we demonstrate that this limitation can be overcome by pre-activation of chronic lymphocytic leukemia cells with bryostatin 1.Primary malignant B cells from chronic lymphocytic leukemia and mantle cell lymphoma patients were used in vitro to assess the therapeutic impact of drug combinations using BL22 and bryostatin 1.We demonstrate that bryostatin 1 sensitizes chronic lymphocytic leukemia cells for the cytotoxic effects of BL22 through activation of protein kinase C and subsequently increased CD22 surface expression. Dose and time response analysis reveals that activation of protein kinase C further activates an autocrine feedback loop degrading protein kinase C-βII protein. Depletion of protein kinase C-βII and upregulation of CD22 persist for several days following pre-stimulation with bryostatin 1. Therefore, our data provide a rationale for the sequential administration of BL22 following bryostatin 1 treatment. In addition to primary chronic lymphocytic leukemia cells, bryostatin 1 also sensitizes diffuse large B-cell lymphoma and mantle cell lymphoma cells to BL22 induced apoptosis.Our data suggest that the combination of bryostatin 1 with antibodies directed against CD22 is a potent drug combination for the treatment of low- and high-grade B-cell lymphoma.
|
[
{
"section_content": "2] [3] The combination of the monoclonal antibody rituximab and fludarabine-based chemotherapies has now become the standard first-line therapy for young and fit CLL patients. However, in spite of excellent overall response rates, the disease still remains incurable with increasing shorter progression free survival (PFS) following salvage therapies. Therefore, new treatment options are needed for patients who relapse after immunochemotherapy and those who are ineligible for such treatments. \n\nBL22 is a recombinant protein composed of the variable region of a monoclonal antibody that binds to CD22 on the surface of normal and malignant B cells and of PE38, a truncated Pseudomonas exotoxin A. 4 BL22 has demonstrated significant in vivo cytotoxicity in patients diagnosed with relapsed hairy cell leukemia following treatment with cladribine. 5 We previously demonstrated that BL22 induces cell death in CLL, involving the intrinsic apoptotic pathway. However, apoptosis induction correlates with the expression of CD22 on the surface of CLL cells and is only moderate in 'CD22 low'-expressing cells. 6 The aim of this study was to increase BL22 cytotoxicity by modulating the surface expression of CD22 on leukemic cells. \n\nBryostatin 1 is a macrocyclic lactone which was isolated from the marine bryozoan Bugula neritina more than 30 years ago. It modulates the family of protein kinase C (PKC) enzymes due to the structural similarities to the PKC-activating second messenger diacylglycerol. 7 Evidence from several groups indicates that PKC activity plays an important role in the pathogenesis of CLL and is crucial for cell survival by regulating anti-apoptotic proteins such as Mcl-1 and Bcl-2. 8, 9 The effects of bryostatin 1 are complex and include induction of differentiation of CLL cells, 8 modulation of Fas/CD95 signaling 10 and downregulation of PKCs. 11 owever, after phase I/II evaluation, it is now evident that bryostatin 1 has minimal single agent activity and, therefore, combined treatments of bryostatin 1 and chemotherapeutics were investigated in clinical trials. 12, 13 he ability of bryostatin 1 to induce a 'hairy cell phenotype' in CLL cells, including the marked upregulation of CD22, prompted us to investigate whether it could enhance the cytotoxicity of BL22. By using dose-response evaluation of bryostatin 1 we demonstrate that the combination of BL22 and bryostatin 1 increases the cytotoxicity of the immunotoxin not only through upregulation of CD22, but also through modulation of PKC-βII. The upregulation of Mcl-1 appears to be an undesirable effect of bryostatin 1 and may account for an impaired activity in CLL cells when used as monotherapy. Notably this upregulation of Mcl-1 was not sufficient to block the cytotoxicity of BL22. Furthermore, we demonstrate in vitro that the combination of bryostatin 1 and BL22 can be separated temporally, allowing enhanced cytotoxicity and potentially decreasing side effects in vivo.",
"section_name": "The cytotoxicity of anti-CD22 immunotoxin is enhanced by bryostatin 1 in B-cell lymphomas through CD22 upregulation and PKC-βII depletion Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Design and Methods",
"section_num": null
},
{
"section_content": "Approval for the study was obtained from the local ethics committee and all patients taking part gave their informed consent. Peripheral blood was obtained from patients with a diagnosis of B-CLL who had not been treated for at least three months. At the time of analysis, all patients were clinically stable, free from infectious complications and undergoing routine clinical outpatient review. \n\nAll cell lines used for the experiments were obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen; GmbH, Braunschweig, Germany). Cells were characterized by morphology, immunphenotyping, cytogenetics and molecular genetics. A detailed description of each cell line can be found on http://www. dsmz. de/",
"section_name": "Cell samples",
"section_num": null
},
{
"section_content": "Data from individual experiments are presented as mean +SEM. Statistical significances were determined using unpaired and paired t-tests as appropriate. P<0. 05 was considered statistically significant. \n\nFurther details of study Design and Methods are available in the Online Supplementary Appendix.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "BL22 is a genetically engineered antibody directed against CD22 and is composed of the variable region of a monoclonal antibody coupled to a truncated pseudomonas exotoxin A. 4 We have previously reported that BL22 is active in a subset of CLL patients, but this activity is reduced in patients expressing only low levels of CD22. 6 In contrast to CLL, BL22 has demonstrated strong in vivo activity in hairy cell leukemia, characterized by high expression levels of CD22. 5 Bryostatin 1 is a PKC-modulator with minimal single agent activity in CLL. Interestingly, bryostatin 1 induces a 'hairy cell'-phenotype in CLL. These morphological changes include cell enlargement and formation of cytoplasmatic extensions and are associated with an upregulation of CD22 14 (Figure 1A ). We, therefore, hypothesized that bryostatin 1 may enhance the cytotoxic effects of BL22. To test this, CLL cells were incubated in the absence or presence of bryostatin 1 (1 and 50 ng/mL) and BL22 (1 µg/mL). In order to reduce spontaneous apoptosis of CLL cells ex vivo and to mimic microenvironment survival signals, CLL cells were cultured on a murine fibroblast cell line Ltk -15 (kindly provided by P. Pérez-Aciego). Leukemic cells were sufficiently protected from spontaneous apoptosis when cultured on Ltk -cells (Figure 1B, bars 1 and 2). Bryostatin 1 showed no cytotoxic effects on CLL cells cultured on feeder cells. However, the cytotoxic effect of BL22 was not abolished in the presence of survival signals derived from bystander cells (Figure 1B ). Importantly, bryostatin 1 significantly sensitized primary CLL cells to the cytotoxic effects of BL22. (Relative apoptosis induction compared to CLL cells cultured on Ltk -cells: BL22 alone, mean 9%, range 0-33%; BL22 + bryostatin 1 (1 ng/mL), mean 28%, range 10-65%; BL22+ bryostatin 1 (50 ng/mL), mean 45%, range 24-78%). The expression of ZAP70 and CD38 in CLL cells is a surrogate marker for poor prognosis. Patients' characteristics according to the samples used for our experiment (Figure 1B ) are shown in Table 1. These show that the combination of BL22 and bryostatin 1 is highly effective in low-and high-risk patients (Online Supplementary Table S1 ). \n\nWe hypothesized that an increased binding and uptake of the immunotoxin occurred due to an increased expression of CD22 by bryostatin 1 on the surface of CLL cells. To test this hypothesis, surface protein expression of CD22 was determined by flow cytometry 24 h after stimulation of leukemic cells with bryostatin 1. An isotype matching, non-specific antibody was used as negative control. We observed a significant upregulation of CD22 at a bryo-statin1 dose of 1 ng/mL (Figure 1C ). Importantly, the surface expression of CD22 varies between CLL patients, ranging from low to moderate expression. 6 By analyzing the expression of CD22 in response to bryostatin 1 (1 ng/mL) we observed that 'CD22-low' and 'high' expressers up-regulate CD22, although the effect was more pronounced in CLL cells with a higher baseline expression of CD22 (Figure 1C ). Surprisingly, higher doses of bryostatin 1 (50 ng/mL) further enhanced the cytotoxic effect of BL22 compared to 1 ng/mL (Figure 1B ), but CD22 upregulation peaked at a dose of 5 ng/mL and was significantly less pronounced with 50ng/mL bryostatin 1 (Figure 1D ). These data indicate that additional mechanisms than enhanced CD22 expression must account for the sensitizing effects of bryostatin 1 on BL22.",
"section_name": "Bryostatin 1 enhances the cytotoxic effects of BL22 in primary CLL cells",
"section_num": null
},
{
"section_content": "As reported above, bryostatin 1-induced differentiation of CLL cells requires both PKC and extracellular signalrelated kinase (Erk)-activity. 14 7] [18] In order to characterize which of these pathways was required for CD22 upregulation, CLL cells were exposed to bryostatin 1 (1 ng/mL) in the presence of the small molecule inhibitors enzastaurin and PD98059. Enzastaurin is an acyclic bisindolylmaleimide that was developed as a selective and specific PKC-β inhibitor. 19 PD98059 is a widely used inhibitor of the Erk1/2 pathway. Leukemic cells were pre-incubated with the inhibitors 2 h before stimulation with bryostatin 1. Blockage of PKC-β abrogated the upregulation of CD22 by bryostatin 1, whereas inhibition of the Erk-pathway had no effect on the expression of CD22 (Figure 2A ). Conclusively, bryostatin 1 up-regulates CD22 using PKC-β as main conduit. \n\nSince our data suggested that PKC-β is a mediator of bryostatin 1-induced upregulation of CD22, we analyzed the expression of PKC-βII 24 h after stimulation with bryostatin 1. We noticed that 'low dose' bryostatin 1 (1 ng/mL) did not affect the expression levels of PKC-βII (Figure 2B, lanes 2 and 3). In contrast, higher doses of bryostatin 1 (50 ng/mL) depleted the expression of PKC-βII in CLL to levels undetectable by immunoblotting (lane 4). Inhibitory effects of PKC activators have been observed on the expression of PKC isoforms and may be related to a negative feedback loop involving proteasomal protein-degradation, proteinproteolysis and vesicle-dependent degradation. 20, 21 mportantly, even though low-dose bryostatin 1 (1 ng/mL) is sufficient to up-regulate CD22 on CLL cells (Figure 1 ), this activation seems to be incapable of activating this feedback loop. The dose-dependency of PKC-βII degradation appeared to be isoform-specific, since the expression levels of PKC-ε were unaltered upon 'low'-and 'high-dose' bryostatin 1 treatment (Figure 2B ). \n\nIn the light of the pro-apoptotic effects of PKC-inhibitors in CLL, 17, 18 we hypothesized that depletion of PKC-βII augmented the cytotoxic effects of BL22. In order to prove this hypothesis and to rule out the possibility that the downregulation of PKC-βII by bryostatin 1 was merely an epiphenomenon, PKC-βII activity was blocked with the small molecule inhibitor enzastaurin. BL22-induced cell death was significantly enhanced in the presence of enzastaurin. The pro-apoptotic effect of the combined treatment of BL22 and enzastaurin exceeded the additive effects of single agent treatment, suggesting a synergism between blockage of PKC-β and BL22 induced cell death (mean apoptosis induction: enzstaurin+BL22 41%; enzastaurin 13. 8%; BL22 18. 7%; Online Supplementary Figure S1 ). \n\nNotably, expression levels of CD22 remained unchanged in enzastaurin-treated CLL cells (data not shown).",
"section_name": "Bryostatin 1 up-regulates the expression of CD22 through PKC-β, but not Erk",
"section_num": null
},
{
"section_content": "To further characterize the kinetics of CD22 upregulation and PKC-βII depletion following bryostatin 1 exposure, bryostatin 1 was removed after 24 h and CD22/ PKC-βII expression was monitored by flow cytometry and immunoblotting. Successful removal of bryostatin 1 from the cell culture was monitored by expression of Mcl-1, a putative downstream target of activated PKC 8 (data not shown). The upregulation of CD22 following a 24-h exposure to bryostatin 1 was maintained for at least 72 h after removal of bryostatin (Figure 3A ). Remarkably, we observed that PKC-βII expression did not recover within 72 h after removal of bryostatin 1 (Figure 3B, lanes 6, 8 and 10). \n\nThis result prompted us to investigate whether sensitizing effects with BL22 could last for several days after bryostatin 1 treatment. From a clinical point of view, sequential therapies offer a strategy to reduce drug-interactions and side effects while increasing the cytotoxic effect. To address whether this was a feasible approach for BL22based therapies, CLL cells were primed with bryostatin 1 (1 ng/mL and 50 ng/mL) for 24 h. Forty-eight and 72 h after removal of bryostatin 1, leukemic cells were exposed to BL22 for an additional 24 h. Priming of CLL cells with bryostatin 1 significantly enhanced the cytotoxic effects of sequentially administered BL22 (Figure 3C ). Due to a significant amount of apoptosis in the medium control after several days of in vitro culture, the pro-apoptotic effect of BL22 was less pronounced than in CLL cells protected by pro-survival factors (Figure 1B ).",
"section_name": "Bryostatin 1 primes primary CLL cells for the cytotoxic effects of BL22",
"section_num": null
},
{
"section_content": "Based on results from phase I/II clinical trials, it is evident that bryostatin 1 has only minimal single agent activity in CLL. 12, 13, 22 Mechanistically this may be related to the fact that bryostatin 1 acts as a mixed antagonist/agonist of PKC. Bryostatin 1 has been shown to induce Mcl-1 expression in CLL cells, thus enhancing apoptosis resistance. 23 ccordingly, we also observed that bryostatin 1 attenuated the cytotoxic effect of the chemotherapeutic drugs fludarabine, doxorubicine and vincristine (data not shown). In order to further evaluate the relative contribution of Mcl-1 induction by bryostatin 1 to BL22 toxicity, we analyzed the expression of apoptosis-regulating proteins after treatment with BL22, bryostatin 1 or a combination hereof. As expected, a strong upregulation of Mcl-1 in CLL cells upon bryostatin 1 treatment was observed. Low-dose (1 ng/mL) bryostatin 1 was significantly more potent in inducing Mcl-1 than high-dose (50 ng/mL) bryostatin 1 (Online Supplementary Figure S2A, compare lane 2 to lanes 7 and 8). Accordingly, single agent treatment with bryostatin 1 significantly reduced spontaneous apoptosis of CLL cells (Online Supplementary Figure S2B ). In addition to the upregulation of Mcl-1, bryostatin 1 induced phosphorylation of Bcl-2 at serine-70. This phosphorylation increases the binding of the pro-apoptotic Bim to Bcl-2 and enhances the anti-apoptotic properties of Bcl-2. 18 In addition, Bim-function was further impaired due to degradation of BimEL in bryostatin 1-treated cells. We observed a reduced electrophoretic mobility of BimL in broystatin 1-treated cells, indicative of posttranslational modifications mediated by PKC. These modifications of Bim were not affected by BL22, excluding the possibility that BL22-mediated apoptotic effects were mediated by Bim. In contrast, single agent BL22 diminished the expression of Mcl-1 and XIAP (Online Supplementary Figure S2A, lanes 2 and 3). The combination of BL22 and bryostatin 1 attenuated the upregulation of Mcl-1 and phosphorylation of Bcl-2 (Online Supplementary Figure S2A, compare lanes 4 and 5 to 7 and 8). Importantly, expression levels of Mcl-1 and phospho-ser70-Bcl-2 were still above baseline-levels (Online Supplementary Figure S2A, compare lanes 4 and 5 to lane 1), indicating that bryostatin 1 has unfavorable effects on the expression of anti-apoptotic proteins. However, BL22 can overcome the anti-apoptotic effects of bryostatin 1 mediated by upregulation of Mcl-1 and phosphorylation of Bcl-2 (Online Supplementary Figure S2B ). The pro-apoptotic effect of BL22 in combination with bryostatin 1 was not related to induction of Noxa or Puma (Online Supplementary Figure S2A ). \n\nUnder co-culture conditions of leukemic B cells and stromal cells (LTK -), bryostatin 1 lacked the anti-apoptotic effect we had observed in the previous experiment (compare Online Supplementary Figure S2B and Figure 1B ). This raised the question as to whether bryostatin also mediated the upregulation of Mcl-1 and downregulation of PKC-βII in the presence of stromal cell-derived survival factors. In order to address this question, primary CLL cells were cultured on LTK -cells for up to 72 h. Continuous administration for 72 h had been used in clinical trials with bryostatin 1 in order to shift the balance from PKC-agonistic to antagonistic properties. 22 Notably, even in the presence of stromal cells, bryostatin 1 significantly up-regulated the expression of Mcl-1 (Online Supplementary Figure S2C ). Continuous exposure to bryostatin 1 for 72 h depleted haematologica | 2012; 97(5) Mcl-1 expression only in cells treated with higher doses of bryostatin 1 (50 ng/mL), indicating that PKC-antagonistic effects of bryostatin 1 are dose-and time-dependent. PKC-βII expression was depleted upon high-dose bryostatin 1 expression according to our results obtained from CLL cells cultured in the absence of feeder cells (compare Online Supplementary Figure S2C and Figure 3B ).",
"section_name": "BL22 overcomes bryostatin 1-induced upregulation of Mcl-1 and p-Ser70-Bcl-2",
"section_num": null
},
{
"section_content": "5] [26] Clinical trials with either 'naked' CD22 antibodies or labeled-antibodies are currently underway in nearly all these B-cell malignancies (see http://www. clinicalTrials. gov). We were, therefore, interested in seeing whether enhanced cytotoxic effects with bryostatin 1 and BL22 could also be achieved in other B-cell malignancies. Karpas422, 27 DB 28 and Sudhl-4 29 are cell lines derived from patients with diffuse large B-cell lymphomas. Cells were treated for 24 h with bryostatin 1 at a dose of 1 ng/mL and then expression of CD22 was analyzed. All cell lines up-regulated surface CD22 in response to bryostatin1 (Figure 4A ). Notably, the upregulation was much stronger in DB and Sudhl-4 cells compared to Karpas422 cells (Figure 4A ). BL22-induced cell death when used as single agent in all cell lines, but its pro-apoptotic effect was dramatic only in Sudhl-4 cells (Figure 4C ). The combined treatment of bryostatin 1 (1 ng/mL) and BL22 displayed synergistic effects in DB cells according to an increase in CD22 expression. Due to the high sensitivity of Sudhl-4 cells to BL22, no further increase in apoptosisinduction could be detected in spite of a significant upregulation of CD22. In contrast, Karpas422 cells were relatively resistant to single agent or combined treatment of BL22 and bryostatin 1 (Figure 4C ). \n\nSimilar to DLBCL cells, mantle cell lymphoma (MCL) cells were equally sensitive to single agent treatment with BL22 or bryostatin 1 (Figure 4D ). However, bryostatin 1 failed to induce an upregulation of CD22 on the surface of Granta 30 cells (Figure 4B ). Accordingly, no increase in BL22 induced toxicity was observed in these cells upon treatment with bryostatin 1. Additionally, high levels of Bcl-2 in Granta cells may also contribute to the lack of synergism between bryostatin 1 and BL22. 31 In contrast, the upregulation of CD22 in Jeko-cells 32 was associated with significantly increased cell death induced by BL22 (Figure 4B and D ). \n\nThese cell lines were suitable models to exclude the remote possibility that free-dissociated pseudomonastoxin from BL22 was responsible for the lymphoma cell death. Therefore, we performed knockdown experiments for CD22 on Sudhl-4 cells. The surface expression of CD22 could be reduced by repeated transfection with an siRNA directed against CD22 (Figure 4E ). Such modified cells were exposed to BL22 for 24 h. Reduced expression of CD22 attenuated the cytotoxic effects of BL22, indicating that surface expression of CD22 determines the efficacy of BL22 (Figure 4F ). \n\nIn addition, we tested whether primary mantle cell lymphoma cells derived from patients with leukemic variants of MCL were susceptible to the drug combination of bryostatin 1 and BL22. Single agent bryostatin 1 had no proapoptotic effect, but rather displayed an anti-apoptotic effect. However, bryostatin 1 enhanced the cytotoxic effects of BL22 on primary MCL cells (Figure 4G ). \n\nSurprisingly, we noticed that single agent bryostatin 1 exerts cytotoxic effects in 5 out of 5 cell lines (Figure 4C and D) in contrast to primary CLL and MCL cells (Figure 1B and Figure 4G ). \n\nThese data suggest that the combination of bryostatin 1 and BL22 may be useful not only for the treatment of CLL, but also in other B-cell malignancies.",
"section_name": "Combined treatment with BL22 and bryostatin 1 is active in diffuse large B-cell and mantle cell lymphomas",
"section_num": null
},
{
"section_content": "Targeting CD20 with monoclonal antibodies directed against various cell surface proteins has become a common approach to treat patients with B-cell lymphomas. However, in CLL, CD20 is a weak target and requires excessively high doses of CD20 directed antibodies such as rituximab 33 or ofatumumab 34 in order to achieve significant clinical responses with monotherapies. Alternative target proteins on CLL cells are CD22, CD23, CD40, CD70 and HLA-DR. Monoclonal antibodies directed against each of these proteins have been developed and demonstrated preclinical activity, and some of them have already been tested in clinical trials. \n\nEpratuzumab is an anti-CD22 humanized antibody and has shown single agent activity in patients with relapsed and refractory B-cell lymphomas. 35 However, its activity in CLL patients was only modest, possibly related to variations in CD22 expression on the surface of leukemia cells. The conjugation of protein toxins to monoclonal antibody is a neat and efficient way of increasing their toxicity. BL22 is a monoclonal antibody directed against CD22 and fused to a truncated Pseudomonas exotoxin. 4 Of 11 CLL patients treated in an early phase I clinical trial with BL22, only one patient had a partial response and 3 patients showed minor responses. 12 In sharp contrast, this drug has demonstrated noteworthy clinical responses in patients with refractory hairy cell leukemia (HCL) with a median progression free survival of 36 months in patients achieving a complete remission. 5 This discrepancy between CLL and HCL in terms of response to BL22 is most likely related to a much lower surface expression of CD22 on CLL cells compared to HCL cells. In addition, all CLL patients included in this trial had been more heavily pre-treated than HCL patients before receiving BL22. Interestingly, in spite of excessive prior treatments, BL22 was surprisingly well tolerated with reversible toxicities not exceeding grade 1-2 at the MTD level. Most common side effects of BL22 included transfusion-related fever, fatigue, elevation of liver enzymes and hypoalbuminemia. 36 Based on these results, BL22 appears to be a potent drug for the treatment of B-cell malignancies, but its efficacy in CLL is hampered by low expression of Figure 3. Sequential treatment of BL22 following bryostatin 1. (A) Primary CLL cells were exposed to either 1 ng/mL or 50 ng/mL bryostatin 1 for 24 h. Cells were then washed three times in PBS/10% FCS in order to remove bryostatin 1 and re-cultured in medium/ FCS. After the time points as indicated, cells were harvested and surface CD22 expression was analyzed by flow cytometry. Histograms show fluorescence intensities following bryostatin 1 treatment (shaded; (Bry), superimposed with that of medium control (solid line; (M)). Experiments were carried out in triplicates: one representative experiment is demonstrated. (B) Cells were treated as described in (A). After the time points as indicated, cells were harvested and analyzed for the expression of PKC-βII or PKC-ε. T0 indicates cell lysates from CLL cells freshly isolated from peripheral blood. Results from 3 experiments revealed the same results. (C) CLL cells were either pre-treated with 1 ng/mL or 50 ng/mL bryostatin 1 or kept in medium alone. After 24 h of incubation, bryostatin 1 was removed by washing cells three times in PBS/ FCS. Cells were re-cultured in medium/ FCS. After 48 or 72 h BL22 was added to pre-treated cells at a dose of 1 μg/mL. Apoptotic cells were determined following Annexin-V/PI staining after an additional 24 h (n=3). Apoptosis-induction by BL22 was calculated by subtracting spontaneous apoptosis of CLL cells cultured in medium. surface CD22 in some cases of CLL. In line with this observation, we have previously reported that the expression of CD22 is low in 50% of CLLs, irrespectively of disease stage, age, sex or leukocyte counts. 6 The pro-apoptotic effect of BL22 in vitro is strongly correlated to the density of CD22 on the surface of CLL cells, indicating that the expression of CD22 is crucial for BL22 toxicity to malignant cells. 6 he purpose of this study was to overcome this limitation by increasing the cytotoxicity of BL22 through an upregulation of CD22. Phorbol esters have been reported to induce differentiation of CLL cells. 37 Bryostatin 1 is a structurally related compound lacking the tumor-promoting effects of phorbol esters. Bryostatin 1 induces morpho-logical changes in CLL, including an increase in cell size, irregularity in cell shape, and formation of cytoplasmatic extensions (Figure 1A ). These changes into a 'hairy cell phenotype' are accompanied by an upregulation of CD22. 14 Bryostatin 1 exerts a wide range of biological activities and differentiation of CLL cells is related to activation of PKCs. The molecular mechanisms of apoptosis induction with bryostatin 1 treatment are complex and little understood. Bryostatin 1 and related compounds were described to act as mixed PKC agonist/antagonists. Shortterm exposure to bryostatin 1 causes activation of PKC and subsequent upregulation of anti-apoptotic proteins. 23 In contrast, pro-apoptotic effects of bryostatin 1 are thought to be related to diminished enzyme activity due to protea-BL22 and bryostatin in CLL haematologica | 2012; 97 (5) 777 Mantle cell lymphoma somal protein degradation following long-term exposure to bryostatin 1. 38 To date, few data are available on the clinical activity of bryostatin 1 in CLL. Over ten years ago, bryostatin 1 was given to refractory or relapsed CLL patients. Of the 3 CLL patients treated with bryostatin 1, 2 showed a significant, but only transient decrease in peripheral-blood lymphocyte counts, whereas the third patient did not respond to therapy. 12 A few years later, the same group conducted a phase II clinical trial. Of the 8 patients treated with bryostatin 1, 2 showed a partial remission and disease stabilized in an additional 2 patients. 22 In an independent trial, NHL patients were treated sequentially with bryostatin 1 and fludarabine (or vice versa). The overall response rate was reported to be 40% with only one patient achieving a complete remission. 13 Based on all available data, bryostatin 1 appears to be a safe drug in CLL patients. The most frequently reported side effects were dose-dependent myalgia that resolved spontaneously after several weeks. There were no reports of severe hematotoxicity or myelosuppression in patients receiving single agent bryostatin 1; 13, 22, 39, 40 however, this might be of greater concern in combination with other chemotherapeutic drugs. Conclusively, these clinical data indicate that bryostatin 1 has only modest activity in CLL and its effect is rather cytostatic than cytotoxic. On a molecular base, this may be explained by the properties of bryostatin 1 not only acting as a PKC inhibitor, but also partially activating PKCs. \n\nOur findings confirm that the combination of bryostatin 1 and BL22 has positive effects on apoptosis induction in CLL cells. Using two arbitrarily chosen concentrations of bryostatin 1, we discovered that two distinct mechanisms contribute to this: at low-dose bryostatin 1 (1 ng/mL), predominate activation of PKC causes a strong upregulation of CD22 on CLL cells (Figures 1 and 2, Online Supplementary Figure S1 ). Subsequently, increased binding of BL22 and uptake of the immunotoxin kills leukemia cells. Interestingly, the upregulation of CD22 following bryostatin 1 treatment occurred in CD22 low-and high-expressing CLL cells (Figure 1C ). In addition, high-dose bryostatin 1 (50 ng/mL) depletes PKC-βII from CLL cells in association with a more moderate upregulation of CD22 41 However, elevated levels of Mcl-1 in BL22 and bryostatin 1-treated CLL cells (Online Supplementary Figure S2A ) suggest that also other mechanisms need to be taken into account. Based on the observation that inhibition of PKC-β by enzastaurin strongly induces apoptosis in CLL cells 17, 18 (Online Supplementary Figure S1 ), we conclude that loss of PKC-βII and increased binding of BL22 following bryostatin 1 treatment is inducing cell death. We demonstrated that the combination of enzastaurin and BL22 strongly induced cell death of CLL cells by an amount exceeding the pro-apoptotic effect of either compound alone (Online Supplementary Figure S1 ). Since CD22 is up-regulated by bryostatin 1 in spite of a loss of PKC-βII (Figure 1C and D, Figure 2B ) it remains unclear whether this is a persistent effect due to a transient activation of PKC-βII (before protein degradation) or dependent on the activation of other PKC-isoforms. \n\nOne intriguing question arising from our work is what plasma levels of bryostatin 1 are achievable in vivo. Applying our findings to in vivo settings, plasma concentrations of at least 1 ng/mL of bryostatin 1 are needed to sensitize cells to the cytotoxic effects of BL22. One problem of the clinical application of bryostatin 1 has been the lack of reliable pharmacological studies. This is related to both a shortage of sensitive analytical methods and to the rapid clearance of bryostatin 1 after intravenous administration. 39 o tackle the problem, PKC activity has been assessed instead in patients receiving bryostatin 1. 40 Based on several reports, either continuous or bolus administration of bryostatin 1 was able to down-regulate PKC in PBMC. Since the concentration of bryostatin 1 required to increase CD22 expression on CLL cells is below the dose necessary to deplete PKC (Figure 1D and 2B), one can envisage that achievable in vivo concentrations will be sufficient to increase BL22 toxicity. Another pharmacokinetic aspect of bryostatin 1 is its ability to accumulate in lipophilic tissues. Therefore, bone marrow concentrations might even exceed plasma levels. \n\nSequential treatment of monoclonal antibodies and cytotoxic drugs is a common way of increasing cytotoxicity and avoiding potentially harmful drug interactions and side effects. For instance, rituximab is given one day before chemotherapy in nearly all patients diagnosed with B-cell lymphomas. Sequential treatment of bryostatin 1 and fludarabine has already been proven to be effective in CLL patients. 13 Here we provide in vitro evidence that priming CLL cells for 24 h with bryostatin 1 significantly increases the cytotoxicity of BL22 administered even several days after (Figure 3C ). The initial treatment with bryostatin 1 increases the expression of CD22 and depletes PKC-βII from CLL cells. Both effects last for several days (Figure 3A and B) allowing administration of BL22 several days after bryostatin 1. Based on our in vitro experiments and the strong clinical activity of BL22 in hairy cell leukemia, we believe that the combination of BL22 and bryostatin 1 could constitute a very effective treatment for relapsed or refractory CLL patients. Both drugs have already been given to CLL patients and displayed moderate side effects when given as monotherapy. \n\nIn addition to CLL, we show that BL22 and bryostatin 1, either given as single agent or in combination, exert strong pro-apoptotic effects in diffuse large B-cell lymphomas and mantle cell lymphoma (Figure 4 ). Therefore, this drug combination may be a promising new treatment option for a variety of B-cell malignancies. However, our study is limited to in vitro experiments and we can only speculate that the synergism between the two drugs can be translated in vivo. Therefore, clinical trials are needed to prove whether or not this is a feasible therapeutic approach in the treatment of CLL and B-cell lymphoma patients.",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "for helping to capture the images. Funding: this research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, USA to RJK and IP. This work was also supported by a grant from the Deutsche Forschungsgemeinschaft, Germany ( DFG-SFB TRR54 TPC3 ) to IR.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Authorship and Disclosures",
"section_num": null
},
{
"section_content": "The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www. haematologica. org. \n\nFinancial and other disclosures provided by the authors using the ICMJE (www. icmje. org) Uniform Format for Disclosure of Competing Interests are also available at www. haematologica. org.",
"section_name": "Authorship and Disclosures",
"section_num": null
}
] |
10.1038/s41416-020-0887-6
|
UGT2B17 modifies drug response in chronic lymphocytic leukaemia
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>High UGT2B17 is associated with poor prognosis in untreated chronic lymphocytic leukaemia (CLL) patients and its expression is induced in non-responders to fludarabine-containing regimens. We examined whether UGT2B17, the predominant lymphoid glucuronosyltransferase, affects leukaemic drug response and is involved in the metabolic inactivation of anti-leukaemic agents.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>Functional enzymatic assays and patients’ plasma samples were analysed by mass-spectrometry to evaluate drug inactivation by UGT2B17. Cytotoxicity assays and RNA sequencing were used to assess drug response and transcriptome changes associated with high UGT2B17 levels.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>High UGT2B17 in B-cell models led to reduced sensitivity to fludarabine, ibrutinib and idelalisib. UGT2B17 expression in leukaemic cells involved a non-canonical promoter and was induced by short-term treatment with these anti-leukaemics. Glucuronides of both fludarabine and ibrutinib were detected in CLL patients on respective treatment, however UGT2B17 conjugated fludarabine but not ibrutinib. AMP-activated protein kinase emerges as a pathway associated with high UGT2B17 in fludarabine-treated patients and drug-treated cell models. The expression changes linked to UGT2B17 exposed nuclear factor kappa B as a key regulatory hub.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Data imply that UGT2B17 represents a mechanism altering drug response in CLL through direct inactivation but would also involve additional mechanisms for drugs not inactivated by UGT2B17.</jats:p> </jats:sec>
|
[
{
"section_content": "Chronic lymphocytic leukaemia (CLL) is the most frequent adult leukaemia in the western world. Cancer growth varies widely with indolent and aggressive forms of CLL. In recent years, there has been substantial progress in the clinical management of CLL patients supported by a better risk stratification and the introduction of a number of novel therapeutic agents. 1 These advances significantly improved clinical outcomes of CLL patients. \n\nPre-treatment evaluation of informative prognostic markers helps the stratification of CLL patients into risk subgroups. These markers include unmutated immunoglobulin heavy chain variable region (U-IGHV), del(17p) or TP53 mutations, which are associated with poor response to treatment and predict earlier relapse after achieving initial haematological remission. 2 For treatment-naïve patients with these high-risk features, the Bruton tyrosine kinase (BTK) inhibitor ibrutinib is indicated as an initial systemic therapy rather than chemoimmunotherapy. 3, 4 The latter is based on the purine analogue fludarabine or alkylating agent bendamustine or chlorambucil backbones combined to the monoclonal antibody rituximab directed against the CD20 B-cell marker. 5 Other therapeutic small molecules include two distinct mechanisms of actions with venetoclax, a BCL-2 inhibitor, 6 idelalisib, a phosphatidylinositol-3-kinase (PI3K) δ inhibitor, and duvelisib, a dual inhibitor of PI3K δ and γ isoforms. 7, 8 Phase 3 trials are ongoing for additional agents such as the second-generation oral BTK inhibitor acalabrutinib and the Syk/Jak inhibitor cerdulatinib. 9, 10 Whilst treatment has significantly advanced patients' survival, it is also associated with incomplete clonal eradication, relapse, refractoriness or transformation to a more aggressive lymphoma (Richter's syndrome). 3 oth innate and acquired resistance represent major challenges for long-term disease control and an intense area of research. Findings reveal that targeted agents have unique mechanisms of resistance compared with chemotherapy. Genetic anomalies associated with fludarabine refractoriness include mutations in TP53, SF3B1, NOTCH1 and BIRC3 genes. 11, 12 Other genetic screens have recently identified further genes that could be implicated in fludarabine sensitivity such as ARID5B and BRAF. 13, 14 As targeted therapy agents have become more established in cancer therapy, recent reports attribute acquired hypermorphic mutations in genes of targeted pathways, including BTK and its immediate downstream effector phospholipase C, γ2 (PLCG2) critical for BCR signalling. 15 Mechanisms of resistance to other targeted agents, so early in clinical use, have not yet been portrayed in CLL patients but work is ongoing in this area. \n\nAdditional contributing mechanisms of reduced drug sensitivity that may be shared by targeted agents and chemotherapy may include increased drug efflux by ATP-dependent transporters and drug inactivation by metabolising pathways such as the glucuronidation pathway by UDP-glucuronosyltransferase enzymes (UGTs). By conjugating lipophilic drugs to glucuronic acid (GlcA), UGTs impair biological activity and enhance water solubility of drugs, driving their elimination and a lack of drug efficacy. 16 8] [19] However, for most drugs used in CLL, the glucuronidation pathway has not been comprehensively studied. Reports have established that high UGT2B17 expression is an adverse prognostic factor in CLL, associated with shorter treatment-free survival, overall survival and patients requiring more treatment. 17, 20, 21 The link between UGT2B17, adverse CLL features and progression appears complex, and precise molecular mechanisms underlying these observations remain to be elucidated. In cohorts of CLL patients, UGT2B17 was associated with unmutated IgHV and predicted poor survival, 17 and was further associated with progressive disease within the group of patients with a more favourable profile (mutated IgHV). 21 A recent study supports that UGT2B17-mediated metabolism would participate in the regulation of signalling pathways critical for CLL progression. 22 igh UGT2B17 expression in leukaemic cells was further linked to a lack of response to fludarabine-containing regimens. 17 UGT2B17 is one of the 19 functional human drug-conjugating UGT enzymes, also involved in regulating homoeostasis of a number of endogenous metabolites, however its role in conjugating antileukaemics remains largely unknown. 16 n the present study, we investigated the response to antileukaemic agents namely fludarabine, ibrutinib, idelalisib, bendamustine, chlorambucil, venetoclax, acalabrutinib, cerdulatinib and duvelisib in relation to UGT2B17 expression levels. We then sought to evaluate the UGT-mediated enzymatic inactivation of drugs relevant to CLL and the possible involvement of the UGT2B17 enzyme. Our data support that high UGT2B17 expression alters drug response by direct inactivation but likely also through other mechanisms. We further evidence that the UGT pathway is involved in the inactivation of the majority of antileukaemic agents used in CLL, suggesting that this pathway may be associated with drug resistance.",
"section_name": "BACKGROUND",
"section_num": null
},
{
"section_content": "",
"section_name": "METHODS",
"section_num": null
},
{
"section_content": "Cryopreserved peripheral blood mononuclear cells (PBMCs) and plasma samples were obtained from CLL patients who were diagnosed between 1987 and 2011 at Vienna General Hospital. Patients provided informed consent. The study was carried out in accordance with the Declaration of Helsinki and was approved by the local ethical research committees of the Medical University of Vienna (Ethics vote 1499/2015) and the CHU de Québec (A14-10-1205). UGT2B17 mRNA analysis was performed in CD19 + -sorted cells of twenty CLL patients before and after the first cycle of treatment with fludarabine-containing regimens as described. 23 lasma samples for quantification of glucuronides in circulation were available for two patients in the 1st week after the first cycle of treatment with fludarabine-chlorambucil-rituximab and for 15 patients during treatment with ibrutinib. The B lymphoblastoid leukaemic cell lines included p53 wild-type (EHEB, JVM-2) and p53 mutated (MEC-1) cell lines purchased from ATCC (Manassas, VA, USA) or DSMZ (Braunschweig, Germany). Cells overexpressing UGT2B17 were previously described. 22 Cell culture components were all purchased from Wisent Bioproducts (Saint-Bruno, QC, Canada).",
"section_name": "Patients, primary CLL cells and cell lines",
"section_num": null
},
{
"section_content": "Glucuronidation assays were conducted using human liver, intestine and kidney microsomes (Xenotech, Kansas City, KS, USA) or supersomes expressing individual human UGT isoforms (Corning, MA, USA). In the absence of commercially available UGT2B11 supersomes, microsomal proteins derived from HEK293 expressing the recombinant UGT2B11 enzyme were used. Substrates included dihydrotestosterone (DHT) purchased from Steraloids (Newport, RI, USA), fludarabine and chlorambucil from Sigma-Aldrich (Oakville, ON, Canada) and bendamustine from Toronto Research Chemicals (North York, ON, Canada). All other compounds were purchased from Selleckchem (Houston, TX, USA). UGT assays were incubated at 37 °C for 6 h in a final volume of 100 µL of assay buffer containing 20 µg of proteins, 0. 5 mM DTT, 10 mM MgCl 2, 50 mM Tris-HCl (pH 7. 5), 20 µg/mL alamethicin, 2 mM UDP-GlcA and 0. 5 µg/mL leupeptin with the indicated substrate. Samples were then stopped by adding 100 µL cold methanol, centrifuged at 16,000×g for 10 min and supernatants analysed by mass spectrometry. The following negative controls were included for each analysis: reaction assays without substrate, without the microsomal fraction and without substrate and microsomal fraction.",
"section_name": "Drug conjugation assays",
"section_num": null
},
{
"section_content": "The establishment of these analytical methods required the optimisation of chromatographic conditions, production of glucuronide standards using pooled liver microsomes and the use of deuterated molecules as internal standards (Clearsynth, Mississauga, ON, Canada). Drugs and their G metabolites were measured on an API 6500 mass spectrometer (Sciex, Concord, ON, Canada), operated in multiple reactions monitoring mode and equipped with a turbo ion-spray source. Electrospray ionisation was performed in the positive ion mode. The chromatographic system consisted of a Nexera (Shimadzu Scientific instruments, Inc, Columbia, MD, USA). For fludarabine-G1 and G2, the chromatographic separation was achieved with an ACE Phenyl 3. 0 µm packing material, 100 × 4. 6 mm (Canadian Life Science, Peterborough, ON, Canada). The mobile phases were solvent A: water with 0. 1% formic acid (v/v) and solvent B: methanol, at a flow rate of 0. 9 ml/min. Fludarabine-G1 and G2 were eluted using the following programme: 0-2. 0 min, isocratic 20% B; 2. 0-2. 1 min, linear gradient 20-65% B; 2. 1-4. 0 min, isocratic 65 % B; 4. 0-4. 1 min, linear gradient 65-90% B; 4. 1-5. 2 min, isocratic 90 % B; 5. 2-5. 3 min, linear gradient 90-20 % B; 5. 3-8. 5 min, isocratic 20% B. For cerdulatinib-G, chlorambucil dechlorinated metabolites G1 and G2, venetoclax-G, and the bendamustine derivative HP2-G, the chromatographic separation was achieved with a Gemini C18 3. 0 µm packing material, 100 × 4. 6 mm (Phenomenex, Torrance, CA, USA). The mobile phases were solvent A: 1 mM ammonium formate in water and solvent B: 1 mM ammonium formate in methanol at a flow rate of 0. 9 ml/min. Cerdulatinib-G and chlorambucil dechlorinated metabolites G1 and G2 were eluted using the following programme: 0-5. 0 min, linear gradient 10-90% B; 5. 0-5. 2 min, isocratic 90% B; 5. 2-5. 3 min, linear gradient 90-10% B; 5. 3-8. 3 min, isocratic 10 % B. Venetoclax-G was eluted using the following programme: 0-2. 0 min, linear gradient 10-90% B; 2. 0-6 min, isocratic 90% B; 6. 0-6. 1 min, linear gradient 90-10% B; 6. 1-9. 2 min, isocratic 10% B. The bendamustine metabolite HP2-G, was eluted using the following programme: 0-5. 0 min, linear gradient 20-90% B; 5. 0-5. 2 min, isocratic 90% B; 5. 2-5. 3 min, linear gradient 90-20% B; 5. 3-8. 3 min, isocratic 20% B. For idelalisib-G1 and G2, the chromatographic separation was achieved with a Gemini C18 3. 0 µm, 100 × 4. 6 mm, using solvent A: 2 mM ammonium formate in water and solvent B: 2 mM ammonium formate in methanol at a flow rate of 0. 9 ml/min. Idelalisib-G1 and G2 were eluted using the following programme: 0-3. 0 min, isocratic 60 % B; 3. 0-3. 1 min, linear gradient 60-90% B; 3. 1-4. 0 min, isocratic 90% B; 4. 0-4. 1 min, linear gradient 90-60% B; 4. 1-7. 0 min, isocratic 60% B. For ibrutinib-G1 and G2, acalabrutinib-G and duvelisib-G, the chromatographic separation was achieved with an ACE Phenyl 3. 0 µm, 100 × 4. 6 mm, using a mobile phase of 75%, 75% and 65% methanol, respectively, eluted with 1 mM ammonium formate in water in isocratic mode at a flow rate of 0. 9 ml/min. The systems were controlled through Analyst Software, version 1. 6. 1 from AB Sciex.",
"section_name": "Mass spectrometry-based analysis of drug conjugation",
"section_num": null
},
{
"section_content": "Cells were plated at 1 × 10 4 cells/well (MEC1) or at 5 × 10 4 cells/ well (JVM2) in 96-well U-bottom tissue culture plates (BD Bioscience, Mississauga, ON, Canada). Drugs were added at time of plating at concentrations ranging from 1 nM to 100 µM (7-9 concentrations per drug), depending on the drug and based on cell viability. MTS cell viability assays (Aqueous One assay, Promega, Madison, WI, USA) were conducted 72 h after treatment initiation according to the manufacturer's instructions. Absorbance was read on a TECAN infinite M1000 plate reader (Tecan Group Ltd., Männedorf, Zurich, Switzerland) at 495 nm. Control cells were treated with corresponding vehicle concentration. To determine half maximal inhibitory concentrations (IC 50 ), MTS readout of control samples treated with vehicle only was set to 100% cell viability, and relative cell viability of drug-treated cells was determined by dividing MTS values of the treated samples by the control. IC 50 were calculated by fitting variable slope non-linear curves to normalised response data from drug treatments using GraphPad Prism v5 (GraphPad Software Inc., La Jolla, CA, USA). Assays were replicated at least three times in triplicates. \n\nCytotoxicity of drug treatments was determined by labelling cells with AlexaFluor 647-conjugated Annexin V (Life Technologies Inc., CA, USA) and propidium iodide (Sigma). Cells plated in 96well plates at 2 × 10 4 cells/well (MEC1) or 3 × 10 4 cells/well (JVM2) were exposed to varying drug concentrations as above for 72 h. Cells were washed, labelled for 12 min with Annexin V (1:200) and PI (2 µg/ml) in Annexin V binding buffer, then immediately analysed by flow cytometry on a FACSCelesta equipped with a high throughput sampler (BD Bioscience). Annexin V labelling assays were replicated at least twice in duplicates.",
"section_name": "Drug sensitivity assays",
"section_num": null
},
{
"section_content": "Total RNA was extracted from MEC1, EHEB and JVM2 cells treated with fludarabine, ibrutinib or idelalisib using RNeasy plus mini spin kits (Qiagen, Toronto, ON, Canada). cDNA was generated using SuperScript IV reverse polymerase (Thermo Fisher Scientific, Waltham, MA, USA). UGT genes were measured by qPCR analysis of 10 ng of cDNA with Power SYBR green master mix (Thermo Fisher Scientific). For measures of other genes including housekeeping genes from normalisation, total RNA was DNase I-treated and purified using the RNeasy MinElute Cleanup kit (Qiagen) per manufacturer's instructions and as described previously. 22 RNA sequencing experiments were performed on MEC1, EHEB and JVM2 cells treated with fludarabine (10 µM and 50 µM), ibrutinib (1 µM and 5 µM) and idelalisib (5 µM and 10 µM) for 48 (MEC1), 72 (JVM2) or 96 (EHEB) hours. Sequencing data was quality trimmed using Trimmomatic v0. 36 and aligned to GRCh38 using HISAT2 v2. 1. 24 Transcriptome assembly and generation of read-count matrices were performed using Ensembl GRCh38 transcriptome annotation with StringTie v1. 3. 4. 25 Differential gene expression analysis was done with the edgeR package for R v3. 5. 1. Isoform quantification was done using kallisto v0. 44. 0 with a custom GTF formatted annotation containing alternative UGT transcript information. 26 Sequencing data are available from the Gene Expression Omnibus (GEO) accession number GSE135030. \n\nPublic expression data from 291 CLL patients was downloaded from the International Cancer Genome Consortium (ICGC) with the project code CLLE-ES. 27 Public microarray data from PBMCs of untreated CLL patients and CD19 + -sorted B-cells from patients treated with fludarabine-containing regimens were obtained from the Gene Expression Omnibus with the accession numbers GSE13159 28 and GSE15490, 23 respectively. In the latter study, responders included complete response or partial response, and non-responders comprised stable disease or progressive disease. Long-read sequencing data was downloaded from the Sequence Read Archive (SRA) using the accession number SRP036136 and aligned with HISAT before visualisation. Identification of possible upstream regulators was carried out using iRegulon 29 for Cytoscape v3. 2. 1. ENCODE ChIP-seq data from samples GM12891, GM12878 and GM19099 was analysed for RELA binding within a 10 kb window centred on the TSS of genes co-expressed with UGT2B17 by bootstrapping. Differential expression analysis for RNA-seq data was done using the edgeR v3. 22. 5, while microarray data was analysed with limma v3. 36. 5 and affy v1. 58. Annotation-Hub v2. 12. 1, Iranges v2. 14. 12 and GenomicRanges v1. 32. 7 were used for analysis of ChIP-seq data. Clustering and functional enrichment analyses were performed using clusterProfiler v3. 8. 1, coseq v1. 4. 0, [32] [33] [34] [35] [36] [37] Luciferase assays The sequence corresponding to 2. 5 kbp of UGT2B17_n2 promoter (including exon 1c) was PCR amplified from LNCaP cell line genomic DNA using Phusion DNA Polymerase (NEB, Whitby, ON, Canada) as recommended, with the following primers (annealing at 63 °C): forward 5′-CTAGCAGACGCGTGAGATCCTAGTAGGAGGTT TTGGC-3′ and reverse 5′-CTAGCAGCTCGAGCAAGTTCCAGATGTC CAGACTC-3′. The PCR fragment was then digested with MluI and XhoI restriction enzymes and inserted in pGL3 basic vector (Promega) digested with the same enzymes, using the Rapid DNA Ligation Kit (Roche). Construct was verified by Sanger sequencing. MEC1 cells were co-transfected with 9. 5 µg pGL3 constructs and 0. 5 µg pRL-null basic renilla (Promega) using the Neon Transfection System (Thermo Fisher Scientific). Cells were then harvested, lysed and assessed for luciferase activity using the dual-luciferase reporter assay kit (Promega), as per manufacturer's instructions. Luciferase activity was calculated as the ratio of firefly luciferase to renilla activity, relative to the pGL3 control. Assays were replicated four times in triplicates.",
"section_name": "Gene expression analyses",
"section_num": null
},
{
"section_content": "Results from glucuronidation assays represent a minimum of two independent experiments. All other results represent at least three independent experiments. Statistics were calculated using Graph-Pad Prism v5 (GraphPad Software Inc., La Jolla, CA, USA) or R v3. 5. 1. P-values were calculated using Student's T-test unless otherwise indicated. Differences in gene expression were considered significant if adjusted P-values were inferior to 0. 05.",
"section_name": "Statistical analyses",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "In 20 CLL patients exposed to fludarabine-based regimens, 23 microarray data analysis of RNA from CD19 + -sorted B-cells (GSE15490) showed that UGT2B17 was induced shortly after treatment initiation in 6 of 11 non-responders whereas it was induced in only 2 responders out of 9 (P = 0. 0007) (Fig. 1a ). Patients showing an induced expression of UGT2B17 after treatment also displayed a 7. 5-fold higher basal expression of UGT2B17 relative to those showing no induction (P = 0. 0006), with all cases showing low expression of all other UGT isoforms. This was sustained by the analysis of RNA sequencing data from 291 CLL cases indicating that UGT2B17 predominates in leukaemic cells (Fig. 1b ). Cells of MEC1 and JVM2 models overexpressing UGT2B17 were more resistant than cells with low expression, with half maximal inhibitory concentration (IC 50 ) values higher by 1. 5 to 4. 3-fold (P < 0. 013) for fludarabine and ibrutinib (MEC1 and JVM2) as well as idelalisib, chlorambucil and venetoclax (MEC1) established by MTS assays (Table 1 ). Similar results were obtained using AnnexinV/PI labelling (data not shown). At clinically relevant concentrations, fludarabine, ibrutinib and idelalisib displayed no cytotoxicity in either cell lines, determined by Annexin V staining (Supplementary Fig. S1 ).",
"section_name": "UGT2B17 expression in B-cells is associated with reduced sensitivity to anti-leukaemic drugs and is induced by short-term drug treatment in patients",
"section_num": null
},
{
"section_content": "As observed in CLL patients (Fig. 1a ), fludarabine significantly induced UGT2B17 expression in MEC1, JVM2, and EHEB cellular models (Fig. 2a-c ). UGT2B17 was also induced by ibrutinib and idelalisib (by 1. 2 to 13. 6-fold; P < 0. 01) and was associated with elevated enzyme activity for DHT glucuronidation, a substrate for the UGT2B17 enzyme (1. 9 to 10. 5-fold; P < 0. 01) (Fig. 2d ). The low expression of UGT1A was also perturbed by each drug treatment (Fig. 2a-c ). Consistent with these changes, glucuronidation activity was induced as demonstrated by increased levels of conjugate of the substrate oestradiol (E 2 ) (Fig. 2d ). In the same line, fludarabine, ibrutinib and idelalisib increased fludarabine glucuronidation (Fig. 2e ). As observed in CLL patients (Fig. 1b ) comparative analysis of relative UGT expression levels further indicated a predominance of UGT2B17 (Fig. 2f ). A more detailed assessment of the isoforms expressed identified UGT1A6 as the most abundant UGT1A isoform (Supplementary Fig. S2 ). \n\nAnti-leukaemic drugs are conjugated to GlcA in CLL-treated patients Given the higher and induced expression of UGT2B17 in patients not responding to fludarabine-containing treatment regimen, we explored whether UGT2B17 may be involved in the glucuronidation of the drug itself. We also examined the conjugation of additional agents relevant to CLL. Our observations in CLL patients treated with fludarabine raised the possibility that UGT2B17 may be involved in 23 a UGT2B17 is preferentially induced in CLL patients not responding to fludarabinecontaining regimen. Expression of UGT2B17 was determined in CD19 + -sorted cells of CLL patients before and three days after the first cycle of fludarabine treatment. Patients received standard doses of fludarabine and cyclophosphamide (FC) or FC with rituximab (FCR). Clinical response was assessed three months after treatment initiation. 23 Responders displayed partial or complete remission, non-responders had stable or progressive disease. Expression of UGT2B17 is significantly enhanced (> 15 %; P = 0. 0007) three days after treatment in the subset of patients not responding to fludarabine-containing regimen. CLL patients were dichotomised on the basis of UGT2B17 induction. Prior to initiation of treatment with a fludarabine-containing regimen, average levels of UGT2B17 were 4. 1 and 7. 0 log 2 -units (P = 0. 0006, after normalisation by robust multi-array average) in patients exhibiting no induction and induction of UGT2B17 expression, respectively. Clinical characteristics of CLL patients are provided in Supplementary Table S6. b UGT2B17 predominates in leukaemic cells. Relative expression levels of UGT isoforms in a cohort of 291 CLL patients from the International Cancer Genomic Consortium (ICGC) demonstrate that UGT2B17 is the main UGT expressed in leukaemic cells. The proportion of patients expressing UGT2B17 and other UGTs is illustrated. Other UGTs include nine UGT1A (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9 and 1A10) and six UGT2B (2B4, 2B7, 2B10, 2B11, 2B15 and 2B28). \n\nthe glucuronidation of the drug itself, and potentially also of B-cell receptor inhibitors ibrutinib and idelalisib, which contain functional hydroxyl and amino groups susceptible for conjugation into inactive glucuronides (G). Glucuronidation assays using pooled human liver microsomes enriched in UGT enzymes led to the formation of polar G derivatives. This was confirmed by their fragmentation patterns assessed by mass spectrometry (MS), namely a loss of the GlcA moiety corresponding to a m/z shift of 176 Da (Fig. 3a ). Fludarabine was conjugated into two glucuronides G1 and G2, named according to their chromatographic resolution (Fig. 3a ). Additional confirmation was achieved by the disappearance of G1 and G2 upon βglucuronidase hydrolysis (Supplementary Figure S3 ). Similarly, ibrutinib and idelalisib each led to two glucuronidated products (Fig. 3a ). \n\nA second set of experiments identified the UGT enzyme(s) involved, based on UGT expressed in metabolic liver, kidney and intestine tissues, and individual recombinant UGT1A (n = 8) and UGT2B (n = 6) enzymes, using quantitative MS methods (Fig. 3b ). UGT2B17 and UGT1A4 were the main conjugating enzymes for fludarabine glucuronidation (Fig. 3b ), with a preferred formation of G2 over G1. This observation mirrored activity in livers, which express these UGTs. The confirmation of fludarabine-G formation in CLL patients was established in plasma of two cases collected in the first week after fludarabine treatment (Fig. 3c ). Both fludarabine-G1 and G2 were measured in the first patient (16. 4 and 11. 7 pg/mL), whereas G2 (24. 1 pg/mL) was detected in the second patient. For ibrutinib and idelalisib, G1 and G2 were formed only in the presence of UGT1A4 and livers, with similar kinetic parameters (Fig. 3b, Supplementary Table S1 ), supporting the implication of this sole enzyme. Their formation was further abolished by nearly 90% in the presence of a specific UGT1A4 inhibitor, hecogenin (Supplementary Table S1 ). The formation of ibrutinib-G was then established in serial serum samples from 15 CLL patients undergoing ibrutinib treatment collected at baseline (T 0 ), 3 weeks to 2 months (T 1 ), and between 4 and 9 months (T 2 ) after treatment initiation (Fig. 3d, Supplementary Tables S2 and S3 ). A predominance of G2 over G1 (ratio of 6. 5) was noted, similar to what was observed in livers and UGT1A4. The formation of ibrutinib-G represented on average 24 % of the parent drug and was strongly correlated to levels of ibrutinib (R 2 = 0. 917, P < 0. 001) but with a considerable patient-to-patient variability (CV = 190%). Lastly, we explored whether glucuronidation may be involved in the conjugation of additional anti-leukaemics and the potential involvement of UGT2B17. MS analysis confirmed the formation of at least one G product by livers following incubations with bendamustine, chlorambucil and targeted agents venetoclax, acalibrutinib, cerdulatinib and duvelisib (Fig. 4 ). The UGT2B17 and UGT1A4 enzymes were predominantly involved in their inactivation by glucuronidation (Fig. 4 ).",
"section_name": "UGT2B17 expression in B-cell models is induced by short-term drug treatment",
"section_num": null
},
{
"section_content": "To gain insights into the cellular pathways associated with high and inducible UGT2B17 expression, we initially established a transcriptional signature associated with elevated UGT2B17 expression in 448 untreated CLL samples (Fig. 5a ). This signature was then examined in cell models expressing high levels of UGT2B17 as well as in drug-treated cells in which UGT2B17 expression was induced. First, clustering revealed transcriptomic changes associated with elevated UGT2B17 expression in untreated CLL patients that resembled those found in overexpression models (Fig. 5b ). K-means clustering of gene expression data designated two clusters with globally up-regulated (cluster 2) or down-regulated (cluster 3) gene expression across all samples, suggesting that these clusters encompass changes connected to UGT2B17 levels rather than those produced by drugs. Cluster 2 contained the UGT2B17 gene whereas several genes of the AMP-activated protein kinase (AMPK) signalling pathway were significantly enriched in cluster 3 (Supplementary Table S4 ), and further validated by quantitative PCR (Fig. 5b, c ). \n\nA second series of analysis focused on drug-related signatures. We observed that kinase inhibitor-treated cells clustered together whereas fludarabine-treated cells had a distinct expression profile, likely owing to different mechanisms of action (Fig. 5b ). An upstream analysis for causal interpretation of the expression changes associated with UGT2B17 exposed an enrichment of nuclear factor kappa B (NF-κB) binding targets (Supplementary Table S5 ). The analysis of NF-κB ChIP-seq data (GM12891, GM12878 and GM19099) derived from tumour necrosis factor α (TNF-α)-treated B-cells further confirmed NF-κB as a key regulatory 'hub point'. \n\nAn analysis of the UGT2B17 transcriptomes of CLL patients and leukaemic cell models revealed that the enzyme is largely expressed from alternative transcripts rather than the canonical v1 transcript (Fig. 5d, e ). Although these alternative transcripts, named UGT2B17_n2, n3 and n4, encode the UGT2B17 enzyme, they are comprised of additional alternative exons that extend the 5' untranslated region (Fig. 5e ). Their expression was also detected by RT-PCR amplification of full-length transcripts from CLL patients and Sanger sequencing of amplicons. Long-read sequencing data using PacBio SMRT technology also confirmed the expression of alternative UGT2B17_n2 in the GM12891 lymphoid cell line. The functionality of the regulatory sequences upstream of the novel exon 1c (P3) and exon 1b (P2) were evidenced in luciferase assays (Fig. 5f ). Compared to the low expression derived from the canonical UGT2B17 promoter P1 (1. 3-fold), P2 and P3 enhanced luciferase gene expression by 2. 5-and 16. 3-fold, respectively, in MEC1 cells (Fig. 5f ).",
"section_name": "Transcriptional changes associated with high UGT2B17 in CLL patients and cell models",
"section_num": null
},
{
"section_content": "Understanding mechanisms that contribute to intrinsic and acquired resistance to therapy is key to finding useful predictive markers and innovative strategies to prevent or overcome treatment resistance in CLL. Previous reports identified UGT2B17 as a prognostic marker and a potential therapeutic target. 17, 20, 21, 38 elalisib-G (pmol/min/mg protein) UGT2B17 modifies drug response in chronic lymphocytic leukaemia EP Allain et al.",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "A potential impact of UGT2B17 expression on clinical outcome in treated patients emerges with observations of poor response in fludarabine-treated patients expressing high UGT2B17 levels whereas drug treatment further induces the UGT2B17-associated metabolic capacity of lymphoid cells according to our findings. \n\nOur observations further support a significant impact of the UGT metabolic pathway on the inactivation of most anti-cancer agents used in CLL, including commonly used treatments fludarabine, ibrutinib, bendamustine, chlorambucil and emerging targeted therapies idelalisib, venetoclax, acalabrutinib, cerdulatinib and duvelisib. This resulted in reduced sensitivity of cells to several anti-leukaemics associated with high UGT2B17 expression. Our. The metabolite of bendamustine HP2 was glucuronidated. The HP2 metabolite has hydroxyl groups in place of chlorine atoms in bendamustine. Chlorambucil undergoes a similar process, generating a dechlorinated metabolite subsequently conjugated. Venetoclax is an orally available, selective, small molecule inhibitor of BCL2 approved by the US Food and Drug Administration for the treatment of patients with CLL. Acalabrutinib is an orally available, irreversible Bruton's tyrosine kinase (BTK) inhibitor in development designed to be more selective than ibrutinib. 10 Cerdulatinib (PRT062070) is an investigational oral, dual spleen tyrosine kinase (Syk), janus kinase (JAK1/3) and tyrosine kinase 2 (TYK2) inhibitor for the treatment of haematological malignancies and approved for the treatment of peripheral T-cell lymphoma. 9 Duvelisib is an oral, dual small molecule inhibitor of phosphatidylinositol 3-kinase (PI3K) δ and γ, approved for the treatment of relapsed or refractory CLL. 7 A second set of experiments identified the UGT enzyme(s) involved, revealing a predominant role for UGT2B17 and UGT1A4. S4. \n\nfindings indicate that this may be caused, at least in part, by direct glucuronidation of the drug by the UGT2B17 enzyme namely for fludarabine but would also involve other mechanisms for ibrutinib and idelalisib not inactivated by UGT2B17. By catalysing the transfer of GlcA from the co-substrate UDP-GlcA, UGT2B17 inactivates and detoxifies its substrates. This is supported by the detection of fludarabine-glucuronides in circulation of CLL patients that recently initiated fludarabine-based treatment. The glucuronidation pathway inactivates other nucleotide analogues such as ribavirin and cytarabine through a glioma-associated oncogene homologue 1 (GLI1)-dependent mechanism involving the regulation of UGT1A protein stability in AML. 18, 19 This differs from our observations in untreated CLL patients, in which UGT2B17 expression predominates, and where high UGT2B17 expression was associated with shorter treatmentfree and overall survival and more patients requiring treatment. 17, 20, 21 This shows that for a significant proportion of high-risk CLL patients, UGT2B17 is expressed in cancer cells prior to treatment initiation with the potential to affect primary response to first line treatment such as fludarabine and ibrutinib. Once fludarabine treatment is initiated, an induction of UGT2B17 expression was observed in B-cells of CLL patients not responding to fludarabine as well as in lymphoid cell models. 17, 23 reatment with targeted agents ibrutinib and idelalisib also resulted in a marked transcriptional up-regulation of UGT2B17, and high UGT2B17 expression was associated with reduced sensitivity to these drugs in B-cell models. While an influence of other drugrelated mechanisms, including transporters and drug metabolising enzymes is possible, CYP3A4, which is another key metabolising enzyme for these drugs, 39 was not perturbed in patients treated with fludarabine (not shown). It raises the possibility that therapeutic pressure induces UGT expression in B-cells. This rapid adaptation of neoplastic cells in the presence of a cytotoxic stressor and targeted therapies support that the UGT metabolic pathway is highly relevant in leukaemia, has the potential to affect drug response locally in malignant cells, and may be useful in predicting response to several CLL therapies. We also demonstrated that the glucuronidation pathway is involved in the conjugation of a number of additional anti-leukaemics (chlorambucil, bendamustine, venetoclax, acalabrutinib, cerdulatinib and duvelisib) and that UGT2B17 plays a role in the inactivation of chlorambucil and cerdulatinib. These two anti-leukaemic agents target different cellular pathways than fludarabine and have distinct modes of action. High UGT2B17 expression may thus lead to lower response to these drugs in CLL patients but this remains to be demonstrated. \n\nCrucial parts of the machinery governing UGT2B17 transcription remain poorly understood and especially in lymphoid cells. We provide the first evidence that UGT2B17 expression in B-cells is driven by a non-canonical UGT2B17 promoter and the use of an alternative noncoding exon 1c coupled to the common proteincoding region leading to the canonical UGT2B17 enzyme. According to our analysis, the gene expression signature associated with high UGT2B17 expression in CLL patients and cell models comprises a number of genes targeted by NF-κB. This promoter may thus be targeted by NF-κB that plays a central role in CLL. 1] [42] [43] [44] The mechanism underlying high UGT2B17 expression in B-cells remains to be fully explored. \n\nOur data also point to other mechanisms of altered drug sensitivity associated with high UGT2B17 expression. The inactivation of ibrutinib and idelalisib is largely dependent on UGT1A4 as the conjugation of other anti-leukaemics tested herein (chlorambucil, bendamustine, venetoclax, acalabrutinib, cerdulatinib and duvelisib). UGT1A4 is far less abundant than UGT2B17 in leukaemic B cells, and undetected at the mRNA level in most untreated CLL patients. Given that ibrutinib is mainly administered as an oral agent and subjected to first-pass hepatic metabolism, this drug is likely conjugated primarily in the liver expressing high levels of UGT1A4. In one of the lymphoid cell models, we showed drug-mediated induction of UGT1A4, but this finding remains to be demonstrated in CLL patients. Induction at the protein level also requires examination, given the recent report in drugresistant AML cells suggesting reduced mRNA expression but an enhanced UGT1A protein expression mediated by increased protein stability upon drug treatment. 18 onsistent with its regulatory function of the levels of endogenous molecules, high UGT2B17 may deplete intracellular metabolites leading to aberrant cell signalling and dysregulated cell functions, 22 which could favour progression in untreated CLL cases and subsequent drug resistance in treated patients. In our transcriptomic analysis, AMPK signalling was negatively associated with high UGT2B17 expression in conditions of both induced and high basal UGT2B17 expression in cell models and CLL patients, potentially linking B-cell metabolism to the glucuronidation pathway and subsequent adverse clinical outcomes. AMPK is a major regulator balancing energy supply and ultimately protects cells from harmful stresses by the coordination of multiple metabolic pathways. 45 The activation of the AMPK pathway has been shown to affect growth and apoptosis in CLL. 46 As a stressresponse molecule mediating drug resistance through different mechanisms, AMPK is further involved in the metabolism reprogramming and induction of autophagy, also regulating the self-renewal ability of cancer stem cells. 47 More recently, ibrutinib resistance was associated with a metabolic rewiring in CLL. 48 ikewise, UGT proteins were shown to be part of complex protein networks. Their functional interaction with other metabolic proteins induced broad changes in cell metabolism and may contribute to tumorigenesis and drug response. 49, 50 he findings of this study have to be seen in light of some limitations, including the fact that drug cytotoxicity and treatment outcome in relation to UGT2B17 expression was investigated in cell models and a limited number of fludarabine-treated patients. However, IC 50 values of tumour cells, namely for ibrutinib, were in the same range as those reported previously in B lymphoblastoid leukaemic cell lines and in primary cells from patients, [51] [52] [53] supporting the relevance of our initial observations. The impact of UGT2B17 expression on response to drug in vivo and ex vivo, as well as the influence of the microenvironment on UGT2B17related responses, remains to be examined in more details. \n\nIn summary, we unveiled a biochemical underpinning of reduced drug sensitivity related to the UGT2B17 metabolic pathway and drug inactivation. The evidence provided should prove useful for understanding and potentially overcoming drug refractoriness. The impact of glucuronidation in the inactivation of Fig. 5 Ascertaining pathways affected by high UGT2B17 expression in B cells and upstream regulators. a Schematic overview of the analysis pipeline. K-means clustering, enrichment and co-expression analyses served to identify pathways associated with high UGT2B17 expression and upstream transcriptional regulators. Detailed analysis of UGT2B17 transcriptome by RNA-seq revealed expression of noncanonical transcripts and alternate regulation. b A transcriptional signature associated with elevated UGT2B17 expression in PBMCs from 448 untreated CLL patients (GSE13159) 28 enabled clustering and identification of pathways altered by elevated UGT2B17 expression. The top 1000 most statistically significant genes were selected as features for clustering RNA-seq samples with log2 fold-change values using the K-means method. Cluster 3 contained genes which were globally down-regulated across all experimental conditions and was enriched with genes belonging to the AMPK signalling pathway. c RT-qPCR validation of down-regulated AMPK pathway-related genes in cell models. d UGT2B17 is predominantly expressed from an alternative UGT2B17_n2 transcript in lymphoid cell models and in CLL patients (n = 6; GSE99724) evaluated by RNA sequencing. e Schematic overview of the UGT2B17 gene and main coding transcripts in leukaemic cells. Only exons included in main leukaemic UGT2B17 transcripts are shown for sake of clarity. The alternative UGT2B17_n2-n4 transcripts include the supplementary exon 1c or 1b previously reported, which extend the 5′ untranslated sequence relative to the canonical UGT2B17_v1 transcript. 26 UGT2B17_n2 and n4 transcripts were validated by RT-PCR in cells of three CLL patients, and in MEC1 and JVM2. These transcripts encode a functional UGT2B17 enzyme. f Luciferase reporter gene expression assays were performed in MEC1 cells. Cells were transfected with pGL3 vectors containing either the canonical promoter of UGT2B17_v1 (P1), or the alternative promoters P2 or P3 upstream of each novel exons 1b or 1c, respectively. Experiments were conducted four times in triplicates. \n\na number of anti-leukaemic drugs is underestimated since we established that most agents are subjected to this metabolic process, including ibrutinib, idelalisib, venetoclax and duvelisib as well as other small molecules under development such as acalibrutinib and cerdulatinib. This may well apply to a number of other cancer therapeutics given the recent report that GLI1inducible glucuronidation imparts resistance to a broad spectrum of compounds including FDA-approved drugs such as methotrexate. 19 Our observations warrant additional studies to appreciate the prevalence and the clinical implications of high UGT2B17 expression on outcomes of leukaemia patients.",
"section_name": "MS Fragmentation Chromatograms",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "We would like to thank Isabelle Laverdière and Dominic Bastien for their help with the FACS analysis of leukaemic cells, and Etienne Audet-Walsh for his helpful advices with expression analyses.",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "The datasets generated during the current study are available in the Gene Expression Omnibus repository with the accession number GSE135030.",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "Study concept: C. G. Performed experiments: E. P. A., S. T., J. V., V. B., P. C., L. V., A. L., V. T. Analysed data: E. P. A., M. R., S. T., J. V., V. B., P. C., L. V., V. T., C. J. B., E. L., C. G. Patient recruitment and clinical data: K. V., T. L., M. S., S. S., D. D., R. H., U. J., P. S. Drafting of the paper: E. P. A., M. R., C. G. Critical revision of the paper for intellectual content: all authors. Obtaining funding: E. L. and C. G.",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "Ethics approval and consent to participate The study was carried out in accordance with the Declaration of Helsinki. Patients provided informed consent and the study was approved by the local ethical research committees of the Medical University of Vienna (Ethics vote 1499/2015) and the CHU de Québec (A14-10-1205). \n\nConsent to publish Not applicable.",
"section_name": "ADDITIONAL INFORMATION",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. Supplementary information is available for this paper at https://doi. org/10. 1038/ s41416-020-0887-6.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Funding information",
"section_num": null
}
] |
10.18632/oncotarget.26043
|
Fludarabine-resistance associates with ceramide metabolism and leukemia stem cell development in chronic lymphocytic leukemia
|
Fludarabine (flu) -containing regimens such as flu, cyclophosphamide and rituximab have been established as one of the standard first line therapy in medically-fit chronic lymphocytic leukemia (CLL) patients. Therefore, flu-refractory (primary flu-insensitivity or flu-caused relapse) remains a major problem causing treatment failure for CLL patients. We isolated the peripheral blood mononuclear cells (PBMCs) from CLL patients and treated with flu to find flu-refractory cases, and established flu-resistant clonal cells to study molecular mechanism of flu-resistance. By comparing parental MEC-2 cells, a human CLL cell line, we found that flu-resistant clonal cells were significantly increased lethal dose 50 of flu concentration, and up-regulated expression of P-glycoprotein, a drug-resistant marker, glucosylceramide synthase (GCS), an enzyme that can convert ceramide to glucosylceramide, and CD34, a leukemia stem cell marker. Overexpression of GCS leads to promptly elimination of cellular ceramide levels and accumulation of glucosylceramide, which reduces apoptosis and promotes survival and proliferation of flu-resistant clonal cells. Furthermore, we demonstrated that the accumulation of glucosylceramide can be blocked by PDMP to restore flu-sensitivity in flu-resistant clonal cells. We also found that elevating glucosylceramide levels in flu-resistant clonal cells was associated with up-regulation of GCS and CD34 expression. Importantly, overexpression of GCS or CD34 was also determined in flu-refractory PBMCs. Our results show that flu-resistance is associated with the alteration of ceramide metabolism and the development of leukemia stem cell-like cells. The flu-resistance can be reversed by GCS inhibition as a novel strategy for overcoming drug resistance.
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[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is classified as a lymphoproliferative disorder characterized by the accumulation of a clonally expanded lymphocytic population with resistance to apoptosis and coexpression of CD5, CD19, and CD23 in B lymphocytes in the peripheral blood, lymph nodes, bone marrow, spleen and liver [1] [2] [3] [4]. Cytogenetic analysis indicates that CLL has many different genetic mutations which are heterogeneous in terms of progression, therapeutic response and outcome [5] [6] [7] [8]. Several biological markers related to CLL outcome have been identified such as deletion of chromosome 17p13, 11q23 and 13q14, trisomy 12, expression of ZAP70, IgVH genomic rearrangement, and aberration of tumor protein 53 gene [5] [6] [7] [8]. These cytogenetic markers allow the stratification of broad prognostic groups of CLL patients; however, underlined mechanisms of drug insensitivity (primary drug refractory and chemo-caused drug-relapse) and the regulation to overcome drug-resistance remain poorly understood.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Since 1995, fludarabine (flu) has been used as one of the chemotherapy agents to treat CLL [9]. Later, rituximab, a monoclonal antibody against B-cell marker CD20, and alemtuzumab, a monoclonal antibody that binds to CD52, were also developed and have been used for immunotherapy in chemo naïve patients [10, 11]. More recently, in order to reduce the relapse rate and increase complete responses, a combination of multiple therapeutic agents such as the combination of flu, cyclophosphamide and rituximab (FCR) has been developed [12] [13] [14]. FCR has been established as one of the current standard first line treatment for medicallyfit CLL patients. The medium survival of FCR treatment is longer than 10 years, however, the survival for flu-refractory patients is only 0-3 years [10, 12]. Furthermore, most of the treated patients will eventually relapse, and about 10% of CLL patients are primary flu-refractory [10, 12]. It is clear that flu-insensitivity (primary flu-refractory and flu-caused relapse) is associated with poor survival, and represents a big challenge for treatment used flu and other purine analogue drug containing regimens. Therefore, it is very important to understand molecular mechanisms of fluresistance, to identify the novel targets, to develop rational therapeutic strategies for overcoming flu-resistance and to provide new therapeutic options. \n\nDrug-resistance is still one of the most pressing problems in treating cancer. Overexpression of some proteins [such as P-glycoprotein (P-gp), also known as ATP-binding cassette sub-family B member, multidrug resistance protein or cluster of differentiation 243 (CD243)] or alteration of some genes (such as p53) leads to the aberrant cell signaling and dysregulation of cell function [15, 16]. Sphingolipids are a class of lipids with important functions involved in a variety of cellular processes such as growth, proliferation, differentiation, senescence, apoptosis, survival and drug-resistance [17] [18] [19] [20]. The metabolism of sphingolipids is one of the important signaling pathways that regulate apoptotic (chemotherapy), survival (drug resistance) and proliferative (cancer progression) activities [17] [18] [19] [20]. Deregulation of sphingolipid metabolism is reflected in various pathophysiological conditions including metabolic disorders and cancers [17] [18] [19] [20]. Ceramide, the central molecule of sphingolipid metabolism, generally mediates anti-proliferative and pro-apoptotic functions, and has important therapeutic potential [21]. A number of anticancer drugs or cytotoxic agents can significantly induce the accumulation of ceramide in response to treatment [19]. On the contrary, ceramide can also be converted to glucosylceramide by glucosylceramide synthase (GCS) which transfers the glucose from uridine diphosphate glucose to ceramide, promptly decreasing ceramide levels and consequently promoting cell survival [18, 19]. It is very important to understand how ceramide metabolism is associated with drug-resistance. \n\nIn the present study, we isolated the peripheral blood mononuclear cells (PBMCs) from 34 CLL patients, treated them with flu, and analyzed cell viability to identify primary flu-refractory and flu-relapsed patients. We used MEC-2 cells, a CLL cell line established from the peripheral blood of a patient with B-chronic lymphocytic leukemia [22], to establish flu-resistant clonal cells and demonstrated that flu-resistance is associated with the alteration of ceramide metabolism and the development of leukemia stem cell (LSC)-like cells, and that the modulation of ceramide metabolism can enhance flu sensitivity and reverse flu resistance.",
"section_name": "Research Paper",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "We isolated the PBMCs from 34 CLL patients: 14 patients are chemo-naïve and 20 patients were treated with either single drug (alemtuzumab, rituximab, ofatumumab, pembrolizumab, bendamustine, ibrutinib or idelalisib) or combinations (bendamustine and rituximab; flu and rituximab; cyclphosphamide, vincristine and prednisone or FCR). The isolated PBMCs were treated with 10 µM flu for 72 hrs and then measured cell viability. Table 1 showed patient prognostic, pretreatment characteristics and cell viability. We found four flu-insensitive patients which cell viability is over 85%. Two are chemo-naïve patients (P7 and P21), one is bendamustine-rituximab-treating patient (P3) and the other is FCR-treating patient (P19). Due to the limited amount of patient blood samples and most of the PBMCs only survive but do not proliferate in vitro, we used MEC-2 cells, a CLL cell line, to establish flu-resistant clonal cells and to study molecular mechanism of flu-resistance.",
"section_name": "Effect of flu on PBMC viability",
"section_num": null
},
{
"section_content": "Using escalating concentrations of flu (from 30 µM up to 200 µM), we have established multiple MEC-2 fluresistant clones (Figure 1A ). Flu-resistant clonal cells did not display any obvious morphological changes, except they grew in large clumps (Figure 1B ). To determine the B cell lineage of flu-resistant clonal cells, we performed immunoblotting to compare expression of CD20, a B-cell marker, in MEC-2 cells and flu-resistant clonal cells (clones 13A and 18A). Figure 1C illustrated that expression of CD20 levels in MEC-2 cells and flu-resistant clonal cells was similar. Next, we determined the effect of flu concentrations on cell viability of MEC-2 cells and flu-resistant clonal cells. Figure 1D shows that the clonal cells are clearly resistant to flu-treatment. The lethal dose 50 (LD 50 ) in MEC-2 cells is 13. 5 ± 2. 1 µM, but >400 µM in flu-resistant clonal cells. To confirm flu-resistance, we analyzed expression of P-gp which is a drug-resistant marker and can pump drug out of cells [15]. Expression of P-gp was significantly up-regulated in flu-resistant clonal cells (Figure 1E ). These two lines of evidence demonstrate that our selected clonal cells are flu-resistant cells.",
"section_name": "Establishment and characteristics of flu-resistant clones",
"section_num": null
},
{
"section_content": "Earlier studies showed the involvement of caspase activation and ceramide accumulation in flu-induced apoptosis of B-cell leukemia cell lines (WSU and JVM-2 cells) and Jurkat lymphoblastic leukemia cells [23, 24]. In order to investigate whether flu-resistance is associated with ceramide metabolism, we firstly determined whether flu induces MEC-2 cell apoptosis",
"section_name": "Flu-treatment induces apoptosis in MEC-2 cells but not in flu-resistant clonal cells",
"section_num": null
},
{
"section_content": "Ceramide, a product of sphingomyelin degradation, can induce cell programmed death [21] and can also be converted to other non-cytotoxic metabolites, such as glucosylceramide, which has the effect of promptly eliminating ceramide level and consequently promoting cell survival [17] [18] [19]. In examining [ 3 H]sphingomyelin degradation, we found similar degradation of [ 3 H] sphingomyelin in flu-treated MEC-2 cells and flu-resistant clonal cells (Figure 3B ) although the accumulation of [ 3 H] ceramide was not observed in flu-resistant clonal cells (Figure 3A ). The results indicate that the formation of ceramide in flu-resistant clonal cells is likely converted to other metabolites. To identify possible metabolites, we analyzed the same samples and found a significant increase of [ 3 H]glucosylceramide in flu-resistant clonal cells (Figure 3C ). To determine whether the accumulation of glucosylceramide is associated with GCS overexpression or activation, we further performed immunoblotting to determine expression of GCS in MEC-2 cells and flu-resistant clonal cells. Figure 3D clearly shows that GCS expression is up-regulated in flu-resistant clonal cells. Next, we treated MEC-2 cells with different concentrations of glucosylceramide for 24 hrs and then determined the effect of glucosylceramide on GCS expression and cell proliferation. The results showed that glucosylceramide enhanced expression of GCS and CD34 (Figure 3E ) and promoted cell proliferation (Figure 3F ).",
"section_name": "Accumulation of glucosylceramide and overexpression of glucosylceramide synthase in flu-resistant clonal cells",
"section_num": null
},
{
"section_content": "Our results indicate that the conversion of ceramide to glucosylceramide is clearly increased in flu-resistant clonal cells. To further confirm whether this conversion is associated with CLL cell flu-resistance, we use PDMP to block the conversion of ceramide to glucosylceramide. PDMP is a ceramide analog and can block the glycosylation of ceramide by inhibiting GCS [25]. The cells were prelabeled with [ 3 H]palmitic acid for 24 hrs, and then incubated with different concentration of PDMP in 100 µM flu-containing medium for 24 hrs. Total cellular lipids were extracted and analyzed for the",
"section_name": "PDMP inhibits the formation of glucosylceramide and restores chemo-sensitivity in flu-resistant clonal cells",
"section_num": null
},
{
"section_content": "Cancer stem cells are a small subpopulation of cancer-initiating cells that tend to be drug resistance and have the capabilities of self-renewal, proliferation, differentiation and tumorigenicity [26, 27] doubling times of ~30 ± 1. 4 hours that were significantly longer than the 22 ± 2. 1 hour doubling time measured for MEC-2 cells. It is clear that flu-resistant clonal cells have slow-growing and self-renewal capacity. We further analyzed expression of CD34, a marker antigen expressed on the surface of LSCs [26, 27], in MEC-2 cells and fluresistant clonal cells. Figure 5B shows that expression of CD34 is significantly up-regulated in flu-resistant clonal cells (Figure 5B ). To confirm that flu-resistant clonal cells are LSC-like cells, we used methylcellulose-based medium for colony formation. Figure 5C -5H showed the colonies of MEC-2 cells and flu-resistant clonal cells in the presence or absence of flu. More colonies were found in flu-resistant clonal cells compared to parental MEC-2 cells, in particular with flu treatment (Figure 5I ). Based on slow-growing, self-renewal capacity, up-regulation of CD34 and colony formation, flu-resistant clonal cells compared to parental cells tend to be more LSC-like cells.",
"section_name": "Flu-resistant clonal cells are LSC-like cells",
"section_num": null
},
{
"section_content": "As shown in Table 1, the PBMCs from four CLL patients are flu-insensitivity. We lyzed multiple CLL patient's PBMCs from chemo naïve, treated with either the combination of bendamustine and rituximab or FCR, and then the samples were processed for immunoblotting to determine the expression of GCS and CD34. We found that expression of GCS or CD34 was significantly upregulated in flu-insensitive samples compared to flusensitive samples (Figure 6 ). These results are similar to flu-resistant clonal cells, and indicate that flu-insensitivity in PBMCs is also associated with the alteration of ceramide metabolism and the development of LSC-like cells.",
"section_name": "Overexpression of GCS and CD34 in fluinsensitive PBMCs",
"section_num": null
},
{
"section_content": "The resistance to flu-based therapies is one of the predominant reasons for treatment failure and is a major challenge for CLL treatment. In analyzing CLL patients resistant to flu, Moussay et al. found various genomic abnormalities (deletion or gain) in more than twenty genes that are involved in p53, DNA damage and repair, cell cycle and apoptosis signaling [28]. Using piggyBac transposon-mediated mutagenesis combined with nextgeneration sequencing, one recent report also found that some new candidate genes such as deoxycytidine kinase and BMP-2-inducible protein kinase could be associated with flu-resistance in HG3 cells, a human modified CLL cell line [29]. Identifying the genes that are involved in fluresistance is important because these cytogenetic mutations may be prognostic markers. However, genomic alterations are not enough because the proteins coded by these genes are involved in multiple different signaling pathways that can play opposite roles in the regulation of cellular functions. Understanding the signaling pathways is a key to develop new strategies for overcoming flu-resistance. \n\nThere are few CLL cell lines available for research. Current several flu-resistant cell lines, such as malignant B-1 cell line (a mouse model of CLL) [30], K562 cells (a cell lines from chronic myelogenous leukemia patient) [31] and HG3 cells (a human modified CLL cell line) [29], are not ideal cell model for defining flu-resistant signaling pathways because these cell lines are either a modified cell line or not human CLL cell lines. Here, we used MEC-2 cells, a cell line established from the peripheral blood of a patient with B-chronic lymphocytic leukemia [22] to establish flu-resistant CLL clonal cells and used the clonal cells as a platform to study molecular mechanism of flu-resistance. Our recent study shows that MEC-2 cells respond to flu treatment similarly to the PBMCs from CLL patients [32]. By comparing parental cells to flu-resistant clonal cells, we found that flu-resistant clonal cells like their parent cells express very high CD 20, a B-cell CD marker, but the flu-resistant clonal cells exist the significant alteration of ceramide metabolism that is associated with overexpression of GCS and the development of LSC-like cells that up-regulates CD34 expression. Importantly, up-regulation of GCS and CD34 expression was also found in flu-resistant PBMCs from CLL patients (Figure 6 ). This could be proved by the fact that the conversion of ceramide to glucosylceramide in CLL cells plays a key role in flu-resistance. \n\nCeramide induced by numerous apoptotic stimuli (e. g. cytokines, anticancer drugs or cytotoxic agents, irradiation and environmental stresses) is recognized as a proapoptotic signaling molecule. Increasing the levels of cellular ceramide can enhance many proapoptotic molecules such as NH 2 -terminal Jun kinase, caspase-3, and reactive oxygen species [20] and suppress antiapoptotic molecules such as phosphatidylinositol 3-kinase, AKT and mTOR [33, 34]. Comparing to parental cells, our data clearly showed that flu-resistant clonal cells altered ceramide metabolism and up-regulated GCS expression (Figure 3 and Figure 6 ). Schwamb et al. . identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide in drug-resistance of primary CLL cells [35]. Earlier reports indicate that glucosylceramide can stimulate DNA synthesis and cell growth (Figure 3F ) [36, 37]. More and more evidence supports the accumulation of glucosylceramide in multidrug resistant cancer cell lines isolated from different solid tumors [38, 39]. Overexpression of GCS was also reported in adriamycin-resistant K562 cells, vincristineresistant HL-60 cells and clinical multidrug resistant samples of acute myeloid leukemia, acute lymphocytic leukemia and chronic myeloid leukemia [40] [41] [42] [43]. Our results and the data from many others [38] [39] [40] [41] [42] [43] indicate the biochemical significance of accumulation of glucosylceramide and overexpression of GCS in drug-resistant cancer cells, and the inhibition of GCS has therapeutic potential for restoration of chemo-sensitivity and reversal of drug-resistance. \n\nOur data demonstrate that overexpression of GCS alters ceramide metabolism and promotes cancer cell survival. P-gp is the first described and most extensively studied multidrug resistant efflux protein that results in resistance to many structurally unrelated drugs [44]. \n\nEarlier studies showed that glucosylceramide is a substrate for P-gp and that both ceramide and glucosylceramide regulate P-gp expression and function [41, [45] [46] [47]. Using a dithionite fluorescence quenching technique, Eckford et al. [45] showed that P-gp is a broad-specificity outwardlydirected flippase which enhances glycosphingolipid translocation. On the other hand, both cyclosporin A and GF120918 (p-gp inhibitors) can increase C8-ceramide mediated apoptosis [46]. Using siRNA to silence GCS, knockdown of GCS expression affects P-gp expression and function [42]. All these data clearly shows that either ceramide or glucosylceramide plays an important role in the regulation of P-gp expression and function [47]. We found up-regulation of P-gp expression in flu-resistant clonal cells (Figure 1E ), but the modulation of glucosylceramide levels by adding or depleting glucosylceramide does not significantly regulate P-gp expression in MEC-2 cells and flu-resistant clonal cells (Figures 3E and 4D ). Whether P-gp expression is regulated by ceramide metabolites and whether P-gp interacts with GCS need to be further studied. \n\nCancer stem cells were first identified in myeloid leukemia with the cell surface marker combination of CD34 + and CD38- [48]. These cells exhibit a slowing growth and pronounced self-renewal and differentiation capacity. Recently, accumulating evidence supports that cancer stem cells are considered as a major source of cancer recurrence and therapeutic resistance [49] [50] [51]. Based on overexpression of CD34, slow-growth and self-renewal capacity and colony formation (Figure 5 ), we conclude that flu-resistant clonal cells are LSC-like cells. One recent study shows that glucosylceramide synthase is enhanced in breast cancer stem cells but not in normal mammary epithelial stem cells [52]. With the accumulation of glucosylceramide and up-regulation of GCS and CD34 expression in fluresistant clonal cells, it indicates that ceramide metabolism is likely associated with the development of LSC. \n\nIn conclusion, our data present signaling pathways that are involved in flu-resistance (Figure 7 ) and show consistent evidence that flu-resistant clonal cells are associated with ceramide metabolism (decreasing ceramide level and increasing glucosylceramide level) and that reducing GCS expression and activity can reverse flu-resistance and restore drug-sensitivity. Moreover, fluresistance is also associated with the up-regulation of CD34 expression which links to the development of LSClike cells.",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "All chemicals were purchased from Fisher Scientific (Pittsburgh, PA) or Sigma Chemicals (St. Louis, MO) unless specified otherwise. Cell culture reagents were provided by HyClone. (Logan, UT). Methylcellulose-base medium (MethoCult H4434 classic) was purchased from Stem Cell Technologies (Cambridge, MA). Flu and PDMP were obtained from Cayman Chemical (Ann Arbor, MI). Glucosylceramide was supplied by Matreya, LLC (College Station, PA). Ceramide was purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama). [ 3 H]palmitic acid (30-60 Ci/mmol) were obtained from American Radiolabeled Chemicals, Inc. (St Louis, MO). Ficoll-Paque Plus was obtained from Amersham Biosciences (Piscataway, NJ, USA). The monoclonal anti-CD20 (L26), anti-GAPDH (0411), and anti-cytochrome c (7H8) antibodies, and the polyclonal anti-UGCG (glucosylceramide synthase, H-300) and anti-CD34 (H-140) antibodies were provided by Santa Cruz Biotechnology, Inc. (Dallas, TX). The monoclonal anti-P-glycoprotein (F4) antibody was supplied by Sigma-Aldrich (Saint Louis, Mo). CellTiter 96 ® Non-Radioactive Cell Proliferation Assay kit (MTT) was purchased from Promega (Madison, WI, USA). Halt protease and phosphatase single-use inhibitor cocktail, SuperSignal West Pico chemiluminescent substrate and BCA protein assay reagent were obtained from Thermo Scientific (Rockford, IL).",
"section_name": "Materials",
"section_num": null
},
{
"section_content": "Blood was obtained from CLL patients as defined by NCI96 criteria 28 [53] following a receipt of written informed consent under an IRB protocol approved by Saint Louis University. PBMCs were isolated from whole blood immediately following donation using Ficoll density gradient centrifugation. Isolated cells were plated in 96-well assay plates at a concentration of 10-50,000 cells (depend on patient cell numbers) per well in 100 µl of RPMI 1640 media with 10% FBS with or without 10 µM flu. The cells were cultured for 72 hrs, and then cell viability was determined using Promega's CellTiter 96 ® Non-Radioactive Cell Proliferation Assay kit (MTT) according to the manufacturer's instructions [32]. Absorbance at 570 nm was recorded using a BioTek Epoch Reader (Winooski, VT). The rest of PBMCs from CLL patients were harvested and lysed for immunoblotting.",
"section_name": "PBMC isolation and treatment",
"section_num": null
},
{
"section_content": "",
"section_name": "Cell culture and establishment of flu-resistant clones",
"section_num": null
},
{
"section_content": "MEC-2 cells were treated with or without 100 µM flu for 3 hrs and flu-resistant clonal cells were maintained in the regular medium containing 100 µM flu. After treatment, cells were harvested and washed once with 1 × PBS. The cells were homogenized in a buffer containing 20 mM Hepes, 2 mM MgCl 2, 1 mM EDTA and 1 mM DTT with protease and phosphatase inhibitor cocktails and centrifuged at 14,500 rpm to yield a pellet and supernatant. Cell lysates and cellular fractions were measured for protein concentration using the BCA protein assay reagent with BSA as a standard, and then adjusted to equal amounts of cellular protein in 1 × loading buffer. The samples were boiled for 10 min and 15 µg/lane were subjected to SDS-PAGE, and processed for immunoblotting with the appropriate antibodies [54]. \n\nIn the experiments for analyzing cell viability, MEC-2 cells and flu-resistant clonal cells were plated in 96-well assay plates at a concentration of 50,000 cells per well in 100 µl of culture medium with different concentrations of flu. The cells were treated for 72 hrs, and then cell viability was determined. MEC-2 cells were incubated with different concentrations of glucosylceramide for 24 hrs, and the samples were further processed for immunoblotting analysis. For PDMP-treated experiments, MEC-2 cells and flu-resistant clonal cells (in \"maintaining\" medium containing 100 µM flu) were cultured in 96-well assay plates with different concentrations of PDMP for 72 hrs and then analyzed for cell viability. In some experiments, flu-resistant clonal cells were cultured in 6-well plates and treated with 50 µM PDMP for 24 hrs, and the samples were used for immunoblotting analysis.",
"section_name": "Cell treatment, immunoblotting and cell viability assay",
"section_num": null
},
{
"section_content": "MEC-2 cells and flu-resistant clonal cells were cultured in 6-well plates containing 0. 5 ml medium with 2 μCi/ml of [ 3 H]palmitic acid and 0. 5 ml medium with or without 100 µM flu. After 24 hr treatment, the cells in the medium were collected and centrifuged at 1,500 rpm for 5 mins. Total cellular lipids in the cells were extracted by chloroform: methanol: water (5. 5:5. 5:5, v/v). In some experiments, the cells were prelabeled with [ 3 H]palmitic acid for 24 hrs in 100 µM flu-containing medium and then treated with different concentrations of PDMP for another 24 hrs. The individual radiolabeled lipid was resolved from the total cellular lipids by thin layer chromatography and identified by co-migration with commercial standards in different solvent systems: I) chloroform: acetic acid (90:10, v/v) for ceramide, II) chloroform: methanol: ammonium hydroxide (40:10:10, v/v) for glucosylceramide, and III) chloroform: methanol: acetic acid: and water (50:25:8:4, v/v) for sphingomyelin. The standards were visualized with iodine vapor, and the areas corresponding to ceramide, glucosylceramide or sphingomyelin were scraped into scintillation vials and quantitated by liquid scintillation spectrometry.",
"section_name": "Cell radiolabeling and lipid metabolite analysis",
"section_num": null
},
{
"section_content": "MEC-2 cells were treated with or without 100 µM flu for 24 hrs and flu-resistant clonal cells were cultured in \"maintaining\" medium containing 100 µM flu. After treatment, cells (dead and alive) were harvested by centrifugation at 1,500 rpm for 2 mins, and the pellets were re-suspended in 0. 5 ml of lysis buffer containing 5 mM Tris-HCl, pH 8. 0, 20 mM EDTA, and 0. 5% Triton X-100 and placed on ice for >60 mins. The samples were then centrifuged at 14,500 rpm for 20 mins, and the supernatant containing DNA cleavage products with the same amount of cellular proteins was precipitated by isopropyl alcohol for 15 hrs. The samples were centrifuged at 14,500 rpm for 20 mins, and the pellets were re-suspended in Tris-EDTA buffer with proteinase K and RNase A for 2-3 hrs at 37° C. DNA fragments were separated on a 1. 2% agarose gel, visualized with ethidium bromide, and photographed using the Bio-Rad Image System.",
"section_name": "Measurement of DNA fragmentation",
"section_num": null
},
{
"section_content": "MEC-2 Cells and flu-resistant clonal cells were harvested, counted and adjusted to 2 × 10 4 cell/ml. To set up colony forming assay, we removed 5 ml MethoCult medium to a set of tubes and then added 0. 5 ml cell suspense to each tube. The cells were mixed with MethoCult medium by vortex, dispensed into 48-well plates and cultured in the 37° C. The 25 μl regular medium or 100 µM flu-containing medium was carefully added to the designed wells in day 1 and day 8. The colonies were photographed by AMG EVOS Core Cell Imaging System. To quantify the colonies, the plates were determined using Promega's CellTiter 96 ® Non-Radioactive Cell Proliferation Assay kit (MTT).",
"section_name": "Colony forming unit setting and analysis",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "This work was supported by the grants from the Elsa U. Pardee Foundation and Saint Louis University.",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "",
"section_name": "Data analysis",
"section_num": null
},
{
"section_content": "The data were analyzed for significance using oneway repeated measures of ANOVA followed by Tukey's test for comparisons between the experimental groups shown in the figures.",
"section_name": "Data analysis",
"section_num": null
},
{
"section_content": "The materials and all data generated or analyzed during this study are available from the corresponding author on reasonable request.",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The authors are responsible for the reported research, and have participated in the concept and design, analysis and interpretation of data, drafting or revising of the manuscript, and have approved the manuscript as submitted.",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "Blood was obtained from CLL patients following a receipt of written informed consent under an IRB protocol (00005304) approved by Saint Louis University.",
"section_name": "Ethics approval",
"section_num": null
},
{
"section_content": "CLL: Chronic lymphocytic leukemia; FCR: fludarabine, cyclophosphamide and rituximab; flu, fludarabine; GCS: glucosylceramide synthase; LSC: leukemia stem cell; PBMCs: peripheral blood mononuclear cells; P-gp: P-glycoprotein.",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "CH conducted the experiments; YT collected the patient blood samples; CH wrote the paper; and all authors participated in the experimental design, result discussion and interpretation, and edited and approved the final draft of the paper.",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
}
] |
10.1007/s00277-024-05627-w
|
Conditional survival to assess prognosis in patients with chronic lymphocytic leukemia
|
<jats:title>Abstract</jats:title><jats:p>Biomarkers in chronic lymphocytic leukemia (CLL) allow assessment of prognosis. However, the validity of current prognostic biomarkers based on a single assessment point remains unclear for patients who have survived one or more years. Conditional survival (CS) studies that address how prognosis may change over time, especially in prognostic subgroups, are still rare. We performed CS analyses to estimate 5-year survival in 1-year increments, stratified by baseline disease characteristics and known risk factors in two community-based cohorts of CLL patients (Freiburg University Hospital (<jats:italic>n</jats:italic> = 316) and Augsburg University Hospital (<jats:italic>n</jats:italic> = 564)) diagnosed between 1984 and 2021. We demonstrate that 5-year CS probability is stable (app. 75%) for the entire CLL patient cohort over 10 years. While age, sex, and stage have no significant impact on CS, patients with high-risk disease features such as non-mutated <jats:italic>IGHV</jats:italic>, deletion 17p, and high-risk CLL-IPI have a significantly worse prognosis at diagnosis, and 5-year CS steadily decreases with each additional year survived. Our results confirm that CLL patients have a stable survival probability with excess mortality and that the prognosis of high-risk CLL patients declines over time. We infer that CS-based prognostic information is relevant for disease management and counseling of CLL patients.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous hematologic malignancy with variable outcomes [1, 2]. While some patients may have a prolonged survival without needing treatment, others experience a rapidly fatal disease course despite receiving highly effective therapies [3] [4] [5]. Improving patient management and treatment strategies requires reliable biomarkers to predict prognosis, disease progression, and treatment responses. \n\nAdvancements in understanding the molecular mechanisms and detailed clinical characterization have led to the identification of numerous prognostic and predictive biomarkers that complement the classic clinical staging classifications [6, 7]. Cytogenetic and molecular genetic aberrations like deletions in chromosomes 11q and 17p, TP53 mutations, and the immunoglobulin heavy chain variable gene (IGHV) mutation status play a crucial role in prognosis estimation and treatment respons [8, 9]. Composite prognostic scores like the CLL International Prognostic Index (CLL-IPI) integrate genetic, biochemical, and clinical parameters to predict survival differences. However, these models have limitations as they fail to consider how prognosis may change over time [10] [11] [12] [13]. From a patient's perspective, the probability of surviving another t years when she/he has already survived s years might be more relevant than a static prediction. Conditional survival (CS) analysis, which takes into account how long an individual has already survived, offers dynamic prognostic information about changes in survival probability over time. For many cancers, CS is reported to increase over time [14]. In contrast, CLL has shown remarkable stable survival estimates even over a 10-year period following diagnosis. However, it is unclear whether this stability applies to all clinical stages or risk groups [15]. \n\nTo address these knowledge gaps, we performed CS analyses in CLL patients with different risk profiles to improve prognosis estimation in patient subgroups by accounting for years already survived [16]. As treatment options evolve, incorporating dynamic prognostic information becomes increasingly crucial for guiding clinical practice and improving patient outcomes in CLL.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Material and methods",
"section_num": null
},
{
"section_content": "Data from CLL patients were collected independently from two German university hospitals, including baseline characteristics, clinical parameters, and biological and molecular markers. Three hundred and sixteen CLL patients were included at the Department of Hematology, Oncology and Stem Cell Transplantation at the University Freiburg Medical Center between December 1984 and April 2014, and 564 CLL patients were included at the Augsburg University Hospital between January 1999 and January 2021. The diagnosis of CLL and response to therapy were assessed according to the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) guidelines [17]. All diagnostic variables were determined at the time of diagnosis or first presentation. Data on demographics, clinical and molecular biological parameters, and disease progression were collected from local clinical information systems, digitized reports from external practices and clinics, and the cancer registries of the Comprehensive Cancer Center Freiburg (CCCF) and Augsburg University Hospital. \n\nData collection was approved by the local ethics committee, and written informed consent was obtained from all patients from the University Freiburg Medical Center. For patients of the Augsburg University Hospital, anonymized retrospective analysis of data is permitted without informed consent according to the Bavarian Hospital Act (BayKrG) in the version of March 28, 2007 (GVBl. S. 288, BayRS 2126-8-G Art. 27 Abs4). The study including data collection and analyses was performed according to the terms of the Declaration of Helsinki.",
"section_name": "Patient cohorts and data collection",
"section_num": null
},
{
"section_content": "Biological and molecular biomarkers were assessed according to local diagnostic standards (described in the Supplement).",
"section_name": "Assessment of biological and molecular biomarkers",
"section_num": null
},
{
"section_content": "Overall survival (OS) was calculated from the date of initial diagnosis until the date of death from any cause. When no event of interest occurred, observations were censored at the time the patient was last seen alive or at the latest on July 1, 2017, and March 1, 2021, for Freiburg and Augsburg, respectively. OS rates were estimated using the Kaplan-Meier method [18]. Conditional survival (CS) estimates stratified by covariables were based on Cox proportional-hazards model in the corresponding landmarked dataset [16]. OS rates were compared using the log-rank test. CS is defined as the probability of surviving additional time t after the patient has already survived a certain time s: CS was calculated using landmarks s = 0 to s = 10 years.",
"section_name": "Survival analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Data collection was performed for a total of n = 880 CLL patients of two community-based cohorts (Table 1 ; Freiburg University Medical Center: n = 316 CLL; Augsburg University Hospital: n = 564). \n\nFor the Freiburg cohort, the median follow-up time was 8. 7 years (range 5. 5-14 years). Patients were predominantly male (66%). The median age at diagnosis was 62 years (interquartile range (iqr) 53-69 years). At diagnosis, 37% and 43% of patients could be assigned to the early, asymptomatic stages Rai 0 or Binet A, respectively. IGHV gene mutation status was available for 289 of the 316 patients. Of these, 114 (36%) had a non-mutated IGHV locus with ≥ 98% sequence homology, and 175 (55%) had a mutated IGHV locus. Cytogenetic analyses were available for 304 out of the 316 patients. In 138 cases",
"section_name": "Baseline patient characteristics",
"section_num": null
},
{
"section_content": ". \n\n(45%), a sole del(13) (q14) was detected; 38 patients (13%) had a del(11) (q22), 47 patients (15%) a trisomy 12, and 21 patients (7%) a del(17) (p13). Serum β2-microglobulin (B2MG) levels were available for 276 of the 316 patients. The median B2MG level was 3. 29 mg/l (IQR 2. 37-5. 08 mg/l). One hundred and twenty-nine patients (41%) had an elevated B2MG level (upper limit of the normal range, 3. 5 mg). According to CLL-IPI, 94 patients (30%) were classified as high (score = 2) or very high (score = 3) risk. Until the end of follow-up, 212 (67%) patients had received therapy. Patients who were registered at the Augsburg University Hospital had a median follow-up time of 4. 4 years (1. 8-8. 4 years). Patients were predominantly male (64%) and median age was 69 years (iqr 61-76 years). More than one-third of the cohort were in an early-stage Binet A or Rai 0 at diagnosis (35% and 63%, respectively). IGHV mutation status, cytogenetics, B2MG levels, and consequently the CLL-IPI score were not available for the majority of patients. Comorbidities were systematically recorded in the Augsburg cohort (summary in Supp. Figure 1 ), and patients were stratified using the Cumulative Illness Rating Scale (CIRS). A relevant comorbidity burden was identified in 327 (58%) patients with a CIRS > 6. Until the end of follow-up, 272 (48%) patients had received therapy. 2E ). \n\nFor IGHV mutation status, we observed significant differences of 5-year survival probability with 91. 4% (CI 87-95. 9%) for mutated patients and 78. 9% (CI 70. 8-86. 9%) for those with unmutated IGHV locus (Fig. 2A ). Median OS was 20. 3 years (CI 17. 89-24. 3) and 9. 3 years (CI 7. 77-10. 9) for mutated and unmutated patients respectively [HR 3. 90 (CI 2. 58-5. 88); p = 9. 8e-11]. Patients with del(17) (p13) formed the patient subgroup with the poorest survival with a 5-year survival probability of 61. 1% (CI 38. 6-83. 6%) compared to 87. 6% (CI 83. 5-91. 8%) in patients with intact TP53 (Supp. Figure 2F ). We were also able to validate the prognostic separation of the composite risk score CLL-IPI in our non-study cohort of CLL patients (Fig. 2B ). Patients with CLL-IPI scores 0 and 1 had a significantly better outcome with a median OS of 27. 3 and 15. 85 years (HR 1vs0 3. 15, CI 1. 77-5. 60, p = 9. 8e-5), respectively, compared to patients of the combined risk group 2/3 who had a median OS of 9. 14 years (HR 2+3vs0 8. 52, CI 4. 84-15. 01, p = 1. 2e-13). Comorbidities as assessed by CIRS impacted significantly on prognosis. Patients with CIRS ≤ 6 had a significantly longer median OS compared to patients with higher comorbidity burden (HR ≤6vs>6 1. 40, CI 1. 07-1. 84, p = 0. 013) (Supp. Figure 2G ).",
"section_name": "CS(t|s) = S(s + t) S(s)",
"section_num": null
},
{
"section_content": "CS was first determined for the entire patient cohort (n = 880) (Fig. 3A, B ). The Kaplan-Meier curves for the landmarks s = 0 to s = 5 years showed parallel shifted comparable shapes, indicating highly similar survival of CLL patients in general, regardless of how long they had survived from diagnosis. The conditional 5-year survival CS(5|s) for s = 0 to s = 10 years after diagnosis ranged from 72. 5% (CI 69-76. 1%) to 74. 9% (CI 71. 9-78%) and remained constant throughout this period, indicating a stable prognostic prediction from baseline to 10 years after diagnosis. As a sensitivity analysis, we estimated CS incorporating center as a co-variable. Observed differences in CS were explained by the different age structures of the two community-based cohorts (Supp. Figure 3. A, B ).",
"section_name": "Conditional survival",
"section_num": null
},
{
"section_content": "When separated by patient age (≤ 65 and > 65 years), a constant 5-year CS probability in the ranges of 79. 4-85. 7% and 60. 2-66. 0% was observed for both patient subgroups over the time span of s = 0,…,10 years after diagnosis. Patients ≤ 65 years at diagnosis (n = 412) had a higher probability for 5-year CS than older patients (Supp. Figure 4A ). Throughout the observation period, we observed no sexassociated differences in CS (Supp. Figure 4B ). Patients in the early, asymptomatic stage Rai 0 (n = 314) at diagnosis had a significantly higher probability of 82. 9%/81. 0% vs. 73. 5%/72. 1% than higher stage patients for 5-year CS for the first 2 years (s = 0 and s = 1 years) after diagnosis (Fig. 4A ). However, the 5-year CS probability of both subgroups converged in further years of the disease course and was almost identical between 73. 8 and 75. 8% between s = 7 and s = 10 years, indicating a loss of long-term prognostic value of assessment of Rai staging at diagnosis over time. An almost identical result was obtained by comparing the early, asymptomatic stage Binet A at diagnosis with the advanced disease stage Binet B/C (Fig. 4B ). \n\nThe strong prognostic value of IGHV mutation status was also reflected in the CS. Patients with a mutated IGHV locus had a stable 5-year CS between 87. 3 and 92. 8% without significant changes from baseline over the time span of 10 years from diagnosis (s = 0 to s = 10 years). In contrast, high-risk patients with unmutated IGHV locus showed a significantly lower 5-year survival probability of 75. 8% at diagnosis (s = 0 years) with an immediate steady decline to 43. 3% over the identical time span (Fig. 5A ). This was also evident in the analysis of CLL-IPI (Fig. 5B ). While patients with CLL-IPI 0 had a stable 5-year CS of 96. 4, 93. 4, and 92. 5% over the landmarks from s = 0, s = 5, to s = 10 years, a trend of a decline from baseline at diagnosis could be observed for CLL-IPI 1 patients with 89. 2, 80. 5, and 81. 8% over the identical landmarks. In high-risk patients with CLL-IPI 2/3, 5-year CS was already significantly lower at 73. 3% at the time of diagnosis and showed a significant decline to 46. 9% over the entire 10-year period after diagnosis. A similar, yet less pronounced, decrease in 5-year CS over the course of 10 years after diagnosis was also observed for high-risk patients defined by del(17) (p13) (Supp. Figure 3C ). The extent of comorbidities proved to be a prognostic factor in the Augsburg cohort with a 5-year CS at the time of diagnosis of 73. 6% and 65. 1% for patients with CIRS ≤ 6 and CIRS > 6, respectively. However, the 5-year CS during the observation period showed almost perfectly parallel curves for both subgroups with no significant changes over the 10-year period (see Fig. 3D ). \n\nFinally, we asked if receiving treatment over the observation period would impact CS. Patients who had received treatment at any time point during the observation period had a worse prognosis at diagnosis than untreated patients, with 56. 8 vs. 76. 2% 5-year survival. However, receiving treatment for CLL was not associated with significant changes in CS over time (Supp. Figure 4 ).",
"section_name": "Conditional survival stratified by prognostic factors",
"section_num": null
},
{
"section_content": "Accurate long-term prognosis in CLL is a challenge. Existing prognostic models, such as the CLL-IPI, are limited as they provide predictions based on a single time point, typically at diagnosis, without taking into account years already survived. This limitation hinders effective disease management and appropriate counseling of CLL patients. Previous analyses of prognostic models in CLL have highlighted this issue, raising uncertainties about which models can be reliably used in clinical practice to predict long-term outcomes [15]. \n\nIn this study, we present a systematic analysis of absolute CS in CLL patients based on data from a non-study cohort comprising individuals diagnosed between 1984 and 2021 at two university medical centers. Our goal was to investigate survival estimates over time, stratified across different CLL patient subgroups. With a mean post-diagnostic follow-up of 7. 3 years, we observed a constant 5-year CS of approximately 75% for the entire patient cohort, demonstrating stable survival over a period of up to 10 years. \n\nNotably, CS remained remarkably stable regardless of age at diagnosis, although patients older than 65 years exhibited approximately 20% lower CS likelihood. These findings are consistent with a Canadian study that demonstrated stable CS up to 5 years after diagnosis [19]. Data from the USA and the Netherlands noted very slight decreases over time [20, 21]. However, it should be noted that the analysis in these studies examined relative CS from an epidemiological perspective, whereas we emphasized the patient-relevant perspective and considered CS. These data suggest that the probability of surviving additional 5 years remains at 75% or slightly below over the disease course, indicating that CLL patients face a constant risk of death with each additional year of survival [22]. This is in contrast to the patterns observed in most hematologic and solid malignancies that are potentially curable [23]. Some aggressive diseases (e. g., pancreatic cancer, malignant melanoma) are associated with increasing CS [14, 17]. For other entities at early stages or with a tendency to recur (e. g., prostate or breast cancer), CS increases slightly over time or remains stable [19, 20, 23]. Constant CS comparable to CLL has been shown for multiple myeloma [24]. Common to both entities is the lack of curative treatment options and the goal of remission maintenance. CLL remains incurable to date with a steady risk of infection, autoimmune complications, secondary malignancies, and conversion to high-grade B-cell lymphoma (Richter's transformation). It is unclear whether the availability of highly effective targeted treatment options (e. g., BTKis, venetoclax, novel CD20 antibodies) might influence these results. \n\nPrevious analyses have mostly not included the clinical and biological heterogeneity of CLL. Because such factors are available for a large proportion of patients in our cohort, we were able to stratify patients by known risk parameters including the composite prognostic index CLL-IPI. For IGHV mutation status, TP53 deficiency, and the CLL-IPI, our CS analyses revealed a clinically meaningful and significant separation of subgroups. IGHV mutation status and TP53 deficiency have known prognostic value and impact on the choice of targeted therapy [25, 26]. Their importance is also emphasized by the weighting in the CLL-IPI scoring system. The much less favorable prognosis of patients with an unmutated IGHV locus and the marked deterioration over time (CS decreases by approximately 30% over 10 years) not seen in IGHV-mutated patients reflect the heterogeneity of CLL and the fundamental biological differences of the disease associated with the IGHV mutation status. While patients with del(17) (p13) had a similarly poor prognosis at diagnosis followed by worsening in CS over time, the small number of patients and possible acquisition of TP53 deficiency during disease progression could obscur prognostic trends over time. Not surprisingly, the composite prognostic score CLL-IPI, whose \"static prognostic significance\" we can excellently reproduce here in a \"real-world\" cohort outside clinical trials, shows a similar prognostic separation of CS over time with a 5-year survival rate ranging from a stable 95% (CLL-IPI = 0) to a decline to 25% (CLL-IPI = 2 + 3) over the 10-year observation period. \n\nPatients in need for treatment over the observation period were less likely to survive, whereas 5-year CS increased slightly over time for untreated patients. This finding aligns with a recent study from the USA, which reported an increase in 5-year CS for untreated CLL patients aged ≥ 66 years based on linked surveillance, epidemiology, and end results-Medicare data [27]. However, given the heterogeneity of the patient cohort, the length of the observation period, and the therapeutic advances that have led to substantial changes in treatment regimens over time, meaningful conclusions are difficult. \n\nIn patients with CLL and other cancers, comorbidity is associated with shorter survival [28] [29] [30] [31]. In CLL, comorbidity has been shown to be an independent predictor of outcome, and different types of comorbidities are associated with increased overall mortality and particularly higher CLL-related mortality [32]. While the extent of comorbidities also had a significant impact on prognosis in our cohort, as previously reported for CLL, CS probability did not show significant changes over the 10-year period when stratified by CIRS score, using a cutoff commonly used in clinical trials to identify patients with relevant comorbidity burden (≤ 6 vs. > 6). This indicates that the prognostic significance of comorbidities remains similarly relevant and does not decline with increasing disease duration. This might be connected to a significant interaction between comorbidities and CLL treatment (in terms of treatment options and treatment tolerance) as previously demonstrated [29]. \n\nThus, in order to profoundly investigate the influence of comorbidities on prognosis and CS in CLL patients in more detail, it is relevant to identify the causes of mortality (CLL related vs. unrelated), as CLL-related deaths also contribute significantly to increased mortality in patients with a high burden of comorbidities. This is mainly due to the fact that increased comorbidities are associated with a reduced chance of sufficient disease control [29]. However, the documentation of causes of death was only very incomplete in our registry-based dataset and therefore does not allow any analyses in this regard. This clearly demonstrates that inclusion and documentation of comorbidities and, in particular, causes of death in cancer registries, are essential for a meaningful prognosis assessment at time of diagnosis and for dynamic CS assessment over the disease course. \n\nThe study has several other limitations. First, the limited number of patients restricts detailed subgroup analyses and statistical power. Additionally, molecular parameters were only available for a subset of patients, potentially limiting the scope of the findings. Second, no reassessment of prognostic parameters, such as clinical stage or genetic changes, was performed. The extended 35-year period of diagnosis and follow-up, with data collected independently in two cohorts, may introduce variability in the analyses. In addition, patient and disease characteristics of patients from the two university hospitals comprising the cohort may not be fully comparable, and there is a risk of a bias towards patients with higher complication rates and more comorbidities. \n\nIn addition, at later time points in the patient observation period, there were newer treatments available that were not approved at the beginning. Thus, the effects of treatment remain largely unclear because of the fundamentally different types of therapies administered. Stratification by type of therapy or by time windows encompassing different modes of disease management (e. g., pre-Rituximab era or post 2014 introduction of BTKis) was not possible because of the cohort size and limited follow-up. As targeted therapies have become increasingly important in treatment regimens in recent years, their impact on current prognostic models remains to be determined. Finally, no information is available on the specific causes of death, which precludes conclusions about the reasons for persistent excess mortality in the entire cohort and increasing mortality in high-risk patients.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Overall, we can demonstrate that CS is relevant for the management of CLL and the assessment of prognosis for physicians and patients. We confirm the previously reported stable prognosis of CLL patients over a long observation period and show that high-risk subgroups undergo dramatic and patient-relevant prognostic changes over time with gradually increasing mortality. In a disease like CLL, which often progresses slowly over decades, this type of prognostic information is clearly superior to a static survival model and of higher relevance to the patient.",
"section_name": "Summary and conclusion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The authors thank the staff of the cancer registries of the University Hospitals of Augsburg and Freiburg, especially Mrs. Vera Gumpp, for collecting and providing patient data. The authors also thank Mrs. Sandra Hild for excellent technical and organizational support.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Funding Open Access funding enabled and organized by Projekt DEAL. This project was supported by intramural funding of the Comprehensive Cancer Center Augsburg. The work of P. S. was supported by DFG Project -ID 431984000-CRC 1453 NephGen, Project-ID 523737608 ( SCHL 2292/2-1 ), and Germany ś Excellence Strategy ( CIBSS -EXC-2189 -Project ID 390939984 ). R. C. was supported by the German Cancer Aid ( DKH 110461 ).",
"section_name": "",
"section_num": ""
},
{
"section_content": "Data availability Data available on request from the authors.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s00277-024-05627-w. \n\nAuthor contribution P Schlosser, A Schiwitza, and R Claus performed the research and wrote the paper. R Claus and M Schumacher designed the resarch study. P Schlosser analyzed the data. J Klaus, S Hieke, K Zirlik, and K Szarc vel Szic contributed to the data collection. K Zirlik, M Trepel, M Schumacher, and J Duyster contributed to data analysis and performed manuscript editing. All authors finally approved the manuscript.",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "Ethics approval Data collection was approved by the local ethics committee, and written informed consent was obtained from all patients from the University Freiburg Medical Center. For patients of the Augsburg University Hospital, anonymized retrospective analysis of data is permitted without informed consent according to the Bavarian Hospital Act (BayKrG) in the version of March 28, 2007 (GVBl. S. 288, BayRS 2126-8-G Art. 27 Abs4). The study including data collection and analyses was performed according to the terms of the Declaration of Helsinki.",
"section_name": "Declarations",
"section_num": null
},
{
"section_content": "Open Access This article is licensed under a Creative Commons Attribution 4. 0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4. 0/. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Competing interests The authors declare no competing interests.",
"section_num": null
}
] |
10.3390/cancers12113142
|
A Molecular Test for Quantifying Functional Notch Signaling Pathway Activity in Human Cancer
|
<jats:p>Background: The Notch signal transduction pathway is pivotal for various physiological processes, including immune responses, and has been implicated in the pathogenesis of many diseases. The effectiveness of various targeted Notch pathway inhibitors may vary due to variabilities in Notch pathway activity among individual patients. The quantitative measurement of Notch pathway activity is therefore essential to identify patients who could benefit from targeted treatment. Methods: We here describe a new assay that infers a quantitative Notch pathway activity score from the mRNA levels of generally conserved direct NOTCH target genes. Following the calibration and biological validation of our Notch pathway activity model over a wide spectrum of human cancer types, we assessed Notch pathway activity in a cohort of T-ALL patient samples and related it to biological and clinical parameters, including outcome. Results: We developed an assay using 18 select direct target genes and high-grade serous ovarian cancer for calibration. For validation, seven independent human datasets (mostly cancer series) were used to quantify Notch activity in agreement with expectations. For T-ALL, the median Notch pathway activity was highest for samples with strong NOTCH1-activating mutations, and T-ALL patients of the TLX subtype generally had the highest levels of Notch pathway activity. We observed a significant relationship between ICN1 levels and the absence/presence of NOTCH1-activating mutations with Notch pathway activity scores. Patients with the lowest Notch activity scores had the shortest event-free survival compared to other patients. Conclusions: High Notch pathway activity was not limited to T-ALL samples harboring strong NOTCH1 mutations, including juxtamembrane domain mutations or hetero-dimerization combined with PEST-domain or FBXW7 mutations, indicating that additional mechanisms may activate Notch signaling. The measured Notch pathway activity was related to intracellular NOTCH levels, indicating that the pathway activity score more accurately reflects Notch pathway activity than when it is predicted on the basis of NOTCH1 mutations. Importantly, patients with low Notch pathway activity had a significantly shorter event-free survival compared to patients showing higher activity.</jats:p>
|
[
{
"section_content": "An increasing number of precision drugs are becoming available for clinical medicine, and many more are in development. These targeted drugs are intended for personalized medicine and aim at targeting the pathophysiological defects underlying specific diseases in individual patients. For cancer, but also for many other diseases including auto-immune or immune-mediated diseases, patient samples may display a similar histopathology, while significant pathophysiological variations can be found at the cellular level [1, 2]. Such variations may be the reason that only a portion of all patients with a specific disease responds to a targeted drug. Matching the right drug to the right patient has therefore become an increasingly important issue. However, developing a diagnostic approach to reliably predict therapy responses has proven difficult. The prime example is oncology, wherein efforts in predicting patient responses to targeted drugs based on cancer genome mutations have generally been disappointing, despite exceptions in select cases [3] [4] [5] [6] [7]. To improve clinical decision-making regarding targeted treatment, and therefore to improve clinical outcomes, assays are needed that accurately characterize and quantify the underlying pathophysiological processes in individual patient samples [1, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]. Cellular signal transduction pathways are evolutionarily conserved, and control fundamental cellular processes such as cell division, differentiation, migration and metabolism [1, [18] [19] [20]. They include nuclear receptor pathways (e. g., androgen and estrogen receptor pathways), developmental pathways (Wnt, Hedgehog, TGFβ and Notch), the highly complex growth factor-and cytokine-regulated signaling pathway network (including JAK-STAT, PI3K-AKT-mTOR and MAPK pathways), and the inflammatory NFκB pathway [18, 21]. The measurement of the functional activity of these pathways in tumor biopsies from individual patients is expected to improve the prediction of therapy response. We have previously described a novel approach to quantitatively measure the activity levels of individual signal transduction pathways in various cell and tissue types [22] [23] [24] [25]. In addition to the development of assays to measure the activity of the estrogen and androgen receptor pathways, the PI3K, JAK-STAT3, Wnt, Hedgehog, TGFβ, NFκB and JAK-STAT1/2 pathways, we now report the development and biological validation of a quantitative Notch pathway activity assay. The human Notch pathway is an evolutionarily highly conserved developmental signaling pathway, activated by the interaction of one of four NOTCH transmembrane receptors with Jagged or Delta-like Canonical Notch ligands on neighboring cells [16]. Upon ligand binding, the receptor is cleaved by two consecutive protease steps that include an ADAM (a disintegrin and metalloproteinase) protease and the gamma-secretase complex. The resulting cleaved intracellular NOTCH (ICN) migrates to the nucleus, where it forms a transcription factor complex with DNA binding factor RBPJ (recombination signal binding protein for immunoglobulin kappa J region) and coactivators of the MAML (Mastermind-like) family, and activates the transcription of its target genes. The Notch pathway plays a role in multiple diseases, including T-cell acute lymphoblastic leukemia (T-ALL) [16, 26]. Notch pathway inhibitors have been developed for multiple potential clinical applications, but their use has generally been associated with severe side effects [27] [28] [29] [30] [31] [32]. In addition, NOTCH inducers have been developed, e. g., for small cell lung cancer [33]. \n\nCancers 2020, 12, 3142 3 of 17 A major clinical challenge is to minimize side effects and identify patients who benefit from Notch pathway-modifying drugs. \n\nTo illustrate the potential utility of the Notch pathway assay for clinical decision-making, a Notch pathway activity analysis was performed in a large cohort of diagnostic samples from pediatric T-ALL patients with known genetic backgrounds and mutation statuses. Activating mutations in the NOTCH1 pathway, including mutations in NOTCH1 and/or FBXW7 (which encodes for a ubiquitin ligase involved in the degradation of active intracellular NOTCH1 (ICN1)), are found in approximately 60% of T-ALL patients [34, 35]. Publications on patient outcomes in T-ALL report different prognostic significances for NOTCH1-activating mutations alone [36]. We present evidence that patients with active Notch pathway signaling have a more favorable long-term outcome when on high-intensity combination treatment protocols [37] [38] [39].",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "2."
},
{
"section_content": "For the development of the Notch pathway assay, we selected high evidence direct target genes of NOTCH. This selection was based on (i) the presence of minimally one binding element in the promoter region, (ii) the functionality of these binding elements that have been assessed, for instance, by gene promoter-reporter studies, (iii) the binding of ICN to the respective response/enhancer element using ChIP and/or Electrophoretic Mobility Shift Assay, (iv) their differential expression upon pathway activation and/or inhibition, and (v) the consistency of evidence as reported by multiple research groups for multiple cell/tissue types. Based on such accumulated experimental evidence as described before [22] [23] [24], we selected 18 direct target genes, CD44, DTX1, EPHB3, HES1, HES4, HES5, HES7, HEY1, HEY2, HEYL, MYC, NFKB2, NOX1, NRARP, PBX1, PIN1, PLXND1 and SOX9 (Table S1 ). This number is sufficient for the robust and sensitive prediction of the pathway activity while comprising only high evidence target genes that enable maximal specificity over multiple cell types.",
"section_name": "Development of the Notch pathway Assay and Selection of NOTCH Target Genes",
"section_num": "2.1."
},
{
"section_content": "We calibrated the Notch pathway assay using data from high-grade serous (HGS) ovarian cancer samples with high Notch pathway activity, and normal ovarian tissue samples with low Notch pathway activity (Figure 1A ). While in healthy ovarian tissue samples the Notch pathway is inactive, HGS ovarian cancer is associated with an active Notch pathway and activating NOTCH3 gene mutations or amplifications in about two third of the patients [83] [84] [85] [86]. Following the freezing of the Notch pathway model, it was validated on various independent datasets from cells of different tissue origins with Notch pathway-activated or gamma-secretase inhibited conditions, including cell types from ectodermal (neuroblastoma) and endodermal (lung cancer cells) origins, in contrast to the mesodermal origin of the ovarian cancer samples the model had been calibrated on (Figure 1B-H ). Two independent clones of a neuroblastoma cell line transfected with ICN3 showed a rapid and persistent quantitative increase in Notch pathway activity score, starting within 4 h and reaching a plateau activity at 12 h after transfection (Figure 1B ). In leukemia, the AF1Q-MLLT11 fusion protein confers sensitivity to ligand-induced Notch pathway signaling [87, 88]. Hematopoietic progenitor cells (CD34 + CD45RA -Lin -) from umbilical cord blood were transduced with the A2M mutant version of this fusion product that sequesters it in the nucleus. Following a three-day exposure to immobilized NOTCH ligand Delta1ext-IgG at two different dose levels, high Notch pathway activity scores were measured for both mock and A2M transduced cells (Figure 1C ). As expected, the Notch pathway activity scores were higher for A2M-transduced cells than control cells. This result provides additional evidence for the ability of the Notch pathway assay to quantify small differences in Notch pathway activity. In other experimental designs in which Notch pathway activity was inhibited by exposure to gamma-secretase inhibitors (GSIs), the robustness of the assay for various additional cell types was validated. A549 lung cancer cells exposed to the GSI RO4929097 for 6 or 24 h scored a significantly lower Notch pathway activity than control A549 cells (Figure 1D ). Similar findings were found for the GSI-exposed Mantle B-cell lymphoma cell line SP-49 and the NOTCH mutant Rec-1 line [89] (Figure 1E ), and for the T-cell lymphoma and leukemia cell lines CUTLL1 and MOLT4 (Figure 1F, G ). In CUTLL1 cells, the wash-out of the GSI resulted in the reactivation of the Notch pathway, which was accurately quantified (Figure 1H ). Furthermore, a dominant-negative form of the NOTCH cofactor MAML1 (DNMAML1) synergized with GSI and resulted in the lowest Notch pathway activity score. Interestingly, in this study the removal of GSI was performed both in the absence and presence of the protein translation inhibitor cycloheximide, so as to exclude any feedback or secondary effects from NOTCH-induced gene products. The measured Notch pathway activity scores were independent of protein translation, confirming that all genes that are part of the computational Notch pathway model are indeed direct target genes (Figure S1 ). In summary, these results demonstrate that the ovarian cancer-calibrated Notch pathway assay can be used to measure Notch pathway activity levels in T-cells, while the limited results available on other cell types suggest that the assay may also be usable in cell types of endodermal and ectodermal origin.",
"section_name": "Calibration and Validation of the Notch Pathway Activity Assay",
"section_num": "2.2."
},
{
"section_content": "Following biological validation of the Notch pathway assay, we measured the Notch pathway activity scores in diagnostic samples from 117 pediatric T-ALL patients. This dataset has been previously used to distinguish four main T-ALL subgroups (ETP-ALL/immature, TLX, Proliferative and TALLMO) based on their differential gene expression profiles that strongly correlate with unique oncogenic rearrangements [98]. Notch pathway activity scores ranged from -8. 59 to 7. 45 on the linear log2 odds scale. To investigate these scores in relation to the presence of specific types of Notch pathway-activating mutations, we categorized NOTCH1 mutations into weak or strong activating mutations, as done before [35, 99, 100]. Weak NOTCH1 activating mutations are considered mutations in the NOTCH1 heterodimerization domain (HD) or PEST domain, or inactivating mutations in FBXW7. Strong NOTCH1-activating mutations are mutations in the juxtamembrane domain, or HD-mutations combined with PEST domain or FBXW7 mutations. Based on this division, the median Notch pathway activity score was lowest for the patient samples without NOTCH-activating mutations, and highest for the samples with strong NOTCH1-activating mutations (p < 0. 001; Figure 2A ). Still, there is considerable overlap in activity scores among these groups. To investigate the potential effect of differences in genetic background among patients, we compared Notch pathway activity levels between the four T-ALL subtypes. The TLX subtype had the highest Notch pathway activity scores compared to the other subtypes, and included 10 out of 23 patient samples with strong NOTCH mutations (Figure 2B ). Various TLX samples without, or with only weak, NOTCH-activating mutations also had high Notch pathway activity scores, further supporting the previous observation that alternative Notch pathway-activating mechanisms may exist. We then related the activity scores to intracellular NOTCH1 (ICN1) levels as measured using reverse-phase protein array for 62 patient samples [35]. We observed a significant relationship between ICN1 levels and the absence or presence of NOTCH1-activating mutations (Figure 2C ), and between ICN1 levels and the Notch pathway activity scores (Figure 2D ). The significance of the correlation between ICN1 levels and Notch pathway activity was mainly attributed to the strong NOTCH1-activating mutations, as the significance was lost for patient samples without, or with only weak, NOTCH1-activating mutations (Figure S2 ). This raised the question of whether those samples could harbor other Notch pathway-activating mechanisms. For this, we assessed NOTCH3 protein levels as an alternative Notch pathway-activating mechanism for various NOTCH1/FBXW7 non-mutated T-ALL patient samples with low ICN1 levels but high Notch pathway activity scores. We did not find expression of NOTCH3 protein in these or other T-ALL samples tested. We then excluded the influence of bone marrow or peripheral blood origin of the T-ALL samples on the Notch pathway activity scores. Therefore, the incidental discrepancy between ICN and Notch pathway activity scores remains unclear. In conclusion, the results show that the Notch pathway assay quantitatively measures Notch pathway activity not only in cell line systems, but also in a cohort of primary T-ALL patient samples. \n\n1F,G). In CUTLL1 cells, the wash-out of the GSI resulted in the reactivation of the Notch pathway, which was accurately quantified (Figure 1H ). Furthermore, a dominant-negative form of the NOTCH cofactor MAML1 (DNMAML1) synergized with GSI and resulted in the lowest Notch pathway activity score. Interestingly, in this study the removal of GSI was performed both in the absence and presence of the protein translation inhibitor cycloheximide, so as to exclude any feedback or secondary effects from NOTCH-induced gene products. The measured Notch pathway activity scores were independent of protein translation, confirming that all genes that are part of the computational Notch pathway model are indeed direct target genes (Figure S1 ). In summary, these results demonstrate that the ovarian cancer-calibrated Notch pathway assay can be used to measure Notch pathway activity levels in T-cells, while the limited results available on other cell types suggest that the assay may also be usable in cell types of endodermal and ectodermal origin. A2M (+ symbol) or control vector-transfected CD34 + CD45RA -Lin -hematopoietic progenitor cells from umbilical cord blood were cultured for 72 h on a surface with 0, 2 or 5µg plastic-immobilized NOTCH ligand Delta1ext-IgG. A2M is a nuclear-trapped mutant of AF1q/MLLT11. (D) GSE36176 [95]. A549 lung cancer cell lines subjected to vehicle control or gamma secretase inhibitor (GSI) RO4929097 for 6 or 24 h. (E) GSE34602 [89]. Rec-1 (containing an activating NOTCH1 mutation) and SP49 Mantle cell lymphoma cell lines subjected to vehicle control or GSI compound E for 24 h.",
"section_name": "Notch Pathway Activity in Pediatric T-ALL Patient Samples",
"section_num": "2.3."
},
{
"section_content": "The prognostic significance of NOTCH-activating mutations is not consistent in various patient studies [36]. Part of this may be due to the mechanisms, other than activating mutations in hotspots Cancers 2020, 12, 3142 7 of 17",
"section_name": "Notch Pathway Activity and T-ALL Patient Survival",
"section_num": "2.4."
},
{
"section_content": "The prognostic significance of NOTCH-activating mutations is not consistent in various patient studies [36]. Part of this may be due to the mechanisms, other than activating mutations in hotspots of NOTCH1 or FBXW7, that activate Notch signaling in T-ALL patients, and which may explain the large overlap in the Notch pathway activity levels for T-ALL patients with and without NOTCH/FBXW7 mutations. In order to investigate outcomes in relation to Notch pathway activity, we divided the T-ALL patients into three groups based on their NOTCH activity scores: a group with the highest NOTCH activity scores (>75th percentile), a group with the lowest activity scores (<25th percentile) and a group with intermediate activity scores (between the 25th and 75th percentiles of activity scores). When assessing the event-free and relapse-free survival curves, we observed that the patients with the lowest activity scores had the shortest event-free survival compared to both of the other groups (p < 0. 05), while relapse-free survival showed the same trend (Figure 3A, B ).",
"section_name": "Notch Pathway Activity and T-ALL Patient Survival",
"section_num": "2.4."
},
{
"section_content": "The group with the lowest NOTCH activity scores contained patients that lacked either PTEN protein and/or had inactivating mutations or deletions in PTEN. We found an increased percentage of patients (11 out of 29, 38%) with functional PTEN loss in the group with the lowest Notch pathway activity, whereas only 12 out of the 84 patients (14%) with intermediate and high Notch pathway activity scores had functional PTEN loss (p = 0. 006, Pearson Chi-Square, 2-sided).",
"section_name": "Relation between Notch Pathway Activity and PTEN Loss",
"section_num": "2.5."
},
{
"section_content": "We have developed an assay to measure Notch pathway activity, consisting of a Bayesian network computational model which calculates a pathway activity score based on target gene levels. The set of NOTCH target genes was selected based on experimental evidence, irrespective of cell type or gene function [22] [23] [24]. The computational model was successfully validated on a variety of samples from different cell types with known Notch pathway activity, i. e., brain, lung, hematopoietic stem cells, and T-ALL cell lines. This suggests that the assay can be used on multiple different cell types without model recalibration, even across cell types originating from different embryonic germ layers. This is to a large extent enabled by the selection of high evidence direct transcriptional target genes of the NOTCH transcription factor family (e. g., NOTCH1, NOTCH2, NOTCH3), eliminating cell type-specific influences on target gene expression as much as possible. In addition, the Bayesian network model is well suited to handling variations in input data, which presents a crucial advantage when analyzing patient samples that are intrinsically highly variable in gene expression regulation [22]. Other RNA-based pathway analysis tools are available, mainly for biomarker discovery applications, and differences have been discussed before [22, 24, [101] [102] [103]. In short, we use a knowledge-based Bayesian modeling approach as opposed to a more generally used data-driven approach, thus avoiding common problems with data-overfitting. This approach improves specificity in measuring signaling pathway activity, and enables development as a diagnostic assay across multiple disease types. \n\nTo explore the clinical utility of the biologically validated Notch pathway model, we have analyzed diagnostic samples from 117 pediatric T-ALL patients. We found that the Notch pathway activity score was related to the presence of NOTCH1-activating mutations and the type of mutations, and was correlated to the levels of ICN protein in these samples. Correspondingly, we found the highest Notch pathway activity scores in the TLX subgroup, a group that we described before as having the highest incidence of NOTCH1-activating mutations [35]. Most T-ALL patients in this T-ALL subgroup (21 out of 30 patients) bear TLX3-BCL11B rearrangements [98]. Moreover, the TLX subgroup is related to gamma-delta T-cell lineage development [104]. Interestingly, human gamma-delta T-cell lineage development especially depends on high Notch pathway activity levels, in contrast to alpha-beta T-lineage development [77]. The proliferative and TALLMO subgroups, which are associated with the early and late cortical stages of the alpha-beta T-lineage, respectively, indeed have lower Notch pathway activity scores. Therefore, the NOTCH dependency in normal development mirrors that of the respective T-ALL subgroups. Remarkably, about half of the ETP-ALL patients seem to have an activated Notch signaling pathway based on measured activity scores, despite their overall lower incidence of NOTCH-activating mutations [105]. We observed that various samples without, or with weak, NOTCH-activating mutations still have high Notch pathway activity scores [35]. This is especially evident for patients from the TLX subgroup, and points to other, as yet unidentified, mutations outside the present hotspot regions or other mechanisms that may activate the Notch pathway in T-ALL. \n\nThe patients with a Notch pathway activity score in the lowest 25th percentile had the worst event-free and relapse-free survival. Interestingly, NOTCH mutations in this cohort were not associated with beneficial outcomes, as reported before [35], while other studies identified activating NOTCH mutations as a favorable prognostic factor [37] [38] [39]. This result suggests that scoring the Notch pathway activity might be a more reliable method for determining prognosis than identifying NOTCH-activating mutations. In addition, the Notch pathway test has the potential to improve the stratification of patients to novel therapies targeting the Notch pathway. \n\nThe patients with the lowest Notch pathway activity scores were more likely to have functional PTEN loss, indicating that Notch pathway activation and PTEN inactivation reflect two distinct T-ALL entities, as we and others have reported before [100, 106]. PTEN aberrancies are often found in the TALLMO T-ALL subgroup, in which they occur mutually exclusively with strong NOTCH1 mutations [100]. Moreover, patients with PTEN aberrancies been shown to have an inferior survival rate [100, 106]. The finding that PTEN aberrancies occurred more often in the patients with the lowest Notch pathway activity helps explain the inferior event-free/relapse-free survival of this group. \n\nOverall, our results indicate that Notch pathway activity cannot be deduced from the presence of activating mutations only, which may provide an explanation for the differences in the prognostic significance of NOTCH-activating mutations in various pediatric and adult patient cohorts [36]. \n\nWhile the here-described Notch pathway assay is expected to be of value for a broad range of diseases, as well as for preclinical research and drug development, the first envisioned clinical application is therapy response prediction, e. g., to NOTCH inhibitors, for T-ALL, small cell lung cancer and other malignancies. To enable the use of the Notch pathway activity assay on formalin-fixed paraffin-embedded tissue samples, which are the standard in pathology diagnostics, the here-described Affymetrix-based Notch pathway activity test has been converted to an RT-qPCR-based test, which can be performed using standard lab equipment (in principle within three hours). To enable the determination of Notch pathway activity from RNA sequencing data, the assay has been converted to an RNAseq-based assay (www. philips. com/oncosignal). The conversion procedure has been described before, and does not involve the addition of new target genes [104, 107]. These assays will be used in future clinical validation studies.",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "4."
},
{
"section_content": "The mathematical approach to developing Bayesian network models for the measurement of signal transduction pathway activity, based on mRNA expression analysis, has been described in detail before [24]. In brief, a causal computational network model for the Notch signal transduction pathway was generated that calculates the probability that NOTCH transcription factors are active, based on the expression levels of direct target genes (Figure 4 ). The Bayesian network describes the causal relation between the up-or downregulation of NOTCH target genes and the presence of an active or inactive NOTCH transcription complex. The parameters that describe this relationship are based on literature evidence, and are calibrated on patient samples with known Notch pathway activity. Target genes for the Notch pathway assay were selected according to the same principles as described before, using the available scientific literature [22, 24]. The probesets of direct target genes from the publicly available Affymetrix (Santa Clara, CA, USA) HG-U133Plus2. 0 microarray datasets were selected using the Bioconductor package, hgu133plus2. db, available in the statistical environment R and manually curated using GRCh38/hg38, available on the UCSC Genome Browser (www. genome. ucsc. edu, last access 9-30-2020) [26, 28, 32, 108, 109]. Probesets representing intronic sequences, probesets on opposite strands, and other chromosomal sequences than the respective target gene, were excluded. Probesets that were missing in Bioconductor were added.",
"section_name": "Development of the Notch Pathway Assay",
"section_num": "4.1."
},
{
"section_content": "The Notch pathway Bayesian model, intended for generic use across different cell and tissue types, was calibrated on a single public dataset containing data from normal (low Notch pathway activity) and high-grade serous ovarian cancer (high Notch pathway activity) tissue samples [86]. Following calibration, the model parameters were frozen. Subsequently, given that the Bayesian model describes how expression and probeset measurements depend causally on pathway activity, it can be used to reason backwards from a set of given measurements to assess the likelihood that the pathway was active. Upon entering new mRNA probeset measurements into the model, this reasoning is performed by Bayesian inference, which yields the odds that the pathway is active vs. not, after which we apply a log2 transformation to obtain a symmetric scale with higher resolution at the extreme ends [22, 23]. The resulting Notch pathway score reflects the amount of evidence delivered by the target gene expression levels for being active, and thus is a read-out of functional Notch signaling pathway activity. In general, higher target gene expression levels will lead to higher Notch activity, and vice versa. The model-based Notch pathway assay was validated using multiple independent Affymetrix datasets containing gene expression data from samples with known Notch pathway activity.",
"section_name": "Calibration and Validation of the Notch Pathway Activity Model",
"section_num": "4.2."
},
{
"section_content": "The Affymetrix HG-U133Plus2. 0 datasets used for Notch pathway model calibration and validation, and for the Notch pathway analysis of T-ALL (GSE26713), are available at the GEO website (www. ncbi. nlm. nih. gov/geo, last access 9-30-2020). GEO datasets have been listed with their associated publications in the figure legends. Before using the microarray data, extensive quality control was performed on the Affymetrix data from each individual sample, based on 12 different quality parameters according to Affymetrix's recommendations and previously published literature [22, 110, 111], and then they were further preprocessed in the statistical environment R using frozen RMA [112] with 'robust weighted average' summarization.",
"section_name": "Microarray Data Source and Quality Control",
"section_num": "4.3."
},
{
"section_content": "The Affymetrix HG-U133Plus2. 0 gene expression profiles (GSE26713) from diagnostic biopsies of 117 T-ALL patients, who were treated according to the German co-operative study group for childhood ALL-97 protocol (COALL-97) or the Dutch Childhood Oncology Group (DCOG) protocols ALL-7,-8 or -9, were used in this study [98]. The patient data used in this study were obtained with informed consent from the subjects' guardians and in accordance with the Declaration of Helsinki.",
"section_name": "Description of the T-ALL Pediatric Patient Cohort",
"section_num": "4.4."
},
{
"section_content": "For the validations of the Notch pathway model, two-sided Wilcoxon signed-rank statistical tests were performed. Other used statistical methods that are more appropriate due to the content of a specific dataset are indicated in the figure legends. For pathway correlation statistics, both Pearson correlation and Spearman rank correlation tests were performed; since the results were similar, only the Pearson correlation coefficient and associated p-value are reported. For outcome analysis, Kaplan-Meier survival curves were calculated together with the associated p-value using the log-rank test.",
"section_name": "Statistics",
"section_num": "4.5."
},
{
"section_content": "Informed consent was given in accordance with the Institutional Review Board of the Erasmus MC Rotterdam and in accordance with the Declaration of Helsinki.",
"section_name": "Ethics Approval and Consent to Participate",
"section_num": "4.6."
},
{
"section_content": "We have developed an assay to measure Notch pathway activity, which calculates a pathway activity score that is based on the expression levels of conserved Notch direct target genes. This assay was successfully validated by detecting the Notch activity in a variety of tumor models of different cellular origins with known Notch pathway activity, i. e., brain, lung, hematopoietic stem cells, and T-ALL cell lines. Our assay is expected to be of value for a broad range of diseases, as well as for preclinical research and drug development. The first envisioned clinical application is therapy response prediction, e. g., to NOTCH inhibitors, for T-ALL, small cell lung cancer and other malignancies.",
"section_name": "Conclusions",
"section_num": "5."
},
{
"section_content": "The following are available online at http://www. mdpi. com/2072-6694/12/11/3142/s1, Table S1 : Scoring of NOTCH target genes with respect to evidence as to direct gene regulation by Notch transcription factors, Figure S1 : Extended validation of the Notch pathway model in CUTLL1 cells, Figure S2 : Correlations of ICN1 level and Notch pathway activity, divided per NOTCH1-activating mutation status.",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
[
{
"section_content": "Funding: K. C. -B and R. H. are funded by the Dutch 'Kinderen Kanker Vrij ' foundation grants KiKa-295 and KiKa-219, respectively. V. C. is funded by the Dutch Cancer Society grant KWF-10355.",
"section_name": "",
"section_num": ""
}
] |
10.3960/jslrt.22047
|
Concurrent development of small lymphocytic lymphoma and lung cancer: A report of two cases and a review of the literature
|
Small lymphocytic lymphoma (SLL) is a rare disease subtype which has the same morphological and immunophenotypic features as chronic lymphocytic leukemia (CLL) but does not demonstrate lymphocytosis and grows mainly in the lymph nodes and spleen. As with CLL, SLL patients tend to present with immune abnormalities, and are associated with an increased risk for developing second primary malignancies. We report here two cases of SLL who developed lung cancer concurrently. The biological and clinical features of these two patients were very similar to each other; they both developed SLL with trisomy 12 and lacked lymphocytosis or cytopenia. SLL cells involved nodal areas adjacent to lung adenocarcinoma which expressed PD-L1. One patient received immunochemotherapy including nivolumab and ipilimumab against lung cancer, and notably, transient deterioration of SLL occurred after the second cycle of immunochemotherapy along with the development of immune related adverse events. Immunohistochemical analysis of the SLL samples of the patient revealed that the tumor cells were positive for CTLA-4, suggesting that ipilimumab might have potentially induced the activation of SLL cells by blocking the inhibitory signal mediated by CTLA-4. These clinical findings indicate the potential biological relationship between SLL and lung cancer. According to these observations, we would like to draw attention to the possibility of deterioration of SLL when immune checkpoint inhibitors are used for the treatment of malignancies developed in SLL patients.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy that is characterized by monoclonal B-cell lymphocytosis in the blood and lymphoid organs. Small lymphocytic lymphoma (SLL) is a rare disease subtype which has the same morphological and immunophenotypic features as CLL but does not demonstrate lymphocytosis and grows mainly in the lymph nodes and spleen. 1 Specific chromosomal abnormalities including 13q, 11q, and 17p deletions and trisomy 12 are often detected in CLL/SLL, and are associated with distinctive clinical presentation and outcomes. \n\nThe clinical course of the patients with CLL/SLL is highly heterogeneous. Although patients typically have an indolent disease and a sometimes do not even require treatment throughout their lifetime, others exhibit an aggressive clinical course and dismal outcome. The mutational status of the immunoglobulin heavy-chain variable region gene (IGHV) of CLL/SLL is also predictive of prognosis, and those without IGHV mutations tend to have poor prognosis. 2 t has been clinically recognized that CLL/SLL patients have an increased incidence of developing second primary malignancies. Although disease-and treatment-related immunosuppression is assumed to be an underlying cause of predisposition to malignancies, the precise mechanisms for developing other neoplasms have not been clarified. We report here the clinical course of two patients who concurrently developed SLL and lung cancer. Clinical and biological similarities in these two patients raise the possibility that there may be a causal relationship between the pathogenesis of these two malignancies.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Kensuke Nakao, 1) Momoko Nishikori, 1) Masakazu Fujimoto, 2) Hiroshi Arima, 1) Hironori Haga, 2) Akifumi Takaori-Kondo 1) Small lymphocytic lymphoma (SLL) is a rare disease subtype which has the same morphological and immunophenotypic features as chronic lymphocytic leukemia (CLL) but does not demonstrate lymphocytosis and grows mainly in the lymph nodes and spleen. As with CLL, SLL patients tend to present with immune abnormalities, and are associated with an increased risk for developing second primary malignancies. We report here two cases of SLL who developed lung cancer concurrently. The biological and clinical features of these two patients were very similar to each other; they both developed SLL with trisomy 12 and lacked lymphocytosis or cytopenia. SLL cells involved nodal areas adjacent to lung adenocarcinoma which expressed PD-L1. One patient received immunochemotherapy including nivolumab and ipilimumab against lung cancer, and notably, transient deterioration of SLL occurred after the second cycle of immunochemotherapy along with the development of immune related adverse events. Immunohistochemical analysis of the SLL samples of the patient revealed that the tumor cells were positive for CTLA-4, suggesting that ipilimumab might have potentially induced the activation of SLL cells by blocking the inhibitory signal mediated by CTLA-4. These clinical findings indicate the potential biological relationship between SLL and lung cancer. According to these observations, we would like to draw attention to the possibility of deterioration of SLL when immune checkpoint inhibitors are used for the treatment of malignancies developed in SLL patients.",
"section_name": "Concurrent development of small lymphocytic lymphoma and lung cancer: A report of two cases and a review of the literature",
"section_num": null
},
{
"section_content": "A 67-year-old male, current smoker, presented with slowly growing cervical tumors. A computed tomography (CT) scan revealed enlarged lymph nodes in the bilateral cervical, axillary, supraclavicular, and paraaortic regions, and a mass lesion in the apex of the left lung (Figure 1A ). An 18-F-fluorodeoxyglucose (FDG)-positron emission tomography (PET)/CT scan demonstrated abnormal FDG uptake in these tumors. Blood examination revealed hemoglobin of 14. 6 g/dL, platelet count of 12. 3×10 4 /μL, and white blood cell count of 5,970 /μL with 47% neutrophils, 40% lymphocytes, 8% monocytes, 4% eosinophils, and 1% basophils. His serum lactate dehydrogenase (LD) was 210 U/L, IgA 210 mg/dL, IgG 1,327 mg/dL, IgM 54 mg/dL, soluble interleukin-2 receptor (sIL-2R) 1,450 U/mL (normal range, 121-613 U/mL), Pro-GRP 35. 4 pg/mL (normal range, < 81. 0 pg/mL), CEA 6. 8 ng/mL (normal range, < 5. 0 ng/mL), NSE 11. 42 ng/ mL (normal range, <12. 0 ng/mL), SCC 0. 7 ng/mL (normal range, < 1. 5 ng/mL), and CYFRA 1. 9 ng/mL (normal range, < 2. 2 ng/mL). Bone marrow examination revealed no involvement of lymphoma cells. An excisional biopsy of the left axillary node led to a diagnosis of SLL. \n\nThe possibility of concurrent lung cancer in the left lung apex was indicated by the PET-CT scan, but it was considered difficult to precisely evaluate its distribution. Since the systemic lymph nodes grew larger in the following 4 months (Figure 1B ), we decided to treat SLL first following consultation with a thoracic physician. The patient was treated with 4 cycles of BR (bendamustine and rituximab), and a PET/CT scan after the completion of chemotherapy showed regression of all tumors except the left lung apex lesion (Figure 1C ). A bronchoscopic biopsy of the residual tumor led to the diagnosis of acinar-predominant lung adenocarcinoma. The tumor was positive for PD-L1 expression in immunohistochemistry and was found to carry an EGFR L858R gene mutation by Oncomine Dx Target Test Multi-CDx system. 3 A detailed chest CT scan demonstrated the presence of visceral pleural invasion. The patient has been treated with osimertinib and is alive with lung cancer for two years.",
"section_name": "CASE PRESENTATION Case 1",
"section_num": null
},
{
"section_content": "A 73-year-old male, former smoker, presented with persisting productive cough. A chest CT scan demonstrated mediastinal lymphadenopathy and a mass lesion in his right lung (Figure 2A, B ). Blood examination revealed hemoglobin of 16. 5 g/dL, platelet count of 16. 7×10 4 /μL, and white blood cell count of 9,320 /μL with 80. 4% neutrophils, 12. 6% lymphocytes, 5. 5% monocytes, 1. 1% eosinophils, and 0. 4% basophils. Serum LD was 228 U/L, IgA 153 mg/dL, IgG 1,022 mg/dL, IgM 30 mg/dL, sIL-2R 683 U/mL, Pro-GRP 83. 1 pg/mL, CEA 9. 1 ng/mL, and CYFRA 10. 6 ng/mL. Bone marrow examination showed no involvement of lymphoma cells. \n\nHistological analysis of bronchoscopic biopsy samples of both the lung tumor and a mediastinal lymph node revealed lung adenocarcinoma. Only a few clusters of carcinoma cells were identified in these biopsy specimens, and histologic subclassification was not possible. Although no obvious lymphocyte clusters were found in these samples, flow cytometric analysis of the lymph node detected a CD20, CD5, CD23, IgM, and Igκ-positive monoclonal B-cell popu- lation accounting for 60% of the nucleated cells, and concurrent development of SLL was suggested. A cranial magnetic resonance imaging (MRI) scan revealed a brain metastasis of lung cancer, and immunochemotherapy consisting of carboplatin, pemetrexed, nivolumab, and ipilimumab was initiated for the treatment of lung cancer. \n\nOn day 9 of the second cycle of chemotherapy, the patient was hospitalized for rapidly progressive hypoxia and thrombocytopenia with fever. A CT scan revealed further enlargement of systemic lymph nodes, although the lung tumors were regressing (Figure 2C, D ), and an axillary lymph node biopsy was performed for the evaluation of his illness. Surprisingly, the biopsied lymph node demonstrated diffuse infiltration of small monotonous B cells with CD20, CD5, and CD23 expression and the absence of cyclin D1 expression, and the patient was diagnosed with progression of SLL. His fever, hypoxia, and thrombocytopenia gradually resolved after the cessation of immunochemotherapy, and they were considered to be compatible with immunerelated adverse events (irAEs) caused by immune checkpoint inhibitors. Notably, systemic lymphadenopathy also regressed along with the resolution of other symptoms. Chemotherapy was resumed without immune checkpoint inhibitors, but the patient died of progression of lung cancer six months later. Figure 3 shows the summary of his clinical course.",
"section_name": "Case 2",
"section_num": null
},
{
"section_content": "Histological analysis of the axillary lymph nodes of both Case 1 and Case 2 demonstrated disrupted architecture and diffuse proliferation of small lymphocytes with CD20, CD5, and CD23 expression, which led to the diagnosis of SLL (Figure 4 ). The Ki-67 labelling index of the tumor cells was 10% in Case 1 and 30% in Case 2. Both tumor cells were positive for IgM-κ in flow cytometric analysis. In Case 2, SLL seemed to be deteriorated along with the occurrence of irAEs during the immune checkpoint inhibitor treatment. We detected CTLA-4 but no obvious PD-1 expression in SLL cells in immunohistochemical analysis (Figure 4N, O), indicating that SLL cells might be activated by blockade of inhibitory CTLA-4 signaling following ipilimumab. \n\nAlthough G-banded chromosomal analysis of SLL cells demonstrated normal karyotypes, trisomy 12 was detected by interphase fluorescence in situ hybridization (FISH) in both patients (Figure 4G, M ). Informed consent was obtained from the patients for further genetic analysis, and the IGHV gene segment usage and somatic hypermutation status of SLL cells were examined. Semi-nested polymerase-chain reaction (PCR) amplification of monoclonal immunoglobulin gene rearrangements 4 and Sanger sequencing of the PCR products were performed using the following 3 primers:",
"section_name": "HISTOLOGICAL AND GENETIC ANALYSIS OF CLL AND LUNG CANCER",
"section_num": null
},
{
"section_content": "were used for the first round PCR, FR2A and VLJH were used for the second round PCR, and VLJH was used for Sanger sequencing. The results demonstrated the usage of VH3-9 and VH1-2 in Case 1 and Case 2 SLL, respectively, both of which are reported as frequently used IGHV segments in CLL/SLL. 5 IGHV was not mutated in Case 1, but was mutated in Case 2. \n\nIn both cases, biopsied lung cancer tissues were histologically evaluated to be adenocarcinoma with PD-L1 expression. The histological and genetic features of SLL and lung cancer in each case are summarized in Table 1.",
"section_name": "T G A G G A G A C G G T G A C C ; V L J H , GTGACCAGGGTNCCTTGGCCCCAG. FR2A and LJH",
"section_num": null
},
{
"section_content": "CLL/SLL patients have an increased risk for comorbid malignancies, 6, 7 a phenomena thought to be associated with their predisposition to immunodeficiency due to the disease itself and/or cumulative immunosuppressive treatment. We reported two patients who developed SLL and lung adenocarcinoma concurrently and demonstrated very similar clinical features between the patients. In both cases, SLL was positive for CD5, CD23, and IgM-κ expression, as well as chromosomal abnormality of trisomy 12. SLL was not accompanied with lymphocytosis or cytopenia, but involved in lymph nodes adjacent to lung cancer, which led to the hypothesis that there might be a causal relationship between these two malignancies. \n\nIn the analysis of second primary malignancies among CLL patients from the Surveillance, Epidemiology, and End Results (SEER) database between 1973 and 2015, 6,487 second primary malignancies were diagnosed with a 20% higher risk compared to the general population. 7 Moreover, patients with lung and various other cancers were shown to have inferior overall survival when developed with a background of CLL. 8 Trisomy 12 is one of the most common cytogenetic abnormalities in CLL/SLL, and is more frequently found in patients with SLL than CLL (28-36% vs. 13-16%). 9 Notably, as high as 9% of CLL/SLL patients with trisomy 12 demonstrate complications of other hematologic and nonhematologic malignancies. 9 t is also known that more than 20% of CLL patients carry a stereotyped B-cell receptor (BCR), 5 and CLL patients with trisomy 12 have a significantly higher incidence of carrying stereotyped BCR than those without (44. 1% vs 27%, p = 0. 01). 10 These findings suggest that persistent BCR activation by certain endogenous antigens plays a role in the pathogenesis of CLL, especially in those with trisomy 12. Although there is only limited information on the antigens bound to CLL/SLL BCRs, reported candidates include cytoskeletal proteins, such as vimentin, filamin B, and cofilin-1, and phosphorylcholine-containing antigens, such as Streptococcus pneumoniae polysaccharides and oxidized low-density lipoprotein. 11 Since these molecules compose motifs exposed on apoptotic cells and bacteria, it is speculated that CLL/SLL cells are derived from B cells that play roles in the elimination of apoptotic cells and/or pathogenic bacteria. 12 IGHV analysis of SLL in Case 2 demonstrated the usage of mutated IGHV1-2, which was previously reported to have an affinity for filamin B. 11 As filamin B is reported to be highly expressed in invasive cancer cells and contributes to their invasiveness, 13 it is possible that BCR expressed on SLL cells in Case 2 recognized the antigen presented by lung cancer. \n\nMoreover, transient SLL progression was observed in Case 2 along with the occurrence of irAEs during immunochemotherapy, and immunohistochemical examination detected CTLA-4 expression in SLL. In T-cell lymphomas, CTLA-4 and PD-1 are known to potentially act as tumor suppressors, and downregulation or blockade of these molecules may induce lymphoma progression in some situations. 14 TLA-4 and PD-1 are also reported to be occasionally expressed in CLL/SLL and Richter transformation; [15] [16] [17] [18] [19] [20] however, the functional roles they play in CLL are not well clarified. A previous report demonstrated that downregulation of CTLA-4 induced proliferation and survival of CLL cells in vitro. 21 Therefore, it can be speculated that ipilimumab might have augmented the immune response of SLL cells against lung cancer in Case 2, which led to the transient SLL progression. \n\nAlthough further investigation is necessary, our observations suggest the possible causal relationship between SLL and lung cancer. We would like to draw attention to the potential risk of deterioration of CLL/SLL when immune checkpoint inhibitors are used for the treatment of second primary malignancies in CLL/SLL patients.",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "CONFLICT OF INTEREST",
"section_num": null
},
{
"section_content": "The authors declare no potential conflict of interest in this work.",
"section_name": "CONFLICT OF INTEREST",
"section_num": null
}
] |
10.21203/rs.3.rs-3186246/v1
|
5-Aza-4’-thio-2’-deoxycytidine induces C&gt;G transversions in a specific trinucleotide context and leads to acute lymphoid leukemia
|
<jats:title>Abstract</jats:title> <jats:p>DNA methyltransferase inhibitors (DNMTi), most commonly cytidine analogs, are compounds that are used clinically to decrease 5’-cytosine methylation, with the aim of re-expression of tumor suppressor genes. We used a murine pre-clinical model of myelodysplastic syndrome based on transplantation of cells expressing a NUP98::HOXD13 transgene to investigate 5-Aza-4’-thio-2’-deoxycytidine (Aza TdCyd or ATC), a thiol substituted DNMTi, as a potential therapy. We found that ATC treatment led to lymphoid leukemia in wild-type recipient cells; further study revealed that healthy mice treated with ATC also developed lymphoid leukemia. Whole exome sequencing revealed thousands of acquired mutations, almost all of which were C > G transversions in a previously unrecognized, specific 5’-NCG-3’ context. These mutations involved dozens of genes well-known to be involved in human lymphoid leukemia, such as <jats:italic>Notch1, Pten, Pax5, Trp53</jats:italic>, and <jats:italic>Nf1</jats:italic>. Treatment of human cells <jats:italic>in vitro</jats:italic> showed thousands of acquired C > G transversions in a similar context. Deletion of <jats:italic>Dck</jats:italic>, the rate-limiting enzyme for the cytidine salvage pathway, eliminated C > G transversions. Taken together, these findings demonstrate that DNMTi can be potent mutagens in human and mouse cells, both <jats:italic>in vitro</jats:italic> and <jats:italic>in vivo</jats:italic>.</jats:p>
|
[
{
"section_content": "Myelodysplastic syndromes (MDS) are a diverse group of blood cancers characterized by peripheral blood cytopenias and dysplastic blood cell morphology 1, 2. Up to 40% of patients with high risk MDS will transform to acute myeloid leukemia (AML) 3, an aggressive blood cancer with signi cant morbidity and an overall 5-year survival of less than 35% 4. MDS, AML, and blood cancers in general are thought to be driven by acquired and inherited mutations [5] [6] [7]. Acquired mutations in MDS, AML, as well as T cell and B cell Acute lymphoid leukemia (ALL) have recently been documented through next generation sequencing studies [8] [9] [10] [11] [12] [13]. Both MDS and AML have been linked to hypermethylation of cytosine residues, a crucial process in normal biology 14. Hypermethylation in the 5' regulatory region can result in epigenetic downregulation of gene expression, and it has been speculated that hypermethylation of tumor suppressor genes can contribute to malignant transformation 15. \n\nDNA Methyl-Transferase Inhibitors (DNMTi), also referred to as hypomethylating agents (HMA), represent a class of drugs that inhibit DNA methylation through inactivation of DNMT1 16. Treatment with DNMTi is hypothesized to restore function to tumor suppressor genes that have become inactivated, slowing the progression of malignancy, often via induction of apoptosis 15. One class of DNMTi are cytidine analogs, designed to mimic cytidine and inhibit the action of DNMT, most commonly DNMT1 16. Two drugs of this class, 5-Azacytidine (5-AZA) and Decitabine (DAC), both of which are US Food and Drug Administration (FDA) have been approved for use in MDS 16. However, the morbidity and mortality for patients with MDS or AML remain high, and thus new drugs are necessary to address this clinical need. 5-Aza-4'-thio-2'-deoxycytidine (Aza-TdCyd or ATC) is a cytidine analog that incorporates an aza modi cation (de ned as a nitrogen in place of a carbon) of the cytosine base and a thioether modi cation of the deoxyribose sugar. ATC is a promising new DNMT1i that has demonstrated e cacy in preclinical studies of solid tumors [17] [18] [19]. Given this demonstration of e cacy in a pre-clinical model, and the use of similar cytidine analogs in MDS, we sought to characterize the potential utility of ATC in the treatment of MDS, using a murine NUP98::HOXD13 chimeric transplantation model.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "ATC Treatment of WT/NHD13 chimeric mice leads to T-ALL in both donor and recipient tissue. To assess the in vivo pre-clinical e cacy of ATC, a newly described DNMT1 inhibitor, we generated a cohort of chimeric mice with both wild-type (WT) and MDS hematopoiesis. The chimeric mice were generated by co-transplantation of 200,000 WT and 1,000,000 NHD13 (MDS) bone marrow nucleated cells (BMNC) into WT recipients, following a myeloablative conditioning regimen of 900 cGy ionizing radiation. The NHD13 BMNC expressed the CD45. 2 allele, while the WT BMNC and WT recipients expressed the CD45. 1 allele, allowing us to distinguish hematopoiesis generated from WT and NHD13 cells using ow cytometry. Chimeric mice successfully engrafted both WT and NHD13 cells, as shown in Extended Data Fig. 1A-B ; in addition, chimeric mice developed peripheral blood abnormalities consistent with MDS, such as macrocytic anemia and leukopenia (Extended Data Fig. 1C-D ). Similar to the ndings with NHD13 transgenic mice, 71% percent of NHD13/WT chimeric mice progressed to myelodysplastic syndrome or acute leukemia of donor origin, most commonly acute myeloid leukemia (AML; Ext Data Fig. 1E-G ). \n\nNHD13/WT chimeric mice were treated with either vehicle control (phosphate buffered saline; PBS) or ATC (1. 0 mg/kg) once daily via intraperitoneal injection for up to 14 cycles of therapy, each cycle being two weeks of treatment followed by one week rest. Pooled results from three independent experiments revealed that ATC treated mice did not show prolonged survival, but instead showed reduced survival compared to PBS treated controls (Fig. 1A ). Surprisingly, further analysis showed that over half of the ATC treated mice had developed leukemia of WT, not donor origin (Fig. 1B ). Notably, leukemias that developed in WT cells were exclusively lymphoid, as opposed to myeloid leukemia that was seen in most NHD13 transformed cells. The most common form of lymphoid leukemia we identi ed was T cell acute lymphoblastic leukemia (T-ALL), also referred to as precursor T-lymphoblastic leukemia/lymphoma (pre-T LBL) 20 (Extended Data Table 1 ). The T-ALL cases typically showed thymic enlargement (Extended Data Table 1 ), along with peripheral blood abnormalities such as anemia, thrombocytopenia, and leukocytosis (Fig. 1C ). More detailed analysis revealed invasion of lymphoblasts in the BM and spleen (Fig. 1D ), and clonal T cell receptor beta (Trb) gene rearrangements (Ext Data Table 2 ; Fig. 1E ). In sum, 72. 7% of mice treated with ATC developed recipient T-ALL. Ionizing radiation, which was used as a preparative regimen, is well-established to be leukemogenic in mice; however, the 72. 7% incidence is signi cantly higher than the 5-10% incidence of T-ALL seen in historical controls from our lab (p = 0. 0001) (Fig. 1F ). \n\nWES of ATC treated mouse leukemia reveals marked increase in C > G transversion. We utilized whole exome sequencing (WES) to search for acquired mutations in the T-ALL samples. Remarkably, we found a dramatic increase in both number (mean +/-standard deviation of 762 +/-642 vs 2 +/-1; Mann-Whitney p = 0. 003189) and percentage (89 +/-5 vs 7. 5 +/-2. 5; Mann-Whitney p =. 003318) of C > G transversions in all ATC treated samples, with up to thousands of acquired mutations per sample (Fig. 2A-B, Supp Data Table 1 ). Further characterization of the acquired mutations using Single Base Substitution (SBS) pro le software (SigPro ler MatrixGenerator, Extractor, Simulator, and Plotter) demonstrated that the C > G transversions occurred almost exclusively in a 5'-NCG-3'context (Fig. 2C ). This signature was not recognized as an existing signature present in the COSMIC database by the SigPro ler software, which led us to regard this as a novel SBS signature [21] [22] [23] [24] [25] ; we have provisionally designated this new signature as SBS-ATC. Figure 2C shows the stark difference between T-ALL in a PBS treated mouse, with 1 C > G transversion, as opposed to T-ALL in an ATC treated mouse, with > 1000 C > G transversions. Additional evidence for the speci city of ATC associated mutations comes from analysis of two mice that developed early thymocyte precursor (ETP) ALL in donor NHD13 cells (Fig. 2D ; Extended Data Fig. 2 ). Close examination of these ETP samples demonstrated that these two leukemias had identical, clonal D-J rearrangements, indicating that a pre-malignant ETP clone had been transplanted to the recipient mice; this model is consistent with a recent report that NHD13/IDH2 R140Q mice frequently develop ETP ALL 26. However, despite the fact that these two donor ETP originated from the same pre-malignant clone, the ETP-ALL clone in a PBS treated mouse acquired only two C > G transversions, whereas an ETP clone from an ATC treated mouse demonstrated over 2000 acquired C > G transversions (Fig. 2D ). The reproducibility of the SBS-ATC signature is evident in Supp Fig. 1A, in which a marked increase in C > G transversions, in a 5'-NCG-3' context, is seen in every leukemia from mice treated with > 0. 1 mg/kg of ATC. \n\nWe employed PCR ampli cation and Sanger sequencing to verify a subset of the acquired C > G transversions that were identi ed by NGS; in all cases tested, the C > G transversions were detected by Sanger sequencing (Ext data Fig. 3A ). In a smaller subset, we veri ed that the C > G transversions were indeed transcribed into mRNA (Ext data Fig. 3B ). The detection of these mutations in both gDNA and cDNA using Sanger sequencing demonstrated that these highly speci c C > G transversions were bona de mutations and not sequencing artefacts. \n\nATC treatment of transplanted mice leads to recipient (r) T-ALL in the absence of ionizing radiation. Given the use of myeloablative ionizing radiation (IR) in generating the NHD13 chimeric model, and the known mutagenic potential of IR, we sought to examine a requirement for IR in the unique C > G transversions characterized above. To avoid IR, we used CASIN as a non-genotoxic conditioning regimen 27. CASIN, for Cdc42 activity-speci c inhibitor, treatment leads to egress of WT hematopoietic stem and progenitor cells (HSPC) from the recipient BM, allowing for engraftment of transplanted HSPC 28. We also varied the dosage of ATC in this experiment to investigate the possibility of a dose-dependent effect on C > G transversion. CASIN conditioning led to successful engraftment of NHD13 HSPC (Fig. 3A, Extended Data Table 2 ). \n\nThe engraftment of NHD13 HSPC varied with ATC dosage, as higher ATC doses were associated with a lower median engraftment; this may have been due to effective treatment of the Cd45. 2 + NHD13 MDS (Fig. 3A ). Similar to the transplants using IR, we noted a highly penetrant phenotype; all mice treated with either 0. 5 mg/kg or 1. 0 mg/kg ATC developed rT-ALL (Fig. 3B ). In contrast, only one of six mice treated with 0. 1 mg/kg ATC developed rT-ALL, and none of six PBS control mice developed rT-ALL. All mice treated with PBS developed donor MDS/AML, con rming the high penetrance of MDS/AML in recipients of NHD13 BMNC (Fig. 3B, Extended Data Tables 1 and 2 ). Two ATC-treated mice developed a concurrent donor AML and a recipient T -lineage ALL; this is not surprising given the highly penetrant nature of both ATC treatment and NHD13 BMNC transplantation (Supp Fig S2 ). \n\nSimilar to the results with transplants that employed IR, WES of leukemia from ATC treated mice following CASIN conditioning showed hundreds to thousands of C > G transversions (Fig. 3C, Supp Data Table 2 ) and that almost all SNV were C > G transversions (Fig. 3D ). In addition, the number of C > G transversions increased with increasing ATC dosage (Fig. 3C ). The unique 5'-NCG-3' context for the C > G transversion was reproduced in all ATC treated mice that received CASIN conditioning (Fig. 3E ; Supp Fig. 1B ). These results demonstrate that the C > G transversion and induction of lymphoid leukemias in ATC treated NHD13 chimeric mice was not dependent on IR.",
"section_name": "Results",
"section_num": null
},
{
"section_content": "There is evidence that both human 29, 30 and murine 31 MDS hematopoietic stem and progenitor cells (HSPC) cells can \"re-shape\" the wild-type BM microenvironment. To eliminate the possibility that a BM microenvironment re-shaped by NHD13 HSPC was required for ATC-induced mutagenesis, we treated non-transplanted WT C57BL6 mice with ATC. Given that the thymus typically involutes with age, we also wished to determine if age effected the susceptibility to ATC-induced T-cell leukemogenesis. Three independent experiments were conducted using WT mice aged 2, 8, or 12-15 months and 1. 0 mg/kg ATC. Sublethal IR (600 cGy) was also included in some experiments to allow for comparison to prior experiments with ATC and IR. \n\nAll three age groups showed signi cantly decreased survival (Figs. 4A-C ) for both ATC alone and ATC + IR treatment, with ATC + IR consistently showing poorer survival. A pooled survival curve along with cause of death is shown in Fig. 4D. All mice that received ATC, with or without IR, were either found dead or developed lymphoid leukemia within 27 weeks of beginning ATC treatment, with the majority of mice developing lymphoid leukemia between 15-20 weeks. This peak corresponds to 5-6 cycles of ATC treatment. \n\nRecipient T-ALL in non-transplanted WT mice treated with ATC was similar to that which developed in the NHD13 transplant recipients, with invasion of T-lymphoblasts in BM and spleen (Ext Data Fig. 4A ), as well as non-hematopoietic tissues such as kidney and liver (Ext Data Fig. 4B ), and clonal Trb gene rearrangements (Ext Data 4C-D). In addition to T-ALL, we also observed B-ALL in a smaller number of mice, suggesting the possibility that ATC treatment could be oncogenic in B as well as T lymphoblasts. These were two independent leukemias, as there were only nine mutations shared between the T-ALL and B-ALL, as compared to 6272 mutations that were not shared (Supp Fig. 3H ). Acquired mutations included those associated with murine T-ALL (Notch1) in the thymus and murine B-ALL (Bcor) in the BM (Supp Fig. 3I ). \n\nWES (Supp Data Table 3 ) of leukemias that arose in ATC treated mice revealed increased number and percentage of C > G transversions (Fig. 4E-F ) similar to prior experiments, once more in a 5'-NCG-3' context (Supp Fig. 1C ). These results demonstrate that neither IR nor NHD13 transplantation was required for the induction of lymphoid leukemia by ATC; and that ATC treatment could induce B-ALL as well as T-ALL in WT mice (Fig. 4D ). \n\nWGS shows correlation of ATC-induced C > G transversions and global CpG density. We used Whole Genome Sequencing (WGS) (Supp Data Table 4 ) to map the location of C > G transversions on a subset of eight ATC-induced T-ALL samples. We detected an average of more than 24,000 mutations per sample, primarily C > G transversions in a 5'-NCG-3' context. As shown in Supp Fig. 1D, despite a considerable difference in the total number of SNV (range 3,865 - 42,867), the mutation pro les of these samples are almost identical. Using the ChromoMap package in R, we mapped pooled C > G transversion density as well as each individual mouse C > G transversion density 32, 33 (Supp Fig. 4A-H ). Given the 5'-NCG-3' context of C > G transversions, we hypothesized that C > G transversion would correspond to known CpG density. We found that while C > G transversion dense areas generally mapped to CpG dense regions, the highest density of C > G transversions did not invariably map to the highest density of CpG dinucleotides (e. g., the distal portion of chromosome 2 in Fig. 5 ). In addition, not every CpG dense region had high C > G transversion density (e. g., chromosome 18 in Fig. 5 ), and there is considerable heterogeneity in C > G transversion mapping when examining individual mouse samples (Supp Fig S4A -H ). We also found increased C > G transversion density in genomic regions that contain known cancer-associated genes, such as Notch1, Ikzf1, and Trp53 (Fig. 5 ), suggesting that C > G transversion may lead to in vivo selection due to mutations that confer a tness advantage. Nonetheless, Supp Data Table 5 reveals that among 193,839 total mutations detected by WGS, only 281 (0. 145%) C > G transversions occurred in the exact same nucleotide position among two different mice, and no exact nucleotide position was mutated more than twice; therefore, it seems ATC does not preferentially mutate at any single nucleotide position, but rather acts preferentially within larger chromosomal regions. \n\nNumerous C > G transversions occur in genes relevant for human lymphoid leukemia. To investigate a relationship between C > G mutagenesis and lymphoid oncogenesis, we determined whether C > G transversions commonly occurred in genes associated with human cancer, especially lymphoid malignancy. To avoid complexities introduced by IR and NHD13 transplant, we focused these studies on 22 lymphoid leukemias (19 T-ALL and 3 B-ALL) that were generated by ATC treatment of WT mice. We compared Tier 1 mutations identi ed in this set to a set of 432 genes commonly associated with cancer that were part of the FoundationOne® Heme Gene panel (Foundation Medicine, Inc. ) used to detect relevant cancer mutations in human hematologic malignancy. This analysis identi ed a total of 612 Tier 1 C > G transversions in genes associated with cancer in the 22 mouse samples for an average of 28 potentially oncogenic mutations per leukemia sample (Fig. 6A, Supp Data Table 6 ). Additionally, the 40 genes most commonly mutated included genes well known to be associated with human lymphoid malignancy, such as Bcl11b, Ikzf1, Trp53, Pten, Kras, Jak3, and Notch1 (Fig. 6A ). Further analysis of C > G transversion position revealed that amino acid mutations often occurred in known oncogenic \"hotspots\", such as Trp53 R270P, homologous to human R273 mutants (Fig. 6B ), Pax5 P80R (Fig. 6C ), and Pten R130G (Fig. 6D ) 34 These results indicate that the induction of C > G transversions in lymphoid cells is the likely proximal cause of leukemic transformation observed in ATC treated mice. \n\nC > G transversions can be generated in human cells after brief ATC exposure in vitro. We treated the human AML cell line U937 with ATC in vitro (Fig. 7A ) to address two questions; 1) were human cells susceptible to the mutagenic effect of ATC, and 2) could we develop an in vitro assay for ATC-induced mutagenesis. To minimize diversity of the initial U937 cell population, we rst single cell cloned the U937 cell line. The cloned U937 parental line was then treated for 6 days in vitro with 25, 50, or 100nM ATC or PBS. Reasoning that DNA harvested at this time may contain multiple populations of mutagenized U937 cells, we then single cell cloned the treated cells, and harvested genomic DNA from both the bulk U937 population as well as the individual single cell clones. \n\nWES ( ltered for VAF > = 0. 2 and alternate allele count > = 5) from bulk ATC treated cells revealed 0 SNV. However, there was a dramatic difference in number and percent of C > G SNV in the ATC treated individual clones. The ATC treated clones had 495 ± 360 C > G transversions per clone (Fig. 7B ), whereas the PBS treated clones had 0. 2 ± 0. 4 C > G transversions per clone, p = 0. 009701, a difference of > 1000fold (Supp Data Table 7 ). There were similar differences in the percent C > G SNV among all mutations (Fig. 7C ). SBS analysis revealed a similar 5'-NCG-3' mutational context in human cells, but without the 5'-TCG-3' peak as observed in mice (Fig. 7D, Supp Fig. 5 ). We refer to this in vitro assay as GEMINI for Genotoxic Mutation Signature Identi ed After Clonal Expansion In Vitro. \n\nThe human T-cell line CEM was also examined using the GEMINI assay. Similar to U937 cells, we noted a marked increase in C > G transversions in ATC treated single cell clones (1172 ± 447 vs 55 ± 38, p = 0. 007937) (Ext Data Fig. 5A ). However, in contrast to treatment of U937 cells, in which the percent of C > G transversions was 86-91% (Fig. 7C ), the percent of mutations that were C > G transversions in ATC treated CEM clones was much lower, only 26-45% (Ext Data Fig. 5B ). Moreover, the total number of variants in the CEM PBS control single cell clones was far higher than the U937 PBS single cell clones (1215 ± 194 vs. 0. 25 ± 0. 4, p = 0. 01193) (Supp Data Table 7 ). However, it has previously been reported that the CEM cell line has a mismatch repair de ciency due to deletion of MLH1 35. Examination of the CEM WES. bam les revealed an almost total absence of MLH1 reads, consistent with a homozygous MLH1 deletion. SBS analysis of the CEM clones identi ed two signatures associated with mismatch repair de ciency (COSMIC signatures SBS15 and SBS21), as well as a novel, previously unreported signature, similar to that seen in U937 cells. Inspection of the SBS pro les for CEM reveals C > G and C > T SNVs, both preferentially in a 5'-VCG-3' context (V indicating not T) (Ext Data Fig. 5C ) (See Supp Fig. 5 for all SBS plots). Taken together, these results demonstrate that ATC can induce C > G transversions in human cells. \n\nIn addition to ATC, we evaluated the DNMTi decitabine (DAC; FDA approved for treatment of MDS) for a potential mutagenic effect, as both molecules are deoxycytidine analogs with Aza moieties in the cytosine base. WES using the protocol outlined in Fig. 7A revealed an increase in the number (10 ± 6 vs. 0. 2 ± 0. 4, p = 0. 009701) (Ext Data Fig. 6A ) and proportion (20-70%, p = 0. 009701) (Ext Data Fig. 6B ) of C > G mutations in DAC treated vs PBS controls (Supp Data Table 7 ). However, this effect was markedly reduced compared to ATC, and no clear 5'-NCG-3' or 5'-VCG-3' signature was observed in DAC treated samples (Ext Data Fig. 6C ) (See Supp Fig. 5 for all SBS plots). These results suggest that DAC may have a similar, but weaker mutagenic effect compared to ATC. \n\nDck is required for C > G transversions induced by ATC. It is unclear why ATC induced only lymphoid malignancy in the in vivo studies. Given that ATC is an unphosphorylated cytidine analog, we reasoned that ATC would need to be phosphorylated to be incorporated into DNA. Phosphorylation of deoxycytidine is mediated by deoxycytidine kinase (Dck), the rate-limiting enzyme in the cytosine \"salvage\" pathway (Fig. 8A ) 36. Dck is most highly expressed in lymphoid tissue 37, 38 and its importance in lymphoid cell development is underscored by the observation that the only phenotype noted in Dck KO mice was in T and B cell precursors 39. We thus hypothesized the expression of Dck in lymphoid tissue allowed for incorporation of ATC into the lymphoid cell genome, leading to C > G mutations and leukemogenesis. \n\nTo assess whether phosphorylation of ATC by Dck was required for the mutagenic effect of ATC, we utilized a murine T-ALL cell line with a homozygous deletion of Dck that had been generated by serial passage in the presence of cytarabine; the parental cell line is designated 7298, while the cytarabine resistant cell line is designated 7298CR (Fig. 8B-C ). A dose-dependent effect on both cell growth (Fig. 8D ) and viability (Fig. 8E ) was evident in the parental 7298 (Dck WT) cell line, whereas the 7298CR (Dck deleted) cell line showed little effect at any concentration of ATC tested. We used the GEMINI assay described in Fig. 7A to generate single cell clones of both the 7298 and 7298CR cell line following treatment with 1000 nM ATC. WES revealed a marked increase in both the number (Fig. 8F ) and proportion (Fig. 8G ) of C > G transversions in the 7298 clones, whereas 7298CR clones had very few C > G transversions (Supp Data Table 8 ). SBS pro les of the 7298 clones demonstrated the same 5'-NCG-3' context that was identi ed in the murine T-and B-ALL samples, whereas pro les of the 7298CR clones had very few C > G transversions (Fig. 8H ) (Supp Fig. 6 ). These results demonstrate that Dck expression is required for ATC induced C > G mutagenesis and suggest that lymphoid leukemia induction in the context of ATC treatment may be due to high Dck expression in lymphoid cells.",
"section_name": "ATC induces C > G transversions and T-ALL in nontransplanted WT mice",
"section_num": null
},
{
"section_content": "The results of this study reveal a highly penetrant mutagenic and carcinogenic phenotype associated with ATC, an investigational DNMTi. The observation of lymphoid leukemias with or without either NHD13 co-transplantation or ionizing radiation demonstrates that ATC exposure is su cient to induce lymphoid leukemia. Furthermore, sequencing of these leukemias revealed C > G transversions in a unique 5'-NCG-3' context. The nding of C > G transversions encoding missense and nonsense mutations within well-known cancer genes, often at residues that are recurrently associated with human lymphoid leukemia, provides compelling evidence that the mutagenic phenotype is directly linked to malignant transformation of murine lymphoblasts. \n\nThe discovery of a previously unrecognized mutational signature associated with ATC treatment highlights the complexity and non-random nature of SBS mutational processes. Other mutational signatures induced by chemotherapy treatment have been characterized, including temozolomide (SBS11) 21, 40, and cisplatin (SBS34, SBS35) 41 ; in addition, the toxin aristolochic acid generates a highly speci c signature (SBS22) 24, 42, 43. However, none of these agents are characterized by a striking C > G transversion preference. \n\nThe unique C > G transversion following ATC treatment offers the potential to study this form of mutation in the context of malignant transformation. Among the six potential types of SBS, the most common form of SBS found in human cancer is a C > T transition, likely caused by spontaneous deamination of a 5'-methylcytosine (designated SBS1) 23. A C > T transition can produce certain amino acid substitutions based on the mammalian genetic code, whereas other amino acid substitutions cannot be produced by a C > T transition; for example, a C > T transition cannot produce a Pro > Arg substitution, whereas a C > G transversion can produce a Pro > Arg substitution. This fact makes it more di cult to study cancers which are driven by less common missense or nonsense mutations that can not be generated by a C > T transition. The ability to induce thousands of C > G transversions makes ATC a potential tool for the detection and validation of less common oncogenic amino acid substitutions that are underrepresented in current databases, which in turn offers promise in understanding oncogenic protein variants. \n\nThe leukemias that developed in WT (including recipient cells for transplant experiments) cells were exclusively of T or B lymphoblast origin. However, despite the absence of WT AML arising in ATC treated cells, AML did occur in NHD13 donor cells of transplant recipients treated with ATC, and these AML samples showed the same unique C > G transversions in a 5'-NCG-3' context, indicating an ATC mutagenic effect in myeloid as well as lymphoid cells. In addition, the in vitro experiments with U937 (myeloid) cells demonstrates that ATC can be mutagenic in myeloid cells as well as lymphoid cells. We speculate that the predilection for lymphoid leukemias may be due to elevated expression of Dck in lymphoid tissues, potentially due to higher usage of the lymphoid salvage pathway in lymphoid precursor cells 39. \n\nThe use of single cell colonies to isolate and amplify a unique mutational pro le which is undetectable by WES of bulk populations of cells treated in vitro demonstrates the power of single cell clones for the detection and study of mutagenic compounds. Current techniques commonly used to evaluate mutagenicity in vitro include the Ames test 44, the HPRT assay 45, and the TK assay 46, and assessment of BigBlue™ 47 mice in vivo. While these tests are capable of detecting SBS and deletions within select genes, none are able to characterize the prevalence of mutations across the genome or a speci c mutational signature associated with a mutagenic agent in question. Thus, we speculate that the in vitro GEMINI assay described in this manuscript could be useful in future studies of mutational processes associated with chemical or biological agents in human cells. \n\nAlthough not the focus of this manuscript, the in vitro assay demonstrated a signi cant 40-fold increase in C > G transversions in U937 cells treated with decitabine vs PBS treatment. This nding is consistent with the observation that some MDS patients show increased C > G transversions following treatment with decitabine or 5-azacytidine 48, 49. Moreover, a small number of patients with either AML 50 or solid tumors 17, 50 have responded to treatment with immune checkpoint inhibitor therapy either with or following 5-azacytidine treatment 50, 17. The interpretation of these observations has been that reactivation of endogenous antigens following 5-azacytidine treatment leads to additional antigenic targets and resultant sensitivity to immune checkpoint inhibitors 51. The data presented in this manuscript suggests a potential alternate mechanism, namely, that increased antigenic targets caused by multiple C > G transversions results in increased sensitivity to immune checkpoint inhibitors. \n\nIn sum, this study demonstrates that ATC is a potent mutagen capable of inducing C > G transversions, in a unique 5'-NCG-3' context, in murine cells or a unique 5'-VCG-3' context in human cells. The collection of C > G transversions is capable of inducing B and T-ALL in mice, regardless of transplant or IR exposure. These mutations arise predominantly within CpG islands, are reproducible in multiple studies, and are con rmed by orthogonal assays such as Sanger sequencing of DNA and RNA. We propose that ATC can be a useful reagent for future studies in characterizing C > G transversions and cancers arising from these transversions and highlight the potential utility of in vitro assays of clonal expansions for identifying mutagenic potential in mammalian cells.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Mouse strains, genotyping, and identi cation. NUP98::HOXD13 (NHD13) transgenic donor mice were generated on a C57BL/6 Cd45. 2 background and genotyped as previously reported 39. Chimeric mice with both wild-type and MDS-derived hematopoiesis were generated using a bone marrow transplantation model as previously reported 52 Flow cytometry. Flow cytometry was performed as described previously 52. Single cell suspensions were prepared from each tissue: bone marrow was ushed from long bones as described above, and spleen and thymus tissue was gently teased apart and ltered through a 40-uM mesh lter into HF2. Cells were counted and resuspended in an HF2 solution containing 5% rat serum solution to block binding to Fc receptors. Cells were then incubated with a cocktail of antibodies for 30 minutes at 4 degrees Celsius. \n\nA Dck deleted cell line is resistant to ATC and does not show C>G transversions.",
"section_name": "Online Methods",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "The authors thank current and former members of the Aplan lab, Andre Nussenzweig, and Michael Kuehl (deceased) for insightful discussions. We thank the NCI Sequencing Minicore for Sanger sequencing, the NCI Transgenic Core for generation of transgenic mice, the NCI Genomics Core for Next Generation Sequencing, the NCI Flow cytometry core for cell sorting, the NCI Pathology/Histotechnology Lab (PHL) for immunohistochemistry, Shelley Hoover and Mark Simpson of the NCI Molecular Pathology Unit for assistance with slide imaging, and Maria Jorge for excellent animal husbandry. This work was supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health (grant numbers ZIA SC 010378 and BC 010983 ) and the NCI Experimental Therapeutics (NExT) program.",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "",
"section_name": "BW17-942E).",
"section_num": null
},
{
"section_content": "Cell culture. Human U937 and CEM cell lines were obtained from American Type Culture Collection (ATCC), cultured as single cell suspensions in Iscoves modi ed Dulbecco Media (IMDM) media supplemented with 10% fetal bovine serum (FBS), glutamine, and penicillin/streptomycin (hereafter referred to as IMDM complete media), and maintained as previously reported 54, 55. The 7298 cell line is a murine T-ALL cell line derived from SCL/LMO1 transgenic mice 54 and was grown in IMDM complete media. The 7298CR cell line is a cytarabine resistant derivative of 7298 that has a biallelic deletion of Dck (D. Cao and P. D. Aplan, unpublished). All cells were maintained at 37 C and 5% CO2. \n\nIn vitro drug treatment (GEMINI assay). Cell lines were expanded and treated in 25 mL asks at a concentration of 50,000 cells/mL in 5mL of IMDM complete media. Cells were treated for 3 days with drug, then washed, resuspended in fresh media, and treated for an additional 3 days. Following a 4-day rest period, single cell clones were expanded in 96 well plates as follows. Treated cells were collected by centrifugation and resuspended at a concentration of 5 cells/mL. 100 uL (or an average of 0. 5 cells/well) were then plated in 96 well plates and expanded for one week. In a typical experiment, 40-50 wells would expand; 5-10 clones were then picked at random to expand for whole exome sequencing (WES). Cell counts and viability were conducted using an automated cell counter (TC20 Automated Cell Counter, BioRad) and Trypan Blue Viability assay (Lonza BioWhittaker Trypan Blue 0. 4%, Fisher Scienti c, Cat#",
"section_name": "",
"section_num": ""
},
{
"section_content": "Nucleic acid extraction, reverse transcription, polymerase chain reaction (PCR), and Sanger sequencing. \n\nGenomic DNA was extracted from each tissue using the DNeasy Blood & Tissue kit (Qiagen) and the manufacturer's suggested protocol. RNA was isolated using TRIzol Reagent and the accompanying RNA extraction protocol (Cat#15596026, Invitrogen). Reverse transcription of mRNA was accomplished using the SuperScript III First-Strand Synthesis System (Invitrogen, Cat#: 18080-051) and accompanying protocol. PCR ampli cation to identify clonal murine T cell receptor beta (Trb) rearrangements used mRNA or DNA templates and primers and protocols as previously described 26. Clonal Igh gene rearrangements were identi ed using primers and protocols as previously described 56. Gene speci c ampli cation to verify mutations used DNA or mRNA templates and primers listed in Supp Data Table 9. Primers for cDNA PCR were designed to reside on differing exons as to avoid false positive signals from possible contaminating gDNA. Ampli cation of the murine Scid locus was used as a quality control for genomic DNA, and ampli cation of the murine Actb gene was used as a quality control for mRNA. All PCR ampli cation assays used LiTaq PCR mix (Cat# M0024, LifeSct) and the manufacturer's recommended protocol. Primers are listed in Supp Data Table 9. Amplicons were size fractionated on agarose gels and puri ed using the Qiagen QIAquick PCR Puri cation Kit (cat#28104). Sanger sequencing of puri ed fragments was performed by the NCI Sequencing Core. Clonal Trb and Igh rearrangements identi ed using PCR were con rmed by inspection of. bam les using an Integrated Genome Viewer (IGV, Broad Institute v 2. 12. 3) for samples in which WES results were available. \n\nWestern Blot. SCL/LMO1 parental 7298 cells and cytarabine resistant 7298 cells were lysed in RIPA lysis buffer (sc-24948A, Santa Cruz) with 1 mM sodium orthovanadate, 1 mM PMSF and protease inhibitor cocktail on ice for 15 min. The protein concentration of sample is determined by Micro BCA™ Protein Assay Kit (Cat#23235, Thermo Fisher Scienti c). The protein sample is prepared with Laemmli buffer (Cat#1610747, BIO-RAD) to a nal concentration of 1 ug/ul. 20 µl protein samples were applied to 10% SDS-PAGE gels, separated by electrophoresis at 80 V for 30 min and 120 V for 90 min. After electrophoresis, protein was transferred to a nitrocellulose membrane using Semi-dry Transfer System (Power Blotter, Thermo Fisher Scienti c). The membrane was then blocked in 5% non-fat dry milk (Blotting-Grade Blocker, BIO-RAD) in TBST (10 mM Tris-Cl, 150 mM NaCl, 0. 05% Tween-20, pH 7. 5) for 1 h. The membrane was then incubated with primary antibody overnight at 4°C followed by incubation with a secondary antibody conjugated to horseradish peroxidase (HRP), and visualized using a chemiluminescence kit (GE Healthcare, RPN2106). Speci c antibodies to DCK (Cat# ab186128, Abcam), β-Actin (Cat# A1978, Sigma) were used to detect protein levels. β-Actin was used as a loading control. \n\nWhole-Exome Sequencing. Genomic DNA (500 ng) was sheared to a mean size of 300 bp on a S2 Covaris Ultrasonicator. Illumina libraries were prepared using a KAPA HyperPrep Kit, according to the manufacturer's instructions, incorporating unique dual indexes (IDT). Mouse Illumina libraries were pooled and enriched using Agilent SureSelect XT System with Mouse All Exon baits according to the manufacturer's instructions. Human Illumina libraries were pooled and enriched using IDT xGen Human Exome Lockdown Probes and Reagents according to the manufacturer's instructions. Enriched libraries were sequenced on an Illumina NextSeq 2000 Sequencing System using P3 (300 cycle) reagents. Data processing and variant calling procedure followed the Best Practices work ow recommended by the Broad Institute. Brie y, the raw sequencing reads were mapped to mouse genome build 10 (mm10) by the Burrows-Wheeler Aligner 57 followed by local realignment using the GATK suite v4. 2. 5. 0 9 58 from the Broad Institute, and the Picard tools marked duplicated reads. The somatic variants were called by MuTect2 within the GATK suite. The somatic variants were rst ltered with the GATK recommended ltering criteria and further ltered by the following criteria: baitRegion= \"TRUE\", FILTER = \"PASS\", TUMVAF > = 0. 2, and AD_TUMOR > = 5. The ltered variants were used for the Mutational Signature analysis, for which the software SigPro lerMatrixGenerator v1. 2 and SigPro lerExtractor v1. 1. 4 59 from AlexandrovLab at UCSD were used. \n\nWhole-Genome Sequencing. Whole genome sequencing was conducted at the NCI Frederick ATRF (Advanced Technology Research Facility). Analysis of mapped sequence reads (percent duplicate reads 12-31%, mean coverage of 62-123X) using Mutect 2 software and parameters was done as described above for WES. Generation of ChromoMaps was conducted using the R package ChromoMap v4. 1. 1 32 using R Statistical Software version 4. 3. 0 33. Statistics and Testing. Data are reported means ± standard deviation unless otherwise noted. Survival curves were generated using GraphPad Prism v8. 4. 3 (GraphPad Software, LLC) and were analyzed using the Mantel-Cox Log-Rank test for signi cance. The Bonferroni correction was used to correct for multiple hypothesis testing where applicable. Comparisons between mutation counts and percentages were conducted using the Mann-Whitney U test. P values of < 0. 05 were considered statistically signi cant in the context of this study. Note the marked difference in total number of substitutions and substitutions in a speci c 5'-NCG-'3 context (\"SBS-ATC\"). D. SBS plots of mouse with dETP treated with ATC vs. mouse with dETP treated with PBS. The dETP in both of these mice was derived from a pre-leukemic clone transplanted from the NHD13 donor, as documented by identical, clonal Trb DJ rearrangements (see inset).",
"section_name": "BW17-942E).",
"section_num": null
},
{
"section_content": "",
"section_name": "Supplementary Files",
"section_num": null
}
] |
10.1038/s41419-022-05052-9
|
Hydroxylation of the NOTCH1 intracellular domain regulates Notch signaling dynamics
|
<jats:title>Abstract</jats:title><jats:p>Notch signaling plays a pivotal role in the development and, when dysregulated, it contributes to tumorigenesis. The amplitude and duration of the Notch response depend on the posttranslational modifications (PTMs) of the activated NOTCH receptor – the NOTCH intracellular domain (NICD). In normoxic conditions, the hydroxylase FIH (factor inhibiting HIF) catalyzes the hydroxylation of two asparagine residues of the NICD. Here, we investigate how Notch-dependent gene transcription is regulated by hypoxia in progenitor T cells. We show that the majority of Notch target genes are downregulated upon hypoxia. Using a hydroxyl-specific NOTCH1 antibody we demonstrate that FIH-mediated NICD1 hydroxylation is reduced upon hypoxia or treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG). We find that a hydroxylation-resistant NICD1 mutant is functionally impaired and more ubiquitinated. Interestingly, we also observe that the NICD1-deubiquitinating enzyme USP10 is downregulated upon hypoxia. Moreover, the interaction between the hydroxylation-defective NICD1 mutant and USP10 is significantly reduced compared to the NICD1 wild-type counterpart. Together, our data suggest that FIH hydroxylates NICD1 in normoxic conditions, leading to the recruitment of USP10 and subsequent NICD1 deubiquitination and stabilization. In hypoxia, this regulatory loop is disrupted, causing a dampened Notch response.</jats:p>
|
[
{
"section_content": "The highly conserved Notch signaling pathway regulates a wide range of biological processes such as immune cell development and function [1] vascular morphogenesis [2, 3] and its deregulation is frequently observed in cancer [4] [5] [6]. Notch signaling is activated by the binding of a Notch ligand to a transmembrane NOTCH receptor. This interaction results in two sequential proteolytic cleavages that lead to the release of the NOTCH intracellular domain (NICD). The NICD subsequently translocates into the nucleus where it interacts with the transcription factor (TF) RBPJ, the coactivator MAML1 (MASTERMIND-LIKE 1), and the acetyltransferase EP300 to drive the expression of Notch target genes [7]. This transcriptional program is regulated by posttranslational modifications (PTMs) of the NICD, including prolyl isomerization [8] [9] [10], influencing the amplitude and duration of the Notch response [11, 12]. \n\nCells have developed mechanisms to cope with oxygen (O 2 ) deprivation (hypoxia) by activating the hypoxia-induced factors (HIFs) [13, 14]. Under normoxic conditions, two different 2-oxoglutarate-dependent oxygenases regulate HIF1α: Prolyl hydroxylases (PHDs), that catalyze prolyl (P) hydroxylation of HIF1α, and factor inhibiting HIF (FIH), which catalyzes of asparagine (N) hydroxylation [15, 16]. Proline hydroxylation of HIF1α promotes its proteasomal degradation by the Von-Hippel-Lindau (VHL)-containing E3 ubiquitin ligase complex [13, 14, 17]. FIH-mediated asparagine hydroxylation in the C-terminal activation domain of HIF1α prevents its interaction with the coactivator EP300 [16]. Limited availability of O 2 leads to inhibition of both PHDs and FIH resulting in the stability of HIF1α (and of other members of the same family of transcription factors) and activation of the hypoxiainducible target genes [13, 14, 16, 17]. Interestingly, FIH has a higher O 2 affinity and still functions under intermediate O 2 levels [18]. In addition, FIH seems to be physiologically important in situations with a rapid onset of hypoxia such as ischemia [19]. \n\nGiven the central importance of Notch and hypoxia pathways not only for development but also for homeostasis, it is not surprising that both signaling cascades regulate each other. For instance, hypoxia has been reported to induce the expression of direct Notch target genes of the Hairy Enhancer of the Split family [20] [21] [22] [23] [24] [25] [26] [27] [28], suggesting that hypoxia increases the Notch pathway activity. Moreover, FIH has been shown to hydroxylate NICD within the ankyrin (ANK) repeats [12, 27, [29] [30] [31], providing a potential molecular mechanism for the direct oxygen-dependent regulation of Notch signaling. However, the functional consequences of NICD hydroxylation are not entirely clear. \n\nHere, we study the consequences of hypoxia on Notch signaling using a mouse progenitor T-cell line, in which the Notch pathway is constitutively active. Unexpectedly, we observe that the majority of Notch target genes are downregulated in conditions of hypoxia correlating with lower NICD1 hydroxylation and protein levels. Using an antibody recognizing site-specific NICD1 hydroxylation, we find that FIH mediates NICD1 hydroxylation. We further show that NICD1 hydroxylation alters its ubiquitination, influencing both degradative and non-degradative ubiquitin chains. Moreover, we observe that the molecular crosstalk between NICD1 hydroxylation and ubiquitination depends on deubiquitinase (DUB) USP10, which modulates Notch responses.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Antibody was raised against the hydroxylated asparagine 1945 (N*) of the NICD1 sequence ASADA N* IQDNM and affinity purified with peptides immobilized on sulfolink beads. The serum was first passed over a column with NICD1 N1945-OH peptides; afterward, unspecific antibody was depleted over a column with unmodified NICD1 peptides. The supernatant containing the NICD1 N1945-OH antibody was recovered and dialyzed overnight in PBS. The specificity of the purified antibody was analyzed by dot blot. Peptides were synthesized at Biosynthan and the antibody was produced by BioGenes.",
"section_name": "MATERIALS AND METHODS Generation of the NICD1 N1945-OH antibody",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "We studied the molecular interplay between Notch signaling and hypoxia using the mouse progenitor T-cell line Beko, derived from T-cell receptor knockout mice, in which the Notch pathway is constitutively active [32] [33] [34]. We treated Beko cells for 24 hours with the hydroxylase inhibitor dimethyloxalylglycine (DMOG) or kept the cells for 12 h under hypoxic conditions (1% O 2 ). We observed increased stability of both HIF1α and HIF2α proteins (Fig. S1A, B ). In addition, both regimens elicited similar transcriptional responses: 780 genes were affected in both conditions (Fig. S1C -G and Tables S1, S2 ). Gene set enrichment analysis using KEGG datasets showed that genes associated with the \"HIF1 signaling pathway\" were significantly enriched in the genes commonly regulated by DMOG and hypoxia (Fig. S1H and Table S3 ). Similarly, \"response to hypoxia\" and \"cellular response to hypoxia\" were among the significantly enriched gene ontology (GO) terms of the overlap of upregulated genes (Table S4 ). Gene set enrichment analysis (GSEA) also demonstrated that genes linked to \"response to hypoxia\" were enriched (Tables S5, S6 ). The upregulation of hypoxia target genes was further validated by qPCR (Fig. S1I, J ). The GSEA analysis also unveiled that genes associated with the \"Notch signaling pathway\" were significantly changed upon both DMOG and hypoxia treatment (Fig. S2A, B and Tables S5, S6 ). \n\nTo further investigate how Notch signaling is regulated by hypoxia in these cells, we made use of RNA-and ChIP-Seq datasets in Beko cells, in which Notch signaling is inhibited by the γsecretase inhibitor (GSI) DAPT [32]. We focused on genes that were downregulated by GSI and bound by the transcriptional effector RBPJ, assuming that these are direct Notch target genes. Using this approach, we defined 34 genes as bona fide direct Notch targets, including Hes1 and Hey1 encodes for transcriptional repressors, Il2ra (IL2 receptor-alpha, also known as CD25) that encodes for a subunit of the IL2 receptor, which is required for cell-autocrine T-cell homeostasis and Ptcra (preT-cell receptor-alpha) which encodes for the essential subunit of the preT-cell receptor (preTCR) (Fig. 1A, B and Table S7 ). Taking advantage of this Notch signature, we observed that canonical Notch targets Hes1 and Hey1 are upregulated in hypoxic conditions, which is in line with previous reports [12, [20] [21] [22] [23] [24] [25] [26] [27] [28]. However, surprisingly, we observed that the majority of the Notch signature genes are downregulated upon hypoxia or DMOG treatment (Fig. 1A, B and Table S2 ), a finding that was also validated by qPCR (Figs. 1C and S2C, D ). To better understand whether the transcriptional regulation is RBPJ/Notchdependent or HIF1α dependent, we performed ChiP-Seq experiments in Beko cells to determine the localization of transcription factors RBPJ and HIF1α in normoxic and hypoxic conditions (Table S7 ). We observed a significant increase in the binding of HIF1α upon hypoxia (Fig. S3A ) but the genomic binding of RBPJ was hardly influenced (Fig. S3B ). The hypoxia-responsive element (HRE) was promptly identified at the HIF1α binding sites (Fig. S3C ) and we could identify several genes that are deregulated by hypoxia or DMOG and bound by HIF1α (Fig. S3D and Table S8 ). When focusing on the bona fide Notch target genes, we observed that both Hes1 and Hey1 are bound by HIF1α upon hypoxia while the binding of RBPJ is reduced, suggesting that their upregulation is HIF1α dependent (Fig. S3E, F ). \n\nTogether, our data reveal that in the Notch-ON state the majority of Notch target genes are downregulated upon hypoxia. In contrast, Notch targets like Hes1 and Hey1, which are also true HIF1α targets, are upregulated upon hypoxia.",
"section_name": "Hypoxia or DMOG-treatment downregulates expression of Notch target genes",
"section_num": null
},
{
"section_content": "PTMs of the NICD1 regulate its stability and activity [11, 12]. Given that FIH is an O 2 -sensitive enzyme known to hydroxylate NICD1 [12, 27, [29] [30] [31], we reasoned that FIH might influence NICD1's stability. Indeed, we observed lower NICD1 protein levels after 12 h of hypoxia incubation or 24 h of DMOG treatment (Fig. 2A ) in Beko cells, consistent with the downregulation of the Notch target gene signature (Figs. 1A-C, S2C, D, and Table S2 ). To determine whether these effects are due to changes in NICD1 protein halflife, we performed cycloheximide (CHX) experiments in Beko cells (Figs. 2B and S4H ). We observed that DMOG treatment reduces the stability of NICD1 compared to the DMSO treated controls (Figs. 2B and S4H ). Given that DMOG inhibits both FIH and PHDs [35] [36] [37] [38] [39], we treated Beko cells with roxadustat, a selective inhibitor of PHDs. We observed that roxadustat did not affect NICD1 protein levels (Fig. S4A ), suggesting that the DMOG-and hypoxia-induced effects on Notch signaling are caused by FIH. In support of a functional relationship between FIH and NICD1, we found that these proteins interact with each other in both HEK293 cells (Fig. 2C ) and in Beko cells (Fig. S4B, C ). We further mapped the domains necessary for the FIH/NICD1 interaction and found that it requires the ankyrin-repeats of the NICD1 (Fig. S4D, E ). Finally, using NICD1 and FIH ChIP we demonstrate in Beko cells that both proteins co-occupy enhancers of Notch target genes that are downregulated in hypoxic conditions (Fig. S4F, G ). Altogether, these data suggest that FIH stabilizes the NICD1.",
"section_name": "FIH positively regulates the NICD1 protein stability",
"section_num": null
},
{
"section_content": "To further understand the regulation of Notch signaling by the FIH-mediated hydroxylation of NICD1, we generated an antibody recognizing hydroxylated NICD1 on N1945 (NICD1 N1945-OH) but not the unmodified protein (Fig. 3A ). To test the specificity of the antibody, we generated HEK293 cells lacking NOTCH1 or just its C-terminal PEST domain important for the ubiquitin-mediated degradation of NICD1 (ΔPEST; Fig. S5A ). Western blotting for NICD1 validated the correct targeting of HEK293 cells showing the truncated protein in the NICD1 ΔPEST cells (Fig. S5A, compare lane 2 to lane 1) but no detectable signal in the NOTCH1 knockout cells (Fig. S5A, compare lanes 3 and 4 to lane 1). Similar results were obtained for the N1945-OH-specific antibody (Fig. 3B, compare lane 2 to lane 1 and lane 3 to lane 1). Importantly, the NICD1 N1945-OH antibody failed to recognize the NICD1 protein when both the NICD1 hydroxylation sites N1945 or N2012 were mutated to alanine residues (NNAA; Fig. S5B lane 3 ) or when only the N1945 was mutated to alanine (N1945A; Fig. S5B lane 4 ). In line with these results, our antibody recognizes a NICD1 mutated only on N2012 (N2012A; Fig. S5B lane 5 ). Altogether, these data validate the specificity of our NICD1 N1945-OH antibody. \n\nUsing this tool, we observed that hypoxia or DMOG but not 24 h of roxadustat treatment lead to a reduction of asparaginylhydroxylated NICD1 in Beko cells (Fig. S5C-E ). To exclude that the reduction of modified NICD1 just reflects a reduction in overall NICD1 protein abundance, we shortened the hypoxia incubation and DMOG treatment. We kept Beko cells for 4 hours under hypoxia or we treated the cells for 4 or 6 h with DMOG. Under these conditions, NICD1 protein levels remained unchanged (Fig. 3C, middle panels) while the levels of N1945 hydroxylated NICD1 were diminished (Fig. 3C, upper panels). These data demonstrate that the reduction in NICD1 hydroxylation precedes the reduction in protein levels and raises the possibility that hypoxia reduces NICD1 stability via its altered hydroxylation. \n\nSimilarly to Beko cells, DMOG treatment or hypoxia in RPMI-8402, a human T-cell acute lymphoblastic leukemia (T-ALL) line, again results in downregulation of Notch target genes (Fig. S6A, B ) associated with decreased NICD1 protein level (S6C, D) and reduced NICD1 hydroxylation (Fig. S6E, F ). \n\nTo assess whether FIH is implicated in this regulation, we generated cells depleted for FIH using CRISPR/Cas9 (Fig. S7A-D ). In FIH-depleted HeLa cells, NICD1 hydroxylation on N1945 was abolished (Fig. 3D, compare lanes 2 and 3 to lane 1). Furthermore, re-expression of wild-type (wt) but not catalytically dead (CD) FIH [40] restored NICD1 hydroxylation (Fig. 3D ). In addition, we also analyzed NICD1 mutants harboring mutations of the hydroxylation acceptor sites N1945 and N2012 (Fig. S7E ). Overexpression of FIH increases NICD1 N1945-OH when the NICD1 wild-type or N2012A mutant but not NICD1 N1945A or NICD1 NNAA mutants are co-overexpressed (Fig. S7E ). Biochemically, the protein stability of the NICD1 NNAA mutant is slightly reduced as shown in CHX assays (Fig. 3E ). Importantly, the mutation of N1945A and N2012A did not impact NICD1's interaction with RBPJ and MAML1 in HEK293 cells (Fig. S8A-D ). Similar to the wild-type NICD1, the mutant also localizes to the nucleus in HeLa cells (Fig. S8E ). In contrast, transactivation of Notch-dependent luciferase reporters was severely compromised (Fig. 3F ). Altogether, these data suggest that the hydroxylation-resistant NICD1 mutant is less stable and less transcriptionally active. \n\nTo further explore the function of hydroxylation-resistant NICD1 in vivo, we tested the NICD1 NNAA mutant in embryonic development using Danio rerio (zebrafish) as well known Notch model system (Fig. 4A, B ). We injected mRNA encoding for N1ΔE wt or NNAA mutant in one-cell-stage zebrafish embryos together with a Notch-dependent GFP reporter plasmid (12x CSLRE-EGFP). In control (vector only) embryos Notch-driven gene expression is on background levels. Wild-type (wt) N1ΔE but not the NNAA mutant is able to drive the expression of the Notch reporter (EGFP) (Fig. 4A ). In line with this, the number of malformed embryos was much higher in presence of the N1ΔE wt compared to the hydroxylation-deficient NICD1 NNAA mutant (Fig. 4B ). Together, these data support the notion that the hydroxylation-defective NICD1 is functionally impaired in vivo in Notch-dependent neurogenesis in D. rerio. \n\nFIH-mediated NICD1 hydroxylation regulates its stability using a ubiquitination-dependent mechanism Ubiquitination of NICD1 is pivotal to limit Notch responses and is guided by other NICD1 PTMs [11, 32, 33, 41, 42]. Therefore, we investigated whether NICD1 hydroxylation is coupled to ubiquitination. To this end, we used tandem ubiquitin-binding entities (TUBEs) assays [43] to assess ubiquitination patterns of wild-type and NNAA-mutated NICD1. We observed that the NICD1 NNAA mutant shows increased ubiquitination compared to its wild-type counterpart in Phoenix TM cells (Fig. 5A, compare lane 3 to 2). Similarly, we observed an increase in ubiquitination of the endogenous NICD1 upon DMOG treatment of Beko cells (Fig. 5B ). Interestingly, the NICD1 ubiquitination was also increased in the NICD1 NNAA mutants that lack the destabilizing OPA-PEST domain in Phoenix TM cells (Fig. 5C, compare lane 7 with 9 and lane 8 with 10), suggesting that there is additional ubiquitination beside of degradative K48-linked ubiquitination that is known to occur within the PEST domain [44, 45]. While K48-linked ubiquitination plays key roles in proteasomal degradation, ubiquitination through K11 or K63 has important signaling functions [46]. To further unravel the link between NICD1 hydroxylation and ubiquitination, we used ubiquitin (Ub) mutants in which specific lysines were mutated to arginines (K11R, K48R or K63R) or mutants in which all the lysines with the exception of a single one were mutated to arginines (K11 only, K48 only and K63 only). In Phoenix TM cells, we observed that the increased ubiquitination of the NICD1 NNAA mutant in comparison to the NICD1 wt was abolished with the K63R Ub mutant (Fig. S9A, compare lane 13 with 12) and slightly reduced with the K11R Ub mutant (Fig. S9A, compare lane 15 with 12) but hardly influenced in presence of the K48R mutant (Fig. S9A, compare lane 14 with 12). In addition, in presence of Ub K63 only, the ubiquitination of the NICD1 NNAA was retained even if slightly reduced (Fig. S9B, compare lane 13 with 12). In presence of the Ub K48 only, the ubiquitination of the NICD1 NNAA mutant was almost completely abolished (Fig. S9B, compare lane 14 with 12) even if still higher than the one of the NICD1 wt in presence of the Ub K48 only (Fig. S9B, compare lane 14 with 10). In presence of the Ub K11 only, the ubiquitination of the NICD1 NNAA mutant was strongly reduced (Fig. S9B, compare lane 15 with 12) but higher than the one of the NICD1 wt in presence of the Ub K11 only (Fig. S9B, compare lane 15 with 11). Altogether, these data suggest that NICD1 hydroxylation prevents its ubiquitination and that the increased ubiquitination of the NICD1 protein is mainly occurring through Ub K63 and to a minor but still significant extent to Ub K11 while there is a minimal increase through Ub K48.",
"section_name": "Characterization of the FIH-mediated asparaginyl hydroxylation of the NICD1",
"section_num": null
},
{
"section_content": "To further explore the link between NICD1 hydroxylation and ubiquitination, we focused on the E3 ubiquitin ligase FBXW7, which targets NICD1 for proteasomal degradation [42, 44]. We hypothesized that NICD1 hydroxylation affects the NICD1/FBXW7 interaction and hence impairs NICD1 ubiquitination. We performed co-immunoprecipitation experiments in Phoenix TM cells co-expressing FBXW7 as well as Flag-tagged NICD1 wild-type (wt) or NNAA mutant (Fig. S10A ). We observed no major changes in the interaction between FBXW7 and NICD1 NNAA when compared to the NICD1 wild-type (Fig. S10A, compare lane 5 with 6), suggesting that the increased NICD1 ubiquitination is not due to enhanced FBXW7 recruitment. Protein ubiquitination is reversed by deubiquitinases (DUBs), which cleave ubiquitin (chains) from their substrates. USP10 has been shown to regulate NICD1 ubiquitination [47]. Of note, we found in our RNA-Seq analysis that the expression of the USP10-encoding gene is downregulated in hypoxic or DMOG-treated Beko cells (Table S2 ). Similar results were obtained at the protein level in Beko cells (Fig. 6A ). However, no expression changes were noted for FBXW7 in Beko cells (Fig. S10B, C ). Furthermore, we observed that USP10 interacted less efficiently with NICD1 NNAA than with the NICD1 wild-type in Phoenix TM cells (Fig. 6B, compare lanes 5 to 6), suggesting that the hypoxia-induced changes in NICD1 ubiquitination and stability result from impaired deubiquitination. These data suggest a model, whereby FIH-mediated hydroxylation determines the ability of NICD1 to interact with USP10, a DUB, whose expression is downregulated by hypoxia. In addition, we observed that Usp10 knockdown in Beko cells leads to reduced NICD1 protein levels (Fig. 6C left) and to reduced expression of Ptcra, Uaca, and Il2ra Notch target genes (Fig. 6C middle). In line with that, GSEA analysis upon Usp10 knockdown in Beko cells followed by RNA-Seq (Fig. S11A and Tables S1, S2 ) demonstrated that genes linked to the \"Notch signaling pathway\" were enriched (Fig. S11B and Table S9 ). In addition, a general downregulation of the Notch response was observed when looking at bona fide Notch target genes in our RNA-Seq analysis (Fig. 6C right, S11C, and Tables S1, S2 ). \n\nTogether, our data strongly suggest that USP10 stabilizes NICD1 in Beko cells through a hydroxylation-sensitive mechanism.",
"section_name": "The interplay between NICD1 hydroxylation and ubiquitination involves the deubiquitinase USP10",
"section_num": null
},
{
"section_content": "Regarding the interplay between Notch signaling hypoxia, we have uncovered that downregulation of Notch target genes is due to reduced protein stability. In normoxic conditions, FIH hydroxylates the NICD1 affecting ubiquitination regulated by E3 ubiquitin ligase FBXW7 and deubiquitinase USP10 (see also model in Fig. S12 ). Upon hypoxia, NICD1 hydroxylation ceases and the USP10 protein level is reduced, leading to perturbed NICD1 ubiquitination and subsequent downregulation of Notch target genes. \n\nSeemingly contradictory to our data, several other studies have suggested hypoxia as an inducer of the Notch signaling pathway [20] [21] [22] [23] [24] [25] [26] [27] [28]. However, these studies focused only on the Hairy Enhancer of Split family of genes as a readout of Notch activation, which have later been shown to be also regulated by HIF1α and other signaling pathways [12, 26, 28]. For example, it was shown that mutations of the RBPJ binding motif do not prevent the hypoxia-mediated induction of Hes1 [28], supporting the notion that this upregulation is Notch-independent. In line with this, we observe the binding of HIF1α upon hypoxia at Hes1 and Hey1. Thus, taking a genome-wide approach we disentangle RBPJ/NICD from HIF1α-regulated target genes. \n\nMechanistically, NICD1 protein stability is affected by hypoxia and postulates a link between NICD1 hydroxylation and ubiquitination. We propose that not only the hypoxia-regulated enzyme FIH but also the deubiquitinase USP10 play a key role in this process resulting in a shifted balance to enhanced NICD1 ubiquitination, in particular, regulatory ubiquitination (see also model in Fig. S12 ). According to our model, NICD1 hydroxylation provides a docking site for USP10, which in turn deubiquitinates NICD1. \n\nIt remains to be determined which E3 ubiquitin ligase counteracts USP10. Apart from FBXW7, known to control degradative ubiquitination, possible candidates are ITCH, MDM2, and/or RNF8, that have been previously linked to NOTCH ubiquitination [48] [49] [50] [51]. \n\nOur data reveal that hypoxia affects not only HIF1α but also the NICD1 coactivator. In our view, this has both physiological and potentially also pathophysiological implications. Physiologically, NOTCH1 is essential in early T-cell development in the thymus [52, 53], which is known to be highly proliferative with low O 2 tension [54, 55]. In our view, this hypoxic condition could contribute to the modulation of Notch responses, which is pivotal for subsequent T-cell maturation. Our results showing reduced expression of Ptcra and Il2ra (encoding for CD25) support this hypothesis. Regarding the pathophysiological context, our findings are particularly relevant to Notch-driven leukemias, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), for which NOTCH1 mutations been previously described [56, 57]. They could also play a role in the setting of solid cancer such as breast cancer and squamous cell carcinoma, where active Notch signaling has been described [4, 9, 58]. Future genome-wide analyses should not only focus on HIF1α but also on Notch-dependent gene regulation. \n\nTogether, our data suggest that NICD1 hydroxylation determines not only the strength but also the duration of Notch responses through an FIH-and USP10-dependent mechanism.",
"section_name": "DISCUSSIONS",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "We are grateful to T. Schmidt-Wöll for excellent technical assistance. The authors wish to acknowledge Centro de Análisis Genómico (CNAG-CRG), Spain, for sequencing the RNA-Seq samples. The authors wish to thank Tobias Zimmermann ( University of Giessen, Germany ) for giving access to the PETRA package and Prof. Dr. L. Schmitz ( University of Giessen, Germany ) for providing reagents.",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation )- TRR81-A12 and BO 1639/9-1, the Behring-Röntgen foundation and Excellence Cluster for Cardio-Pulmonary System (ECCPS) in Giessen to TB. Funding for the open access charge was provided by the DFG collaborative research TRR81. This work was further supported by the DFG ( JU2859/2-1 ) to SJ, ( SFB1074/A3, GRK2254/C4, and OS 287/4-1 ), the Deutsche Krebshilfe (# 70114289 ) to FO, (# 70114291 ) to DM, and the German Federal Ministry of Education and Research (BMBF) (e:Med-SYMBOL-HF grant # 01ZX1407A ) to SJ. LP is a participating member of the International Graduate School in Molecular Medicine at Ulm University (IGradU ), which is supported by the DFG (grant number GSC 270 ). BDG is supported by a research grant from the University Medical Center Giessen and Marburg (UKGM) and by a Prize from the Justus Liebig University Giessen.",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "",
"section_name": "DATA AVAILABILITY",
"section_num": null
},
{
"section_content": "ChIP-Seq and RNA-Seq developed in the current study have been deposited at GEO under the accession number GSE194003. Detailed Materials and Methods are available in the supplement file.",
"section_name": "DATA AVAILABILITY",
"section_num": null
},
{
"section_content": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "FF, BDG, LP, and FO performed experiments and analyzed data. BDG, TF, and MB performed the bioinformatic analysis. TS and MP provided reagents. DM and SK generated the HEK293 cells depleted of the NOTCH1 gene. BMG and SJ performed the in vivo experiments. FF, BDG, and TB designed experiments and wrote the manuscript with contributions from other authors.",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The authors declare no competing interests.",
"section_name": "COMPETING INTERESTS",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi. org/10. 1038/s41419-022-05052-9. \n\nCorrespondence and requests for materials should be addressed to Tilman Borggrefe. \n\nReprints and permission information is available at http://www. nature. com/ reprints Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "ADDITIONAL INFORMATION Supplementary information",
"section_num": null
}
] |
10.1371/journal.pone.0046627
|
A Novel Anticancer Therapy That Simultaneously Targets Aberrant p53 and Notch Activities in Tumors
|
Notch signaling pathway plays an important role in tumorigenesis by maintaining the activity of self-renewal of cancer stem cells, and therefore, it is hypothesized that interference of Notch signaling may inhibit tumor formation and progression. H101 is a recombinant oncolytic adenovirus that is cytolytic in cells lacking intact p53, but it is unable to eradicate caner stem cells. In this study, we tested a new strategy of tumor gene therapy by combining a Notch1-siRNA with H101 oncolytic adenovirus. In HeLa-S3 tumor cells, the combined therapy blocked the Notch pathway and induced apoptosis in tumors that are p53-inactive. In nude mice bearing xenograft tumors derived from HeLa-S3 cells, the combination of H101/Notch1-siRNA therapies inhibited tumor growth. Moreover, Notch1-siRNA increased Hexon gene expression at both the transcriptional and the translational levels, and promoted H101 replication in tumors, thereby enhancing the oncolytic activity of H101. These data demonstrate the feasibility to combine H101 p53-targted oncolysis and anti-Notch siRNA activities as a novel anti-cancer therapy.
|
[
{
"section_content": "Most forms of cancer chemotherapy are unable to eradicate all malignant cells, and they often are highly toxic because of their lack of selectivity to cancer cells. As a result, new efforts have focused on developing interventions that include tumor-specific replicating viruses and siRNA. \n\nA virus-based strategy takes advantage of the fact that the intracellular replication and production of adenoviral progeny requires the cell cycle gatekeeper p53 to be in an inactive status, and in many tumors, p53 is either mutated or epigenetically silenced. The viral early gene E1B, which encodes a 55-kDa protein (E1B 55K), is essential to viral replication. E1B interacts with cellular p53 and inactivates it to allow viral replication. ONYX-015, a modified adenovirus lacking the E1B 55K gene, can only replicate and lyse tumor cells that have inactivated p53, sparing the normal cells that retain wild-type p53 function [1]. Clinical trials in patients with recurrent head and neck cancer, metastatic colorectal cancer, or pancreatic cancer have shown that ONYX-015, when used alone or in combination with chemotherapy, is safe and has significant antitumor activity in a subset of patients [2, 3, 4]. \n\nIn China, an oncolytic adenovirus called H101 has been clinically approved for the treatment of several malignancies [5]. This virus selectively infects and kills only those cells that lack active p53 viral oncolysis because the viral proteins E1B and E3 are deleted [6]. Without E1B to inactivate p53, this H101 adenovirus cannot replicate and lyse normal cells where p53 is active. In addition, the deletion of a 78. 3-85. 8 mm gene segment in the E3 region, which encodes the adenovirus death protein, may enhance the safety of the product [5]. However, H101 has limited efficacy as monotherapy in clinical practice. In order to increase its effectiveness, it is often combined with radiotherapy or chemotherapy. \n\nNotch signaling plays a pivotal role in cellular differentiation, proliferation, and apoptosis [7]. The Notch proteins constitute a family of transmembrane proteins that form heterodimeric transmembrane receptors. Following ligand binding, the receptor catalyzes the cleavage of its own intracellular domain (ICN), which can then enter the nucleus to regulate target genes involved in regulating cell growth, cell differentiation and cell apoptosis [8, 9]. \n\nThe Notch signaling pathway is disrupted in several malignancies, offering a potential target for therapeutic intervention. There is aberrant activation of Notch signaling in glioblastoma (GBM) cell lines and in human GBM-derived neurospheres. Inhibition of Notch signaling via the expression of a dominant negative form of the Notch co-activator, mastermind-like 1 (DN-MAML1) or the treatment of an c-secretase inhibitor (GSI) MRK-003 resulted in a significant reduction in GBM cell growth in vitro and in vivo [10]. While there is abundant evidence that Notch signaling can stimulate the growth of wide range of tumors, the precise molecular mechanisms underlying alterations of this pathway during carcinogenesis are yet to be identified. \n\nNotch is also critical in maintaining the ability of cancer stem cells (CSCs to self renew) (see reviews [11, 12, 13] ). CSCs are a subpopulation of tumor cells that possess stem cell properties, including indefinite self-replication, pluripotency, and resistance to chemotherapeutic agents. CSCs are associated with tumor relapse and metastasis, and may also account for the ultimate failure of conventional cancer therapies [14, 15]. Cancer cures may require the complete elimination of the small cancer stem cell population of the tumor as well as of the non-CSC majority of cancer cells. Consequently, the idea of selectively targeting CSCs with novel therapeutics, e. g. those attacking Notch signal pathway, is gaining considerable interest. \n\nCervical cancer cell line Hela-S3 was deficient in p53, and preclinical studies demonstrated that Hela-S3 was very sensitive to H101 oncolytic treatment. We have previously shown that knockdown of the Notch 1 gene could inhibit the proliferation and growth of HeLa cells both in vitro and in vivo [16]. In this study, we test a dual therapeutic approach by simultaneously targeting p53 mutations and aberrant Notch signal activity in tumors. To accomplish this, we combined a Notch1 siRNA with the oncolytic adenovirus H101. It is assumed that H101 replication specifically lyses the bulk of cancer cells that are p53-inactive. At the same time, Notch1 siRNA targets both the Notch-pathway mutated tumors and the minority CSC population of the tumor. As a first step to prove this concept, in this communication we report the in vitro and in vivo therapeutic effects of H101/Notch1-siRNA combined therapy in HeLa-S3 tumor cells.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Among the Notch family genes, Notch1 is the best validated target in malignancies, with the highest activating mutations identified in tumors. Our previous in vitro and in vivo studies demonstrated that knockdown of the Notch 1 gene inhibited the proliferation and growth of HeLa cells. We examined the expression of the Notch family genes in HeLa-S3 cells that lack the activity of p53. Using RT-PCR, we found that Notch1 was expressed in HeLa-S3 cells, while other three family members Notch2, Notch3, and Notch4 were barely detectable (Fig. S1 ). We thus focused on the well-validated Notch1 in this study. \n\nWe then tested the suppression of Notch1 by its siRNA in HeLa-S3 tumor cells. Notch1-siRNA and control NC-siRNA were used to transfect HeLa-S3 cells, respectively, and the efficiency of siRNA on Notch1 expression was examined by RT-PCR and Western blot. As expected, no change in the abundance of Notch1 mRNA was detected in the H101 group. As compared with the NC-siRNA control, Notch1-siRNA, used either alone or with H101, suppressed Notch1 expression (Fig. 1A ). Suppression of Notch1 by Nocth1-siRNA was also confirmed at the protein level by Western blot analysis. (Fig. 1B ).",
"section_name": "Suppression of Notch1 by siRNA in Tumor Cells",
"section_num": null
},
{
"section_content": "Having established that Notch1-siRNA inhibited Notch1 expression, we used the MTT method to detect the effects of combined treatment of Nocth1-siRNA and H101 on cell growth (Fig. 2 ). In the siNotch1 group, a significant degree of proliferative inhibition was observed after 72 hours, suggesting that RNA interference blocked the endogenous Notch pathway and showed a delayed inhibition of cell growth. Similarly, inhibition of cell growth was detected 72 hours after cells were infected with H101 virus (MOI 100). In the combined treatment group, however, cell growth was significantly inhibited as early as 48 hours after the treatment, indicating an augmentation of growth inhibition. Similar data were also obtained in other tumor cell lines A549, OCM1 and VUP (Fig. S2 ). We used the normal cervical keratinocytes as the control. The data showed that the cell prolifercation was unaffected by H101 treatment (MOI = 100) in normal cervical keratinocytes (Fig. S3 ).",
"section_name": "Enhanced Cytotoxicity by the Combined Treatment of Notch1-siRNA and H101",
"section_num": null
},
{
"section_content": "To examine whether the enhanced in vitro tumor cytotoxicity could be translated into in vivo animal testing, we first initiated a pilot study by treating animals when HeLa-S3 tumor xenografts reached 200-300 mm 3. Based on the data from this pilot study, we modified the protocol by initiating the treatment at an early stage when the average tumor volume reached about 100 mm 3. At this stage, animals began to receive an intra-tumor injection of Notch1-siRNA, H101 or PBS every three days, for a total of four injections. \n\nAs compared with the PBS control group, the Notch1-siRNA or H101 monotherapy showed similar inhibition of tumor growth. The Notch1-siRNA/H101 group, however, had an enhanced anti-tumor effect (Fig. 3A ). Marked differences were seen in the degrees of inflammation and necrosis in the tumor specimens. Tumors from the H101-Notch1-siRNA treated group were more differentiated than those from the PBS treated group (Fig. 3B ).",
"section_name": "Improved in vivo Antitumor Activity by the Combined Treatment of Notch1-siRNA and H101",
"section_num": null
},
{
"section_content": "To examine the mechanism underlying the augment of antitumor effect by the combined Notch1-siRNA/H101 treatment, cell apoptosis was measured using an Annexin V-FITC apoptosis kit and flow cytometric analysis 48 hours after the cells were transfected with Notch1-siRNA and H101. As seen in Figure 4, the combined treatment of Notch1-siRNA and H101 induced 20. 7% apoptosis in treated cells as compared with 10. 9% in Notch1-siRNA-treated cells and 9. 6% in H101-treated cells. These data suggest an augment effect of Notch1-siRNA and H101 in inducing tumor apoptosis.",
"section_name": "Enhanced Apoptosis by the Combined Treatment of Notch1-siRNA and H101",
"section_num": null
},
{
"section_content": "We then used Western blot analysis to detect the activity of caspase-3, a critical component in cell apoptosis pathway. We found that the expression of caspase-3 did not change significantly among the treated groups (Fig. 5A, middle panel). Neither did we detect a significant amount of the cleaved caspase-3 (active form) in treated tumor cells. Using a more sensitive Caspase-3 Colorimetric Activity Assay Kit, we still could not detect a significant change of caspase-3 in the experimental groups (Fig. S4 ), suggesting that the combined therapy enhanced tumor apoptosis by a non-caspase-3 pathway. \n\nWe were also curious whether the combined Notch1-siRNA/ H101 therapy would affect the expression of endogenous p53, an important component that affect H101 oncolysis and apoptosis. Three days after transfection with Notch1-siRNA and H101, HeLa-S3 cells were collected and total cellular protein was extracted. We found that the treatment of Notch1-siRNA, whether used alone or combined with H101, did not have significant effect on p53 protein level in treated cells (Fig. 5A, top panel). \n\nWe then used Western blot analysis to examine the expression of MDM2, a downstream target gene of p53. Three days after transfection with Notch1-siRNA and H101, both the H101 treatment and the combined H101/Notch-siRNA treatment significantly affected MDM2 protein expression in treated cells. Although the H101/siRNA combined therapy showed a slightly better effect than the H101/siNC control (Fig. 5B ), it seems that the downregulation of MDM2 was primarily derived from the H101 treatment. We also used the Western blot to measure the expression of p21, another p53 target gene. Similarly, we only detected a low level of p21 protein in the siNotch1 group (Fig. S5 ), indicating that the p53/p21 pathway may not be a significant factor in cell apoptosis induced by the combined therapy.",
"section_name": "Activation of Caspase-3 and Expression of Endogenous p53 and MDM2 after Combined Treatment of Notch1-siRNA and H101",
"section_num": null
},
{
"section_content": "We then focused on whether the Notch-siRNA alters viral replication in H101-infected tumors. Hexon protein is a component of the adenovirus capsid and is synthesized after cell infection. The synthesis of hexon protein marks the packaging of virus particles in the final replication stage. Thus, the amount of protein synthesized is considered to be a reliable indicator of viral replication. \n\nTo test whether Notch1-siRNA would affect H101 DNA replication, the expression of the late gene hexon was determined by real-time RT-PCR. We found that after Notch1-siRNA interference, the H101/Notch1-siRNA group had a significant increase of hexon mRNA expression compared with the H101 group (P,0. 05, Fig. 6A ). Similarly, Western blot analysis also showed an approximately two-fold increase in Hexon protein when the combination of Notch1-siRNA and H101 were used (P,0. 05, Fig. 6B ). Taken together, these data suggest that the silencing of Notch1 actually enhanced DNA synthesis of H101.",
"section_name": "Notch1-siRNA Enhanced H101 DNA Replication",
"section_num": null
},
{
"section_content": "The Notch signaling pathway plays an important role in the regulation of cell growth and differentiation, tissue renewal, and cell homeostasis, and the pathway may be disregulated in several carcinomas [17, 18]. Notch1 antisense RNA treatment may lead to growth inhibition and even cell death if stably transfected in cervical cancer cells [19]. Notch signaling promotes cell survival, and the increment in Notch1 activity promotes tumor growth in lung adenocarcinoma when cultured under hypoxic conditions [20]. Synthetic triterpenoids inhibit growth and induce apoptosis in human glioblastoma and neuroblastoma cells through inhibition of Notch signaling [10, 21]. Notch1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network promoting leukemic cell growth [22]. Down-regulation of Notch-1 contributes to cell growth inhibition and apoptosis in pancreatic cancer cells, including BxPC-3, HPAC, and PANC-1 [23]. Studies have demonstrated the existence of a novel intracellular mechanism for Notch1 regulation mediated by DDR1, in which deregulated DDR1 activation results in persistent autonomous activation of a Notch signaling and subsequent induction of Notch-dependent pro-survival neoplastics [24]. Our previous study demonstrates that blocking Notch1 signaling by RNA interference can induce growth inhibition in HeLa cells [16]. Furthermore, Notch is involved in the maintenance of self-renewal of cancer stem cells (CSCs) [11, 12, 13], contributing to tumor relapse, metastasis, and drug resistance [14, 15]. Thus, the Notch signaling pathway is a promising target for the development of new anti-cancer therapeutics. \n\nThere is also a functional link between Notch and p53 activities. Notch1 is a p53 target gene involved in human keratinocyte tumor suppression through negative regulation of ROCK1/2 and MRCK kinases [25]. Notch1 is induced upon p53-dependent UVB exposure in skin cells [25, 26]. p53 is a tumor suppressor gene that is often mutated in tumors [27]. Restoration of p53 expression in a human cancer cell line up-regulates the expression of Notch1 [28]. Interference of p53 activity by either the E6 protein of human papillomavirus or p53 siRNA leads to a reduction of Notch1 at the transcriptional level in cervical cancer cells [29]. In this communication, we tested combined therapy of Notch1 siRNA with a p53-targeted oncolytic adenovirus H101, in order to target two common abnormalities in cancer cells. We demonstrated the augmented tumor-killing of the combined therapy both in vitro and in vivo, confirming the feasibility of this combined modality for future studies. \n\nH101, which lacks E1B55-kDa, can specifically lyse tumor cells. However, H101 has limited potential to eradicate tumors when used as monotherapy. Thus, H101 is often used in combination with traditional modalities, such as chemotherapy. In this communication, we studied the antitumor efficacy of H101 in conjunction with siRNA to Notch1. As demonstrated in Figure 1, Notch1-siRNA efficiently inhibited the expression of Notch1 mRNA and protein. Interestingly, the RNAi activity was not affected by the infection of oncolytic adenovirus H101. The combined action of Notch1 knockdown and H101 oncolysis significantly inhibited tumor growth in vitro, suggesting an additional effect of the combined tumor therapy. In the animal studies, we used direct intratumoral injections of high concentrations of Notch1-siRNA to increase the efficiency of intracellular transport of siRNA in the animal models. Direct intratumoral injection of H101 suspension also showed significant inhibition of growth in the nude mouse tumor model. Compared with monotherapy with either agent, the combined treatment of Notch-siRNA and H101 showed better tumor inhibition and prolonged the survival of animals bearing the tumor. \n\nWe also tested this combined therapy in other three tumor cell lines that had different status of p53 mutations, including lung cancer cells (A549) and uveal melanoma cells (OCM1 and VUP). Both OCM1 and VUP cell lines contain a common mutation (C. 797G. A, P. Gly133Glu) in the exon 7 of p53 [30], thus serving as a ideal therapeutic target for H101. However, we also noticed that A549, a cell line known to harbor a wild type p53, also responded to the H101 monotherapy (Fig. S2 ). Other two groups [31, 32] also reported that a second oncolytic adenovirus ONYX-015 was also able to replicate in A549 cells. Theoretically, the mutant virus with the deleted E1B, like H101, is able to replicate only in p53defifcient cells. The molecular basis for such p53 status-independent effect of H101 in certain tumor cells, like A549, remains to be determined [31]. We also examined the cytotoxic effect of this combined therapy on cancer stem cells (CSCs). We first infected HeLa S3 cells with H101 and/or siNotch1. After 24 hours, cancer cells were cultured in CSC sphere culture medium and CSC spear numbers were recorded [24]. Because CSCs in HeLa-S3 cells were very low, we observed only a few CSC spears in control cells, but none in the group treated with the combined therapy. An ongoing study is under the way to isolate CSCs first and then to treat them with H101 and/or siNotch1. \n\nThe mechanism underlying the additive effect of the combined therapy remains uncharacterized. In tumor cells treated by the combined Notch1-siRNA and H101, we found an enhanced induction of apoptosis in HeLa-S3 cells, but we did not detect significant alteration of caspase-3 or activated caspase-3 expression. Neither Notch1-siRNA nor H101 appears to induce apoptosis by a non-caspase-3 pathway, and neither agent alters p53 expression. We also examined the expression of MDM2 and p21, which are the downstream targets of p53. MDM2 is an E3 ubiquitin ligase that targets p53 for ubiquitination and degradation. Both the MDM2 C-terminal region including the RING finger and the acidic domain are essential for p53 ubiquitination [33, 34]. MDM2 ablation in mice results in early embryonic lethality due to elevated levels of p53-induced apoptosis, and this phenotype is reversed by the simultaneous deletion of p53, demonstrating the importance of MDM2 in suppressing p53 [35, 36]. In our study, we did not detect a significant alteration of MDM2 from the Notch1-siRNA therapy. Similarly, no significant changes were noticed for p21, a second p53 target gene, in the combined therapy group. \n\nNumerous studies have shown that the efficiency of adenovirus infection is related to the tumor cell surface receptor CAR. However, the expression of CAR in some tumor cells is relatively low. Extensive studies have attempted to alter viral tropism to increase infection rates and improve the anti-cancer effect [37, 38]. Adenovirus group C that lacks E1B 55-kDa protein is replicable, and its replication efficiency is also related to the expression of p53 in host cells [39]. E1B55-kDa protein affects oncolysis of adenovirus by several mechanisms, including the inhibition of p53 and pRB expression, regulation of the RNA output, turningoff of host protein synthesis, release of E2F, and inhibition of apoptosis [40, 41]. Interestingly, we found that Notch1 knockdown increased the amplification of the adenovirus as measured by late gene Hexon protein. Thus, promotion of viral replication by Notch1-siRNA may partially explain the increased antitumor efficacy in our combined therapeutic approach. \n\nIn an attempt to improve H101 anti-tumor efficacy, we previously demonstrated that H101 therapy was potentiated by concomitant use of a Bcl2 siRNA. In mice bearing human xenograft tumors, all treated animals survived, and that some animals were tumor-free following the combined therapy [42]. In this extended study, we targeted the Notch signaling pathway that plays an important role in tumorigenesis by maintaining the activity of self-renewal of cancer stem cells. We hypothesized that interference of Notch signaling may inhibit tumor formation and progression by cutting off the source for cancer stem cells, therefore enhancing the therapeutic efficacy of oncolytic H101. It would be interesting in future studies to examine whether these strategies can be combined to offer a ''three punch'' approach by targeting p53 deficiency, Bcl2 overexpression, and cancer stem cell. \n\nIn summary, this study provides support for the combined use of an oncolytic adenovirus and Notch1-siRNA as a promising approach in cancer gene therapy. We demonstrated an anticancer augmentation of the combined therapy of Notch1-siRNA and H101. Future studies will combine two therapies as a single adenoviral agent by integrating Notch1-siRNA into the H101 viral backbone. In addition, it will be interesting to examine whether the strategy used here would be more potent to target cancer stem cells (CSCs), particularly those CSCs cultured from clinical surgery or biopsy tumors.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Cervical cancer cell line HeLa-S3 is deficient in p53, and preclinical studies demonstrated that HeLa-S3 was very sensitive to H101 oncolytic treatment [43]. Our previous in vitro and in vivo studies also demonstrated that knockdown of the Notch 1 gene inhibited the proliferation and growth of HeLa cells [16]. We thus tested our combined p53 and Notch therapy in this p53-deficent cervical cancer cell line. In addition, three tumor cell lines with different status of p53 mutations, including lung cancer A549 (wild type p53), uveal melanoma OCM1 and VUP (mutated p53), were also used for the study. \n\nTumor cell lines HeLa-S3 (cervical cancer) and A549 (lung cancer cells) were obtained from American Type Culture Collection (Manassas, VA, USA). OCM1 and VUP (uveal melanoma were kindly provided by Professor John F. Marshall (Tumor Biology Laboratory, Cancer Research UK Clinical Center, John Vane Science Centre, London, UK) [44]. HeLa-S3 cells were cultured at 37uC under 5% CO 2 in Dulbecco's modified Eagle's medium (Gibco, Carlsbad, CA, USA) supplemented with 10% newborn calf serum (PAA Laboratories GmbH, Co ¨lbe, Germany). Recombinant adenovirus H101 was kindly provided as a gift by Shanghai Sunway Biotech (Sunwaybio, Shanghai, China) and was maintained under conditions recommended by the manufacturer.",
"section_name": "Cell Culture and Recombinant Adenovirus H101",
"section_num": null
},
{
"section_content": "The Notch1 siRNA duplexes were produced by Shanghai Genepharma Co. Inc. (Genepharma, Shanghai, China) against human Notch1 (59-AAG GUG UCU UCC AGA UCC UGA-39). Scrambled fluorescent-labeled siRNA (59-AAA UGU GUG UAC GUC UCC UCC-39) (siNC) [30] was also designed and used as the negative control in the study.",
"section_name": "RNA Interference",
"section_num": null
},
{
"section_content": "HeLa-S3 cells at 30-50% confluence in 24-well plates were transfected with 80 nmol/l Notch1 siRNA using Lipofectamine 2000 following the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). After overnight incubation, cells were infected with H101 at a multiplicity of infection of 100 MOI. Control groups included cells that were transfected with siNC or PBS.",
"section_name": "Co-treatment of Tumor Cells with Notch1 siRNA and Oncolytic Adenovirus H101",
"section_num": null
},
{
"section_content": "Total RNA was isolated from treated cells using Trizol reagent (Invitrogen) following the protocol provided by the manufacturer. Total RNA (1mg) was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen) [45, 46]. The cDNA was used to amplify the Notch1 fragments. For normalization of RNA, the housekeeping gene b-actin was also amplified from each sample. The primer sequences were as follows: Notch1 (forward primer: 59-TTCCCTGAGGGCTTCAAAGT-39, reverse primer: 59-CCCGCTACTCACGCTCTGAT-39, 522 bp), Notch2 (forward primer: 59-TTGCTGTTGCTGTTGTCATCA-39, reverse primer: 59-AAGGTGCTGCTGTGTCCAT-39, 338 bp), Notch3 (forward primer: 59-CTGTCTTGCTGCTGGTCATTC-39, reverse primer: 59-GTGTCATCTGCCTCATCCTCT-39, 496 bp), Notch4 (forward primer: 59-TGCTGCTGCTGCTGCTAT-39, reverse primer: 59-CTGCTCACCTGTCCATCCA-39, 428 bp ), GAPDH (forward primer: 59-GGATTTGGTGGTATTGGG-39, reverse primer: 59-GGAAGATGGTGATGGGATT-39, 428 bp) and b-actin (forward primer: 59-CCTTCCTGGGCATG-GAGTCCT-39, reverse primer: 59-GGAGCAATGATCTT-GATCTT-39, 202 bp). RT-PCR amplification was performed using the following conditions: 95uC for 5 min, 1 cycle; 94uC for 45 sec, 56uC for 45 sec and 72uC for 45 sec, 30 cycles. After amplification, 10 ml of PCR product was run on a 1. 5% agarose gel and visualized by ethidium bromide staining. \n\nQuantitative real-time RT-PCR amplification was carried out using Real-Time MIX (SYBR Premix Ex TaqTM, TaKaRa, Tokyo, Japan). Specifically, total RNA was extracted by Trizol reagent (Invitrogen), and cDNA was synthesized with RNA reverse transcriptase. The C T (threshold cycle) value of Hexon was quantitated by Q-PCR in triplicate using an ABI Prism 7900 HT sequence detector (AB Applied Biosciences, CA, USA) following the manufacturer's protocol and was normalized over the C T of the b-actin control [47, 48].",
"section_name": "RNA Extraction and Reverse Transcription-PCR Analysis",
"section_num": null
},
{
"section_content": "Cells were harvested at the indicated time, and proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis in 10% SDS-polyacrylamide Tris-glycine gels for protein expression. The immunoblotting was performed with the Notch1 (Epitomics, CA, USA), p53 (Cell Signaling Technology, Inc., Danvers, MA, USA), Caspase 3 (Thermo Fisher Scientific, Loughborough, UK), Hexon (Abcam, Cambriadge, UK) and mouse b-actin (Sigma-Aldrich, St. Louis, MO, USA), followed by detection with a horseradish peroxidase-conjugated secondary antibody [42].",
"section_name": "Western Blot Analysis",
"section_num": null
},
{
"section_content": "Cells were seeded at 2610 5 cells per well in flat-bottomed 6-well plates. At the end of the incubation time, the cells were harvested and the Caspase-3 Colorimetric Activity Assay Kit was used to detect the activity of caspase-3 [49].",
"section_name": "Caspase-3 Activity Assay",
"section_num": null
},
{
"section_content": "The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed to assess the effect of H101 in combination with RNA interference on cell proliferation [42]. Cells were seeded in 96-well plates at a concentration of 5610 3 cells/well. At the end of the incubation, 20 ml of 5 mg/ml MTT (Sigma-Aldrich) in PBS was added to each well. Each experiment was repeated five times. Absorbance was measured after a further 4 h incubation at 37uC with a solution of MTT (0. 33 mg/ml) that contained 12. 5mM Dimethyl sulfoxide (DMSO). The absorbance was measured on a spectrophotometer microplate reader at a wavelength of 492 nm.",
"section_name": "MTT Assay",
"section_num": null
},
{
"section_content": "Apoptosis was determined by dual staining with annexin-Vfluorescein isothiocyanate and propidium iodide and analyzed by flow cytometry [42]. Cells were prepared according to the manufacturer's instruction provided in the Annexin V-FITC apoptosis kit (BD Biosciences, San Diego, CA, USA). Apoptosis was quantified on a fluorescence-activated cell sorter (Becton Dickinson, Sunnyvale, CA), and data from 10,000 events were collected for further analysis.",
"section_name": "Apoptosis Analysis",
"section_num": null
},
{
"section_content": "Animal experiments were performed in accordance with institutional guidelines for animal care by Jiao Tong University. Specifically, HeLa-S3 cell tumor xenografts were established by s. c. injection of 1610 6 cells into the right flank of 4-6-week-old male athymic nude mice. Based on the data from a pilot study, we initiated an early treatment when the tumor volume reached about 100 mm 3 (volume = length6width 2 60. 5). Animals were randomly assigned into four groups. The Notch1-siRNA plus H101 group received intratumoral injections of 10 mg Notch1-siRNA on day 1, 4, 8, 11, 15, and H101 adenovirus at 1610 8 plague-forming units on day 2, 5, 9, 12, and 16. The Notch1-siRNA group received five intratumoral injections of 10 mg Notch1-siRNA. The H101 adenovirus group received five intratumoral injections of H101. The control group mice received five injections of PBS. The tumor size was measured by vernier calipers every 4 days. Mice from each group were selected randomly and killed on day 7 after treatment for hematoxylin-eosin staining. \n\nSpecimens were dehydrated in ethanol series (80, 85, 80, 90, 95 and 100%), embedded in paraffin, and cut into 5 mm-thick sections. Sections were then deparaffinized, stained with hematoxylin/eosin (H-E) following standard protocol, and observed using a light microscope.",
"section_name": "Tumor Xenograft Model in Nude Mice",
"section_num": null
},
{
"section_content": "All experiments were performed in triplicate, and the data were expressed as mean 6 SD. The data were analyzed with one-way analysis of variance (ANOVA), and results were considered statistically significant at P#0. 05.",
"section_name": "Statistical Analysis",
"section_num": null
}
] |
[
{
"section_content": "This study was supported by The National Key Program for Basic Research of China ( 2010CB529902 ), The National Natural Science Foundation of China ( 10979034 and 81001008 ) to G. Q. ; The Science and Technology Commission of Shanghai ( 10JC1409100 ), The Shanghai Leading Academic Discipline Project ( S30205 ) to S. G. ; National Institutes of Health grant ( 1R43 CA103553-01 ) and Department of Defense Grant ( W81XWH-04-1-0597 ) to J. F. H. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Supporting Information",
"section_num": null
}
] |
10.3324/haematol.2012.069369
|
Clinical implications of the molecular genetics of chronic lymphocytic leukemia
|
Genetics and molecular genetics have contributed to clarify the biological bases of the clinical heterogeneity of chronic lymphocytic leukemia. In recent years, our knowledge of the molecular genetics of chronic lymphocytic leukemia has significantly broadened, offering potential new clinical implications. Mutations of TP53 and ATM add prognostic information independently of fluorescence in situ hybridization cytogenetic stratification. In addition, next generation sequencing technologies have allowed previously unknown genomic alterations in chronic lymphocytic leukemia to be identified. Mutations of NOTCH1, SF3B1 and BIRC3 have been associated with short time to progression and survival. Each of these lesions recognizes a different distribution across different clinical phases and biological subgroups of the disease. The clinical implications of these molecular lesions are in some instances well established, such as in the case of patients with TP53 disruption, who should be considered for alternative therapies/allogeneic stem cell transplant upfront, or in patients with ATM disruption, who are candidates to rituximab-based immunochemotherapy. On the contrary, NOTCH1, SF3B1 and BIRC3 mutations appear to have a specific significance, the clinical value of which is currently being validated, i.e. association to Richter syndrome transformation for NOTCH1 mutations, and short progression-free survival after treatment for SF3B1 mutations. Certainly, these new lesions have helped clarify the molecular bases of chronic lymphocytic leukemia aggressiveness beside TP53 disruption. This review covers the recent advancements in our understanding of the molecular genetics of chronic lymphocytic leukemia and discusses how they are going to translate into clinical implications for patient management.
|
[
{
"section_content": "The clinical course of chronic lymphocytic leukemia (CLL) is extremely heterogeneous. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] The Rai and Binet clinical staging systems still remain the cornerstone for identifying CLL patients with advanced disease stages for whom treatment-free survival (TFS) and overall survival (OS) are usually short. 2, 5, 6 However, these staging systems do not provide risk stratification in early stage disease, that nowadays includes most cases of newly diagnosed CLL, and also fail to identify those patients who will develop chemorefractoriness. [11] [12] [13] [14] [15] Understanding CLL genetics may help clarify the molecular bases of the clinical heterogeneity of this leukemia. In the 1990s, Juliusson et al. 16 applied conventional karyotype banding analysis to systematically assess the prevalence and prognostic impact of chromosomal abnormalities associated with CLL. Despite its technological limitations, this approach revealed that more than half of CLL patients had clonal chromosomal changes. Importantly, this pivotal analysis indicated that chromosomal abnormalities affect CLL outcome and served as a proof of concept to document that genetic alterations in CLL may be prognostically relevant in a hierarchical order. 16 n 2000, this notion was unequivocally documented by the seminal study by Döhner et al. 17 that established interphase fluorescence in situ hybridization (FISH) analysis as a standard technique to evaluate cytogenetic lesions in CLL, detecting chromosomal abnormalities in over 80% of patients, thus overcoming the limited applicability and resolution of conventional karyotyping. By correlating FISH lesions with the course of the disease, a hierarchical model based on five risk categories was established. CLL cases harboring the 17p13 deletion independent of concomitant abnormalities (prevalence 7%) had the worst prognosis (median survival 32 months), followed by cases carrying the 11q22-q23 deletion (prevalence 18%, median survival 79 months), trisomy 12 (prevalence 16%, median survival 114 months), normal karyotype (prevalence 18%, median survival 111 months) and 13q14 deletion (prevalence 55%, median survival 133 months). 17 ytogenetic lesions, however, do not entirely explain the genetic basis of the clinical heterogeneity of CLL. Additional information has come from the detailed definition of the molecular correlates of CLL chromosomal aberrations. In fact, TP53, the tumor suppressor gene affected by 17p13 deletion, and ATM, the gene targeted by 11q22-q23 deletion, are not only deleted, but also recurrently mutated in CLL. ] [20] [21] [22] [23] [24] [25] In recent times, the improvements in next generation sequencing technologies have provided a novel opportunity to examine the CLL genome, and have allowed previously unknown genomic alterations to be identified, such as mutations of NOTCH1 (neurogenic locus notch homolog protein 1), SF3B1 (splicing factor 3B subunit 1) and BIRC3 (baculoviral",
"section_name": "Genetic heterogeneity of chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "Genetics and molecular genetics have contributed to clarify the biological bases of the clinical heterogeneity of chronic lymphocytic leukemia. In recent years, our knowledge of the molecular genetics of chronic lymphocytic leukemia has significantly broadened, offering potential new clinical implications. Mutations of TP53 and ATM add prognostic information independently of fluorescence in situ hybridization cytogenetic stratification. In addition, next generation sequencing technologies have allowed previously unknown genomic alterations in chronic lymphocytic leukemia to be identified. Mutations of NOTCH1, SF3B1 and BIRC3 have been associated with short time to progression and survival. Each of these lesions recognizes a different distribution across different clinical phases and biological subgroups of the disease. The clinical implications of these molecular lesions are in some instances well established, such as in the case of patients with TP53 disruption, who should be considered for alternative therapies/allogeneic stem cell transplant upfront, or in patients with ATM disruption, who are candidates to rituximab-based immunochemotherapy. On the contrary, NOTCH1, SF3B1 and BIRC3 mutations appear to have a specific significance, the clinical value of which is currently being validated, i. e. association to Richter syndrome transformation for NOTCH1 mutations, and short progression-free survival after treatment for SF3B1 mutations. Certainly, these new lesions have helped clarify the molecular bases of chronic lymphocytic leukemia aggressiveness beside TP53 disruption. This review covers the recent advancements in our understanding of the molecular genetics of chronic lymphocytic leukemia and discusses how they are going to translate into clinical implications for patient management.",
"section_name": "Clinical implications of the molecular genetics of chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "",
"section_name": "ABSTRACT",
"section_num": null
},
{
"section_content": "",
"section_name": "Pattern and distribution of genetic lesions affecting chronic lymphocytic leukemia outcome",
"section_num": null
},
{
"section_content": "Molecular defects of TP53 and ATM are well-established genetic lesions carrying clinical relevance in CLL. The tumor suppressor gene TP53 maps on the short arm of chromosome 17 (17p13) and codes for a central regulator of the DNA-damage-response pathway. 34 Activation of TP53 leads to cell-cycle arrest, DNA repair, apoptosis, or senescence via both transcription-dependent and transcriptional-independent activities. Consistently, TP53 plays a central role in mediating the pro-apoptotic and antiproliferative action of several DNA-damaging chemotherapeutic agents, including alkylators and purine analogs. 34 8] [19] [20] [21] Most cases with 17p13 deletion also carry TP53 mutations on the second allele (~70%), while the remaining cases have a monoallelic 17p13 deletion in the absence of TP53 mutations (~20%), or TP53 mutations in the absence of 17p13 deletion (~10%). 35 In line with the genetic instability associated with defective DNA-damage checkpoints, TP53 abnormalities frequently couple with complex cytogenetic abnormalities, particularly with unbalanced translocations. 21 t the molecular level, approximately 75% of all mutations are missense substitutions, while the remaining lesions (~25%) are represented by truncating events, including frameshift insertions or deletions, non-sense substitutions and splice site mutations. 35, 36 Most missense mutations are localized within exons 5-8, which encode the central DNA-binding domain of TP53, thus impairing DNA binding and target gene transactivation (Figure 1 ). 35 he ATM gene is a member of the phosphatidylinositol-3 kinase (PT3K) gene family and encodes a nuclear serine/threonine kinase whose activity is induced by chromosomal double-strand breaks that arise endogenously or after exposure to DNA-damaging agents, including ionizing radiations and chemotherapeutic drugs. 37 ATM protects the integrity of the genome by regulating the cellcycle arrest at G1/S and G2/M to prevent processing of damaged DNA, and by activating DNA-repair pathways and inducing apoptosis if the DNA damage cannot be repaired. Many of these effects are mediated by the activation of both TP53-dependent and TP53-independent cellular pathways. 37 TM is a large gene of 62 coding exons that maps on haematologica | 2013; 98(5)",
"section_name": "Molecular characteristics of clinically relevant genetic lesions of chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "chromosome 11q22-q23, which is a minimally common deleted region in CLL. As for TP53, the ATM gene in CLL may be inactivated by both deletion and/or somatic mutations. 22, 23 At the molecular level, ATM mutations consist in a mixture of missense substitutions distributed across the ATM coding sequence, with no hotspots. 22, 23 Only a proportion of ATM mutated CLL (10%-20%) show a concomitant 11q22-q23 deletion. 37] [38] [39] Insights into the pathogenicity of ATM mutations in CLL are provided by the observation that two recurrent mutations (p. R2691C and p. P2699S) localize within functionally relevant sites, including the ATP-binding pocket of ATM, and critically impair ATM kinase activity. 25 ATM mutants with an impaired kinase activity sequester ATM wild-type proteins with a dominant negative effect and inhibit their activation in response to physical and chemical insults. 25 ecent studies based on next generation sequencing have revealed new genes implicated in CLL and potentially carrying clinical relevance. NOTCH1 encodes a transmembrane protein that acts as a ligand-activated transcription factor and regulates multiple target genes, including MYC, TP53 and molecules of the NF-κB pathway. 40, 41 In 2009, it was shown that the constitutive NOTCH signaling activation was implicated in CLL cell survival and apoptosis resistance. In an attempt to identify the underlying mechanism, the first identification of the NOTCH1 PEST domain mutation in CLL patients was reported. 42, 43 t the molecular level, NOTCH1 mutations in CLL are mainly represented by frameshift or non-sense events clustering within exon 34, and including the highly recurrent c. 7544_7545delCT deletion (approx. 80%-95% of all mutations) (Figure 1 ). 26, 27, 31, 32 NOTCH1 mutations in CLL are selected to disrupt the C-terminal PEST domain of the protein, that is responsible for the proteosomal degradation of the activated form of NOTCH1. Indeed, truncation of the PEST domain is predicted to result in NOTCH1 impaired degradation, stabilization of the active NOTCH1, and deregulated NOTCH1 signaling. 44 onsistent with this notion, a number of cellular pathways, including those controlling cell metabolism and cell cycle progression, are deregulated in CLL harboring NOTCH1 mutations. NOTCH1 is preferentially targeted in specific biological groups of CLL. 27, 45 In fact, NOTCH1 mutations are significantly more common in CLL with unmutated immunoglobulin heavy variable (IGHV) genes and are enriched in CLL harboring +12, where they identify a distinct clinico-molecular subgroup characterized biologically by deregulated cell cycle and clinically by short survival. 26, 27, 31, 32, 46, 47 9] [50] SF3B1 mutations in CLL are almost always represented by missense substitutions affecting the HEAT domains of the SF3B1 protein and recurrently target five hotspots (codons 662, 666, 700, 704 and 742), with the K700E substitution accounting for approx. ] [30] Among CLL genetic events, SF3B1 lesions have shown a preferential, though not consistent, association with 11q22-q23 deletion and ATM mutations. 29, 30 The functional consequences of SF3B1 mutations in CLL are currently under scrutiny. 5] [56] [57] BIRC3 is also involved in maintaining wildtype TP53 levels by preventing NF-κB-mediated transcriptional and post-translational modifications of MDM2 expression and function. Consistently, BIRC3 knockdown contributes to cancer promotion through downregulation of the TP53 protein via MDM2. 58 IRC3 is recurrently disrupted in CLL by mutations, deletions or a combination of both. 33 BIRC3 inactivating mutations are mainly represented by frameshift or nonsense substitutions causing the truncation of the C-terminal RING domain of the BIRC3 protein (Figure 1 ), whose E3 ubiquitin ligase activity is required to prime MAP3K14 towards proteosomal degradation. ] [29] [30] 64",
"section_name": "A B D C",
"section_num": null
},
{
"section_content": "CLL course may proceed through distinct clinical phases, ranging from a pre-malignant condition known as monoclonal B-cell lymphocytosis (MBL) to overt CLL, and even to transformation into an aggressive lymphoma known as Richter's syndrome (RS). 1, 2 6] [67] [68] [69] [70] This observation is consistent with the notion that 13q14 deletion and +12 represent first step genetic abnormalities in CLL. All the other genetic events accumulate in the more advanced phases of the disease, suggesting that they represent second hit lesions that are progressively selected or acquired during the evolution of the clone. \n\nAs in other pre-malignant conditions, also MBL frequently harbor some of the genetic lesions that can be observed in the overt phases of the disease. ] [67] [68] [69] [70] [71] [72] Two categories of MBL exist. 73 One is represented by clinical MBL, which are detected in the context of a lymphocytosis investigated with laboratory techniques. The second category is represented by low count MBL, which are discovered while screening normal individuals of the general population for research purposes, and in whom the absolute number of lymphocytes is not increased. 73 While the impact of high-risk genetic lesions on clinical MBL survival is currently unknown, their occurrence associates with an increased rate of progression to overt CLL. 67, 69 igh-risk cytogenetic abnormalities have been occasionally described also in low count MBL, but the clinical implications of this observation are currently unknown. 66, 68 hree major clinical phases can be distingushed in overt CLL, including: i) newly diagnosed CLL; ii) progressive CLL; and iii) relapsed and fludarabine-refractory CLL. \n\nDeletion of 17p13 occurs below 5% in newly diagnosed CLL. 11, 14 21] [74] [75] [76] [77] [78] [79] [80] Deletion of 11q22-q23 occurs in less than 10% newly diagnosed CLL, while its prevalence rises to ~20% at the time of first treatment and ~20% at the time of fludarabine-refractoriness. 11,14,74-77 9 These frequencies make ATM alterations the most common genetic lesion predicting poor outcome at CLL presentation and at the time of treatment requirement (Table 1 ). \n\nNOTCH1, SF3B1 and BIRC3 mutations follow the same distribution across CLL clinical phases as other high-risk abnormalities. NOTCH1 mutations characterize ~5%-10% newly diagnosed CLL, while their prevalence increases to 13%-20% in progressive CLL requiring first treatment and in relapsed cases. 26, 27, 31, 32, 64 30] 64 Though occurring at low rates in newly diagnosed CLL (~5% of cases), BIRC3 lesions are enriched among relapsed and fludarabine-refractory CLL (~25% of cases). 33 Due to the lack of information from clinical trials, the precise rate of occurrence at BIRC3 lesions at the time of first treatment requirement still remains to be clarified (Table 1 ). \n\nRS transformation is a very aggressive and an almost always lethal complication of CLL that combines the effects of both chemoresistance and rapid disease kinetics. 1 The genetics of RS strongly influences its clinical behavior. The high rate of TP53 abnormalities (~60% of cases) accounts for the chemoresistant phenotype that is commonly observed in RS. 70 NOTCH1 mutations and MYC network abnormalities are the second most frequent genetic lesions in RS, where they occur in ~30% of cases. 26 In RS, NOTCH1 mutations are largely mutually exclusive with MYC oncogenic activation by translocation/amplification of the gene or by disruption of MGA, its negative regulator. 26, 81 This finding is consistent with the observation that NOTCH1 directly stimulates MYC transcription and suggests that activation of oncogenic MYC may be one common final pathway selected for tumorigenesis in ~60% RS. 26, 81 Lesions affecting ATM, BIRC3 and SF3B1, that are otherwise frequent at the time of chemorefractory relapse, occur at low rates in RS, thus corroborating the notion that RS is molecularly distinct from chemorefractory progression without transformation. 29, 33, 70",
"section_name": "Molecular lesions at different clinical phases of chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "",
"section_name": "Clonal evolution of chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "17p13 deletion 11,14, 72,74,75 4-5 5-7 8-10 1. 5-2 TP53 mutation [19] [20] [21] 77, 78 5-10 5-6 8-11 1. 5-3 11q22-q23 deletion 11, 14, 72, 74, 75 8-9 8-9 21-23 3-5 ATM mutation 24, 25, 39 10-15 7-8 15 4 NOTCH1 mutation 26, 27, 29, 30, 32, 62 5-11 4-8 10 4. 5 SF3B1 mutation [28] [29] [30] 62 6-9 4-9 17 4. 5 BIRC3 disruption 33 4 3. 5 na na ease, CLL may undergo clonal evolution in a substantial fraction of cases. Compa son of the profile of cytogenetic lesions from primary and relapsed CLL samples has revealed differences in ~20-40% cases, illustrating the dynamic nature of clonal evolution in this leukemia. 70, [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] The risk of developing new genetic lesions during the course of the disease is positively correlated with the duration of the follow up. 82, 83 Additional factors contributing to clonal evolution in CLL are the IGHV mutation status and the selective pressure of treatments given during the disease history. 82, 83 From a cl ical standpoint, clonal evolution has been associated with poor outcome, treatment resistance and transformation. 70, 82, 83 Based on conventi l and FISH cytogenetic analysis, clonal evolution mainly consists in the development of 17p13 or 11q22-q23 deletions. 82, 83 Mu tion analysis of sequential samples has revealed that the development of new TP53 mutations also contributes to clonal evolution, especially in chemorefractory patients and in patients complicated by RS. 18, 70 Consistent with this observation, current guidelines recommend to repeatedly test for TP53 lesions at treatment requirement also in cases that were previously wild-type. 2, 92 NOTCH1, SF3B1 and BIRC3 lesions may emerge during the course of CLL, thus expanding the spectrum of genetic events currently associated with clonal evolution. 93 imilar to TP53 abnormalities and 11q22-q23 deletion, also the development of new NOTCH1, SF3B1 and BIRC3 lesions may occur at the time of shift to a more aggressive clinical phenotype. In this context, a fraction of NOTCH1 mutations may develop at the time of transformation to RS. 26 Consistently, the acquisition of high-risk genetic lesions over time, including NOTCH1, SF3B1 and BIRC3 mutations, affects survival in a manner that is independent of modifications of other time-varying factors, such as patient age and disease stage. 93 Open issues in the field of clonal evolution of CLL are: i) whether mutations detected from a certain timepoint onward were already present at subclonal levels in earlier disease phases and are thus subsequently selected (as demonstrated for a few cases), or whether they are acquired de novo during the course of the disease; and ii) whether the presence of small subclones harboring highrisk genetic lesions from the early phases of the disease may affect CLL outcome. A conclusive demonstration of the precise timing of mutations in CLL awaits studies aimed at tracking these lesions with high sensitivity techniques in sequential disease phases.",
"section_name": "Diagnosis First treatment Prevalence % Overall survival (years) Prevalence (%) Overall survival (years)",
"section_num": null
},
{
"section_content": "",
"section_name": "Clinical relevance of genetic lesions in chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "TP53 abnormalities represent strong predictors of poor survival and refractoriness in CLL and, for these reasons, they have direct implications for the clinical management of this leukemia. \n\nAmong newly diagnosed CLL, patients harboring 17p13 deletion show a median overall survival (OS) of only 5-7 years. 17 Although the outcome of patients with 17p13 deletion is generally considered poor, it is important to underline that there is a small subgroup of cases, generally expressing mutated IGHV genes, who may exhibit a slowly progressive disease without treatment indications for years. 94 t the time of treatment requirement, the outcome of patients with 17p13 deletion is almost always very poor. Patients with 17p13 deletion respond poorly (5% of complete response vs. ~50% in non-17p13 deleted CLL) to fludarabine-cyclophosphamide-rituximab (FCR), that is the most effective regimen available today for CLL. The poor response rate in 17p13 deleted patients translates into very short progression-free survival (PFS) (11. 2 months vs. 51. 8 months) and OS (38. 1% at 36 months). 77 This is in line with the central role of the wild-type TP53 protein in priming CLL cells to apoptosis and in mediating the cytotoxic effect of DNA-damaging agents, including purine analogs. \n\nRecently, by assessing the impact of CLL genetics on disease outcome, a number of prospective studies have consistently documented that TP53 mutations, even in the absence of 17p13 deletion, represent a predictor of poor response to treatment and short survival in CLL. In the GCLLSG CLL4 trial (F vs. FC), 74 none of the TP53 mutated CLL achieved a complete response, and the median PFS (23. 3 vs. 62. 2 months) and OS (29. 2 vs. 84. 6 months) were significantly shorter in patients with TP53 mutations compared to TP53 wild-type cases. 79 In the GCLLSG CLL8 trial (FC vs. FCR), the presence of TP53 mutations associated with the lowest complete and overall response rates (6. 9% vs. 36. 4% and 62. 1% vs. 95. 3%, respectively), the shortest PFS (12. 4 months vs. 45 months) and the shortest OS (39. 3 months vs. not reached in all other patients) compared to TP53 wild-type cases. 95 In the UK LRF CLL4 trial (chlorambucil vs. F vs. FC), TP53 mutated patients showed a complete remission rate of only 5%, a 5-year PFS of 5%, and a 5-year OS of 20%. 80 lthough clinical trials have consistently shown a clear association between TP53 mutations and poor outcome in CLL, some controversial issues still remain to be clarified in this field. Indeed, it is currently a matter of debate whether monoallelic TP53 abnormalities have the same poor prognostic effect as biallelic TP53 lesions, and whether the TP53 mutation type and position in the protein might impact on patient oucome. In fact, in contrast to the GCLLSG CLL4 trial, cases from the LRF CLL4 trial harboring isolated TP53 mutations or deletions showed a longer PFS and OS after treatment compared to cases harboring biallelic TP53 disruption. 79, 80 Also, in a retrospective study, patients harboring truncating TP53 mutations or missense substitutions mapping outside the DNA binding domain seem to have a longer OS from diagnosis than cases harboring mutations within the DNA-binding motifs. 96 n these bases, at least three main clinical implications need to be considered: 2,92 i) patients with TP53 abnormalities should not be treated until disease progression, since they can occasionally experience a prolonged TFS; ii) alongside 17p13 deletion, TP53 mutation analysis should be performed in all CLL patients before treatment initiation, since cases with TP53 disruption should be considered for alternative therapies upfront (see below); iii) a thorough search for TP53 mutations/deletions should be performed repeatedly before each line of therapy. \n\nChemorefractoriness is due to TP53 disruption in ~40% of CLL patients failing treatment, but the molecular basis of this aggressive clinical phenotype remains unclear in a sizeable fraction of patients (~60%). 78 In order to optimize the early diagnosis of chemorefractory CLL, it is crucial to understand the molecular basis of chemorefractoriness beyond TP53 disruption. The new molecular lesions recently identified may shed some light on this (see below).",
"section_name": "Clinical relevance of TP53 abnormalities in chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "Deletion of 11q22-q23 was historically associated to CLL progression, poor response to alkylator-and fludarabine-based chemotherapy and, ultimately, short survival. 17, 74, 76 The introduction of chemo-immunotherapy based regimens has changed the prognosis of patients with this genetic abnormality. In fact, in the GCLLSG CLL8 trial, treatment with FCR significantly improved the complete response rate in CLL patients with 11q22-q23 deletion compared to FC alone (51% vs. 15%, P<0. 0001), making these patients more similar to standard-risk patients in terms of response and PFS. 77 he prognostic impact of ATM mutations in CLL has been investigated in few studies, due to the complexity of the DNA sequencing procedure of such a large gene in the absence of well-defined mutational hotspots, and also because of the difficulty in discriminating somatic mutations and damaging germline mutations from single nucleotide polymorphisms. ] [24] [25] The impact of ATM mutations on response to treatment and chemorefractoriness is still an open issue. In vitro, CLL cells with mutations affecting either one or both ATM alleles show defective apoptosis in response to radiation and chemotherapy-induced DNA damage. On the contrary, 11q22-q23 deleted CLL with a normal residual ATM allele preserve the DNA damage response, suggesting that loss of a single ATM allele might not be sufficient to induce chemorefractoriness. 97 Consistent with these pre-clinical observations, in the UK LRF CLL4 trial, patients with both ATM mutation and 11q22-q23 deletion showed a significantly reduced PFS (median 7. 4 months) compared to those with wild-type ATM (28. 6 months), 11q22-q23 deletion alone (17. 1 months), or ATM mutation alone (30. 8 months). Consistently, OS for patients with biallelic ATM alterations was significantly reduced compared to those with wild-type ATM or ATM mutations alone (median 42. 2 vs. 85. 5 vs. 77. 6 months, respectively). 39 On these bases, at least in the context of CLL treated with alkylating agents or purine analogs, only the co-occurrence of 11q22-q23 deletion and ATM mutation associated with poor outcome. Importantly, in the UK LRF CLL4 trial, the PFS of CLL harboring biallelic ATM lesions was similar to that of patients with TP53 alterations. 39 Therefore, when using chemotherapy alone, ATM mutation could be one mechanism that accounts for a fraction of chemorefractory CLL in which no aberrations of TP53 are detected. \n\nIn conclusion, ATM disruption represents an independent marker of poor prognosis in CLL patients, particularly if they are treated with chemotherapy regimens not containing rituximab. Since the addition of rituximab to intensive combination chemotherapy (i. e. FCR) leads to an improved outcome in CLL harboring 11q22-q23 deletion, this regimen represents the treatment of choice in clinically fit patients belonging to this genetic subgroup. Nonetheless, even in the immunochemotherapy era, 11q22-q23 deleted patients have a short PFS and, therefore, may be particularly suited for investigational agents combined with immunochemotherapy or maintenance strategies aimed at prolonging disease remission. 98",
"section_name": "Clinical relevance of ATM abnormalities in chronic lymphocytic leukemia",
"section_num": null
},
{
"section_content": "Beside their pathogenetic role, NOTCH1, SF3B1 and BIRC3 lesions may also represent new biomarkers for the identification of poor-risk CLL patients. \n\nRetrospective studies have consistently shown the impact of NOTCH1 and SF3B1 mutations on newly diagnosed CLL outcome. NOTCH1 mutated patients have a more rapidly progressive disease and a significantly shorter OS probability (21%-45% at 10 years) compared to NOTCH1 wild-type cases (56%-66% at 10 years). 26, 27, 32 9] [30] In a retrospective analysis of newly diagnosed CLL, BIRC3 disruption identifies patients with a poor survival (median OS 3 years) similar to that associated with TP53 abnormalities. 33 ata from prospective studies and clinical trials validate the clinical importance of NOTCH1 and SF3B1 mutations in CLL. 64, 99 In a well-characterized population-based cohort of newly diagnosed CLL patients who were included in the Scandinavian Lymphoma Etiology study, the presence of NOTCH1 or SF3B1 mutations was strongly associated with poor outcome, both in terms of shorter time to treatment and decreased survival. 99 In fact, time to treatment was only 4. 8 months in patients harboring NOTCH1 mutations and 2. 4 months in patients harboring SF3B1 mutations. This higher propensity to progression translated into a short OS of 66 months in NOTCH1 mutated patients and 63 months in SF3B1 mutated patients, that was significantly reduced compared to that of wild-type cases. 99 In addition, NOTCH1 and SF3B1 mutations in this study had a similarly poor impact on prognosis as TP53 aberrations. 99 In the UK LRF CLL4 trial, patients harboring NOTCH1 and SF3B1 mutations have an OS (55 and 54 months, respectively) that is significantly shorter compared to wild-type patients (83 months) and longer than that of patients carrying TP53 abnormalities (26 months). 64 verall, these data document that, at the time of treatment requirement, patients with NOTCH1 and SF3B1 mutations display an outcome that is intermediate between the one marked by TP53 abnormalities and the one characterizing wild-type cases. While both NOTCH1 and SF3B1 mutations are independent predictors of OS by multivariate analysis in this trial, only SF3B1 mutations, but not NOTCH1 mutations, significantly correlate with a short PFS. 64 These data point to chemoresistant progression as a potential reason for the poor outcome in SF3B1 mutated patients, while NOTCH1 mutations apparently have no impact on disease sensitivity to treatment. The short survival associated with NOTCH1 mutations can be explained, at least in part, by a substantial risk (~50% at 15 years) of developing RS in patients harboring this genetic lesion. 100 he GCLLSG is currently exploring the role of new mutations in CLL patients treated with first-line FC or FCR (CLL8 trial), as well as the role of alemtuzumab in overcoming NOTCH1 and SF3B1 alterations in relapsed/refractory patients (CLL2H trial). Preliminary data from the GCLLSG CLL8 trial 77 indicate that both SF3B1 and NOTCH1 mutations represent independent predictors of short PFS after treatment with FCR. 101 In particular, in this trial, NOTCH1 mutations appear to identify a subset of CLL patients that may not benefit from the addition of rituximab to FC. 101 Conversely, based on a preliminary analysis of the GCLLSG CLL2H trial, 75 patients harboring NOTCH1 mutations may have a superior PFS after alemtuzumab treatment compared to NOTCH1 wild-type cases, at least in the relapsed/refractory setting. 102 hough information on the impact of BIRC3 lesions on response to treatment is currently lacking, their enrichment among fludarabine-refractory CLL might suggest an association of these molecular defects with chemorefractory progression. 33 he integration of the most recurrent and clinically relevant new molecular lesions into the backbone of the FISH hierarchical model has allowed a better understanding of the genetic basis of CLL heterogeneity and a significant improvement in patient prognostication. 93 According to a proposed model, four genetic groups of patients are hierarchically classified: 93 i) high-risk patients, harboring TP53 and/or BIRC3 abnormalities independent of co-occurring genetic lesions, that account for approximately 15%-20% newly diagnosed CLL and show a 10-year survival of 29%; ii) intermediate-risk patients, harboring NOTCH1 and/or SF3B1 mutations and/or del11q22-q23 in the absence of BIRC3 and TP53 abnormalities, that account for ~15%-20% newly diagnosed CLL and show a 10-year survival of 37%; iii) low-risk patients, harboring +12 or a normal genetics, that account for approximately 40% of newly diagnosed CLL and showed a 10-year survival of 57%; and iv) very low-risk patients, harboring del13q14 only in the absence of any additional abnormality, that account for ~20%-25% newly diagnosed CLL and a nearly normal life expectancy with a 10-year survival (69%) that did not significantly differ from a matched general population (Figure 2 ). 93",
"section_name": "Clinical relevance of novel molecular lesions",
"section_num": null
},
{
"section_content": "TP53 disruption is at present the only molecular marker that changes the therapeutic approach to CLL patients. The median OS of TP53 disrupted CLL treated with intensive immunochemotherapy regimens (i. e. FCR) is within 2 to 3 years, 77, 95 thus resembling the outcome of acute leukemias. As a consequence, the occurrence of TP53 abnormalities represents a strong indication for treating patients with drugs with a TP53-independent mechanism of action and for performing an allogeneic stem cell transplant (SCT) consolidation in eligible cases. \n\nThe anti-CD52 monoclonal antibody alemtuzumab has so far been the single agent with the highest efficacy in CLL with 17p13 deletion. 4] [105] [106] [107] [108] [109] [110] [111] [112] At first-line, patients with TP53 disruption and treated with alemtuzumab-based regimens can achieve a response rate of 60%-90%, a complete response rate of 20%-60% and a PFS of 10-17 months. ] [105] [106] [107] [108] [109] [110] [111] [112] The efficacy of alemtuzumab is increased by its combi-nation with high-dose steroids. In the UK CLL206 trial, alemtuzumab combined to high-dose methylprednisolone was administered to 39 CLL with 17p13 deletion (17 untreated and 22 previously treated). 111 This combination resulted in a response rate of 85%, a complete response rate of 36%, a median PFS of 11. 8 months and a median OS of 23. 5 months. Based on these results, alemtuzumab combined to high-dose methylprednisolone represents the most effective cytoreductive therapy to be considered for fit patients with TP53 disruption as a bridge to allogeneic SCT, which is still required because otherwise responses are of short duration. 4] [115] [116] On these bases, according to the EBMT guidelines, young CLL patients requiring treatment and harboring TP53 abnormalities have an indication for allogeneic SCT. 117 4] [115] [116] Consistently, the actual strategy for fit patients with TP53 abnormalities is to induce the disease at least in partial remission to perform a reduced intensity conditioning allogeneic SCT. \n\nDespite these advances, a number of unmet clinical needs remain in the setting of CLL patients requiring treatment and harboring TP53 abnormalities, including: i) the design of alternative and safer approaches for remission induction, as remission rates with the current regimens are reached only in approximately one-third of patients with relatively high costs in terms of toxicity and infections; and ii) the design of alternative strategies for those patients who are not eligible to transplant procedures because of age and comorbidities. \n\nNovel compounds that act through mechanisms completely different from chemotherapy may overcome the refractoriness induced by TP53 abnormalities. Among fludarabine-refractory CLL with 17p13 deletion, ofatumumab, a new anti-CD20 antibody, led to a response rate of 41%; however, responses were of short duration. 118 reliminary results obtained from ongoing clinical trials employing novel drugs interfering with the B-cell receptor signaling suggest that these novel agents may circumvent the negative impact of 17p13 deletion. 119, 120 urrently, data on genetic alterations of NOTCH1, SF3B1 and BIRC3 are not sufficient to substantiate a role, if any, for these mutations in guiding therapeutic choices. This issue still awaits clarification by the analysis of these new mutations in the context of multiple clinical trials.",
"section_name": "Molecular genetics as a guide for choosing therapy",
"section_num": null
},
{
"section_content": "In recent years, our understanding of the complexity of the molecular genetics of CLL has broadened profoundly and this has translated into important implications for an optimized management of patients. Indeed, a workup at diagnosis for TP53 and ATM disruption, and NOTCH1, SF3B1 and BIRC3 mutations enables a more refined prognostic stratification of patients. None of these markers, however, represents per se an indication for early treatment, but prompts a closer clinical follow up. For patients Molecular genetics of CLL haematologica | 2013; 98 (5) with disease progression, it is mandatory to perform TP53 mutation analysis alongside 17p13 deletion, since cases with TP53 disruption should be considered for alternative therapies upfront, including alemtuzumab plus steroids followed by an allogeneic SCT in young and fit patients. Although there are currently no treatment strategies for TP53 disrupted CLL in the elderly, the use of low doses of alemtuzumab has proven feasible. 109 ATM disruption represents a strong indication for the use of the FCR scheme in clinically fit patients, whilst in elderly/frail patients with this lesion rituximab-based alternative approaches should be explored. \n\nNOTCH1, SF3B1 and BIRC3 mutations at present do not guide therapeutic choices. However, they represent markers of short time to progression and survival, with potentially different clinical implications that need to be conclusively validated. NOTCH1 mutations apparently do not impact on CLL chemosensitivity, but are associated to a substantial risk of RS transformation, particularly if associated to TP53 disruption; therefore, a close follow up and early node biopsy is recommended in patients with this/these lesion(s). SF3B1 mutations are associated to a chemoresistant progression of the disease after alkylating agents and/or fludarabine therapy. BIRC3 mutations are also associated to a chemorefractory disease, although their impact on response to treatment has not yet been proven in the context of clinical trials. Thus, the molecular bases of CLL aggressiveness have been extended beyond TP53 disruption. Due to the clonal evolution of the disease, a thorough search for TP53 mutations/deletions should be repeatedly performed before each line of therapy in view of the clinical relevance of these abnormalities; we know that 11q22-q23 deletions can be also acquired over time and there is evidence of the acquisition of NOTCH1 mutations. Finally, the efficacy of new drugs needs to be tested according to the presence of these molecular lesions for a future personalized medicine approach based on the genetic profile of CLL patients.",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "The work by the authors described in this review was supported by AIRC Special Program Molecular Clinical Oncology, 5 x 1000, n. 10007, Milan, Italy (to RF and to GG); Compagnia di San Paolo, Torino, Italy (RF) ; Progetto FIRB-Programma \"Futuro in Ricerca\" 2008 (to DR); PRIN 2008 (to GG) and 2009 (to DR), MIUR, Rome, Italy ; Progetto Giovani Ricercatori 2008 (to DR), Ministero della Salute, Rome, Italy; and Novara-AIL Onlus, Novara (to GG), Italy.",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "",
"section_name": "Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.",
"section_num": null
}
] |
10.30977/at.2219-8342.2019.44.0.5
|
Research and diagnostics of electric car BMW I3 electric systems
|
The problem. The article considers the problem of increasing the efficiency of the use of electric vehicles. This is done by studying and diagnosing electrical equipment of the BMW i3 electric car. Goal. The purpose of the work is to increase the efficiency of the use of electric vehicles by studying and diagnosing electrical equipment of BMW i3 elec-tric vehicle. Methodology. Systematic, theoretical analysis of the methods applied to an electric car. The methodology is based on the integrated approach to studying the BMW i3. The research of the main components of the electrical equipment of the electric vehicle was carried out, its technical characteristics and the principle of operation of the power system were analyzed, issues of charging a high-voltage battery were considered. System power control functions were investigated. The diagnostics of BMW i3 power system in the conditions of the service station was performed. Results. It is established that the purpose of the power control program is to provide sufficient level of the battery charge in different conditions of car operation. It is determined that for the BMW i3 you can use 4 different types of charging. It has been discovered that an emergency situation caused by a discharged battery or problems in the on-board power grid may have various causes, which in most cases do not relate to a high-voltage rechargeable battery. Originality. In the article a comprehensive study of the BMW i3 electric vehicle was conducted. Practical value. New original knowledge has been gained that will improve the performance of BMW i3 electric vehicle.
|
[
{
"section_content": "Для досягнення поставленої мети необхідно вирішити такі завдання:\n\n-проаналізувати технічні характеристики електромобіля BMW i3 та дослідити його основні компоненти електрообладнання. До таких компонентів належать: система електроживлення, тягова акумуляторна батарея (ТАБ) з контролером керування, системи зарядки, генераторна установка тощо;\n\n-здійснити дослідження систем зарядки високовольтної акумуляторної батареї BMW i3 та додаткового обладнання для цього. Провести аналіз міжнародних стандартів для визначення сприятливих методів зарядки електромобіля BMW i3 в різних країнах;\n\n-провести практичне виконання діагностики електрообладнання BMW i3 в умовах СТО «Баварія Моторс» м. Харків.",
"section_name": "",
"section_num": ""
},
{
"section_content": "На сучасному етапі розвитку науки та техніки проблема створення енергозберігаючих транспортних засобів вирішується за рахунок створення електричних транспортних засобів: електромобілів, гібридних електромобілів або гібридних транспортних засобів [3, 4]. Перспективними є гібридні транспортні засоби, що мають режим «тільки електрика» та здатні накопичувати енергію в тяговій акумуляторній батареї від стаціонарних джерел електричної енергії [5]. Для живлення електропривода використовуються акумуляторні батареї літій-іонного типу, важливою проблемою яких є балансування їх елементів під час заряду від зовнішньої електричної мережі [6, 7]. Важливою проблемою є порівняння енергетичної ефективності гібридних, електричних та звичайних транспортних засобів, а також розроблення методики визначення витрат енергоносіїв електричних та гібридних транспортних засобів у процесі експлуатації в різних країнах [8] [9] [10] [11].",
"section_name": "Аналіз публікацій",
"section_num": null
},
{
"section_content": "Електромобіль BMW i3 оснащений новітніми системами управління, зокрема системою супутникової навігації Professional і дистанційного керування, клімат-контролем, відеокамерами заднього виду, підігрівом скла і сидінь, системою посиленого живлення, датчиками дощу, парктроником і мультимедійною розважальною системою з Bluetooth, USB, радіо та іншими елементами комфорту. \n\nЗахист водія та пасажирів в автомобілі забезпечується бічними та фронтальними подушками безпеки, системою ABS, АЕВ, активним круїз-контролем, сигналізацією аварійного зближення під час паркування PDC та іншими активними й пасивними системами. \n\nОсновні технічні характеристики електромобіля BMW i3 зведені в табл. 1. Проведемо аналіз зниження споживання потужності або відключення споживачів електроенергії. Відключення окремих споживачів або зниження споживання потужності слугує для оптимального розподілу потужності, яка є залежною від стану автомобіля та ступеня заряду акумуляторної батареї:\n\n-забезпечення достатнього заряду акумуляторної батареї під час руху (зарядний баланс); -зниження споживання потужності під час розпізнавання зниженої напруги;\n\n-зниження споживання потужності у випадку вимоги мінімальної напруги для електромеханічного підсилювача рульового управління;\n\n-зменшення споживаної потужності для зниження розряду акумуляторної батареї на стоянці (зупинка двигуна автоматичною системою пуску й зупинки двигуна MSA). \n\nСистема управління електроживленням керує зниженням споживання потужності та відключенням окремих споживачів за допомогою запитів (повідомлень), які направляються відповідним ЕБК. Якщо ступінь заряду виходить із критичної ділянки або ступінь заряду поліпшується, функції знову відновлюються.",
"section_name": "Технічні характеристики BMW i3",
"section_num": null
},
{
"section_content": "Для зарядження електромобіля потрібні додаткові електричні компоненти. В автомобілі потрібні роз'єм для зарядки й силові електронні пристрої для трансформації напруги. Для автомобіля, крім мережі змінної напруги і зарядного кабелю, потрібна ще і станція зарядки (наприклад Wallbox). \n\nЗарядна станція Wallbox має захисну функцію й управляє зарядженням ТАБ. Напруга мережі змінного струму може становити від 110 до 240 В. На практиці для повного заряду тягової акумуляторної батареї потрібно її заряджати від електричної мережі змінного струму 220 В за час від 7 до 10 год. \n\nУсі компоненти для зарядження високовольтної акумуляторної батареї електромобіля BMW i3 стандартизовані у частині конструкцій і функцій. У країнах Європи діє відповідний стандарт IEC 61851 [12] [13] [14]. \n\nEC 61851 -це міжнародний стандарт для систем провідного електропостачання електричних дорожніх транспортних засобів та електричних вантажних автомобілів промисловості, частина яких на цей час все ще розробляється. Компоненти для зарядження високовольтної батареї електромобіля BMW i3 працюють в описаних у стандарті режимах. \n\nПриклади режимів зарядки згідно зі стандартом IEC 61851:\n\n-режим зарядки 2: підключення до стандартної побутової розетки з додатковою лінією передачі даних; -режим зарядки 3: підключення до стаціонарної станції зарядки Wallbox з лінією передачі даних. \n\nДля США діє стандарт SAE J1772 [15]. SAE J1772 (IEC -історія останніх збільшень інерційних фаз роботи контакту 30 B (споживачі струму спокою); -історія останніх причин пробудження. До запам'ятовувального пристрою історії енергосистеми записується різна інформація, яка може допомогти у пошуку причин проблем із бортовою енергетичною мережею. Інформація, збережена в запам'ятовувальному пристрої історії енергетичної системи, -це, зокрема:\n\n-огляд поїздок за останні 5 тижнів. Огляд поїздок зберігається в пам'яті історії електроживлення за допомогою 6 наборів даних. Кожен набір даних містить таку інформацію: час початку реєстрації набору даних, пройдений шлях у км під час реєстрації, кількість поїздок на різних ділянках;\n\n-новий набір даних запускається, коли часовий інтервал між поточним часом і часом початку реєстрації поточного набору даних перевищує 7 днів. Таким чином, проміжок часу аналізу становить, як правило, приблизно 35 днів, якщо тільки автомобіль не знаходився в режимі спокою більшу кількість часу;\n\n-старий набір даних перезаписується, як тільки всі 6 наборів даних у приої заповнюються;\n\n-максимальне число активізацій протягом періоду спокою за відповідні останні 5 тижнів. \n\nСистема управління двигуном або система електричного блока управління двигуном (EDME) зберігає різні діагностичні дані, що використовуються під час діагностики електроживлення:\n\n-результати останніх 24 перевірок струму спокою;\n\n-остання зареєстрована заміна акумуляторної батареї;\n\n-ступінь заряду ТАБ за останні 5 днів; -пробіг за останні 5 днів; -час і тривалість останніх 4 обмежень або відключення споживачів струму;\n\n-дані вимірювань для контролю стану акумуляторної батареї з розширеним інтелектуальним датчиком акумуляторної батареї: розпізнавання несправних елементів акумуляторної батареї, залишкова ємність. \n\nПроведемо дослідження пам'яті помилок у системі керування двигуном та в EDME. Система управління двигуном, або система EDME, зберігає код помилки за умови перевищення струму спокою, глибокого розрядження акумуляторної батареї й обмеження або відключення споживачів струму. \n\nЗапам'ятовувальний пристрій помилок у системі управління світлом працює таким чином. У разі вимкненого контакту R система управління світлом вимикає стоянкові або паркувальні вогні, якщо вимірювана напруга в бортовій мережі нижча, ніж 10,6 В, протягом мінімум 2 хв. За умови відключення в запам'ятовувальному пристрої несправностей записується відповідний код несправності. \n\nПід час ускладненого переходу до стану спокою або несанкціонованої активації послідовно проводяться різні заходи, такі як відключення контактів, щоб не допустити глибокого розрядження акумуляторної батареї і забезпечити можливість пуску автомобіля.",
"section_name": "Зарядження високовольтної акумуляторної батареї BMW i3",
"section_num": null
},
{
"section_content": "Проведений аналіз публікацій свідчить, що перспективними видами автотранспортних засобів є електромобілі та гібридні електромобілі. Тому для дослідження обраний саме BMW i3, який випускається в цих двох модифікаціях. \n\nДослідження технічних характеристик BMW i3 демонструє його високу економічність та екологічність. За проведеними дослідженнями основних компонентів електрообладнання та системних функцій управління електроживленням сформульовано висновок, який свідчить, що метою програми управління електроживленням є забезпечення достат-нього рівня заряду акумуляторної батареї в різних умовах експлуатації та зберігання автомобіля. \n\nНа основі дослідження різноманітних систем зарядки електромобілів у різних країнах визначено, що для BMW i3 можна застосовувати чотири різних типи зарядки:\n\n-зарядка змінним струмом потужністю 3,7 кВт (базове виконання); -зарядка змінним струмом потужністю 7,4 кВт (додаткове обладнання SA4U8); -комбінована зарядка змінним струмом потужністю 3,7 кВт і постійним струмом потужністю 50 кВт (додаткове обладнання SA4U7); -комбінована зарядка змінним струмом потужністю 7,4 кВт і постійним струмом потужністю 50 кВт (додаткове обладнання SA4U7 і SA4U8). \n\nУнаслідок діагностики електрообладнання електромобілів BMW i3, яка проходила в умовах СТО «Баварія Моторс», м. Харків, було виявлено їх несправності та зроблено висновок, що аварійна ситуація, викликана розрядженою батареєю або проблемами в бортовій енергетичній мережі, можуть мати різні причини, які здебільшого не відносяться безпосередньо до високовольтної акумуляторної батареї.",
"section_name": "Висновки",
"section_num": null
},
{
"section_content": "Abstract. The problem. The article considers the problem of increasing the efficiency of the use of electric vehicles. This is done by studying and diagnosing electrical equipment of the BMW i3 electric car. Goal. The purpose of the work is to increase the efficiency of the use of electric vehicles by studying and diagnosing electrical equipment of BMW i3 electric vehicle. Methodology. Systematic, theoretical analysis of the methods applied to an electric car. The methodology is based on the integrated approach to studying the BMW i3. The research of the main components of the electrical equipment of the electric vehicle was carried out, its technical characteristics and the principle of operation of the power system were analyzed, issues of charging a highvoltage battery were considered. System power control functions were investigated. The diagnostics of BMW i3 power system in the conditions of the service station was performed. Results. It is established that the purpose of the power control program is to provide sufficient level of the battery charge in different conditions of car operation. It is determined that for the BMW i3 you can use 4 different types of charging. It has been discovered that an emergency situation caused by a discharged battery or problems in the on-board power grid may have various causes, which in most cases do not relate to a high-voltage rechargeable battery. Originality. In the article a comprehensive study of the BMW i3 electric vehicle was conducted. Practical value. New original",
"section_name": "Research and diagnostics of electric car BMW I3 electric systems",
"section_num": null
}
] |
[] |
10.3389/fgene.2022.1001364
|
Super-Enhancer–Associated nine-gene prognostic score model for prediction of survival in chronic lymphocytic leukemia patients
|
<jats:p>Chronic lymphocytic leukemia (CLL) is a type of highly heterogeneous mature B-cell malignancy with various disease courses. Although a multitude of prognostic markers in CLL have been reported, insights into the role of super-enhancer (SE)–related risk indicators in the occurrence and development of CLL are still lacking. A super-enhancer (SE) is a cluster of enhancers involved in cell differentiation and tumorigenesis, and is one of the promising therapeutic targets for cancer therapy in recent years. In our study, the CLL-related super-enhancers in the training database were processed by LASSO-penalized Cox regression analysis to screen a nine-gene prognostic model including TCF7, VEGFA, MNT, GMIP, SLAMF1, TNFRSF25, GRWD1, SLC6AC, and LAG3. The SE-related risk score was further constructed and it was found that the predictive performance with overall survival and time-to-treatment (TTT) was satisfactory. Moreover, a high correlation was found between the risk score and already known prognostic markers of CLL. In the meantime, we noticed that the expressions of TCF7, GMIP, SLAMF1, TNFRSF25, and LAG3 in CLL were different from those of healthy donors (<jats:italic>p</jats:italic> &lt; 0.01). Moreover, the risk score and LAG3 level of matched pairs before and after treatment samples varied significantly. Finally, an interactive nomogram consisting of the nine-gene risk group and four clinical traits was established. The inhibitors of mTOR and cyclin-dependent kinases (CDKs) were considered effective in patients in the high-risk group according to the pRRophetic algorithm. Collectively, the SE-associated nine-gene prognostic model developed here may be used to predict the prognosis and assist in the risk stratification and treatment of CLL patients in the future.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL), a mature and monoclonal CD5+ CD23+ B cell malignancy, proliferates and accumulates in the bone marrow, blood, and lymphoid nodes (Hallek et al., 2018). It is often asymptomatic in the early stage. It is often found that painless lymphadenopathy or the absolute value of lymphocytes is increased for unknown reasons. Patients have mild fatigue, fatigue, and other non-specific manifestations. Once they enter the advanced stage, they can present with weight loss, repeated infection, bleeding, and anemia in addition to systemic lymph nodes and splenomegaly. CLL cases are fewer in Asia than those in the Western world, and it is reasonable to assume that genetic and environmental factors play roles in pathogenesis (Burger, 2020). During 2014-2018, the rate of new cases of CLL was 4. 9 per 100,000 per year and the median age at diagnosis is 72 years, the death rate was 1. 1 according to the aforementioned survey [The Surveillance Epidemiology and End Results (SEER) Program of the National Cancer Institute. Cancer fact sheets: chronic lymphocytic leukemia (CLL). https://seer. cancer. gov/ statfacts/html/clyl. html (accessed 22 September 2021)]. \n\nCLL is widely known as a heterogeneous disease that exhibits variable clinical symptoms, time-to-treatment (TTT), easily progression and difficult prognosis. CLL patients are often diagnosed with incidental findings, and the clinical course ranges from an asymptomatic, indolent disease that requires no treatment to a rapidly progressive and chemotherapy-resistant disease until death within a short period (Burger, 2020). The indications for treatment mainly include the clinical stage and symptoms of patients, and the standard therapy is chemoimmunotherapy. Unfortunately, the majority of CLL patients are too old to tolerate intensive standard chemotherapy; therefore, an effective prognostic model is needed to predict the individual clinical courses and to improve the outcome. Over the past few decades, great advances have been made in figuring out the molecular and genetic biology of CLL to identify the indicators of progression and survival. These indicators include cytogenetics, age, IGHV gene mutation status, β2-microglobulin (β2-MG), clinicalstage (RAI/BINET stage), and so forth (Bosch and Dalla-Favera, 2019). In CLL, 13q14, 11q22-23, trisomy of 12q, and 17p deletions are found in 80% of the cases. 11q22-23 and 17p deletions are associated with poor survival, whereas 13q14 deletions and trisomy of 12q have a longer TTT and survival time (Dohner et al., 2000). TP53 aberrations (Zenz et al., 2010) indicate a more aggressive disease progression and extensive drug-resistant and worse outcome, and the same role applies to IGHV genes (Damle et al., 1999) and ZAP-70 (Crespo et al., 2003). Unmutated IGHV and high-expression of ZAP-70 have a comparatively aggressive disease course too, and other relevant risk markers include expression of CD38 (Rassenti et al., 2008), CD49d (Bulian et al., 2014), lipoprotein lipase (LPL) (Prieto and Oppezzo, 2017), serum concentrations of thymidine kinase (Hallek et al., 1999), and β2-microglobulin (Hallek et al., 1996). \n\nIn this article, a SE-associated gene list was used to carry out LASSO-penalized Cox regression analysis, and construct a nine SE-associated gene prognostic model, namely, TCF7, VEGFA, MNT, GMIP, SLAMF1, TNFRSF25, GRWD1, SLC6AC, and LAG3. Meanwhile, this model was verified by testing GEO datasets and the ICGC-CLL dataset, respectively. Univariate and multivariate Cox regression analyses, and the ROC curve were analyzed to evaluate the prognostic accuracy of this ninegene model. Moreover, the aforementioned validated steps, the role of the nine-gene prognostic model, and the nine hub genes were further explored in CLL genesis and the relationship between this prognostic model and other known risk markers, such as IGHV status, FISH abnormality, and ZAP70 expression level. It was indicated that the model demonstrated predictive power and had an expected relationship with known risk markers. In addition, an interactive nomogram based on the nine-gene risk score and clinical traits was constructed. Finally, paired pre-and post-treatment datasets were used to examine the effects of treatments on the risk score or each of the nine hub genes' expression, and we predicted 25 clinical drugs that may be more sensitive to high-risk patients. The improved nine-gene prognostic model of this work provided a bright future for the diagnosis, disease stratification, and therapy of patients with CLL.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The flowchart featured the construction and validation of the SE-associated gene-based prognostic model of CLL and the correlation with other known risk markers (Figure 1 ). An 831 primary B-CLL cell-related SE list was downloaded from the website and the 18,887 gene matrix in CLL patients was provided in the GSE22762 column, and a 587 SE-associated gene matrix for CLL was gained via overlapping the aforementioned two gene sets. Immediately after, the gene matrix was done by LASSOpenalized Cox regression to screen the prognosis-related genes with potential. Figure 2A shows the coefficient values for each at various penalty levels as long as genes with non-zero coefficients had prognostic value in the LASSO-penalized regression model. Tenfold cross-validation obtained the maximum lambda value, and we selected one model which produced a group of nine genes (Figure 2B ). Principal component analysis (PCA) showed highrisk patients separate from low-risk ones evidently (Figure 2C ). Also, the obvious distinction between survival and death was calculated by using the nine gene-based prognostic model, implying that the prognostic model functioned smoothly in the prediction on the OS of patients with CLL (Figure 2D ). \n\nTo validate the LASSO-penalized Cox regression model, univariate Cox proportional hazard regression analysis determined that these genes affected the OS of patients with CLL independently, and all log-rank p-values of the nine genes were < 0. 01 (Supplementary Figure S1A ). Multivariate Cox proportional hazard regression analysis was also performed, and the global p-value of our model was only 2. 64e-16 (Supplementary Figure S1B ), with an AIC of 124. 96 and a C-index of 0. 95. These indexes suggest that the nine genes are possibly prognostic markers for OS in CLL patients. Meanwhile, the results of K-M survival analysis showed that GRWD1, SLC6A3, and MNT had no significant association with survival (Supplementary Figure S2 ). Furthermore, we concluded that SLAMF1, TCF7, TNFRSF25, MNT, and VEGFA were protective factors, whereas GRWD1, SLC6A3, GMIP, and LAG3 appeared to be harmful factors in CLL, based on the aforementioned hazard ratios of univariate and multivariate regression analyses. Thus, the nine-gene SEassociated model by LASSO-penalized Cox regression possibly predicted the OS of CLL patients.",
"section_name": "Construction of a nine-gene LASSOpenalized cox regression model and validation of independent prognostic factors",
"section_num": null
},
{
"section_content": "A total of 107 patients in the training dataset of GSE22762 (HGU-133plus2) were divided into high-risk (risk score > 0. 7) and low-risk groups (risk score < 0. 7) (Figure 3A ). Figure 3B shows that death was more frequently observed in the high-risk group than in the low-risk group. The K-M survival analysis presented a much worse outcome in the high-risk group than that of the low-risk group (logrank test, p = 3. 561e-09) (Figure 3C ). Also, the AUCs of a time-dependent ROC curve of 1, 3, and 5 years calculated by the nine gene-based risk score model were 0. 997, 0. 958, and 0. 996, respectively (Figure 3D ), suggesting that the prediction was highly sensitive and specific. The testing column (GSE22762, N = 44, HGU-133A) verified the predictive values of the nine gene-based risk scores. The K-M curves of the high-and low-risk groups were noticeably different (logrank test, p < 0. 05) and the AUCs of 1-, 3-, and 5-year ROC curves were 0. 738, 0. 679, and 0. 628, respectively; these results showed that this prognostic model might be a potential predictor to judge the OS of patients with CLL (Supplementary Figure S3 ). \n\nGSEA was carried out in two datasets on exploring enriched KEGG pathways in which the analysis suggested that vital enrichment was concentrated in the high-risk cohort, including base and nucleotide excision repair, DNA replication, and valine-leucine and isoleucine degradation (Supplementary Figures S4A, B ). Other pathways including homologous recombination, oxidative phosphorylation, mismatch repair, RNA degradation, RNA polymerase, and one carbon pool by folate and lysine degradation were enriched in the high-risk group of the two cohorts.",
"section_name": "Establishment and validation of the nine gene-based risk score model",
"section_num": null
},
{
"section_content": "In addition to survival, we also investigated the nine-gene prognostic model on TTT, and the results demonstrated that the nine-gene risk model performed well on predicting TTT in the training dataset (GSE22762). Low-risk patients showed a longer TTT than high-risk patients, and the p-value < 0. 001 (Figure 4A ). Additionally, the time-dependent ROC curve analysis prompted that the AUCs of 1-, 3-, and 5-year TTT were 0. 818, 0. 840, and 1. 000, respectively (Figure 4B ). These results were in accordance with testing datasets (GSE39671) (Figures 4C, D ), and it indicated that the prognostic model was equally effective in predicting TTT.",
"section_name": "The prediction of the nine-gene model on time-to-treatment",
"section_num": null
},
{
"section_content": "In addition to the prognostic value, we also expected a relationship between the nine-gene model and tumorigenesis. WGCNA was another statistical method for the analysis of finding the different genes between normal and CLL patients. As shown in Figure 5A, the best soft-thresholding value via prediction of the scale independence was β = 6. Then, genes were divided into 9 different modules with 9 different colors, and a heatmap was developed according to Pearson's correlation coefficient (Figure 5B ). An intersection between the SE matrix and the nine modules which presented a higher correlation with CLL showed that TCF7 and LAG3 appeared in the interaction genes between module purple, yellow, and SE-associated genes (Figure 5C ). Simultaneously, TCF7, GMIP, SLAMF1, TNFRSF25, and LAG3 were found to express differently in normal and CLL patients when we compared the individual expression of nine SE-related hub genes in CLL (Figure 5D ). The data indicated that the five genes may play a vital role in regulating the genesis of CLL.",
"section_name": "Identification of SE-related hub genes in chronic lymphocytic leukemia using weighted gene co-expression network analysis",
"section_num": null
},
{
"section_content": "The performance of the nine-gene prognostic model was additionally evaluated in different subgroups defined by confirmed risk factors. Patients with mutated IGVH genes, 13q14 or single deletion or trisomy 12 on FISH analysis, presented a favorable outcome, whereas patients with unmutated IGVH status, 17p13 or a 11q23 deletions, had an unfavorable prognosis. Unmutated IGHV patients had a higher risk score than mutated IGHV patients in three independent datasets (GSE9992, GSE16746, and GSE28654) (Figures 6A-C ). Simultaneously, we analyzed the correlation between IGHV mutation status and each gene in the nine-gene prognostic model. The results reported that the expression of TCF7 and SLAMF1 had a strong positive correlation, and LAG3 showed a negative correlation with IGHV mutation (Figure 6D ). Similarly, patients with del17p13 had a higher risk score compared to other chromosome types (p < 0. 001, Figure 6E ). The risk score of ZAP70-high patients was higher than that of ZAP70-low patients; the expression of MNT and SLAMF1 had a negative association, and LAG3 had a positive association with ZAP70, respectively (Figures 6F, G ). Additionally, the variation of risk score and each gene expression before and after treatment was provided in Supplementary Figure S5A. The risk score was downregulated after processing with HDAC inhibitory in vitro, and VEGFA and MNT were upregulated accompanied by downregulated GMIP and TGFRSF25. In the other two in vivo treatment experiments, no significant change was found except LAG3, the LAG3 gene was upregulated consistently after lenalidomide and thalidomide treatment respectively (Supplementary Figures S5B, C ).",
"section_name": "The validated nine-gene prognostic model and other risk factors",
"section_num": null
},
{
"section_content": "International Cancer Genome Consortium (ICGC, http:// daco. icgc. org/), which collected multiple genetic mutations, copy number variants, epigenetic modifications, and clinical data covering 50 tumor types, and we extracted 255 CLL patient data for following analysis. Again, high risk scores were significantly associated with shorter survival time, p < 0. 001 (Supplementary Figure S6A ), and the AUCs of ROC curves of the 3-, 5-, and 10-year survival were 0. 731, 0. 718, and 0. 800, respectively (Supplementary Figure S6B ). CLL patients could be divided into two molecular subtypes according to the mutational status of the IGHV, with cases carrying unmutated IGHV (U-CLL) having more aggressive behavior than patients with mutated IGHV (M-CLL). Consistent with the most accepted view, the nine-gene risk score median value was obviously lower in the indolent CLL subtype (M-CLL) compared to the aggressive one (U-CLL) (Supplementary Figure S6D ). The nine-gene risk score was associated with the evolution of M-CLL with a median OS of 6. 57 versus 8. 87 years for patients with high and low risk scores, respectively (p = 0. 005, Supplementary Figure S6C ), while no differences were seen in U-CLL patients in relation to highand low-risk scores (data not shown). Moreover, on the basis of the obtained sample clinical characteristics, we performed univariate as well as multivariate Cox survival analyses. Age, IGHV mutated status, and risk were identified to be independent prognostic factors for patients with CLL (p < 0. 05; Figures 7A, B ). Based on the nine-gene risk score and clinical traits, a nomogram was constructed to accurately predict CLL patients' 1-, 3-, 5-, and 10-year survival rates by the using aforementioned clinical indicators and the nine-gene risk score. The C-index of this model was 0. 82 (Figure 7C ).",
"section_name": "Validation of nine-gene prognostic model in ICGC and construction of a nomogram to predict OS",
"section_num": null
},
{
"section_content": "According to the pRRophetic algorithm, we predicted the IC50 of 130 chemotherapeutic agents and pathway inhibitors in both of high-and low-risk patients and found that 25 drugs had lower IC50 in high-risk patients (p < 0. 05, additional file 1), which indicated that the high-risk patients were more sensitive to these 25 drugs. Among these compounds, some have been reported to have pre-clinical anti-tumor activity in CLL, such as thapsigargin, which was found to be a potent cytotoxin that induced apoptosis by inhibiting the sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase (SERCA) pump, which was necessary for cellular viability. Some have not been reported in CLL before, and therefore the therapeutic effect is still unknown. Interestingly, there were three kinds of compounds which could inhibit the mTOR pathway and CDKs in CLL, respectively, and these have been researched in CLL before and CDK inhibitors have entered clinical trials in patients with relapsed or refractory chronic lymphocytic leukemia. These results could be helpful for the precise treatment of CLL (Figure 8 ).",
"section_name": "Response of high-and low-risk patients to chemotherapeutic compounds",
"section_num": null
},
{
"section_content": "CLL is considered to have a highly heterogeneous clinical course, with time to first treatment varying from months to years and many patients eventually progressing and requiring chemotherapy, although initially, CLL is reported as an indolent malignancy. A review of the data so far, disease stratification, IGHV mutation status, 17p, and ZAP70 expression are the validated predictors of overall survival. Beyond that, gene expression analysis was carried out on various surrogate markers for genetic features and prognosis. A total of six surface antigens (CD62L, CD54, CD49c, CD49d, CD38, and CD79b) and prognostic risk models were put in place to diagnose and predict the OS for CLL (Zucchetto et al., 2006). Moreover, some large-scale gene expression profiling analyses generate different prognostic factors (Kienle et al., 2010; Herold et al., 2011a; Schweighofer et al., 2011). But the previous studies constructed no prognostic model according to SE-associated genes which regulate the expression of hub genes related to CLL tumorigenesis. \n\nA super-enhancer is a new concept developed in recent years; a growing body of evidence indicates an explicit relationship between increasing tumorigenesis and malignancy of cancer and SEs. SEs drive not only the expression of genes but also non-coding RNA that regulates biological functions directly and indirectly. LASSOpenalized Cox regression has become popular in recent years because it could minimize overfitting (Ma et al., 2019). Hence, in our article, we use this novel bioinformatic strategy and the Cox proportional hazard regression models to screen and optimize hub genes related to survival. \n\nIn our research, the LASSO-penalized Cox regression analysis was carried out by filtering out the potential SEassociated genes and yielding a nine-gene prognostic model to foresee the OS of CLL patients. All of the individual markers in the nine-gene model associated with OS of CLL by Cox regression analysis were identical. K-M survival analysis also indicated that the majority of the nine genes correlated to OS. Beyond that, the nine-gene prognostic model was highly significant in the multivariate analysis of patients without treatment. The AUCs and C-index showed that our model performed well in the prediction of survival. The effectiveness of this prognostic model could be validated by an independent patient cohort. Moreover, this risk model was another indicator of TTT. We utilized the nine-gene risk score in the GSE22762 and GSE39671 datasets, and the results also indicated that the nine-gene model could be applied to predict TTT. The high-risk patients had less time-to-treatment than the low-risk patients. These data strongly indicated that the ninegene prognostic model was a significant and valid risk forecaster. \n\nWe not only evaluated the data by a rigorous training and validation design, but also concentrated on the connection between individual genes and selected disease characteristics, such as IGHV mutation status, FISH abnormality, and ZAP70 expression level. The results of three of the markers (TCF7, SLAMF1, and LAG3) detected according to the association with IGHV status were expected. The lack of a public database that included both survival data and mutation information limited further research on a correlation between the nine-gene model and ZAP70, a FISH abnormality. But in the poor prognosis groups, like ZAP70-high and 17q-patients, the nine-gene risk score was significantly higher than that in the lowrisk group, and we found that the low expression of SLAMF1 in CLL was associated with ZAP70-high expression. The quantitative relationship between TCF7, LAG3, and SLAMF1 expression and inferior overall survival was an accurate finding and indicated that these genes had a pathogenic role in CLL. Additionally, the nine-gene prognostic model also played an important role in CLL etiopathogenesis. The WGCNA of the GSE50006 dataset revealed that TCF7 and LAG3 belonged to two gene modules, respectively. In addition to this, the expression of GMIP, SLAMF1, and TNFRSF25 were also significantly different in normal and CLL patients. Therefore, the five genes contained in our model were possibly functionally vital in the pathogenesis of CLL. In the present study, SLAMF1, TCF7, TNFRSF25, MNT, and VEGFA were protective factors, whereas GRWD1, SLC6A3, GMIP, and LAG3 appeared to be harmful factors in CLL; we subsequently discussed each gene in the prognostic model. \n\nTranscription factor 7 (TCF7), the T-cell-specific transcription factor required for T-cell development in animal models, suggests that it probably functions as a tumor suppressor (Roose et al., 1999). TCF7 over-expression in mice led to a disease resembling CLL, indicating that it was probably involved in the CLL transformation in a direct way (Bichi et al., 2002). In CLL, TCF7 expression provided a high rate (74%) of correct assignment of patients at genetic risk (IGHV unmutated, V3-21 usage, 11q-, or 17p-) (Kienle et al., 2010). The aforementioned results are consistent with ours, and this indicates TCF7 plays an important role in CLL. \n\nSignaling lymphocytic activation molecule family member 1 (SLAMF1), also known as CD150, regulates hematopoietic stem cell differentiation, leukocyte adhesion and activation, and humoral immune responses. SLAMF1 comparatively overexpress in normal peripheral blood B cells according to the meta-analysis of three gene expression profiling studies. Recently, researchers found lower levels of SLAMF1 expression in cases with ZAP70-high (p < 0. 001), IGHV-unmutated (p < 0. 001), and 17q-(p = 0. 003). In past studies, we believed that loss of SLAMF1 expression in CLL modulates genetic pathways regulating chemotaxis and autophagy and that potentially affects drug responses, suggesting that the effects underlie unfavorable clinical outcomes experienced by SLAMF1-low patients (Bologna et al., 2016). Together, SLAMF receptors, the vital modulators of the BCR signaling axis, improve immune control in CLL by potentially interfering with NK cells (von Wenserski et al., 2021). In our research, the univariate and multivariate analyses presented that downregulated SLAMF1 levels had an independent negative prognostic impact on overall survival (p < 0. 05). We subsequently discovered that SLAMF1 is relatively overexpressed in IGHV-mutated and ZAP70-low CLL patients. The strict correlation among low levels of it and high-risk genetic features indicated that it probably represented a marker of surrogate genomic complexity; however, the mechanism of this correlation is still unknown. \n\nLymphocyte activating 3 (LAG3), the immune inhibitory checkpoint receptor, is one of the immunoglobulin superfamily with about 20% amino acid homology with CD4. The expression of it activates and exhausts T, NK cells, B cells, dendritic cells, and regulatory T (Treg) cells. LAG3 high expression in CLL cells correlates with unmutated IGHV (p < 0. 0001) and decreased treatment-free survival (p = 0. 0087) (Kotaskova et al., 2010). Increased LAG-3 expression on leukemic cells correlates with shorter time-totreatment and poor outcome in CLL; moreover, treatment with relatlimab, a novel anti-LAG-3 blocking monoclonal antibody currently under clinical trial for different solid and hematological malignancies including CLL, restored, at least in part, NK and T-cell-mediated anti-tumor responses (Sordo-Bahamonde et al., 2021). CART cell generation with the showing of ibrutinib created enhanced cell viability and expansion of CLL patient-derived CART cells. Also, ibrutinib enriched the mentioned cells with the less-differentiated naïve-like phenotype and declined expression of exhaustion markers (PD-1, TIM-3, and LAG-3) (Fan et al., 2021). \n\nVascular endothelial growth factor A (VEGFA) is a member of the PDGF/VEGF growth factor family. The angiogenesis process makes a significant contribution to the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL), the levels of VEGFA and bFGF being higher in patients than in healthy people (Ballester et al., 2020). Whereas, in our research, VEGFA has a protective role in CLL. The high expression of VEGFA indicated a good prognosis by the K-M survival analysis, and in normal samples, the level of VEGFA was higher even though it was not statistically significant. \n\nThe TNF receptor superfamily member 25 (TNFRSF25), the receptor expressed preferentially in the tissues of lymphocytes, possibly plays functions vital to the regulation of lymphocyte homeostasis. The receptor stimulates sNF-kappa B activity and regulates cell apoptosis. TNFRSF25 was differentially expressed, activating CLL cells and predominantly detected in those with early clinical stage disease (Cavallini et al., 2015) and probably alters the balance between cell proliferation and death, influencing CLL physiopathology and results in the clinic. \n\nA total of three genes (GRWD1, GMIP, and SLC6A3) have not been described in the context of CLL before, and all of them were upregulated in high-risk CLL patients. The results of the univariate and K-M survival curves were not completely consistent with multivariate analysis. Glutamate-rich WD repeat containing 1 (GRWD1) was identified as one of the ribosomal/nucleolar proteins that promote tumorigenesis (Takafuji et al., 2017). Meanwhile, GRWD1 was also viewed as having histone-binding activity and regulating chromatin openness to specific chromatin locations (Sugimoto et al., 2015). Overexpression in colon carcinoma tissues was related to pathological grading, tumor size, N stage, TNM stage, and poor survival; knockdown of GRWD1 function as an inhibitor on cell proliferation and colony formation, and induced cell cycle arrest and more drug susceptibility, and suppressed the migration and invasion (Zhou et al., 2021). GEM interacting protein (GMIP), a RhoA-specific GAP, in a proteomics screen for proteins interacting with Girdin (Girders of actin), an actin-binding protein critical for neuronal migration to the olfactory bulbs, is identified as one of the major regulators of neuronal migration in the postnatal brain (Ota et al., 2014). Solute carrier family 6 member 3 (SLC6A3) involving in the metabolism of dopamine and catecholamine is the potential gene for Parkinson's disease and alcoholism. The significance of the aforementioned three genes in CLL remains to be further studied. \n\nIn GSE14973, the risk score was significantly downregulated after the valproic acid (VPA) treatment in vitro; meantime, protective factors (VEGFA and MNT) were highly expressed, and pathogenic genes (GMIP) were less expressed than in the previous treatment, except TNFRSF25, and these results were almost consistent with our previous conclusion. VPA is a welltolerated anti-epileptic drug with HDAC inhibitory activity. HDAC1 and HDAC3 inhibition or knockdown results could be figured out in HDAC7 downregulation, which was related to a decline in histone 3 lysine 27 acetylation (H3K27ac) at transcription start sites (TSS) and super-enhancers (SEs) prominently in stem-like BrCa cells. In GSE112953 and GSE15913, the only upregulated gene was LAG3, and it may suggest that combination drug treatment with an anti-LAG3 monoclonal antibody would have a better outcome. \n\nIn the present study, a nomogram based on the nine-gene risk score and other clinical traits was constructed, and to determine the predictive effect, we applied the nomogram to a specific patient in the ICGC project; moreover, the predictive model containing the nine-gene risk score was more accurate than the nomogram model containing only four clinical traits. Meanwhile, the risk score was strongly correlated with some known prognostic indicators, such as IGHV mutation state and chromosomal abnormalities. While, a further dissection of the nine-gene risk score on OS in the IGHV mutation state could identify that the nine-gene risk score value was apparent only in the less aggressive M-IGHV subtype, and this predicted trait corresponded to what Frontiers in Genetics frontiersin. org 10 has been reported in an article which studied the relationship between the ENDOG expression and prognostic study of CLL. The reason why this situation occurred needed further exploration. \n\nThe introduction of fludarabine, fludarabine/cyclophosphamide, and either of these combined with rituximab has improved the outcome for younger patients with CLL. Treatment options available for patients in the setting of relapsed disease following receipt of chemoimmunotherapy are limited where most patients have highrisk genomic findings including IgVH un-mutated disease, del (17p13. 1) and del (11q22. 3) associated with poor treatment response (reviewed in Rassenti et al., 2008). Identifying therapies with novel mechanisms of action for this patient group is important (Johnson et al., 2012). In our research study, all patients were divided into two risk subtypes based on the nine-gene prognostic model, and we endeavored to estimate the drug response of each patient based on IC50 according to the activation of different pathways. ADZ8055 was a dual mTOR kinase inhibitor with inhibition of both mTORC1 and mTORC2 that preferentially decreased cell viability of poor prognostic CLL subsets like with del (11q) or del (17p). One class of drugs that has promise for the treatment of relapsed CLL is the cyclin-dependent kinase (CDK) inhibitors (Seftel et al., 2017). Interestingly, one research study has described that the pan-CDK inhibitor dinaciclib has potent pre-clinical in vitro activity against CLL cells independent of high-risk genomic features (Johnson et al., 2012). In our drug sensitivity prediction, there are three kinds of CDK inhibitors which seemed to be more effective for high-risk CLL patients. The reasons that could account for this difference may include: 1) Different drugs have different mechanisms of action, although they are all one class of inhibitor. 2) The criteria of stratifying patients into \"High-risk\" and \"Low-risk\" were not consistent. 3) The most important point is the lack of experimental validation in our research.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "To sum up, it was the initial study using the LASSO model to screen prognostic indicators from the profile of SE-associated genes in CLL. A useful prognostic score for OS in untreated CLL patients was presented, and the determination of the score can be achieved via the measurement of the expression levels of nine genes. It also could be done easily in a routine diagnosis. These nine SE-associated genes in this model were not only vital in the development and progression of CLL, but also could assist in guiding the development of alternative treatments.",
"section_name": "Conclusion",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "The microarray data and clinical data of GSE22762 (Herold et al., 2011a) and GSE39671 (Chuang et al., 2012), which contain 107 and 130 CLL patients, respectively, were downloaded from the Gene",
"section_name": "Data source and microarray analysis",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This work was supported by the major subject of science and technology of Anhui province: (Grant number 201903a07020030 ).",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "LASSO-penalized cox regression analysis",
"section_num": null
},
{
"section_content": "Expression Omnibus (GEO) database. These data were conducted by GPL570 and GPL96/GPL97. Here, 9 other datasets were also analyzed for different purposes, and the details were presented in Table 1 (Fabris et al., 2008; Stamatopoulos et al., 2009a; Stamatopoulos et al., 2009b; Giannopoulos et al., 2009; Mosca et al., 2010; Herold et al., 2011b; Trojani et al., 2011). In the meantime, the International Cancer Genome Consortium (ICGC) CLL sequencing data were extracted from the European Genome-Phenome Database (EGA).",
"section_name": "",
"section_num": ""
},
{
"section_content": "Super-enhancer-related genes list figured from the primary B-CLL cell was downloaded from SEA version 3. 0, which was enriched with a post-translational modification histone mark, H3K27ac ChIP-seq signal. The gene matrix for subsequent analysis was obtained from the overlapping set of genes in the GSE22762 dataset and the SE-associated genes in the primary B-CLL cell. For narrowing and selecting the prognostic genes with potential, the overlapping gene matrix was weighted by the relative coefficients through the LASSO-penalized Cox regression. Tenfold cross-validation derived the best-fit lambda value to decrease the mean cross-validated error as much as possible via the R package \"glmnet\". We chose one median parameter to establish an ideal prognosis model. Then, we measured time-dependent ROC curves and calculated the area under the ROC (AUC).",
"section_name": "LASSO-penalized cox regression analysis",
"section_num": null
},
{
"section_content": "After LASSO-penalized Cox regression analysis was carried out, a risk score model was built using the aforementioned nine genes and could calculate a risk score for each sample through this formula: Risk score = GRWD1 *3. 69 -TCF7*2. 09 -VEGFA*0. 90-MNT* 2. 14 + GMIP*1. 23 -SLAMF1*0. 91 -TNFRSF25*2. 29+SLC6A3* 1. 79 + LAG3*0. 44 + 8. 67. Patients were separated into high-and low-risk cohorts (median risk score) using the R software \"survival\" and \"survminer\" packages, and a t-test was used to distinguish death and survival events according to the risk score.",
"section_name": "Risk score model establishment on predicting patient overall survival",
"section_num": null
},
{
"section_content": "Univariate Cox hazard regression analysis validated the correlation among the expression levels of nine genes and OS of each patient by the R package \"survival\" and \"survminer\". At the same time, multivariate Cox hazard regression analyses were performed too. We foresaw the regression coefficient (β-value) and HR. The K-M survival curve and log-rank test of every single gene were also performed by the R package referred previously.",
"section_name": "Cox proportional hazard regression model",
"section_num": null
},
{
"section_content": "Weighted gene co-expression network analysis (WGCNA) screened SE-associated hub genes differentially expressed between healthy donors and CLL patients. We counted out the optimal soft-threshold value under the scale independence and mean connectivity analyses. CLL-related genes were clustered into various modules and gained an intersection of significant models and SE-related gene lists via Venn diagrams.",
"section_name": "Weighted gene co-expression network analysis",
"section_num": null
},
{
"section_content": "Under the standard of risk score, we separated the participants into high-and low-risk group sets. Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis revealed a potential signaling pathway underlying the two sets via gene set enrichment analysis (GSEA v4. 1. 0 software). p < 0. 05 and a false discovery rate q < 0. 25 were thought to be vital in the statistic.",
"section_name": "Gene set enrichment analysis",
"section_num": null
},
{
"section_content": "A nomogram based on independent prognostic factors of clinical traits and the polygenic risk score was constructed to predict the probability of 1-, 3-, 5-, and 10-year OS of patients with CLL. Subsequently, the discrimination of the nomogram was verified using the C-index obtained through a bootstrap method with 1,000 resamples.",
"section_name": "Predictive nomogram for prognostic prediction",
"section_num": null
},
{
"section_content": "To predict the half-maximal inhibitory concentration (IC50) of chemotherapy drugs in the high-and low-risk groups of CLL patients and to infer the sensitivity of the different patients, we used the \"pRRophetic\" package in R.",
"section_name": "Evaluation of the sensitivity of chemotherapeutic agents",
"section_num": null
},
{
"section_content": "SPSS software vision 25. 0 (SPSS, Inc., Chicago, IL, United States) and R software vision 3. 6. 3 (R Foundation for Statistical Computing, Vienna, Austria) were used to analyze the data in statistics. A two-sided p < 0. 05 was thought vital in a statistic.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.",
"section_name": "Data availability statement",
"section_num": null
},
{
"section_content": "All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. XL, CL, and ZZ have wrote the main manuscript text. YM, LL, LH, and YW have prepared figures 2, 3, 4, 5, 6 and figures s1, s2, s3, s4, s5. LP and QL have prepared figure 1, figures 7, 8, figure s6 and table1.",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.",
"section_name": "Conflict of interest",
"section_num": null
},
{
"section_content": "All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.",
"section_name": "Publisher's note",
"section_num": null
},
{
"section_content": "The Supplementary Material for this article can be found online at: https://www. frontiersin. org/articles/10. 3389/fgene. 2022. 1001364/full#supplementary-material",
"section_name": "Supplementary material",
"section_num": null
}
] |
10.1186/1471-213x-8-75
|
Msx1 and Msx2are required for endothelial-mesenchymal transformation of the atrioventricular cushions and patterning of the atrioventricular myocardium
|
<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p> <jats:italic>Msx1</jats:italic> and <jats:italic>Msx2</jats:italic>, which belong to the highly conserved <jats:italic>Nk</jats:italic> family of homeobox genes, display overlapping expression patterns and redundant functions in multiple tissues and organs during vertebrate development. <jats:italic>Msx1</jats:italic> and <jats:italic>Msx2</jats:italic> have well-documented roles in mediating epithelial-mesenchymal interactions during organogenesis. Given that both <jats:italic>Msx1</jats:italic> and <jats:italic>Msx2</jats:italic> are crucial downstream effectors of Bmp signaling, we investigated whether <jats:italic>Msx1</jats:italic> and <jats:italic>Msx2</jats:italic> are required for the Bmp-induced endothelial-mesenchymal transformation (EMT) during atrioventricular (AV) valve formation.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>While both <jats:italic>Msx1-/-</jats:italic> and <jats:italic>Msx2-/-</jats:italic> single homozygous mutant mice exhibited normal valve formation, we observed hypoplastic AV cushions and malformed AV valves in <jats:italic>Msx1-/-; Msx2-/-</jats:italic> mutants, indicating redundant functions of <jats:italic>Msx1</jats:italic> and <jats:italic>Msx2</jats:italic> during AV valve morphogenesis. In <jats:italic>Msx1/2</jats:italic> null mutant AV cushions, we found decreased Bmp2/4 and <jats:italic>Notch1</jats:italic> signaling as well as reduced expression of <jats:italic>Has2</jats:italic>, <jats:italic>NFATc1</jats:italic> and <jats:italic>Notch1</jats:italic>, demonstrating impaired endocardial activation and EMT. Moreover, perturbed expression of chamber-specific genes <jats:italic>Anf</jats:italic>, <jats:italic>Tbx2</jats:italic>, <jats:italic>Hand1</jats:italic> and <jats:italic>Hand2</jats:italic> reveals mispatterning of the <jats:italic>Msx1/2</jats:italic> double mutant myocardium and suggests functions of <jats:italic>Msx1</jats:italic> and <jats:italic>Msx2</jats:italic> in regulating myocardial signals required for remodelling AV valves and maintaining an undifferentiated state of the AV myocardium.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>Our findings demonstrate redundant roles of <jats:italic>Msx1</jats:italic> and <jats:italic>Msx2</jats:italic> in regulating signals required for development of the AV myocardium and formation of the AV valves.</jats:p> </jats:sec>
|
[
{
"section_content": "A complex series of morphogenetic events and hemodynamic influences are required for cardiogenesis [1] [2] [3]. Malformations of cardiac valves constitute the most prevalent form of human birth defects, appearing in nearly one percent of newborn infants [4, 5]. The formation of cardiac valves requires two major consecutive steps: cardiac cushion formation and valve remodeling [1, 4, 6]. After cardiac looping, the cardiac cushions in the regions of the atrioventricular (AV) canal and distal outflow tract (OFT) are formed through an endothelial-mesenchymal transformation (EMT), a remarkably complex event initiated by the specification and activation of a subset of endothelial cells in the cushion-forming regions. This event is followed by cell delamination from the endocardium and cell migration into the extracellular matrix between the endocardium and myocardium (referred to as the cardiac jelly) [4] [5] [6]. Concomitant with migration into the cardiac jelly, the endothelial cells transdifferentiate into mesenchymal cells and proliferate to form multiple layers, resulting in the expansion of the cushion crests toward each other [6]. AV cushion formation is followed by a series of morphogenetic events, including elongation, outgrowth and remodeling, which result in the conversion of the thick cushions into thin valve leaflets [1, [6] [7] [8]. Several signaling molecules have been implicated in regulating EMT during cardiac valve formation, including the Nuclear Factor in Activated T cells (NFAT) [9] [10] [11] [12], Vascular Endothelial Growth Factor (VEGF) [11, 13], and members of the Epidermal Growth Factor (EGF) [1, 6, 14, 15], Bone Morphogenetic Protein (Bmp) [16] [17] [18] [19], Notch [20] [21] [22], Transforming Growth Factor-β (TGF-β) [4, 18, 19, 23], and Wnt/β-catenin families [24, 25]. \n\nMsx1 and Msx2, closely related members of the Nk-family of homeobox transcription factors, have well-documented roles as both downstream effectors and upstream regulators of Bmp signalling [26] [27] [28] [29]. Msx1 and Msx2 function redundantly in multiple tissues and organs during vertebrate development, including the heart [30, 31]. We showed previously that Msx1-/-; Msx2-/-mutants exhibit malalignment defects of the developing outflow tract including double outlet right ventricle and pulmonary atresia or stenosis [30, 32]. These defects are associated with excessive proliferation of cardiac neural crest, endothelial and myocardial cells in the mutant outflow tract between E10 and E11 [32]. \n\nReduced expression of Msx1 and Msx2 was observed in the AV cushions deficient in Bmp signaling. Such cushions also displayed immature cardiac jelly, compromised AV myocardium and hypoplastic AV valves [17, 33]. However, no valve defects have been reported in mice deficient in either Msx1 or Msx2 [34, 35]. \n\nIn the present study, we focused on AV cushion and valve formation in mice with combined deficiencies of Msx1 and Msx2, and compared marker gene expression in Msx1-/-; Msx2-/-double mutant AV cushions with that in Msx1-/-and Msx2-/-single mutant AV cushions. We observed hypoplastic AV cushions and deformed AV valves in Msx1-/-; Msx2-/-double mutants but not in Msx1-/-or Msx2-/single mutants, and no discernable difference in the expression of AV cushion markers between wild-type and single mutant mice. On the other hand, there was a reduced level of NFATc1 immunostaining in the Msx1/2 double mutant AV endocardium, and decreased expression of α-smooth muscle actin, Notch1, Has2, Bmp2/4 and Pitx2 in the Msx1/2 double mutant AV cushion mesenchyme, indicating impaired EMT and cushion formation [4, 9, 10, 12, 16, 17, 21, 22, [36] [37] [38] [39] [40] [41]. In addition, perturbed expression of Bmp2/4, Tbx2, Anf, Hand1 and Hand2 in the Msx1/2 null mutant AV myocardium suggests impaired myocardial patterning during chamber formation [17, [42] [43] [44] [45] [46] [47] [48] [49]. Taken together, we conclude that Msx1 and Msx2 function redundantly in regulating the expression of genes required for AV canal (AVC) morphogenesis.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Histological sections of hearts between E14. 5 and E16. 5 revealed atrial septal defect as well as deformed AV cushions and valves in Msx1-/-; Msx2-/-double mutants (n = 8), but not in Msx1-/-or Msx2-/-single mutants (n = 22), nor in Msx1-Msx2 homozygous-heterozygous compound mutants (n = 20). All eight Msx1/2 null mutants examined had hypoplastic AV cushions and shorter but thickened AV valves, indicating a failure to undergo proper valve outgrowth and remodeling (Fig. 1B, C, E and 1F). \n\nTo evaluate the functional redundancy of Msx1 and Msx2 in AV cushion morphogenesis, we examined expression patterns of Msx1 and Msx2 in the AV canal (AVC) between E9. 5 and E11. 5, while EMT of the endocardial cushions was actively taking place [4, 16, 18]. At E9. 5 and E10. 5, Msx1 and Msx2 displayed overlapping expression in the endocardial and cushion mesenchymal cells, while Msx2 was also expressed in the AV myocardium, where no Msx1 expression was detected (Fig. 2A-D ). By E11. 5, Msx1 expression extended to the lateral cushion mesenchyme adjacent to the AV myocardium as well as the membranous parts of the interatrial and interventricular septa (Fig. 2E ). Our observations suggest that Msx1 and Msx2 may function redundantly during AV endocardial EMT and AV cushion morphogenesis. \n\nWe then studied whether endocardial cell maturation and activation were perturbed in Msx1/2 double mutants. Immunostaining for NFATc1 (nuclear factor in activated T cells), which was previously shown to be upregulated in activated endothelial cells within the AVC [10, 12, 17], indicated that NFATc1 was present in the Msx1/2 double mutant AV endocardium at E10. 5, but with a significantly lower staining intensity compared to the control AV endocardium (compare the regions pointed by red arrows in Fig. 3A and 3B ). In contrast, the intensity of NFATc1 immunostaining in the OFT endocardium was comparable between controls and Msx1-/-; Msx2-/-mutants (compare the regions indicated by red asterisks in Fig. 3A and 3B ). At the same developmental stage, the expression of Notch1, an essential inducer of EMT [4, 21, 22], was greatly diminished in the Msx1/2 double mutant AV cushions compared with the control (compare the regions pointed by red arrows in Fig. 3C and 3D ). In contrast, Notch1 expression in the Msx1/2 double mutant OFT cushions was comparable with that in the control OFT (compare the regions marked by red asterisks in Fig. 3C and 3D ). On the other hand, the expression of another endocardial marker gene Twist1 in Msx1-/-; Msx2-/-double mutants was comparable with that in control embryos (data not shown). \n\nWe also analysed expression of Has2, which is the principle synthase of the extracellular matrix component hyaluronan (HA) [1, 36], in the AV cushion mesenchyme of the Msx1-/-; Msx2-/-mutants. HA has been demonstrated to mediate endocardial cushion expansion by interacting with water and other extracellular matrix components to create a hydrated low-resistance macromolecular environment that promotes loss of cell contact inhibition and increased cell motility, which is a prerequisite of cardiac cushion expansion and subsequent EMT [1, 36, [50] [51] [52]. Our results indicated normal expression of Has2 in spite of a greatly decreased number of Has2-expressing cells in the Msx1/2 double mutant AV cushion mesenchyme (compare the regions pointed by red arrows in Fig. 3E and 3F ). Statistical analyses of multiple sections across the AV cushions revealed that, in spite of normal cell proliferation and apoptosis, there was a significant reduction in the total number of mesenchymal cells in Msx1-/-; Msx2-/-mutants compared with their littermate controls (Fig. 4 and data not shown). In addition, we found that there were only sparse mesenchymal cells expressing α-smooth muscle actin (α-SMA) in the Msx1/2 double mutant AV cushions, whereas the majority of mesenchymal cells in the control AV cushions are α-SMA-positive (compare the regions indicated by red asterisks in Fig. 4C, 4D, 4G and 4H ). As α-SMA was previously shown to be a marker of EMT [39], our results further support impaired EMT in the Msx1/2 null mutant AV cushions, which may contribute to a decreased total number of cushion mesenchymal cells.",
"section_name": "Hypoplastic AV cushions and impaired endocardial signaling in Msx1-/-; Msx2-/-mutant hearts",
"section_num": null
},
{
"section_content": "",
"section_name": "Expression of Msx1 and Msx2 in the developing AV canal",
"section_num": null
},
{
"section_content": "A variety of Bmps (bone morphogenetic proteins), including Bmp2, Bmp4, Bmp5, Bmp6 and Bmp7, have been implicated in regulating EMT during OFT and AV valve formation [16] [17] [18] 33, 40, 41, 53, 54]. As Msx1 and Msx2 both have been shown to be upstream regulators of Bmp2, Bmp4 and Bmp7 during organogenesis [30, [55] [56] [57], we asked whether the EMT-regulating Bmp signals were deficient in Msx1/2 mutant AV cushions. Immunostaining for Bmp2/4 and phosphorylated Smad1/5/8 at E10. 5 and E11. 5 revealed significantly decreased Bmp2/4 signaling (more than 50% decrease at E10. 5 and more than 30% decrease at E11. 5) in the AV cushion mesenchyme of Msx1-/-; Msx2-/-mutants compared with their littermate controls (Fig. 5E-H, 5M-P, 5Q and 5R; AV cushions are marked by red and yellow asterisks). In addition to the AV cushions, we also found decreased Bmp2/4 signaling in the atrial and ventricular myocardium of Msx1/2 double mutants (indicated by white triangle arrowheads in Fig. 5A-D and 5I-L ). Therefore, in contrast to the local upregulation of Bmp2/4 signaling in the Msx1/2 null mutant OFT and pharyngeal mesoderm (white arrows in Fig. 5A-D, and our previous study) [32], down-regulation of Bmp2/4 signaling in Msx1/2 double mutants is restricted to the AV cushions and chamber myocardium. \n\nPrevious studies have demonstrated that Bmp2 induces Tbx2 (T-box transcription factor 2) expression in the AV myocardium, which in turn inhibits the expression of chamber-specific genes including Anf (atrial natriuretic factor), Chisel and Cx40 in the AVC, and thus establishes the identity of chamber myocardium [17, 40, 42, 43, 49]. To investigate whether reduced Bmp2/4 signaling in Msx1/2 mutant AVC perturbed gene expression in the AV myocardium, we compared expression of Tbx2 and Anf in controls and Msx1/2 double mutants. In Msx1-/-; Msx2-/mutants, Tbx2 expression was dramatically reduced in the AV myocardium (compare the regions pointed by red arrowheads in Fig. 6A and 6B ). This is a local down-regulation, as Tbx2 expression in the pharyngeal mesoderm was comparable between controls and Msx1/2 double mutants (compare the regions pointed by black arrows in Fig. 6A and 6B ). In agreement with decreased Tbx2 expression, Msx1/2 double mutants displayed increased and ectopic expression of Anf in the AV myocardium (compare the regions pointed by red arrowheads in Fig. 6C and 6D ). Anf expression was also increased in the myocardium of the double mutant right ventricle (marked by the red asterisk in Fig. 6D ). \n\nIn addition to Tbx2, other transcription factors that have been implicated in regulation of Anf expression, including Hand1 [46], Hand2 [47], and Pitx2 [58], also exhibited dramatically reduced expression in Msx1-/-; Msx2-/-Impaired expression of endocardial and cushion mesenchy-mal genes in Msx1-/-; Msx2-/-mutant AV cushions mutant AVC (Fig. 6E -H and Fig. 7 ). Hand1 is normally expressed in the left AV myocardium [45, 48, 59], but this was almost undetectable in Msx1/2 double mutants (compare the regions pointed by red arrows in Fig. 6E and 6F ). In addition, Hand1 expression was significantly decreased in the left ventricular myocardium of the Msx1/2 null mutants compared with the controls (marked by a red asterisk in Fig. 6F ; three pairs of Msx1-/-; Msx2-/-mutants and littermate controls were analysed). Similar to Hand1, there was an overall decrease of Hand2 expression in the Msx1/2 double mutant myocardium (compare Fig. 6G and 6H ). In fact, we have previously demonstrated reduced Hand1 and Hand2 expression in the Msx1/2 null mutant OFT and secondary heart field, suggesting that Hand1 and Hand2 are target genes regulated by Msx1 and Msx2 [32]. We found that, in contrast to the control AV cushions, where approximately 40-60% of mesenchymal cells expressed Pitx2 (Fig. 7C, 7G and 7I; AV cushions are marked by yellow asterisks), there were only sparse Pitx2expressing mesenchymal cells (average %10%) in the Msx1/2 double mutant AV cushions (Fig. 7D, 7H and 7I ). In addition, there was a substantial reduction of Pitx2 immunostaining signal in the myocardium of the Msx1/2 double mutant left atrium shown in Fig. 7B and 7F (indicated by white arrows; compare with Fig. 6A and 6E ). Multiple lines of evidence have shown that loss of Hand1 or Pitx2 expression in the AVC cause AV valve defects [37, 38, 45, 60]. Myocardium-specific deficiency of Hand1 was shown to cause hyperplastic AV cushions and thickened AV valves [45], implicating Hand1 in post EMT valve remodelling [6]. Pitx2 may be required for both EMT and post EMT cushion morphogenesis, as Pitx2 descendents were reported to be indispensable for late AV cushion formation and AV valve remodelling [38].",
"section_name": "Loss of function of both Msx1 and Msx2 perturbs myocardial signaling in the AV canal",
"section_num": null
},
{
"section_content": "Previous studies have demonstrated that Msx1 and Msx2 are both expressed in the AV endocardium, while Msx2 is also expressed in the cushion mesenchyme and AV myocardium [17, 33, 61, 62]. In this study, we demonstrated overlapping expression of Msx1 and Msx2 in not only the AV endocardium but also the cushion mesenchyme (Fig. 2 ). In support of the overlapping expression patterns, Msx1 and Msx2 exhibited redundant functions in the AV endocardium and cushion mesenchyme during EMT. We found that Msx1 and Msx2 function redundantly to upregulate NFATc1 expression in the AV endocardium and maintain Notch1 and Bmp2/4 expression in the AV cushions during EMT (Fig. 3A -D and Fig. 5 ). Previous studies demonstrated that NFATc1 signaling during valve morphogenesis is dispensable for EMT (between E8. 5 and E10. 5) but is required for remodeling of the endocardial cushions into mature valve leaflets (after E11) [9, 10, 12, 63]. NFATc1 was found to regulate valve remodeling by transcriptional activation of matrix degrading enzymes including cathepsin K, whose substrates are collagen and elastin, via the RANKL signaling pathway [63]. Notch signaling in the AV endocardium was reported to promote loss of cell-cell contact via down-regulation of endocardial VE-cadherin and induce EMT via upregulation of myocardial TGF-β2 [4, 22]. To initiate EMT, Bmp2 acts synergistically with TGF-βs to enhance the TGF-β-induced phenotypic changes associated with EMT [16, 18, 19, 40]. \n\nWe failed to detect any significant change of expression of Twist1, which has been implicated in down-regulation of E-cadherin and VE-cadherin during epithelial-or endothelial-to-mesenchymal transition [17, 64, 65]. It is likely that the expression levels of other transcription factors which also repress E-cadherin/VE-cadherin expression, including Snail and Slug [4, 64], are decreased in the Msx1-/-; Msx2-/ -mutant AV endocardium. In fact, Snail expression is activated by Notch signaling [22]. It remains to be determined whether the expression of Snail and VE-cadherin are perturbed in the Msx1/2 double mutant AV endocardium. \n\nIn agreement with impaired EMT, we observed significantly decreased total numbers of mesenchymal cells in the Msx1-/-; Msx2-/-mutant AV cushions (Fig. 4I ). Furthermore, only a small proportion of mesenchymal cells in the Msx1/2 double mutant AV cushions expressed α-SMA, suggesting impaired differentiation during EMT (Fig. 4D and 4H ). On the other hand, both cell proliferation and cell survival were normal in Msx1-/-; Msx2-/-mutant AV cushions during EMT (Fig. 4A, B, E, F and data not shown). \n\nInterestingly, we found decreased Bmp2/4 signaling and expression of Tbx2, Hand1 and Hand2 in the Msx1-/-; Msx2-/-mutant myocardium, including the AV myocardium, which expresses Msx2 but not Msx1 in wild-type embryos, and the chamber myocardium, where neither Msx1 nor Msx2 is normally expressed. One possible explanation is that perturbed gene expression in the double mutant myocardium is a secondary effect of hemodynamic changes due to the absence of normal AV cushions. Reduced expression of both Hand1 and Hand2 in the Msx1/2 null mutant myocardium further supports our previous hypothesis that Hand1 and Hand2 are candidate target genes regulated by Msx1 and Msx2 [32]. Decreased Hand1 expression in the Msx1/2 double mutant AV myocardium may be associated with defects in remodeling of the AV cushions into mature valve leaflets, since myocardium-specific Hand1 deficiency led to thickened AV valves [45], which were previously shown to be associated with impaired valve remodeling [1, 6, 15]. \n\nIt is noteworthy that, in contrary to decreased Bmp2/4 signaling in the Msx1/2 null mutant AVC and chamber myocardium, our previous study demonstrated increased Bmp2/4 signaling in the double mutant OFT myocardium and cushion mesenchyme [32]. It has been shown that Bmp2 expression is normally switched from the OFT myocardium to the AVC and atrial myocardium between E9. 5 and E10. 5 [16, 61, 66], suggesting that an alternative explanation for perturbed Bmp signaling in Msx1-/-; Msx2-/mutant hearts is that Bmp2 expression does not shift from the OFT to the AVC in the double mutants. Locally reduced Bmp2 expression in the Msx1/2 null mutant AVC may be insufficient to maintain normal expression of Tbx2 and in turn lead to reduced Tbx2 and increased Anf expression in the double mutant AV myocardium (Fig. 6A-D ) [17, 40, 42, 43, 49]. Perturbed Bmp2 expression may also disrupt normal Pitx2 expression in the Msx1-/-; Msx2-/-mutant AVC and atrial myocardium, as Bmp2 has been shown to be a positive regulator of Nodal signaling and Pitx2 [67]. In addition, there may be altered expression patterns of other Bmp molecules, including Bmp6, that contribute to the abnormal distribution of Bmp signals in the Msx1-/-; Msx2-/-mutant OFT and AV cushions. Interestingly, Bmp6 exhibits an asymmetric (left-sided) expression in the OFT myocardium at E10. 5, reminiscent of the expression pattern of Pitx2 [53]. Bmp6 expression undergoes a transition from the AV cushion mesenchyme to the OFT cushion mesenchyme between E10. 5 and E12. 5, indicating that it plays a critical role in both the EMT of the AV cushions and the development of the OFT cushions [16, 53]. Further analyses of the expression patterns of the aforementioned Bmp ligands will determine which Bmp molecules are critical contributors to the impaired development of the Msx1-/-; Msx2-/-mutant OFT and AV cushions.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In this study, we documented redundant functions of Msx1 and Msx2 genes in distinct aspects during AV cushion morphogenesis: endocardial activation prior to EMT (via upregulation of NFATc1 expression), induction of EMT (via upregulation of Notch1 and Bmp2/4 signaling in the AVC), as well as post-EMT valve remodelling (via upregulation of Hand1 expression in the AV myocardium).",
"section_name": "Conclusion",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "All mice used in this study were maintained in a mixed genetic background of BALB/c and CD-1. Msx1-/-and Msx2-/-single knockout and Msx1-/-; Msx2-/-double knockout mice were described previously [30, 34, 35]. The noon copulation plug was counted as embryonic day 0. 5 (E0. 5). Genomic DNA was extracted from yolk sac (embryos) or tails (postnatal mice) for genotyping. PCR primers and conditions for Msx1 and Msx2 knockout alleles as well as the Wnt1-Cre and R26R transgenes were as described [34, 35, 68].",
"section_name": "Mouse strains and genotyping",
"section_num": null
},
{
"section_content": "For histology, embryos at E15. 5 were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 14 hours at 4°C, dehydrated through graded ethanol and embedded in paraffin wax. Sections were cut at 7-8 μm and stained with Hematoxylin and Eosin. For cryostat sectioning, fixed embryos at E10. 5 were dehydrated through graded methanol and stored in 100% methanol at -20°C till use. Stored embryos were rehydrated through graded methanol, washed in PBS, and cryopreserved in sucrose solutions with increasing concentrations, then frozen in O. C. T Sections were cut at 10-μm thickness and RNA section in situ hybridization was performed using the In Situ Hybridization kit (BioChain) with the protocol modified according to Dijkman et al. [69]. All RNA probes were generated as described: Anf [17], Hand1 [45], Hand2 [45], Has2 [17], Notch1 [17], Pitx2 [38], and Tbx2 [17]. \n\nFor immunostaining, the following primary antibodies were used: biotinylated goat polyclonal anti-BMP-2/4 (1:100, R&D Systems), mouse monoclonal anti-bromodeoxyuridine (BrdU) (1:100, Sigma), mouse monoclonal anti-NFATc1 (1:200, BD Pharmingen; a kind gift from Dr. Kaartinen), guinea pig polyclonal anti-Pitx2 (1:200, a kind gift from Dr. Kioussi [70] ), rabbit polyclonal antiphospho-Smad1/5/8 (1:50, Cell Signaling). \n\nLittermates of each Msx1-/-; Msx2-/-mutant embryo were used as the controls, and no discernable variation in either gene expression or cell proliferation/survival/differentiation was detected between the controls from the same litter and with different genotypes. \n\nReduced Bmp2/4 signaling in Msx1-/-; Msx2-/-mutant AV cushions and myocardium during EMT",
"section_name": "Histology, section in situ hybridization and immunostaining",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We are grateful to Dr. Chrissa Kioussi for the kind gift of anti-Pitx2 antibody, Dr. Vesa Kaartinen for the kind gift of anti-NFATc1 antibody, and Drs. James F. Martin and Eric N. Olson for the cRNA probes. This work was supported by NIH grants DE12941 and DE12450 to RM.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "Anf: Atrial natriuretic factor; AV: Atrioventricular; Bmp: Bone morphogenetic protein; EMT: Endothelial-mesenchymal transformation; NFAT: Nuclear factor in activated T cells; PBS: Phosphate-buffered saline; PFA: Paraformaldehyde; TGF: Transforming growth factor.",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "Y-HC designed most of the experiments, carried out mouse dissections at E10. 5 and E11. 5, analyzed the results and drafted the manuscript. MI performed mouse dissections and histological analyses at E15. 5, and first observed the phenotype of malformed AV valves in Msx1-/-; Msx2-/-double mutants. HMS diagnosed the AV cushion and valve defects in Msx1/2 double mutant mice. REM conceived, funded and supervised the project, which was carried out in his laboratory. Both HMS and REM critically read and revised the manuscript. All authors have read and approved the final manuscript.",
"section_name": "Authors' contributions",
"section_num": null
}
] |
10.1186/s13046-019-1076-4
|
Combination of Enzastaurin and Ibrutinib synergistically induces anti-tumor effects in diffuse large B cell lymphoma
|
Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma. Although durable remissions can be achieved in more than half of these patients, DLBCL remains a significant clinical challenge, with approximately 30% of patients not being cured. BCR-associated kinases (SYK, BTK, and PI3K) inhibitors have exhibited encouraging pre-clinical and clinical effects, as reported by many researchers. Early studies demonstrated that protein kinase C-β (PKCβ) inhibitors alter phosphorylation level the Bruton's tyrosine kinase (BTK), which leads to enhanced BTK signaling. Here, for the first time, we investigate whether the combination of PKCβ inhibitor enzastaurin and BTK inhibitor ibrutinib has synergistic anti-tumor effects in DLBCL.In vitro cell proliferation was analyzed using Cell Titer-Glo Luminescent Cell Viability Assay. Induction of apoptosis and cell cycle arrest were measured by flow cytometry. Western Blotting analysis was used to detect the essential regulatory enzymes in related signaling pathways. RNA-seq was conducted to evaluate the whole transcriptome changes brought by co-treatment with low doses of enzastaurin and ibrutinib. The synergistic anti-tumor effects of enzastaurin and ibrutinib were also evaluated in vivo.Combination of enzastaurin and ibrutinib produced a lasting synergistic effect on the survival and proliferation of DLBCL cells, including reduction of proliferation, promoting apoptosis, inducting G1 phase arrest, preventing cell invasion and migration, and down-regulating activation of downstream signaling. More importantly, whole-transcriptome changes results showed that combination therapy worked synergistically to regulate whole-transcriptome expression compared with enzastaurin and ibrutinib alone. Co-treatment with low doses of enzastaurin and ibrutinib could effectively downregulate BCR, NF-κB, JAK and MAPK related signaling pathway. Furthermore, the mRNA expression analysis further indicated that co-treatment significantly decreased the mRNA levels of NOTCH1. The combination effect in inhibiting proliferation of DLBCL cells probably was realized through suppression of NOTCH1 expression. Finally, the anti-tumor activity of co-treatment also was demonstrated in vivo.Combination of enzastaurin and ibrutinib had synergistic anti-tumor effects in DLBCL, independent of molecular subtype. These results provided a sound foundation for an attractive therapeutic treatment, and the simultaneous suppression of BTK and PKCβ might be a new treatment strategy for DLBCL.
|
[
{
"section_content": "Diffuse large B cell lymphoma (DLBCL), the most common form of lymphoma, is characterized by a heterogeneous tumor entity that can vary in morphologic, biological, immunophenotypic, and clinical presentation, as well as therapeutic outcome [1]. Gene expression profiling can be used to differentiate two subtypes of DLBCL, germinal center B-cell like (GCB) and activated B-cell-like (ABC) subgroups of DLBCL, leaving approximately 10~20% of cases \"unclassified\" [1]. ABC and GCB DLBCL are characterized by activation of different cellular pathways, posing a major barrier for developing a clear understanding of tumor development, maintenance, and response to therapy [2]. Although durable remissions are achieved in more than half of DLBCL patients, the disease remains a major clinical challenge, with approximately 30% of patients not being cured [3]. Especially as relapsed/refractory DLBCL patients involve poor survival, novel and effective therapeutic strategies are urgently needed. \n\nAbnormal B-cell receptor (BCR) signaling has been implicated in the pathogenesis of B-cell malignancy, which is widely appreciated as one of the primary mechanisms underlying disease progression [4, 5]. Continuous activation of BCR in ABC-type DLBCL leads to the phosphorylation and activation of regulatory and adaptor proteins, such as spleen tyrosine kinase (SYK), Bruton's tyrosine kinase (BTK), and protein kinase C-β (PKCβ), especially in ABC-type DLBCL [2, [6] [7] [8]. By contrast, oncogenic signaling in GCB DLBCL is typically initiated and reinforced by sharing a dependence on PI3K/mTOR signaling, which is independent of nuclear factor κB (NF-κB) [9, 10]. In recent years, an increasing number of studies have focused on the therapeutic inhibition of BCR signaling, especially combination-based therapeutic regimens for treating DLBCL [6, 11]. \n\nEnzastaurin, a potent and selective oral inhibitor of several PKC isoforms, has been shown to regulate the PI3K/ AKT/mTOR, MAPK, and JAK/STAT pathways in solid and hematological malignancies [12] [13] [14]. Although enzastaurin showed promising result in preclinical studies and Phase I/II clinical trials in DLBCL, recent Phase III clinical trials did not meet the primary end point [15] [16] [17]. Interestingly, some researchers have found that PKCβ works as a feedback loop inhibitor of BTK activation, which modulates signaling pathways via altering BTK membrane localization [18, 19]. PKCβ downregulates BTK activation via both transphosphorylation at Tyr551 and autophosphorylation at Tyr223. Thus, enzastaurin-mediated inhibition of PKCβ leads to enhanced membrane targeting of BTK, increased phosphorylation of PLCγ2, and amplified BCR-mediated Ca 2+ signaling [19]. \n\nIbrutinib is an irreversible small molecule BTK inhibitor that has clearly demonstrated promising therapeutic effects in a variety of B cell malignancies [2, [20] [21] [22]. Therefore, we aimed to investigate whether the combination of PKCβ inhibitor enzastaurin and BTK inhibitor ibrutinib has synergistic anti-tumor effects in DLBCL. We demonstrated that low doses of enzastaurin and ibrutinib act synergistically to suppress growth of both ABC and GCB DLBCL cells in vitro and in vivo. These results provide support for future investigation of the combination of enzastaurin and ibrutinib as an attractive therapeutic option for patients with both subtypes of DLBCL.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "Cell lines and cell culture HBL-1, TMD8, OCI-LY7 cell lines were generously provided by Dr. Fu, University of Nebraska Medical Center (Omaha, NE, USA). SU-DHL-2 and SU-DHL-6 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Cells were grown in RPMI 1640 medium (Gibco, Life Technologies, CA, USA) supplemented with 10-20% fetal bovine serum (Gibco, Life Technology, CA, USA), penicillin/ streptomycin, glutamine, beta-mercaptoethanol. Except for OCI-LY7, which was maintained in IMDM (Gibco, Life Technology, CA, USA) supplemented with beta-mercaptoethanol, penicillin/ streptomycin, and 20% heparinized human plasma. All cell lines were maintained in a humidified 5% CO 2 incubator at 37 °C. Identification of all DLBCL cell lines was confirmed by short tandem repeat DNA fingerprinting analysis (Applied Biosystems, Foster City, CA, USA).",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Enzastaurin was a gift from Denovo Biopharma (San Diego, USA), and ibrutinib was purchased from Medchem Express (NJ, USA). It was initially dissolved in 100% DMSO (Sigma-Aldrich, Darmstadt, Germany) at a concentration of 10 μM and stored in -80 °C. Primary and secondary antibodies were listed in additional file (Additional file 1: Table S1 ).",
"section_name": "Drugs and reagents",
"section_num": null
},
{
"section_content": "Cells were seeded in a 96-well culture plate at a density of 3000 cells per 100 μl and treated with different concentrations of enzastaurin and ibrutinib for 72 h. Cells were counted and viability was assessed using Cell Titer-Glo Luminescent Cell viability assay system (Promega, Madison, WI, USA). Luminescent signals were measured by LMax II (Molecular Devices, Sunnyvale, CA, USA). Inhibition rates were calculated following the formula: inhibition rates = (1-dosing/control) × 100%.",
"section_name": "Analysis of cell proliferation",
"section_num": null
},
{
"section_content": "Cells were treated with vehicle or indicated concentrations of enzastaurin and ibrutinib for 48 h for apoptosis and cell cycle analysis. For apoptosis assays, cells were stained with annexin V-APC (Biolegend, CA, USA) according to the protocol. For cell cycle assays, cells were stained with PI staining buffer (Sigma-Aldrich, Darmstadt, Germany) according to the manufacturer's protocol. Finally, the labeled cells were analyzed using BD Accuri C6 flow cytometer (BD, Biosciences, San Jose, CA).",
"section_name": "Apoptotic cells and cell-cycle assays",
"section_num": null
},
{
"section_content": "Total cellular RNA was extracted using Trizol reagent (Life Technologies, Carlsbad, CA) and cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). qRT-PCR analysis was performed using Go Taq qPCR Master Mix (Promega Corporation, Madison, USA). Specific primers for NOTCH1 (Forward: 5′-TCCACCAGTTTGAATGGTCAAT-3′; Reverse:\n\n5′-CGCAGAGGGTTGTATTGGTTC-3′) and GAPDH (Forward: 5′-GCACCGTCAAGGCTGAGAAC-3′; Reverse: 5′-TGGTGAAGACGCCAGTGGA-3′) were used to perform qRT-PCR. All reactions were run in Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Woburn, MA, USA), mRNA expression data were calculated using the following equation: RQ = 2 -ΔΔCt.",
"section_name": "Real-time reverse transcription-PCR (qRT-PCR) assay",
"section_num": null
},
{
"section_content": "Harvested cultured cells were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA) with protease/ phosphatase inhibitor (Roche, Mannheim, Germany). Signaling proteins were detected by western blot as previously described [23]. Immunopositive bands were visualized using chemiluminescence detection system (Alpha Innotech, San Leandro, CA, USA) according to the manufacturer's instructions.",
"section_name": "Western blotting and signaling assays",
"section_num": null
},
{
"section_content": "Cells were treated with vehicle or indicated concentrations of enzastaurin and ibrutinib for indicated time in FBS-free RPMI 1640. For cell invasion assays, cells were placed into Matrigel basement membrane matrix-coated upper chambers in a transwell plate with 8. 0 μM pores (Corning Costar, NY, USA). For cell migration assays, cells were seed into transwell with 8. 0 μm pore polycarbonate membrane insert (Corning Costar, NY, USA). The lower portion of the chamber contained 30% FBS for use as a chemoattractant. After 24 h (48 h), the number of cells migrating (invading) into the lower chamber were counted using Cell Titer-Glo Assays. Invasive and migration abilities were determined by the number of viable cells in the lower chamber.",
"section_name": "Invasion and migration assay",
"section_num": null
},
{
"section_content": "Cells were treated with the indicated drug alone or in combination for 24 h, and then total RNA was isolated. Total RNA (3 μg) was converted to cDNA using TransScript First-Strand cDNA Synthesis SuperMix. RNA quantification and qualification, library preparation, clustering and sequencing, read mapping and data processing were performed in Novogene Bioscience (Beijing, China). Differential expression analysis of two groups (two biological replicates per group) was performed using the DESeq2 R package (1. 16. 1). Corrected P-value of 0. 05 and absolute foldchange of 2 were set as the threshold for significantly differential expression. To analyze the underlying mechanism of the sets of genes which were differentially expressed following each treatment, we used clusterProfiler R package to test the statistical enrichment of differential expression genes in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.",
"section_name": "Gene expression profiling and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis",
"section_num": null
},
{
"section_content": "Lentiviral vectors (GV493) containing green fluorescent protein (GFP) (shControl) or NOTCH1-specific short hairpin RNA (shNOTCH1, sequence #1: 5′-TGCCAACATCCAGG ACAACAT-3′) were constructed, packed, and purified by Genechem (Shanghai, China). Cells were infected with shControl, shNOTCH1, at MOI 1: 100 and cultured for > 72 h to be used for the downstream experiments. The depletion efficiency was assessed by western blot analysis.",
"section_name": "Lentivirus packing and infection",
"section_num": null
},
{
"section_content": "All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals and in accordance with the ethical guidelines of CrownBio (Beijing, China). Female immune-deficient NPG mice (NOD-Prkdcscid Il2rgnull), six to eight weeks old, were obtained from HFK Bioscience Co. Ltd. (Beijing, China). HBL-1 tumor cells (5 × 10 6 ) in serum-free medium with matrigel (1:1 ratio) were injected subcutaneously into the area under the right flank of each mouse. When the tumor reached 100-150 mm 3, mice were randomly divided into four groups (control, treated with enzastaurin, treated with ibrutinib, treated with both enzastaurin and ibrutinib). Enzastaurin (125 mg/kg, dissolved in 10% Acacia) was administered twice daily orally and/or ibrutinib (12 mg/kg, dissolved in 1% methylcellulose, 0. 4% Cremophor® EL) was administered once daily orally for 21 days. Tumor volume (V) and body weight were monitored two to three times per week. The tumor volume (V) was calculated as V = (length×width 2 ) /2. Tumor tissue samples were collected from all groups at 4 h after the last dose.",
"section_name": "Detection of treatment efficacy in vivo",
"section_num": null
},
{
"section_content": "TUNEL is a method for detecting DNA fragmentation by labeling the 3′-hydroxyl termini in the double-strand DNA breaks generated during apoptosis. HBL-1 tumor samples were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 5 μm sections. A TUNEL assay was then conducted to examine DNA fragmentation using an in situ cell death detection kit (Cat No. 11684795910, Roche, Mannheim, Germany) according to the manufacturer' s instructions.",
"section_name": "In situ apoptosis quantification by TUNEL",
"section_num": null
},
{
"section_content": "Immunohistochemistry stains for Ki-67, p-BTK and p-PKCβ were performed in the department of pathology of Peking University Cancer Hospital using the standard streptavidin-biotin-peroxidase immunostaining procedure. The slides were incubated with primary antibody overnight at 4 °C and then with HRP-conjugated secondary antibody at room temperature for 30 min. DAB was used for staining. The intensity and density of the staining were examined in a double-blinded manner by two independent pathologists from the department of pathology in Peking University Cancer Hospital & Institute. Primary antibodies were listed in Additional file 1: Table S1.",
"section_name": "Immunohistochemistry",
"section_num": null
},
{
"section_content": "All experiments in vitro were independently done more than three times. The SPSS 22. 0 statistical software (IBM, New York, NY, USA) was used for all analyses. Data were analyzed using paired or unpaired Student's t test comparisons or one-way ANOVA. P values <0. 05 were accepted as statistically significant. The combination index (CI) for drug combination was determined according to the Chou-Talalay method using the CalcuSyn software (version 2, Biosoft, Cambridge, UK). CI values <1, =1, and > 1 indicates synergism effects, additive effects, and antagonism effects, respectively.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To determine the effect of enzastaurin on the survival of DLBCL cell lines, we cultured nine cell lines in the presence of enzastaurin (0 to 20. 0 μM) for 72 h. As shown in Fig. 1a, treatment with enzastaurin resulted in a dose-dependent inhibition of cell proliferation, with a 50% inhibitory concentration (IC50) values ranging between 6. 7 and 15. 6 μM (Fig. 1a ). We confirmed that treatment with enzastaurin effectively reduced the viability of DLBCL cells, and there was no statistical difference between ABC and GCB cells lines (p = 0. 48). \n\nPKCβ is a common signaling target that lies downstream of BTK. Surprisingly, we observed that HBL-1 and TMD8 cells exhibited notable upregulation of phosphorylated BTK (p-BTK) upon treatment with enzastaurin (Fig. 1b ). These results suggest that although inhibition of PKCβ is therapeutically effective in DLBCL cells, it also leads to positive regulation of BCR signal pathway. Thus, while pharmacological inhibition of enzastaurin attenuated some branches of BCR signaling pathways, inactivation of these pathways can be compensated by upregulation of other pathways (Fig. 1c ). These compensatory pathways greatly limit the effectiveness of enzastaurin in DLBCL, especially as a monotherapy.",
"section_name": "Enzastaurin inhibited proliferation of ABC and GCB cell lines in a dose-dependent manner and upregulates BTK phosphorylation",
"section_num": null
},
{
"section_content": "Our initial results suggested that simultaneous inhibition of PKCβ and BTK would block BCR signaling and induce cell death in DLBCL cells. Based on the cytotoxicity of enzastaurin and ibrutinib, we exposed the GCB (SU-DHL-6 and OCI-LY7) and ABC (HBL-1, TMD8 and SU-DHL-2) lymphoma cells to minimally toxic concentration of enzastaurin, together with increasing concentrations of ibrutinib in combination for 72 h. The toxicity of each treatment was assessed by measuring the rate of growth inhibition. Notably, DLBCL cells (SU-DHL-2 and SU-DHL-6) that responded poorly to enzastaurin or ibrutinib as a single-agent therapy were exquisitely sensitive to combination treatment with these two drugs (Fig. 2a ). Combination therapy with enzastaurin and ibrutinib greatly increased the inhibition rate of DLBCL cell growth irrespective of the molecular subtype or the level of responsiveness to ibrutinib monotherapy (Fig. 2a ). \n\nTo further confirm the synergistic effect of enzastaurin and ibrutinib in DLBCL, CI values were calculated (Fig. 2b ). The combined therapy showed a strong synergistic inhibitory effect on the growth of HBL-1, TMD8, SU-DHL-2, SU-DHL-6 and OCI-LY7 cells at all tested doses, with CI value ranging from 0. 239 to 0. 686. The synergistic effects in SU-DHL-2 were weak, with a CI range of 0. 608-0. 923. Overall, the combinations of enzastaurin and ibrutinib thus exhibited synergistic effects in GCB and ABC subtypes of DLBCL cell lines at all doses examined (CI < 1, Fig. 2b ). \n\nTime-course analysis of cell death further indicated that that prolonged exposure to the combination had an even greater effect on inhibition of cell proliferation (Fig. 2c ). Thus, the combination of enzastaurin and ibrutinib demonstrated long-term synergistic effects on the survival and proliferation of DLBCL cells, independent of their subtype.",
"section_name": "Synergistic effects of enzastaurin and ibrutinib on the induction of cell death in DLBCL cell lines",
"section_num": null
},
{
"section_content": "To determine whether inhibition of cell growth by co-treatment with enzastaurin and ibrutinib was associated with apoptosis and/or cell cycle arrest, we analyzed levels of apoptosis in four cells lines after 48 h exposure to the indicated concentrations of enzastaurin and/or ibrutinib. In HBL-1, the combination of enzastaurin with two different doses of ibrutinib induced 43. 8 ± 8. 7% or 51. 4 ± 5. 9% apoptosis respectively, as measured by annexin V staining; these values were greater than those cells treated with each single agent alone (enzastaurin = 25. 5 ± 5. 4%, ibrutinib = 15. 9 ± 6. 0% and 19. 0 ± 6. 7%, Fig. 3a ). Thus, co-treatment with enzastaurin and ibrutinib has a synergistic effect on promoting apoptosis. Consistent with the results of annexin V staining, expression of proteins associated with apoptosis also changed in response to co-treatment in HBL-1 cells (Fig. 3b ). Treatment with either enzastaurin or ibrutinib slightly increased expression the active forms of poly-ADP ribose polymerase (PARP) and caspase-3, but co-treatment dramatically increased these effects (Fig. 3b ). Treatment with the combination also induced a sharp decrease in the expression of anti-apoptotic Bcl-2 family members, including Mcl-1, XIAP, and Bcl-2. Similar results were observed in TMD8, SU-DHL-6 and OCI-LY7 cells (Fig. 3a, b ). Taken together, these results show that the con-administration of enzastaurin and ibrutinib promotes apoptosis through activation of the caspase-dependent and mitochondrial pathway in DLBCL cells, ultimately resulting in cytotoxicity. \n\nIn order to assess the effects of enzastaurin and ibrutinib on the cell cycle, we used flow cytometry to analyze the cell cycle profiles of treated cells (Fig. 3c ). The percentage of HBL-1 cells in G1 phase increased from 28. 5 ± 0. 05% in the control group to 46. 4 ± 0. 84% and 47. 2 ± 3. 12% in the combination treatment groups. A corresponding decrease of cells in S phase also occurred. Similar results were observed in TMD8, SU-DHL-6 and OCI-LY7 cells (Fig. 3c ). Consistent with these results, expression of CDK2, CDK4, CDK6 and Cyclin D1 substantially decreased in cells co-treated with enzastaurin and ibrutinib, whereas treatment with single agents only mildly affected the expression of these proteins known to play essential roles in the G1/S transition. Similar trend were observed in the other three cell lines (Fig. 3d ). These data demonstrate that the combinations of enzastaurin and ibrutinib induced G1 phase arrest and the combination therapy suppressed cell proliferation by inducing both cell cycle arrest and initiation of apoptotic.",
"section_name": "The combination of enzastaurin and ibrutinib promoted apoptosis and induced G1 arrest in DLBCL cells",
"section_num": null
},
{
"section_content": "In order to assess the possible effects of treatment with low doses of enzastaurin and ibrutinib on cell motility, we performed cell migration and invasion assays using DLBCL cells. For invasive abilities, treatment with enzastaurin or ibrutinib alone slightly suppressed invasive of HBL-1 cells, with 97. 0 and 85. 0% cells exhibiting invasion, respectively. In contrast, invasion was notably suppressed in cells treated with the combination of enzastaurin and ibrutinib, with only 32. 8% of cells invading relative to the control group (Fig. 4a ). Analysis of migration revealed that treatment with the single agent reduced migration to 79. 0 and 70. 2% of HBL-1 cells, respectively. In contrast, the number of co-treated cells passing through the membrane was only approximately 25. 5% of the control cells (Fig. 4b ). Similar trends were observed in TMD8, SU-DHL-6 and OCI-LY7 cells, and detailed results are shown in the invasion and migration histogram (Fig. 4c ). These findings demonstrate that enzastaurin and ibrutinib synergistically decrease cell migration and invasion, which are essential for DLBCL cell motility.",
"section_name": "Treatment with low doses of enzastaurin and ibrutinib synergistically inhibits migration and invasion in DLBCL",
"section_num": null
},
{
"section_content": "To gain insight into the mechanism underlying the anti-proliferative effects of co-treatment with enzastaurin and ibrutinib in DLBCL models, we next investigated the changes of signal transduction pathways in treated cells. As shown in Fig. 5a, HBL-1 cells treated with low doses of enzastaurin monotherapy for 60 min and 120 min showed clearly reduced the phosphorylation of glycogen synthase kinase 3β (GSK3β), which serves as a biomarker for enzastaurin activity. Short-term and low-dose enzastaurin treatments had not significantly affect on the PKCβ phosphorylation (data not show), and increased expression of p-BTK, p-ERK. Similarly, treatment with Ibrutinib alone reduced levels of BTK phosphorylation, which was accompanied by a mild effect on phosphorylation of mTOR, PLCγ2, and ERK. However, co-treatment with enzastaurin and ibrutinib resulted in a greater reduction in phosphorylation of ERK, mTOR, PLCγ2, compared to each monotherapy alone (Fig. 5a, b ). These results were also confirmed in TMD8 and SU-DHL-6 cells (Fig. 5b, Additional file 2: Figure S1 ). Overall, in contrast to single treatment, the combination of enzastaurin and ibrutinib appears to more effective inhibit signal transduction in both ABC and GCB cell models, indicating that co-treatment successfully suppress multiple signaling pathways downstream of BCR.",
"section_name": "Co-demonstration of enzastaurin and ibrutinib synergistically inhibit downstream signaling pathways",
"section_num": null
},
{
"section_content": "In order to better understand the effects of combination therapy with low doses of enzastaurin and ibrutinib in DLBCL cells, we assayed whole-transcriptome changes by RNA-sequencing. Several hundred transcripts observed to be either up or down regulated by different treatments. Because the upregulated genes were not closely associated with these inhibitors, only the downregulated genes were further analyzed. Venn diagram was used to depict the number of downregulated genes associated with the different treatments (< 2 fold, p < 0. 05). Enzastaurin and ibrutinib were less efficient as single agents, with 399 and 336 transcripts significantly downregulated, respectively, compared with 605 downregulated transcripts for the combination treatment. Approximately 91% of transcripts (365 genes) repressed by enzastaurin and 73% of transcripts (246 genes) repressed by ibrutinib were included in the combination group. Additionally, the combination treatment efficiently downregulated an additional 163 transcripts that had not been downregulated by either drug alone. Similar results were observed in TMD8 cells (Fig. 6a ). Thus, co-treatment with enzastaurin and ibrutinib resulted in the downregulation of a broader set of genes compared to the treatment with either of the compounds alone. \n\nFurther analysis of downregulated genes showed that compared with vehicle treatment control, significantly downregulated genes from top ranked pathways (by KEGG) are represented in the heat map (Fig. 6b ). Co-treatment with low doses of enzastaurin and ibrutinib effectively downregulated genes associated with BCR, NF-κB, JAK-STAT and MAPK signaling pathways. These pathway analysis results were also confirmed in TMD8 cells (Additional file 2: Figure S2 ), which consistent with those from Western blot results (Figs. 3, 5 ). Thus, combination therapy appeared to synergistically regulate whole-transcriptome changes. \n\nTo further assess the synergistic anti-tumor effects of enzastaurin and ibrutinib, we analyzed the expression of transcripts downregulated by the combination treatment using qRT-PCR. Compared with enzastaurin and ibrutinib monotherapy, combination treatment was able to decrease the mRNA expression of NOTCH1 more significantly (Fig. 6e ). A strong body of evidence underscores the important oncogenic role of NOTCH1 in promoting changes in cellular metabolism, cell growth and proliferation, and enhanced the activity of signaling pathways [23] [24] [25] [26]. Furthermore, aberrant NOTCH1 activity has emerged as an important oncogenic regulator of hematological malignancy [26]. The NOTCH1 mRNA and protein were expressed at medium-to-high levels in DLBCL cells (Fig. 6c ). Thus, the anti-proliferative effects of the combination of enzastaurin and ibrutinib in DLBCL cells are likely due to suppression of NOTCH1 expression. \n\nTo validate the role of NOTCH1 downregulation in DLBCL cell survival and proliferation, we used shRNA transfection to knock-down NOTCH1expression (Fig. 6d ). Silencing of NOTCH1 in DLBCL cells had anti-proliferative effects, indicating that NOTCH1 expression is important for the survival of DLBCL cells. Similar proliferation effects and timing were observed in NOTCH1 shRNA treatment and co-treatment with enzastaurin and ibrutinib, suggesting that the synergistic effects of the combination treatment may occur through downregulating NOTCH1 expression (Fig. 6f ).",
"section_name": "Whole-transcriptome changes in DLBCL occur in response to the combination of enzastaurin and ibrutinib",
"section_num": null
},
{
"section_content": "Finally, we assessed the ability of enzastaurin, alone and in combination with ibrutinib, to reduce tumor growth in a lymphoma model, in which ABC-DLBCL HBL-1 cells were engrafted in NPG mice (Fig. 7 ). Enzastaurin or ibrutinib monotherapy resulted in a smaller reduction in tumor volume relative to the control. Compared with control and monotherapy, tumor volumes were significantly smaller in mice treated with the combination treatment (p < 0. 05, Fig. 7a ). Treatment was well tolerated, and no mice lost weight obviously or died (Fig. 7b ). At the end of the experiment, neither enzastaurin (811. 28 ± 182. 10 mg) nor ibrutinib (719. 25 ± 156. 71 mg) significantly inhibited tumor growth compared with that of the vehicle group (1075. 29 ± 152. 56 mg), while the co-treatment robustly suppressed tumor growth and restrained tumor weight (444. 65 ± 87. 64 mg, Additional file 2: Figure S3 ). To further evaluate the apoptosis, proliferation, and BCR signal status of tumor tissue post different treatments, TUNEL, Ki-67, p-BTK and p-PKCβ was investigated and quantified in paraffin sections of tumor samples collected from HBL-1 xenografts. As shown in Fig. 7c and d, combination therapy of enzastaurin and ibrutinib induced a notable increase of apoptosis compared with each agent alone. Moreover, co-treatment with enzastaurin and ibrutinib produced a more significant decrease of Ki-67, p-PKCβ and p-BTK expression than the monotherapy achieved (Fig. 7e, f ). Thus, these results demonstrate that the co-treatment of enzastaurin with ibrutinib has synergistic activity in preclinical models, confirming our in vitro findings.",
"section_name": "Enzastaurin and ibrutinib have synergistic antitumor effects in a DLBCL models in vivo",
"section_num": null
},
{
"section_content": "DLBCL is a heterogeneous lymphoma, and although the introduction of rituximab has greatly improved clinical outcomes, it still proves incurable in 30%~40% of all cases [27]. One of the most important reasons underlying negative outcomes is that ABC and GCB DLBCLs exhibit activation of different signaling pathways. The ABC subtype is characterized by mutations in MYD88, CARD11, CD79A and CD79B, and constitutive activation of NF-κB signaling, features associated with less favorable clinical outcome [6, 7]. In contrast, GCB subtype is more frequently characterized by activation of the PI3K/AKT pathway, rather than NF-κB pathway [10]. These differences in signaling translate into different levels of tumor aggressiveness and differential response to therapy [28]. The crucial role played by the BCR signaling pathways in DLBCL has prompted the development of targeted kinase inhibitors, including inhibitors of BTK, PI3K, SYK and PKCβ, representing promising potential therapeutic strategies for DLBCL patients [29]. Here, for the first time, we demonstrate that combination treatment with enzastaurin and ibrutinib augments anti-tumor effects of the single agents in DLBCL in vitro and in vivo. These effects may be due to inactivation of related signaling pathways and downregulation NOTCH1 expression. \n\nEnzastaurin is a relatively well-studied anti-tumor agent. Preclinical evaluation of enzastaurin has shown promising results in cutaneous T-cell lymphoma, B-cell lymphoma, multiple myeloma (MM), Waldenstrom's macroglobulinemia (WM) and other solid tumors [30] [31] [32]. With respect to DLBCL, 22% of DLBCL tumor samples have been found to be positive for PKCβ expression, defined as immunostaining of > 50% of cells [33]. Furthermore, PKCβ expression is a useful marker of poor prognosis in DLBCL. Phase I/II studies of enzastaurin have shown that it is well tolerated in DLBCL patients, 15% (8/55) of the patients experienced prolonged freedom from progression (FFP ≥ 4 cycles) and 7% (4/55) of patients experienced FFP (See figure on previous page. ) Fig. 3 The combination of enzastaurin and ibrutinib promoted apoptosis and induced G1 phase arrest. a Combination treatment prompted apoptosis in DLBCL cells. Cells pre-treated with indicated concentrations of ibrutinib in the presence or absence of fixed concentration of enzastaurin for 48 h were stained with annexin V-APC, then apoptosis was assessed using flow cytometry. Apoptosis cells were determined by APC + cells. b The combination treatment mediated expression of proteins associated with apoptosis in DLBCL cells. After 48 h of exposure to enzastaurin and/or ibrutinib in combination, proteins were extracted from cells of different groups and proteins associated with apoptosis were analyzed by western blot. c Co-treatment with enzastaurin and ibrutinib induced G1 phase arrest in DLBCL cells. Cells were treated with different concentrations of ibrutinib in the presence or absence of fixed concentration of for enzastaurin 48 h, and then stained with propidium iodide (PI). Cell cycle was assessed using flow cytometry. d The combination treatment group mediated alterations in proteins associated with G1/S transition in DLBCL cells. After 48 h of exposure to indicated concentration of enzastaurin and ibrutinib in combination, proteins were extracted from cells of different groups and proteins were analyzed by western blot. Error bars represent the result and SD of three different experiments. * p < 0. 05, ** p < 0. 01, *** p < 0. 001, compared with control group; # p < 0. 05, ## p < 0. 01 compared with enzastaurin group 20~50 months [16, 17]. However, similar results have not be observed in a phase III clinical trial (PRELUDE), which showed that enzastaurin monotherapy did not significantly improve disease-free survival (DFS) in high-risk DLBCL patients after remission of B cell lymphoma. These results have essentially halted the development of enzastaurin as a monotherapy in DLBCL. A large phase 3 global clinical trial was launched to assess enzastaurin plus R-CHOP in DLBCL patients with the genomic biomarker DGM1, identifying a novel genetic biomarker related to drug efficacy, which could improve the efficacy and outcomes of enzastaurin. \n\nAnalysis of failed therapeutics presents an opportunity for improvement through both preclinical and clinical investigations of therapeutic combinations. Prior studied have noted the combination treatment with HDACi and enzastaurin exhibit a synergistic effect in DLBCL. HDACi may increase the expression of PKCβ, leading to activation of survival signals [14]. Additionally, therapeutic regimens composed of enzastaurin with other agents, such as lenalidomide, NVP-BEZ235 (PI3K inhibitor), and bortezomib, have been evaluated in non-Hodgkin lymphoma B-cell lines [13, 34, 35]. These studies can be considered as examples of new innovative attempts to identifying logical therapeutic combination. \n\nIbrutinib (PCI-32765) is an orally active inhibitor of BTK that binds Cysteine-481 on the kinase domain, leading to an irreversible inhibition at Tyr-223. Remarkable progress has been made in the development of ibrutinib in recent years, and the drug demonstrated considerable efficacy in a variety of B-cell malignancies. In ABC and GCB DLBCL, differences in activation of signaling pathways translate to differences in response to BTK inhibition, which have largely been confirmed in a Phase II trial of ibrutinib in relapsed DLBCL patients. Results from this trial revealed an overall response rate (ORR) of 37% (14/38) of patients with ABC-DLBCL, but only 5% (1/20) of patients with GCB-DLBCL [2]. Furthermore, ABC-DLBCL patients harboring CD79A/B mut, CARD11 mut, TNFAIP3 mut, or MYD88 mut showed primary resistance to ibrutinib [2, 6, 36]. On the other hand, as a result of activating mutations in BTK or PLCγ2, a subset of patients with an initially response to ibrutinib eventually relapse, underscoring the need for developing new target agents and combination treatments to improve the outcomes of such resistant patients [37]. Recent attention has been focused on potential drug combinations in DLBCL, particularly co-treatment with a BTK inhibitor and lenalidomide, bortezomib, PI3K inhibitor, or Pan-SRC kinase inhibitors in DLBCL [6, 29, [38] [39] [40]. Addition of ibrutinib to DLBCL cells treated with these agents resulted in synergistic cytotoxic effects on cells. There is also clinical data supporting the use of ibrutinib in combination with other agents, as combination therapy with rituximab and ofatumumab has been shown to be effective for the treatment of relapsed or refractory CLL/SLL [41]. Current on-going trials will further define the role of ibrutinib as upfront therapy and/ or as a combination treatment in B-cell lymphoid malignancies. In the present study, analysis of the combination of PKCβ inhibitor enzastaurin and the BTK inhibitor ibrutinib has shown synergistic anti-tumor effects in DLBCL, thereby providing a rational basis for future preclinical/ clinical investigations that may allow for the development of specific, well tolerated and efficient cancer therapeutics for relapsed or refractory DLBCL patients. \n\nAnother critical reason for supporting the combination treatment of enzastaurin with ibrutinib is that early studies have demonstrated the role of PKCβ in the negative regulation of BTK. Also, treatment with PKCβ inhibitors alter phosphorylation of BTK, leading to enhanced BTK signaling [18, 19]. Consistent with these previous works, our study also revealed that the expression of p-BTK was markedly increased by treatment of enzastaurin. Thus, PKCβ potently activates negative feedback signals of BTK, indicating that PKCβ inhibitors upregulate BTK's activation thereby altering oncogenic signals downstream of BCR. Based on this mechanism, we investigated whether the combination of PKCβ inhibitor enzastaurin and BTK inhibitor ibrutinib had synergistic anti-tumor effects in DLBCL. Our findings revealed synergistic effects of these two agents on reduction of proliferation, promoting apoptosis, inducting G1 phase arrest, inhibition of cell invasion and migration, and downregulation activation of downstream signaling in GCB and ABC lymphoma cell lines. \n\nCombination treatment of enzastaurin with ibrutinib has also been shown to trigger time-dependent inhibition of NOTCH1 mRNA expression, whereas treatment with either drug alone only slightly affected NOTCH1expression. The oncogenic role of NOTCH1 has been verified in a number of hematological diseases, including T-cell acute lymphoblastic leukemia, multiple myeloma, Hodgkin and anaplastic large cell lymphoma [23, 24, 26]. Many recent studies also have shown that a large number of DLBCL patients harbor NOTCH1 mutations and aberrations, validating the oncogenic role of NOTCH1 as the genetic drivers of DLBCL [42] [43] [44]. Moreover, NOTCH1 promotes the activation of the PI3K-AKT-mTOR and NF-κB signaling pathway, which plays a pivotal role in accelerating cell growth and promoting cell apoptosis not only in T-cell (See figure on previous page. ) Fig. 6 Whole-transcriptome changes in DLBCL occur in response to the combination of enzastaurin and ibrutinib. HBL-1 and TMD8 cells were exposed for 24 h with enzastaurin (HBL-1 2 μM, TMD8 4 μM) and/or ibrutinib (HBL-1 0. 02 μM, TMD8 0. 003 μM). RNA was collected for RNA sequencing. a Venn diagram illustrating the number of overlapping downregulated gene between different groups. b Significantly downregulated genes from top ranked pathways (by KEGG) are represented in the heatmap. Colors scale bar represents from higher (red) to lower (blue) expression. Gene expression levels are expressed in FPKM values, differences shown in color scale after Z-score transformation. Downregulated genes were determined by log2foldchange<0. FPKM, fragments per kilo base of exon per million fragments mapped. c The expressions of NOTCH1 in DLBCL cell lines were detected using qRT-PCR and western blot. d NOTCH1 knockdown by shRNA was validated by western blot in HBL-1, TMD8, OCI-LY7, SU-DHL-6 cells. β-actin is shown as a loading control. e The expressions of NOTCH1 gene were further confirmed in DLBCL cells after pre-treated with enzastaurin and/or ibrutinib. f The DLBCL cells were transfected with shRNA targeting NOTCH1, or treated with enzastaurin and ibrutinib for 48 h, 72 h. The cell viability of tumor cells was determined using the Cell Titer-Glo luminescent cell viability assay. Results are expressed as mean ± SD, data are representative of three independent experiments. * p < 0. 05, ** p < 0. 01, *** p < 0. 001 compared with control group; # p < 0. 05, ## p < 0. 01 compared with enzastaurin group but also in B-cell neoplasms [24, 25]. In our works, treatment of DLBCL with a combination of enzastaurin and ibrutinib significantly reduced expression of NOTCH1, and shRNA mediated reduction in NOTCH1 expression dramatically inhibited DLBCL cell proliferation. These data indicated that downregulation of NOTCH1 could be a crucial biological mechanism by which the synergistic effect of co-treatment with enzastaurin and ibrutinib in suppressing cell growth. The precise mechanism in detail is likely to be a promising direction of further research.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "We have evaluated the combination of enzastaurin and ibrutinib in DLBCL in vitro and in vivo, demonstrating the co-treatment had synergistic anti-tumor effects in DLBCL, independent of molecular subtype. These results provide a sound foundation for further evaluation of an attractive therapeutic combination, suggesting that simultaneous inhibition of BTK and PKCβ may represent a novel, effective therapeutic approach for ABC and GCB DLBCL.",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We thank Dr. Fu from the University of Nebraska Medical Center in USA for the kind gifts of the DLCBL cell lines. We also thank Dr. Luo Wen from Denovo Biopharma for provide guidance for experimental design, Dr. Huirong Ding and Dr. Xijuan Liu from the Peking University Cancer Hospital & Institute for the analysis of flow cytometry. Further thanks are due to Drs Yunfei Shi and Bin Dong from Peking University Cancer Hospital & Institute for analysis of Immunohistochemistry stains.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This work was financially supported by NSFC (Nos. 81870154, 81470368, 81670187 and 81600164 ), Beijing Natural Science Foundation (No. 7172047 and 7172046 ), Capital's Funds for Health Improvement and Research (No. 2018-1-2151 ), Beijing Municipal Administration of Hospitals' Ascent Plan (No. DFL20151001 ), Beijing Municipal Administration of Hospitals Clinical Medicine Development of special funding support (No. XMLX201503 ) and Beijing Municipal Administration of Hospitals Incubating Program (Code. PX2017001 ).",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "Additional file 1: Table S1.",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "This study was approved by review board of the Peking University Cancer Hospital & Institute. Animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, which were approved by the Peking University Cancer Hospital & Institute.",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Not applicable.",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.7892/boris.126363
|
Comprehensive Analysis of Alternative Splicing Across Tumors from 8,705 Patients
|
Our comprehensive analysis of alternative splicing across 32 The Cancer Genome Atlas cancer types from 8,705 patients detects alternative splicing events and tumor variants by reanalyzing RNA and whole-exome sequencing data. Tumors have up to 30% more alternative splicing events than normal samples. Association analysis of somatic variants with alternative splicing events confirmed known trans associations with variants in SF3B1 and U2AF1 and identified additional trans-acting variants (e.g., TADA1, PPP2R1A). Many tumors have thousands of alternative splicing events not detectable in normal samples; on average, we identified ≈930 exon-exon junctions ("neojunctions") in tumors not typically found in GTEx normals. From Clinical Proteomic Tumor Analysis Consortium data available for breast and ovarian tumor samples, we confirmed ≈1.7 neojunction- and ≈0.6 single nucleotide variant-derived peptides per tumor sample that are also predicted major histocompatibility complex-I binders ("putative neoantigens").
|
[
{
"section_content": "und für die Berichterstattung an die Fakultäten der Universität und den Kanton Bern verwendet. (if necessary after consultation with the submitters, cf. below, 6. 4). Members of the Editorial Board are appointed by the Open Science Team of the University Library of Bern. \n\n• Roles of researchers: Submitters can pick the roles of researchers whose work they submit to BORIS Portal. A role is a combination of name, institute/department, and duration of employment. The role of a researcher is necessary to unambiguously identify contributors to research outputs. • Eligibility: authorization on a technical level to perform certain actions in BORIS Portal (e. g., submit or review items). The eligibility criteria are determined by the Open Science Team of the University Library of Bern. On a technical level, the system administrator can authorizes users (granting of rights). • Open -The datasets are accessible without restrictions and can be downloaded. \n\n• Embargo -The records are freely accessible after the expiration of an embargo period. The embargo end date is determined by the submitter. \n\n• Restricted -Access to records must be requested from a contact person. \n\n• Closed -Records cannot be uploaded to the repository but metadata can be entered. \n\n• Metadata Only -If data are already uploaded to another repository, the metadata on BORIS Portal can be used to verify the existence of the dataset.",
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"section_name": "Access restriction for datasets",
"section_num": "5.2."
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"section_num": "5.3."
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"section_name": "Disclaimer of warranty and liability",
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] |
[] |
10.3389/fgene.2021.747344
|
Sequence Divergence and Functional Specializations of the Ancient Spliceosomal SF3b: Implications in Flexibility and Adaptations of the Multi-Protein Complex
|
<jats:p>Multi-protein assemblies are complex molecular systems that perform highly sophisticated biochemical functions in an orchestrated manner. They are subject to changes that are governed by the evolution of individual components. We performed a comparative analysis of the ancient and functionally conserved spliceosomal SF3b complex, to recognize molecular signatures that contribute to sequence divergence and functional specializations. For this, we recognized homologous sequences of individual SF3b proteins distributed across 10 supergroups of eukaryotes and identified all seven protein components of the complex in 578 eukaryotic species. Using sequence and structural analysis, we establish that proteins occurring on the surface of the SF3b complex harbor more sequence variation than the proteins that lie in the core. Further, we show through protein interface conservation patterns that the extent of conservation varies considerably between interacting partners. When we analyze phylogenetic distributions of individual components of the complex, we find that protein partners that are known to form independent subcomplexes are observed to share similar profiles, reaffirming the link between differential conservation of interface regions and their inter-dependence. When we extend our analysis to individual protein components of the complex, we find taxa-specific variability in molecular signatures of the proteins. These trends are discussed in the context of proline-rich motifs of SF3b4, functional and drug binding sites of SF3b1. Further, we report key protein-protein interactions between SF3b1 and SF3b6 whose presence is observed to be lineage-specific across eukaryotes. Together, our studies show the association of protein location within the complex and subcomplex formation patterns with the sequence conservation of SF3b proteins. In addition, our study underscores evolutionarily flexible elements that appear to confer adaptive features in individual components of the multi-protein SF3b complexes and may contribute to its functional adaptability.</jats:p>
|
[
{
"section_content": "Several proteins in the cell perform vital functions as a component of specialized molecular complexes that are usually dedicated to carrying out sophisticated multistep biochemical events (Pieters et al., 2016). Like individual proteins, molecular complexes are also subject to evolutionary pressures. While evolution of single proteins has been studied extensively (Pál et al., 2006), the influence of such forces on the evolution of protein complexes is yet to be explored extensively. Advancements in cryo-electron microscopy (cryo-EM) and quantitative mass spectrometry-based proteomics, paired with affinity purification and coimmunoprecipitation, have begun to elucidate the molecular evolution of protein complexes (Hyung and Ruotolo, 2012; Stengel et al., 2012; Skiniotis and Southworth, 2016; Vimer et al., 2020) and identified distinct patterns in protein association networks between species (Wan et al., 2015). These studies have demonstrated unequivocally that protein complexes evolve through accruement of contemporary proteins, loss of primordial proteins, and modulation of protein composition and their physical connections. These phenomena are evident in multi-protein molecular machines, which perform highly complex cellular events (Marsh et al., 2013; Marsh and Teichmann, 2015; Phanse et al., 2016). A fine example of such molecular systems is the spliceosome. \n\nThe spliceosome is a eukaryote-specific molecular assembly that processes intron-containing nascent mRNA through a series of events called splicing (Collins and Penny, 2005). Intron excision by slicing and splicing of exons involves orchestrated (dis)assembly of five small ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs) and scores of spliceosomal proteins on to pre-mRNA. All these multi-protein/RNA complexes together form a spliceosome (Matera and Wang, 2014). The overall steps in splicing and the mechanism of two transesterification reactions are conserved among eukaryotes (Wahl et al., 2009). However, the number of protein players integrated as spliceosome in each splicing step varies remarkably from lower to higher-order eukaryotes (Jurica and Moore, 2003; Will and Lührmann, 2011). For example, pre-catalytic B spliceosome assembly has ∼110 proteins in humans while the yeast assembly contains only 60 proteins (Fabrizio et al., 2009), indicating evolutionary innovations in a selected set of eukaryotic species. \n\nIn addition to understanding the evolution of the protein complex, the observed differences in the number of players in orthologous spliceosomes attracts the question as to why such innovation occurs despite a conserved function and what adjustments ancestral components incur to achieve the changes. To address this, information on the contribution and essentiality of each component to the functions of the protein complex and interplay between components is crucial. Acquiring this information demands complementary approaches involving biochemical characterization of the function, gene manipulations, 3-D structure, sequence conservation and phylogeny of all components of the complex. Currently, such comprehensive information is unavailable for the whole spliceosome. However, SF3b, a multi-protein spliceosomal subcomplex, which functions as an integral part of U2 snRNP, has been well characterized in terms of 3-D structure and biochemical function (Das et al., 1999; Cretu et al., 2016). \n\nThe SF3b participates in both major and minor spliceosome assemblies (Golas et al., 2003). In the splicing event mediated by major spliceosomes, the SF3b helps recognize branch-point adenosine in the nascent pre-mRNA, stabilizing U2 snRNA/ pre-mRNA duplex and preventing pre-mature cleavage (Will and Lührmann, 2011). The complex has seven proteins, viz. SF3b1, SF3b2, SF3b3, SF3b4, SF3b14b, SF3b5 and SF3b6. In yeast, the homolog of SF3b6 is absent, and hence yeast SF3b performs its function with only six components. Our recent study demonstrates that SF3b6 may play an allosteric role in the SF3b complex in a specific set of eukaryotes (Yazhini et al., 2021). This study also showed that in comparisons of yeast and human SF3b proteins, individual SF3b proteins differ substantially in length and in functional and structural domain compositions. In addition, we found significant differences in the 3-D structure and dynamics of SF3b proteins. These observations suggested considerable divergence of SF3b complex among species and invite a study on the conservation of their sequences across diverge lineages of eukaryotes. \n\nIn this study, we have undertaken a comprehensive comparative study to investigate the conservation pattern of SF3b protein components. Using a diverse set of homologs from >2000 eukaryotic species, separated by billion years of evolution (Chernikova et al., 2011), we have identified patterns of conservation and diversity in the SF3b proteins. Further, phylogenetic distribution analysis was employed to determine trends in the distribution profiles among individual protein components. These trends were then coupled with studies of multiple sequence alignments to characterize signatures of sequence divergence at the level of both the complete protein and local regions. The local regions include intermolecular interfaces, proline-rich motifs, cancer mutation sites and anticancer drug binding site in the individual protein components of the SF3b. Our studies show the influence of protein location within the complex and subcomplex formation patterns on the sequence conservation of SF3b proteins. The association of such patterns with taxonomic lineages reveals that Saccharomycetales and pathogenic protists, namely Candida, Entamoeba and Trypanosoma species, have diverged extensively. We find that physiochemically non-conservative residue substitutions in cancer mutation sites and anti-cancer drug binding site, as well as the lack of proline-rich motifs at the C-terminus of SF3b4, discriminate these fungi and pathogens from the rest of eukaryotes. Although the biological implications of these observations are unclear, our study unveils the signatures of sequence divergence of SF3b proteins across eukaryotes and the taxa-specific regions serving add-on functional roles that may be essential for organismal adaptation.",
"section_name": "INTRODUCTION",
"section_num": "1"
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": "2"
},
{
"section_content": "Our study is aimed at studying conservation/divergence of functionally conserved SF3b protein complex sequences across eukaryotes. The SF3b complex is well characterized in yeast and humans and hence we considered the complex in these two species as references for this study. For the recognition of SF3b protein sequences in the entire eukaryotic domain, human and yeast SF3b proteins were considered as bait sequences and searched in the OMA database (Altenhoff et al., 2018) to retrieve orthologs. We chose to initially select orthologs because they are likely to be involved in a similar function (Koonin, 2005). Each resultant ortholog was queried, one at a time, against the OMA database to collect more distant orthologs. In addition, orthologs of each SF3b protein were collected from KEGG Orthology (Kanehisa et al., 2016) and EGGNOG databases (Huerta-Cepas et al., 2019). The use of multiple resources that are formed based on different approaches expanded the taxa sampled for ortholog identification. A union set of orthologs obtained from all three resources (one per species) was further taken as a query set and searched against the NCBI non-redundant protein sequences and the UniProt database (Uniclust30, 2018), using BLASTP (Camacho et al., 2009) and HHblits algorithms (Remmert et al., 2012) respectively. Hits from BLASTP search were parsed using an E-value threshold (0. 0001) and sequence coverage threshold (70%) for both query and hits. Likewise, hits from HHblits were parsed using the E-value threshold (0. 0001) and query coverage (70%) with 90% probability. Care was taken to exclude paralogs, primarily because paralogs are known to diversify in function (Koonin, 2005). We also verified that their inclusion will not significantly impact the diversity of the sequences considered for this analysis (data not shown). \n\nHits with less than 70% sequence coverage in both searches were further examined for the presence of functional and structural domains that are known to be associated with SF3b proteins. Domain assignment was performed using hmmscan (Eddy, 2009) against PFAM (El-Gebali et al., 2019) for functional domains and against SUPERFAMILY databases (Gough et al., 2001) for structural domains. Proteins with domain composition similar to yeast or human homolog were included in the data set. Further, domain information was used to identify potential false positives in the dataset. This filtration, using domain composition, was especially useful for multi-domain proteins viz. SF3b2, SF3b3 and SF3b4. Only one sequence per species was selected. This was done by mapping protein ids to species taxonomy ids through cross-referencing NCBI and UniProt databases. Subsequently, poor-quality sequences such as partial/fragment proteins, uncharacterized genomic contig sequences, proteins with segments of unknown residues and obsolete entries were discarded.",
"section_name": "Mining Homologues of the SF3b Complex",
"section_num": "2.1"
},
{
"section_content": "For conservation analysis, homologs were clustered at 60% sequence identity to obtain representative sequences of reasonably diverged homologs across eukaryotes, using CD-HIT (Li and Godzik, 2006). Multiple sequence alignments were performed using MAFFT-DASH algorithm with the default option for single-domain proteins (SF3b1, SF3b14b, SF3b5 and SF3b6) and E-INS-I option for multi-domain proteins (SF3b2, SF3b3 and SF3b4) (Rozewicki et al., 2019). \n\nSequence alignment was guided by pairwise alignment of yeast and human SF3b protein structures obtained from cryo-EM structures of B act spliceosome assembly, to attain reliable multiple sequence alignment (PDB codes: 5GM6 for yeast and 5Z58 for human) (Yan et al., 2016; Zhang et al., 2018). Sequences that lack functional regions (such as HEAT repeats in SF3b1 that interact with pre-mRNA/U2 snRNA as well as other SF3b proteins and two RRM domains in SF3b4) were subsequently pruned and the alignment protocol was reiterated. Alignments were manually refined to avoid gaps that interrupt proteinprotein interface regions or secondary structural regions as predicted by PSIPRED method for non-human/non-yeast homologs (Buchan and Jones, 2019). Statistics of refined alignments were obtained from \"alistat\" and \"esl-alipid\" programs in HMMER package (Eddy, 1998). Conservation of each residue position was calculated using Jensen-Shannon divergence (JSD) (Capra and Singh, 2007). JSD score is an information theory-based measure that is built on the notion that the probability distribution of amino acids at residue positions evolving under \"evolutionary pressure\" is different from those of residue positions evolving under no pressure. It uses the BLOSUM62 matrix to derive background amino acid distribution.",
"section_name": "Multiple Sequence Alignment and Conservation Analysis",
"section_num": "2.2"
},
{
"section_content": "To study the conservation of protein-protein interactions within the SF3b complex, interface residues were identified using protein interactions calculator or PIC (Tina et al., 2007). We used multiple available cryo-EM structures of complex A (PDB code: 6G90), pre-B (5ZWM, 6AH0 and 6QX9), B (5NRL and 5ZWO) and B act (5GM6, 5Z56, 5Z57 and 5Z58) spliceosome assemblies from both human and yeast. The inclusion of multiple structures from distinct biological states that belong to different species, captures interactions that are conformation-specific and species-specific. Residues involved in hydrogen bonding and interactions with pre-mRNA and U2 snRNA were recognized using HBPLUS (McDonald and Thornton, 1994) and NUCPLOT programs (Luscombe et al., 1997). The extent of interface residue conservation was analyzed and compared among different protein-protein interfaces of the SF3b complex using the JSD score.",
"section_name": "Interface Residue Identification",
"section_num": "2.3"
},
{
"section_content": "Phylogenetic profiling is a technique that infers coupling between two proteins based on the profile of joint presence/ absence across a large set of species (Pellegrini et al., 1999). In addition to our homology searches in the protein databases, we probed for homologues of SF3b proteins in the genomic sequences of species covered in this study. We created a database of genome sequences of species for which assembly information is available for full genome representation or at least assembled as scaffolds. This filter was employed to consider only genomes of reasonable coverage. The details of genome availability were retrieved from the ftp site of NCBI database (https://ftp. ncbi. nlm. nih. gov/genomes/ASSEMBLY_ REPORTS/, June 2021). In total, 26,123,526 genomic sequences belonging to 1838 eukaryotic species formed our nucleotide search space. For the query search, we used the reference sequence sets of recognized homologs of SF3b proteins (refer Section 2. 1), clustered at 40% sequence identity. We searched our query protein sets against the prepared nucleotide database using TBLASTN algorithm (Camacho et al., 2009). The results were parsed using an E-value threshold of 10 -12 and the query sequence coverage of at least 75%. Based on the recognition of homologs at the level of both proteins and nucleotides, we generated phylogenetic distribution profiles of all the seven SF3b proteins. The profiles were then clustered based on the presence/absence patterns using SciPy hierarchical clustering package in python programming language (Virtanen et al., 2020). In the clustering, the pairwise distance calculation was performed using \"correlation\" metric with the \"average\" linkage method to compute correlated pattern between two profiles. The heatmap figure was generated using seaborn \"clustermap\" function (Waskom, 2021).",
"section_name": "Phylogenetic Distribution of SF3b Complex",
"section_num": "2.4"
},
{
"section_content": "",
"section_name": "RESULTS AND DISCUSSION",
"section_num": "3"
},
{
"section_content": "The ancestral SF3b complex is constituted by 7 proteins in humans and 6 in yeast. We surveyed the distribution of individual components of this complex across eukaryotes. An earlier report suggests that SF3b is likely to have been present in the last common ancestor of extant eukaryotes (Collins and Penny, 2005). Although nearly ∼1. 6 million eukaryotic species are known thus far (according to the NCBI taxonomy database, June 20, 2021), the knowledge of their genome sequence is minuscule (0. 6%), indicating that only limited data is available. To collect homologs from as many representative species as possible, we have employed rigorous homology searches in the known protein sequences of eukaryotic species (refer Materials and Methods). As a result, we find that 2142 eukaryotic genomes possess homologs of one or more SF3b proteins (Supplementary Table S1 ). Figure 1 shows the NCBI common taxonomy tree for FIGURE 1 | Distribution of SF3b homologs across eukaryotes. Shown is the NCBI common taxonomy tree of 2142 species that have the recognizable homolog for at least one of the seven SF3b proteins. Square boxes at the node tip in the tree indicate the presence of homologs for SF3b1 (red), SF3b2 (green), SF3b3 (yellow), SF3b4 (blue), SF3b14b (maroon), SF3b5 (dark cyan) and SF3b6 (orange). The major clades in the tree namely Metazoa and Fungi are highlighted in light brown and light green background, respectively, for their node labels. The enlarged version of the figure with legible labels can be accessed in https://doi. org/10. 6084/m9. figshare. 16866493. v1. The species tree was obtained from NCBI taxonomy database (Schoch et al., 2020) and the figure was generated using iTol tool (Letunic and Bork, 2021). \n\n2124 species that we have covered in our study, with detailed representation of the distribution of SF3b homologs. Figure 1 shows that SF3b homologs are recognized in diverse eukaryotic lineages indicating that protein components are well conserved in a large variety of species. These range from microbial eukaryotes such as phytoplankton and protists to complex multicellular organisms such as humans. At the higher taxonomic level, we find that SF3b homologs are recognized across 10 major \"supergroups\" of eukaryotes (Supplementary Figure S1A ). The species group corresponds to 1070 genera. Animals, fungi (Opisthokonta), plants (Viridiplantae) and protists (Sar) groups are the predominant members of the taxa, as seen in Figure 1. \n\nThe highlighted example on the right panel of Supplementary Figure S1A illustrates that 183 SF3b homologs were recognized in 109 and 15 genera from Streptophyta and Chlorophyta clades of Viridiplantae respectively, of which 9 belong to the Oryza genus. \n\nFurthermore, we find that homologs of all seven SF3b proteins are observed in 578 eukaryotic species. For individual SF3b proteins, the distribution shows that 1756, 1684, 1797, 1318, 1057, 1235 and 1308 species possess homologs of SF3b1, SF3b2, SF3b3, SF3b4, SF3b14b, SF3b5 and SF3b6, respectively. The comparison of species counts among SF3b proteins shows that the number of homologs that we have recognized in each of the 10 supergroups of eukaryotes is similar for all seven SF3b proteins (Supplementary Figure S1B ). However, in \"Opisthokonta,\" we observed considerable variations in the number of homologs of each SF3b protein. We reason that the observation could be due to non-availability of data as only 52 of 1722 \"Opisthokonta\" species that we covered in this study have complete genome assembly information. In addition, only 20 out of the 52 completely sequence genomes have all seven SF3b proteins and were found to be human-like while 7 species had only 6 SF3b proteins and were yeast-like. Therefore, these trends are likely to change with the availability of more completely sequenced genomes. In the case of \"Apusozoa,\" which is a eukaryotic microbial flagellate with only limited genome sequence data available (incomplete) for only one species (Thecamonas trahens), we were able to recognize homologs for SF3b1 and SF3b2 proteins. Hence, we could include such a distant eukaryotic member in our analysis.",
"section_name": "Distribution of SF3b Homologs Across Eukaryotes",
"section_num": "3.1"
},
{
"section_content": "We then studied the overall sequence conservation using the homologues of SF3b proteins recognized in our searches. Based on the available structures of the complex, we know that in the SF3b complex, SF3b1 acts as a hub protein with the maximum number of interacting protein partners viz. SF3b2, SF3b3, SF3b14b, SF3b5 and SF3b6. SF3b14b and SF3b5 that reside in the interior have ∼27% and ∼49% of their residues involved in inter-component interfaces and form the core of the complex (Figure 2 and Supplementary Table S2A ). The structure of the complex shows that the remaining SF3b proteins surround the core. SF3b1, SF3b2, SF3b4 and SF3b14b directly interact with pre-mRNA or U2snRNA duplex. As SF3b is an RNA interacting protein complex, the interactions with proteins and RNA molecules can both influence the evolution of individual protein components. To determine the overall sequence conservation of individual SF3b proteins, we used two sequence conservation measures based on sequence identity and conservative residue substitution patterns: 1) average pairwise sequence identity and 2) JSD score. These scores were calculated from structure-guided multiple sequence alignments of representative homologs of individual SF3b proteins, clustered at 60% sequence identity. Average pairwise sequence identity among homologs shows that SF3b1 (41%) and SF3b14b (40%) have the highest percentage of residues that remain the same across homologs. This is in agreement with their contribution to the function of the SF3b complex, as they serve to be the major components for pre-mRNA and U2 snRNA binding within the SF3b complex (Supplementary Table S2B ). The same values for the other proteins such as SF3b2 (32%), SF3b3 (29%), SF3b4 (30%), SF3b5 (33%) and SF3b6 (33%) are found to be lower. Such trend is also reflected in the distribution of pairwise sequence identity among homologs. SF3b1 and SF3b14b show relatively greater number of pairs sharing above average sequence identity (Supplementary Figure S2 ). Whereas SF3b2, SF3b3, SF3b4, SF3b5 and SF3b6 homologs have more pairs with sequence identities below the average value. \n\nThe second conservation measure that we employed was the JSD score, where higher values indicate better conservation of residues. Here, we observe that SFb14b holds the highest average JSD score (0. 49), followed by SF3b5 (0. 42) and SF3b1 (0. 4). SF3b6 has moderate conservation, as indicated by the average JSD score of 0. 37 (Figure 2 ). The peripheral components SF3b2, SF3b3 and SF3b4 show lower JSD scores of 0. 27, 0. 28 and 0. 28, respectively, among which SF3b2 and SF3b4 have RNA-binding roles. Although SF3b1 is a peripheral protein with only ∼10% of its sequence being at the interface in the context of SF3b complex, it has higher sequence conservation than the other peripheral proteins (SF3b2, SF3b3 and SF3b4) (Supplementary Table S2 ). When we analyze the cryo-EM structures of spliceosome assemblies, we observed that SF3b1 has ∼5% more interface residues by forming interactions with other components in the spliceosome (Supplementary Figure S3 and Supplementary Table S2C ). This value is higher than the percentage of increased interface residues for SF3b2 (1. 6%), SF3b3 (0. 6%) and SF3b4 (2. 6%) in the spliceosome assembled form. This indicates that SF3b1 acts as a core component having added interactions in the spliceosome assembled form. Hence, these additional interactions could further influence sequence evolution leading to a better conservation of SF3b1 compared to the other peripheral components of the SF3b complex. \n\nTogether, our observation suggests that the RNA-binding role results in a well conserved sequence and protein-protein interactions allow for conservative substitutions. In both scoring measures employed here, peripheral proteins show poorer conservation than the core proteins suggesting that the extent of sequence conservation is linked to the spatial location of proteins within the complex. It is especially evident in the RNAbinding peripheral components (SF3b2 and SF3b4). Together, these observations suggest that constraints due to protein-protein interactions profoundly affect the overall sequence conservation. Therefore, within a complex, proteins residing inside the complex are observed to be better conserved than the peripheral proteins. Indeed, it would be interesting to determine if similar trends are observed in other multi-protein complexes. Also, our observation is useful for inferring possible associations between proteins in the multi-protein assemblies of unknown structures and 3-D structure modeling using cryo-EM experiments.",
"section_name": "Components Present in the Core of the SF3b Complex are Better Conserved Than Peripheral Components",
"section_num": "3.2"
},
{
"section_content": "Our observation of varied conservation between core and peripheral components prompted us to perform focused analysis in interface regions. In total, the SF3b complex comprises 12 protein-protein interfaces and 8 protein-RNA interfaces. Since some interactions are specific to a functional state or species, we analyzed multiple structures of humans and yeast to identify interface residues that might have otherwise been missed, if only one structure of SF3b complex structure was studied. We observed that SF3b1 and SF3b2 share the largest interface region in the SF3b complex, which involves >100 residues in the interface (Supplementary Table S2B ). On the contrary, SF3b2/SF3b5 interface is the smallest, with only six residues involved. The interaction between SF3b6 and SF3b1 is mediated by 40 interface residues. The analysis of interfaces for pre-mRNA and U2 snRNA shows that SF3b1 has the largest interface regions for both RNAs. The average conservation score of interface regions reveals that SF3b1/SF3b2 interface is the most conserved interface region among the 12 protein-protein interfaces in the SF3b (Figure 3A ). On the contrary, SF3b3 shows the least interface conservation, despite having a considerable number of interface residues for all its interacting partners (Supplementary Table S2B ). In the case of protein-RNA interfaces, the sequence conservation varies among different RNA-binding SF3b proteins. SF3b1 and SF3b2 show high sequence conservation for the pre-mRNA interface, with a conservation score of 0. 53 and 0. 62 respectively than the SF3b4 (0. 49) and SF3b14b (0. 45). Likewise, U2 snRNA interfaces in SF3b1 (0. 62) and in SF3b2 (0. 55) are better conserved compared to the interfaces in the SF3b4 (0. 38) and SF3b14b (0. 38) (Figure 3A ). \n\nOverall, the results of interface conservation show three key observations. First, the extent of residue conservation significantly varies among different protein-protein interfaces. For instance, SF3b1 interacts with five SF3b proteins and the interface with SF3b2 is better conserved than the interfaces with other proteins, namely SF3b3, SF3b14b, SF3b5 and SF3b6 (Figure 3A ). Second, a notable difference is observed in the extent of residue conservation between the interface region of two protein partners in the complex. For instance, in the SF3b3/SF3b14b interface, SF3b3 binding region in SF3b14b (JSD: 0. 51) is better conserved than the SF3b14b binding region in the SF3b3 (JSD: 0. 29). Third, within an interface, one part is more conserved than the other, S2B ). To simplify representation of the bidirectional network, and an edge from a protein that shares a smaller percentage of sequence at the interface is indicated by an arrow (dark grey), while the edge from its partner having a higher percentage is indicated by a line without an arrow (light grey). Total percentage of protein sequence involved in the interface is indicated within brackets next to the node labels. Core components have higher percentages (SF3b14b and SF3b5) than the peripheral components (SF3b1, SF3b2, SF3b3, SF3b4 and SF3b6). \n\nas observed in the interface region of SF3b2 for the SF3b3 partner (Figure 3B ). These observations emphasize that within a multi-protein SF3b complex, residue conservation markedly varies among different protein-protein interfaces, between interfaces of the same protein for two interacting partners and within an interface for a single partner. Interestingly, we observed that interface residues involved in bifurcated interactions with two different protein partners (overlapping interface region) are better conserved than the interface residues involved with only one protein partner (non-overlapping interface region) (Supplementary Text S1 and Supplementary Figure S4 ). This result supports our earlier observation of the variation in the extent of residue conservation within an interface and emphasizes that location and interactions with multiple protein partners dictate the nature of overall sequence conservation in a protein. \n\nFurthermore, to understand the rationale for differential conservation of protein-protein interfaces of the SF3b complex and between interfaces of the same protein, we performed phylogenetic distribution analysis of the seven SF3b proteins. Typical usage of this technique is to determine correlation in the distribution profiles of proteins with the implication that proteins are functionally related show similar profiles. In the present analysis, we have adapted this technique to recognize distinct clusters between interacting partners within the SF3B complex (refer Materials and Methods section). Here, we observe that the profiles of SF3b1 and SF3b3 are similar as they clustered into a distinct group (Figure 3C ). Similarly, SF3b2 and SF3b4 share similar profiles. Further, both sets jointly form a separate group from the other proteins of the SF3b complex. SF3b6 has a profile that is distinct from the cluster of SF3b14b and SF3b5. Together, these results show that SF3b1, SF3b2, SF3b3 and SF3b4 have similar profiles among themselves and that it is markedly different from the cluster formed by SF3b14b, SF3b5 and SF3b6. This observation is intriguing, especially since the SF3b14b and SF3b5 are core components of the human SF3b complex that interacts physically with SF3b1 (Golas et al., 2003; Cretu et al., 2016). \n\nEarlier biochemical studies on the SF3b complex have demonstrated that SF3b1, SF3b2, SF3b3 and SF3b4 can form an assembly that can bind pre-mRNA (Das et al., 1999). This suggests that the assembly of these four SF3b proteins can occur independent of other SF3b components and also perform an RNA binding function. Our results on their phylogenetic distribution profiles lend support to this finding. Further, the study on individual SF3b proteins has already shown that SF3b1 and SF3b3 can associate to form a protein complex even in the absence of other SF3b proteins. Notably, our findings show that the interface conservation of SF3b3 for SF3b1 is higher than the same for other SF3b partners (Figure 3A ). Likewise, it has been shown that SF3b2 and SF3b4 can interact independent of other proteins (Fromont-Racine et al., 1997; Igel et al., 1998; Das et al., 1999). We also observe that SF3b4 shows better residue conservation for the interface region with SF3b2 than the interface region for SF3b3. Therefore, our clustering results based on phylogenetic distribution analysis lend support to earlier biochemical findings that suggest that these proteins can form subcomplexes (Figure 3C ). The observations point to the inherent modularity within the SF3b complex and offer clues on the nature of potential subcomplexes formed by the protein components. These results also corroborate our findings on the differential conservation of interface regions observed in the analysis of multiple sequence alignments. The lack of complete genome sequence information and inability to recognize extremely diverged homologs are factors that can influence the outcomes of such analysis. We hope that with the improvements in genome sequencing and annotation efforts more accurate estimates of such interactions may be gathered in future. \n\nIt is worth noting that SF3b proteins interact with diverse partners and play multiple roles (Sun, 2020; Yazhini et al., 2021). We show that such interactions contribute significantly to our observations on the differences in the extent of conservation at protein-protein interfaces within a complex. In addition, our earlier report has revealed that the interactions of SF3b3 with the SF3b1 vary substantially between human and yeast SF3b complexes (Yazhini et al., 2021). Similarly, the conformation of SF3b5 component differs between humans and yeast homologs within the SF3b assembled form (Yazhini et al., 2021). Collectively, these observations of differential conservation of interfaces and species-specific interaction patterns imply that the inter-protein interactions of the well conserved SF3b complex are flexible.",
"section_name": "Interface Residue Conservation and Phylogenetic Distribution Reveal Correlated Patterns Between Proteins Forming Subcomplexes",
"section_num": "3.3"
},
{
"section_content": "We next probed into the association between the extent of conservation for proteins within the complex and their known roles in terms of biological function or disease. Below we discuss our observations in SF3b4 and SF3b1 that show taxa-specific sequence features.",
"section_name": "Case Studies on the Conservation Patterns of Specific Regions in the SF3b Components",
"section_num": "3.4"
},
{
"section_content": "SF3b4 has recently been discovered to be a versatile player. It participates in transcriptional and translational regulation of multiple genes (Watanabe et al., 2007; Ueno et al., 2019; Xiong et al., 2019) and acts as an oncogene in hepatocellular carcinoma (Iguchi et al., 2016) and as a suppressor in pancreatic cancers (Zhou et al., 2017). It comprises two RRM domains, a linker region connecting them and the C-terminal region. Based on domain assignments in all homologs, we find that both RRMs are present uniformly in all homologs as also shown elsewhere (Xiong et al., 2019). However, we observe that conservation patterns differ considerably between the two RRM domains (Figure 4A ). The N-terminal RRM (average JSD: 0. 47) domain is better conserved than the C-terminal RRM (average JSD: 0. 4). To probe this observation at the nucleotide-level, we calculated dN/dS ratio for the two RRM domains (refer Supplementary Text S2 for method, Supplementary Table S3 and Supplementary Figure S5 ). Calculations based on codon substitution Model 0, a basic one ratio model (Goldman and Yang, 1994; Yang and Nielsen, 1998) show that the N-terminal RRM has the dN/dS ratio of 0. 0107 while the C-terminal RRM has the value of 0. 034. We further examined if the trend holds true using other codon substitution models namely Model 2a and Model 8 that allow variation in selection among sites (Nielsen and Yang, 1998; Yang et al., 2000; Yang et al., 2005). We find using Model 2a estimation that the ratios are 0. 0723 and 0. 1218 for N-terminal and C-terminal RRMs respectively. Likewise, the ratios are 0. 0201 and 0. 0452 for N-and C-terminal RRMs, respectively based on Model 8. Overall, such low dN/dS ratios of both RRM domains indicate that they evolve under evolutionary constraints. However, considerable variation (∼2 fold) between them suggests that the extent of positive selection in C-terminal RRM is relatively higher compared to the N-terminal RRM domain. It is important to note that between the two RRM motifs, the C-terminal RRM is involved in other protein-protein interactions and helps SF3b4 to perform diverse functions, independent of its role as an integral component in the SF3b complex (Watanabe et al., 2007; Ueno et al., 2019). Our observation of poor conservation in this domain implies that the amenability of C-terminal RRM to adaptive evolution may be driven by its interactions with a diverse set of proteins. \n\nFurthermore, careful analysis of the multiple sequence alignment shows that RNP motifs, present in both RRMs that directly interact with RNA, are well conserved (Figure 4A ). However, the conservation profile of key functional residues in the RNP motifs namely Tyr56 and Phe58 in the N-terminal RRM as well as Tyr156 and Phe158 in the C-terminal RRM shows that Tyr56 and Tyr156 allow considerable residue substitutions. Predominantly these involve substitutions with another hydrophobic residue phenylalanine. Also, we observed Tyr156 of C-terminal RRM is substituted by cysteine in species from 11 genera that includes Saccharomyces and Candida. This shows that these two sites are poorly conserved in comparison with Phe58 and Phe158 (highlighted in yellow star, Figure 4A ). It has been shown that mutations of these two tyrosine residues impairs the RNA binding function of SF3b4 (Igel et al., 1998). Nevertheless, their poor conservation suggests that they evolve under positive selection and are evolutionarily more flexible with constraints operating on the physicochemical properties of the sites, than the other key functional residues (Phe58 and Phe158) in the RNP motifs that show conservation at the level of residue type. \n\nAmong homologs, the linker region connecting the RRMs varies from 10 to 40aa, while the C-terminal tail ranges from 1 to 477aa among homologs. This substantial variation in the length of the C-terminal tail has prompted us to study this region in detail. We find that human SF3b4 comprises prolinerich regions at the C-terminal tail. To understand their conservation, we screened all the SF3b4 homologs identified in our study (i. e., 1318 proteins) for the presence of prolinerich motifs viz. PPRxxP, PPPPP, PxPPxR, PPLP and PPxY in which x indicates any residue type. These motifs are reported to mediate protein-protein interactions (Ingham et al., 2005). We recognized them in the homologous sequences of SF3b4 using MAST algorithm of the MEME suite (Bailey and Gribskov, 1998). To find disordered regions in the C-terminal tail, we used InterproScan, which comprehensively integrates many protein functional sites prediction tools and maximizes the in silico functional characterization of proteins (Jones et al., 2014). As a result, 804 homologs were found to possess proline-rich motifs, and 40 of them comprise disordered regions. Together, we observed that 64% (844 protein) of the identified homologs possess added functional regions at the C-terminal tail akin to human SF3b4 (Figure 4B ). The remaining 36% of the SF3b4 homologs (474 proteins) lack functional motifs and are similar to the yeast homolog. \n\nWhen we delineated their distribution across the genomes in our dataset, we find that a considerable proportion of SF3b4 homologs have proline-rich motifs in all kingdoms except Bangiophyceae, Parabasalia and Pyrenomonadales (Figure 4B and Supplementary Table S4 ). Since only a few homologs were identified in these kingdoms, a conclusive inference on the implications of the absence of proline-rich motifs could not be made in these kingdoms. In the case of fungi, in which we find that yeast SF3b4 homolog (Hsh49) lacks the motifs, 55% of the identified homologs have prolinerich motifs. These observations suggest that many kingdoms of eukaryotes have a considerable proportion of SF3b4 homologs harboring proline-rich motifs as also homologs lacking such motifs (Figure 4B ). Notably, we observe that the motifs are absent in SF3b4 homologs of multiple taxonomic clades, namely Saccharomycetales, Trypanosoma, Candida, Streptophytina, Parabasalia, as well as few metazoans (Supplementary Table S4 ). It is possible that these are distant homologs of the other eukaryotes with no recognizable functional features in the C-terminal tail. Further, it is possible that the SF3b4 in these species might not play versatile roles in translation and cell signalling, as observed in specific eukaryotes having SF3b4 with functional regions in the C-terminal tail (Xiong and Li, 2020). Our largescale screening on SF3b4 homologs reveals that proline-rich motifs in SF3b4 are present in a majority of eukaryotes but selectively absent in few specific groups of pathogenic fungi, plants, protists, and parasites. This suggests that the C-terminal tail of SF3b4 is an evolutionarily flexible region and incurs taxa-specific molecular signatures. This may well be attributed to their functional adaptations and contribute to their versatility in other eukaryotes, although this remains to be experimentally demonstrated and verified.",
"section_name": "Conservation Patterns of Functional Regions in the Versatile Player SF3b4",
"section_num": "3.4.1"
},
{
"section_content": "",
"section_name": "Residue Conservation of Key Functional Sites in the SF3b1",
"section_num": "3.4.2"
},
{
"section_content": "SF3b1 is directly involved in stabilizing pre-mRNA/U2snRNA duplex. Somatic mutations in the protein are observed to be associated with several cancer conditions. Lys700Glu (or K700E) substitution is the most frequently recurring mutation across various cancers, including myelodysplastic syndromes (Seiler et al., 2018). Structural analysis shows that Lys700 physically interacts with the phosphate ion and sugar moiety of the uracil base of pre-mRNA through electrostatic interactions. Our earlier work has identified Lys700 to be a critical residue in the structural network of SF3b1 and that its perturbation affects the residue motions of the entire structure (Yazhini et al., 2021). Here, we examined residue substitution patterns among SF3b1 homologous sequences for this residue. Our analysis shows that Lys700 is fully conserved in metazoans, plants and Sar groups (protists), indicating that the residue is preserved in these eukaryotic groups (Figure 5A ). In several other eukaryotic groups, we observe that the lysine has been substituted by other residues. For example, the Saccharomyces genus has proline, while the pathogenic Candida genus contains glutamine. Entamoeba and Parabasalia genus have asparagine, proline and serine residues. It is important to note from the cryo-EM structure (Yan et al., 2016) that the equivalent residue Pro369 in the yeast homolog is not involved in pre-mRNA interaction (Figure 5B ). Moreover, a previous biochemical study has shown that Lys700Glu substitution does not interfere with the affinity for RNA binding or have any effect on SF3b1 interaction with other proteins (e. g., U2AF65) (Cretu et al., 2016). This observation suggests that lysine is selected mainly in the three supergroups of eukaryotes and its selectivity in these taxa is perhaps enforced by a role in pre-mRNA binding. On the contrary, genus-specific substitution in other supergroups, which are predominantly pathogens, hints that the position is susceptible to adaptive evolutionary force and might not have been selected for pre-mRNA binding, as evident in the yeast complex structure. Thus, our finding reveals and highlights taxa-specific residue substitutions at lysine 700. \n\nWe also extended our analysis to screen a comprehensive list of sites that were reported to be associated with cancers in humans. 89 residue positions are observed to be mutated in one or many cancer conditions (Cretu et al., 2016; Seiler et al., 2018). Cancer-causing substitutions at these hotspot sites is associated with aberrant splicing patterns (Dziembowski et al., 2004; Alsafadi et al., 2016). We analyzed the multiple-sequence alignments of the protein at these positions and find that they are predominantly located in the HEAT repeats 4-12. When we compute the JSD score, we find that 79% of these 89 sites have a score above 0. 4, suggesting that the sites that undergo mutation in various cancers are conserved (Supplementary Figure S6A ). From the structures of human and yeast SF3b complexes, we find that 11 of these mutation sites are involved in pre-mRNA and/or U2 snRNA binding (green boxes highlighted at the bottom, Figure 5C ). Two other residues viz. Glu592 and Cys1204 are engaged in protein-protein interactions of SF3b1 with SF3b14b and SF3b3, respectively. This shows that mutations at such sites may affect SF3b1 interactions with RNAs and other SF3b components. Further, we find that in 9 sites which are located in the helical regions of HEAT repeats, residue substitutions that cause cancers in humans are naturally observed in SF3b1 homologs in other eukaryotes (grey star symbols, Figure 5C ). Notably, of these 9 sites, Asn626 functions to interact with pre-mRNA. Therefore, cancer-causing residue substitutions might have influence on the pre-mRNA binding in species having such substitutions. Interestingly, when cancer-causing mutations of some of these sites (Leu822, Glu862, Glu902 and Arg957) are introduced experimentally in yeast cells, they do not show any growth defect (Kaur et al., 2020). Therefore, the observed substitution patterns suggest that although specific residue types at these positions are critical in human SF3b1 and their mutations may lead to cancers, we observe that they are not uniformly selected across eukaryotes. Furthermore, we analyzed the conservation of these sites in 490 metazoans covered in our dataset, to understand how well these sites are conserved in closely related species of humans. We observe that 40 residues are highly conserved (JSD >0. 7), of which Arg590, Gln669, Gly676, Arg775, Asp781, Glu862 and Gly1146 have no substitutions in the metazoan homologs (Supplementary Figure S6B ). Among these 40 residues, six residues interact with pre-mRNA and Glu592 is involved in protein-protein interactions with SF3b14b. Interestingly, when we compare these results with the overall conservation pattern across eukaryotes, we find that a pre-mRNA binding residue Glu622, showing cancer-causing substitution Glu622Asp in humans, harbours the same substitution in the SF3b1 homologs present in a few clades. These includes species from Brettanomyces, Ophiocordyceps, Tolypocladium and Zygosaccharomyces of \"Fungi\" clade and Paramecium of \"Alveolata\" clade (Glu622Asp) (Supplementary Table S5 ). Likewise, another pre-mRNA binding residue, Asn626 possessing a cancer-causing substitution Asn626Asp in (B) Shown is the cartoon representation of the interaction of Lys700 (or P369 in yeast homolog) with pre-mRNA in human (left) and yeast SF3b complex (right), as observed in the cryo-EM structures corresponding to PDB codes 5Z58 and 5GM6, respectively (Yan et al., 2016; Zhang et al., 2018). (C) Shown is a sequence logo representing residue substitution patterns of 89 cancer mutation sites (Crooks et al., 2004). Substitution patterns of residues were obtained from 1756 homologs of SF3b1. Interface residues for protein-protein interactions (light green) and protein-RNA interactions (dark green) are highlighted by square boxes at the bottom. Yellow boxes mark hotspot positions that frequently get mutated in cancers. Taxa-specific substitutions are highlighted with arrows. Positions in which the type of substitution is natural in other eukaryotes but causes cancers in humans are indicated by the star symbol. \n\nhumans, shows the same substituion in two species (Tortispora caseinolytica and Thecamonas trahens). The observed trend suggests that these sites that are universally conserved in metazoans and play pre-mRNA binding roles are not well preserved in specific groups of species in other taxonomic clades and flexible enough to allow radical residue substitutions. Given that cancer-causing substitutions at these sites lead to alternative splice site selection (cryptic 5′ and 3′ splice sites) and result in defective or alternative splice variants (Darman et al., 2015; Alsafadi et al., 2016; Shiozawa et al., 2018; Liu et al., 2020), our observation of taxa-specific substitution patterns invites a detailed investigation on the link between the nature of residue type at these sites and splicing pattern. We anticipate that such a study will unravel a regulatory mechanism of gene expression mediated by splice site selection (Cooper and Mattox, 1997).",
"section_name": "Cancer Mutation Sites",
"section_num": "3.4.2.1"
},
{
"section_content": "As SF3b1 mutations are associated with cancers, SF3b1 has been used as a target for anti-cancer therapy. Thus far, a few splicing modulators, namely Spliceostatin A, Pladienolide B and Herboxidiene have been designed against SF3b1 (Cretu et al., 2018). These drugs occlude pre-mRNA binding site and stymie SF3b1 interaction with the branch site sequence. In addition, they hamper the conformational transition of the \"open\" to \"close\" state required for SF3b1 to assemble into the spliceosome. Conservation of drug binding residues located in HEAT repeat domains 15-16 indicates that all of them are well conserved. They all have a JSD score above 0. 5 in homologs across 10 eukaryotic supergroups (Figure 6A ). Of the 8 residues at the binding site, three positions (Lys1071, Arg1074 and Val1078) are well preserved across eukaryotes. Of these, Lys1071 and Val1078 (Robert and Gouet, 2014). (C) The cartoon representation shows SF3b1 (light grey) and SF3b6 (dark grey) in the complexed form. Shown in the ball and stick representation are core interface residues proposed as critical residues that stabilize the SF3b1/SF3b6 complex. Residues are recognized based on the conservation pattern and contributions to energy and the geometry of the complex. The color code of these residues is the same as in (A). Hydrogen bond formed at the core interface is shown by the red dashed line. \n\nare directly involved in pre-mRNA binding. In addition, the mutation of Arg1074 (Arg1074His) which is present in the helical region of 14th HEAT repeat, was observed to show in phenotypic resistance for anti-cancer drug treatment (Yokoi et al., 2011; Cretu et al., 2018). This suggests that the residue potentially aids in pre-mRNA binding of neighbouring residues (His1069-Lys1071, Arg1075 and Val1078). Our observation on the absolute conservation of Arg1074 across all eukaryotes establishes its critical role in anti-cancer drug binding. In the remaining binding sites, we observed physicochemically nonconservative residue substitutions exclusively in two \"Metamonada\" parasites and nine fungal pathogens. For example, Val1078 is substituted by asparagine in yeast and the same position has other polar residues in selected fungal pathogens (Figure 6B ). Our observation of non-conservative residue substitutions in pathogenic parasites indicates that the SF3b1 of these pathogens is distinct from human SF3b1 at these sites. Although the exact physiological implications are unclear and beyond the scope of the current study, we believe that such observations will be useful and can be exploited to appropriately modify and repurpose existing drugs, to selectively target SF3b1 proteins of such fungal pathogens and treat infectious diseases caused by them. Such findings gain significance since SF3b1 is currently being considered as an effective drug target (Bonnal et al., 2012).",
"section_name": "Pathogenic Parasites Have Unique Residue Features in the Anti-Cancer Drug Binding Site of SF3b1",
"section_num": "3.4.2.2"
},
{
"section_content": "Previous studies on protein interfaces defined two categories of regions within an interface: 1) \"core\" wherein the surface exposed residues become highly buried, i. e., relative solvent accessibility ≤0. 7% upon complex formation and 2) \"rim\" covering the rest of the interface with residues having slightly higher solvent accessibility (between 7 and 10%) in the complexed form. The core region is indispensable for protein-protein interactions and generally, its conservation is higher than that of the \"rim\" region (Chakrabarti and Janin, 2002; Guharoy and Chakrabarti, 2005). In the SF3b complex, SF3b6 component is not well conserved across eukaryotes (Yazhini et al., 2021). To study the extent of conservation of interactions formed between SF3b6 component and the SF3b complex, we analyzed conservation pattern of interface residues and identified core interface residues essential for the SF3b6 association with the complex. The SF3b6 interacts solely with SF3b1 in the SF3b complex. Our analysis on multiple sequence alignments of SF3b6 homologs reveals that 5 out of 19 interface residues are highly conserved (average JSD: 0. 56 and Supplementary Figure S7 ). The conservation score of corresponding interface residues in the SF3b1 shows an average JSD value of 0. 48 (Supplementary Table S6 ). To predict core interface residues that form critical interactions between SF3b1 and SF3b6, we probed for a complementary conservation pattern at the interface regions between SF3b1 and SF3b6. For this purpose, we considered a residue pair to lie in the core only when one partner has JSD score >0. 5 and the other partner residue has the JSD score of at least 0. 4. Based on this criterion, we identified 9 residues in total viz. Asp405, Phe408, Leu415, Pro417 and Tyr421 from SF3b1 and Try36, Arg49, Tyr61 and Val63 from SF3b6 as the most critical interface residues. When we analyzed the available structures, we note that association between these residues is contributed by four hydrophobic interactions, one ionic and one side-chain and mainchain non-bonded interactions (Figure 6C and Supplementary Table S6A ). In addition, by using PPCheck and KFC2 servers that employ energy-based and geometry-based principles, respectively, for hotspot interface residues prediction (Zhu and Mitchell, 2011; Sukhwal and Sowdhamini, 2013), we recognized that Phe408 and Leu415 in SF3b1 and all the four residues in the SF3b6 are hotspots for stabilizing SF3b1/SF3b6 interactions (Supplementary Table S6B ). The conserved residues that we report in our study are observed to confer essential interactions for SF3b1/SF3b6 binding and any perturbations to them potentially impede their association. In our earlier study based on structural features and dynamics of the SF3b complex, we predict that SF3b6 is a potential allosteric regulator of the SF3b1 (Yazhini et al., 2021). We anticipate that our current funding will help in the design of in vitro mutagenesis experiments, to study the biological significance of SF3b1/SF3b6 association and validate our hypothesis of SF3b6 mediated allosteric regulations in pre-mRNA splicing. We believe that these observations will also be relevant to the other eukaryotic species in which SF3b6 is observed.",
"section_name": "Identification of the Most Critical interactions for SF3b6 Association With SF3b1",
"section_num": "3.4.2.3"
},
{
"section_content": "The growth of biochemical and 3-D structural data on macromolecular complexes is accelerating with the advent of large-scale proteomics and cryo-EM techniques. These data form the basis to characterize molecular complexes apropos of the nature of components, their topology, architecture etc (Marsh et al., 2015; Marsh and Teichmann, 2015). Concomitantly, there is considerable interest in the evolutionary aspects of molecular complexes, to understand how evolutionary force brings about new functions and sophisticated regulatory mechanisms in protein complexes of higher-order organisms. Studies have shown that at the coarse level, a complex evolves through rewiring of intermolecular association within the complex and addition or loss of components (Wan et al., 2015). In this context, our work provides insights on the evolution of a molecular complex by showcasing diversity in the sequence of each component among their homologs and the biological links associated with its sequence diversity in the ancient spliceosomal SF3b complex. Our findings reveal that the location and the formation of subcomplexes can have a strong influence on the sequence conservation of individual protein components. Further, their demography across eukaryotes, residue conservation patterns of key functional sites collectively showcase the greater divergence of fungal species. Specifically, species belonging to Saccharomyces and pathogens infecting humans from the Candida, Entamoeba and Trypanosoma genus are observed to have diverged extensively. We foresee that our results have potential applications in the 1) accurate structure modeling of multi-protein complexes and assemblies of such complexes from various species, 2) functional characterization of protein-protein associations between SF3b proteins through genetic manipulations and 3) detailed investigations on the role of the unique sequence signatures in the SF3b proteins of the pathogens that we have reported here.",
"section_name": "CONCLUSION",
"section_num": "4"
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "The authors thank Prof. R. Sowdhamini for her timely inputs in reviewing the revised manuscript. SS acknowledges MSRUAS for use of their facilities during final revisions of the manuscript.",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "This research is supported by Mathematical Biology program and FIST program sponsored by the Department of Science and Technology and also by the Department of Biotechnology, Government of India in the form of IISc-DBT partnership programme. Support from the Bioinformatics and Computational biology Centre, DBT and support from UGC, India -Centre for Advanced Studies and Ministry of Human Resource Development, India is gratefully acknowledged. SS was a postdoctoral fellow supported by IISc-DBT partnership programme. NS is a J. C. Bose National Fellow.",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "AY performed the data analysis. SS, NS and AY designed the study. SS and NS conceptualized and closely supervised the project. AY wrote the first version of the manuscript that was revised by SS and NS. All authors read, wrote and approved the final manuscript.",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The Supplementary Material for this article can be found online at: https://www. frontiersin. org/articles/10. 3389/fgene. 2021. 747344/ full#supplementary-material Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. \n\nPublisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.",
"section_name": "SUPPLEMENTARY MATERIAL",
"section_num": null
}
] |
10.1186/s10020-021-00413-0
|
Microfibrillar-associated protein 5 regulates osteogenic differentiation by modulating the Wnt/β-catenin and AMPK signaling pathways
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Dysfunctional osteogenesis of bone marrow mesenchymal stem cells (BMSCs) plays an important role in osteoporosis occurrence and development. However, the molecular mechanisms of osteogenic differentiation remain unclear. This study explored whether microfibrillar-associated protein 5 (MFAP5) regulated BMSCs osteogenic differentiation.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>We used shRNA or cDNA to knock down or overexpress MFAP5 in C3H10 and MC3T3-E1 cells. AR-S- and ALP-staining were performed to quantify cellular osteogenic differentiation. The mRNA levels of the classical osteogenic differentiation biomarkers Runx2, Col1α1, and OCN were quantified by qRT-PCR. Finally, we employed Western blotting to measure the levels of Wnt/β-catenin and AMPK signaling proteins.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>At days 0, 3, 7, and 14 after osteogenic induction, AR-S- and ALP-staining was lighter in MFAP5 knockdown compared to control cells, as were the levels of Runx2, Col1α1 and OCN. During osteogenesis, the levels of β-catenin, p-GSK-3β, AMPK, and p-AMPK were upregulated, while that of GSK-3β was downregulated, indicating that Wnt/β-catenin and AMPK signaling were activated. The relevant molecules were expressed at lower levels in the knockdown than control group; the opposite was seen for overexpressing cell lines.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>MFAP5 regulates osteogenesis via Wnt/β‑catenin- and AMPK-signaling; MFAP5 may serve as a therapeutic target in patients with osteoporosis.</jats:p> </jats:sec>
|
[
{
"section_content": "Osteoporosis is very common worldwide, and is associated with bone fragility caused by osteopenia, reduced bone mass, and an increased fracture risk (Coughlan and Dockery 2014; Kanis 1994; Wang et al. 2009). \n\nOsteoporosis affects motor function and is also associated with secondary complications, including fracture and skeletal deformities, which affect health and the quality of life (Black and Rosen 2016; Kanis 1994). Accidental trauma often triggers bone malformation or even non-healing, which are associated with poor prognosis (Hegde et al. 2016). It is accepted that reduced proliferation and osteogenesis of bone marrow mesenchymal stem cells (BMSCs) are closely associated with osteoporosis occurrence and progression (Liu et al. 2021; Luo et al. 2019). But due to the unclear mechanism of osteogenic differentiation, few methods could be applied to treat osteoporosis in clinical by targeting osteoblastic cells. More effective genes that could regulating osteogenesis need to be identified which could provide potential targets and develop drugs for patients suffering from osteoporosis. \n\nThe Wnt/β-catenin signaling pathway plays an important role in osteogenic differentiation and bone regeneration (Chen et al. 2021; Leucht et al. 2019). When Wnts bind to the low density lipoprotein receptor-associated proteins (LRPs) and Frizzled transmembrane receptors, the classic Wnt/β-catenin signaling pathway would be activated (Siracusa et al. 2021). After that, the β-catenin migrates into the nucleus and regulates osteogenic gene transcription (Huybrechts et al. 2020 ). Previous study found that Morusin could promote osteogenic differentiation of BMSCs via the activation of Wnt/β-catenin signaling pathway (Chen et al. 2021). Similarly, vasoactive intestinal peptide (VIP) could also increase the BMSCs through activating the Wnt/β-catenin pathway (Shi et al. 2020). Recently, the AMPK signaling was found could positively regulate osteogenesis. Compared with wildtype and AMPK+/-mice, the AMPK-/-mice showed a retardation of postnatal bone development (Kanazawa et al. 2018). Metformin could directly contribute to osteogenesis by activating AMPK and expression of Runx2 (Molinuevo et al. 2010). All these findings suggested the crucial function of Wnt/β-catenin and AMPK pathways in regulating BMSCs osteogenesis. \n\nMicrofibrillar-associated protein 5 (MFAP5) was a component of extracellular matrix (ECM) genes. The N-terminal of MFAP5 contains an Arg-Gly-Asp sequence (RGD) domain, which united with the elastic fibers in ECM and regulating the function of it (Deford et al. 2016). MFAP5 is crucial in regulating cell motility and signal transduction (Albig et al. 2008). Bioinformatics analyses indicate that, in osteoblasts, MFAP5 is highly expressed (Zhu et al. 2021 ). In the meanwhile, it is found that, during the process of osteoblastic differentiation, the MFAP5 has an increasing expression pattern with many other osteogenesis biomarkers, including Runx2, type I collagen, Msx-2, Dlx-5, etc. (Burns et al. 2010). But the role of MFAP5 in osteogenic differentiation is still not clear. Through analyzing bioinformatic data, we found that MFAP5 expression was downregulated in BMSCs in osteoporosis patients. In the meanwhile, during osteogenesis, MFAP5 expression tended to increase. We thus hypothesized that MFAP5 might regulate osteogenic differentiation. Through silencing or overexpressing MFAP5 in mouse osteoblastic C3H10 and MC3T3-E1 cells, the role of MFAP5 in regulating osteogenesis was tested. We stained cells for alkaline phosphatase (ALP) and Alizarin Red S (AR-S) and measured the expression levels of the osteogenic biomarkers Runx2, Col1α1, and OCN. It's shown that MFAP5 strongly promoted osteoblastic differentiation. Also, the expression patterns of key proteins in the Wnt/β-catenin and AMPK signaling pathways were affected by MFAP5 knockdown or overexpression. The results indicated that MFAP5 served as an osteogenic factor. This improves our understanding of bone metabolism; moreover, a potential therapeutic target for osteoporosis was identified.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "GSE156508 and GSE80614 gene expression profiles were obtained from the GEO database (https:// www. ncbi. nlm. nih. gov/). GSE156508 reflects the gene expression pattern of primary osteoblasts (OBs) in women with osteoporotic fractures or severe osteoarthritis; sequencing was performed using GPL16686 of the Human Gene 2. 0 ST Array (Affymetrix, Santa Clara, CA, USA). GSE80614 reflects the gene expression pattern of osteogenically differentiated hMSCs at 0, 1, 2, 3, and 4 days. For sequencing, GPL6947 of the Human HT-12 V3. 0 expression head chip was used (Illumina, San Diego, CA, USA). The data were analyzed using the GEO2R online tool.",
"section_name": "Microarray data",
"section_num": null
},
{
"section_content": "AR-S (catalog no. A553), dimethyl sulfoxide (DMSO; D2650), ascorbic acid (AA; A4403), β-glycerophosphate (β-GP; G9422), and dexamethasone (DXMS; D4902) were purchased from Sigma-Aldrich (St. Louis, MO, USA). AR-S, AA, and β-GP were dissolved in phosphatebuffered saline (PBS) to concentrations of 40 mM (pH 4. 2), 10 mM, and 1 M, and stored at 4 °C. DXMS was dissolved in DMSO (to 1 mM) and stored at -20 °C. The primary antibodies used were anti-MFAP5 (DF13146, Affinity, USA), -β-catenin (sc7199; Santa Cruz Biotechnology, Santa Cruz, CA, USA), -phospho-GSK-3β (5558; Cell Signaling Technology, Danvers, MA, USA), -GSK-3β (12456; Cell Signaling Technology), -AMPK (9158; Cell Signaling Technology), -phospho-AMPK (5759; Cell Signaling Technology), -Notch1 (4380; Cell Signaling Technology), and -GAPDH (G9295; Sigma). The secondary antibodies were goat anti-rabbit or -mouse antibodies (7074, 4410; Cell Signaling Technology).",
"section_name": "Reagents and antibodies",
"section_num": null
},
{
"section_content": "C3H10 and MC3T3-E1 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and grown in high-glucose Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) or α-MEM (Gibco) with 10% (v/v) fetal bovine serum (FBS; Gibco) at 37 °C under 5% (v/v) CO 2 in a humid atmosphere. The effect of MFAP5 on proliferation of cells were tested by a Cell Counting Kit 8 (Dojindo, Kumamoto, Japan) according to the instructions. The osteogenic induction medium was 10 mM β-GP, 50 mM AA, and 100 mM DXMS in growth medium. The growth or induction medium was replaced every other day.",
"section_name": "Cell culture, cell counting kit 8 assay and osteoblastic differentiation",
"section_num": null
},
{
"section_content": "The plasmids psPAX2, pMD2. G, and pLKO. 1-EGFPpuromycin were purchased from GeneChem (Shanghai, China) and used to deliver lentivirus-expressing short hairpin RNA (shRNA). Three shRNAs (shRNA1: 5′-CCG GCG GGA TGA GAA GTT TGC TTG TCT CGA GAC AAG CAA ACT TCT CAT CCC GTT TTTTG-3′, shRNA2: 5'-AAA ACA CCA GTT TAC GAC GTA TGT ATT CGT ACA TAC GTC GTA AAC TGG TGC-3′, and shRNA3: 5′-CCG GGA GAT GAT GTG CCT GAG ACA TCT CGA GAT GTC TCA GGC ACA TCA TCT CTT TTTTG-3′) were used to knock down MFAP5 expression. The full-length DNA coding sequence of MFAP5 was amplified and inserted into the lentiviral vector pLKO. 1-EGFP-puromycin to allow MFAP5 overexpression in target cells. The recombinant lentiviruses were used to infect cells at about 60% confluence for 2 days. Transfected cells were selected by growth in puromycin (Sigma) for 2 weeks.",
"section_name": "Plasmid and viral infections",
"section_num": null
},
{
"section_content": "Seeded C3H10 and MC3T3-E1 cells were allowed to grow for 0, 3, 7, and 14 days. At each time point, the cells were rinsed twice in PBS and fixed in polyformaldehyde for 15 min at 37 °C. An ALP staining kit (DE0004; Leagene, Beijing, China) was used as instructed by the manufacturer. Cells were stained with an AR-S solution for 35 min at 37 °C. PBS was used to wash away excess stain. The stained cells were then photographed, with the exposure time and white balance held constant.",
"section_name": "ALP and AR-S staining",
"section_num": null
},
{
"section_content": "AR-S, used for staining cells, was dissolved in 10% (w/v) cetylpyridinium chloride in PBS, and absorbance was measured at 562 nm. To measure ALP levels, stained cells were collected from plates and lysed in lysis buffer (Beyotime, Shanghai, China). The protein-containing supernatants were collected, and the total protein concentrations were normalized using the BCA method (23228; Thermo Scientific, Waltham, MA, USA). A test kit from Jiancheng Biotechnology (Nanjing, China) was used to measure ALP activities.",
"section_name": "AR-S and ALP assays",
"section_num": null
},
{
"section_content": "Adherent cells at various time points were washed twice with cold PBS; 65 μL of RIPA buffer with 1% (w/v) phenylmethylsulfonyl fluoride (PMSF) was added to each 60-mm dish and the cells scraped into Eppendorf (EP) tubes. The lysates were placed on ice for 20 min with vortexing for 3 s every 5 min, and then centrifuged (10 min, 12,000 rpm, 4 °C); the supernatant protein levels were normalized using a BCA Protein Assay Kit (Beyotime) according to the manufacturer's instructions. The proteins were subjected to gel electrophoresis and transferred to poly (vinylidene fluoride) (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with skim milk and incubated for 15 h at 4 °C with primary antibodies. Goat anti-rabbit or -mouse secondary antibodies (7074, 4410; Cell Signaling Technology) were used to probe the membranes at room temperature for 2 h. An ECL kit (Zhejiang Share Bio, Zhejiang, China) was employed to detect bands, and images were obtained using the Fluor Chem E system (ProteinSimple, Santa Clara, CA, USA). ImageJ (1. 8. 0) for Windows was applied to quantify the results of western blotting.",
"section_name": "Western blotting",
"section_num": null
},
{
"section_content": "Total cellular RNA was extracted into RNAiso Plus (9108; Takara, Shiga, Japan) according to the manufacturer's protocol. An Infinity 200-Pro multi-well plate reader (Tecan, Männedorf, Switzerland) was used to assess the concentrations and qualities of RNA samples. RNA was reverse-transcribed to cDNA using the PrimeScript RT Master Mix (RR036A; Takara). cDNA samples were mixed with SYBR Premix Ex Taq (RR420A; Takara), and forward and reverse primers for quantitative real-time PCR (qRT-RCR) performed as follows: 95 °C for 10 min followed by 95 °C for 10 s, 60 °C for 115 s, and 72 °C for 15 s (40 cycles). The data were analyzed using the 2 -△△Ct method. All qRT-PCR primers are listed in Table 1.",
"section_name": "Quantitative real-time PCR",
"section_num": null
},
{
"section_content": "All experiments were repeated at least three times. The results were analyzed using GraphPad Prism for Windows (ver. 8. 0; GraphPad Software Inc., La Jolla, CA, USA) and are presented as means ± standard deviations. Groups were compared using the two-tailed Student's t-test; differences among more than two groups were analyzed by one-way ANOVA. P-values of < 0. 05, < 0. 01 and < 0. 001 were considered statistically significant.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To explore the potential role played by MFAP5 in osteogenesis, we analyzed the GSE156508 database, which includes data on the primary osteoblasts of women with osteoporotic fractures (n = 6) and severe osteoarthritis (n = 6). MFAP5 expression was significantly decreased in the primary osteoblasts of the osteoporosis compared to osteoarthritis group (Fig. 1A ). As the control group contained patients with osteoarthritis who are different from healthy individuals, data interpretation was not quite convincible. Thus, we obtained data on osteogenically differentiated hMSCs at 0, 1, 2, 3, and 4 days, and found that MFAP5 expression was positively correlated with osteogenesis (Fig. 1B ). We then evaluated MFAP5 expression during osteogenesis of the mouse osteoblastic cell lines C3H10 and MC3T3-E1 (Fig. 1C, G, H ). MFAP5 expression increased during osteogenesis in both protein and mRNA. We determined the extent of osteogenic differentiation by staining the cells and performing qRT-PCR of mRNAs encoding osteogenic biomarkers. As osteogenesis progressed, mineralization and osteogenesis were enhanced, and ALP and AR-S staining Table 1 The sequences of qRT-PCR primers became more intense (Fig. 1D-F ). Also, the expression levels of the osteogenic biomarkers Runx2, Col1α1, and OCN increased (Fig. 1G and H ), suggesting a positive correlation between MFAP5 expression and osteoblast differentiation.",
"section_name": "MFAP5 expression correlated positively with osteogenesis",
"section_num": null
},
{
"section_content": "",
"section_name": "Gene",
"section_num": null
},
{
"section_content": "MFAP5-knockdown C3H10 and MC3T3-E1 cell lines were established using a lentivirus transfection system. Three shRNAs targeting MFAP5 were used. MFAP5 expression was evaluated in protein and mRNA. As shown in Fig. 2A and B, MFAP5 expression was significantly reduced in the MFAP5-shRNA3 C3H10 and MC3T3-E1 cell lines.",
"section_name": "Establishment of MFAP5 knockdown cell lines",
"section_num": null
},
{
"section_content": "After establishing MFAP5 knockdown lines, we assessed the role played by MFAP5 in osteogenic differentiation. \n\nAs shown in Fig. 2C, ALP and AR-S staining were used to qualitatively assess the extent of osteogenesis and mineralization in negative control (NC) and MFAP5-shRNA (shRNA) groups of C3H10 and MC3T3-E1 cells; dye staining was less intense in the two shRNA groups. The quantitative ALP levels and AR-S absorbance were consistent with the staining data (Fig. 2D and E ). For the cell numbers would affect the degree of staining, the proliferation abilities of cells were tested and there were no significant differences (see Additional file 1). We extracted mRNAs at various times during osteogenesis; qRT-PCR indicated that the mRNA expression levels of the osteogenic biomarkers Runx2, Col1α1, and OCN were lower in the shRNA groups than the NC group (Fig. 2F-H ). Thus, MFAP5 knockdown significantly inhibited osteogenic differentiation.",
"section_name": "MFAP5 knockdown inhibited osteogenic differentiation",
"section_num": null
},
{
"section_content": "We explored how MFAP5 regulated osteogenesis using Western blotting to quantify key proteins in osteogenic differentiation-related signaling pathways in the NC and shRNA groups. Figure 3A shows that the β-catenin, p-GSK-3β, AMPK, and p-AMPK levels increased, while that of GSK-3β decreased, during osteogenic induction, indicating that the Wnt/β-catenin and AMPK signaling pathways were activated. Also, Wnt/β-catenin and AMPK signaling were inhibited in the shRNA groups compared to the control during osteogenic differentiation of both C3H10 and MC3T3-E1 cells. In the meanwhile, we also quantified the results of western blotting which were shown in Fig. 3B-D. The protein level of β-catenin was higher in the control groups in both two cells lines. The trends of p-GSK-3β /GSK-3β was consistent to the result of β-catenin. As for the AMPK signaling pathway, when the osteogenic differentiation started, the p-AMPK level sudden increased, and lead the increase of AMPK. Because there was a positive feedback relationship between p-AMPK and AMPK, there was no clear trend of the value of p-AMPK/AMPK. But, the expression of AMPK, p-AMPK and p-AMPK/AMPK are significantly higher in the control group which indicating that the AMPK signaling was suppressed in the shRNA group. Therefore, the Wnt/β-catenin and AMPK signaling pathways were active when MFAP5 promoted osteogenic differentiation.",
"section_name": "MFAP5 knockdown inhibited Wnt/β-catenin and AMPK signaling",
"section_num": null
},
{
"section_content": "To confirm that MFAP5 regulated osteogenesis, we established MFAP5-overexpressing C3H10 cell lines. The MFAP5 sequence was amplified and transfected into cells, and MFAP5 expression was measured at both the protein and mRNA levels. As shown in Fig. 4A and B, compared to the controls, MFAP5 expression increased significantly. AR-S and ALP staining were more intense during osteogenesis, indicating greater mineralization and osteogenesis in MFAP5-overexpressing (from cDNA) cells (Fig. 4C ). The ALP activity assay and qualitative AR-S staining intensity supported this conclusion (Fig. 4D and E ). Protein quantification during osteogenesis indicated that MFAP5 overexpression activated the Wnt/β-catenin and AMPK signaling pathways (Fig. 4F-I ). In summary, all of the data indicated that MFAP5 positively regulated osteogenic differentiation by activating Wnt/β-catenin and AMPK signaling The Runx2, Col1α1, and OCN levels were higher in the test cells than the control during osteogenesis (Fig. 4J-L ).",
"section_name": "Overexpression of MFAP5 promoted osteogenic differentiation and activated Wnt/β-catenin and AMPK signaling",
"section_num": null
},
{
"section_content": "Osteoporosis is a major public health problem worldwide; there is no satisfactory therapy (Bone et al. 2017; Eastell and Szulc 2017; Kanis 1994; Naylor et al. 2016). Dysfunctional osteogenic differentiation of BMSCs is associated with osteoporosis initiation and development, accompanied by a significant decline in osteogenic differentiation and increased adipogenic differentiation (Chen et al. 2016; Li et al. 2018). BMSCs can develop into cells of different types; the mechanism that determines the direction of differentiation remains unclear (Guo et al. 2020; Liu et al. 2015). A better understanding of BMSC osteogenesis is needed. In this study, we investigated the role of MFAP5 in regulating BMSCs osteogenic differentiation by activating the Wnt/β-catenin and AMPK signaling pathways. We examined the GEO database and found that MFAP5 expression in the BMSCs of osteoporosis patients was lower than in controls, implying that MFAP5 might play a role in BMSC osteogenic differentiation. By inducting the C3H10 mouse mesenchymal stem cell line and MC3T3-E1 mouse embryonic osteoblast progenitor cell line into osteoblasts, we found the expression of MFAP5 was upregulated during this process. After silencing it in these two cells lines, the osteogenic differentiation ability was declined. Many signaling pathways play essential roles in osteogenesis and bone formation. Previous studies have shown that the canonical Wnt/β-catenin signaling pathway positively regulates osteogenic differentiation (Hong et al. 2019; Wang et al. 2018). In normal cytoplasm atmosphere, the expression of glycogen synthase kinase 3β (GSK-3β) brakes the stability of β-catenin, inhibiting its further function of cellular metabolic regulation. When Wnt signaling is activated (activation this pathway lies on the cell membrane binding of Wnt and frizzled receptors and the LRP co-receptor), the degradation of GSK-3β is enhanced, eventually promoting β-catenin stabilization and nuclear translocation (Wang et al. 2018). Notably, the phosphorylated form of GSK-3β is degraded (Kim et al. 2017; Li et al. 2012; Oh et al. 2014). Based on the character of this signaling pathway, we detected the expression of p-GSK-3β, GSK-3β and β-catenin. We found that, during osteogenic differentiation, β-catenin and p-GSK-3β were upregulated, while GSK-3β was downregulated in the shRNA groups, indicating that Wnt/β-catenin signaling was suppressed after knocking down MFAP5. \n\nAMPK was recently shown to play a role in osteogenesis by promoting Runx2, ALP, and OCN (Kim et al. 2018; Wang et al. 2013; Wang et al. 2016). We found that the AMPK and p-AMPK levels gradually increased during osteogenesis, consistent with previous reports. However, the p-AMPK level fell after MFAP5 knockdown, suggesting that AMPK signaling was involved in the MFAP5 regulation of osteogenesis. Of note, MFAP5 activated Notch 1 signaling in certain tumors, promoting tumor invasion and migration (Chen et al. 2020; Li et al. 2019). Notch 1 signaling is also involved in osteogenesis (Díaz-Tocados et al. 2017; Fan et al. 2016; Fan et al. 2021 ). However, the Notch 1 levels of the MFAP5 knockdown and control groups did not differ significantly (see Additional file 2). Ann et al. found that Runx2 inhibited Notch 1 signaling (Ann et al. 2011). The Runx2 expression level of MFAP5-knockdown BMSCs was lower than that of control cells. The opposing effects of MFAP5 and Runx2 on Notch 1 signaling in BMSCs might cancel out their activities; this possibility should be studied further. \n\nThe main finding of this study was that MFAP5 knockdown decreased the levels of β-catenin, phosphorylated GSK-3β, AMPK, and downstream osteogenic biomarkers, while MFAP5 overexpression had the opposite effects. Thus, MFAP5 regulates osteoblast differentiation via the Wnt/β-catenin and AMPK signaling pathways. Interestingly, Wnt/β-catenin and AMPK pathways were found significantly suppress adipogenic differentiation (Chen et al. 2014; Takada et al. 2009 ). Previous study found that MFAP5 is high expressed is adipose tissue (Vaittinen et al. 2011). But during the process of adipogenic differentiation, the MFAP5 expression significantly decreased (more than 80%) from the 6th day of adipogenesis (Vaittinen et al. 2015). This finding suggests MFAP5 might also participate in regulating adipogenic differentiation. Based on the results of this study, it may trigger a switch from adipogenesis to osteogenesis as BMSCs differentiate; we are currently investigating this possibility. Further studies on how MFAP5 regulates BMSC differentiation might identify other potential therapeutic targets, or useful small-molecule drugs. MFAP5 is small (25 kDa) and the recombinant protein is easy to synthesize. The protein is expressed mainly on microfibrils of the extracellular matrix. Thus, exogenous MFAP5 may be compatible with cell surfaces. Yeung et al. used an immunological approach to successfully block MFAP5; this enhanced the chemosensitivity of ovarian and pancreatic cancer (Yeung et al. 2019). Similar methods might be used to treat osteoporosis.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "We found that MFAP5 promoted osteogenic differentiation of MSCs by activating the Wnt/β-catenin and AMPK signaling pathways, which is a potential therapeutic target for bone metabolism diseases. \n\n• fast, convenient online submission • thorough peer review by experienced researchers in your field\n\n• rapid publication on acceptance\n\n• support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year",
"section_name": "Conclusion",
"section_num": null
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"section_num": null
},
{
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] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Thanks to Zhenxu Wang from the Shanghai Children's Hospital for her enlightening advices of this research design.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This work was supported by the Key Department of Minhang District ( 2020MWTZB03 ), the Key Department of the Fifth People's Hospital of Shanghai ( 2020WYZDZK03 ), the Fifth People's Hospital of Shanghai, Fudan University ( 2018WYZT01 ), the Fifth People's Hospital of Shanghai, Fudan University ( N123E5 ) and the Minhang District Leading Talent Development Funds.",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "The data that support the findings of this study are available from the corresponding author upon reasonable request.",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "BMSCs: Bone marrow stromal cells; MFAP5: Microfibrillar-associated protein 5; ALP: Alkaline phosphatase; AR-S: Alizarin red S; qRT-PCR: Quantitative real-time polymerase chain reaction; DXMS: Dexamethasone; AA: L-ascorbic acid; β-GP: β-Glycerophosphate; DMEM: Dulbecco's modified eagle's medium; FBS: Fetal bovine serum; DMSO: Dimethyl sulfoxide; shRNA: Short hairpin RNA; LRPs: Lipoprotein receptor-associated proteins; ECM: Extracellular matrix; NC: Negative control; Runx2: Runt-related transcription factor 2; Col1α1: Pro-alpha1 chains of type I collagen; OCN: Osteocalcin; GSK-3β: Glycogen synthase kinase 3β.",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s10020-021-00413-0.",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "Additional file 2. The expressions of Notch1 signaling in different groups.",
"section_name": "Additional file 1. Proliferation capacity of cells in different groups.",
"section_num": null
},
{
"section_content": "HRL and WLZ: The acquisition, analysis, interpretation of the data for the work. SWS and HTH: Substantial contributions to the conception and design of the work. TLZ: Revising this work critically for important intellectual content. MHW: Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "Ethics approval and consent to participate Not available.",
"section_name": "Declarations",
"section_num": null
},
{
"section_content": "Not available.",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.3390/cancers15020507
|
Characteristics and Clinical Outcomes of Patients with Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Receiving Ibrutinib for ≥5 Years in the RESONATE-2 Study
|
<jats:p>Primary results from the phase 3 RESONATE-2 study demonstrated superior efficacy and tolerability with ibrutinib versus chlorambucil in patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Here, we describe characteristics and outcomes of patients who received ibrutinib treatment for ≥5 years in RESONATE-2. Patients aged ≥65 years with previously untreated CLL/SLL, without del(17p), were randomly assigned 1:1 to once-daily ibrutinib 420 mg until disease progression/unacceptable toxicity (n = 136) or chlorambucil 0.5–0.8 mg/kg for ≤12 cycles (n = 133). Baseline characteristics in ibrutinib-randomized patients (n = 136) were generally similar between patients on ibrutinib treatment for ≥5 years (n = 79) versus those on treatment for <5 years (n = 57). In patients on ibrutinib treatment for ≥5 years, complete response rates improved over time, reaching 42% by 5 years. Estimated 7-year progression-free survival and overall survival rates were 82% and 94%, respectively. Adverse events (AEs) led to dose reductions in 16/79 patients (20%); these AEs were resolved for 13/16 patients (81%). AEs led to dose holds (≥7 days) in 45/79 patients (57%); these AEs were resolved for 43/45 patients (96%). More than half (58%) of ibrutinib-randomized patients benefitted from ibrutinib treatment for ≥5 years regardless of baseline characteristics. Dose modification resolved AEs for most patients, thereby facilitating continued treatment.</jats:p>
|
[
{
"section_content": "Ibrutinib is a once-daily oral Bruton tyrosine kinase (BTK) inhibitor that is approved as first-line treatment for patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) in the United States, Europe, and other countries. Ibrutinib has the longest follow-up of any targeted therapy across multiple randomized phase 3 studies and is the only therapy to date that has demonstrated a significant overall survival (OS) benefit compared with chemotherapy/chemoimmunotherapy in patients with previously untreated CLL/SLL [1] [2] [3] [4]. Initial approval of ibrutinib in the first-line setting was supported by results from the primary analysis of the phase 3 RESONATE-2 study, which demonstrated that ibrutinib was superior to chlorambucil with respect to both efficacy and tolerability [5]. With up to 8 years of follow-up (median: 82. 7 months; range, 0. 1-96. 6 months) in the RESONATE-2 study, the majority of ibrutinib-randomized patients remained progression-free; median progression-free survival (PFS) was not yet reached at the latest data cut [1]. \n\nPrevious studies suggest that patients who continue treatment with single-agent ibrutinib experience better survival outcomes than patients who discontinue treatment within the first few years [6] [7] [8] [9]. Additionally, real-world evidence suggests that dose management (dose reduction or temporary dose holds for up to 1-2 weeks) results in improvement in or resolution of adverse events (AEs) [10] without impacting disease outcomes [6, [10] [11] [12] [13] [14] [15] [16] [17] [18]. Therefore, active management of AEs by dose modification might facilitate continued ibrutinib treatment and maximize clinical outcomes [12]. As of May 2022, the US prescribing information for ibrutinib includes updates to recommended dose modifications for AEs [19]. \n\nAs it is the only BTK inhibitor with long-term follow-up for up to 8 years, we have the opportunity to examine efficacy and safety outcomes in patients with longer-term experience on ibrutinib treatment. Here, we describe characteristics and outcomes of patients who received treatment with ibrutinib for ≥5 years in the RESONATE-2 study.",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "",
"section_name": "Study Design and Patients",
"section_num": "2.1."
},
{
"section_content": ") is a multicenter, international, randomized, open-label, phase 3 study designed to compare the efficacy and safety of first-line treatment with ibrutinib versus chlorambucil in patients aged ≥65 years with previously untreated CLL/SLL who required therapy per the 2008 International Workshop on CLL (iwCLL) criteria [20]. Patients with del(17p) were excluded. Detailed methods were previously reported [5]. Briefly, eligible patients were randomly assigned in a 1:1 ratio to receive oral ibrutinib 420 mg once daily until occurrence of progressive disease or unacceptable toxicity, or chlorambucil 0. 5 mg/kg, escalated to a maximum of 0. 8 mg/kg as tolerated, on days 1 and 15 of each 28-day cycle for up to 12 cycles. After confirmation of progressive disease per iwCLL criteria [20, 21], patients who were randomly assigned to the chlorambucil arm could cross over to second-line treatment with ibrutinib. Per protocol, ibrutinib was temporarily held for any unmanageable grade ≥ 3 AE that was considered by the investigator to be potentially related to the study's treatment. Other AEs, including AEs of grade 2 in severity, could be managed with a one-level dose reduction of ibrutinib if the AE was considered to be potentially manageable by dose reduction as judged by the investigator. \n\nThis study was performed in accordance with International Conference on Harmonisation Guidelines for Good Clinical Practice and the principles of the Declaration of Helsinki. The study protocol was approved by institutional review boards of each participating institution, and all patients provided written informed consent before participation in the study. This study was registered with ClinicalTrials. gov, numbers NCT01722487 and NCT01724346.",
"section_name": "RESONATE-2 (PCYC-1115 [NCT01722487]/PCYC-1116 [NCT01724346]",
"section_num": null
},
{
"section_content": "The current exploratory analysis evaluated baseline demographics and clinical characteristics, overall response rates (per iwCLL criteria [20, 21] ), PFS, OS, prevalence of AEs over time, and AEs leading to dose modifications (per protocol) for ibrutinib-randomized patients who were on ibrutinib treatment for ≥5 years. Since the study protocol provided flexibility for dose reductions based on an investigator's judgment, additional analyses were performed to retrospectively determine the incidence of dose reductions due to AEs for which dose reductions are recommended in the recently updated US prescribing information (grade 2 cardiac failure, grade 3 cardiac arrhythmia, grade 3 or 4 nonhematologic AEs [excluding cardiac failure and cardiac arrhythmia], grade 3 or 4 neutropenia with infection or fever, and grade 4 hematologic AEs) [19]. \n\nBaseline characteristics were also evaluated as potential predictors for remaining on treatment for ≥5 years using a multivariate logistic regression model including the following baseline characteristics: age group, sex, Eastern Cooperative Oncology Group performance status, Cumulative Illness Rating Scale score, creatinine clearance, TP53 mutation status, IGHV mutation status, del(11q) status, disease histology, bulky disease, β-2 microglobulin, Rai stage, any history of cytopenia, lactate dehydrogenase level, and geographic region. Additionally, PFS and OS were analyzed in subgroups of patients with and without dose reductions in the overall population of all ibrutinib-treated patients; these exploratory post hoc analyses were not powered for significance, and comparative statistics are provided for descriptive purposes only. PFS and OS were estimated using the Kaplan-Meier method.",
"section_name": "Analysis",
"section_num": "2.2."
},
{
"section_content": "In RESONATE-2, 269 patients were randomly assigned to receive ibrutinib (n = 136) or chlorambucil (n = 133). Of the 136 patients in the ibrutinib arm, 79 (58%) received ibrutinib treatment for ≥5 years. Median follow-up duration for patients who were on ibrutinib treatment for ≥5 years (n = 79) was 89. 2 months (range: 61. 3-96. 6). Of these 79 patients, 22 subsequently discontinued ibrutinib in years 5-6 (n = 9), 6-7 (n = 10), or 7-8 (n = 3); reasons for discontinuation in these 22 patients were progressive disease (n = 10), death (n = 4), AEs (n = 3), physician decision (n = 3), and patient withdrawal (n = 2).",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "Within ibrutinib-randomized patients in the intention-to-treat population (n = 136), baseline characteristics in the subset of patients who were on ibrutinib treatment for ≥5 years (n = 79) were generally similar to those in the subset of patients who were on ibrutinib treatment for <5 years (n = 57) (Table 1 ). Compared with the subset of patients who were on ibrutinib treatment for <5 years, the subset of patients who were on ibrutinib treatment for ≥5 years were more likely to be in the youngest age group (65-69 years; 37% vs. 19% of patients) and had a longer interval between initial diagnosis and initiation of study treatment (median 35 vs. 26 months).",
"section_name": "Baseline Characteristics",
"section_num": "3.1."
},
{
"section_content": "In multivariate analysis, several baseline characteristics showed a trend toward continuation of ibrutinib treatment for ≥5 years (age ≤ 73 years, female sex, creatinine clearance ≥60 mL/min, TP53 mutated, del(11q), CLL histology, absence of bulky disease [<5 cm], β-2 microglobulin >3. 5 mg/L, Rai stage III/IV, absence of cytopenia, and lactate dehydrogenase ≤250 U/L), but none reached statistical significance (Figure 1 ).",
"section_name": "Predictors of Ibrutinib Treatment for ≥5 Years",
"section_num": "3.2."
},
{
"section_content": "Responses deepened over time, as indicated by the improvement of complete response (CR) rates from 10% (8/79 patients) at 1 year to 42% (33/79 patients) by 5 years and 46% (36/79 patients) by 7 years (Figure 2a ). In patients who were on ibrutinib treatment for ≥5 years, 23/79 (29%) had a documented response of partial response with lymphocytosis (PR-L); of these patients, 9/23 (39%) achieved a best response of partial response (PR), 1/23 (4%) achieved nodular PR (nPR), and 13/23 (57%) achieved CR. In the overall population of ibrutinib-randomized patients, 30/136 (22%) had a documented response of PR-L; of these patients, 13/30 (43%) achieved a best response of PR, 1/30 (3%) achieved nPR, and 15/30 (50%) achieved CR. In patients who were on ibrutinib treatment for ≥5 years, the median time to PR was 4. 6 months (95% CI: 3. 8-7. 4), whereas the median time to CR was 32. 3 months (95% CI: 19. 7-37. 7) for those patients achieving CR. With up to 8 years of follow-up, complete response was achieved in 44 patients in the overall population, 36 of whom received ibrutinib treatment for ≥5 years. \n\nIn patients who were on ibrutinib treatment for ≥5 years, median PFS and OS were not yet reached; 7-year PFS and OS rates were 82% (95% CI: 71-89) and 94% (95% CI: 86-97), respectively (Figure 2b, c ).",
"section_name": "Efficacy in Patients on Ibrutinib Treatment for ≥5 Years",
"section_num": "3.3."
},
{
"section_content": "In patients who were on ibrutinib treatment for ≥5 years, the median duration of ibrutinib treatment was 89. 2 months (range: 60. 4-96. 6). Median relative dose intensity of ibrutinib for these patients was 98% (range: 47-100). The most frequent AEs of any grade across the entire study period were diarrhea (42/79 patients; 53%), cough (34/79; 43%), and upper respiratory tract infection (33/79; 42%). Prevalence of the most frequent AEs of any grade and of grade ≥ 3 were generally highest in years 0-1 and decreased over time thereafter (Figure 3a, b ). Prevalence of AEs of clinical interest of any grade over time are shown in Supplementary Figure S1. AEs of any grade (occurring in ≥25% of patients overall) by yearly interval; (b) Most frequent grade ≥ 3 AEs (occurring in ≥5% of patients overall) by yearly interval. Prevalence was determined by the proportion of patients with a given AE (existing event or new onset of an event) during each yearly interval. Multiple onsets of the same AE term within a specific yearly interval were counted once, and the same AE term continuing across several yearly intervals was counted in each of the intervals. Abbreviations: AE, adverse event; UTI, urinary tract infection; URTI, upper respiratory tract infection.",
"section_name": "Prevalence of AEs over Time",
"section_num": "3.4."
},
{
"section_content": "AEs led to dose reductions in 16/79 patients (20%) who were on ibrutinib treatment for ≥5 years and in 31/135 patients (23%) in the overall population of all ibrutinib-treated patients (Table 2 ). Most patients (12/16; 75%) experienced only one AE leading to dose reduction. \n\na Denominator is patients with dose reductions because of any AE. b The same patient may be counted in more than one category because of multiple AE events leading to dose reduction. c Of 12 AEs that recurred at same/higher grade at any point during treatment, 3/13 were infections, 2/13 were hematologic, 2/13 were cardiac, 1/13 was gastrointestinal, and 4/13 were other. Abbreviations: AE, adverse event; NR, not reached; SOC, system organ class. \n\nAmong patients who were on ibrutinib treatment for ≥5 years, the lowest ibrutinib dose for most patients with dose reductions was 280 mg once daily (10/16 patients). At data cutoff, 3/16 patients were receiving ibrutinib 420 mg once daily, 10/16 were receiving 280 mg once daily, and 3/16 were receiving 140 mg once daily. The median duration of treatment with ibrutinib at a reduced dose was not reached (range: 8. 4-84. 0+ months) for patients who were on ibrutinib treatment for ≥5 years compared with 36. 1 months (range: 0. 0-84. 0+) in all ibrutinib-treated patients with dose reductions (n = 31). \n\nFollowing dose reduction, 13/16 patients (81%) had a resolution of the initial AE. Three patients had AEs that were not resolved at data cutoff (grade 3 malignant lung neoplasm, grade 2 fatigue, and grade 1 contusion in 1 patient each). When considering the subset of AEs for which dose reductions are recommended in the updated ibrutinib US prescribing information (as of May 2022), such AEs led to dose reductions in 4/79 patients (5%) (Table 3 ). Among these patients, AEs did not recur or recurred at a lower grade in 3/4 patients; 1 patient had recurrence at the same grade AE 3 years after initial resolution (grade 3 atrial fibrillation), that resolved without further dose reduction. Patients who were on ibrutinib treatment for <5 years (n = 56) experienced similar rates of AEs leading to dose reduction (15/56; 27%). Most common reasons for dose reduction by system organ class in this subgroup were hematologic (n = 3), cardiac (n = 3), and dermatologic (n = 3). Dose reductions were more common in response to grade 3 AEs (n = 8); however, 100% of AEs (15/15) were initially resolved. Six patients (40%) experienced a recurrence of their AE at the same or higher grade. \n\nAEs led to dose holds of ≥7 days in 45/79 patients (57%) who were on ibrutinib treatment for ≥5 years and in 79/135 patients (59%) in the overall population of all ibrutinibtreated patients (Table 4 ). \n\nAmong patients who were on ibrutinib treatment for ≥5 years, ibrutinib was restarted at 420 mg once daily after dose holds of ≥7 days for most patients (42/45 patients). Following a dose hold of ≥7 days, 43/45 patients (96%) had resolution of the initial AE. \n\nAmong patients who were on ibrutinib for ≥5 years, the frequency of AEs leading to dose reductions was highest in years 0-2 and lower in subsequent years, whereas the frequency of AEs leading to dose holds of ≥7 days remained relatively consistent across the first 6 years of treatment (Supplementary Figure S2 ). a The same patient may be counted in more than one category because of multiple AE events leading to dose holds; b Denominator is patients with dose holds ≥7 days because of any AE. Abbreviations: AE, adverse event; SOC, system organ class.",
"section_name": "Dose Management with Ibrutinib Treatment",
"section_num": "3.5."
},
{
"section_content": "In the overall population of all ibrutinib-treated patients, median PFS for patients who had dose reductions (n = 31) was 87. 7 months (95% CI: 56. 9-NE) and was not reached (95% CI: 81. 9-NE) for those without dose reductions (n = 104) (hazard ratio 0. 96 [95% CI: 0. 50-1. 84]; p = 0. 9011; Figure 4a ). Estimated 7-year PFS rates were 59% (95% CI: 39-74) and 59% (95% CI: 48-68) for patients with and without dose reductions, respectively. With up to 8 years of follow-up, median OS was not reached in either group (hazard ratio 1. 28 [95% CI: 0. 58-2. 83]; p = 0. 5363; Figure 4b ); estimated 7-year OS rates were 74% (95% CI: 54-86) and 79% (95% CI: 69-86) in patients with and without dose reductions, respectively.",
"section_name": "Exploratory Post Hoc Analysis of Outcomes in Patients with Dose Reductions",
"section_num": "3.6."
},
{
"section_content": "Concomitant medications of clinical interest in patients who were on ibrutinib treatment for ≥5 years are shown in Supplementary Table S1. Anticoagulants and antiplatelet agents were frequently used during the treatment period (33% and 65%, respectively), as were antihypertensive medications, including agents acting on the renin-angiotensin system (61%), beta-blocking agents (46%), calcium channel blockers (35%), and other antihypertensives (10%). Overall, 67% of patients received medications to treat acid-related disorders, including proton pump inhibitors in 58% of patients.",
"section_name": "Concomitant Medications",
"section_num": "3.7."
},
{
"section_content": "Results of the current analysis demonstrate that more than half of patients with previously untreated CLL/SLL were able to receive treatment with single-agent ibrutinib for ≥5 years. While real-world studies have suggested an increased risk of discontinuation of targeted therapies in patients with older age, higher comorbidity burden, higher tumor burden, and/or worse performance status at baseline [13, 22, 23], no individual baseline characteristics were identified as significant predictors for continuation of long-term ibrutinib treatment in the current study. \n\nAmong patients who were on ibrutinib treatment for ≥5 years, responses deepened over time. This subgroup of patients had a higher CR rate over the course of the study (46%) relative to the overall population of ibrutinib-randomized patients (34%) [1], suggesting that patients with favorable responses may be more likely to continue on ibrutinib treatment. In line with this, a higher PFS rate at 7 years was seen in patients who remained on long-term ibrutinib treatment for ≥5 years (82%) relative to the overall ibrutinib-randomized population (59%) [1]. These findings are consistent with those of previous studies, suggesting that continuation of ibrutinib treatment is associated with improved efficacy outcomes [6] [7] [8]. \n\nSafety results in patients who were on ibrutinib treatment for ≥5 years were consistent with those seen in the overall population of ibrutinib-treated patients, including incidences of AEs of clinical interest (hypertension, atrial fibrillation, and major hemorrhage) [1]. AEs generally decreased over time with continued ibrutinib treatment, and no new safety signals emerged in patients who received ibrutinib treatment for ≥5 years. Treatment with ibrutinib was well tolerated irrespective of the frequent use of concomitant antithrombotics, antihypertensives, and acid-reducing agents. Since AEs are the most common reason for discontinuation of ibrutinib in the first-line setting [1, [23] [24] [25] [26] [27] [28], optimization of AE management is crucial to enabling patients to remain on long-term therapy. In the subgroup of patients who were on ibrutinib treatment for ≥5 years, active management of AEs with dose reductions or dose holds was associated with AE resolution in the majority (>80%) of patients. Additionally, dose reductions helped to prevent recurrence or worsening of AEs for most patients, facilitating continued benefit from ibrutinib treatment. \n\nIn the current study, disease assessments were performed at regularly scheduled intervals based on iwCLL criteria [20, 21]. With up to 8 years of follow-up in the RESONATE-2 study, PFS and OS were similar between patients with and without dose reductions in the overall population of ibrutinib-randomized patients. Patients who had dose reductions received reduced doses of ibrutinib for extended periods of time (median of 3 years in all ibrutinib-treated patients with dose reductions). Together, these results suggest that patients experiencing AEs leading to dose reduction continue to benefit from ibrutinib at the reduced dose. While two real-world studies found significantly worse PFS in patients receiving ibrutinib at reduced doses (<420 mg once daily), this finding from RESONATE-2 is consistent with several other studies that have found no significant difference in efficacy outcomes between patients with dose reductions due to AEs compared to patients without dose reductions [10, [12] [13] [14] [15] [16] 29, 30].",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "Regardless of demographic and disease characteristics at baseline, more than half (58%) of the patients randomly assigned to the ibrutinib arm in the RESONATE-2 study continued to benefit from ibrutinib treatment for ≥5 years. With up to 8 years of follow-up, the subset of patients who received ibrutinib treatment for ≥5 years experienced sustained efficacy benefits as evidenced by improved depth of response over time and high PFS rates. \n\nFor patients who received ibrutinib treatment for ≥5 years, the safety profile was consistent with previous reports of long-term ibrutinib treatment and no new or unexpected AEs were observed. Dose modification (dose reduction or dose hold) was effective in resolving AEs for most patients, likely facilitating continuation of ibrutinib treatment.",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "We thank the patients who participated in the study and their supportive families, as well as the investigators and clinical research staff from the study centers. Editorial support was provided by Melanie Sweetlove, and funded by Pharmacyclics LLC, an AbbVie Company.",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "",
"section_name": "Institutional Review Board Statement:",
"section_num": null
},
{
"section_content": "Funding: This research was funded by Pharmacyclics LLC, an AbbVie Company.",
"section_name": "",
"section_num": ""
},
{
"section_content": "The study was conducted according to the guidelines of the Declaration of Helsinki, and was approved by the Institutional Review Boards or Independent Ethics Committees of each participating institution. \n\nInformed Consent Statement: Informed consent was obtained from all patients involved in the study.",
"section_name": "Institutional Review Board Statement:",
"section_num": null
},
{
"section_content": "Data Availability Statement: Requests for access to individual participant data from clinical studies conducted by Pharmacyclics LLC, an AbbVie Company, can be submitted through Yale Open Data Access (YODA) Project site at http://yoda. yale. edu.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "The following are available online at https://www. mdpi. com/article/ 10. 3390/cancers15020507/s1: Figure S1, Adverse events of clinical interest of any grade by yearly interval; Figure S2, AEs leading to dose modifications over time in patients on long-term ibrutinib treatment for ≥5 years; Table S1 honoraria from Gilead, Janssen, Novartis, TG Therapeutics, and Pharmacyclics LLC, an AbbVie Company; consulting/advisory role and speakers bureau for BeiGene, Gilead, Janssen, TG Therapeutics, and Pharmacyclics LLC, an AbbVie Company; research funding from AstraZeneca, BeiGene, and Pharmacyclics LLC; an AbbVie Company; travel/accommodations/expenses from Gilead, Janssen, Novartis, TG Therapeutics, and Pharmacyclics LLC; an AbbVie Company. This study was sponsored by Pharmacyclics LLC, an AbbVie Company. The sponsor was involved in study design, data analysis, data interpretation, writing/review of the manuscript, and the decision to publish the results. The sponsor had no role in data collection.",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
10.7150/jca.51275
|
Ethanol extracted from radix of Actinidia chinensis inhibits human colon tumor through inhibiting Notch-signaling pathway
|
<jats:title>Abstract</jats:title> <jats:p>Abstract: Background Colorectal cancer is one of the most common tumors, and its five-year survival is still very low despite of the advance of treatment strategies. The antitumor effect of ethanol extracted from radix of Actinidia chinensis (EERAC) were identified in human colon cancer cells, but the underlying mechanism remains unclear. Methods Cell proliferation, migration, and invasion were measured with CCK-8, wound healing, and transwell assays. Cell apoptosis and cycle were detected by flow cytometry. Western blotting and qRT-PCR were used to measure expression of target molecules. Xenograft tumor assay was applied to detect the influence of EERAC on tumor growth. Results In the present study, we found that EERAC inhibited the cell viability, migration, and invasion of SW480 cells in a concentration dependent manner, but promoted apoptosis and the cell percentage in S phase significantly. The suppression of notch-signaling pathway molecules (Notch1, Jagged1, and c-Myc) by EERAC was confirmed using western blotting and immunohistochemical staining. The significant inhibition of tumor growth by EERAC was also observed. Meanwhile, EERAC remarkably reversed the effects of MAML1 (activator of notch-signaling pathway) on cell survival of SW480. Conclusions Therefore, EERAC might be a promising chemotherapeutic agent for CRC treatment.</jats:p>
|
[
{
"section_content": "Colorectal cancer (CRC) is a common public health problem, being one of the most common gastrointestinal tumors in the world [1, 2]. The bad prognosis induced tumor metastases and invasion leads to the low five-year survival rate of CRC patients [3]. Although chemotherapy is accepted as standard treatment, large scale of patients suffers from the severe side effects, and drug resistance is commonly observed after long-term treatment. These adverse effects have greatly limited its clinical application [4]. Therefore, in order to improve CRC survival rates, searching for a better therapeutic agent with enhanced activity is imminent. \n\nSeveral Traditional Chinese Medicines isolated from natural plants have been widely used to treat tumors. For example, taxol and docetaxel were used to treat breast cancer and ovarian cancer, and irinotecan was applied for advanced CRC treatment [5] [6] [7]. Several types of Traditional Chinese Medicines have been proved to be a potent anti-tumor agent [8]. Flavonoids in Ageratum conyzoides and Chinese herbal formulas Miao-Yi-Ai-Tang have presented significant inhibition on cervical carcinoma and lung cancer, respectively [9]. \n\nActinidia chinensis is an important type of raw materials in the fields of Traditional Chinese Medicines. Actinidia chinensis has presented promising therapeutic effect on several types of cancers including breast, liver, and gastric cancer [10, 11]. The antitumor effects of radix Actinidia chinensis",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "International Publisher is that its root contains a large amount of terpenes, including ursolic acid, oleanolic acid and their derivatives [12] [13] [14]. Besides, many studies have proved that ethanol extract from radix of Actinidia chinensis (EERAC) has an obviously curative effect on several types of tumors including hepatocellular carcinoma and lung cancer, [15] [16] [17]. However, the specific regulatory mechanism remains unknown. \n\nActivation of Notch-signaling pathway was related with angiogenesis, cell migration, invasion, differentiation, and differentiation [18, 19]. Notchsignaling pathway contains notch receptors, notch ligands, and some key downstream proteins [20, 21]. Notch 1, Jagged1, and c-Myc are the main notch receptor, notch ligand, and downstream molecule, respectively. Our previous study indicated that Jagged1 and Notch1 played a vital role during the prognosis, recurrence, metastasis of CRC [22]. Therefore, notch-signaling pathway could regulate the occurrence, development, and prognosis of CRC. However, whether the EERAC can inhibit the proliferation and metastasis of CRC by down-regulating the Notch signal pathway has not been clearly described. \n\nIn the present study, we demonstrated that EERAC could potently suppress the CRC via down-regulating notch-signaling pathway in vivo and vitro. This is the first report implying the inhibition role of EERAC on CRC. Our results may provide new thought about the therapeutic method of CRC. Meanwhile, this research suggests that EERAC may have a good application value.",
"section_name": "Ivyspring",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "The roots of Actinidia chinensis were purchased from Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine. The EERAC (ethanol extract from radix of Actinidia chinensis) was isolated in our laboratory according to extraction technology of effective anti-cancer active ingredient of Actinidia chinensis root from Institute of Chemistry, Chinese Academy of Sciences (Patent publication No. : CN1977869A). The main active chemical components of EERAC were triterpene saponins, which identified by Libermannn Burchard reaction and foam test. Phosphate buffer saline (PBS), Fetal bovine serum (FBS), and tryspin were purchased from KeyGEN Biotech (Nanjing, Jiangsu, China).",
"section_name": "Reagents",
"section_num": null
},
{
"section_content": "Colon cancer cell lines SW480 were obtained from Chinese Academy of Science (Beijing, China). Cells were cultured in DMEM medium (Invitrogen, Carlsbad, California) containing 5% FBS (Invitrogen, Carlsbad, California), and cultivated in the incubator at 37°C with 5% CO 2. After passages, cells were incubated with ERRAC, and applied for different experiments.",
"section_name": "Cell culture",
"section_num": null
},
{
"section_content": "EERAC was dissolved to the concentration of 400 μg/mL with 0. 1% DMSO for stock (DMSO administration concentration <0. 1%). The stock was diluted to 50, 100, 150, 200 μg/mL, respectively. A blank control group (PBS buffer) and a solvent control group (DMSO<0. 1%) were used.",
"section_name": "Drug preparation",
"section_num": null
},
{
"section_content": "Cells (2×10 3 cells/well) were maintained in 96-well plates. For cell viability assessment, transfected cells were incubated for 48 h. After treatment, CCK-8 reagent (Nanjing Jiancheng, Nanjing, China) was added, and OD at 450 nm was detected. The experiment was repeated 3 times.",
"section_name": "CCK-8 assay",
"section_num": null
},
{
"section_content": "The transwell chamber without matrigel (Keygen, Nanjing, China) was used for cell invasion experiment. Cells (1×10 6 ) suspended with 200 μL medium were seeded in the top chamber. The bottom chamber was supplemented with 500 μL medium containing 5% FBS. After 24 h incubation, cells on the lower chamber were fixed using 4% polyoxymethylene for 15 min. Then, 0. 1% crystal violet was used to stain cells for 20 min. Invasive Cells were calculated by capturing 3 fields using an inverted microscope (BX53, Olympus, Tokyo, Japan) at 400× magnification.",
"section_name": "Transwell assay",
"section_num": null
},
{
"section_content": "The horizontal lines were drawn evenly on the back of the 6-well plate with ruler and marker pen. The interval between each two lines is 0. 5-1. 0 cm and the lines crossed the holes. Each well was seeded approximately 5×10 5 cells and incubated overnight. Using a 100 μL pipette tip made scratches in the six-well plate. After scratching, the cell status at 0h was recorded by taking photos. Remove the original cultured medium and wash cells twice with 1 mL PBS. The prepared drug was added to the plate. Cells were cultured on the condition of 37°C and 5% CO2. Cells were recorded after 48 h by taking pictures.",
"section_name": "Wound healing assay",
"section_num": null
},
{
"section_content": "SW480 cells were firstly treated with EERAC for 48 h. Then, cells were collected and lysed using lysis buffer (KeyGEN, Nanjing, China). Same amount of protein was loaded for 12% SDS-PAGE. Then, the gels were transferred to a PVDF membrane (Nanjing Jiancheng, China) electrophoretically. 5% non-fat milk was used for blocking. After 2 h of blocking, membrane was incubated with primary antibodies at 4°C for 12 h. After washing twice, secondary antibodies (1:2000) were applied for incubation for 4 h. TBST washing buffer was used to remove secondary antibodies, and Image J software was used to analyze protein bands. The primary antibodies used were listed as follows: Notch1 (1:800, #194123, Abcam, Cambridge, UK), Jagged1 (1:800, #109536, Abcam, Cambridge, UK), c-Myc (1:1000, #32072, Abcam, Cambridge, UK); beta-actin (1:1500,#16891, Abcam, Cambridge, UK).",
"section_name": "Western blot",
"section_num": null
},
{
"section_content": "RNA was isolated using trizol reagent (TaKaRa, Beijing China). cDNA from different groups were measured by real time PCR with ChamQ TM SYBR ® qPCR Master Mix (Vazyme, California, USA). The information of primers was listed as follows: Jagged1 (F: CGAGTCCTTTACGTGCGTCT, R: CAGACACA CCGGTAGCCATT); Notch1 (F: GAGGCTTGAGATG CTCCCAG, R: ATTCTTACATGGTGTGCTGAGG); c-Myc (F: GAGGAGGAACGAGCTAAAAC, R: TGCT TGGACGGACAGGATG); GAPDH (F: ATGGGGAA GGTGAAGGTCG, R: TCGGGGTCATTGATGGCAA CAATA). GADPH was used as internal control. 2 -ΔΔCT method was used to analyze the change of target gene expression.",
"section_name": "qRT-PCR",
"section_num": null
},
{
"section_content": "Cells (4 ×10 5 ) were plated and cultivated in an incubator. After different treatments, cells were digested, and centrifuged to get cell pellet. Then, cells were suspended using 700 µl binding buffer containing 10 μl propidium iodide (Sigma, St. Louis, Missouri, USA) and 10 μl Annexin V-FITC (Life Technologies, Carlsbad, California, China). After incubation for 30 min in dark, apoptosis was detected using flow cytometric.",
"section_name": "Flow cytometry",
"section_num": null
},
{
"section_content": "3% formalin was used for tissue fixation. After 24 h, tissues were embedded using OCT (Sigma, US). Tissues were sectioned in 10-μm thickness. Heating for 5 min using microwave for antigen repair, and then tissues were washed with PBS. Blocking was applied using goat serum. Then, primary antibody (1:1000) was applied to incubate tissues overnight, and secondary antibody was used to incubate sections for 3 h. DAB reagent was applied to culture tissues, and sections were analyzed with Olympus BX41 microscope (Tokyo, Japan).",
"section_name": "Immunohistochemical staining",
"section_num": null
},
{
"section_content": "The xenograft tumor assay was approved by the Institutional Animal Care of the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University (2019-066). Male nude mice (C57BL/6) were purchased from GemPharmatech (Nanjing, China), and randomly divided into different groups (3 mice/group). HT29 cells (2× 10 5, 0. 1mL) were injected subcutaneously into the back of mice. Animals were fed with EERAC (200 mg/kg) or sterile PBS. All mice were sacrificed after 5 weeks, and tumor weights were analyzed.",
"section_name": "Xenograft tumor assay",
"section_num": null
},
{
"section_content": "The data were shown as mean ±SD, and analyzed with SPSS software (22. 0, IBM, Armonk, USA). t-test was applied to compare the data of two groups. p <0. 05 was believed statistically difference.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The influence of EERAC on SW480 cells growth was measured with CCK-8, wound healing, and transwell methods. We found that EERAC suppressed the proliferation of SW480 cells on a dose-dependent mode (Figure 1A ). Meanwhile, the migration and invasion of SW480 cells were also suppressed remarkably after treatment with various concentrations of EERAC (50-200 μg/mL) (Figure 1B-E ). Therefore, EERAC might present potential ability of anti-colorectal cancer cells.",
"section_name": "EERAC significantly inhibited the proliferation, migration, and invasion of SW480 cells",
"section_num": null
},
{
"section_content": "We found that the apoptosis of SW480 cells was markedly increased after treatment with EERAC (Figure 2A-B ), and the promotion of apoptosis was dose-dependent manner. Meanwhile, results of cell cycle indicated that cell percentage in the S phase were significantly increased by EERAC, but the cells in the G2 and G1 phases were increased (Figure 2C-D ). These findings indicated that treatment with EERAC induced remarkable S phase arrest of SW480 cells.",
"section_name": "EERAC remarkably increased the apoptosis rate of SW480 cells, and increased the cells percentage of S phase",
"section_num": null
},
{
"section_content": "To investigate the potential mechanism how EERAC affects SW480 cells, we measured the expression of some key molecules in Notch signaling pathway after treatment with various concentrations of EERAC. We found that both the protein and mRNA expression of c-Myc, Jagged1, and Notch1 were suppressed by EERAC (Figure 3A-C ). High concentration of EERAC (200 μg/mL) presented a stronger inhibition effect on Notch signaling pathway.",
"section_name": "EERAC significantly inhibited the Notch signaling pathway",
"section_num": null
},
{
"section_content": "After xenograft tumor, no mice death was observed after 3 weeks. We found that EERAC treatment significantly suppressed the tumor weight (Figure 4A-C ). Meanwhile, we detected the levels of c-Myc, Jagged1, and Notch1 in the tumor tissues by IHC staining. The levels of c-Myc, Jagged1, and Notch1 were markedly suppressed by EERAC (Figure 4D ), which was similar to the findings in vitro.",
"section_name": "EERAC remarkably inhibited the xenograft tumor in vivo",
"section_num": null
},
{
"section_content": "MAML1 has been believed to be the activator of Notch signaling pathway, and the overexpression cell model of MAML1 was established in this study. After overexpression of MAML1, the cells were denser compared with control using light microscope, but simultaneous treatment with EERAC significantly decreased cells dense (Figure 5A ). Similar results were observed about the cell proliferation, migration, invasion and apoptosis. Overexpression of MAML1 remarkably promoted the proliferation, migration, and invasion, but suppressed apoptosis (Figure 5B-H ). While, simultaneous treatment with EERAC and MAML1 significantly reversed the effects of MAML1. The proliferation, migration, and invasion of SW480 cells were inhibited, but cell apoptosis was increased remarkably by EERAC (Figure 5B-H ). These findings further confirm the evidence that EERAC might affect colorectal cancer through targeting Notch signaling pathway.",
"section_name": "EERAC remarkably reversed the influence of MAML1 on the survival of SW480 cells in vitro",
"section_num": null
},
{
"section_content": "CRC has become the most frequent gastrointestinal tumor in the world. In recent years, adverse side effects and drug resistance of chemotherapy has reduced success rate of CRC treatment. Nowadays, several types of plant extracts have been proved to be effective for anti-tumor with fewer side effects [23]. EERAC is extracted from the radix of Actinidia chinensis, and exhibited antitumor activity. However, if EERAC could be a potential therapeutic agent for CRC and the specific mechanism remain unclear. Notch-signaling pathway plays a vital role in influencing cell proliferation, apoptosis and differentiation [18]. In recent years, overexpression of NICD1 and Jagged1 was detected in multiple types of cancer, such as CRC, prostate cancer, breast cancer and several types of lymphomas [21, 24, 25]. NF-κB, DLL4, Hes-1and c-Myc are critical downstream molecules in the notch-signaling pathway, and could service as biomarkers for the recurrence, metastasis and prognosis of CRC. Therefore, notch-signaling pathway acts an important role in the occurrence and development of CRC. In this study, we elaborated the relationship between EERAC and notch-signaling pathway in vivo and vitro. \n\nIn this study, the growth inhibition of EERAC in CRC cancer cells depends on the dose-dependent. The cell viability of SW480 cells were decreased from 91. 25% to 23. 97% with various concentrations of EERAC (50-200μg/mL), respectively. In a word, EERAC has significantly inhibitive effects on the growth of SW480 cells. The invasion and migration of tumor cells are the key factors determining the malignancy of cancer. We studied the migration capacity of SW480 in vitro. We found that the invasion and migration capacity of SW480 cells was decreased with the increasing EERAC concentration. \n\nThe antitumor mechanism of EERAC was evaluated on SW480 cells in vitro by western blot. The results indicated that notch-signaling pathway downstream protein Notch1, Jagged1, and c-Myc expression were decreased in SW480 cells after treatment with EERAC suggesting that EERAC might exerted its anti-proliferative activity by targeting these downstream proteins of notch signaling pathway. In order to further confirm the suppression of EERAC on downstream proteins of notch signaling pathway, we measured the expression of c-Myc, Jagged1, and Notch1 in the tumor tissues, and similar findings were achieved. These results showed that EERAC might inhibit the levels of downstream molecules in the notch-signaling pathway to suppress the growth of tumor cells. \n\nOur results demonstrated that EERAC had a potent inhibitory effect on the proliferation, invasion, and migration of SW480 cells. The significant suppression of EERAC on tumor growth in vivo was also observed. Remarkable suppression of EERAC on the downstream proteins of notch-signaling pathway (Notch1, Jagged1, and c-Myc) might be the regulatory mechanism how EERAC inhibits CRC. This study indicates that EERAC may a promising chemotherapeutic agent for CRC.",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The study was supported by National Natural Science Foundation of China (Grant NO. 81704076 ).",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "Data supporting this study has been presented in the manuscript; the data required by editor, reviewer and reader could be provided by the corresponding author.",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "Extracted from radix of Actinidia chinensis (EERAC); colorectal cancer (CRC); Traditional Chinese Medicines (TCM); notch intracellular domain (NICD); phosphate buffer saline (PBS); fetal bovine serum (FBS); Cell counting kit-8 (CCK-8).",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "WH and CZ conceived and designed the experiments; CW, CY, MC, and CJ performed the experiments, WH and CZ wrote the paper.",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "The authors have declared that no competing interest exists.",
"section_name": "Competing Interests",
"section_num": null
}
] |
10.17159/caj/2011/20/1.7180
|
Air Quality and Human Health among a Low Income Community in the Highveld Priority Area
|
<jats:p>Human exposure to poor air quality is linked to adverse health effects. The largest burden of air pollution-related diseases is in developing countries where air pollution levels are also among the highest in the world. In South Africa, two geographic areas, the Vaal Triangle and the Highveld, have been identified for air quality managementinterventions to ensure compliance with National Air Quality Management Standards and to control potential harmful air pollution impacts on human health. The Highveld Priority Area (HPA) is characterised by intense mining, coal-fired power plants, industries, including iron and steel manufacturing, chemical plants, agricultural activity, motor vehicles and domestic fuel burning. Apart from two previous studies, no respiratory health studies have been carried out in the HPA. This paper describes the results of a recent, comprehensive study of ambient air quality, potential exposure to air pollution and air-related human health among a low income community living in the HPA in order to better understand the impact of air pollution on human health in South Africa.</jats:p>
|
[
{
"section_content": "Human exposure to poor air quality is linked to adverse health effects, ranging from acute symptoms, such as nose and throat irritation, to chronic and debilitating illness and disease. The World Health Organization (WHO) states that chronic respiratory diseases, (i. e. asthma, chronic obstructive pulmonary disorder, and pneumonia), are among the leading causes of mortality and morbidity (WHO, 2007; WHO, 2010), and while not all of these adverse health effects are caused by air pollution, they are all exacerbated by the presence of poor air quality. Empirical evidence suggests that the largest burden of air pollution-related diseases is in developing countries where air pollution levels are also among the highest in the world (Krzyzanowski, and Cohen, 2008). In some developing countries, the burden of disease is said to have 'quadrupled' due to the HIV/AIDs epidemic, emerging diseases (especially as they relate to climate change), crime and poverty-driven environmental health outcomes such as diarrhoea. Furthermore, observations in developing countries have indicated that effects of air pollution on these nations may be greater because they are more vulnerable as a result of their nutritional status and lifestyle (Romieu and Hernandez-Avila, 2003). \n\nIn South Africa, the National Environmental Management: Air Quality Act (Act 39 of 2004) allowed for the development of priority areas for air quality management interventions to ensure compliance with national air quality management standards and to control potential harmful air pollution impacts on human health. To date, the Vaal Triangle Air-Shed Priority Area (DEAT, 2006) and the Highveld Priority Area (HPA) (DEAT, 2007) have been declared as priority areas (Figure 1 ). The main towns in the HPA are eMalahleni (previously Witbank) and Middelburg in the Mpumalanga province. The HPA is characterised by intense mining, coal-fired power plants, industries, including iron and steel manufacturing, chemical plants and agricultural activity. In addition to industrial sources, motor vehicles and domestic fuel burning also contribute to air pollution. Since these two sources emit pollutants at ground level, they may contribute more to human exposure than other sources. \n\nTo date, two studies have assessed the human health risks posed by air pollution potentially impacting upon communities living in the HPA. In 1990, the respiratory health of children living in the HPA was compared to that of children living further east in an environment deemed to have less air pollution (Zwi et al., 1991). The 'Highveld' children were found to be more likely to have a morning cough, wheeze, chest colds and asthma compared to the other group. Factors that were identified to increase the risk of children developing these symptoms included attendance of school in the exposed area, cigarette smoking, and not using electricity for cooking in the home. A smaller, cross-sectional study (involving 377 households) was also undertaken in 2006 in eMbalenhle, near Secunda (John et al., 2008). One of the aims of this study was to identify risk or vulnerability factors that may be significantly associated with respiratory health outcomes. The prevalence of health outcomes (including asthma, chronic asthma and pneumonia) were surprisingly low. Apart from these two studies, no further respiratory health studies have been performed thus far in the HPA. \n\nTo fill this gap, and to build onto knowledge generated in the previous studies, a more comprehensive study was initiated to understand the potential impacts of environmental pollution, both air and water pollution, on communities in the HPA. This paper describes one part of this study pertaining to ambient air quality and addresses potential exposure to air pollution and airrelated human health among a low income community living in the HPA in order to better understand the impact of air pollution on human health.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "This paper reports on air quality, personal exposure risk and air-related human health outcomes of a dual arm, cross-sectional study that also examined waterrelated health in a sample of households in a low income community in the HPA. Participating households answered a questionnaire about demographics, possible exposure to water and air pollutants, use of the local river, sanitation, solid waste removal, nutrition, energy use, health and healthcare, personal hygiene and socio-economic factors. A pilot study among 96 households was done prior to implementation of the full study. Ambient air quality monitoring was done at a safe location in the community measuring ambient levels of particulate matter, sulphur dioxide (SO ), nitrogen dioxide (NO ), 2 2 lead (Pb) and transition metals: mercury (Hg) and manganese (Mn). The first three pollutants are National Criteria Pollutants for which recent National Standards have been set under the Air Quality Act (Act No. 39 of 2004) (DEA, 2009). It was decided to include some transition metals in the analysis, since it is known that these metals may cause oxidative and inflammatory reactions in cells (Di Pietro et al., 2009). \n\nMercury was included since it is emitted through the combustion of coal. There are coal-fired power plants, industries using coal, as well as domestic coal use in the area. Manganese was included, because methylcyclopentadienyl manganese tricarbonyl (MMT) is a fuel additive, while Pb is being phased out. \n\nThere are also industries in the area emitting Mn. This air quality monitoring coincided with the questionnaire survey. Ethical approval for this study was granted by the CSIR Research Ethics Committee (No 04/2010).",
"section_name": "Study design",
"section_num": null
},
{
"section_content": "The study area constituted a geographical area of 2 approximately 10 km and is referred to as the town of KwaGuqa (25° 51' 46. 23\" S 29° 07' 13. 88\" E) in the Mpumalanga Province (Figure 1 ). The HPA Baseline Assessment (DEA, 2010) showed that the area with the most air pollution in the HPA was the Witbank / Ferrobank area including the low-income communities of KwaGuqa. KwaGuqa town comprises informal dwellings and low cost housing. Some sections of the town are electrified, have water reticulation and water-borne sewerage, while other sections, especially newly inhabited sections, have no electricity or services (Balmer, 2007)",
"section_name": "Study area",
"section_num": null
},
{
"section_content": "A multi-stage probability sample was implemented. \n\nAn equal number of household interviews was planned for each of the three suburbs (n = 334). Street blocks were randomly selected and therein a random starting point was selected (i. e. North-east, North-west, South-east or South-west). A random starting house was selected choosing one of the first three houses using a random number list. From the starting house, the fieldworker walked around the block in a clockwise direction and interviewed every third house. When respondents refused to participate or were not present, the next house in the sequence was selected. Inclusion criteria stipulated that participants must be older than 18 years of age and not be under the influence of any substance at the discretion of the fieldworker. \n\nSixteen fieldworkers were recruited from the three study suburbs and attended a 6-day training period, with the last day used for piloting. A training manual used in similar studies was adapted and applied. \n\nF i e l d w o r k e r s w e r e f a m i l i a r i s e d w i t h t h e questionnaire, protocol for administering the questionnaire and the electronic device used to ask the questions and record responses. Fieldwork was carried out by the fieldworkers using consistent principles from the protocol from 11 -26 October 2010.",
"section_name": "Participants and procedures",
"section_num": null
},
{
"section_content": "The household questionnaire was derived from previously applied questionnaires (Terblanche et al., 1992; Richards et al., 1996). The questionnaire was transferred to mobile electronic devices beforehand for ease of data capture and data quality assurance. \n\nEach participant was read an information sheet about the study and asked to give written consent prior to administering of the household questionnaire. The questionnaire asked each respondent about demographics, possible exposure to water and air pollutants, use of the local stream, sanitation, solid waste removal, nutrition, energy use, health and healthcare, personal hygiene and socio-economic factors. The instrument has an internal data logger that records the mass concentration every second and averages the particle mass concentrations at 15minute intervals. A pump continuously draws an air sample (flow rate = 0. 6 L/min) through the instrument, and the particle size and mass concentrations were measured using a light-scattering technique that analysed the individual particles as they passed through a laser beam. These same particles were then collected on a reference filter. After the first two weeks of monitoring, the filter was analysed for Mn (a neurotoxic compound) and Pb by atomic absorption spectroscopy. Monitoring then continued for another two weeks, after which the filter was again analysed for Mn and Pb.",
"section_name": "Questionnaire",
"section_num": null
},
{
"section_content": "The gases SO and NO were sampled using Radiello (Trüe et al., 2010).",
"section_name": "Ambient AQ monitoring",
"section_num": null
},
{
"section_content": "Quality control and data assurance for the survey data was performed by means of follow-up interviews with participants. A random sample (15%) of records was drawn from the full dataset to achieve a realised sample of 10% for telephonic follow-up interviews. Systematic sampling was performed by using a random record in the full dataset as the starting point th and selecting every 7 record thereafter. A shortened version of the original structured interview was repeated over the phone and 50 key variables were captured. This was done by a single evaluator fluent in multiple languages to ensure consistency. A total of 174 participants were telephoned. Of these, 127 answered, 121 completed the telephonic shortened version of the questionnaire (6 of those who answered the telephone call did not participate in the face-to-face interview). Only 10% of the follow-up records contained one deviation in the 50 fields checked, demonstrating sound intra-and inter-rater reliability. In cases where deviations from original responses were found during quality control, corrective action was taken to update records accordingly. \n\nThe air quality monitoring equipment was installed and maintained by the supplier according to standard protocols. The applicability of SO and NO analyses 2 2\n\nas sulphate and nitrate determination by ion chromatography was guaranteed by the use of independent reference materials (certified standards) as well as participation in the World Meteorological Organisation-Global Atmospheric Watch (WMO-GAW) inter-comparison scheme. \n\nCertified standards were also used during metal analyses.",
"section_name": "Quality control and data assurance",
"section_num": null
},
{
"section_content": "Questionnaire data were imported into STATA and analysed to determine descriptive statistics. The primary unit of analysis was the household.",
"section_name": "Data analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results and Discussion",
"section_num": null
},
{
"section_content": "The distribution of households selected for the survey by sub-area is indicated in Table 1. A total of 1 639 household interviews were attempted by the 16 fieldworkers. From these, 1 180 interviews were possible because in 375 instances no one was at home, 74 people refused to participate and 10 people did not feel safe to answer the questions. Following quality control and data assurance procedures, the final workable dataset comprised 1 003 households (final response rate = 61. 2%) reflecting a total number of 4 190 respondents. \n\nThe oldest person-age recorded was 98 years, with the average age being 26. 3 years. There were slightly more males (53%) than females in the study sample. About 16% of respondents had not completed any schooling while 5% indicated that they had a tertiary education. Individuals who were unemployed and looking for work constituted 20% of the sample, 19% had full-time paid employment and 31% were school pupils or full-time students. \n\nThe main source of water supply was piped water into the house (70%), or a stand pipe or borehole in the yard (21%). A communal pipe or borehole outside the yard was used by 4% of households. About 14% of those households which used a communal source of water indicated that it was used by more than 75 households. Where water was collected in containers (i. e. no piped water into the house) these were cleaned with detergent by 92% of households, with 80% of households having cleaned them during the past week. Just over 20% of all households indicated that they treated their water, mostly by boiling (66%). A total of 72% of all households interviewed indicated that they were storing drinking water in their house. \n\nOf the 78% of households who indicated that they used a scoop to take water from the storage container, 77% indicated that they may drink from the scoop, too. \n\nResponses indicated that 97% of households had at least one toilet in the yard. Almost 70% of these toilets were a flushed-to-pipe sewer system. Refuse was collected from 63% of households, of which 82% had weekly-collection. Where refuse was collected, 85% of households paid for the removal service. If refuse was not collected, 63% of households indicated that refuse was mostly disposed of outside the yard, of which 91% disposed in a waste pit or dump site. \n\nHouses were mostly constructed of brick or blocks (80% of households), with 23% of households having 3 bedrooms, 45% having 2 bedrooms and 19% having 1 bedroom in the house. The majority of respondents (76%) indicated that there were between 1 and 6 people living in the house on a dayto-day basis.",
"section_name": "Community demographics, services and amenities",
"section_num": null
},
{
"section_content": "Survey results suggested that approximately 77% of households perceived their health to be good. This corroborates the results of a previous study in which 90% of respondents considered the health of family members living in their household to be good (John et al., 2008). The reported prevalence of health outcomes was generally low. Sinus-reported symptoms were the most frequent (42% of households had at least one case), followed by high blood pressure (36% of households) and arthritis (18% of households). Approximately 10% of households reported the presence of at least one case of asthma in their household. According to the South African Demographic Health Survey (SADHS, 1998), the prevalence of asthma in the South African adult (age 15+) population was 7% for men and 9% for women at the time. A total of 17% of respondents indicated that a member of the household has been absent from work or studies because of an illness in the past month. Almost 23% of households indicated that at least one member had access to medical care through medical aid or through a clinic at work. Regarding transport and health facility accessibility, most households were close to a transport mode but not close to a hospital. About 90% (n = 1 217) of children (aged 15 years or less) had been immunised. \n\nAlmost 45% of households indicated that their members usually ate fruit and vegetables daily and most produce had been bought from a nearby shop. Protein such as fish and chicken were being consumed daily by 61% of households. Of the 65% of households with school-going children, 32% indicated that children got food at school regularly. point was divided by its respective daily average. Thus, a value of 1 in Figure 4 indicates the daily average. The PM mass concentration during this 10 campaign peaked at midday (12:00-13:00), but also had smaller peaks in the early morning (1:00-2:00) and evening (17:00). The minimum occurred in the late evening (22:00). The diurnal variation for the duration of the study, suggests that during this time period, local sources of PM contributed throughout 10 the day. Since there is no build-up of pollution in the evening and decrease in the day, the PM mass 10 concentration for this month of measurements was not dictated by daytime convective mixing followed by the formation of a night time boundary layer.",
"section_name": "Health and wellbeing",
"section_num": null
},
{
"section_content": "A pollution rose (Figure 5 ) compiled from the monitored 15-minute averaged PM mass given in Table 2. The averages of the two-week SO 2 and NO concentrations analysed were relatively low. \n\n2\n\nThe maximum concentration for SO and NO were 10 exposure showed a concentration of 14. 7 µg/m of Mn while Pb concentrations were below the detection 3 limit of 0. 01 µg/m. Analysis of the second filter (after the second two-week period) showed that both Mn and Pb concentrations were below the detection limit 3 of 0. 01 µg/m. It is possible that the difference in these results could have been caused by changes in predominant wind direction between the two-week periods. There is no South African National standard for Mn in ambient air, while an annual standard of 0. 5 3 µg/m exists for Pb. In the absence of a National 3 standard for Mn, the WHO guideline of 0. 15 µg/m may be used (WHO, 1999). This guideline was based on a no observed effect level (NOEL) (for 3 neurological effects in workers) of 30 µg/m. However, this guideline is for an annual average and the monitoring period for this study was one month 3 Total Hg concentrations were 2. 0 ng/m during the 3 first two-week period and 2. 4 ng/m during the second two-week period. The main source of Hg in the area is likely to be coal burning. These concentrations correlate with results of a study by Trüe et al. (2010) where Hg was monitored on a weekly basis between 29 October 2009 and 3 December 2009 in eMalahleni town; the average Hg concentration for that 3 monitoring period was found to be 1. 8 ng/m. South Africa does not have a National standard for Hg. The WHO guideline (WHO, 1999) for inorganic mercury in 3 3\n\nair is 1000 ng/m (1 µg/m ), which is an annual guideline based on a lowest observed adverse effect 3 level (LOAEL) of 20 µg/m due to renal effects in humans. This guideline was not exceeded in this study or in the study by Trüe et al. (2010). \n\nIn terms of indoor air pollution exposure, tobacco smoking was prevalent in 24% of households. Coal was used for cooking and heating in 6% and 29% of households, respectively. In a study by Balmer (2007) coal used in this area was found to be of a low quality. The 'Basa njengo Magogo' or top-down method of lighting a fire was used at 13% of households. Electricity was mostly used for cooking (75%) and heating (52%).",
"section_name": "Ambient air quality",
"section_num": null
},
{
"section_content": "From the relatively limited ambient air monitoring campaign measuring PM SO, NO, Hg, Pb and Mn in, 2 2 the study area, preliminary evidence suggests that ambient air quality during September -October 2010 was highly variable with a variety of local sources. In addition, the ambient air quality was not in excess of National standards. This may be because these months fall within spring when temperature inversions are less common and ambient temperatures are milder than during winter months, hence less coal is burnt for household heating. In Josipovic et al. (2009), SO and NO were monitored 2 2\n\nby passive sampling at 37 different sites over the HPA to determine the spatial distribution of these gases and their seasonality. Monitoring sites were situated mostly in rural areas away from urban centres, industrial point sources, main roads or other influences of air pollution. They found the highest concentrations directly over the Mpumalanga industrial Highveld. In the same study, a seasonal trend was more obvious for SO than for NO 2 2, although NO was slightly elevated in summer. The 2 concentrations of SO were higher in winter 2 (Josipovic et al., 2009). The higher concentrations of SO in winter were most probably as a result of an 2 increase in domestic coal use for heating during the cold winter months. Lourens et al. (2011) Descriptive community demographic and health status statistics suggested that household members tended to be relatively young (i. e. average = 26 years of age) and perceived themselves to be in good health. Some individuals did suffer from sinusitis and asthma; however, further statistical analyses will determine risk factors associated with these health outcomes. Together with a better understanding of local issues, which includes stakeholder concerns and community inputs, these results will improve understanding of risks in the community and provide useful information for improved decision-making.",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This project was supported by a CSIR Parliamentary Grant. The communities, clinics and local councillors are thanked for their support and cooperation. The NOVA Institute are acknowledged for developing the survey plan and carrying out the fieldwork in collaboration with the CSIR.",
"section_name": "Acknowledgements",
"section_num": null
}
] |
10.18632/oncotarget.19534
|
NOTCH1 activates the Wnt/β-catenin signaling pathway in colon cancer
|
The translocation of β-catenin/CTNNB1 to the nucleus activates Wnt signaling and cell proliferation; however, the precise mechanism underlying this phenomenon remains unknown. Previous reports have provided evidence that NOTCH1 is involved in the Wnt signaling pathway. Therefore, we sought to determine the mechanism by which NOTCH1 influences the Wnt/β-catenin pathway. We constructed a vector expressing the NOTCH1 intracellular domain (NICD1) and transfected the vector into HCT116 which has low expression of NICD1. Furthermore, inhibition of NOTCH signal pathway in SW480 which has abundant NICD1 expression, was performed by transfection of siNICD1 or DAPT, gamma secretase inhibitor, treatment. In addition, we evaluated NICD1 and β-catenin localization in colon cancer cell lines and in 189 colon cancer tissue samples and analyzed the correlation between the nuclear localization of NICD1 and the clinicopathological features of colon cancer patients.Immunohistochemical assays demonstrated that NICD1 and β-catenin exhibited a similar localization pattern in colon cancer tissues. In addition, we found that NICD1 induced the translocation of β-catenin to the nucleus and that NICD1 and β-catenin co-localized in the nucleus. Overexpression of NICD1 increased luciferase activity of Wnt signal pathway. On the other hand, reduction of NICD1 reduced luciferase activity of Wnt signaling pathway. In the 189 analyzed colon cancer cases, multivariate COX regression analysis demonstrated the independent prognostic impact of nuclear localization of NICD1(p=0.0376).NOTCH1 plays a key role in the Wnt pathway and activation of NOTCH1 is associated with the translocation of β-catenin to the nucleus.
|
[
{
"section_content": "Recent progress in cancer research has revealed that β-catenin/CTNNB1, which functions in cell-tocell adhesion and Wnt signaling, is a key contributor to carcinogenesis in various tissues, including the colon, liver, ovary, and skin [1] [2] [3] [4] [5] [6]. Cellular β-catenin is normally degraded by complexes composed of glycogen synthase kinase-3β (GSK-3β), Axin, and adenomatous polyposis coli (APC) [5, [7] [8] [9]. Mutations in APC, Axin, or β-catenin promote the accumulation of β-catenin and the formation of complexes composed of β-catenin and Tcf/Lef [10] [11] [12] [13]. The β-catenin and Tcf/Lef complex translocates to the nucleus where it transactivates downstream genes [5, 10, 11] that promote the transformation of a normal cell into a tumor cell. Although several genes targeted by this complex, including c-myc and cyclin D1, have been identified, the molecular mechanism underlying β-catenin-Tcf/Lef signaling has yet to be fully characterized [14] [15] [16]. Notably, the mechanism mediating the critical event of β-catenin/ CTNNB1 translocation to the nucleus remains unclear. \n\nThe NOTCH signaling pathway plays a critical role in tissue development and homeostasis by regulating cell fate, proliferation, differentiation, and apoptosis [17, 18]. NOTCH1 has been reported to act as a transcriptional activator that plays essential roles in the development of multiple types of cancers [17, [19] [20] [21]. The NOTCH family includes 4 receptors, NOTCH1-4, whose ligands include JAG1, JAG2, DLL1, DLL3, and DLL4. All of the NOTCH receptors have an extracellular domain containing multiple epidermal growth factor-like repeats and an intracellular region composed of a RAM domain, ankyrin repeats, and a C-terminal PEST domain [22]. NOTCH receptors and their ligands have been shown to be up-regulated in cervical, lung, colon, renal, and pancreatic cancers as well as in acute myeloid leukemia and Hodgkin and large-cell lymphomas [17, [19] [20] [21]. \n\nIn this study, we evaluated the involvement of NOTCH1 in the Wnt/CTNNB1 pathway using a vector expressing the NOTCH1 intracellular domain (NICD1). Several reports have provided evidence suggesting that NOTCH1 functions as a negative regulator of the Wnt signaling pathway [23]. However, there are also recent reports suggesting that NOTCH1 is overexpressed in patients with colon cancer [24] [25] [26] and that a reduction in NOTCH1 expression induces apoptosis in pancreatic cancer cells [21]. \n\nStudies investigating whether NOTCH1 negatively or positively regulates the Wnt signaling pathway have presented conflicting results. Here, we demonstrated that NOTCH1 acts as an oncogene in colon cancer by activating Wnt signaling.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "First, we examined the distribution of β-catenin and NOTCH1 in colon cancer tissue samples using coimmunohistochemistry. Interestingly, the distribution of the 2 proteins was similar in colon cancer cells (Figures 1A and 1B ). In the majority of the colon cancer tissue samples, NOTCH1 and β-catenin were co-localized.",
"section_name": "NOTCH1 and β-catenin exhibited a similar localization pattern in colon cancer cells",
"section_num": null
},
{
"section_content": "We examined the localization of NOTCH1 in 189 colon cancer tissue samples using immunohistochemistry. In approximately 50% of the colon cancer samples, NOTCH1 was strongly expressed in the nucleus (Figure 1A ). Intracellular domain of NOTCH1 is called NICD1 that localizes in the cytoplasm or nucleus of the cells. \n\nIn addition, we observed a statistically significant association between nuclear NICD1 expression and TNM staging (Table 1 ). Nuclear NICD1 expression in colon cancer tissue samples was associated with a significantly greater prevalence of stage T3-4 disease compared with stage T1-2 disease (p=0. 0013) (Table 1 ).",
"section_name": "Association between NOTCH1 localization and clinicopathological parameters",
"section_num": null
},
{
"section_content": "Next, we examined whether NICD1 nuclear localization was associated with patient survival after surgery. Kaplan-Meier survival curves demonstrated that the survival rate was significantly lower in patients with nuclear NICD1 expression (p=0. 0027 by logrank test) (data not shown). The prognostic value of various clinicopathological factors was evaluated using univariate Cox regression analysis (Table 2 ). In addition to the correlation of NICD1 nuclear localization with the T factor (p=0. 0237) and N factor (p=0. 0167) in the TNM staging system, nuclear NICD1 expression was significantly associated with a poor prognosis (p=0. 0217). In contrast, lymphatic invasion and venous invasion were not significantly associated with survival (p=0. 2235 and p=0. 6507, respectively) (Table 2 ). \n\nThe associations of the T factor, N factor, lymphatic and venous invasion, and nuclear expression of NICD1 with prognosis and survival were further analyzed using Cox proportional hazards modeling. Nuclear NICD1 localization and the T factor (p=0. 0086) were identified as significant and independent prognostic indicators (p=0. 0376) of survival in postoperative colon cancer patients. \n\nThe hazard ratio for reduced survival associated with positive nuclear NICD1 expression compared with negative nuclear NICD1 expression was 2. 181 (95% confidence interval: 1. 046-4. 549) (Table 2 ). These findings strongly suggest that nuclear NICD1 expression have an impact on the survival of postoperative colon cancer patients.",
"section_name": "Nuclear NICD1 expression correlates with poor prognoses in colon cancer patients",
"section_num": null
},
{
"section_content": "To determine the cell line to transfect NICD1 expression vector, we examined the status of NICD1 expression in the four colon cancer cell lines (Figure 2A ). In HCT116, NICD1 expression was lower than other cell lines. On the other hand, in SW480, NICD1 expression was abundant among four colon cancer cell lines. Next, to evaluate the effect of NICD1 expression in colon cancer cells, we constructed an NICD1 expression vector. We confirmed the expression of the NICD1 expression vector (pcDNA3. 1-NICD1) in the HCT116 colon cancer cell line using western blot analysis (Figure 2B ). And we inhibited NICD1 expression in SW480 colon cancer cells using siRNA. Western blotting using extracted whole cell lysate showed that NICD1 levels were decreased in siNICD1transfected SW480 cells compared with cells transfected with the control siRNA (Figure 2C ). \n\nFurthermore, we analyzed HCT116 and SW480 colon cancer cells using immunohistochemical assays with antibodies against NICD1 and β-catenin. NICD1 localized to the cell membrane and cytoplasm of HCT116 (Figure 3A ) transfected with control vector. Similarly, β-catenin/CTNNB1 was strongly expressed in the cytoplasm and at the cell membrane in HCT116 cell lines (Figure 3A ). In addition, we observed low levels of endogenous NICD1 expression in the nucleus of HCT116 (Figure 3A ). To further analyze the effect of NICD1 expression, we compared the localization patterns of β-catenin in HCT116 cells transfected with the pcDNA3. 1-NICD1(+) or pcDNA-Mock(-) (as a control) vector (Figure 3A ). Nuclear β-catenin levels were increased in NICD1-transfected HCT116 cells compared with cells transfected with the control plasmid (Figure 3A ). \n\nOn the other hand, endogenous NICD1 localized to the nucleus and cytoplasm of SW480 transfected with control siRNA (Figure 3B ). Similarly, β-catenin/CTNNB1 was strongly expressed in the cytoplasm and at the nucleus in SW480 cell lines (Figure 3B ). To determine the effect of siNICD1 expression, we compared the localization patterns of β-catenin in SW480 cells transfected with the siNICD1 or negative control siRNA (as a control) (Figure 3B ). Nuclear β-catenin levels were decreased in siNICD1-transfected SW480 cells compared with cells transfected with the control siRNA (Figure 3B ). In summary, nuclear β-catenin were observed in NICD1-expressing colon cancer cells, but not in cells transfected with the control vector (Figure 3A ). Reduction of nuclear NICD1 decreased nuclear β-catenin in the colon cancer cells.",
"section_name": "NICD1 induced β-catenin translocation to the nucleus and cell proliferation in colon cancer cells",
"section_num": null
},
{
"section_content": "To determine the effect of NICD1 on genes activated downstream of Wnt signaling, we induced NICD1 expression in HCT116 colon cancer cells using NICD1expressing vector. The results of Western blotting using extracted nuclear protein revealed that nuclear β-catenin levels were increased in pcDNA NICD1(+)-transfected HCT116 cells compared with cells transfected with the control vector (Figure 4A ). Furthermore, CyclinD1, a downstream target of the Wnt signaling pathway, increased in NICD1-induced HCT116 cells (Figure 4A ). \n\nGiemsa staining demonstrated that cell proliferation was enhanced in HCT116 colon cancer cells compared with the control cells (Figure 4B ). Moreover, TCF/βcatenin activity increased in HCT116 cells transfected pcDNA NICD1(+) compared with control vector using luciferase assay (Figure 4C ). Next, to inhibit the translocation of NICD1 to the nucleus in SW480 colon cancer cells with abundant nuclear NICD1, we used DAPT which is a gamma-secretase inhibitor. DAPT inhibits the cutting of the site between NICD1 and NECD (NOTCH1 extracellular domain). The results of Western blotting using extracted nuclear protein revealed that nuclear β-catenin and Cyclin D1 levels were decreased in DAPT-treated SW480 cells compared with cells transfected with DMSO as a control (Figure 5A ). Cell proliferation was reduced in SW480 colon cancer cells compared with the control cells (Figure 5B ). TCF/β-catenin activity decreased in SW480 cells treated DAPT compared with control using luciferase assay (Figure 5C ).",
"section_name": "NICD1 controlled Wnt signal in colon cancer cells",
"section_num": null
},
{
"section_content": "NOTCH1 activation is mediated by gamma secretasemediated cleavage of the NOTCH S3 site [31]. S3 cleavage releases the NOTCH1 intracellular domain (NICD1) from the membrane, and NICD1 subsequently translocates to the nucleus, where it functions as a transcriptional activator [32, 33]. Until recently, it was unclear whether NOTCH1 functions as an oncogene or a tumor suppressor in cancer. Nicolas et al. reported that NOTCH1 acts as a tumor suppressor in mammalian skin [34], and other groups have reported that NOTCH1 acts as a tumor suppressor in esophageal cancer [35] and hepatocellular carcinoma [36]. In contrast to these findings, there have been recent reports that NOTCH1 functions as an oncogene in melanoma [37], breast cancer [38, 39], pancreatic cancer [21], and lymphoma [40]. \n\nThe expression of NOTCH1 has been reported to be up-regulated in colon cancer tissue [25, 26]. Furthermore, NOTCH1 knockdown significantly inhibits proliferation and colony formation in colon cancer cell lines [25]. These findings suggest that activation of the NOTCH signaling pathway plays a role in colon cancer and that NOTCH1 possesses oncogenic activity. Our data revealed that translocation of NICD1 into the nucleus was correlated with a poor prognosis in colon cancer patients (Table. 2). In addition, colony formation assays demonstrated that translocation of NICD1 into the nucleus enhanced cell proliferation in colon cancer cells. Consistent with these findings, other proteins involved in NOTCH signaling, including JAG1-2, HES1, DLL1 and DLL3, are activated in colon adenocarcinoma [24, [41] [42] [43]. Together, these findings suggest that NOTCH signaling is activated in colon cancer. Figure 2A revealed that most colon cancer cell lines expressed NICD1. We used SW480 with especially abundant NICD1 expression and HCT116 with lower expression of NICD1 compared with other colon cancer cell lines. \n\nFurther investigation into the role of NOTCH1 in colon cancer might help to elucidate its role in the Wnt signaling pathway. \n\nThere are many reports describing the crosstalk between the Wnt/β-catenin and NOTCH signaling pathways [23, 35, [44] [45] [46] [47]. Nevertheless, the role of NOTCH1 in Wnt signaling remains controversial. One hypothesis is that NOTCH interacts with β-catenin at the cell membrane in stem and colon cancer cells and inhibits the accumulation of β-catenin [23]. However, β-catenin accumulation in the nucleus and cytoplasm is frequently observed in colon cancer cases with APC mutations [48, 49]. These findings suggested discrepancies related to the hypothesis that NOTCH1 and β-catenin localize to the nucleus in colon cancer cells. Our data revealed that NOTCH1 and β-catenin co-localize in the colon cancer cells. (Figure 1A and 1B )\n\nMembrane-bound NOTCH1 binds to and inhibits unphosphorylated (active) β-catenin [23]. However, once NOTCH1 (NICD1) is activated by gamma-secretase, β-catenin transcriptional activity might be activated by interacting with the activated NICD1. \n\nTransfecting colon cancer cells with the NICD1 expression vector promoted the translocation of β-catenin into the nucleus and induced cell proliferation. As β-catenin lacks a nuclear localization signal (NLS), it might translocate to the nucleus by interacting with NICD1, which contains an NLS. In fact, luciferase assay using TOPFLASH indicated that NICD1 induced TCF/ β-catenin activity. Thus, we hypothesize that NICD1 promotes Wnt signaling by mediating the translocation of β-catenin to the nucleus and that NICD1 functions as an oncogene in colon cancer. Interestingly, the accumulation of NICD1 in the nucleus induces the transformation of rat kidney cells (RKE cells) by up-regulating cyclin D1 expression [50]. Several genes, including cyclin D1 and c-myc, are known to be up-regulated by the activation of β-catenin [14] [15] [16]. As cyclin D1, in conjunction with retinoblastoma protein, is involved in cell cycle regulation, the up-regulation of cyclin D1 expression might promote uncontrolled cell proliferation [15, 16]. Moreover, there are some reports indicating that c-Myc is a transcriptional target of NOTCH1 [51] [52] [53]. \n\nOverexpression of components of the NOTCH signaling pathway is often associated with poor outcomes or tumor metastasis [54]. Together, these findings indicate that NOTCH signaling is oncogenic in a variety of human tumors. Consistent with these findings, our data suggest that NOTCH1 exerts oncogenic activity in colon cancer, as the nuclear translocation of NOTCH1 correlated with the T factor in TNM staging and with a poor prognosis. \n\nOur data indicate that the activation of the NOTCH pathway promotes colon cancer. Therefore, we hypothesize that NOTCH1 acts as an oncogene in colon cancer. \n\nNOTCH1 mutations have been observed in various cancers [55, 56]. The observation that β-catenin and NOTCH1 do not always co-localize in immunohistochemical assays might be attributed to mutations in NOTCH1 itself. However, to the best of our knowledge, there have been no reports of NOTCH1 mutations in colon cancer. \n\nThe association between NOTCH1 and colon cancer was confirmed by the results of experiments using SW480 human colon cancer cells, which express high levels of β-catenin. siRNA-mediated NICD1 depletion in SW480 cells reduced Wnt signaling activity mediated by the β-catenin-Tcf/Lef complex, thereby significantly reducing cyclin D1 expression. Together, the results of our experiments using the NICD1 expression vector strongly suggest that NOTCH1 plays an important role in the β-catenin signaling pathway. \n\nUpon the activation of gamma secretase, NOTCH1 is cleaved at its S3 site, resulting in the release of activated NICD1. In our experiments, activated NOTCH1 (the cleaved NICD1) interacted with β-catenin and translocated to the nucleus. Moreover, cell proliferation was induced in pcDNA3. 1 NICD1-transfected HCT116 colorectal cancer cells expressing low levels of β-catenin. However, additional molecular functions of NOTCH1 in colon cancer have yet to be characterized. We subsequently investigated the association of NOTCH1 expression in resected tumor tissues samples with the clinicopathological features and prognosis of colon cancer patients. \n\nOur findings indicate that the role of NOTCH1 in colon cancer progression merits further investigation. Although the precise molecular mechanisms underlying the up-regulation of NOTCH1 expression and the activation of NICD1 have yet to be elucidated, our data suggest that NOTCH1 is a candidate prognostic molecular marker and a promising molecular target for the development of effective therapeutic options for patients with colon cancer.",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "The HCT116, SW480, LoVo and WiDr human colon cancer cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD). SW480 cells, LoVo, WiDr and HCT116 cells were cultured in L15, Ham F12, EMEM and RPMI medium, respectively, supplemented with 5-10% fetal bovine serum and a 1% antibiotic/antimycotic solution (Sigma Chemical Co., St. Louis, MO) at 37°C in a humidified atmosphere. An antibody ab8925 (Abcam, Cambridge, UK) which actually recognizes the active form of Notch1 receptor, exposed after cleavage by γ-secretase in the methods, was obtained as previously described [27]. \n\nAnti-β-catenin was purchased from BD Transduction Laboratories (Japan) and anti-GAPDH was purchased from Cell Signaling Technology, Inc. (Dancers, MA).",
"section_name": "Cells and reagents",
"section_num": null
},
{
"section_content": "Colorectal cancer tissue samples were obtained from 189 patients who had undergone surgery at the Nagoya City University Hospital (Nagoya, Japan) between January 1998 and December 2007. The resected specimens were staged by pathological evaluation according to the UICC guidelines for clinical and pathological studies of colon cancer (Table 1 ). Colon cancer was confirmed in all of the tumor tissue samples by the Clinicopathology Department. The samples were used after obtaining written consent from the patients.",
"section_name": "Patients and tumor samples",
"section_num": null
},
{
"section_content": "Total cell lysates were prepared in 2% SDS lysis buffer with 330 mM Tris-HCl (pH 8. 8), 2% SDS, 10% glycerol, and 1 Mini Protease Inhibitor Cocktail Tablet (Roche Diagnostics Corp., Tokyo, Japan). Cytoplasmic and nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher scientific Inc., Waltham, MA, USA) according to the manufacturer's instructions. Equal amounts of protein were separated by electrophoresis on 10% Tris-glycine gels. Western blot analyses were performed as previously described [28], and the immunoreactive protein bands were visualized using a chemiluminescence detection reagent. To harvest the cells for immunocytochemistry staining, cytospin slides were prepared with a Cytospin cytology centrifuge. NOTCH1 and β-catenin were detected as previously described [27, 29].",
"section_name": "Protein isolation and immunoblot analysis",
"section_num": null
},
{
"section_content": "The NICD1 cDNA expression vector pcDNA3. 1-NICD1 was constructed to express the region of NOTCH1 between the codon encoding Ser-1748 and the stop codon. To examine the effects of NICD1 on Wnt transcriptional activity, NICD1-expressing cells were plated in 6-well plates and transiently transfected with 3 μg of the pcDNA3. 1-NICD1 plasmid with 9 μL of FuGENE 6 (Promega Corporation, Madison,WI, USA) according to the manufacturer's protocol. The cells were harvested 72 h after transfection, and the localization of NICD1 was analyzed using immunohistochemistry and western blot assays.",
"section_name": "Plasmids and transient transfection",
"section_num": null
},
{
"section_content": "For inhibition of Notch1 in HCT116 cells and SW480, Notch1-siRNA and negative control-siRNA and Lipofectamine® RNAiMAX transfection Reagent were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Cells were plated in 10cm or 6cm dish and transiently transfected with Lipofectamine® RNAiMAX transfection Reagent according to the manufacturer's protocol. The cells were incubated with transfection mixtures containing 20 nM of Notch1-siRNA or negative control-siRNA for 72 hours.",
"section_name": "SiRNA transfection",
"section_num": null
},
{
"section_content": "Cells were treated with 25 μmol/L DAPT (D5642: Sigma-Aldrich, USA) and 0. 1% dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, United States) as a control. After treatment for 72 h, all cells were collected for protein extraction.",
"section_name": "DAPT treatment",
"section_num": null
},
{
"section_content": "We transfected HCT116 and SW480 cells with the pcDNA3. 1-NICD1 expression vector. The transiently transfected cell lines were plated on chamber slides and fixed with 4% paraformaldehyde in PBS. The cells were subsequently permeabilized using 0. 1% Triton X-100 in PBS for 3 min at 4°C and incubated with blocking solution (2% BSA in PBS) for 30 min at room temperature to block nonspecific antibody-binding sites. Then, the cells were incubated with a mouse antibody against NOTCH1 (diluted 1:100 in blocking solution). The primary antibodies were detected using a goat anti-rabbit secondary antibody conjugated to rhodamine, and the stained cells were imaged using a laser scanning confocal imaging system, BZX-700(KEYENCE Corp., Osaka Japan). We confirmed that the transfected cells expressed the NICD protein using western blotting as previously described [30].",
"section_name": "Immunocytochemistry analysis",
"section_num": null
},
{
"section_content": "Equal amounts of total cell lysate solubilized in Laemmli's sample buffer were separated using SDS-PAGE and transferred to Immobilon-P filters (Millipore Corp., Bedford, MA). The filters were incubated with anti-NOTCH1 and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies [anti-mouse IgG and anti-rabbit IgG (Cell Signaling Technology, Beverly, MA) and anti-goat IgG (MBL, Nagoya, Japan)]. The reactions were visualized using an enhanced chemiluminescence system, Amersham Imager 600 (GE Healthecare UK Ltd. Buckinghamshire, UK).",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "Colon cancer sections were obtained from Nagoya City University Hospital (Nagoya, Japan). For antigen retrieval, deparaffinized sections were boiled in citrate buffer (10 mM sodium citrate buffer, pH 6. 0) prior to incubation with the primary antibodies. NOTCH1 and β-catenin protein levels were examined using rabbit polyclonal antibodies (ab8925) and mouse monoclonal antibodies (Abcam, Cambridge, UK). Nuclear CyclinD1 and HistonH3 protein levels were examined using rabbit monoclonal antibodies (Cell Signaling Technology, Inc. Dancers, MA, USA)\n\nAntibody staining was conducted using the peroxidase-based DAKO EnVision System.",
"section_name": "Immunohistochemistry",
"section_num": null
},
{
"section_content": "Cell proliferation was analyzed in the HCT116 and SW480 cancer cell lines using colony formation assays. The cells were plated in 10 cm dishes (2×10 6 cells/dish) for 24 h and subsequently transfected with pcDNA3. 1-NICD1 or pcDNA3. 1-Mock plasmid using 10 μg of plasmid DNA and 30ul of FuGENE6® Transfection Reagent (Promega Corporation, Madison, WI, USA). After the cells were transfected for 24 h, they were diluted 1:8 and cultured for 14 days in the presence of 800 μg/ml geneticin (G418).",
"section_name": "Colony formation assay",
"section_num": null
},
{
"section_content": "HCT116 and SW480 cells were grown on Lab-Tek chamber slides (Nalge Nunc International K. K., Rochester, NY). The cells were washed once with phosphate-buffered saline, fixed with 4% paraformaldehyde for 15 min, and permeabilized using 0. 2% Triton X-100 on ice for 5 min. Cells were then incubated with blocking solution (3% BSA in PBS) for 60 min at room temperature. The cells were subsequently incubated for overnight at 4°C with rabbit anti-NOTCH1 (ab8925) (1:400 dilution) in Tris-buffered saline with 3% bovine serum albumin. Goat anti-rabbit Cy3® (IgG H&L)-preadsorbed ab6939 (Abcam) was visualized in the red channel, and goat anti-mouse Alexa Fluor® 488 (IgG H&L) (Abcam) was visualized in the green channel. Microscopy analysis and image acquisition were conducted using a laser scanning confocal imaging system, BZX-700(KEYENCE Corp., Osaka Japan).",
"section_name": "Immunofluorescence",
"section_num": null
},
{
"section_content": "Cells were seeded onto 4-chamber slides before being fixed and permeabilized. The fixed cells were incubated with anti-NOTCH1and anti-β-catenin (all diluted 1:100) for 1 h at room temperature. The cells were subsequently incubated with the Cy3-conjugated goat anti-rat and FITC-conjugated goat anti-rabbit secondary antibodies diluted 1:100 for 30 min at room temperature. The cell nuclei were stained with ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Protein expression and localization were examined using a confocal microscopy system (FluoView FV500, Olympus).",
"section_name": "Indirect immunofluorescence staining and confocal laser microscopy",
"section_num": null
},
{
"section_content": "TOP-Flash reporter and pTK-RL plasmids were transiently co-transfected into colon cancer cells (5×10 4 ) in 24well plates, and the activities of both firefly and Renilla luciferase reporters was determined at 72 hours after transfection using a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. The TOP-FLASH reporter activity is presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. All experiments were performed three times in triplicate.",
"section_name": "TOP FLASH/FOP-FLASH reporter assay",
"section_num": null
},
{
"section_content": "The biostatistical analyses were conducted using Stat-View software (Abacus Concepts, Berkeley, CA). Student's t-test was employed to determine the optimal cut-off value for comparing gene expression levels between 2 groups. The associations between various clinicopathological characteristics and the localization of NOTCH1 and β-catenin were evaluated using Fisher's exact test. Kaplan-Meier estimates of overall survival were compared via the log-rank test. Cox regression analysis of potential prognostic indicators of survival was used to identify independent factors that significantly affect survival. All tests were two-tailed, and p<0. 05 was considered statistically significant.",
"section_name": "Statistical analysis",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "The authors would like to thank Ms. Seiko Inumaru for her excellent technical assistance.",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
},
{
"section_content": "The authors have no proprietary or commercial interest in any product or concept discussed in this manuscript.",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
},
{
"section_content": "This work was supported by a \"Scientific Research (C)\" Program Grant (26461987) from the Japan Society for the Promotion of Science.",
"section_name": "GRANT SUPPORT",
"section_num": null
}
] |
10.1101/000992
|
Mutated SF3B1 is associated with transcript isoform changes of the genes UQCC and RPL31 both in CLLs and uveal melanomas
|
<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Genome sequencing studies of chronic lympoid leukemia (CLL) have provided a comprehensive overview of recurrent somatic mutations in coding genes. One of the most intriguing discoveries has been the prevalence of mutations in the HEAT-repeat domain of the splicing factor<jats:italic>SF3B1</jats:italic>. A frequently observed variant is predicted to cause the substitution of a lysine with a glutamic acid at position 700 of the protein (K700E). However, the molecular consequences of the mutations are largely unknown.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>To start exploring this question, we sequenced the transcriptomes of six samples: four samples of CLL tumour cells, of which two contained the K700E mutation in<jats:italic>SF3B1</jats:italic>, and CD19 positive cells from two healthy donors. We identified 41 genes that showed differential usage of exons statistically associated with the mutated status of<jats:italic>SF3B1</jats:italic>(false discovery rate of 10%). These genes were enriched in pathways related to interferon signaling and mRNA splicing.</jats:p><jats:p>Among these genes, we found<jats:italic>UQCC</jats:italic>and<jats:italic>RPL31</jats:italic>; notably, a similar effect on these genes was described in a previously published study of uveal melanoma. In addition, while this manuscript was under revision, another study independently reported the common splicing signature of the gene<jats:italic>UQCC</jats:italic>in different tumour types with mutations in<jats:italic>SF3B1</jats:italic>.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Our results suggest common effects of isoform deregulation in the genes<jats:italic>UQCC</jats:italic>and<jats:italic>RPL31</jats:italic>upon mutations in<jats:italic>SF3B1</jats:italic>. Additionally, our data provide a candidate list of potential isoform consequences of the SF3B1 (K700E) mutation in CLL, some of which might contribute to the tumourigenesis.</jats:p><jats:p>Validation studies on larger cohorts and model systems are required to extend these findings.</jats:p></jats:sec>
|
[
{
"section_content": "Several DNA sequencing studies of chronic lymphocytic leukemia (CLL) revealed that the splicing factor SF3B1 accumulated somatic point mutations in about 10% of patients [1] [2] [3]. In most cases the mutations were located in the genomic regions coding for the C-terminal HEAT-repeat domain and in many cases, the mutations gave rise to specific amino acid substitutions. For instance, the substitution of a lysine to a glutamic acid at amino acid 700 of the protein (K700E) was prevalent in the tumour cells. In addition, the affected amino acids seemed to be clustered spatially in the 3D structure of the protein. These observations suggest that specific changes to the function of the protein could be one of the main drivers of tumour progression in CLL. Additionally, DNA sequencing studies have found recurrent mutations in SF3B1 in other malignancies, including myelodysplasia (with high incidence in a particular subgroup, RARS) [4, 5] and uveal melanomas [6, 7]. \n\nmRNA splicing is the process by which introns are removed from pre-mRNA molecules in order to produce fully mature transcripts. A crucial step of the splicing process is the recruitment of the U2 small nucleolar ribonucleic particle (U2 snRNP) to the branch point sequence: this results in base pairing between U2 snRNP and the pre-mRNA that allows the first chemical reaction of splicing to occur [8]. This recruitment is preceded by the binding of the protein A2AF to the pyrimidine tract and subsequent recruitment of SF3b 155 (the protein encoded by SF3B1 ) [9, 10]. In fact, either blocking the interaction of SF3b 155 to the pre-mRNA sequences using the anti-tumour drug spliceostatin A (SSA) or the knockdown of SF3B1 results in unstable recruitment of U2 snRNP, which leads to changes in splicing [11]. \n\nAdditional studies have shown the relevance of SF3B1 in the regulation of splicing in different biological contexts. For instance, it has been shown that the interaction between SF3b 155 and the proteins Xfp144 and Rnf2 from the Polycomb group of genes is required for the repression of Hox genes during mouse development [12]. In a similar manner, it was shown that the loss of interaction between the proteins coded by the genes PQBP1 and SF3B1 alters alternative splicing in mouse neurons and leads to neurite outgrowth defects [10]. These lines of evidence suggest that SF3B1 is necessary for the correct splicing of pre-mRNAs. \n\nThe presence of mutations in SF3B1 is correlated with adverse prognosis and shorter survival of CLL patients [13]. But despite their usefulness as clinical markers, the functional consequences of the mutations in SF3B1 are presently not well understood. It has been hypothesized that the mutations in the HEAT-repeat domain might affect the interaction of SF3b 155 with other co-factors and thus, splicing fidelity. Consistent with that hypothesis, it has been observed that mutations in SF3B1 are associated with the activation of abnormal 3' acceptor sites of specific genes in CLL tumour cells [1]. In a similar manner, transcriptome analyses of myelodysplastic syndromes and uveal melanomas have identified sets of genes with differential exon usage between tumours with mutations in SF3B1 and tumours with no mutations in this gene [7, 14]. \n\nHere, we aimed to start addressing the potential consequences on isoform regulation of the mutation predicted to cause the K700E substitution in the protein coded by SF3B1 in CLL tumour cells. We generated transcriptome data from cells of two tumours with mutations in SF3B1, two tumours without mutations in SF3B1 and from cells from two healthy donors. We identified differences in isoform regulation that were associated with the SF3B1 mutation in 41 genes. We compare our results to previous studies of myelodysplastic syndromes and uveal melanomas with mutations in SF3B1.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Transcriptome-wide data reveal potential isoform regulation associated with mutations in SF3B1 in CLL tumour cells We isolated RNA from B-CLL cells of four patients, of which two contained mutations in the SF3B1 gene (predicted to lead to the K700E substitution in the protein), and two had no mutation in SF3B1. Mutation status of each patient was determined by 454 pyrosequencing. We extracted RNA from CD19-purified cells isolated from the peripheral blood of two healthy donors and prepared cDNA libraries for sequencing (see Table S1 for detailed information regarding the samples). We used Illumina HiSeq 2000 to sequence 50 nt paired-end reads using a strand-specific protocol and obtained a total of 275,000,664 sequenced fragments. We mapped the sequencing reads to the human reference genome (ENSEMBL release 68) using GSNAP (version 2013-05-09), allowing split alignments for exon-exon junctions [15]. We considered only uniquely mapping fragments for further analysis. To observe the expression of the SF3B1 alleles, we counted the number of mapped fragments in each sample supporting the evidence for the mutation. Based on this, we estimated that when the mutation was present, around half of the transcripts were transcribed from the variant allele (Figure 1 ). This estimate was consistent with the variant heterogeneity quantification of the tumours' DNA, as assessed by 454 genomic sequencing (also around 50%, Table S1 ). \n\nWe asked, transcriptome-wide, whether specific differences in isoform regulation in the CLL tumour cells were associated with the presence of the SF3B1 K700E mutation. Therefore, we used DEXSeq to test for differences in exon usage (DEU) [16] between the tumour cells with the mutation K700E in SF3B1 compared to the tumour cells without the mutation and the healthy donors. Briefly, DEXSeq considered, for each exon, the ratio between the number of transcripts originating from the gene that contain the exon and the number of all transcripts originating from the gene. This allowed us to identify changes in relative exon usage independently from the fact that a gene could be differentially expressed. Using this approach, we identified a set of 50 exons in 41 genes with DEU at a false disovery rate (FDR) of 10%. This represents an initial candidate list, and analysis of larger cohorts of patients is needed to confirm these associations. \n\nTo explore the functions of the genes whose isoform regulation was associated with the mutant SF3B1 samples, we mapped these genes to pathways annotated in REACTOME [17]. We found a statistically significant overrepresentation compared to a background set of genes that were also expressed in these cells, of pathways associated with mRNA splicing and translation at a false discovery rate of 10% (see Table S2 ). Interestingly, we also found a significant overrepresentation of the interferon signaling pathway, which is known to inhibit cell proliferation and whose aberrant regulation has been linked to aggressive cases of CLL [18]. These results are consistent with the notion that the SF3B1 K700E mutation could preferentially affect the isoform regulation of genes in particular biological pathways. \n\nThe SF3B1 mutation is associated with differential exon usage patterns seen both in uveal melanoma and CLL Next we compared our results with those of two previously published transcriptomes. Notably, these studies used the same sequencing technology, had a similar study design (but in different malignancies) and also used the DEXSeq method to test for differences in exon usage. \n\nFurney et al. [7] compared three SF3B1 mutant and nine SF3B1 wildtype uveal melanoma tumours and identified 34 exons differentially used in 21 genes (10% FDR). Remarkably, we observed significant overlap between their list of differentially spliced genes and those identified in our study (p = 2 • 10 -3, Fisher's exact test). Specifically, the genes UQCC and RPL31 overlapped with our hits. Furthermore, one out of the two 3' untranslated regions with DEU that Furney et al. reported for RPL31, a gene coding for a ribosomal protein belonging to the 60S ribosomal complex, was also seen as differentially used in our data (Figure 2 ). This, in principle, could have consequences for the localisation, stability or folding of the RNAs from this locus. Additionally, three out of the four exonic regions that we detected as significant for the chaperone UQCC were also detected to be differentially used in the uveal melanoma study. Its authors reported a decrease in the expression of the 3' end of this gene in uveal melanomas with mutated SF3B1, and we observed the same in the CLL tumour cells with mutated SF3B1 (Figure 3 ). Interestingly, this region partly codes for a chaperone domain that is conserved with yeast, where it appears to be required for the assembly of the protein ubiquinol-cytochrome C reductase [19]. In humans, genome-wide association studies have linked this gene to body growth [20]. \n\nEven though we are considering two rather different tumours, and the analyses were done in different laboratories, similar patterns of alternative isoform regulation of the UQCC and RPL31 genes were found to be associated with the mutated status of SF3B1 in both cases. We estimated that the probability of finding by chance that these same exon regions are differentially used was low (p = 1. 4 • 10 -11 ). Hence, our results suggest a link between the mutations in SF3B1 and the differential exon usage of these two genes that holds across tumour types. Additionally, while this manuscript was under revision, an independent publication validated (using RT-qPCR) the common transcript isoform signature of UQCC upon SF3B1 mutations in both tumour types [21] Visconte et al. reported 423 exons in 350 genes to be differentially used at an FDR of 5% between myelodysplasia patients with mutations in SF3B1 and one healthy donor [14]. However, the overlap of their list of genes with our list of genes was not larger than what would be expected by chance, and no common pattern was apparent.",
"section_name": "Results and Discussion",
"section_num": null
},
{
"section_content": "As a data set relevant to the study of the effects on isoform regulation of the expression of mutant SF3B1 in CLL, we report transcriptome data from two CLL patients harboring the K700E mutation in SF3B1, two CLL patients without the mutation, as well as two healthy donor cells. Our results provide an initial list of DEU events that appear associated with the K700E mutation in SF3B1 in CLL (see Supporting Dataset S1). Our data rely on a very limited sample of tumours; substantially larger cohorts (e. g. tens or hundreds of tumours with and without the mutation) will be needed for a more reliable, more comprehensive list of events. A notable result of our analysis is the overlap of events seen here with those in a previous study of uveal melanomas [7], namely, differences in the usage of specific exonic regions of the genes UQCC and RPL31. This effect was further confirmed for the gene UQCC by another independent study using RT-qPCR [21]. These effects could be a prevalent consequence of the mutations in the HEAT-repeat domain of SF3B1. The question of whether or not these effects play a causal role in tumorigenesis is not addressed by our analysis, but may merit further study.",
"section_name": "Conclusion",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Samples were acquired by informed written consent in accordance with the Declaration of Helsinki. Ethical and Institutional Board Review (IRB) approvals were obtained from the University Hospital of Heidelberg.",
"section_name": "Ethics Statement",
"section_num": null
},
{
"section_content": "Peripheral blood samples from four patients matching standard diagnostic criteria for CLL and featuring a high lymphocyte percentage (median: 98%) were obtained from the University Hospital of Heidelberg. Mononuclear cells (MNCs) were isolated by centrifugation over Ficoll-Paque Premium (GE healthcare, Freiburg). MNCs from buffy coats of healthy donors obtained from the blood bank of the University Hospital of Heidelberg were further CD19-purified by magnetic activated cell sorting (MACS) according to the manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach) resulting in purities of ≥ 95% CD19+ cells. Clinical and laboratory data are summarized in Supporting Table S1. Exons of SF3B1, TP53, BRAF, MYD88 and NOTCH1 containing mutation hot spots were amplified and subjected to nextgeneration sequencing on the GS Junior 454 platform (Roche, Penzberg) as in [22]. In brief, genomic regions of interest were amplified from 30 ng genomic DNA in two multiplex-PCRs with 11 primer pairs each. In the same reaction, linker tails were added for ligation of multiplex identifiers and 454-specific adaptors by a second PCR step on the combined pools. Primer sequences are available [22]. Bidirectional sequencing was performed using an emPCR Lib-A kit according to manufacturer's instructions with adaptations. Sequencing data were processed with GSRunProcessor (v. 2. 5/v. 2. 7), performing image and signal processing via the amplicon pipeline (Roche). Variants were identified with Amplicon Variant Analyzer (v. 2. 5/v. 2. 7) and annotated manually according to cDNA references from ENSEMBL Genome Browser. Mutations in SF3B1 were validated by conventional Sanger sequencing and were confirmed to be somatic mutations using whole exome sequencing.",
"section_name": "Sample preparation",
"section_num": null
},
{
"section_content": "Total RNA was isolated from 1 • 10 8 to 5 • 10 8 cells (depending on the sample) via standard trizol extraction. Strand specific RNA-Seq libraries were prepared as described in [23]. Briefly, polyadenylated RNA was isolated from 10 µg of total RNA using Dynabeads Oligo (dT)25 (Invitrogen) according to the manufacturer's protocol. The poly(A) enriched RNA was fragmented by incubating the samples at 80 • C for 4 minutes in the presence of RNA fragmentation buffer (40 mM Trisacetate, pH 8. 1, 100 mM KOAc, 30 mM MgOAc). The fragmented RNA was purified using 1. 8X (v/v) Ampure XP Beads (Beckman Coulter Genomics) and eluted in 25 µl Elution Buffer (EB) (10 mM Tris-HCl, pH 8) according to manufacturer's protocol. 24 µl of eluted RNA was reverse transcribed using 1 µl of random hexamers (30 ng/µl, Invitrogen). The samples were denatured at 70 • C for 5 minutes and transferred to ice. Two µl dNTPs (10 mM), 8 µl 5X first strand buffer (Invitrogen), 4 µl DTT (0. 1 M), 0. 5 µl actinomycin D (1. 25 mg/µl) and 0. 5 µl RNaseOut (40 U/µl, Invitrogen) were added to each sample, and the samples were then incubated at 25 • C for 2 minutes. Following this, 0. 5 µl Superscript III reverse transcriptase (200 U/µl, Invitrogen) was added. The retrotranscription was carried out at 25 • C for 10 minutes, at 55 • C for 60 minutes, and inactivated at 75 • C for 15 minutes. The samples were purified using 1. 8X of Ampure XP beads and eluted in 20 µl EB. For producing the second cDNA strand, 19 µl of sample was mixed with 2. 5 µl of 10x NEBNext Second Strand Synthesis (dNTP-free) Reaction buffer (NEB), 1. 5 µl of dNTPs (containing dUTPs instead of dTTPs, 10 mM), 0. 5 µl of RNaseH (10,000 U/ml) and 0. 5 µl of E. coli DNA polymerase I (10 U/µl, Fermentas). The samples were incubated at 16 • C for 2. 5 hours, 80 • C for 20 minutes and purified with 1. 8X Ampure XP beads, and eluted in 17 µl EB. Two µl end repair buffer and 1 µl end repair enzyme mix (NEBNext DNA Sample Prep Master Mix Set 1, NEB) were added, and the samples were incubated at 20 • C for 30 minutes. The samples were purified using 1. 8x Ampure XP and resuspended in 17 µl EB. Two µl dA tailing buffer (10X NEBuffer 2 from NEB and 0. 2 mM dATP) and 1 µl Klenow Fragment 3' -5' exonuclease (5 U/µl, NEB) were added and the samples incubated at 37 • C for 30 minutes. The samples were purified using 1. 8x Ampure XP and resuspended in 20 µl EB. 2. 5 µl 10X T4 DNA ligase buffer (NEB), 0. 5 µl multiplexed PE Illumina adaptors (7 µM, Supporting Table S3 ) and 2 µl T4 DNA ligase were added (2000 U/µl, NEB) and incubated at 16 • C for 1h. The dUTPs of the second strand were hydrolyzed by incubating the samples at 37 • C for 15 min with 1 µl USER enzyme (1 U/µl, NEB) and 5 minutes at 95 • C. The samples were purified using 0. 9 X Ampure XP beads and eluted in 11 µl EB. Enrichment PCR was performed using 5 µl of sample, 25 µl Phusion Master Mix 2x (NEB), 0. 5 µl each of oligos PE1. 0 and PE2. 0 (10 µM, Illumina) and water up to 50µl final. The PCR program was 30 seconds at 98 • C, 15 cycles of (10 seconds at 98 • C, 30 seconds at 65 • C and 30 seconds at 72 • C) and 5 minutes at 72 • C. The PCR product was size-selected (average of 290bp), and the libraries were submitted for Illumina sequencing.",
"section_name": "Strand-specific RNA-Seq library preparation",
"section_num": null
},
{
"section_content": "We mapped the read fragments to the human reference genome from ENSEMBL (release 68) using GSNAP (version 2013-05-09) [15, 24]. For each sample, we tabulated the number of uniquely aligned fragments that overlapped with exon annotations from ENSEMBL release 68 using scripts based on the python HTSeq library [25]. We used the generalized linear model framework implemented in DEXSeq version 1. 9. 1 to test for differences in exon usage between the samples containing the mutations in SF3B1 and the wild type allele samples [16]. \n\nTo avoid biases associated with gene expression strength in further enrichment analysis, we generated a background set of genes that contained at least 600 sequenced fragment counts. We mapped the ENSEMBL gene identifiers to pathways annotated in REACTOME [17] and tested for overrepresentation of our hits compared to the background using Fisher's exact test. We corrected for multiple testing using the method of Benjamini and Hochberg [26]. We used the ENSEMBL Perl API to convert protein domain coordinates annotated in PFAM to genomic coordinates [27]. Genomic ranges operations were performed using the Bioconductor package GenomicRanges [28], and visualizations of the genomic ranges were done using ggbio [29]. We visualized the coverage vectors and the expression of variants of SF3B1 using the Bioconductor package h5vc [30]. We provide Supporting File S1 with a documented R session with the code that was used to analyse the RNA-Seq data and to produce the figures. The y-axis shows the counts for each sample normalised for sequencing depth. The lines from the samples with the mutation in SF3B1 are coloured in green, and the values from the wild-type samples are coloured in blue. The middle panel shows the flattened gene model (set of non-overlapping exon regions) along the genome (x-axis). This flattened gene model was derived from the ENSEMBL annotated transcripts (lowest panel) as described in [16]. The exon detected to be significant for DEU in both our study (CLL) and in the uveal melanoma study [7] is coloured in magenta. The exon detected as significant only by the uveal melanoma study is coloured in orange. These exons correspond to 3' untranslated regions of transcripts (see lower panel). \n\n. Figure 3 Differential exon usage of gene UQCC. The upper panel shows each exon region of the gene represented along the x-axis. The y-axis shows the counts for each sample normalised for sequencing depth. The lines from the samples with the mutation in SF3B1 are coloured in green, and the values from the wild-type samples are coloured in blue. The middle panels depicts two copies of the flattened model, as defined in [16], derived from the transcripts annotated in ENSEMBL. In the upper flattened model, the exon regions detected to have significant DEU in our study are coloured in magenta. In the lower flattened model, the significant exon regions from the uveal melanoma study are coloured in magenta. The lowest panel presents the genomic regions coding for the protein domain PF03981 annotated in PFAM [27]. This domain is a highly conserved region of the protein Ubiquinol-cytochrome C chaperone.",
"section_name": "Bioinformatics",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We would like to thank EMBL's Genomics Core Facility for the RNA sequencing service and the Information Technology (IT) Core Facility for provision of computational infrastructure. WH and AR acknowledge funding from the European Commission through the Collaborative Research Project RADIANT.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of supporting data",
"section_num": null
},
{
"section_content": "The RNA count data are available in the ArrayExpress database (www. ebi. ac. uk/ arrayexpress) under accession number E-MTAB-2025 and in the Bioconductor data package CLL. SF3B1.",
"section_name": "Availability of supporting data",
"section_num": null
},
{
"section_content": "",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "List of abbreviations DEU -differential exon usage; CLL -chronic lympoid leukemia; FDR -false disovery rate",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare that they have no competing interests. \n\nAuthor's contributions WH, TZ and LMS designed the research. CB, VP, PJ and AR performed the research. TZ and CB selected the patients, isolated the biological samples and performed the mutational analysis. LMS, PJ and VP generated the RNA libraries for sequencing. AR and WH analysed the data and wrote the manuscript with input from all the authors. All authors read and approved the final manuscript. \n\nAuthor details 1 European Molecular Biology Laboratory, Genome Biology Unit, 69117, Heidelberg Germany. 2 Department of Translational Oncology, National Centre for Tumour Diseases and German Cancer Research Centre, Heidelberg Germany. 3 Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA. 4 Department of Medicine V, University Hospital Heidelberg, Heidelberg Germany.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Table S1. Clinical and laboratory data of the CLL patients studied. Table S2. Selected pathways enriched among genes with differential exon usage associated with the SF3B1 mutation (FDR 10%). Table S3. Oligonucleotide sequences used per sample. Dataset S1. HTML report of the genes with DEU associated with the mutations in SF3B1 File S1. Documented R session with the program code needed to reproduce our analysis of the data and to generate the figures",
"section_name": "Additional Files",
"section_num": null
}
] |
10.1186/1749-8104-6-35
|
Basal progenitor cells in the embryonic mouse thalamus - their molecular characterization and the role of neurogenins and Pax6
|
<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>The size and cell number of each brain region are influenced by the organization and behavior of neural progenitor cells during embryonic development. Recent studies on developing neocortex have revealed the presence of neural progenitor cells that divide away from the ventricular surface and undergo symmetric divisions to generate either two neurons or two progenitor cells. These 'basal' progenitor cells form the subventricular zone and are responsible for generating the majority of neocortical neurons. However, not much has been studied on similar types of progenitor cells in other brain regions.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>We have identified and characterized basal progenitor cells in the embryonic mouse thalamus. The progenitor domain that generates all of the cortex-projecting thalamic nuclei contained a remarkably high proportion of basally dividing cells. Fewer basal progenitor cells were found in other progenitor domains that generate non-cortex projecting nuclei. By using intracellular domain of Notch1 (NICD) as a marker for radial glial cells, we found that basally dividing cells extended outside the lateral limit of radial glial cells, indicating that, similar to the neocortex and ventral telencephalon, the thalamus has a distinct subventricular zone. Neocortical and thalamic basal progenitor cells shared expression of some molecular markers, including <jats:italic>Insm1</jats:italic>, Neurog1, Neurog2 and NeuroD1. Additionally, basal progenitor cells in each region also expressed exclusive markers, such as Tbr2 in the neocortex and Olig2 and Olig3 in the thalamus. In <jats:italic>Neurog1</jats:italic>/<jats:italic>Neurog2</jats:italic> double mutant mice, the number of basally dividing progenitor cells in the thalamus was significantly reduced, which demonstrates the roles of neurogenins in the generation and/or maintenance of basal progenitor cells. In <jats:italic>Pax6</jats:italic> mutant mice, the part of the thalamus that showed reduced Neurog1/2 expression also had reduced basal mitosis.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Our current study establishes the existence of a unique and significant population of basal progenitor cells in the thalamus and their dependence on neurogenins and Pax6. These progenitor cells may have important roles in enhancing the generation of neurons within the thalamus and may also be critical for generating neuronal diversity in this complex brain region.</jats:p> </jats:sec>
|
[
{
"section_content": "The immense number of neurons in the mammalian neocortex is thought to be determined during development by a prominent progenitor cell population that shows a distinct pattern of migration and division. Unlike the predominant progenitor cell type in other brain regions, the radial glial cells (RGs), these cells divide basally away from the ventricular surface and undergo a symmetric division that generates two neurons or two progenitor cells. It is thought that the six-layered mammalian neocortex is largely dependent on division of these basal progenitor cells that serve as transit amplifying cells or intermediate progenitor cells (IPCs), and that the evolution of the mammalian cortex is correlated with the emergence of progenitor cell populations that enhance the generation of neurons [1] [2] [3] [4]. \n\nBasally dividing progenitor cells have been identified not only in the cerebral cortex, but also in ganglionic eminences, thalamus, hindbrain and spinal cord [5] [6] [7] [8] [9]. However, only the cerebral cortex and ganglionic eminences have been shown to harbor a robust enough population of basal progenitor cells to form a distinct domain, the subventricular zone (SVZ), above the domain of RGs that comprises the ventricular zone (VZ). Recent studies identified a number of molecular markers of basal progenitor cells in the developing neocortex. In addition, genes such as Tbr2 [10, 11], Insm1 [12] and AP2g [13] or inhibition of Notch signaling [14, 15] are found to be essential for the generation of basal progenitor cells from RGs. Time-lapse analysis of fluorescently labeled cortical progenitors in slices elucidated the unique migratory patterns and modes of division of neocortical basal progenitor cells and showed that these cells function as transit amplifying progenitor cells, or IPCs [9, 16, 17]. \n\nThe mammalian thalamus has an extremely complex organization with several dozen distinct neuronal populations called nuclei [18]. During embryogenesis, the thalamus is composed of two molecularly distinct domains of neural progenitor cells, pTH-C and pTH-R, located across rostro-caudal and dorso-ventral axes [19]. pTH-C is a larger, caudo-dorsally located domain that expresses the basic helix-loop-helix (bHLH) transcription factors neurogenin 1 (Neurog1) and neurogenin 2 (Neurog2) and gives rise to all of the thalamic nuclei that project to the cortex. pTH-R is a smaller domain that expresses another bHLH protein, Ascl1 (also known as Mash1), and lies between pTH-C and the zona limitans intrathalamica (ZLI), the boundary population that abuts the thalamus and the prethalamus [19]. pTH-R likely contributes to the majority of GABAergic neurons in the thalamus, including part of the ventral lateral geniculate nucleus and intergeniculate leaflet. Recent studies have unveiled critical roles of secreted signaling molecules in the formation of positional diversity of thalamic progenitor cells [20] [21] [22] [23] [24]. \n\nDespite the finding that there are basally dividing cells in embryonic mouse thalamus [6], their molecular characteristics and the mechanisms for their generation have not yet been determined. Considering the extensive connections between the thalamus and neocortex, we anticipated that the mammalian thalamus has diversified its progenitor cell populations during evolution to allow generation of a larger number of neurons comparable to those found in the six-layered neocortex. \n\nIn the study described here, we explored this possibility by performing a detailed characterization of thalamic basal progenitor cells in mouse embryos. We found that the thalamus contains a remarkably large number of basal progenitor cells, some of which form the SVZ similar to that found in the neocortex and ventral telencephalon. Thalamic basal progenitor cells do not express the same molecular markers as neocortical IPCs, such as Tbr2, but they do share many other aspects with their putative cortical counterpart, including the expression of the bHLH transcription factors NeuroD1 and neurogenins (Neurog1 and Neurog2). We also show the first evidence that Neurog1 and Neurog2 are required for the normal number of basally dividing cells, which demonstrates the critical role of these transcription factors in the formation and/or the maintenance of thalamic basal progenitor cells.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "The embryonic mouse thalamus contains a large number of basally dividing cells\n\nWe found numerous cells away from the surface of the third ventricle that express the M-phase marker phosphorylated histone H3 (PH3), which we define as dividing basal progenitor cells (Figure 1A-E, arrowheads ). These cells were found as early as at embryonic day (E)10. 5 (Figure 1A, B ) and persisted until at least E14. 5 (Figure 1E ). Double/triple immunohistochemistry showed that most of these basal progenitor cells are within the progenitor domain pTH-C, which gives rise to all of the thalamic nuclei that project to the cerebral cortex [19] (Figure 1B, marked as 'C'). Within the pTH-C domain, the ratio of basal PH3-positive cells to total PH3-positive cells was highest at E12. 5 and declined at E14. 5, when thalamic neurogenesis is largely complete, except in the most dorsal location (Figure 1E -G) [25]. In contrast, fewer PH3-positive cells were found in the progenitor domain pTH-R, which produces neurons that do not project to the cortex [19] (Figure 1B, marked as 'R') and in the ZLI, the border cell population abutting the thalamus and the prethalamus (Figure 1B, marked as 'ZLI'). The ratio of PH3-positive cells in the basal location to the total PH3-positive cells was significantly higher in pTH-C than the other two domains analyzed (pTH-R and ZLI; Figure 1H ). Figure 1I shows the average number of PH3-positive cells in each of the 20 μm-wide medial-lateral bins within pTH-C at E12. 5. In addition to the high peak at the ventricular (apical) surface (bin 1), there was another peak of PH3-expressing cells away from the third ventricle (bin 6), indicating the presence of a discrete population of thalamic progenitor cells (Figure 1I ). These initial analyses demonstrate the presence of basally dividing progenitor cells in the thalamus throughout neurogenesis and that they are particularly enriched in the progenitor domain pTH-C.",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We next asked if the basally dividing cells comprise a distinct zone in the mouse thalamus that is not populated by RGs. Such a zone, the SVZ, emerges in mice by E14. 5 in the neocortex, and by E13. 5 in the ganglionic eminences [26] ; however, it has not been evaluated in other brain regions. We used NICD (intracellular domain of Notch) and Pax6 as markers of RGs. NICD is a cleavage product of Notch1 [27], which is co-expressed with Nestin within the neocortical VZ, but not in the SVZ [28]. Notch activity inhibits the formation of IPCs from RGs in the neocortex, indicating that the presence of NICD is a marker for RGs within the VZ. Pax6 is also highly expressed in neocortical RGs in the VZ [29], although low-levels of Pax6 expression are detectable in many IPCs [29] and a recent report identified a new class of Pax6-expressing progenitor cells that divide away from the lateral ventricle in the mouse neocortex [30]. \n\nWe found that NICD is expressed in a cluster of cells near the third ventricle at both E11. 5 (Figure 2A,C,C', left of dashed line) and E12. 5 (Figure 2E,G,G', left of dashed line). Basal PH3-positive cells were located on both sides of the lateral margin of this NICD cluster at E11. 5 and E12. 5 (Figure 2A, E ; arrows indicate the outside population and arrowheads indicate the inside population). The outer population became more evident at E12. 5 (Figure 2E ). PH3-positive cells were also observed on both sides of the Pax6 domain (Figure 2B,F, arrowheads and arrows). Similar to the neocortex, more laterally located basal progenitor cells expressed low levels of Pax6 (Figure 2B,D,D' at E11. 5; 2F,H,H' at E12. 5). In addition, Pax6 expression was generally lower in the rostral part of the pTH-C domain at both E11. 5 and E12. 5 (Figure 2B,F, bracket). Labeling of S-phase cells with a 0. 5-hour ethynyl deoxyuridine (EdU) pulse showed that some thalamic progenitor cells reside outside the NICD + /Pax6-high zone (Figure 2C', D'G'H' ). Based on these results, we propose that, as early as E11. 5, a molecularly distinguishable SVZ exists in the pTH-C domain of the thalamus, which we define as the zone where progenitor cells exist outside of the NICD + /Pax6-high VZ. Thalamic basal progenitor cells populate both the VZ and SVZ.",
"section_name": "The embryonic mouse thalamus has a defined subventricular zone",
"section_num": null
},
{
"section_content": "We then examined the expression patterns of previously characterized genes that are expressed in thalamic progenitor cells in order to determine the progenitor zone (VZ or SVZ) and progenitor cell types (RGs or basal progenitor cells) in which each gene is expressed (Figures 3, 4 and 5 ). Thalamic progenitor cells ubiquitously express the bHLH transcription factor Olig3 [19], but the neocortex does not. Double staining with a 0. 5-hour EdU pulse showed that the domain of Olig3 expression in the thalamus encompassed the entire medial-lateral extent of thalamic progenitor cells, indicating that Olig3 is expressed in both the VZ and SVZ of the thalamus (Figure 3A, F ). In addition, we found that Olig3 heavily overlaps with NICD (Figure 5A ), demonstrating that Olig3 is expressed in RGs. Together, these results show that Olig3 is expressed in both the VZ and SVZ and in both RGs and basal progenitor cells in the thalamus. \n\nWe next determined if NeuroD1 and Insm1 (insulinoma-associated 1), markers for neocortical IPCs, are also expressed in the thalamus. NeuroD1 is a bHLH transcription factor that is expressed in the upper SVZ and lower intermediate zone of the neocortex, presumably being induced following Tbr2 expression [31]. In the pTH-C domain of the thalamus, a densely packed population of NeuroD1-positive cells was found in the middle portion of the diencephalic wall (Figure 3B, arrow). In addition, some NeuroD1-expressing cells were scattered within the VZ. Double immunostaining for NeuroD1 and a 0. 5-hour pulse of EdU showed that NeuroD1 is expressed in basally located progenitor cells in S phase of the cell cycle (Figure 3G, arrowheads). These NeuroD1 + /EdU + cells seemed to be predominantly located in the SVZ. NeuroD1 was also clearly expressed outside of the NICD-expressing VZ (Figure 5D, arrow) and the scattered NeuroD1-positive cells within the VZ did not express NICD (Figure 5D, arrowheads), indicating the lack of NeuroD1 expression in RGs. Double staining experiments also showed that some basal progenitor cells co-expressed NeuroD1 and PH3 (not shown). These results together demonstrate that NeuroD1 is expressed in thalamic basal progenitor cells at least through S phase to M phase of the cell cycle, but not in NICDexpressing RGs. \n\nInsm1 is a zinc-finger transcription factor expressed broadly in progenitor cells within the embryonic brain and spinal cord located away from the ventricular surface [32]. It is required for the generation of basal progenitor cells in the neocortex [12]. We found that Insm1 is strongly expressed in a lateral band of cells within the thalamus. Comparison of Insm1 with PH3 on the same section shows that Insm1 is indeed expressed in thalamic basal progenitor cells (Figure 3C, H ). \n\nOlig2 is a bHLH transcription factor expressed in the pTH-C domain of the thalamus in a rostro-ventral high to caudo-dorsal low gradient at E11. 5 and E12. 5 [19]. We found that Olig2 is not only expressed in the VZ (Figure 3D, arrowhead) but also in a more lateral region (Figure 3D, arrow). Olig2 expression overlapped with a 0. 5-hour EdU pulse (Figure 3I ), and extended further laterally (Figure 3I, arrow; Figure 5E ). Within the VZ, Olig2 colocalized with NICD (Figure 5E, arrowheads), suggesting that it is expressed in RGs. Thus, similar to Olig3, Olig2 is expressed in both RGs and basal progenitor cells. Olig2 also appeared to be expressed lateral to the SVZ, most likely in the mantle zone. \n\nFinally, Lhx2 and Lhx9 are LIM-homeodomain transcription factors expressed in the thalamus [33, 34]. In the neocortex, Lhx2 is expressed in neural progenitor cells and Lhx9 is expressed in the marginal zone [35, 36]. We found Lhx2/9-positive cells are largely confined outside the VZ, with only a minimum overlap with a 0. 5-hour EdU pulse (Figure 3J ), indicating that they are expressed mostly in postmitotic cells. \n\nInterestingly, a well-established IPC marker in the neocortex, Tbr2, a T-box transcription factor [15, 29], was undetectable in the thalamus at E11. 5 and E12. 5 (data not shown). \n\nThese results collectively show that although the thalamus has a histologically identifiable SVZ populated by basal progenitor cells and these cells share expression of some genes, such as Insm1 and NeuroD1, with neocortical IPCs, they are clearly distinct from their putative neocortical counterpart. Thalamic basal progenitor cells do not express Tbr2 and express additional markers such as Olig2 and Olig3 that are not expressed in the neocortex.",
"section_name": "Thalamic and neocortical basal progenitor cells share some molecular properties",
"section_num": null
},
{
"section_content": "To further characterize the thalamic basal progenitor cells, we examined the expression of two bHLH proteins, Neurog1 and Neurog2, both of which are expressed in neocortical progenitor cells. In the neocortex, expression of Neurog2 is initiated soon after the division of RGs, preceding the induction of Tbr2 [15]. Britz et al. [37] reported that at E12. 5, 95% of Neurog1-expressing progenitor cells in the cortical VZ also express Neurog2, and at E15. 5, both Neurog1 and Neurog2 are expressed in the VZ as well as the SVZ. \n\nWe previously showed that Neurog1 and Neurog2 are expressed in the pTH-C thalamic progenitor domain [19]. In this study, we examined the patterns of their expression in more detail. Comparison 5B, dashed line). Thus, in contrast to the neocortex, Neurog1 expression in the thalamus is confined to the VZ. Within the VZ, Neurog1 and Neurog2 showed partially overlapping but distinct expression patterns (Figure 4C ). Similar to the neocortex [28], neither of these two transcription factors co-localized with NICD within the VZ (Figure 5B, C, arrowheads ). This result is consistent with the hypothesis that neurogenin-expressing VZ cells are basal progenitors translocating laterally towards the SVZ. In contrast, Olig2 and Olig3 were expressed in both the thalamic VZ and SVZ and had extensive overlap with NICD within the VZ (Figure 5A, E ).",
"section_name": "Proneural bHLH proteins Neurog1 and Neurog2 are expressed in overlapping but different progenitor populations in the thalamus",
"section_num": null
},
{
"section_content": "We next examined the cell cycle properties of thalamic basal progenitor cells. First, we pulsed the progenitor cells with an S-phase marker, EdU, and analyzed the distribution of PH3-positive cells at various times after EdU injection. We detected EdU and PH3 on the same section of E11. 5 and E12. 5 embryos to estimate the time it takes progenitor cells to enter M phase (Figure 6 ). In E11. 5 embryos that had been pulsed with EdU 0. 5 hours prior to sacrifice, we detected a large, single cluster of EdUpositive cells that encompassed a broad medial-lateral region of the thalamic progenitor domain, suggesting the close proximity of RGs and basal progenitor cells during S phase (Figure 6A ; black curve in Figure 6F, G ). As expected, very few mitotic cells expressing PH3 are labeled by EdU. \n\nAt 2 hours after EdU injection, we detected some EdU-positive cells at the ventricular surface and the region closer to the ventricle (Figure 6B, arrow; red curve in Figure 6F, G ). Many PH3-positive cells both at the ventricular surface and in the basal location were also EdU-positive (Figure 6B, arrowheads). This indicates that, particularly at E11. 5, cells start to enter M phase about 2 hours after S phase. \n\nAt 4 hours, as many as 60 to 75% of PH3-expressing cells were positive for EdU at both the apical and basal locations (Figure 6C, arrowheads; Figure 6E ). In addition, we found two dense clusters of EdU-positive cells that were now separated from each other. One was located close to the ventricle. The other population was located more laterally (green curve in Figure 6F, G ). This separation implies a distinct migratory behavior of thalamic basal progenitor cells, which stay in the basal location from S phase to M phase. Conversely, RGs translocate their nuclei medially from S phase to M phase by interkinetic nuclear migration. \n\nAt 8 hours, we again detected only a small overlap between EdU and PH3, indicating that a majority of progenitor cells labeled 8 hours before have already divided. A broad cluster of EdU-positive cells was found in the middle of the diencephalic wall (Figure 6D, between the dashed lines), and additional EdU-positive cells were found far laterally, which are likely to be postmitotic cells (blue curve in Figure 6F, G ). \n\nIn summary, the EdU pulse experiment distinguishes RGs and basal progenitor cells because of their distinct patterns of migration during their cell cycle.",
"section_name": "Cell cycle properties of basal progenitor cells in the thalamus",
"section_num": null
},
{
"section_content": "By taking advantage of the EdU pulse labeling, we next examined the expression of NeuroD1, Lhx2/9, Neurog1 and Neurog2 in more detail with regard to the cell cycle status of basal progenitor cells. As already shown in Figure 3 NeuroD1 also partially co-localized with p27 (Figure 8H ), a cyclin-dependent kinase inhibitor expressed in differentiating neural progenitor cells as well as postmitotic neurons [38, 39], but it did not co-localize with NeuN (Figure 8C ), a marker for a subset of postmitotic neurons, suggesting that NeuroD1 expression is transient. \n\nLhx2/9 was expressed in the lateral part of the thalamus, and showed only a minor overlap with EdU at each of the pulse times (Figure 7E-H,7V ). The overlap with neuronal markers NeuN and p27 was robust (Figure 8E,J), indicating that Lhx2/9 expression persists in postmitotic neurons, consistent with a previous study showing widespread expression of Lhx2 and Lhx9 in postmitotic thalamic nuclei [33]. \n\nAs shown in Figure 4 8F,G) showed that neurogenins overlap with p27 but not with NeuN. Thus, the expression of neurogenins is transient.",
"section_name": "Expression of basal progenitor markers at different stages of the cell cycle",
"section_num": null
},
{
"section_content": "In the neocortex, Neurog1 and Neurog2 together play a role in neuronal differentiation and, at the same time, in the specification of the dorsal telencephalic fate of neural progenitor cells [40]. Microarray analysis shows that the expression levels of Tbr2 and NeuroD1 in the neocortex are decreased in Neurog1/2 double knockout mice [40]. Although histological analysis of cortical IPCs with immunohistochemistry for Tbr2 and NeuroD1 has not been reported in these mutant mice, both PH3-positive mitotic cells and bromodeoxyuridine-labeled S-phase progenitor cells are increased in the SVZ and decreased in the VZ in Neurog2 single as well as Neurog1/2 double knockout mice [37], suggesting that these transcription factors are likely to play an important role in IPC specification and/or differentiation. \n\nIn order to determine if neurogenins play a role in the formation or maintenance of basal progenitor cells in the thalamus, we analyzed Neurog1/2 double knockout mice and Neurog1 and Neurog2 single knockout mice in comparison with double heterozygous controls. We found that double knockout mice (Neurog1 -/-; Neurog2 -/-) have fewer PH3-positive, dividing basal progenitor cells in the pTH-C domain at E12. 5 (Figure 9D, E ). Both the absolute number and the ratio against the total PH3-positive cell number were significantly reduced from the controls. In contrast, the number of apical PH3-positive cells or the total PH3positive cells did not show a significant difference. The Neurog2 single (Neurog1 +/-; Neurog2 -/-) mutant showed reduction in absolute number of basal PH3-positive cells but not in the ratio against the total PH3-positive cells (Figure 9C, E ). The Neurog1 single mutant did not show any significant difference from the control (Figure 9B, E ). These results indicate that neurogenins are required for the normal number of basally dividing progenitor cells in the thalamus, and that the role of Neurog2 is only partially compensated by Neurog1. \n\nAs already shown previously [41], another bHLH transcription factor, Ascl1 (also known as Mash1) is induced in the neocortex of Neurog2 single and Neurog1/2 double mutant mice. Ascl1 is normally expressed at a high level in the ventral telencephalon, suggesting a role for neurogenins in specifying dorsal telencephalic fate and suppressing ventral telencephalic fate. It has also been shown that neurogenins are required to suppress Ascl1 expression in the thalamus [41, 42]. Consistent with these previous findings, we found robust Ascl1 induction in the thalamus of Neurog1/2 double mutant mice (Figure 9H ), whereas Neurog2 single mutants (Neurog1 +/-; Neurog2 -/-) showed much less severe induction of Ascl1 (Figure 9G ). Ascl1 was not induced in Neurog1 single mutants (Neurog1 -/-; Neurog2 +/-; data not shown). These results demonstrate that neurogenins, of which Neurog2 is the prominent one, suppresses Ascl1 expression. Reduction of the basal progenitor cell number in the thalamus of neurogenin mutant mice indicates that Ascl1 does not compensate for the function of neurogenins in this cell type. Interestingly, Tbr2, a cortical IPC marker, was normally not expressed in the thalamus but was ectopically induced in the mantle zone of the thalamus of the Neurog1/2 double mutant (Figure 9K, L ). Considering the fact that SVZ mitosis was increased in the neocortex [37] but decreased in the thalamus (Figure 9 ) of Neurog1/2 double knockout mice, we conclude that the roles of neurogenins in basal progenitor cells in the thalamus are likely different from those in the neocortex. \n\nThe paired-/homeo-domain transcription factor Pax6 is known to play a critical role in thalamic development [43]. As already shown in Figure 2, high-level expression of Pax6 was detected in the thalamic VZ, although the expression decreased in the rostro-ventral part of the pTH-C domain at E11. 5 and later. In Pax6 mutant mice, we detected reduction of Neurog2 expression (Figure 10E, G) and ectopic induction of Ascl1 (Figure 10F, H ) in the ventral part of the pTH-C domain, but not in the dorsal part (Figure 10A-D ). The ratio of basal PH3-positive cells was specifically reduced in ventral sections, where a large number of Ascl1-expressing cells were intermingled with Neurog2-expressing cells (Figure 10G-I ). The decrease in the number of basal PH3-positive cells was accompanied by an increase in the number of apical PH3-positive cells (Figure 10J ), indicating the role of Pax6 in generating basal progenitor cells from apical progenitor cells. The total number of basal plus apical PH3-positive cells did not change between wild-type and mutant embryos, at both dorsal and ventral levels (data not shown).",
"section_name": "Neurogenins are required for the formation and/or maintenance of basal progenitor cells in the thalamus",
"section_num": null
},
{
"section_content": "In this study, we showed that, throughout thalamic neurogenesis, a high proportion of progenitor cells divide away from the third ventricle and some of these basal cell divisions occur outside of the VZ. We found that basal progenitor cells are most abundant in the thalamic progenitor domain that expresses the bHLH transcription factors Neurog1 and Neurog2, which are also expressed in the neocortex where basal progenitor cells abound. The thalamus and the neocortex share some of the molecular markers expressed in these cell populations, including Neurog1, Neurog2, NeuroD1 and Insm1, but each also expresses a unique set of genes. For example, Tbr2 is expressed only in the neocortex and Olig2 and Olig3 are, n = 13 for Neurog1 -/-;Neurog2 +/-, n = 13 for Neurog1 +/-;Neurog2 -/-, n = 4 for Neurog1 -/-;Neurog2 -/-). One sample is one section. Numbers of basal, apical and basal plus apical PH3-positive cells as well as the ratio of basal PH3 + cells/total PH3 + cells were compared between the genotypes. One-way ANOVA for Neurog1 +/-;Neurog2 +/-, Neurog1 -/-;Neurog2 +/-and Neurog1 +/-;Neurog2 -/-embryos; F = 11. 96 for basal PH3, F = 1. 744 for apical PH3, F = 4. 517 for basal + apical PH3, F = 5. 881 for basal/total ratio. N. s., not significant; **P < 0. 01. expressed only in the thalamus. We then characterized various transcription factors that are differentially expressed at different cell cycle stages of thalamic progenitor cells. We further showed that two bHLH transcription factors, Neurog1 and Neuorg2, as well as the paired-/homeo-domain transcription factor Pax6, are required for the normal number of thalamic basal progenitor cells.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Our current study has identified a domain of progenitor cells outside the thalamic VZ. We term this external zone of progenitor cells the thalamic SVZ. The term thalamic SVZ has been used before (for example, [44] ), but no reports have shown the presence of progenitor cells outside of the VZ that are either dividing or in S phase of the cell cycle. In this paper, we used the presence of cleaved Notch1 (NICD) as the landmark of the VZ [28]. In the neocortex, Notch signaling plays a critical role in maintaining RG fate and inhibiting the expression of Neurog2 and Tbr2, and thus the formation of IPCs [14, 15, 45]. Within the pTH-C domain of the thalamus, progenitor cells outside the VZ start to be detectable at E11. 5 and become more prominent at E12. 5, although basal cell division occurs not only within the SVZ, but also in the VZ as early as E10. 5, when thalamic neurogenesis has just started [25]. Based on these results, we propose a classification of thalamic progenitor cells into three types based on where they divide. The first population (type I) is the RGs that divide at the apical surface of the third ventricle. If we define them as producing NICD (although the signal is weaker near the ventricular surface), they also express Olig3 and Olig2. The second (type II) and third (type III) populations divide away from the ventricular surface; type II cells divide within the VZ and type III cells divide in the SVZ. Based on our gene expression analysis, Neurog1 differentiates type II and type III cells because it is expressed by NICD-negative cells in the VZ, but not in the SVZ. Among the markers expressed in both types of progenitor cells, Neurog2 is expressed evenly between the VZ and SVZ, while Neu-roD1-expressing cells are distributed much more densely in the SVZ than the VZ. Two other bHLH factors, Olig2 and Olig3, are expressed in all three progenitor types (summarized in Figure 11 ). Interestingly, a recent study analyzed gene expression profiles of single progenitor cells in E14 mouse cortex and classified the progenitor cells into three clusters that likely correspond to RGs, VZ basal progenitors and SVZ basal progenitors [45]. Since it is unclear whether the two basal progenitor cell clusters in the cortex represent the difference in their state of cell cycle phase (for example, G1/S phases for VZ basal progenitor cells and G2/M phases for SVZ progenitor cells) or the location of mitosis, it remains to be determined how our two progenitor populations (types II and III) differ in overall gene expression profiles and how they are related with regard to cell lineage. \n\nWhat is the significance of the large number of basal progenitor cells in the thalamus?\n\nOur current study shows that the thalamus is one of the brain regions where basal progenitor division is most prominent. In comparison with a similar quantification study, the ratio of basal PH3-positive cells in E12. 5 thalamus is comparable to the peak ratio of basal divisions in neonatal cortex [46]. As in the neocortex, basal progenitor divisions occur in both the VZ and SVZ. Although basal division of neural progenitor cells has been described in many regions in the central nervous system, including the spinal cord and the hindbrain [5] [6] [7] [8] [9], the existence of the embryonic SVZ, which is populated exclusively by basal progenitor cells and not by RGs, has been described only for the neocortex and the ventral telencephalon, where basal progenitor cells are dominant and a large number of neurons are generated. We propose that the thalamus belongs to this group of brain regions. Considering the extensive interconnections between the mammalian thalamus and the neocortex, it is intriguing to speculate that these two brain regions have evolved together to produce a balance in the large numbers of neurons needed to connect these regions. \n\nRecent lineage tracing studies show that both Neurog1and Neurog2-expressing progenitor cells produce neurons of all thalamic nuclei that project to the neocortex [19, 47]. Because Neurog1 is expressed in basal progenitor cells in the VZ but not in the SVZ, whereas Neurog2 is expressed in both populations, it will be interesting to determine if the basal progenitor cells in the VZ and those in the SVZ generate different sets of neurons in each nucleus. The potentially distinct postmitotic cell fates of Neurog1-and Neurog2-expressing progenitor cells might result in specific thalamic phenotypes in Neu-rog2 single knockout mice. Seibt et al. [48] showed normal expression of Lhx2 and Gbx2, both of which are widely expressed in postmitotic thalamic neurons at E12. 5, in Neurog2 single knockout mice. We have also obtained similar results in Neurog1 +/-; Neurog2 -/-mice (data not shown). Thus, analysis of later embryonic stages with nuclei-specific markers would be necessary to reveal the specific roles of Neurog2 in thalamic neurogenesis. \n\nAlthough our study does not provide information on how the thalamic basal progenitor cells migrate, divide and produce progeny in real time, it is possible they have similar properties to neocortical IPCs, which divide symmetrically to self renew or produce two neurons, and that thalamic basal progenitor cells contribute to the diversity and the large neuronal number of thalamic nuclei. Our analysis of gene expression combined with EdU pulsing support this hypothesis (summarized in Figure 11 ). It is also possible that some thalamic basal progenitor cells later generate oligodendrocytes and/or astrocytes. Future lineage tracing studies using live imaging and genetic fate mapping will be able to test these possibilities.",
"section_name": "Medial-lateral organization of the thalamic progenitor domain",
"section_num": null
},
{
"section_content": "We observed a decreased number of basal PH3-positive cells in the thalamus of Neurog2 single and Neurog1/2 double knockout embryos. In the neocortex, the level of Tbr2 mRNA is decreased in neurogenin mutant mice [37], and in vivo over-expression of Neurog2 increased Tbr2expressing cells in the cortex 24 hours after electroporation [15]. However, basal PH3-positive cells were increased at the expense of apical mitosis in the neocortex of Neurog2 single and Neurog1/2 double knockouts [37]. In contrast, we saw a decrease in basal progenitor cells and an unchanged number of apical progenitor cells in the thalamus of these knockout mice. Our data suggest that Neurog2, once induced in one of the daughter cells after the radial glial division in the neocortex and perhaps in the thalamus, plays a cell-autonomous role in specifying (5). Type III cells divide basally in the SVZ and generate two neurons (6) or two type III cells (7). The actual lineage relationship between the three progenitor cell types in the thalamus is a topic of future investigation. basal progenitor fate [15]. Differences in the genes regulated by neurogenins in the thalamus and neocortex and the functions of these genes may account for the different phenotypes in the SVZ of neurogenin knockout mice. \n\nThere are many other regions in the central nervous system where neurogenins are expressed, but, among them, only the neocortex and the thalamus appear to have prominent populations of basally dividing cells. Conversely, the ventral telencephalon expresses Ascl1 and not neurogenins, but still contains a large number of basally dividing progenitor cells. Therefore, expression of neurogenins alone is not sufficient or absolutely necessary for a large population of basal progenitor cells. Further study is needed to determine what other molecules play a shared role in the neocortex and thalamus. \n\nOur study also showed that Pax6 mutant mice have reduced numbers of basal progenitor cells in the ventral part of the thalamus where there was a severe reduction of neurogenin expression and massive induction of Ascl1. Unlike in the neurogenin mutants, however, we saw increased apically dividing cells in this region of the thalamus. The similarities and differences between the neurogenin and Pax6 mutants indicate that although Pax6 regulates the formation of basal progenitor cells in the thalamus by regulating the normal expression of neurogenins, it may also have distinct roles in RGs, which control the balance between the apical and basal progenitor cells.",
"section_name": "Roles of neurogenins and Pax6 in the generation/ maintenance of thalamic basal progenitor cells",
"section_num": null
},
{
"section_content": "Our study provides evidence for the presence of a prominent population of basal progenitor cells in the embryonic mouse thalamus, part of which forms the SVZ. Combined analysis of transcription factor expression and cell cycle status revealed that these basal progenitor cells may be divided into two populations: one that divides in the VZ and another that divides in the SVZ (summarized in Figure 11 as a working hypothesis). We also found that neurogenins and Pax6 are required for the formation and/ or maintenance of basal progenitor cells in the thalamus. Our study implicates the importance of this special progenitor cell population in enhancing the generation of neurons within the thalamus and may also be critical for generating neuronal diversity in this complex brain region.",
"section_name": "Conclusions",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Care of and experimentation on mice were done in accordance with the Institutional Animal Care and Use Committee of the University of Minnesota. Noon of the day on which the vaginal plug was found was counted as E0. 5. Stages of early embryos were confirmed by morphology [49]. Timed-pregnant CD1/ICR mice (Charles River) were used for gene expression analysis of wild-type mice. Pax6 mutant mice [50] were obtained from G Lanuzo and M Goudling at the Salk Institute and were kept in CD1 background. Neurog1 [51] and Neurog2 [48] mutants were established in F Guillemot's lab (National Institute for Medical Research, London), produced by J Johnson's lab, and were kept in CD1 background.",
"section_name": "Animals",
"section_num": null
},
{
"section_content": "Axial and anatomical nomenclatures are described in [19]. The two progenitor domains of the thalamus, pTH-C and pTH-R, as well as the ZLI were identified by the expression of marker genes Olig3, Ascl1 and Neurog2 [19].",
"section_name": "Axial and anatomical nomenclature",
"section_num": null
},
{
"section_content": "Immunohistochemistry was performed as described [19, 30]. Additional antibodies used were: anti-NICD (rabbit, 1:100; Cell Signaling, Danvers, MA, USA), anti-NeuroD1 (goat, 1:100; Santa Cruz, Santa Cruz, CA, USA), anti-PH3 (mouse and rabbit, 1:100; Millipore, Temecula, CA, USA) and anti-Lhx2 (goat, 1:100; Santa Cruz, sc-19344). Antibody sc-19344 appears to detect a broad postmitotic region in the thalamus at E14. 5 (data not shown), consistent with the possibility that it recognizes both Lhx2 and Lhx9 [33]. For NICD detection, we extended the boiling time to 10 minutes to enhance the antigen retrieval, and also used a Tyramide Signal Amplification System (Perkin Elmer, Waltham, MA, USA). Detailed protocols for the entire procedures are available upon request.",
"section_name": "Immunohistochemistry",
"section_num": null
},
{
"section_content": "In situ hybridization was performed as described [19]. Insm1 cDNA was obtained from J Garcia-Anoveros (Northwestern University).",
"section_name": "In situ hybridization",
"section_num": null
},
{
"section_content": "EdU was dissolved at 0. 5 mg/ml in PBS, and injected intraperitoneally into pregnant female mice at 10 μg/g body weight. Embryos were dissected after varying amounts of time (0. 5 hours, 2 hours, 4 hours or 8 hours). EdU was detected with a protocol based on that reported in [52]. For simultaneous detection of EdU and various other antigens, cryosectioned brains on slides were first treated with primary and secondary antibodies. Slides were then washed once with 1× PBS and permeabilized with 0. 5% Triton, then rinsed twice with 1× PBS. EdU labeling was detected with the Click-iT EdU Imaging Kit (Invitrogen, Carlsbad, CA, USA); detection solution was applied directly to slides and incubated for 15 minutes. Slides were then rinsed and coverslipped according to our previous immunostaining protocol [19].",
"section_name": "Cell cycle analysis",
"section_num": null
},
{
"section_content": "Images were collected with a Nikon E800 microscope or Olympus FluoView 1000 confocal microscope and assembled by Image J (NIMH) and Photoshop CS3 or CS5 (Adobe).",
"section_name": "Image analysis",
"section_num": null
},
{
"section_content": "For single counts of PH3-expressing cells, images of 20μm-thick frontal sections were taken with a Nikon E800 fluorescent microscope. The embryonic mouse thalamus was delineated using specific markers characteristic of the pTH-C and pTH-R domains and the ZLI [19]. For each section, the thalamus was divided into 20-μm bins from the ventricular surface to the lateral surface. Counts were taken for PH3-positive cells per bin. The cell counts from all sections were summed for each half brain. The proportion of PH3-positive cells away from the ventricular surface (>40 μm or >2 bins) was calculated from the total number of PH3-positive cells in the thalamus. The average proportion of PH3-positive cells was calculated with four thalami per embryonic stage. \n\nFor EdU and PH3 co-localization, images were acquired by an Olympus FluoView 1000 confocal microscope. Each section was divided into bins similar to those described above, and the proportion of PH3-positive cells that co-localized with EdU was calculated from the total PH3-positive cell count. Five to six 3-μm-thick optical slices were obtained for each field of view, and two of them were taken for cell counts. \n\nFor EdU-positive cell count and co-localization of EdU and basal progenitor markers, images were acquired and analyzed as described for EdU and PH3 co-localization. However, only a portion of the thalamus (the first 200 μm from the pTH-C/pTH-R border) was analyzed. \n\nFor PH3-positive cell count in Pax6 mutants, two or three adjacent sections of a 300-μm-long column of the pTH-C domain were analyzed separately for dorsal and ventral levels. The ratio of basal PH3-positive cells was calculated for each of the 14 sections counted for each genotype. In addition, the absolute numbers of PH3-positive cells per section were also counted and compared. \n\nCell count data were analyzed and graphed using Prism 4 Software (GraphPad). A one-way ANOVA test was used to determine statistical significance, where P < 0. 05 indicated significance. A post-test -the Tukey multiple comparison's test -was used to determine significance among groups. Double asterisks in indicate P < 0. 01, triple asterisks indicate P < 0. 001.",
"section_name": "Cell counting of PH3-and EdU-expressing cells",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We thank A Hanson and M Simon for their excellent technical assistance, J Johnson ( University of Texas Southwestern Medical Center ) for providing neurogenin knockout mouse embryos and comments on the manuscript, P Kofuji and Vision Core Facility of University of Minnesota ( P30 EY011374 ) for the use of a confocal microscope, and Tim Cherry ( Harvard Medical School ) for the EdU detection protocol. Part of this research was supported by NINDS ( R01 NS049357 to YN). LW was supported by University of Minnesota Undergraduate Research Opportunities Program (UROP), and KKB was supported by NICHD training grants ( T32HD007480, T32HD060536 ) and University of Minnesota Graduate School. We thank S McLoon, P Letourneau and T Vue for comments on the manuscript and members of Nakagawa Lab for discussion.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Authors' contributions LW carried out experiments on wild-type embryos and drafted the manuscript. KKB carried out experiments on neurogenin and Pax6 mutants and drafted the manuscript. LD generated and genotyped neurogenin mutant embryos. YN conceived the study, and participated in its design and coordination and wrote the manuscript. All authors read and approved the final manuscript.",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "Competing interests",
"section_num": null
}
] |
10.3390/molecules22122083
|
Lipoprotein Lipase Expression in Chronic Lymphocytic Leukemia: New Insights into Leukemic Progression
|
<jats:p>Lipoprotein lipase (LPL) is a central enzyme in lipid metabolism. Due to its catalytic activity, LPL is involved in metabolic pathways exploited by various solid and hematologic malignancies to provide an extra energy source to the tumor cell. We and others described a link between the expression of LPL in the tumor cell and a poor clinical outcome of patients suffering Chronic Lymphocytic Leukemia (CLL). This leukemia is characterized by a slow accumulation of mainly quiescent clonal CD5 positive B cells that infiltrates secondary lymphoid organs, bone marrow and peripheral blood. Despite LPL being found to be a reliable molecular marker for CLL prognosis, its functional role and the molecular mechanisms regulating its expression are still matter of debate. Herein we address some of these questions reviewing the current state of the art of LPL research in CLL and providing some insights into where currently unexplored questions may lead to.</jats:p>
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[
{
"section_content": "Lipoprotein lipase (LPL, EC 3. 1. 1. 34) is a N-glycosylated protein [1] that forms homodimers and is able to hydrolize triglycerides from chylomicrons [2] and very low-density lipoproteins [3]. The first evidence of its existence was serendipitously found when studying circulating red blood cell mass in dogs. In those experiments, it was found that the administration of heparin as an anticoagulant was able to counteract alimentary lipemia in five minutes or less [4]. LPL plays a central function in lipid metabolism and has been subject of intense and meticulous studies ever since. General aspects of LPL biology have already been reviewed elsewhere [5, 6].",
"section_name": "",
"section_num": ""
},
{
"section_content": "LPL active dimer consists of two antiparallel subunits [7] whose formation and trafficking rely on a series of post-translational modifications. Interaction with calcium-dependent chaperones of the N-glycosylated polypeptide chain has been proven essential to the correct folding of LPL [8]. Furthermore, a lipase chaperon-Lipase-maturation factor 1 (Lmf1)-has been suggested to be required for dimer assembly and activity, as mutations in LMF1 cause lipase deficiency in mice [9]. A mouse model overexpressing LMF1 has increased LPL activity [10], and LPL has been co-immunoprecipitated with Lmf1 and Sortilin-related receptor (SorLA) [11]. It has been shown that LPL intracellular localization is regulated by SorLA, which directs its trafficking from the trans-Golgi network to endosomes [11]. LPL internalization by receptor-mediated endocytosis has been studied [12] either through LDL receptor-related protein [13] or by an LDL-receptor independent pathway [14].",
"section_name": "LPL Synthesis and Trafficking",
"section_num": null
},
{
"section_content": "Chronic lymphocytic leukemia (CLL) is the most frequent form of leukemia among adult populations of Caucasian origin [15]. CLL is a malignancy of mature clonal B lymphocytes that accumulate in the blood, bone marrow and other lymphoid tissues, and is diagnosed upon the presence of ≥5000 clonal B lymphocytes per microliter of peripheral blood persisting for more than 3 months [16]. This leukemia is characterized by the accumulation of long-lived circulating clonal leukemic B-cells resulting from a complex balance between cell proliferation and apoptotic death. Increasing evidence suggests that CLL B-cells in lymph nodes (LN) and bone marrow (BM) that interact with stromal cells receive proliferative signals and are protected from cell death. These data led to the view that CLL is a dynamic process composed of cells that also proliferate and die, often at appreciable levels [17]. This crosstalk with accessory cells in specialized tissue microenvironments favors disease progression, by promoting malignant B-cell growth and the emergence of new genetic alterations which will lead to drug resistance [18]. Disease prognosis and the heterogeneous clinical evolution in CLL are probably related at least in part to these microenvironmental signaling, and although available treatments often induce remissions, CLL remains an incurable disease [19]. \n\nIn CLL one third of the patients have an indolent disease with long survival and never require treatment, another third have an aggressive disease from onset and need to be immediately treated, and the last third have an indolent disease at onset which may last for years but then invariably progress to an aggressive disease [20]. It is because of this latter group that the search for strong prognostic markers in CLL predicting disease evolution has been of capital importance, and a number of them have been developed, the most reliable and universal still being the mutational status of the variable region of the heavy chain of immunoglobulin (IgHV) genes [21, 22]. Patients carrying somatic hypermutation in their IgHV genes-mutated CLL (Mut)-display a better prognosis than patients with unmutated (Um) IgHV genes",
"section_name": "Chronic Lymphocytic Leukemia",
"section_num": "2."
},
{
"section_content": "",
"section_name": "LPL in Chronic Lymphocytic Leukemia",
"section_num": "3."
},
{
"section_content": "Gene expression profiling analyses comparing Um and Mut patients were performed during the first decade of the century. We and others have performed these studies and described that LPL is differentially overexpressed in Um patients [23] [24] [25]. With these results in mind we selected and validated two genes, LPL for Um and ADAM29 for Mut CLLs, as candidates to propose a novel prognostic method. This methodology was tested in a cohort of 127 CLL patients, and correlated to clinical outcome and IgHV mutational status. Finally, we demonstrated that quantification of LPL and ADAM29 gene expression ratio is a strong prognostic indicator in CLL, providing better prognostic assessment than serologic markers in advanced stages of the disease [26]. A body of evidence has confirmed that the expression of LPL mRNA is associated to bad prognosis, and that it is the most robust of the molecular markers in CLL [27] [28] [29] [30] [31] [32] [33]. \n\nThe elevated expression of LPL gene in Um CLL B-cells is a very remarkable observation, because there is no expression of LPL in normal B cells. This specific and ectopic expression constitutes not only a suitable prognostic marker to study disease evolution, but could also be helpful to understand the heterogeneous proliferative behavior in CLL. Despite the prognostic value of LPL expression is well established, the functional role of LPL overexpression in CLL pathogenesis as well as the molecular mechanisms regulating its expression are still open questions. \n\nConcerning the functional role of LPL in CLL cells, increasing evidence supports the idea that LPL expression could help the leukemic clone to increase survival and proliferative signaling, leading to disease progression. We have also shown that microenvironmental signaling can induce LPL expression and proliferative phenotype in primary CLL B-cells [34, 35]. Supporting this idea Rozovski, Grgurevic, et al. demonstrated that LPL confers CLL a survival advantage, since shRNA knockdown of LPL increases apoptotic death [36]. Accordingly, it has recently been reported that NOTCH1 gene mutations which are associated with disease progression and treatment refractoriness [37] are directly related to LPL expression in CLL [38]. \n\nConcerning the molecular mechanism that regulates LPL expression we previously demonstrated that abnormal expression of LPL gene in Um CLL patients results from the lack of methylation in the CpG island involving the whole exon 1 and the first nucleotides of intron 1 of LPL [34]. This epigenetic mechanism appears to be mainly triggered by proliferative T-cell-dependent signals and, in some patients, through the cross-linking of the B-cell receptor (BCR). By contrast, signaling through TLR9 or TLR1/2 pathways are unable to induce demethylation of the CpG island, LPL expression and B-cell proliferation [35]. Rozovski, Grgurevic, et al. have shown that LPL expression can also be transcriptionally regulated by STAT3 phosphorylation, and nuclear translocation where it can bind LPL promoter [36]. Additionally, it is necessary to mention that LPL expression can be regulated post-transcriptionally by miR-29 [39, 40]. It has been reported that miR-29 expression is down-regulated in high-risk Um CLL patients [41] [42] [43] [44]. In a more recent study of the microRNAome of a large patient cohort, down-regulation of miR-29c was the feature better related to IgHV Um profile [45]. In fact, Santanam et al. have developed a mouse model of early onset indolent CD5+ B-CLL by targeted overexpression of miR-29 in B-lymphocytes under control of the Eµ enhancer [46]. The authors focused on the effect on leukemogenesis by the interaction of miR-29 and TCL1 [44, 47] and did not evaluate LPL expression, which would be expected to be low. Deregulation of miR-29 is known to have important effects in diverse hematological disorders (reviewed in [48] ), to respond to cellular signaling processes such as BCR or CD40 stimulation, and to engage NF-κB activation through TCL1 [47]. Linking these microenvironmental signaling to the epigenetic changes described by us in Um patients as well as their correlation with miR-29 and LPL expression could be an interesting issue that is still awaiting to be studied in CLL progression.",
"section_name": "LPL As a Prognostic Marker of Disease Progression",
"section_num": "3.1."
},
{
"section_content": "LPL has been shown to mediate lipolysis and subsequent fatty acid (FA)-mediated fueling of cell proliferation in several solid tumors [49], and it has recently been shown that low-density lipoproteins may enhance proliferative responses of CLL cells to inflammatory signals [50]. PPARα protein levels in CLL B-cells have been shown to correlate with leukocytosis and clinical Rai stages, which suggests a metabolic switch to oxidation of fatty acids via PPARα [51] and PPARδ [50]. These findings are supported by the observation that CLL B-cells have lipid vacuoles in their cytoplasm, and that incubation with free fatty-acids (FFAs) increased their metabolic rate in terms of oxygen consumption [36]. Furthermore, the incidence of hyperlipidemia has been found to be higher in CLL patients, and treatment of hyperlipidemia with statins benefited them in terms of a delayed time to first treatment [52]. The same group expanded their initial study to a cohort of >2000 CLL patients in a retrospective analysis and found that both lipid-lowering drugs, as well as statin treatment prolonged overall survival by 3. 7 years [53]. These findings suggest that a second mechanism mediated by LDL may be converging in STAT3 phosphorylation and generating an activated state in CLL B-cells [50]. \n\nTranscriptional profiling identified a metabolic shift into a muscle or adipose tissue-like strategy with lipid oxidation in poor prognosis Um IgHV and LPL expressing B CLL cells [54]. How this metabolic reprogramming ends up in a worse outcome for patients is only beginning to be understood. Long chain fatty acids, free cholesterol and vitamin E-increase STAT3 phosphorylation directed either by IL-10, IFNα or phorbol esters in CLL cells [50]. STAT3 phosphorylation in turn drives LPL expression directly, by binding to a GAS-like element 280 bp upstream of the LPL transcription start site and activating its transcription [36]. LPL expression favors FA oxidation, and this seems to result in higher cell survival as LPL knockdown or chemical inhibition reduced CLL cell viability [36, 55], which might be explained in part by a transcriptional response [32]. Accordingly, microenvironmental induction of LPL expression stimulates CLL cell proliferation [35]. These findings indicate that LPL expression can be regulated by the microenvironment, either by autocrine or paracrine signaling and that it reflects a metabolic switch in CLL B-cells which confers an adaptive advantage. A positive feedback loop may maintain LPL expression and worsen the scenario for Um patients. In CLL, STAT3 is constitutively activated which also activates LPL transcription [36]. LPL breaks down very low-density lipoproteins (VLDL) and chylomicrons and liberates FFAs, generating a proinflammatory state which in turn activates STAT3 [51] and further activation of LPL transcription. This would further increase the levels of FFAs, thus exacerbating CLL cells responsiveness to cytokine signaling. More general aspects of metabolic pathways in CLL have been nicely reviewed recently [56].",
"section_name": "LPL in CLL B-Cell Metabolism",
"section_num": "3.2."
},
{
"section_content": "Many studies have reported an increased expression of LPL in poor prognosis CLL, and several metabolic pathways could be involved in cancer progression as discussed above. However, attempts to determine metabolic activity of LPL directly have failed to correlate higher expression to higher metabolic activity. A seminal study with 33 CLL patients reported lower catalytic activity in Um patients than in their Mut counterpart [30]. Another report analyzing data from 42 patients did not find differences between CLL groups and reported that LPL activity was comparable to that of healthy individuals [32]. \n\nLPL can mediate lipoprotein uptake by cells [57], chylomicron attachment to cell surface through LDL-related receptor [58], and lipoprotein margination in small blood vessels, by binding on the one hand to the extracellular surface of endothelium via GPIHBP1, and on the other to triglyceride-rich lipoproteins [59]. Besides its canonical role in lipid metabolism, an interesting-yet quite unexplored-non-metabolic function of LPL has been known for 20 years. LPL can act as a bridging molecule between cells, as in the adhesion of monocytes to endothelial cells mediated by heparan sulfate proteoglycans (HSPGs) and LPL [60], whose interaction has recently been shown to be dynamic [61]. Provided that CLL cells display HSPGs on their surface [62] and that LPL forms homodimers, it could occur that a bridging between leukemic B-cells and other cells expressing surface HSPGs or GPIHBP1 such as endothelial cells would be mediated by LPL. Although several groups have already speculated about it, a cell-cell bridging role for LPL in CLL pathogenesis still has to be demonstrated [30, 35, 63]. If such a bridging actually occurred, LPL would be pivoting between surface HSPGs on the B-CLL cell side, and either HSPGs or GPIHBP1 on their counterpart. \n\nRombout et al. have found that two SNPs commonly found in LPL, rs328 (premature stop codon) and rs13702 were significantly associated with CLL outcome [63]. Although both SNPs are well-known gain-of-function mutations [64, 65], the authors of the aforementioned study reported not to have been able to detect significant differences in LPL mRNA, protein levels, or enzymatic activity in patients carrying the SNPs [63]. How these mutations affect clinical outcome in CLL is still unclear, but whether these SNPs might have a role-if any-in LPL non-metabolic functions has not been explored yet. Furthermore, at least nine isoelectric point isoforms of LPL have been described in human blood of healthy individuals [66], thus opening a new dimension of studies to come for LPL in CLL and other pathologies.",
"section_name": "Non-Metabolic Roles of LPL in CLL",
"section_num": "3.3."
},
{
"section_content": "LPL is a protein located on the luminal side of the blood vessel wall, where it is anchored to heparan sulfate proteoglycans and contains binding sites for both heparan sulfate chains and apoproteins [67]. LPL is overexpressed in B-cells of unmutated IgHV CLL patients, and its expression can be used to predict their clinical outcome [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33]. Accordingly, LPL could have a bridging function in the formation of a trimolecular complex including a lipoprotein particle, LPL and heparan sulfate proteoglycans from different cells [67]. The fact that CLL B-cells display heparan sulfate proteoglycans on their surface [62], invites to speculate about whether LPL localization on the cellular membrane could affect the biological behavior of CLL cells, by favoring cell spreading, migration and intracellular signaling following activation of the tumoral clone by an activated microenvironment. If it is the case, LPL might also act as a crosstalk factor facilitating specific interactions with accessory cells in tissue microenvironments. LPL might then be added to the list of proteins implicated in the activation of CLL proliferative pool together with integrins such as CD49d, metalloproteinases (MMP-9), antiapoptotic molecules (BCL2) as well as chemokines (CCL3, CCL4, CXCL12) [68, 69]. Thus, LPL could be contributing to leukemic progression either per se through metabolic reprogramming, or through the synergistic contribution to an activating microenvironment in which the leukemic clone is continuously nourished (Figure 1 ). reprogramming, or through the synergistic contribution to an activating microenvironment in which the leukemic clone is continuously nourished (Figure 1 ). The role that abnormal LPL expression could have in disease evolution, has also been addressed by previous work from Pallasch et al., demonstrating that lipase associated genes and triglyceridespecific lipase activity were significantly increased when comparing CLL B-cells to normal CD5+ Bcells [55]. The same authors reported that incubation of CLL tumoral cells with the lipase inhibitor orlistat resulted in increased apoptosis, which, could suggest that lipid metabolism and lipase activity could be functionally relevant in aggressive CLL [55]. Phenotypic analyses have shown that CLL Bcells expressing LPL are also enriched in FA degradation genes [54]. Recently, LPL has been shown to mediate lipolysis and subsequent FA-mediated fueling of cell proliferation in several solid tumors [49], and it has recently been shown that low-density lipoproteins may enhance proliferative responses of CLL cells to inflammatory signals [50]. \n\nA big amount of information is known nowadays about LPL some of which relates to CLL. Still, our understanding whether LPL overexpression in poor outcome CLL is a cause or consequence is poor. Many questions are still open and more answers will certainly come in next years.",
"section_name": "Concluding Remarks",
"section_num": "4."
},
{
"section_content": "The authors thank Uruguayan Agencia Nacional de Investigacion e Innovacion (ANII) for the graduate fellowship of DP and for the grants FMV_2_2011_1_7323; FMV_1_2014_1_104397 and FOCEM (MERCOSUR Structural Convergence Fund), COF 03/11. Open access publishing fees have been partially funded by Programa de Desarrollo de las Ciencias Básicas (PEDECIBA)-Uruguay.",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. The role that abnormal LPL expression could have in disease evolution, has also been addressed by previous work from Pallasch et al., demonstrating that lipase associated genes and triglyceride-specific lipase activity were significantly increased when comparing CLL B-cells to normal CD5+ B-cells [55]. The same authors reported that incubation of CLL tumoral cells with the lipase inhibitor orlistat resulted in increased apoptosis, which, could suggest that lipid metabolism and lipase activity could be functionally relevant in aggressive CLL [55]. Phenotypic analyses have shown that CLL B-cells expressing LPL are also enriched in FA degradation genes [54]. Recently, LPL has been shown to mediate lipolysis and subsequent FA-mediated fueling of cell proliferation in several solid tumors [49], and it has recently been shown that low-density lipoproteins may enhance proliferative responses of CLL cells to inflammatory signals [50]. \n\nA big amount of information is known nowadays about LPL some of which relates to CLL. Still, our understanding whether LPL overexpression in poor outcome CLL is a cause or consequence is poor. Many questions are still open and more answers will certainly come in next years.",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "The authors thank Uruguayan Agencia Nacional de Investigacion e Innovacion (ANII) for the graduate fellowship of DP and for the grants FMV_2_2011_1_7323 ; FMV_1_2014_1_104397 and FOCEM (MERCOSUR Structural Convergence Fund), COF 03/11. Open access publishing fees have been partially funded by Programa de Desarrollo de las Ciencias Básicas (PEDECIBA)-Uruguay.",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.1038/s41598-023-45893-8
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Evaluating anti-GPL-core IgA as a diagnostic tool for non-tuberculous mycobacterial infections in Thai patients with high antibody background
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<jats:title>Abstract</jats:title><jats:p>Diagnosis of non-tuberculous mycobacterial (NTM) infection is difficult due to low sensitivity and time-consuming laboratory tests. Current serological assays fail in tropical countries due to high antibody background. This study aimed to investigate an appropriate method for detecting anti-glycopeptidolipid (GPL)-core antibodies to diagnose NTM infection in Thailand. Heparinized plasma samples were collected from 20 patients with NTM-pulmonary disease (NTM-PD) and 22 patients with disseminated NTM (dNTM) for antibody detection by ELISA. The results were compared with those from patients with tuberculosis, other bacterial pulmonary infections and healthy controls. Among the different antibody isotypes, anti-GPL-core IgA exhibited the highest suitability. Therefore, anti-GPL-core IgA and its subclass IgA2 were further investigated. A significant increase in antibody levels was observed during the active infection stage, whereas NTM-PD with culture conversion at the 6-month follow-up showed reduced IgA levels. The diagnostic cut-off for IgA and IgA2 was newly defined as 1.4 and 1.0 U/ml, respectively. Using our IgA cut-off, the sensitivity and specificity for diagnosing NTM-PD were 77.3% and 81.4%, respectively. The new IgA cut-off demonstrated significantly improved specificity compared to the manufacturer's cut-off. Thus, serological detection of anti-GPL-core IgA, with a cut-off of 1.4 U/ml, can be a valuable tool for supporting NTM diagnosis in Thailand.</jats:p>
|
[
{
"section_content": "Non-tuberculous mycobacteria (NTM) are commonly found in the environment, and their infections have become a global public health problem due to the increasing number of cases worldwide 1. NTM infections can occur in both immunocompetent and immunocompromised patients 2, 3. The most common clinical manifestation is NTM pulmonary disease (NTM-PD), although disseminated NTM (dNTM) infections have also been reported 4. Currently, the diagnosis of NTM-PD relies on chest radiography and positive NTM cultures from clinical specimens 5. Bacterial culture is considered the gold standard for laboratory investigations; however, it has two major drawbacks: time consumption and lack of sensitivity 6. \n\nTo improve the diagnostic efficiency of NTM infection, several molecular approaches have been introduced, such as polymerase chain reaction, multi-locus sequence typing, nucleic acid amplification tests, line probe assays, and next-generation sequencing. However, the overall sensitivity of these approaches is only 29-76% 7, 8. Moreover, serological diagnosis of NTM-PD, particularly infection with the Mycobacterium avium complex (MAC), has been introduced using the detection of IgA antibodies against a component on the surface of NTM called the glycopeptidolipid (GPL) core 9. This assay is based on an ELISA and is simple with a high throughput. Several studies conducted in Japan, Taiwan, South Korea, and the United States have reported a sensitivity of approximately 60-90% and a specificity of 91-100% [9] [10] [11] [12] [13]. However, the distribution of NTM-infected cases in previous studies differs from that in Thailand. In Thailand, the distribution of NTM is non-MAC group-dominant, similar to that in China and other Southeast Asian countries 14. A study on NTM infection in northeastern Thailand reported that the most common causative agent is Mycobacterium abscessus, a rapidly growing mycobacterium (RGM) 15. Considering the differences in NTM distribution, the efficiency of the test kit should be evaluated in specific populations to substantiate its validity in Thailand and other countries with RGM dominance. \n\nAnother challenge when applying serological tests for diagnosing infectious diseases in tropical countries is the high background of antibodies. Therefore, this study aimed to (1) investigate an appropriate antibody isotype and the cut-off for Thai patients with NTM infection and (2) evaluate the diagnostic efficacy of plasma anti-GPLcore for Thai patients with NTM-PD and dNTM in comparison with Mycobacterium tuberculosis pulmonary disease (MTB-PD) and other bacterial pulmonary diseases. Our results highlight that anti-GPL-core IgA at a cut-off of 1. 4 U/ml can be applied for the diagnosis and monitoring of Thai patients with NTM-PD or dNTM.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Eighty-eight patients with bacterial infections and 25 healthy controls were enrolled. Bacterial culture-positive patients were classified into four groups: 20 with NTM pulmonary disease (NTM-PD), 22 with disseminated NTM infection (dNTM), 14 with Mycobacterium tuberculosis pulmonary disease (MTB-PD), and 32 with other bacterial pulmonary diseases (Oth-PD). The general demographic characteristics of the participants are presented in Table 1. The median age of the patients was 57 years, with an interquartile range of 16 years. Comparisons among the sample groups revealed a significant difference in age only between the MTB-PD and Oth-PD groups (P-value = 0. 0097). The distribution of sex was not balanced, with varying proportions of males and females across the subgroups. \n\nIn the NTM-PD group, the most common causative agent was M. abscessus (8/20, 40%). Similarly, the majority of patients in the dNTM group were infected with M. abscessus (15/22, 68. 2%). All participants in the MTB-PD group tested positive for M. tuberculosis in sputum samples. Finally, patients with Oth-PD in this study were primarily infected with K. pneumoniae (11/32, 34. 4%), followed by E. coli (10/32, 31. 3%), P. aeruginosa (7/32, 21. 9%), and B. pseudomallei (1/32, 3. 1%).",
"section_name": "Prevalence of M. abscessus in Thai patients with NTM pulmonary disease and disseminated NTM infection",
"section_num": null
},
{
"section_content": "Our previous study demonstrated that both anti-GPL-core IgA and IgG could be used for the diagnosis of disseminated NTM (dNTM), with IgG performing better than IgA 16. To determine the most suitable antibody isotype for Thai patients with NTM-PD, plasma levels of anti-GPL-core antibodies were measured in the first eight enrolled patients. The study quantified three plasma antibody isotypes, namely IgG, IgM, and IgA, in patients with pulmonary disease, as shown in Fig. 1. All four groups exhibited similarly high levels of plasma anti-GPLcore IgG (Fig. 1A ). Conversely, the anti-GPL-core IgM isotype in the NTM-PD group was significantly higher than that in the Oth-PD group but not significantly higher than that in the MTB-PD group or healthy controls (Fig. 1B ). As anticipated, the IgA isotype proved to be the most effective discriminative antibody among all pulmonary disease groups and HCs (Fig. 1C ).",
"section_name": "Effectiveness of plasma anti-GPL-core IgA in distinguishing the Thai NTM-PD group from other pulmonary infection and non-infection groups",
"section_num": null
},
{
"section_content": "We have previously identified IgA as the most promising isotype for NTM-PD, which also suggests its potential usefulness for dNTM 16. In this study, we analysed 113 plasma samples for anti-GPL-core IgA and its subclasses, including IgA1 and IgA2. However, we were unable to detect any signals for the IgA1 subclass (data not shown). The distribution of plasma anti-GPL-core IgA and IgA2 concentrations for each participant is presented in Fig. 2. Plasma anti-GPL-core IgA levels were significantly higher in the NTM-PD group than in the MTB-PD (P = 0. 0398), Oth-PD (P = 0. 0026), and HC (P = 0. 0153) groups (Fig. 2A ). Notably, among the 20 patients with NTM-PD, five showed negative results. Among these patients, three were infected with M. abscessus, one with M. avium, and one with M. intracellulare. Patients with dNTM infection exhibited significantly higher levels of plasma anti-GPL-core IgA than those with MTB-PD (P = 0. 001), Oth-PD (P < 0. 0001), or HC (P = 0. 0001) (Fig. 2A ). Four false-negative results were observed for M. abscessus, and one was observed for M. kansasii. Regarding IgA2 distribution, more interference from the controls was noted. Thus, only dNTM could be distinguished from MTB-PD (P = 0. 0038), Oth-PD (P = 0. 0003), and HC (P < 0. 0001), while NTM-PD was significantly higher than HC (P = 0. 0103) (Fig. 2B ). \n\nFurthermore, the correlation analysis between plasma anti-GPL-core IgA and IgA2 levels revealed no significant correlation in patients with NTM-PD (P = 0. 0923) (Fig. 2C ). Notably, patients with dNTM showed a significant association between IgA and IgA2 levels, with a correlation coefficient of R 2 = 0. 5828 (Fig. 2D ). Additionally, there was no correlation between plasma anti-GPL-core IgA and anti-hIFN-γ autoantibody titres in dNTM patients (Supplementary Fig. S1 ). \n\nSurplus plasma samples from seven patients with dNTM who visited the hospital annually for treatment follow-up were analysed for the decrease in anti-GPL-core levels. We observed a decrease in plasma anti-GPLcore IgA levels in the second year of follow-up (P = 0. 0694), and a significant decrease was observed in the third year (P = 0. 0078) (Fig. 2E ). Similarly, a significant decrease in IgA2 levels was observed in the third year of follow-up (P = 0. 0455) (Fig. 2F ).",
"section_name": "Measurement of plasma anti-GPL-core IgA and IgA2 for distinguishing the Thai NTM-PD group from other pulmonary infection and non-infection groups",
"section_num": null
},
{
"section_content": "The clinicians reviewed the clinical data of our patients before comparing the differences in plasma anti-GPL-core antibodies among different clinical outcome groups. In the comparison of NTM-PD, a significant reduction in plasma anti-GPL-core IgA was observed in NTM-PD patients with culture conversion at the 6-month follow-up (Fig. 3A ). However, NTM-PD patients with non-progressive or progressive outcome after antimicrobial treatment, exhibited similar levels of anti-GPL-core antibodies. Regarding the dNTM group, all cases included in this study were clinically active infections. When comparing anti-GPL-core antibodies between dNTM patients who were non-progressive versus progressive after treatment, no statistically significant difference was observed (Fig. 3B ).",
"section_name": "Plasma anti-GPL-core IgA shows a statistically significant reduction in pulmonary infection with culture conversion at the 6-month follow-up",
"section_num": null
},
{
"section_content": "We analysed the diagnostic efficacy of anti-GPL-core IgA and IgA2. Applying the manufacturer's recommended cut-off of 0. 7 U/ml for anti-GPL-core IgA in our specimen population resulted in 75. 0% sensitivity, 69. 0% specificity, 40. 5% PPV, and 90. 7% NPV for the diagnosis of NTM-PD (Supplementary Table S1 ). For the diagnosis of dNTM, the cut-off of 0. 7 U/ml for anti-GPL-core IgA showed 81. 8% sensitivity, 69. 0% specificity, 45. 0% PPV, and 92. 5% NPV (Supplementary Table S1 ). \n\nReceiver operating curve (ROC) analysis was conducted to determine the most appropriate cut-off level for the plasma anti-GPL-core antibody concentration. The analysis results from 20 patients with NTM-PD compared to all control groups (MTB-PD, Oth-PD, and HC; n = 71) revealed an area under the curve (AUC) of 0. 788 (95% CI = 0. 656-0. 921) for IgA and 0. 740 (95% CI = 0. 616-0. 864) for IgA2 (Supplementary Fig. S2A ). Conversely, the analysis results from 22 patients with dNTM compared to all control groups showed an AUC of 0. 848 (95% CI = 0. 733-0. 962) for IgA and 0. 843 (95% CI = 0. 753-0. 932) for IgA2 (Supplementary Fig. S2B ). Based on the ROC analysis for both NTM-PD and dNTM, we determined the concentration cut-off of 1. 4 U/ml for IgA and 1. 00 U/ml for IgA2 for further analysis. We then applied our cut-off of 1. 4 U/ml for anti-GPL-core IgA based on the ROC analysis above (Supplementary Fig. S2 ). The diagnostic efficacy for patients with NTM-PD (n = 20) in distinguishing them from all control groups (MTB-PD, Oth-PD, and HC; n = 71) demonstrated 75. 0% sensitivity, 81. 4% specificity, 53. 6% PPV, and 91. 9% NPV (Table 2 ). Similarly, patients with dNTM (n = 22) showed a sensitivity of 77. 3%, specificity of 81. 4%, PPV of 56. 7%, and NPV of 91. 9% (Table 2 ). With respect to the IgA2 cut-off at 1. 0 U/ml, the diagnostic efficacy for patients with NTM-PD (n = 20) in distinguishing them from all control groups demonstrated 75. 0% sensitivity, 63. 4% specificity, 36. 6% PPV, and 90. 0% NPV (Table 2 ). Patients with dNTM (n = 22) showed 81. 8% sensitivity, 63. 4% specificity, 40. 9% PPV, and 91. 8% NPV (Table 2 ).",
"section_name": "Application of plasma anti-GPL-core IgA with a higher cut-off at 1.4 U/ml for diagnosis of Thai NTM-PD and dNTM",
"section_num": null
},
{
"section_content": "According to a recent report on the prevalence of mycobacterial infections, there has been a decrease in MTB cases but an increase in NTM infections 17. Although rare, NTM infections have been neglected. Laboratory investigations for NTM infection have poor sensitivity and are time-consuming, taking several days to months [6] [7] [8]. In this study, we evaluated the diagnostic efficacy of an anti-GPL-core ELISA kit for Thai patients with NTM-PD and dNTM. Both patient groups were confirmed as NTM culture positive. Additionally, all patients with dNTM tested positive for anti-interferon-gamma (IFN-γ) antibodies, while all patients with NTM-PD tested negative. The pathogenesis of these two types of infections differs according to the literature 18. Anti-IFN-γ autoantibodies bind to IFN-γ and inhibit its functions, impairing phagocytosis and leading to opportunistic infections, including NTM 19. In contrast, NTM-PD reportedly develops through different mechanisms, such as a history of MTB infection or fibrosis 18. \n\nSeveral studies on NTM infections, including NTM-PD and dNTM, have reported the most prevalent NTM species in Japan, Taiwan, and countries outside of Asia as the Mycobacterium avium complex (MAC), whereas in China, the Philippines, and Thailand, RGM predominates 20. Consistent with our findings in Thai NTM-infected patients, M. abscessus was the most common species. \n\nSerological diagnosis using an anti-GPL-core IgA ELISA kit has been introduced and commercialized, with a recommended cut-off of 0. 7 U/ml. With this cut-off, we found a sensitivity comparable to that of previous studies but with lower specificity. Several studies in Japan have reported sensitivity and specificity above 90% 9, 12. A study in South Korea showed a sensitivity of 77. 5-85% and specificity of 100% 10. A study in Taiwan reported diagnostic efficacy with 60% sensitivity and 91% specificity 13. Another study in the USA reported a sensitivity of 70. 1% and specificity of 93. 9% 11. A more recent study in the US proposed a new cut-off of 0. 178 U/ml for serological diagnosis in the American population, with 84% sensitivity, 72% specificity, 81% PPV, and 76% NPV 21. The difference in these results may be explained by the possibility that Thai individuals have naturally developed high antibody background to endemic microbes 22. This phenomenon has been demonstrated in a melioidosis model, where a serological test failed to diagnose owing to a high antibody background 23. \n\nTo investigate the usefulness of serological diagnosis with anti-GPL-core antibodies for NTM infection in the Thai population, we identified the most promising antibody isotype (IgA) in patients with NTM-PD. Combined with our previous study, plasma anti-GPL-core IgA measurements showed diagnostic efficacy for patients with dNTM, with 91. 18% sensitivity and 70. 15% specificity 16. ROC analysis was performed to determine the most appropriate diagnostic cut-off, resulting in 1. 4 U/ml for anti-GPL-core IgA and 1. 0 U/ml for IgA2. Using the new IgA cut-off, we observed a significant improvement in specificity compared to the manufacturer's recommended cut-off. Moreover, NTM-PD showed no correlation between anti-GPL-core IgA and IgA2, whereas dNTM had a very high correlation. Serum IgA and IgA2 are mainly produced from bone marrow plasma cells 24, which are more likely to associate with systemic infection rather than localized NTM-PD. Furthermore, in the follow-up samples of patients with dNTM, anti-GPL-core IgA and IgA2 levels decreased in the second year, with statistical significance reached in the third year. \n\nAdditionally, we analysed anti-GPL-core antibodies for clinical outcomes after antimicrobial treatment in both patients with NTM-PD and dNTM. Most active infections, whether NTM-PD or dNTM, tested positive for anti-GPL-core antibodies. However, the significant reduction of anti-GPL-core IgA was observed only in NTM-PD patients with culture conversion at the 6-month follow-up, the result was in line with a previous report 25. These data suggest the presence of anti-GPL-core antibodies persisting for 6 months after infection before declining upon clearance of the infection. \n\nThis study had several limitations, including small sample size, lack of samples with NTM colonization for comparison, lack of follow-up samples for NTM-PD, and not covering the entire spectrum of NTM infection types. HCs were enrolled using a questionnaire without laboratory investigation. Of note, the samples used in this study were heparinized plasma which were not the same as in the manufacturer's procedure. However, in a small set of samples, the results of anti-GPL-core IgA and IgA2 from serum versus plasma samples collected from five dNTM patients showed no statistically significant difference (Supplementary Fig. S2 ). Due to the low sensitivity of bacterial culture, the result could not be obtained from dNTM patients during the follow-up. Moreover, we could not identify individuals with NTM colonization or infection who did not present with clinical symptoms. The patients in this study were recruited from Srinakarind Hospital, a medical hub and tertiary care centre in northeastern Thailand. Thus, this area was reportedly dominated by M. abscessus and MAC, with variations in their distribution among provinces 15. Interestingly, NTM-PD patients were distinguished from other infections by IgA, but not IgA2. This implies that IgA1 may be an important subtype in NTM-PD which may require further studies. Therefore, more studies in other areas of Thailand are required to ensure the diagnostic efficacy of anti-GPL-core IgA detection. \n\nIn conclusion, this study provides an alternative serological diagnostic method for NTM infections, both pulmonary and disseminated. Using a new cut-off of 1. 4 U/ml, we can improve the specificity of this test for Thai patients with a high background of antibodies while maintaining comparable sensitivity to previous studies. \n\nAdditionally, previous study suggested the diagnosis of NTM-PD using single bacterial isolation combined with anti-GPL-core IgA detection 26. Recently, the concept of using either culture independent markers or combination of traditional culture method with other markers has been reviewed to increase accuracy of diagnosis and monitoring of patients with NTM-PD or cystic fibrosis 27. Detection of anti-GPL-core IgA should be performed to increase the rate of NTM infection detection and facilitate differentiation from MTB, enabling patients to receive more rapid and appropriate antimicrobial treatments. The association between anti-GPL-core IgA/ IgA2 and clinical outcomes has been investigated in this study, but not yet fully defined. Further studies in large clinical sample sizes are the challenges to provide more diagnostic capability of this clinical test.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Peripheral heparinized venous blood samples were collected from adult participants (≥ 18 years of age) at Srinagarind Hospital, Khon Kaen, Thailand, between July and December 2022. The study was conducted with the approval of the Khon Kaen University Ethics Committee in Human Research (HE654007), and all participants in the NTM-PD (n = 20), M. tuberculosis pulmonary disease (MTB-PD; n = 14), other bacterial pulmonary disease (Oth-PD; n = 32), and healthy control (HC; n = 25) groups provided handwritten informed consent. \n\nPatients with pulmonary disease were diagnosed by clinicians following the guidelines of the American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) 5. Cases without immune deficiency with positive sputum cultures for Mycobacterium tuberculosis were classified as MTB-PD, while those with positive cultures for any Mycobacterium other than M. tuberculosis were classified as NTM-PD. Patients with positive bacterial sputum cultures for any bacteria except Mycobacterium were classified into the Oth-PD group. HCs were enrolled based on the blood donation guidelines of the Blood Bank of Srinagarind Hospital, Khon Kaen. \n\nBlood sample of NTM-PD patients was collected only once. Clinical outcome at the 3-month and 6-month follow-up was reviewed by clinicians according to ATS/IDSA guidelines for monitoring of NTM disease during therapy and treatment endpoint. The goals include symptomatic, radiographic, and microbiologic improvement 5. In more details, patients were subcategorised into (1) Culture conversion: conversion of sputum culture and/ or acid fast bacilli (AFB) test to negative with symptomatic and radiographic improvement at the 3-month or 6-month follow-up, but maintained antibiotic treatment for 12 months according to the guideline, (2) Nonprogressive NTM-PD: no changes in radiographic features and symptoms compared to the preceding examination with positive sputum cultures and/or AFB test, (3) Progressive NTM-PD: positive sputum cultures and/or AFB test with new radiographic abnormalities and patient presented with productive cough, fever, and dyspnea. \n\nSurplus plasma samples from patients with dNTM and positive anti-IFN-γ autoantibody titres were obtained from routine service at Srinagarind Hospital, Khon Kaen, Thailand, between 2020 and 2022. All dNTM samples in this study were originally collected from patients with active infection who presented the signs of infection including lymphadenopathies with or without reactive skin disease, and all patients required antimicrobial drug treatment over the duration of monitoring 28. \n\nTreatment outcomes of dNTM on the day of sample collection were classified as (1) non-progressive dNTM group exhibited stable disease symptoms and continued receiving antimicrobial treatment without requiring hospitalization for parenteral therapy, while (2) progressive dNTM group experienced worsen clinical outcomes after treatment and required hospitalization 29.",
"section_name": "Sample enrolment and definitions",
"section_num": null
},
{
"section_content": "The concentration of anti-GPL-core IgA antibodies in plasma samples was measured using a GPL-core IgA ELISA kit (Capilia MAC Ab ELISA, Cat. CAMC8170, Tauns Laboratory Inc., Shizuoka, Japan) following the manufacturer's instructions. \n\nA modification was made in the plasma dilution step for the detection of anti-GPL-core antibodies: a dilution of 1:400 for IgM and IgG subtypes, 1:40 for IgA, and 1:20 for either IgA1 or IgA2. The diluted plasma was added to the GPL-core IgA ELISA kit and incubated at room temperature (23-25 °C) for 1 h, according to the manufacturer's instructions. After washing the plates four times with the wash solution, instead of the IgA detection antibody provided in the kit, biotinylated mouse anti-human IgM (Clone G20-127, Cat. 555781, BD Biosciences) or biotinylated mouse anti-human IgG (Clone G18-145, Cat. 555785; BD Biosciences) and HRP-conjugated streptavidin (Cat. 554066; BD Biosciences) were added. For IgA1 detection, horseradish peroxidase-conjugated mouse anti-human IgA1 (Clone B3506B4, Cat. 9130-05, Southern Biotech, USA) was used, and for IgA2 detection, HRP-conjugated mouse anti-human IgA2 (Clone A9604D2, Cat. 9140-05, Southern Biotech, USA) was used. The plates were then incubated at room temperature for 1 h. After washing, the chromogen solution was added following the manufacturer's instructions, and the reaction was stopped with the stop solution. The optical density was measured at 450 nm using an ELISA reader (Tecan Magellan, Switzerland). The concentration of each antibody subtype was interpolated by comparing it with the standard curve generated from the standard samples in the kit, with a lower limit of detection of 0. 5 U/ml.",
"section_name": "Measurement of anti-GPL-core antibodies in human plasma samples",
"section_num": null
},
{
"section_content": "Statistical analyses and visualizations were conducted using GraphPad Prism version 9. 5. 1 (GraphPad Software, San Diego, California, USA). The normal distribution of data was assessed using the D' Agostino and Pearson tests. The Kruskal-Wallis test was employed to compare non-normally distributed data, while paired samples were compared using one-tailed paired t-tests. The chi-square test was used to assess differences in categorical data. \n\nTo analyse the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), GraphPad Prism version 9. 5. 1 (GraphPad Software, San Diego, California, USA) was used. Statistical post hoc power analysis was conducted using MedCalc Software Ltd. McNemar's chi-squared test was utilized to examine",
"section_name": "Data analysis",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This research was funded by the National Research Council of Thailand (NRCT) (Contract No. N42A650205 ), Young Researcher Development Project of Khon Kaen University ( Year 2022 ), and Chiang Mai University (CMU) Presidential Scholarship for Postdoctoral Fellowships (Contract no. 25/2021 ) and partially supported by grants from the Japan Agency for Medical Research and Development (AMED) (Contract nos. 23fk0108683 and 336 23fk0108673 ) We thank the medical staff at Srinagarind Hospital, Khon Kaen University, Thailand for assisting and reviewing patient records.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "All data in this article are available in the paper or the supplementary material, and also are available from the corresponding author on reasonable request.",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "www. nature. com/scientificreports/ statistical differences between the diagnostic criteria. All experiments in this study exhibited > 90% power and 95% confidence level for detecting differences between the groups.",
"section_name": "",
"section_num": ""
},
{
"section_content": "V. M. and A. N. acquisition of data, analysis and drafting the manuscript. A. S., P. C. and K. F. interpretation of data and reviewing the manuscript. M. A., A. N. and G. L. designed the study and revising the manuscript. All authors have read and approved the final version of the manuscript.",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare no competing interests.",
"section_name": "Competing interests",
"section_num": null
}
] |
10.3390/ijms22115784
|
Molecular Genetics of Conjunctival Melanoma and Prognostic Value of TERT Promoter Mutation Analysis
|
<jats:p>The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.</jats:p>
|
[
{
"section_content": "Conjunctival melanoma (CM) comprises 5-10% of all ocular melanoma [1] [2] [3]. The majority derives from primary acquired melanosis with atypia (PAM), but infrequently, CM develops from a pre-existing nevus or de novo [1, [3] [4] [5] [6]. CM has an incidence of 0. 2-0. 8 per million [3, 6, 7], with an increasing trend [3, 8]. The 5-and 10-years cumulative incidence of CM-related mortality is 17-31% and 22-59%, respectively [5, 7, [9] [10] [11]. The prognosis of ocular melanoma, including CM and uveal melanoma (UM), depends on clinical and histopathological features, as well as the molecular genetic make-up [3, 12, 13]. During the past decade, the molecular make-up of UM has been well-characterized, with UM harboring recurrent mutations in guanine-nucleotide-binding protein-Q (GNAQ), guaninenucleotide-binding protein-alpha 11 (GNA11), BRCA-associated protein 1 (BAP1), splicing factor 3 subunit 1 (SF3B1), and eukaryotic translation initiation factor 1A (EIF1AX). BAP1 and SF3B1 mutations are associated with the development of metastasis in UM. After the diagnosis of metastatic disease, patients with UM have a survival between 2-9 months [12]. When CM has metastasized, there are also very limited treatment options [1, 13]. Yet, although CM as well as UM are ocular melanoma, CM certainly do show overlapping features, including molecular abnormalities with cutaneous melanoma [1, 3, 6, 13, 14]. For example, in 25-40% of the CM driver v-raf murine sarcoma, viral oncogene homolog B1 (BRAF) V600E/K mutations are described [1, 2, 6, 13, 15]. This incidence is higher as compared to other mucosal melanoma, which harbor a BRAF mutation in only 12% of cases. Although a correlation between BRAF mutations and poor prognostic factors has been described in cutaneous melanoma, no predictive value is yet reported for mucosal melanoma [16, 17]. Other genes in which mutations have been identified in CM are the neuroblastoma RAS viral oncogene homolog (NRAS), Kirsten RAS oncogene homolog (KRAS), neurofibromin 1 (NF1), telomerase reverse transcriptase (TERT), tyrosine protein kinase (c-KIT), TP53, and BAP1 [3, 6, 15, 18]. Mutations in GNAQ/GNA11 have also been described, but these are not the known activating hotspot mutations at amino acid Q209 or R183, which occur in UM [15, 19]. The genetic background of the melanoma originating from these different locations, emphasizes the differences between UM and CM, and the similarities between CM and cutaneous melanoma. Furthermore, in contrast to UM, some of the mutations frequently found in CM are amenable to targeted therapies. However, the prognostic value of these molecular abnormalities in CM is largely unclear. The aim of this study was to further elucidate the genetic background of CM within the spectrum of melanoma and to correlate these findings with the development of recurrences and metastasis.",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "2."
},
{
"section_content": "Clinical and histopathological characteristics are listed in Table 1. Based on the availability of sufficient formalin-fixed paraffin-embedded (FFPE) tissue for DNA isolation, twenty-eight cases could be included. Gender was equally divided with 50% males and 50% females. The median age at the time of diagnosis was 64 years (range 16-89 years). Based on the clinical information, most tumors were (at least partly) located on the bulbar conjunctiva (16 cases, 57%) with involvement of the palpebral conjunctiva in 10 cases (36%), the fornix in 5 cases (18%), and the caruncle in 1 case (4%). The tumors had a median diameter of 0. 7 cm (range 0. 05-1. 8 cm), with a median tumor thickness of 3. 0 mm (range 0. 18-7. 70 mm). According to the Eighth Edition of the American Joint Committee on Cancer (AJCC) Cancer Staging [20], twelve cases (43%) were pathological tumor (pT) stage pT1, including six pT1a cases (21%) and five pT1b cases (18%), and thirteen cases were pT2 cases (46%), comprising one pT2a case (4%), eleven pT2b cases (39%), and two cases (7%) with unknown tumor thickness. In three cases (11%), the pT status was unknown. In eighteen cases (64%), the melanoma were derived from PAM, four melanoma (14%) developed from a nevus, and three melanoma (11%) were de novo lesions. In three cases (11%), the origin could not be reliably determined, based on the pathology reports and the available clinical information. Local recurrent disease occurred in eight patients (29%), between 6. 8-156. 8 months (median 29. 3 months) after treatment. Nine patients (32%) developed metastatic disease between 1. 7-49. 2 months (median 14. 3 months). Metastatic sites included lymph nodes (solitary or within the parotid gland) in all patients (n = 9), with metastatic disease in the orbit (n = 1), thyroid (n = 1), breast (n = 1), lung (n = 1), brain (n = 1), and spleen (n = 1). The thyroid and breast metastases were present in one patient, and the orbit and brain metastases were identified in one patient as well. The spleen and brain metastases were not histologically confirmed. The mean overall survival was 77. 4 months (range 3. 85-257. 2 months), with a median of 62. 8 months.",
"section_name": "Clinical and Histopathological Characteristics",
"section_num": "2.1."
},
{
"section_content": "The specific mutations found per case are listed in Supplementary Table S1, with a summary of the mutations including correlation with metastatic and recurrent disease in Table 2. Fifteen CM cases (54%) showed a TERT promoter mutation. A mutation in the BRAF gene was identified in thirteen CM (46%), mostly affecting amino acid V600. NRAS mutations were seen in six cases (21%) and mutations in BAP1 were identified in five CM (18%). A PTEN mutation was found in four CM (14%), and in two CM (7%), a mutation in c-KIT was identified. Interestingly, a p. Arg625His mutation in SF3B1 was detected in one CM (4%). The diagnosis was unequivocally a CM in terms of both clinical and pathological reports. It was located in the nasal superior in the bulbar conjunctiva (Figure 1 ). None of the CM cases carried a mutation of GNAQ, GNA11, or EIF1AX. The metastasis-free survival (MFS) of patients with a TERT promoter mutation was The metastasis-free survival (MFS) of patients with a TERT promoter mutation was significantly shorter as compared to patients without a TERT promoter mutation in the tumor (p = 0. 008, Table 2, Figure 2 ). No correlation between metastasis-free survival and mutation status of BRAF, BAP1, SF3B1, NRAS, c-KIT, and PTEN could be observed. No correlation was found between the presence of any mutations and the development of recurrences (Table 2 ). We also analyzed whether the mutations were correlated with sex, age, location (bulbar only versus involvement of the palpebral/caruncular/forniceal conjunctiva), pT status (pT1 versus pT2), tumor thickness, origin (PAM-derived melanoma versus non-PAM-derived melanoma). We did find an association between the presence of a TERT promoter mutation and the origin of the lesion (p-value = 0. 005), with most cases (54%) developing either de novo or from a melanocytic nevus (Table 3 ). 25 ) 0 (0) 0 (0) P = p-value calculated with either the Pearson's χ 2 test or Fisher's exact test. In bold, the association between the presence of a TERT promoter mutation and origin of the lesion (p-value = 0. 01), with most cases (54%) developing either de novo or from a melanocytic nevus. None of the cases showed GNAQ, GNA11, or EIF1AX mutations; therefore, these mutations are not included in the table. pT status = pathological tumor status.",
"section_name": "Mutation Analysis",
"section_num": "2.2."
},
{
"section_content": "In five CM cases that revealed a BAP1 mutation using molecular testing, there was enough material available for testing the presence of a BAP1 mutation using immunohistochemistry. Four of these cases did not show loss of expression of BAP1 using immunohistochemistry, while one CM case did show loss of expression using BAP1 immunohistochemistry, with presence of positive (internal) control tissue.",
"section_name": "Immunohistochemistry",
"section_num": "2.3."
},
{
"section_content": "Pathways involved in the pathogenesis of CM included the MAPK/ERK pathway and the PI3K/AKT pathways, and these pathways overlap with the pathways involved in cutaneous melanoma [6]. \n\nThe mutation that we found most frequent in CM is a TERT promoter mutation, congruent with other studies concerning ocular melanoma [6, 13, 14 ] and cancer originating from other sites. These mutations result in a new consensus binding site for E-twenty-six (ETS) transcription factors and this may contribute to increased TERT. The ETS transcription factors are downstream targets of the RAS-RAF-MAPK pathways, and TERT promoter mutations are suggested to have synergistic effects with activating BRAF or NRAS mutations to promote tumor cell proliferation [21]. TERT is involved in the AKT pathway, and plays an important role in cellular immortality [6]. TERT mRNA overexpression does not completely explain all effects of the TERT promoter mutations in tumorigenesis, and the role of immunohistochemistry in determining the TERT status is still a topic of debate [22]. Consequently, other undefined or epigenetic mechanisms of TERT-upregulating are expected to exist [21, 23, 24]. While a TERT promoter mutation is not found in conjunctival nevi, it is found in both PAM [14] and CM [6, 14], with increased TERT expression leading to tumor progression [6]. In this context, the C>T or CC>TT nucleotide changes in these mutations are of interest, since this is the typical UV signature, in line with the UV-exposed location of most CM, as seen in our study and as compared to the molecular make up of cutaneous melanoma [6]. UM usually do not harbor mutations in or near the TERT gene [14, 18, 25]. It indicates that different pathways are involved in the development of CM and UM, as is also suggested by the differences in the presence of mutations in BRAF, NRAS, and GNAQ/GNA11. Since TERT promoter mutations are relatively common in CM, these mutations are of special interest with respect to clinical consequences. We did not find a correlation between the presence of any of the investigated mutations in this study and the well-known adverse histopathological parameters, as has been described for cutaneous melanoma, such as increasing tumor thickness and more advanced pT stage [26]. Previous studies reported an association between PAM with atypia and PAM-derived melanoma, with the presence of a TERT promoter mutation [13, 14]. Remarkably, in the current study, we found a significant association with the presence of a TERT promoter mutation and non PAM-derived melanoma. This difference needs to be clarified by testing larger cohorts. The presence of a TERT promoter mutation in the tumor could have important clinical consequences, including the correlation of mutation status of this gene and follow-up. We found a correlation between the presence of a TERT promoter mutation and MFS, with a lower MFS in patients with a CM with a TERT promoter mutation, congruent with the findings in our previous study [13]. TERT promoter mutations have also been described as an independent prognostic factor in cutaneous melanoma. From this perspective, it is important to mention that most lesions in our cohort concerned relatively large tumors located at prognostic adverse locations (palpebra, fornix, or caruncle) [6], suggesting a bias. Patients with a TERT-promoter-mutated CM might benefit from an intensified follow-up program. \n\nIn addition to TERT promoter mutations, CM frequently harbors BRAF mutations, which are known to activate the downstream kinases MEK1/2 and ERK1/2, resulting in tumor proliferation [1, 6]. In this study, we identified BRAF mutations in almost half the cases, almost all resulting in V600E mutations. This is in line with the literature in which 30-40% of all CM harbor mutations in BRAF, almost all being V600E mutations [3, 6, 13, 27, 28]. These mutations, and specifically the V600E mutation, are also present in about half of all patients with cutaneous melanoma [29], whereas this mutation is not frequently involved in other mucosal melanoma or UM [6]. \n\nIn cutaneous melanoma, the presence of a TERT promoter mutation in addition to a BRAF mutation is associated with unfavorable clinicopathological characteristics, such as large tumor thickness and a high mitotic rate [26]. Unfortunately, the number of cases in the current cohort was too small to render any conclusions concerning these correlations in CM. \n\nDetermining the mutation status of the tumor could be useful with regards to therapeutic consequences, since several studies have shown an improved progression-free survival and overall survival, in patients with metastasized cutaneous melanoma harboring a BRAF mutation, using BRAF inhibitors [30]. BRAF mutations are also attractive as a target for adjuvant therapy in CM [6, [31] [32] [33]. \n\nNRAS mutations are described in 27% of cutaneous melanoma, with a Q61K mutation as the most common mutation followed by Q61R [34]. NRAS-mutated cutaneous melanoma have an unfavorable prognosis as compared to BRAF mutated or wild-type melanoma [34]. We identified NRAS mutations in 21% of all CM in our cohort, which is in line with the 17% previously reported [15] and is somewhat lower compared to other literature [6]. Due to the small numbers of NRAS-mutated cases in our cohort, no correlations to prognosis could be determined. NRAS mutations are mutually exclusive with BRAF mutations [6]. NRAS mutations are amenable to MEK inhibitor therapy, as has been shown for cutaneous melanoma [35]. MEK inhibitors reduce the growth of NRAS mutant CM cell lines [1]. As yet, no cases of NRAS-mutated metastatic melanoma treated with MEK inhibitors have been published. \n\nInterestingly, we detected an SF3B1 mutation at the hotspot R625, which is well-known in UM [3, 28], and was reported in one CM case. The presence of a SF3B1 mutation was reported previously in CM, however, this concerned a p. C1123Y mutation and not a hotspot mutation [36], and another study reported a missense mutation [15]. Although R625 SF3B1 mutations are very rare in most melanoma, they have been identified in UM, including iris melanoma [19], and are less frequent in cutaneous melanoma as well as in vulvovaginal mucosal melanoma [36] [37] [38] [39]. The occurrence of SF3B1 mutations in mucosal melanoma other than CM is higher, with a prevalence of 42% and hotspot mutations in 30-37% [39, 40]. The clinical significance of this mutation in CM is unknown, whereas in UM, SF3B1 mutation is correlated to late metastatic disease [41]. The CM with this mutation was treated with excision. This case also included PAM and showed local recurrence, three and eight years after primary treatment. No metastasis developed in the follow-up period of 6. 8 years. However, metastasis in SF3B1-mutated UM was described even after 10 years [41]. \n\nThe CM cases in our cohort also harbored mutations in c-KIT, PTEN, and BAP1. These findings of mutations in c-KIT, NRAS, and PTEN are congruent with other literature [1, 6], with c-KIT mutations reported in 39% of mucosal melanoma and being feasible for targeted therapy [42]. Of interest is the finding of mutations in BAP1, which is a common hemizygous mutation in UM [12, 43]. BAP1 is a tumor suppressor gene and individuals with cutaneous melanocytic neoplasm with a germline BAP1 mutation, often have BRAF mutations, with these lesions reported to have a benign clinical course [43]. However, UM with somatic BAP1 mutations are correlated to loss of chromosome 3 and early metastatic disease. CM has also been described in a patient with the BAP1 tumor predisposition syndrome [44]. We identified heterozygous BAP1 mutations that can be explained as passenger mutations without consequences, due to expression of the remaining non-affected allele. \n\nThe genetic profile of CM differs from UM, another subtype of ocular melanoma, in which mutations in GNAQ/GNA11 are frequently described [45]. In this study, none of the CM harbored an activating hotspot mutation in GNAQ or GNA11. These findings are congruent with other studies analyzing mutations in CM [15, 46]. BRAF and NRAS mutations are extremely rare in UM [37]. Therefore, these mutations can be useful in distinguishing CM from UM. This may be of interest in the identification of the primary tumor site in the case of metastatic melanoma with unknown primary. It also warrants the need for exploration of the genetic background of metastatic melanocytic lesions. However, such molecular results need to be interpreted with care, since we describe BAP1 and SF3B1 mutations in CM in the current cohort. \n\nWe did not find a correlation concerning the presence of any of the mutations and the development of recurrent disease. Cases with recurrent disease harbored the most frequently found mutations only in a (very) low number of cases. This may imply that recurrence and metastasis relate to different molecular or physical processes. \n\nIn conclusion, based on our molecular findings, CM comprises a separate entity within the ocular melanoma group, although there certainly are overlapping molecular features with UM, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data in the light of clinical findings. About three-quarter of CM contain drug-targetable mutations in BRAF, NRAS, or c-KIT, supporting the relevance of molecular genetic testing in CM for therapeutic reasons. Within this study, we confirmed that TERT promoter mutations are frequently found in CM and are correlated to metastatic disease, supporting the relevance of molecular genetic testing for prognostic reasons.",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "4."
},
{
"section_content": "We collected twenty-eight CM, diagnosed between 1987 and 2016 at the Erasmus MC-University Medical Center (Rotterdam, The Netherlands) and The Rotterdam Eye Hospital (Rotterdam, The Netherlands). Ethics Committee approval was obtained by the Medical Ethics Committee, Erasmus MC-University Medical Center, Rotterdam, The Netherlands (4 October 2018) and was registered with reference 67865. The study was performed according to the tenets of the Declaration of Helsinki. Samples were included when sufficient FFPE material was available for testing. Data regarding gender, age at the time of diagnosis, location, tumor thickness, the origin of the lesion, and information of development of recurrences and metastasis were collected from the patient records and information was obtained from the pathology reports and the nationwide-pathology network and registry system (Pathologisch-Anatomisch Landelijk Geautomatiseerd Archief ). Recurrence was defined as histopathological proven CM at the same location, either after complete excision of the primary lesion or a tumor-free mapping biopsy, after a first incomplete excision of the primary tumor. Recurrence-free survival was defined as the time from the primary treatment to the date of recurrence or last date of follow-up. Metastasisfree survival was defined as time from the primary treatment to the date of metastatic disease or last date of follow-up.",
"section_name": "Material Selection",
"section_num": "4.1."
},
{
"section_content": "DNA from FFPE tissue was isolated using lysis buffer (Promega, Madison, WI, USA) and 5% Chelex (Bio-Rad, Hercules, CA, USA), as described previously [27] and stored at -20 • C. DNA concentrations were measured with the Quant-iT™ PicoGreen™ ds DNA Assay Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA).",
"section_name": "DNA Isolation",
"section_num": "4.2."
},
{
"section_content": "The Ion Personal Genome Machine and Torrent Server (Thermo Fisher Scientific, Waltham, MA, USA) was used for targeted next-generation sequencing (NGS), according to the manufacturer's protocol. An input of DNA was used depending on the available amount of DNA. An extended gene panel covering GNAQ, GNA11, EIF1AX, SF3B1, BAP1, BRAF, NRAS, c-KIT, PTEN, and TERT was used, as described previously [27].",
"section_name": "Targeted Next-Generation Sequencing",
"section_num": "4.3."
},
{
"section_content": "Mutation analysis was performed independently by an ophthalmology resident (NvP) and a fellow in ophthalmic pathology (JvI), trained in the evaluation of NGS data. All data were analyzed manually using Integrative Genomics Viewer (IGV) Version 2. 3. 68 (97) (Broad Institute, Cambridge, MA). Furthermore, an automatic filtering of the variant calling files (vcf) was done according to the following criteria-inclusion of the hotspots at GNAQ/GNA11 (R183 and Q209) and SF3B1 (R625), and other variants meeting the following criteria-coverage of at least 50 reads and an allele frequency of at least 10%. Single nucleotide pleomorphisms (SNP's), synonymous, intergenic, and intronic variants were excluded, but intronic variants with possible splice effects were scored. Subsequently, the filtered mutations were verified using IGV (Broad Institute, Cambridge, MA, USA), and compared to the mutations that were detected manually.",
"section_name": "Mutation Analysis",
"section_num": "4.4."
},
{
"section_content": "The presence of a mutation in the BAP1 gene was also evaluated using BAP1 immunohistochemistry, clone sc-28383, 1:50 dilution (Santa Cruz Biotechnology, Dallas, TX, USA). The samples were scored through masked screening, by an experienced ophthalmic pathologist (RVE).",
"section_name": "Immunohistochemistry",
"section_num": "4.5."
},
{
"section_content": "All statistical analysis was performed using IBM SPSS Statistics Version 25 (IBM, Armonk, NY, USA). Kaplan Meier estimates were used to compare survival between groups. Log-rank test was used to test the null hypothesis that there was no difference in survival. A p-value < 0. 05 was considered to be statistically significant. For the purpose of analyzing age related to the mutation, age was categorized into three groups: <50 years, 50-65 years, >65 years, analogous to other literature [28]. Fisher's exact test was used to analyze whether a specific mutation was correlated with a specific clinical or histopathological parameter.",
"section_name": "Survival Analysis",
"section_num": "4.6."
},
{
"section_content": "The following are available online at https://www. mdpi. com/article/10. 3390/ijms22115784/s1. Supplementary Table S1 : Overview of mutations detected in conjunctival melanoma.",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "We would like to thank R. van Marion for his help performing next-generation sequencing.",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Funding: This research was funded by Stichting Nederlands Oogheelkundig Onderzoek (SNOO). Grant no: 2013-2021, Stichting Wetenschappelijk Oogheelkundig Onderzoek (SWO). Grant no: 2016-2017 and KWF Dutch Cancer Society. Grant no: 6905.",
"section_name": "",
"section_num": ""
},
{
"section_content": "Data Availability Statement: Data can be provided upon request.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest.",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.1038/bcj.2016.9
|
Lenalidomide treatment and prognostic markers in relapsed or refractory chronic lymphocytic leukemia: data from the prospective, multicenter phase-II CLL-009 trial
|
<jats:title>Abstract</jats:title><jats:p>Efficacy of lenalidomide was investigated in 103 patients with relapsed/refractory chronic lymphocytic leukemia (CLL) treated on the prospective, multicenter randomized phase-II CLL-009 trial. Interphase cytogenetic and mutational analyses identified <jats:italic>TP53</jats:italic> mutations, unmutated <jats:italic>IGHV</jats:italic>, or del(17p) in 36/96 (37.5%), 68/88 (77.3%) or 22/92 (23.9%) patients. The overall response rate (ORR) was 40.4% (42/104). ORRs were similar irrespective of <jats:italic>TP53</jats:italic> mutation (36.1% (13/36) vs 43.3% (26/60) for patients with vs without mutation) or <jats:italic>IGHV</jats:italic> mutation status (45.0% (9/20) vs 39.1% (27/68)); however, patients with del(17p) had lower ORRs than those without del(17p) (21.7% (5/22) vs 47.1% (33/70); <jats:italic>P</jats:italic>=0.049). No significant differences in progression-free survival and overall survival (OS) were observed when comparing subgroups defined by the presence or absence of high-risk genetic characteristics. In multivariate analyses, only multiple prior therapies (⩾3 lines) significantly impacted outcomes (median OS: 21.2 months vs not reached; <jats:italic>P</jats:italic>=0.019). This analysis indicates that lenalidomide is active in patients with relapsed/refractory CLL with unfavorable genetic profiles, including <jats:italic>TP53</jats:italic> inactivation or unmutated <jats:italic>IGHV</jats:italic>. (ClinicalTrials.gov identifier: NCT00963105).</jats:p>
|
[
{
"section_content": "Single-agent lenalidomide has clinical activity in chronic lymphocytic leukemia (CLL), both in treatment-naive patients, 1, 2 and in those with relapsed and refractory disease [3] [4] [5] [6] or unfavorable characteristics. 2, 4, 5 8] [9] Multivariate analysis established del(17p), TP53 mutation or unmutated IGHV were each important independent prognostic factors for survival. 7, 9 TP53 mutation without del(17p) is also of prognostic importance, with both markers demonstrating independent prognostic significance in multivariate analyses. 10 tients with CLL having del(17p) had reduced overall response rate (ORR) and progression-free survival (PFS) in a study involving unselected CLL patients treated in routine clinical practice. 11 urthermore, the presence of del(17p) has been associated with significantly inferior outcome in the context of novel, noncytotoxic treatments, such as ibrutinib. 12 e investigated the efficacy of lenalidomide in subgroups of relapsed and refractory CLL patients with high-risk genetics and clinical characteristics at baseline.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "The study design and patient population are described elsewhere. 13 In brief, patients were randomized 1:1:1 to receive a double-blinded starting dose of oral lenalidomide (5, 10 or 15 mg per day) on days 1-28 of each 28-day treatment cycle. Subject to tolerability, doses were escalated to a maximum of 25 mg per day, with dose modifications applied as required. All patients received appropriate prophylaxis for tumor lysis syndrome and thrombosis. Treatment was continued until disease progression or unacceptable toxicity. Institutional Investigational Review Board of each participating site approved this study, which was conducted according to good clinical practice and the ethical principles outlined in the Declaration of Helsinki. All patients provided written informed consent. \n\nSeveral exploratory analyses were conducted as part of the trial. Clinical and demographic characteristics of interest were age, disease stage, number of prior treatments, presence of bulky disease or constitutional symptoms and purine analog response status. \n\nBlood samples for IGHV and TP53 mutation analysis, and fluorescence in situ hybridization studies for interphase cytogenetic assessment were collected pre-dose on day 1. Descriptive statistics were used to describe continuous demographic and baseline variables for each patient; categorical variables were summarized using frequency tabulations for treatment groups separately and combined. Efficacy analyses were performed on the intention-to-treat population and included all patients with genetic data available. For all efficacy end points, determination of responses (including progression of disease) was based on the investigator's assessment of CLL response data using International Workshop on CLL guidelines for diagnosis and treatment of CLL. 15 Responses by presence or absence of pretreatment characteristics were compared using logistic regression stepwise selection. Differences were considered significant at the Po0. 05 level. Logistic regression was done to assess the relationship of patient response (responder vs non-responder) using stepwise selection. The following baseline characteristics were included: relapsed vs refractory to last prior therapy; IGHV mutation status; bulky disease; del(17p) and del(11q) status; serum β 2 -microglobulin level; disease stage; and number of prior therapies (o3 vs ⩾ 3).",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Celgene Corporation funded the study. All authors had full access to all data in the study and had final responsibility for the decision to submit for publication.",
"section_name": "Role of the funding source",
"section_num": null
},
{
"section_content": "Of 104 patients enrolled, 103 received treatment; baseline characteristics are described elsewhere and the primary results demonstrated that lower starting doses of lenalidomide could facilitate dose escalation, with indication of improved efficacy in patients who escalated to higher doses. 13 ased on the intent-to-treat safety population (n=103), data on TP53 mutations, IGHV mutational status or del(17p) were available for 96, 89 or 93 patients, respectively. TP53 mutations were identified in 36 (37. 5%) patients, unmutated IGHV in 68 (77. 5%) patients and del(17p) in 23 (24. 7) patients. \n\nMost patients with TP53 mutations also harbored unmutated IGHV (27/36; 75. 0%), whereas around half had del(17p) (17/36; 47. 2%). In the absence of TP53 mutation, del(17p) was found in 5/60 (8. 3%) patients. A majority of patients with del(17p) also had TP53 mutations (17/22; 77. 3%) or unmutated IGHV (16/22; 72. 7%; Supplementary Table 1 ). Patients with TP53 mutations, compared with those without, were more likely to be 465 years (55. 6% vs 33. 3%), have del(17p) (47. 2% vs 8. 3%), have Rai high-risk/Binet C disease (55. 6% vs 39. 3%) or to have a reduced ( o1 50 000/mm 3 ) platelet count (75. 0% vs 45. 0%; Supplementary Table 1 ). Patients with unmutated IGHV were more likely than patients with mutated IGHV to have TP53 mutation (39. 7% vs 20. 0%) or bulky disease (45. 6% vs 25. 0%). Patients with del(17p), compared with those without, were more likely to have TP53 mutation (36. 8% vs 15. 0%), Rai high-risk/Binet C disease (45. 6% vs 35. 0%) or a reduced (o 1 50 000/mm 3 ) platelet count (77. 3% vs 50. 0%; Supplementary Table 1 ). \n\nInvestigator-assessed ORR was 40. 4% (42/104) for all patients (Supplementary Table 2 ). Median time to first response to lenalidomide for all patients was 3. 3 months (range: 1. 9-34. 9). The median response duration was 22. 8 months (range: 16. 6-29. 3). ORRs for patients with and without TP53 mutation were 36. 1% (13/36) and 43. 3% (26/60; P = 0. 526); for patients with and without mutated IGHV, ORRs were 45. 0% (9/20) and 39. 7% (27/68; P = 0. 796). ORR for patients with del(17p) was lower than for those without deletions with borderline significance, using Fisher's exact test (21. 7% vs 47. 1%, P = 0. 049; odds ratio: 0. 31; 95% confidence interval: 0. 10 and 0. 93). No other significant differences were observed for any other characteristic assessed at baseline. At a median follow-up time of 24 months, significant survival differences were found between responders and patients with stable disease (median PFS: 26. 5 vs 7. 2 months, P o 0. 001; median overall survival (OS): not estimable vs 19. 8 months; P = 0. 011; Table 1 ). The median PFS and median OS were 9. 7 and 33. 0 months, respectively, in the overall population. Median PFS in patients with TP53 mutations, compared with those without, was short with 11. 0 vs 9. 5 months (P = 0. 665; Figure 1a ); median OS was 19. 4 vs 35. 4 months (P = 0. 249; Table 1 ). For patients with mutated vs unmutated IGHV, median PFS was 6. 5 vs 10. 4 months (P = 0. 607; Figure 1b ); median OS was 31. 9 months vs not estimable (P = 0. 293). In patients with del(11q) vs those without, median PFS was 7. 3 vs 17. 6 months (P = 0. 401; Figure 1c ); median OS was 21. 3 vs 35. 4 months (P = 0. 435). In patients with del(17p) vs those without, median PFS was 4. 9 vs 11. 0 months (P = 0. 171; Figure 1d ); median OS was 18. 9 vs 34. 9 months (P = 0. 318; Table 1 ). Of note, although several of the observed differences between risk groups were sizeable, no significant differences were observed as the study was not powered to detect such differences between risk groups. Multivariable analyses were performed for PFS and OS including baseline del(11q), del(17p), TP53 mutation, unmutated IGHV, disease stage, relapse/refractory to prior purine analog therapy, baseline β2 microglobulin, bulky disease and number of prior CLL treatments as potential variables. Backward deletion was performed at a significance level of 0. 05 and the main effects with P-values of ⩽ 0. 05 were retained in the final model and were identified as independent prognostic factors. Regarding PFS, none of the factors were selected into the final model. Regarding OS, only extensive pretreatment (⩾3 lines) significantly impacted outcomes (median OS: 21. 2 months vs not reached; hazard ratio: 0. 51; 95% confidence interval: 0. 28-0. 90; P = 0. 019). \n\nOur data reveal that ORR and survival outcomes are similar and relatively poor in relapsed and refractory patients with CLL following lenalidomide treatment irrespective of the presence of TP53 or IGHV mutations, suggesting that lenalidomide activity may not be affected by loss of functional TP53 or unmutated IGHV. Purine analog refractory status and disease stage, both the clinical features associated with high-risk disease, did not appear to impact ORR or survival outcomes following lenalidomide treatment (Supplementary Table 2 ; Table 1 ). \n\nIn conclusion, our data indicate that a relatively modest lenalidomide activity is seen in relapsed and refractory CLL patients with unfavorable cytogenetic profiles, with ORRs of 36. 1% and 39. 1% observed in patients with TP53 mutations and unmutated IGHV, respectively. In patients with del(17p), ORR was lower (21. 7%) yet still apparent. However, in some patients, these responses were durable as highlighted in the PFS and OS curves (Figure 1 ). PFS and OS outcomes were similar irrespective of highrisk genetic characteristics. The trial was not powered to detect subtle differences between small subgroups, for example, with del (17p) vs TP53 mutation. The pleiotropic effects of lenalidomide observed on the tumor microenvironment 16 or leukemia cell proliferation 17 and new insights into the various mechanisms of action of lenalidomide are of increasing interest. These insights may provide a rationale for specific combination regimens, including lenalidomide plus ibrutinib, or other agents with distinct mechanisms of action.",
"section_name": "RESULTS AND DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "We received editorial support from Excerpta Medica ( Ronald van Olffen, PhD, CMPP) in the preparation of this manuscript, funded by Celgene Corporation. We are fully responsible for all content and editorial decisions for this manuscript. The work was partly funded by the DFG ( SFB1074, project B2 ). This study was funded by Celgene Corporation.",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "",
"section_name": "CONFLICT OF INTEREST",
"section_num": null
},
{
"section_content": "C-MW receives research funding, consultancy and honoraria by Celgene; MH receives research support from Celgene; TJK has served as an advisor to Celgene and received research funding from Celgene; GAMF has received honoraria from Celgene; PH receives honoraria from Celgene; JD has received a research grant and honoraria from Celgene; JGG receives honoraria from Celgene, Roche, Pharmacyclics, Mundipharma and Abbvie, and has received research support grant funding from Celgene; BP is an employee of Celgene and has equity; JZ is an employee of Celgene and has equity; SDB is an employee of Celgene and has equity; JM is an employee of Celgene and has equity; SS has received a research grant and honoraria from Celgene; the remaining authors declare no conflict of interest.",
"section_name": "CONFLICT OF INTEREST",
"section_num": null
},
{
"section_content": "All authors participated in the clinical trial reported in this paper, or in the analysis of data from this study. All authors directed development, review and This work is licensed under a Creative Commons Attribution 4. 0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons. org/licenses/ by/4. 0/ Supplementary Information accompanies this paper on Blood Cancer Journal website (http://www. nature. com/bcj)",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
}
] |
10.1186/s13148-018-0476-1
|
Dynamics of DNMT3A mutation and prognostic relevance in patients with primary myelodysplastic syndrome
|
DNMT3A gene mutation has been associated with poor prognosis in acute myeloid leukemia, but its clinical implications in myelodysplastic syndrome (MDS) and dynamic changes during disease progression remain controversial.In this study, DNMT3A mutation was identified in 7.9% of 469 de novo MDS patients. DNMT3A-mutated patients had higher platelet counts at diagnosis, and patients with ring sideroblasts had the highest incidence of DNMT3A mutations, whereas those with multilineage dysplasia had the lowest incidence. Thirty-one (83.8%) of 37 DNMT3A-mutated patients had additional molecular abnormalities at diagnosis, and DNMT3A mutation was highly associated with mutations of IDH2 and SF3B1. Patients with DNMT3A mutations had a higher risk of leukemia transformation and shorter overall survival. Further, DNMT3A mutation was an independent poor prognostic factor irrespective of age, IPSS-R, and genetic alterations. The sequential study demonstrated that the original DNMT3A mutations were retained during follow-ups unless allogeneic hematopoietic stem cell transplantation was performed, while DNMT3A mutation was rarely acquired during disease progression.DNMT3A mutation predicts unfavorable outcomes in MDS and was stable during disease evolutions. It may thus be a potential biomarker to predict prognosis and monitor the treatment response.
|
[
{
"section_content": "Alterations of epigenetic regulation may result in aberrations of gene expression and malignant transformation of cells [1, 2]. DNA methylation, one of the mechanisms for epigenetic control of gene expression, regulates important physiological development, such as gene imprint and Xchromosome inactivation [3, 4]. In mammalians, three DNA methyltransferase (DNMT), namely DNMT1, 3A, and 3B have been identified [5]. Mutation of DNMT3A gene has been reported in patients with myeloid malignancies, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) [6] [7] [8] [9] [10] [11] [12] [13]. \n\nMDS represent a clinically heterogeneous hematologic neoplasm characterized by variable degrees of cytopenias and risk of leukemia transformation [14]. The incidence (2. 6 to 20. 2%) of DNMT3A mutation in MDS varied widely, possibly due to different patient population and methods used [12, 13, 15, 16]. Regarding the prognostic relevance, DNMT3A mutation has been reported to predict poor prognosis in AML patients [7] [8] [9] [10] [11]. However, the prognostic implications of DNMT3A mutation in MDS are still controversial [12, 13, 15, 17]. Walter et al. [12] and Thol et al. [13] reported that DNMT3A mutation was associated with higher risk of leukemia transformation and shorter survival, but the other studies failed to find these associations [15, 17]. Besides, sequential studies to evaluate the dynamic changes of DNMT3A mutations during disease evolution in MDS are limited. In the present study, we investigated the DNMT3A mutation in 469 patients with de novo MDS and analyzed its associations with the clinical characteristics, outcomes, and other genetic alterations. We also performed sequential analysis of the DNMT3A gene mutation for 431 samples from 148 patients to evaluate the stability of DNMT3A mutation during the clinical course.",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "This study was approved by the Institutional Review Board/Ethical Committee of the National Taiwan University Hospital (NTUH). Diagnosis and classification of MDS were made according to the French-American-British (FAB) Cooperative Group Criteria and the WHO 2016 classification [18, 19]. From May 1985 to December 2010, a total of 469 adult patients with newly diagnosed MDS at the NTUH who had enough cryopreserved cells for analysis were enrolled. Patients with secondary or therapy-related MDS were excluded. The disease of 362 patients fulfilled the criteria of MDS according to the 2016 WHO classification. Most patients (77. 4%) received only palliative treatment, including transfusions, hematopoietic growth factors, immunosuppressive therapy, and lowintensity chemotherapy. Thirty (6. 4%) patients received intensive chemotherapy, 7. 2% received hypomethylating agents (HMA), and 9. 0% received allogeneic hematopoietic stem cell transplantation (HSCT).",
"section_name": "Subjects",
"section_num": null
},
{
"section_content": "Mutational analysis of DNMT3A gene exons 2-23 by PCR and direct sequencing was done as described previously [9]. Analysis of the mutations in other genes involving in activated signaling pathways, such as FLT3-ITD [20], NRAS [21], KRAS [21], JAK2 [21], and PTPN11 [22] ; the transcription factor, such as RUNX1 [23] ; splicing factors, including SRSF2, U2AF1, and SF3B1 [24] ; and epigenetic modifications, including MLL/PTD [25], ASXL1 [26], EZH2 [27], IDH1 [28], IDH2 [29], and TET2 [30], as well as SETBP1 [21], WT1 [31], NPM1 [32], and TP53 [33], were performed as previously described. To detect DNMT3A mutation, we used DNA amplified in vitro from bone marrow (BM) cells with the Illustra GenomiPhi V2 DNA-amplification kit (GE Healthcare, UK). All mutations detected were verified in the original non-amplified samples [34]. Abnormal sequencing results were confirmed by at least two repeated analyses. All nonsense or frameshift mutations were regarded as true mutations. Missense mutations were regarded as true only if they were documented in other studies or could be verified by sequencing of matched normal somatic tissues. Serial analyses of DNMT3A mutations during the clinical course were also performed in 431 samples from 148 patients.",
"section_name": "Analyses of mutations",
"section_num": null
},
{
"section_content": "For the patients with discrepancy of the mutation status of the DNMT3A in sequential samples, TA cloning was performed in the samples without detectable mutation followed by direct sequencing. More than 30 clones were selected for sequencing as previously described [9]. \n\nIllumina next generation sequencing (NGS) for serial studies of patients with DNMT3A mutation Serial analyses of mutations at diagnosis, disease progression, and/or remission were further performed using Illumina next generation sequencing in 32 samples from 13 patients with DNMT3A mutation at diagnosis and one during follow-up. Genomic DNA extracted from BM mononuclear cells was analyzed for mutations in 54 genes involved in myeloid malignancies by TruSight Myeloid Panel (Illumina, San Diego, CA, USA). HiSeq platform (Illumina) was used for sequencing with a median reading depth of 12,000×.",
"section_name": "TA cloning analysis",
"section_num": null
},
{
"section_content": "The discrete variables of patients with and without gene mutations were compared using the χ 2 tests, and the Fisher's exact test was used if the expected values of contingency tables were smaller than 5. The continuous variables of patients with and without gene mutations were compared using Student's t test. If the data were not normally distributed, Mann-Whitney U tests were used to compare continuous variables and medians of distributions. Overall survival (OS) was measured from the date of first diagnosis to the date of last follow-up or death from any cause. Time to leukemia transformation was measured from the date of MDS diagnosis to the date confirmed of acute leukemic change. Kaplan-Meier estimation was used to plot survival curves, and logrank tests were used to calculate the difference of OS and time to leukemia transformation between groups. Multivariate Cox proportional hazard regression analysis was used to investigate independent prognostic factors for OS and time to leukemia transformation. All tests were 2-tailed, and P < 0. 05 was considered statistically significant. All statistical analyses were performed with SPSS Version 17 software.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "A total of 469 patients with de novo MDS according to the FAB classification were included for mutational analysis. Among them, 171 (36. 5%) patients had refractory anemia (RA), 32 (6. 8%) had RA with ring sideroblasts (RARS), 159 (33. 9%) had RA with excess blasts (RAEB), 53 (11. 3%) had RAEB in transformation (RAEB-T), and 54 (11. 5%) had chronic myelomonocytic leukemia (CMML) (Table 1 ). Nineteen different DNMT3A mutations were Good: normal karyotype, isolated -Y, del(5q), or del(20q); Poor: complex (≧ 3 abnormalities) or chromosome 7 anomalies; Intermediate, other abnormalities § IPSS (international prognosis scoring system): low, 0; intermediate (INT)-1, 0. 5-1; INT-2, 1. 5-2; and high, ≥ 2. 5 ζ IPSS-R (revised international prognostic scoring system): very low, ≦1. 5; low, > 1. 5-3; intermediate, > 3-4. 5; high, > 4. 5-6; and very high, > 6 identified in 37 (7. 9%) of the 469 patients, including 7 missense mutations, 2 nonsense mutations, and 10 frameshift mutations (Fig. 1 ). In addition to the 13 single-nucleotide polymorphisms without amino acid residue alterations, 9 missense mutations with uncertain biologic significance were excluded because they were not reported previously and could not be verified for lack of matched normal somatic tissues or remission BM samples. Thirty-six of the 37 DNMT3A-mutated patients had single heterozygous mutation, and the remaining one patient had double mutations. The most common mutation was R882H (n = 11), followed by R882C (n = 8), G543C (n = 2), and Y735C (n = 2). All other mutations were detected in only one patient each (Fig. 1 ; Additional file 1: Table S1 ). According to the 2016 WHO classification, DNMT3A mutations were identified in 28 (7. 7%) of the 362 patients (Table 1 ). \n\nCorrelation of DNMT3A mutations with clinical features DNMT3A-mutated patients had higher platelet counts at diagnosis than DNMT3A-wild patients (Table 1 ). According to the FAB classification, DNMT3A mutation was highly associated with RARS subtype. Patients with RARS had the highest incidence (18. 8%, P = 0. 031) of DNMT3A mutations, whereas those with RA had the lowest incidence (2. 3%, P = 0. 001). By the 2016 WHO classification, patients with MDS with multilineage dysplasia (MDS-MLD) had lower incidence of DNMT3A mutations (2. 8%, P = 0. 029). No association between age or gender of patients and DNMT3A mutation status was found (Table 1 ). There was also no difference in the distribution of risk groups according to the international prognostic scoring system (IPSS) or revised IPSS (IPSS-R) between patients with and without DNMT3A mutations (Table 1 ). \n\nChromosome data were available in 437 (93. 2%) patients at diagnosis, and clonal chromosomal abnormalities were detected in 193 (44. 2%) patients. There were no association of the DNMT3A mutations with common chromosomal abnormalities, including loss of Y, -20/ del(20q), -5/del(5q), + 8, and -7/del(7q) (Additional file 1: Table S2 ), or risks of karyotype (Table 1 ).",
"section_name": "DNMT3A mutations in patients with de novo MDS",
"section_num": null
},
{
"section_content": "Among the 37 patients with DNMT3A mutations, 33 (89. 2%) patients had additional molecular abnormalities at diagnosis, including SF3B1 (n = 11), TET2 (n = 7),\n\n, and IDH1 mutations (n = 1) (Additional file 1: Table S1 ). Fifteen patients had 1 additional mutation, 13 had 2, and 5 had 3 (Additional file 1: Table S1 ). Patients with DNMT3A mutations had a significantly higher incidence of SF3B1 and IDH2 mutations than those without DNMT3A mutations (P < 0. 001 and P < 0. 001, respectively; Table 2 ).",
"section_name": "Association of DNMT3A mutation with other genetic mutations",
"section_num": null
},
{
"section_content": "We could not find the difference in treatment regimens between the patients with DNMT3A mutations and those without. With a median follow-up of 43. 9 months (range 0. 1-250. 7 months), patients with DNMT3A mutations had a higher risk to transform to AML (5-year AML transformation rate, 34. 4 versus 22. 5%, P = 0. 013; Fig. 2 ). MDS patients, based on either the FAB or the 2016 WHO classification, had a significantly shorter OS if they harbored DNMT3A mutation than those who did not (15. 0 versus 32. 5 months, P = 0. 024, and 16. 3 versus 41. 6 months, P = 0. 011, respectively; Figs. 3 and 4 ). Further, we could not find the survival difference between the patients with frameshift and non-frameshift mutations. Interestingly, patients with DNMT3A mutations had a better OS if they received allogenic HSCT than those who did not (P = 0. 038, Additional file 1: Figure S1 ). Because DNMT3A mutation was closely associated with SF3B1 mutation, a good prognostic factor in MDS patients [35, 36], we divided the whole cohort to two subgroups, SF3B1-mutated and SF3B1-wild type, to evaluate the prognostic significance of DNMT3A mutation independent of SF3B1 mutation. In the SF3B1-wild patients, DNMT3A mutation predicted worse prognosis (OS, 14. 6 ± 4. 7 months versus 30. 9 ± 3. 2 months, P = 0. 005). On the other hand, in the 48 SF3B1-mutated patients, DNMT3A mutation had no prognostic implication (OS, 17. 7 ± 11. 0 months versus 39. 7 ± 4. 2 months, P = 0. 858) (Additional file 1: Figure S2 ). \n\nIntriguingly, the impact of DNMT3A mutation on OS and time to leukemia transformation remained significant after adjusting the effects of age, gender, IPSS-R [37, 38], and mutations with prognostic significance in multivariate Cox regression analysis (FAB defined patients: OS: hazard ratio, HR 1. 733, 95% CI 1. 118-2. 688, P = 0. 014; time to leukemia transformation: HR 3. 088, 95% CI 1. 574-6. 056, P = 0. 001; 2016 WHO classification defined patients: OS: HR 1. 800, 95% CI 1. 080-3. 000, P = 0. 024; time to leukemia transformation: HR 2. 360, 95% CI 1. 129-4. 933, P = 0. 022; Table 3 ).",
"section_name": "Correlation of DNMT3A mutation with clinical outcome",
"section_num": null
},
{
"section_content": "To investigate the role of DNMT3A mutation in clinical evolution, DNMT3A gene mutation status was sequentially tested during the clinical course in 431 samples from 148 patients, including 13 patients with DNMT3A mutations at diagnosis and 135 patients without the mutation. In the 13 DNMT3A-mutated patients, 8 had disease progression, including 6 [unique patient numbers (UPNs) 1, 5, 7, 13, 24, and 37] with AML transformation. Four patients (UPNs 17, 24, 30, and 36) lost the original DNMT3A and other concurrent mutations/cytogenetic abnormalities when complete remission (CR) was achieved following curative-intent chemotherapy and/or allogeneic HSCT (Table 4 ). On the other hand, the other 9 patients with DNMT3A mutations at diagnosis retained their mutations during follow-ups. Among the eight with disease progression, one (UPN 37) acquired a novel RUNX1 mutation when the disease transformed to AML. \n\nAmong the 135 patients without DNMT3A mutation at diagnosis, 1 (0. 7%) patient (UPN 47) acquired a novel DNMT3A mutation during sequential follow-up. This patient had MDS with excess blasts-1 (MDS-EB1) at diagnosis when no DNMT3A mutation was detectable even using more sensitive cloning method and next generation sequencing. He acquired GNAS, ASXL1, and ZRSR2 mutations in addition to DNMT3A mutation in the 19th month and died of progressive cytopenia in the 29th month. \n\nWe further analyzed the variant allele frequencies of the mutations in the 48 DNMT3A-mutated patients by NGS (Table 4 ). The mutant burden of DNMT3A mutations at diagnosis ranged from 8. 4 to 45. 24% with a median of 31. 1%. Among the 13 patients with serial studies during the clinical courses, the mutation burden at subsequent follow-ups, compared to that at diagnosis, was increased in 3 patient (UPNs 5, 13, and 24), decreased in 6 patients (UPNs 1, 7, 10, 17, 30, and 36, Table 4 ) and stationary in 4 patients (UPNs 21, 23, 27, and 37). All of the three patients with increased DNMT3A mutation burden had leukemia transformation. Their variant allele frequencies of DNMT3A and other co-occurring mutations were increased at least 10% (10. 0-347. 1%) at leukemia transformation compared with those at baseline. The patient (UPN 37) who had least increase in variant allele frequency of DNMT3A mutation during disease progression acquired RUNX1 mutation at leukemia transformation. In contrast, the variant allele frequencies of DNMT3A and other concurrent mutations were relative stationary or even decreased during follow-up in the patients without leukemia transformation.",
"section_name": "Sequential studies of DNMT3A mutations",
"section_num": null
},
{
"section_content": "In the present study, we identified 19 different DNMT3A mutations in 37 (7. 9%) of the 469 FAB-defined and 7. 7% of the 2016 WHO-defined MDS patients. Similar to previous studies on AML or MDS cohorts [7-10, 12, 13, 17], most mutations are located in the MTase domain, especially at amino acid R882 locus. Of these 19 mutations, 10 are frameshift and 2 are nonsense mutations. They generate truncated peptides with complete or partial deletion of the MTase and are expected to abolish the normal function of DNMT3A gene. The R882 mutations result in impaired gene function [7, 39], but the influence of the remaining missense mutations on the enzyme activity are unclear. In this study, the prevalence of DNMT3A mutation is 7. 9 and 7. 7% in MDS according to the FAB and 2016 WHO classification, respectively (Table 1 ), similar to most of the previous reports (7. 8 to 10%) [12, [40] [41] [42] but higher than that of Thol et al. (2. 6%) [13]. \n\nThe reports with detailed demographics of MDS patients with DNMT3A mutation in literature are limited. In the report of Walter et al., but not in the current study and other studies [40, 42], DNMT3A mutations were associated with older age; in contrast, DNMT3A mutations were associated with higher platelet count in our study but not in other studies [12, 40, 42]. The association of DNMT3A mutations with higher platelet count was also shown in AML in previous studies [8, 9]. No comparison of age and hemogram between patients with and without DNMT3A was done in the study of Thol et al. [13] in which only five patients were found to have DNMT3A mutation. The causes of differences in the incidence of DNMT3A mutation and the clinical characteristics of DNMT3A-mutated patients might result from the differences in patient population recruited, detection platform used, sample size, and DNMT3A regions screened. In the study of Thol et al. [13], exons 15-23 instead of exons 2-23 of DNMT3A gene were analyzed in most patients (173 of 193 patients). Therefore, some patients harboring DNMT3A mutations might not be detected, and this might partially explain the lower incidence of DNMT3A mutation in their cohort (2. 6%). \n\nIn this study, DNMT3A mutations were positively associated with IDH2 and SF3B1 mutations (Table 2 ). The close association of DNMT3A and IDH2 mutations was also shown in AML [9]. Mutations of DNMT3A and SF3B1, a component of spliceosome complex frequently mutated in RARS, have been reported to occur concurrently more often than expected by chance in lower-risk MDS patients [17]. In our cohort, the positive association of these two genetic alterations could also be found in lower-risk MDS patients (P < 0. 001; Table 2 ). In addition, we could find a trend of positive correlation between these two mutations in higher-risk MDS patients (P = 0. 098; Table 2 ). The close associations between DNMT3A mutation and RARS and between DNMT3A and SF3B1 mutations in this study (Table 1 ) might be related with each other. To investigate the associations among the RARS subtype, DNMT3A mutation, and SF3B1 mutation, we divided the whole cohort to RARS and non-RARS patients. The close association of DNMT3A and SF3B1 mutations retained in both subgroups. In contrast, no association between DNMT3A mutation and RARS subtype was found when we divided the whole population to SF3B1-mutated and SF3B1 wild-type patients. In the studies of more than 100 genes by high-throughput DNA sequencing, Haferlach et al. [43] and Papaemmanuil et al. [44] also found a positive correlation between DNMT3A and SF3B1 mutations, indicating that interaction between these two gene mutations may play a role in the pathogenesis of MDS, but further investigations are needed to elucidate its mechanism, especially in RARS subtype. No data regarding the association between DNMT3A mutation and RARS were shown in these two studies. \n\nDNMT3A mutation has been identified as a poor prognostic factor in AML patients [7] [8] [9] [10] [11]. However, its prognostic impact on MDS patients remains uncertain. Walter et al. demonstrated DNMT3A mutations were associated with shorter survival and higher risk of leukemia transformation in univariate analysis [12], and Thol et al. also reported a higher rate of transformation to AML in patients with this mutation [13]. However, three other studies did not reveal significant impact of DNMT3A mutations on survival [15, 17, 44]. In this study, we showed that DNMT3A mutation was associated with poor outcomes, including higher risk of AML transformation and shorter OS. Bejar et al. [17] had speculated that the negative prognostic effect of DNMT3A mutation might be mitigated by the co-existence of SF3B1 mutation. In their cohort, 22% patients had SF3B1 mutation and they did not find the prognostic significance of DNMT3A mutation. The same was also true in another study, in which 24% of patients had SF3B1 mutation [44]. Both cohorts had significantly higher incidence of SF3B1 mutation than ours (10. 2%). It may be possible that DNMT3A mutation would have prognostic effect only in MDS cohorts with low prevalence of SF3B1 mutation. Nevertheless, we distinctly showed that DNMT3A mutation was an independent poor prognostic factor for OS irrespective of the status of SF3B1 mutation and other prognostic factors. \n\nBased on the finding of higher risk of AML transformation and shorter survival in DNMT3A-mutated patients, as shown in current study, it would be interesting to investigate the effect of allogenic HSCT in these patients. We found that patients with DNMT3A mutations had a better OS if they received allogenic HSCT than those who did not. It implied that HSCT might ameliorate the poor survival impact of the adverse-risk genotype. Further prospective studies with more patients recruited are needed to verify this point. In a study of 46 decitabine-treated AML patients, Metzeler proposed that DNMT3A-mutated patients might have better treatment response and longer OS [45]. Subsequently, Traina et al. reported DNMT3A mutation as an independent predictor of better response and improved progression-free survival in MDS patients treated with DNMT inhibitors [41]. In our study, only 2 of 36 patients treated with HMA had DNMT3A mutation. These two patients had treatment response and OS similar to others. The influence of DNMT3A mutation on the treatment response to DNMT inhibitors was not evaluated because of the small number of DNMT3A-mutated patients. \n\nDNMT3A mutation was found quite stable during disease evolution in AML patients [9, 46], but to the best of our knowledge, the dynamic change of this mutation in MDS patients has not been reported yet in literature. Here we showed that DNMT3A mutation was also quite stable in the clinical course of MDS patients; all DNMT3A-mutated patients retained the original mutations during sequential follow-ups unless CR was achieved after allogeneic HSCT or intensive chemotherapy. On the other hand, DNMT3A mutation was rarely acquired during disease evolution; only one (0. 7%) of the 145 DNMT3A-wild patients acquired the mutation subsequently (Table 4 ). \n\nIt is well known that age-related clonal hematopoiesis is associated with increase in the risk of hematologic cancer and the majority of the variants occurred in three genes: DNMT3A, TET2, and ASXL1 [47] [48] [49]. Hematologic cancers were more common in persons with a variant allele fraction of 0. 10 or greater. Therefore, it was proposed that DNMT3A mutation is relevant for initiating hematopoietic stem cell clonal expansion and an early initiation event for hematological malignancies. Our finding that DNMT3A mutation was retained unless CR was achieved was consistent with this hypothesis. In patients who failed to achieve remission, the clone harboring DNMT3A mutation survived and may contribute to subsequent relapse. Persistence of DNMT3A mutation in some AML patients in CR was described by us and other researchers [9, [50] [51] [52] [53] [54]. In a recent study of Gaidzik et al., DNMT3A mutant transcript levels in CR did not predict outcome in AML patients [54]. In contrast, Thol et al. showed that patients with DNMT3A-mutated lympho-myeloid clonal hematopoiesis (LM-CH) in CR had a higher cumulative incidence of relapse at 10 years compared with those without DNMT3Amutated LM-CH (75 versus 27%) [55]. In the present study, we aimed to delineate the dynamic pattern of DNMT3A mutation in MDS development and progression. By NGS, the only patient (UPN 13) who retained his original DNMT3A mutation after high intensity chemotherapy finally relapsed. On the other hand, none of the patients in CR who lost their original DNMT3A mutation after allogeneic HSCT experienced disease relapse. Our data suggested that DNMT3A mutation might be used to assess the treatment response and the risk of relapse after curative-intent treatments in MDS patients. Together, whether retaining of DNMT3A mutations after curativeintent treatment is informative for the assessment of the relapse risk in MDS patients remains unclear. It should be cautious to interpret in clinical decision-making and more large-scale studies in MDS patients are warranted to clarify this point.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "We identified associations of DNMT3A mutations with distinct clinical features and mutations of SF3B1 and IDH2 genes. In addition, we demonstrated that DNMT3A mutations independently predicted poor outcomes and were stable in the clinical course. It may be used as a biomarker to monitor the response after curative-intent treatment. Additional file 1, is available at Clinical Epigenetics' website. \n\nMDS-EB1: Myelodysplastic syndrome with excess blasts-1; MDS-MLD: Myelodysplastic syndrome with multilineage dysplasia; NGS: Next generation sequencing; NTUH: National Taiwan University Hospital; OS: Overall survival; RA: Refractory anemia; RAEB: Refractory anemia with excess blasts; RAEB-T: Refractory anemia with excess blasts in transformation; RARS: Refractory anemia with ring sideroblasts; UPN: Unique patient number",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We would like to acknowledge the service provided by the DNA Sequencing Core of the First Core Laboratory, National Taiwan University College of Medicine.",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This work was partially sponsored by grants MOST 103-2628-B-002-008-MY3, 103-2923-B-002-001, MOST 103-2314-B-002-130-MY3, 103-2314-B-002-131 MY3, 104-2314-B-002-128-MY4, and 106-2314-B-002-226-MY3 from the Ministry of Science and Technology (Taiwan), National Taiwan University Hospital-National Taiwan University joint research grant ( UN103-051 ), and MOHW 105-TDU-B-211-134004 from the Ministry of Health and Welfare (Taiwan), NTUH 102P06, from the Department of Medical Research, National Taiwan University Hospital, and Taiwan Health Foundation.",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "The datasets generated and/or analyzed during the current study are not publicly available due to individual privacy but are available from the corresponding author on reasonable request.",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional file",
"section_num": null
},
{
"section_content": "Additional file 1: Table S1. The mutation patterns in 37 MDS patients with DNMT3A mutations at diagnosis. Table S2. Cytogenetics between MDS patients with and without DNMT3A mutation. Abbreviations AML: Acute myeloid leukemia; BM: Bone marrow; CMML: Chronic myelomonocytic leukemia; CR: Complete remission; FAB: French-American-British; HMA: Hypomethylating agents; HSCT: Hematopoietic stem cell transplantation; IPSS: International prognostic scoring system; IPSS-R: Revised international prognostic scoring system; MDS: Myelodysplastic syndrome;\n\nAuthors' contributions M-EL was responsible for the data management and interpretation, mutation analysis, statistical analysis, and manuscript writing; H-AH was responsible for the study design, study plan and coordination, data management and interpretation, mutation analysis, statistical analysis, and manuscript writing; S-JW contributed patient samples and clinical data and was responsible for the data interpretation; C-HT and Y-YK were responsible for the mutation analysis and interpretation; J-LT, MY, C-CL, W-CC, S-YH, B-SK, S-CH, C-TL, and C-YC contributed patient samples and clinical data; M-HT, C-WL, and M-CL performed the gene mutation and chromosomal studies; H-FT designed and coordinated the study over the entire period and wrote the manuscript. All authors read and approved the final manuscript.",
"section_name": "Additional file",
"section_num": null
},
{
"section_content": "This study was approved by the Institutional Review Board/Ethical Committee of the National Taiwan University Hospital (NTUH20150709RINA).",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "",
"section_name": "Consent for publication Not applicable",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests.",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.",
"section_name": "Publisher's Note",
"section_num": null
}
] |
10.18632/oncotarget.12139
|
Ibrutinib synergizes with MDM-2 inhibitors in promoting cytotoxicity in B chronic lymphocytic leukemia
|
The aim of this study was to investigate the anti-leukemic activity of the Bruton tyrosine kinase inhibitor Ibrutinib in combination with the small molecule MDM-2 inhibitor Nutlin-3 in preclinical models.The potential efficacy of the Ibrutinib/Nutlin-3 combination was evaluated in vitro in a panel of B leukemic cell lines (EHEB, JVM-2, JVM-3, MEC-1, MEC-2) and in primary B-chronic lymphocytic leukemia (B-CLL) patient samples, by assessing cell viability, cell cycle profile, apoptosis and intracellular pathway modulations. Validation of the combination therapy was assessed in a B leukemic xenograft mouse model.Ibrutinib exhibited variable anti-leukemic activity in vitro and the combination with Nutlin-3 synergistically enhanced the induction of apoptosis independently from the p53 status. Indeed, the Ibrutinib/Nutlin-3 combination was effective in promoting cytotoxicity also in primary B-CLL samples carrying 17p13 deletion and/or TP53 mutations, already in therapy with Ibrutinib. Molecular analyses performed on both B-leukemic cell lines as well as on primary B-CLL samples, while confirming the switch-off of the MAPK and PI3K pro-survival pathways by Ibrutinib, indicated that the synergism of action with Nutlin-3 was independent by p53 pathway and was accompanied by the activation of the DNA damage cascade signaling through the phosphorylation of the histone protein H2A.X. This observation was confirmed also in the JVM-2 B leukemic xenograft mouse model.Taken together, our data emphasize that the Ibrutinib/Nutlin-3 combination merits to be further evaluated as a therapeutic option for B-CLL.
|
[
{
"section_content": "Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase member of the Tec kinase family, plays a significant role in B-cell development. BTK is a key component of the B cell receptor (BCR) signaling that regulates B cell proliferation and survival and is involved in signaling pathways downstream of other receptors [1]. BTK is also known to be important for B cell migration and homing and is activated upon chemokine binding to CXCR4 and CXCR5 through direct interaction with the chemokine receptor G protein subunits [2, 3]. For all these reasons, BTK represents an unique therapeutic target in B-cell malignancies [1]. Inhibition of BTK in B chronic lymphocytic leukemia (B-CLL) disrupts integrin-mediated adhesion to fibronectin, diminishes cellular response to tissue homing chemokines, counteracts NFκB DNA binding, inhibits DNA synthesis and induces moderate apoptosis, thus affecting cell survival, proliferation and migration [4] [5] [6].",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Among BTK inhibitors actually in preclinical development and clinical trials, Ibrutinib is the most advanced molecule for the treatment of B-cell malignancies [1]. Ibrutinib is an orally available, selective and irreversible inhibitor of BTK that covalently binds to Cys481 [7]. In particular, BTK inhibition by Ibrutinib clearly has significant activity across all subtypes of CLL including patients with poor prognostic risk disease [8]. Ibrutinib, as single agent, has been used in elderly patients (≥65 years) for treatment of naïve B-CLL as well as for those with relapsed and refractory disease [8, 9]. An unanticipated finding was that treatment with Ibrutinib induced a prompt lymphocytosis in the peripheral blood. In particular, in Ibrutinib-treated CLL patients the lymphocytosis is usually seen by 7 days, peaking within 4 weeks and then slowly decreasing with time. In contrast, however, lymph node size diminishes rapidly in CLL, with the majority of effects seen in the first 2 months of therapy [9]. The present indication is that there are very few complete responses with Ibrutinib (2% in previously treated CLL, 13% in treatment of naïve CLL) and when treatment is interrupted disease progression is rapidly seen. Therefore, it is intensely debated the search of combination therapy to improve responses, with the aim of attaining a minimal residual disease (MRD) negative response, without significant toxicity for the patients. Moreover, another evidence suggesting the importance of searching new therapeutic combinations with Ibrutinib is the emergence of resistance to Ibrutinib monotherapy [10] [11] [12] [13]. \n\nIn this respect, we and other investigators have demonstrated the potential efficacy of MDM-2 inhibitors, used either alone or in combinations, as anti-leukemic agents [14] [15] [16] [17] [18]. Interestingly, a number of studies have shown the potential p53-independent synergism of Nutlin-3 with different anti-cancer drugs [16, 17, [19] [20] [21]. On these bases, the aim of this study was to investigate the potential anti-leukemic activity of Ibrutinib/Nutlin-3 combination in preclinical models consisting of a panel of p53 wild-type and p53 mutated B leukemic cell lines and primary B-CLL patient samples as well as a B leukemic xenograft mouse model.",
"section_name": "Research Paper",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "In the first group of experiments, Ibrutinib was tested on a panel of p53 wild-type (EHEB, JVM-2, JVM-3) and p53 mutated (MEC-1, MEC-2) B lymphoblastoid leukemic cell lines. As documented by the IC 50 values, Ibrutinib showed a variable cytotoxicity independently from the p53 status of the cell line (range 14. 8-19 μM and 5. 4-14. 8 μM, respectively at 24 and 48 hours; Table 1 ). Analysis of the cytotoxicity induced by treatment with Ibrutinib was then evaluated on primary cells derived from a cohort of B-CLL patients (n=22; Table 2 ). Analysis of IC 50 mean values indicated that also B-CLL patient cell samples were susceptible to Ibrutinib independently from the p53 status (Table 2 ). On the other hand, treatment with Ibrutinib (used at concentrations up to 10 μM) of PBMC isolated from healthy blood donors (n=5) did not significantly affect cell viability (data not shown). \n\nOn these bases, in order to enhance the antileukemic activity of Ibrutinib, we have then assessed the in vitro effect of Ibrutinib used in combination with Nutlin-3. As shown in Figure 1, the combined treatment induced a reduction of cell viability significantly higher with respect to the single drugs in all B leukemic cell lines analyzed. Moreover, experiments performed treating cells with serial concentrations of Ibrutinib and Nutlin-3 at constant ratio, and analyzed with the method of Chou and Talalay [22], revealed that the Ibrutinib/Nutlin-3 combination promoted a synergistic (average CI<1) cytotoxicity both in p53 wild-type (EHEB, JVM-2, JVM-3) and in p53 mutated (MEC-1, MEC-2) B lymphoblastoid leukemic cell lines (Table 3 ).",
"section_name": "Treatment with Ibrutinib/Nutlin-3 combination exhibits a synergistic anti-leukemic activity in both p53 wild-type and p53 mutated B leukemic cells",
"section_num": null
},
{
"section_content": "Based on the cell viability data, we have next investigated the effect of Ibrutinib/Nutlin-3 combination both on the cell cycle progression (Figure 2A -2B) as well as on apoptosis modulation (Figure 2C-2D ). As reported in Figure 2A, treatment with Ibrutinib alone induced a significant reduction of S phase accompanied by a concomitant increase in G0/G1 phase in all B lymphoblastoid cell lines. In the p53 wild-type leukemic cell lines, the combination of Ibrutinib/Nutlin-3 further enhanced the accumulation of cells in G0/G1 phase due to the marked cytostatic activity of Nutlin-3 in these cells (Figure 2A-2B ). On the other hand, the cell cycle profile of p53 mutated B leukemic cell lines was very similar in cells treated with Ibrutinib alone or Ibrutinib/Nutlin-3 combination since Nutlin-3 was not effective in these cells (Figure 2A-2B ). Therefore, both in the p53 wild-type and p53 mutated leukemic cell lines, the effect of the drug combination on cell cycle was masked by the predominant effect of each single drug. At the opposite, the analysis of apoptosis showed that Ibrutinib alone induced modest levels of apoptosis in all cell lines, while the Ibrutinib/ Nutlin-3 combination significantly increased the percentage of apoptosis with respect to the treatment with the single drugs used alone, in both p53 wild-type and p53 mutated B leukemic cell lines (Figure 2C-2D ). These data clearly suggest that the induction of apoptosis, rather than the cell cycle block mainly accounted for the synergistic antileukemic activity of the Ibrutinib/Nutlin-3 combination. \n\nTo ascertain the potential clinical relevance of the data obtained in B leukemic cell lines, we have investigated the effect of Ibrutinib/Nutlin-3 combination in experiments performed in primary B-CLL samples. As for B leukemic cell lines, treatment of primary B-CLL cells with serial concentrations of Ibrutinib and Nutlin-3 at a constant ratio revealed a synergistic (average CI<1) cytotoxic activity in 10 out of the 11 primary B-CLL samples analyzed (Table 4 ). The synergistic effect of the Ibrutinib/Nutlin-3 combination was independent from the p53 status of the patient's cells, confirming the data obtained in B-cell lines. Moreover, also in primary B-CLL cells the cytotoxicity induced by the Ibrutinib/Nutlin-3 combination was mainly due to the increase of apoptosis with respect to the treatment with either Ibrutinib or Nutlin-3, used alone (Figure 3A-3B ). As a matter of fact, effects on cell cycle were not considered because B-CLL primary cells derived from peripheral blood and cultured ex vivo are mostly in G0/G1 phase and quiescent not replicating. In light of the well-known pro-survival role of the microenvironment on leukemic cells [23], we have then assessed the effect of Ibrutinib/Nutlin-3 combination on B-CLL cells co-cultured on a monolayer of stromal cells (mimicking the disease microenvironment). Despite the protective effect resulting in general reduction of apoptosis levels respect to culture of B-CLL cells alone, the combined treatment still exhibited a synergistic effect in apoptosis induction (Figure 3C ). \n\nA potential pitfall of our study is represented by the fact that Nutlin-3 showed poor in vivo bioavailability [24], a finding that hampered its potential clinical use. In this regard, a novel MDM-2 inhibitor (RG7112) with superior bioavailability with respect to Nutlin-3 is currently under evaluation in clinical trials in patients with hematologic malignancies [25]. Of note, similarly to Ibrutinib/Nutlin-3 combination, also Ibrutinib/RG7112 combination promoted synergistic cytotoxicity of B leukemic cells (Supplementary Figure S1 ), having average CI values <1 for both p53 wild- type EHEB (0. 82 and 0. 21 at 24 and 48 hours of treatment, respectively) as well as p53 mutated MEC-2 cells (0. 99 and 0. 59 at 24 and 48 hours of treatment, respectively).",
"section_name": "The synergistic anti-leukemic activity of the Ibrutinib/Nutlin-3 combination is mainly due to induction of apoptosis",
"section_num": null
},
{
"section_content": "It is well known that the pathogenesis of B-CLL is characterized by alterations of cellular signaling pathways. Therefore, in the next group of experiments, we have evaluated if the cellular responses observed after treatment with Ibrutinib/Nutlin-3 combination correlated with modification of intracellular signaling events, with particular attention to pathways regulated by BTK and/or p53 that are targets of Ibrutinib and Nutlin-3, respectively (Figure 4 ). In line with previous studies [26, 27], by western blotting analyses we found that exposure of B leukemic cell cultures to Ibrutinib resulted in inhibition of BTK auto-phosphorylation at tyrosine 223 (caused by the inhibition of kinase activity by the binding of Ibrutinib) coupled to a significant reduction of the phosphorylation of the MAPK survival pathway modulators ERK1/2, starting at early time points post drug exposure (Figure 4A ). Moreover, by assessing phosphorylation levels through magnetic-plex assays, we observed a rapid downmodulation of the PI3K survival pathway, as documented by the reduction of both Akt and m-TOR phosphorylation, which was validated also by western blotting (Figure 4B ). Independently by the p53 status of the tested cells, superimposable results were observed also after treatment with Ibrutinib/Nutlin-3 combination, indicating that Nutlin-3 treatment did not significantly affect these pathways (Figure 4A-4B ). On the other hand, while Nutlin-3 treatment induced activation of the p53 pathway in p53 wild-type B-leukemic cells, as documented by the significant increase of the expression levels of CDKN1A, MDM2 and BAX genes, no effects on this pathway were observed upon treatment with Ibrutinib used either alone or in combination (Figure 4C ). No significant modulation of the p53 pathway was observed in p53 mutated B cells following any treatment (Figure 4C ). Finally, considering the emerging role of the histone protein H2A. X for the clinical validation of anticancer candidate drugs [28, 29], we have then evaluated if the anti-leukemic activity in our setting was associated with DNA damage response (DDR). Western blotting analysis on cellular lysates from B-leukemic cell lines highlighted that the exposure to Ibrutinib was associated to upregulation of phospho-H2A. X, responsible for the DDRsignal amplification, and that this response was further enhanced by the combination with Nutlin-3 (Figure 4D ). Of note, the activation of DDR at early time points, before the onset of the apoptosis, was observed both on leukemic cell lines (independently from the p53 status) as well as on primary cells derived from B-CLL patients (Figure 4D ).",
"section_name": "Intracellular mechanisms responsible for the anti-leukemic effect of Ibrutinib/Nutlin-3 combination",
"section_num": null
},
{
"section_content": "To preliminarly validate the in vitro results in an in vivo model, we adopted a JVM-2 xenograft subcutaneous model generated in SCID mice. When tumors reached 50 mm 3, JVM-2 xenograft mice were treated s. c. with control vehicle, Ibrutinib, Nutlin-3 or Ibrutinib/Nutlin-3 combination. Only the combination treatment was associated to significant (p<0. 05) increase in survival as compared to control xenografts (Figure 5A ). Moreover, immunohistochemistry analysis for phospho-H2A. X performed on the tumoral mass of sacrificed mice showed low background in mice inoculated with either vehicle or Nutlin-3, and a strong phosphorylation of H2A. X protein in the nucleus of cells forming the tumoral tissue of mice treated with Ibrutinib (used either alone and in combination with Nutlin-3), with higher signals localized in proximity of areas of necrosis (Figure 5B ).",
"section_name": "The Ibrutinib/Nutlin-3 combination promotes survival and is associated to induction of phospho-H2A.X in mouse tumor tissues",
"section_num": null
},
{
"section_content": "In B-CLL, the FCR regimen (fludarabine, cyclophosphamide, rituximab) continues to represent the 'standard of care', and an existing weight of evidence demonstrates a survival advantage for FCR over historical approaches, at least for younger patients (<65 years) without TP53 aberrations [30]. Nevertheless, Ibrutinib represents currently a substantial therapeutic advance in B-CLL [7]. Several ongoing clinical trials are evaluating Ibrutinib broadly as first-line treatment, alone or in combination with anti-CD20 monoclonal antibodies, as compared with chemo-immunotherapy regimens (FCR, bendamustine-rituximab, obinutuzumab-chlorambucil) [31, 32]. However, since resistances to Ibrutinib therapy are emerging [10] [11] [12] [13], innovative combinations of Ibrutinib with small molecules that block adaptive signaling responses are starting to be investigated in the preclinical setting [33, 34]. \n\nIn this context, we have explored the effects of the combination of Ibrutinib with the small molecule Nutlin-3, based on the rationale that in clinical approaches Ibrutinib would \"mobilize\" B-CLL cells from their protective microenvironment [35], and together with Nutlin-3 would target them in the circulation where they are more susceptible to apoptotic stimuli. Our in vitro studies on leukemic cell models and primary cells from B-CLL patients have confirmed this hypothesis, showing a synergistic cytotoxic effect of Ibrutinib/ Nutlin-3 combination in both p53 wild-type and p53 mutated cells. In addition, a synergistic mechanism of action, thought apoptosis induction, was also documented on B-CLL cells co-cultured with cells mimicking the tumor microenvironment [36], further strengthening the potential therapeutic significance of our current data. \n\nWith respect to the molecular mechanism underlying the Ibrutinib/Nutlin-3 combination, it has been clearly established that an activated B-cell receptor signaling pathway and a disturbed DNA damage response (DDR) play a major role in promoting B-CLL cell survival [22]. External stimuli that lead to activation of the MAPK and PI3K/AKT pathways are similarly essential for B-CLL cell survival [23]. Therefore, while confirming that Ibrutinib (either alone or in combination with Nutlin-3) marked counteracts the MAPK and PI3K/AKT pathways in B-CLL, we have provided evidence that the synergistic anti-leukemic activity of the Ibrutinib/Nutlin-3 combination have a convergence point in regulating cell survival/death through the activation of the DDR signaling. This observation was documented in vitro, in both cell lines as well as in patient cells cultures, and was preliminarily confirmed in mice xenograft, where the Ibrutinib/Nutlin-3 combination induced a survival advantage over the single treatments and activation of the H2A. X histone protein in the tumoral tissues. Certainly, additional experiments, performed on NSG xenotransplant B-CLL mice generated using patient cells [37], will be needed to further clarify this point in in vivo models. Interestingly, activation of H2A. X has recently been involved also in mediating the anti-tumoral activity of 5-fluorouracil-based combinations in a model of colon cancer [38]. \n\nOverall, our data suggest that the Ibrutinib/MDM-2 inhibitor combination merits further investigation for its therapeutic potential. The first non-genotoxic specific small-molecule antagonist of the MDM-2-p53 binding interaction, Nutlin-3, has been used extensively as a probe compound in preclinical and mechanistic studies, but it did not enter into clinical use due to its poor in vivo bioavailability [24]. Anyhow, the second generation MDM-2 inhibitors with superior potency and oral bioavailability, such as RG7112 [25], will enter into clinics. In particular, RG7112 showed promising therapeutic activity in phase I clinical trial in hematological malignancies, including B-CLL [25]. Considering these evidences and the fact that not only the p53 wild-type but also some p53 mutated patients of the clinical trial responded to RG7112 [25, 39], it is noteworthy that also RG7112 synergizes with Ibrutinib in promoting cytotoxicity of B leukemic cells. This preliminary data is indeed encouraging for the advance of the drug combination towards the clinic.",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "The B leukemic cell lines EHEB, JVM-2 and JVM-3 (p53 wild-type ) as well as MEC-1 and MEC-2 (p53 mutated ) were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). EHEB, JVM-2 and JVM-3 were routinely cultured in RPMI-1640, while MEC-1 and MEC-2 were maintained in IMDM, both media supplemented with 10% FBS, L-glutamine and penicillin/streptomycin (all from Gibco, Grand Island, NY) [40]. \n\nFor experiments with primary cells, peripheral blood samples were collected in heparin-coated tubes from B-CLL patients, and from healthy blood donors used as controls, following informed consent, in accordance with the Declaration of Helsinki and in agreement with institutional guidelines (University-Hospital of Ferrara). The diagnosis of B-CLL was made by peripheral blood morphology and immunophenotyping. The main clinical parameters of the B-CLL patients were abstracted from clinical records and all patients had been without prior therapy at least for three weeks before blood collection. Peripheral blood mononuclear cells (PBMC) were isolated by gradient centrifugation with lymphocyte cell separation medium (Cedarlane Laboratories, Hornby, ON). T lymphocytes, NK lymphocytes, granulocytes and monocytes were negatively depleted from B-CLL PBMC with immunomagnetic microbeads (MACS microbeads, Miltenyi Biotech, Auburn, CA), with a purity >95% of resulting CD19+ population. Freshly isolated and thawed primary cells were cultured in RPMI-1640 medium containing 10% FBS, L-glutamine and penicillin/ streptomycin (Gibco), as previously described [41].",
"section_name": "B leukemic cell lines and primary B-CLL patient samples",
"section_num": null
},
{
"section_content": "For in vitro treatments with Ibrutinib (PCI-32765) (Selleckchem, Houston, TX), used either alone or in combination with Nutlin-3 (Cayman Chemicals, Ann Arbor, MI), cells were seeded at a density of 1x10 6 cells/ mL. In selected experiments, Ibrutinib was assessed also in combination with the RG7112 MDM2 inhibitor (Selleckchem). At different time points after treatment, cell viability was examined by Trypan blue dye exclusion and MTT (3-(4, 5-dimethilthiazol-2yl)-2, 5-diphenyl tetrazolium bromide) colorimetric assay (Roche Diagnostics Corporation, Indianapolis, IN) for data confirmation, as previously described [41, 42]. In order to investigate the concentration required to induce death in 50% of cells respect to control, IC 50 values were calculated from dose-response curves constructed by plotting cell survival (%) versus drug concentration. The cell cycle profile was analyzed by 5-bromodeoxyuridine (BrdU) incorporation assessed by flow cytometry, as previously described [43]. Levels of apoptosis were quantified by Annexin V-FITC/propidium iodide (PI) staining (Immunotech). To avoid non-specific fluorescence from dead cells, live cells were gated tightly using forward and side scatter, as described [44].",
"section_name": "Culture treatments and assessment of cell viability, cell cycle profile and apoptosis",
"section_num": null
},
{
"section_content": "For western blotting analysis, cells were lysed as previously described [45]. Protein determination was performed by BCA Protein Assay (Thermo Scientific, Rockford, IL). Equal amounts of protein for each sample were migrated in SDS-polyacrylamide gels and blotted onto nitrocellulose filters. The following Abs were used: anti-Btk (C82B8), anti-phospho-Btk (Tyr223), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-histone H2A. X and anti-phospho-histone H2A. X (Ser139) all from Cell Signaling (Danvers, MA); anti AKT/PKBα from Becton-Dickinson; anti-phospho-Akt1/PKBα (Ser473) from Merck Millipore (Darmstadt, Germany); anti-p44/42 MAPK (ERK1/2) and anti-phospho-Thr202/Tyr204 ERK1/2 from Promega (Madison, USA). After incubation with anti-mouse or anti-rabbit IgG horseradish peroxidaseconjugated secondary antibodies (Sigma-Aldrich), specific reactions were revealed with the ECL Lightning detection kit (Perkin Elmer, Waltham, MA) [46]. Images acquisition was performed using the ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare, Buckinghamshire, UK) and densitometry values were estimated by the ImageQuant TL software (GE Healthcare). \n\nIn selected experiments, cell lysates were analyzed for the detection of phosphoproteins and relative total target proteins by using fluorescently died magnetic bead-based immunoassays (Bio-Plex Pro Phosphoprotein magnetic 8-plex and Total Target magnetic 7-plex, BioRad Laboratories, Hercules, CA), accordingly to the manufacturer's instructions. Data were acquired using a MAGPIX® system (Luminex, Austin, TX), analyzed with the xPONENT® software (Luminex) and reported as Median Fluorescence Intensity (MFI).",
"section_name": "Protein analyses",
"section_num": null
},
{
"section_content": "Total RNA was extracted from cells using the QIAGEN RNeasy Plus mini kit (QIAGEN, Hilden, Germany), accordingly to the supplier's instructions. Total RNA was transcribed into cDNA and amplified using the Express One-Step Superscript qRT-PCR Kit (Invitrogen, Carlsbad, CA). Analysis of human CDKN1A, MDM2 and BAX gene expression was carried out with validated TaqMan Gene Expression Assays specific PCR primers sets (Invitrogen). All samples were run in triplicate using the real time thermal analyzer Rotor-Gene™ 6000 (Corbett, Cambridge, UK), as previously described [47]. Expression values were normalized to the housekeeping gene POLR2A amplified in the same sample.",
"section_name": "RNA analyses",
"section_num": null
},
{
"section_content": "Female cb17/SCID mice (5 weeks-old) were purchased from Charles River Laboratories (Hollister, CA) and maintained in accordance with the guide for the care and use of laboratory animals at the animal facility of the University of Ferrara. Mice were housed in vented cabinet with food and water ad libitum. The procedures involving animals and their care were approved by the institutional animal ethical care committee of the University of Ferrara (OBA) and by the Italian Ministry of Health. JVM-2 (10 7 ) B leukemic cells were harvested, washed and suspended in PBS before subcutaneous injection (in a volume of 100 μL) into the right dorsum of 6-week-old mice, as previously described [48]. Tumor growth was determined by caliper measurements of two orthogonal axes and the tumor volume was calculated by the formula: L×l 2 ×0. 5, wherein l is the shorter and L is the longer axis; the tumor density was assumed to be equal to one. When tumors reached 50 mm 3 of volume, leukemia xenograft mice were randomized into groups (of at least 8 mice each) receiving every other day for a total of five times subcutaneous intra-tumoral injections (in 100 μL PBS/30% DMSO) of Ibrutinib (2. 2 mg/Kg), Nutlin-3 (2. 9 mg/Kg) or Ibrutinib/Nutlin-3 combination (2. 2 and 2. 9 mg/Kg, respectively). Control group was represented by mice injected with vehicle (PBS/30% DMSO). Animals were monitored daily for changes in weight, side effects or signs of sickness. Survival was calculated as the duration of the animal life span from the inoculation of first treatment until sacrifice when excessive signs of sickness were observed. For histological analysis, the subcutaneous masses were fixed in 10% buffered-formalin solution and embedded in paraffin. Five-μm-thick sections were cut from paraffin blocks and stained with hematoxylin-eosin and/or used for immunohistochemistry with the Ab antiphospho-histone H2A. X (Ser139) (Cell Signaling) and the anti-rabbit HRP-DAB tissue staining kit (R&D System, Minneapolis, MN). In each slide, a negative control was obtained carrying out the immunohistochemistry procedure without the primary antibody. Sections were acquired with an Aperio ScanScope® slide scanner by using the Aperio ImageScope v11. 1. 2. 760 software (Leica Biosystems, Nussloch, Germany).",
"section_name": "Experiments in mouse models",
"section_num": null
},
{
"section_content": "Results were evaluated by using analysis of variance with subsequent comparisons by Student's t-test and with the Mann-Whitney rank-sum test. Statistical significance was defined as p<0. 05. In order to investigate the effect of drug combinations, leukemic cells were treated with serial doses (range 1-10 μM) of each drug, used individually or in combination, using a constant 1:1 ratio. Results were analyzed with the method of Chou and Talalay [22] to determine whether the combined treatment greater effect than expected from summation: a combination index (CI) of 1 indicates additive effect, while a CI below 1 indicates synergism. For this purpose, cell viability datawere analyzed with the CalcuSyn software and reported as CI values. \n\nFor the experiments in mice, analysis of survival data was carried out with GraphPad Prism version 5 (GraphPad Software). In particular differences in survival between treatment groups were calculated using the Kaplan-Meier curve and survival distribution of the treated and control groups was compared using the Gehan-Breslow-Wilcoxon test. Differences were considered significant when p value was <0. 05.",
"section_name": "Statistical analysis",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "Authors would like to thank Matteo Carantoni for his excellent technical work.",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "",
"section_name": "GRANT SUPPORT",
"section_num": null
},
{
"section_content": "This study was supported by grants from: Italian Association for Cancer Research (AIRC IG 11465 to G. Z. ) and from MIUR-FIRB (RBAP11Z4Z9_002 to G. Z. ).",
"section_name": "GRANT SUPPORT",
"section_num": null
},
{
"section_content": "",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
},
{
"section_content": "All authors declare no conflicts of interest.",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
}
] |
10.1158/0008-5472.22395708
|
Supplementary Methods from miRNA-130a Targets <i>ATG2B</i> and <i>DICER1</i> to Inhibit Autophagy and Trigger Killing of Chronic Lymphocytic Leukemia Cells
|
<jats:p><p>PDF file - 69K</p></jats:p>
|
[
{
"section_content": "(TO-PRO-3) and 520 nm (GFP) for each sample. The acquired images were analyzed with IDEAS software (Amnis). First, a scatter plot of aspect ratio/area was used to gate for single cells. Second, a gradient RMS (root mean square for image sharpness) histogram was used to gate for cells in focus. Third, the intensity of TO-PRO-3 staining was used to exclude dead cells. On the remaining living, single cells in focus, image analysis was performed to determine the formation of autophagosomes by the extent of GFP-LC3 clustering. To do that, a spot mask was generated to automatically recognize the cellular regions containing GFP clusters. The level of fluorescence clustering was extracted from the individual cellular images and was represented by the bright detail intensity R3 feature. This feature computes the intensity of localized bright spots within the masked area in the image. The bright detail intensity was quantified at the cell population level for each sample and the data was analyzed using MATLAB (Mathworks, Inc., Massachusetts, USA). First, a lillietest for normality distribution was performed on the cellular bright detail intensities, showing that the data are non-normally distributed. Therefore, the data were analyzed using a non-parametric description derived from the Kolmogorov-Smirnov (K-S) statistics. The bright detail intensity distributions were shown as cumulative distribution functions. We defined that the autophagic flux can be described at the population level by the difference between the cumulative distribution functions between bafilomycin-treated cells and untreated cells. The difference between 2 cumulative distributions was calculated according to K-S statistics, by subtracting the values for each feature channel (histogram bin value). The resulting autophagic flux distributions were normalized to the maximum channel of the sample with the highest autophagic flux (set as 100%). Normalized autophagic flux distributions of 4 independent experiments were averaged and the data shown as mean ± SEM for each channel. In addition, a Student's t-test was performed for each channel in order to determine the statistical significance of the population frequency changes in function of the autophagic flux.",
"section_name": "",
"section_num": ""
}
] |
[] |
10.3390/ijms23116260
|
Venetoclax Induces Cardiotoxicity through Modulation of Oxidative-Stress-Mediated Cardiac Inflammation and Apoptosis via NF-κB and BCL-2 Pathway
|
<jats:p>Cardiovascular damage induced by anticancer therapy has become the main health problem after tumor elimination. Venetoclax (VTX) is a promising novel agent that has been proven to have a high efficacy in multiple hematological diseases, especially acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). Considering its mechanism of action, the possibility that VTX may cause cardiotoxicity cannot be ruled out. Therefore, this study was designed to investigate the toxic effect of VTX on the heart. Male Sprague-Dawley rats were randomly divided into three groups: control, low-dose VTX (50 mg/kg via oral gavage), and high-dose VTX (100 mg/kg via oral gavage). After 21 days, blood and tissue samples were collected for histopathological, biochemical, gene, and protein analyses. We demonstrated that VTX treatment resulted in cardiac damages as evidenced by major changes in histopathology and markedly elevated cardiac enzymes and hypertrophic genes markers. Moreover, we observed a drastic increase in oxidative stress, as well as inflammatory and apoptotic markers, with a remarkable decline in the levels of Bcl-2. To the best of our knowledge, this study is the first to report the cardiotoxic effect of VTX. Further experiments and future studies are strongly needed to comprehensively understand the cardiotoxic effect of VTX.</jats:p>
|
[
{
"section_content": "Drug-induced toxicity effects on cardiovascular function or tissues are not only a serious health issue, but they are often detected after the introduction of the drug in clinical practice [1]. This reflects that these high-risk cardiovascular events are either not detected in earlier clinical trials, or those that arise when drugs are administrated for long periods of time to larger patient population are not considered to be biologically significant [1]. Such a high incidence of cardiovascular adverse drug reactions in late-stage clinical development can lead to additional pre-and/or postapproval monitoring, prescribing restrictions, doselimiting toxicity, or ultimately drug discontinuation or withdrawal [1]. Importantly, these drug-induced cardiovascular toxicity events are considered the primary cause of drug withdrawal from the market [1, 2]. \n\nThe list of anticancer therapy drugs that can potentially cause cardiotoxicity-related adverse effects is growing [3]. This raises an important issue in cancer treatment, as it can influence the mortality and morbidity of patients with cancer by causing a delay or discontinuation of chemotherapy [4]. Over the past two decades, anticancer therapy has resulted in remarkable advances in both the survival rate and quality of life of cancer patients [5]. Although anticancer agents have shown efficacy against different types of tumors, many reports have demonstrated its cardiotoxic effect [5, 6]. Cardiotoxicity represents the most feared adverse reaction to chemotherapy, with a growing incidence of up to 30% of patients receiving chemotherapy developing a cardiovascular side effect during their life, which leads to an increase in morbidity and mortality [7] [8] [9]. It is worthy to note that the cardiotoxic side effects induced by long-term use of anticancer therapy has been one of greatest challenges after tumor elimination [10]. Therefore, it is essential for oncologists to know the cardiotoxicity profile of newer agents and determine the etiology and most appropriate management of these various effects so they can consider the risks and benefits of eliminating the tumor and preservation of cardiac function [3, 11]. \n\nVenetoclax (VTX), or ABT-199, is a promising novel agent that has been proven to have a high efficacy in multiple hematological diseases, especially chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) [12]. Of all newly diagnosed cancer patients, 6. 5% have blood cancers such as acute leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, and multiple myeloma [12]. Apoptosis resistance of CLL cells is mediated through Bcl-2 overexpression [13]. When Bcl-2 is overexpressed, this leads to tumor formation, as in CLL and follicular lymphoma, inappropriate cell survival, and chemotherapy resistance [14, 15]. Apoptosis can be initiated by BH3-only proteins in response to significant stresses, such as genetic damage [14]. Thus, higher levels of Bcl-2 overcome the BH3-only proteins and result in evading apoptosis [16]. VTX targets the BH3 domain of Bcl-2 as a BH3 mimetic (Bcl-2 inhibitor) that can restore apoptosis in malignant cells [17]. Indeed, VTX have a high affinity to the BH3-binding groove of Bcl-2, which leads to displacement of the bounded proapoptotic BH3-only proteins [18]. Therefore, these free BH3-only proteins lead to displacement and activation of apoptotic effectors (e. g., Bax) from binding to antiapoptotic members [18]. Eventually, VTX induces the release of proapoptotic from Bcl-2 and restores apoptosis in tumor cells [18]. In the clinical sitting, it has been reported that VTX caused cardiomyopathy and cardiac arrhythmia [19] [20] [21]. Therefore, we hypothesized that VTX treatment can cause toxic effects to the heart. It is well known that the Bcl-2 family of proteins are essential regulators of apoptosis [22]. Moreover, it has been reported that inhibition of Bcl-2 can lead to apoptosis in different organs, especially the heart, and consequently resulting in organ toxicity [21, 23]. Therefore, in the current study, we investigated the toxic effects of VTX on the heart, and examined the signal and molecular mechanism of its toxicity.",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "2."
},
{
"section_content": "It has been demonstrated that cardiac enzymes increase in blood when there are some injuries or damage to the heart. Therefore, we measured the serum level of CK-MB and cardiac troponin I (cTn-I) in rats after 21 days of VTX treatment. Surprisingly, we found that the treatment of VTX increased the serum levels of both CK-MB (Figure 1A ) and cTn-I (Figure 1B ), but the increase in cTn-I was not significant. These results indicated that VTX treatment induced cardiac damage.",
"section_name": "Effect of VTX Treatment on Cardiac Enzymes",
"section_num": "2.1."
},
{
"section_content": "Numerous studies have shown that cardiac hypertrophy can be a maladaptive process in response to intrinsic or extrinsic stresses. Therefore, we measured the body weight (BW), heart weight (HW) and heart weight/body weight ratio (HW/BW), which we used as an indicator of cardiac hypertrophy, and measured the expression of three genes that are known to be involved in this process. As shown in Figure 2A,B, no significant changes were observed in the BW or HW between all groups. However, a significant increase in the HW/BW ratio was observed in VTX-treated groups compared to the control group (Figure 2D ). Furthermore, we found that the gene-expression level of α-Mhc decreased in VTX-treated groups compared to the control group, while the gene-expression levels of β-Mhc and the β-Mhc to α-Mhc ratio increased significantly in the VTX-treated groups compared to the control group (Figure 2A-C, respectively). Furthermore, the gene-expression level of Bnp was substantially increased in the VTX-treated groups (Figure 2D ). Overall, these results indicated that VTX treatment induced cardiac hypertrophy and damage. \n\nG) Bnp were measured using RT-PCR. Data are presented as mean ± SD (n = 5). * p < 0. 05, ** p < 0. 01; n. s., no significant changes were observed (p > 0. 05). Data were normalized to β-actin as a housekeeping gene and one-way analysis of variance (ANOVA), followed by Tukey-Kramer multiple-comparisons tests. Cont, control; LD, low-dose venetoclax (50 mg/kg); HD, high-dose venetoclax (100 mg/kg); α-Mhc, alpha myosin heavy chain; α-Mhc, beta myosin heavy chain; Bnp, brain natriuretic peptide.",
"section_name": "VTX-Induced Cardiac Hypertrophy",
"section_num": "2.2."
},
{
"section_content": "It has been demonstrated that changes in and damage to cardiac cell morphology correlate with many diseases and toxicity. Therefore, we examined the heart architecture of rats after 21 days of VTX treatment. We observed a normal and parallel myocardial fiber with cross-striation and regular nuclei in the myocardium sections obtained from the control group (Figure 3A, D ). Sections of myocardium obtained from the second group, which received a low dose of VTX, also showed a normal appearance of the myocardial fibers, but with minimal nuclear enlargement (Figure 3B, E ). Interestingly, heart sections from the third group, which received a high dose of VTX, showed the presence of a focus of myocardial damage associated with chronic inflammatory reaction (arrowhead) (Figure 3C, F ). Figure 3F shows the arrowhead area at 600× magnification, and indicates the presence of a focus of subendocardial myxoid degeneration. This feature is not well understood, but could indicate myocardial damage in this area. Furthermore, we observed that VTX treatment increased the cardiomyocytes' cross-sectional area considerably in a dose-dependent pattern (Figure 3H ). Taken together, VTX treatment induced morphological changes in cardiac tissues.",
"section_name": "Effects of VTX on Cardiac Architecture",
"section_num": "2.3."
},
{
"section_content": "Several studies have linked myocardial dysfunction and toxicity with apoptosis. Therefore, we measured the gene and protein expressions of multiple apoptotic markers to examine the induction of apoptosis in heart tissues. We found that 21 days of VTX treatment induced the gene expression of Bax compared to the control group (Figure 4A ). Moreover, a significant decline was observed in the Bcl-2 gene and protein expressions in VTX-treated rats compared to the control group (Figure 4B,C, respectively). Nonetheless, a Western blot analysis revealed that treatment with a high dose of VTX resulted in a notable rise in the protein levels of cleaved caspase-3 (Cleaved Cas-3) compared to the control group (Figure 4D ).",
"section_name": "VTX-Induced Apoptosis in the Heart",
"section_num": "2.4."
},
{
"section_content": "Cas-3 protein levels. Data are presented as mean ± SD (n = 5). * p < 0. 05, ** p < 0. 01; n. s., no significant changes were observed (p > 0. 05). Cont, control; LD, low-dose venetoclax (50 mg/kg); HD, high-dose venetoclax (100 mg/kg); Bax, Bcl-2 associated X; Bcl-2, B-cell lymphoma 2; Cleaved Cas-3, cystinyl aspartate-specific proteases 3; β-actin, beta actin.",
"section_name": "(D) Representative Western blot analysis of Cleaved",
"section_num": null
},
{
"section_content": "It has been demonstrated that oxidative stress and inflammation are the main mechanisms that induce cardiac toxicity. Therefore, we measured the gene and protein expressions of different inflammatory and oxidative stress markers. We found that the gene-expression levels of Ifn-γ (Figure 5A ) and Tgf-β (Figure 5B ) sharply rose in the VTX treatment group compared to the control group. Furthermore, the gene and protein levels of Nf-κb-p-65 in VTX-treated rats were significantly increased in the high-dose VTX group compared to the control group (Figure 5C,F, respectively). Moreover, the levels of gene and protein expressions of Tnf-α (Figure 5D,G, respectively) and Il-6 (Figure 5E,H, respectively) were remarkably increased in the treatment groups compared to the control group in a dosedependent manner. Strikingly, the level of the antioxidant protein, Sod-2, significantly declined in rats treated with a high dose of VTX compared to the control group (Figure 5I ). Taken together, these results provided important insight regarding the involvement of inflammation and oxidative stress in cardiac toxicity in VTX treatment. (A-E ) The mRNA levels of Ifn-γ, Tgf-β, Nf-κb-p-65, Tnf-α, and Il-6 were measured using RT-PCR. Data were normalized to β-actin as a housekeeping gene and one-way analysis of variance (ANOVA), followed by Tukey-Kramer multiple-comparisons tests. (F-I) Representative Western blot analysis of protein levels of Nf-κb-p-65, Tnf-α, Il-6, and Sod-2. Data are presented as mean ± SD (n = 5). * p < 0. 05, ** p < 0. 01, *** p < 0. 001; n. s., no significant changes were observed (p > 0. 05). Cont, control; LD, low-dose venetoclax (50 mg/kg); HD, high-dose venetoclax (100 mg/kg); Ifn-γ, interferon gamma; Tgf-β, transforming growth factor beta; Nf-κb-p-65, nuclear factor kappa-B; Tnf-α, tumor necrosis factor alpha; Il-6, interleukin-6; Sod-2, superoxide dismutase-2; β-actin, beta actin.",
"section_name": "VTX-Induced Oxidative Stress and Inflammation in the Heart",
"section_num": "2.5."
},
{
"section_content": "ROS production is involved in the cardiotoxicity of many anticancer drugs. It can cause oxidative damage to many vital components of the cell, including DNA and proteins, as well as mitochondrial dysfunction and cell death. It had been found that increased levels of lipid peroxidation led to an increase in cardiotoxicity. Moreover, decreased levels of antioxidants in the heart have been associated with the production of ROS and cardiotoxicity. Therefore, we measured the levels of malondialdehyde (MDA), catalase (CAT), and glutathione (GSH). We found that the VTX-treated groups had increased levels of MDA compared to the control group (Figure 6A ). Moreover, the levels of CAT (Figure 6B ) and GSH (Figure 6C ) were remarkably diminished in both VTX-treatment groups compared to the control group. In summary, these results confirmed that VTX treatment induced oxidative stresses.",
"section_name": "Effect of VTX on Oxidative Stress Status",
"section_num": "2.6."
},
{
"section_content": "In the present study, we investigated whether VTX treatment could induce toxic effects in the heart. We found that VTX treatment induced cardiotoxicity that was manifested by changes in the histological architecture of cardiomyocytes, an increase in cardiac enzymes and the expression of relevant genes of cardiac injury, induction of apoptosis markers, alterations in oxidative stress markers, and an increase in inflammatory markers. \n\nVTX is a recently approved anticancer drug that is used for the treatment of multiple hematological cancers [12]. VTX acts as a selective inhibitor of the BH3 domain of Bcl-2 that can restore apoptosis in cancer cells [17]. In the clinical sitting, it has been reported that VTX caused cardiomyopathy and cardiac arrhythmia [19] [20] [21]. Therefore, we hypothesized that VTX treatment could cause toxic effects to the heart. It is well known that the Bcl-2 family of proteins are essential regulators of apoptosis [22]. Moreover, it has been reported that inhibition of Bcl-2 can lead to apoptosis in different organs, resulting in organ toxicity [21, 23]. However, to date, there are no preclinical reports that investigated the cardiotoxicity of VTX. Therefore, in the current study, we investigated the toxic effects of VTX on the heart, and examined the signal and molecular mechanisms of its toxicity. \n\nCardiac enzymes, such as myocardial muscle creatine kinase (CK-MB) and cardiac troponin (cTn-I), are key tools for evaluating heart damage, as well as histopathological examination of cardiomyocytes, which have been found to be a major marker of the cardiotoxic effect of anticancer drugs [24, 25]. Furthermore, alterations in the enzymatic reaction, such as CK-MB and cTn, represent a key early response to toxicant exposure in the heart [26]. In the present study, we found that levels of both CK-MB and troponin I had increased significantly in the VTX-treated rats compared to the nontreated controls. These results indicated that VTX treatment was associated with cardiomyocyte damage. Moreover, our histopathological findings in rats that received VTX treatment showed the presence of minimal nuclear enlargement and focus of myocardial damage associated with a chronic inflammatory reaction. Moreover, we showed that VTX treatment substantially increased the size of cardiac myocytes in a dose-dependent manner. These histopathological findings also confirmed that VTX treatment caused cardiomyocytes injuries. \n\nCardiac hypertrophy is thought to be a maladaptive compensatory mechanism of the heart in response to toxic insult [27]. Changes in cardiac hypertrophic markers such as Bnp, α-Mhc, and β-Mhc have been found to be associated with cardiac toxicity, and were reported for several anticancer drugs, such as doxorubicin and sunitinib [28, 29]. In the current study, we found that VTX treatment increased the gene expression of β-Mhc, Bnp, and the β-Mhc:α-Mhc ratio compared to the control group, while α-Mhc gene expression was decreased. These results suggested a VTX-mediated hypertrophy and toxicity on the heart. \n\nCardiomyocyte death is considered the main cause of cardiotoxicity [30]. The most common form of cell death in drug-induced cardiotoxicity is apoptosis [30, 31]. Many cytotoxic insults, such as DNA damage and oxidative stress, can activate the intrinsic apoptotic pathway, which is controlled by the Bcl-2 family of proteins [32, 33]. This family can be divided into proapoptotic and antiapoptotic members. The Bcl-2 protein is present in the outer mitochondrial membrane, and acts as an antiapoptotic protein by preventing the release of cytochrome c into the cytosol. On the other hand, intrinsic cell apoptosis can also be regulated by Bax, a proapoptotic member of the Bcl-2 family that can cause cytochrome c release and activate multiple caspases, eventually leading to cell death. Thus, the balance between Bcl-2 and Bax can influence the cell survival or death [23, 30, 34]. In this study, we found that 21 days of treatment with VTX led to cardiomyocyte death, as evidenced by reduced levels of the gene and protein expressions of Bcl-2, increased levels of Bax gene expression, and elevated cleaved caspase-3 (Cleaved Cas-3) protein expression levels. \n\nIt is well known that cardiomyocyte death induced by ROS production is involved in many cardiac pathological conditions, such as cardiac hypertrophy and HF [35, 36]. Moreover, overproduction of oxygen free radicals and induction of oxidative stress can lead to chemokine production, recruitment of inflammatory cells, and activation of transcription factors such as Nf-κb [36]. Inflammation as a result of oxidative stress is associated with a plethora of pathological diseases, including diabetic cardiomyopathy, congestive cardiomyopathy, and hypertensive heart disease [37]. Increased levels of cytokines in the blood or cardiomyocyte have been found in diseases that lead to cardiomyocyte death. Nf-κb is a transcription factor that upregulates the production of downstream inflammatory mediators, including Tnf-α, Il-6, Ifn-γ, and Tgf-β [38]. Moreover, Ifn-γ and Il-1 are both proinflammatory cytokines that can induce Tnf-α production by cardiomyocytes [38]. Nevertheless, Il-6, which is a proinflammatory cytokine, was found to be elevated in patients with heart failure [26]. Tnf-α is one of the inflammatory mediators that has an important role in the induction of myocardial cell apoptosis [26]. Furthermore, Tgf-β plays an important role in apoptosis, wound healing, and immune regulation [39]. It has been reported that Tgf-β overexpression was associated with fibrosis and hypertrophy [40]. Our findings revealed that 21 days of treatment with VTX resulted in ROS production and inflammation that led to apoptosis of cardiomyocytes, which was confirmed by the increase in the gene and protein expression of Nf-κb-p-65 and the decrease in the Sod-2 protein level. Additionally, the induction of Ifn-γ gene levels, as well as Tnf-α and Il-6 gene-and protein-expression levels, further confirmed the toxic consequences of VTX in the heart. Lastly, increased Tgf-β gene-expression levels confirmed our histopathological findings, in that VTX treatment induced maladaptive cardiac hypertrophy and eventually cardiac damage. \n\nTo further confirm the oxidative stress production, we measured the levels of malondialdehyde (MDA), catalase (CAT), and glutathione (GSH) activity. MDA is a lipid peroxidation end-product and a gold standard marker of lipid peroxidation and oxidative stress. Moreover, GSH and CAT are key endogenous antioxidants that are critical to maintaining cellular homeostasis and ROS levels in response to different toxic insults [41, 42]. In this study, we observed a significant dose-dependent reduction in GSH and CAT in response to VTX treatment. Furthermore, our results also demonstrated a significant dosedependent increase in MDA levels in the VTX-treated groups compared to the control group. These results confirmed our previous findings that VTX treatment induced oxidative stress in the heart.",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "4."
},
{
"section_content": "Male Sprague-Dawley rats weighing 180-200 g were used. The rats were obtained from Prince Naif Bin AbdulAziz Health Research Center, King Saud University, Riyadh, Saudi Arabia. All experiments on rats were conducted according to the standard guidelines and approved by the Animal Care and Use Committee at King Saud University, Saudi Arabia (Approval# KSU-SE-19-86). Animals were housed under normal laboratory conditions (temperature of 25 ± 1 • C) of a 12 h light/dark cycle with free access to water and a normal chow diet.",
"section_name": "Animals",
"section_num": "4.1."
},
{
"section_content": "Rats were arbitrarily divided into three groups, with 8 rats in each group. These groups were as follows:\n\nGroup 1: Rats in this group were treated daily via oral gavage with 0. 9% NaCl for 21 days, and it served as the control group. \n\nGroup 2: Rats in this group were treated daily with a low dose of VTX (50 mg/kg) via oral gavage for 21 days (LDV). \n\nGroup 3: Rats in this group were treated daily with a high dose of VTX (100 mg/kg) via oral gavage for 21 days (HDV). \n\nVTX doses were selected based on previous published studies [43] [44] [45] [46] [47]. For all groups, rats were weighed daily to calculate the dose and monitored for any changes in weight, as well as for any signs of toxicity. On day 21, rats were anesthetized using ketamine 100 mg/kg and xylazine 10 mg/kg intraperitoneally [48]. Blood samples were collected from all rats. Then, the serum was separated for further measurement of cardiac enzymes. Consequently, heart tissues were harvested and washed twice with ice-cold phosphatebuffered saline; some tissues were fixed in formaldehyde solution (4%) for histopathology studies, while other tissues were stored at -80 • C to conduct different biochemical, gene, and protein studies. The ratio of heart weight to body weight (HW/BW) was used as an indicator of myocardial mass, as described previously [49, 50].",
"section_name": "Study Design",
"section_num": "4.2."
},
{
"section_content": "Coagulated blood was centrifuged at 2000× g for 10 min at 4 • C to separate the serum from the whole blood. Serum creatine kinase MB isoenzyme (CK-MB) and cardiac troponin I (cTn-I) were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) (ABBEXA, Cambridge, UK) as per the manufacturer's protocol.",
"section_name": "Measuring the Cardiac Enzymes",
"section_num": "4.3."
},
{
"section_content": "Total cellular RNA was isolated from the heart tissues by using TRIzol reagent (Ambion, Austin, TX, USA) following the manufacturer's protocol. The isolated RNA was assessed by using a NanoDrop™ 8000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) to verify the quality and quantity. Then, the isolated RNA (1 µg) was reverse transcribed to cDNA by using a reverse transcription kit (BIMAKE, Houston, TX, USA). Gene expressions were quantified by using the appropriate primers listed in Table 1, a 7500 Fast Real-Time PCR system (ThermoFisher Scientific, Waltham, MA, USA), and SYBR green master mix (BIMAKE, Houston, TX, USA). Data were normalized to β-actin as a housekeeping gene. The forward and reverse primers used are listed in Table 1. The data were acquired during the extension step. All primers were obtained from Integrated DNA Technologies (IDT, Leuven, Belgium). The DDCt method was used to calculate the relative levels of mRNA expression [51].",
"section_name": "Gene Expression Studies",
"section_num": "4.4."
},
{
"section_content": "A Western blot was used to determine the protein levels of Nf-κb-p-65, Sod-2, Cleaved Cas-3, Bcl-2, Tnf-α, and Il-6. In brief, previously collected heart tissues were homogenized using Dounce homogenizer in ice-cold RIPA lysis buffer (ThermoFisher Scientific, Waltham, MA, USA) that was supplemented with protease and phosphatase cocktail inhibitor (Ther-moFisher). After that, an equal amount of proteins (25-50 µg) were electrophoresed using SDS-PAGE, then transferred onto a PVDF membrane. Following the transfer, membranes were blocked for one hour at room temperature with 5% nonfat dry milk with gentle rocking. Membranes were incubated overnight in the primary antibody at 4 • C with gentle rocking (dilution at 1:1000). These primary antibodies were rabbit anti-nuclear factor kappa B-p-65 (Nf-κb-p-65) antibody, rabbit anti-superoxide dismutase-2 (Sod-2) antibody, rabbit anti-cleaved caspase-3 (Cleaved Cas-3) antibody, rabbit anti-B-cell lymphoma-2 (Bcl-2) antibody, rabbit anti-tumor necrosis factor (Tnf-α) antibody, rabbit anti-interleukin 6 (Il-6) antibody, and mouse anti-B-actin antibody. Thereafter, membranes were incubated for one hour at room temperature with the appropriate horseradish peroxidase (HRB) conjugated secondary antibody (dilution at 1:5000) (ABclonal, Wuhan China). Finally, membranes were visualized using chemiluminescence reagent (Millipore, Burlington, MA, USA) and imaged using a Bio-Rad gel-imaging system (Bio-Rad, Hercules, CA, USA).",
"section_name": "Protein-Expression Studies",
"section_num": "4.5."
},
{
"section_content": "Lipid peroxidation was measured in cardiac tissues by adding thiobarbituric acid (TBA) and trichloroacetic acid (TCA) to tissue homogenates. Then, this mixture was incubated for 30 min in a shaking water bath at 90 • C. Then, the samples were placed on ice for 10 min. Thereafter, the samples were centrifuged for 15 min at 3000× g in a refrigerated centrifuge, and the supernatant was measured at 540 nm. The values of results were expressed in nmol of MDA formed per mg of protein [52].",
"section_name": "Measurement of Lipid Peroxidation",
"section_num": "4.6."
},
{
"section_content": "The amount of GSH in tissues was measured using a previously described method [53]. In brief, 5,50-dithio bis (3-nitrobenzoic acid) was added to the reaction mixture. Then, the absorbance was immediately recorded at 412 nm. The values of GSH were expressed as nmol/mg of protein.",
"section_name": "Measurement of Reduced Glutathione",
"section_num": "4.7."
},
{
"section_content": "The postmitochondrial supernatant (PMS) from heart tissue was used to estimate the CAT activity by using a previously described method [54]. In brief, the reaction mixture, in a total volume of 3 mL, consisted of 1. 95 mL (0. 1 M, pH 7. 4) phosphate buffer, 1 mL (0. 019 M) hydrogen peroxide, and 0. 05 mL PMS. The absorbance was recorded for 5 min at 240 nm at an interval of 1 min. To calculate the activity of CAT, the difference in the absorbance was used as the amount of moles of H2O2 changed per min per mg of protein.",
"section_name": "Measurement of Catalase Activity",
"section_num": "4.8."
},
{
"section_content": "Heart tissues from all groups were collected, fixed in 4% formaldehyde, and embedded in paraffin. Then, thin 3 mm sections were prepared using a microtome and stained with hematoxylin and eosin (H&E) to examine the heart morphology. The morphology of the cardiac cells and the nucleus of myocardial fiber cells were compared using an optical microscope to evaluate the severity of cardiac damage (Olympus BX microscope and DP72 camera, Melville, FL, USA). The damage quantification from at least 10 areas corresponding to the myocardial tissue was graded using the following parameters: nuclear enlargement and inflammation based on a four-score evaluation system (0, histopathological changes = 1-25%; 1, histopathological changes = 26-50%; 2, histopathological changes = 51-75%; and 3, histopathological changes =76-100%). This procedure was conducted in at least 10 random areas in each heart section, in three animals from each group, at 400× magnification. The mean score for each parameter was calculated and subjected to statistical analysis. The cardiomyocyte cross-sectional size was estimated and evaluated using the H&E stained slides.",
"section_name": "Histopathology",
"section_num": "4.9."
},
{
"section_content": "All data are presented as mean ± SD and analyzed using GraphPad Prism version 6. 01 (San Diego, CA, USA). Different results between groups were analyzed using a oneway or two-way analysis of variance (ANOVA), followed by a Tukey-Kramer multiplecomparisons test with significance values of p < 0. 05.",
"section_name": "Statistical Analysis",
"section_num": "4.10."
},
{
"section_content": "To the best of our knowledge, this was the first study to report that VTX treatment could induce cardiotoxicity in a dose-dependent manner. This cardiotoxicity caused by VTX treatment was manifested in different ways, including modification or changes in the histological architecture of cardiomyocytes, increases in cardiac enzymes such as CK-MB and troponin I, and alteration of cardiac hypertrophic genes markers such as Bnp, α-Mhc, and β-Mhc (Figure 7 ). Our findings revealed that VTX treatment induced apoptosis in cardiac tissues as a result of Bcl-2 reduction and Cleaved Cas-3 and Bax induction. Moreover, increased levels of MDA in cardiac tissues and reduced levels of GSH, CAT, and Sod-2 caused oxidative stress that led to activation of Nf-κb-p-65 and induction of the inflammatory response, which was observed as the increases in the expressions of Ifn-γ Tgf-β, Tnf-α, and Il-6. The results of this study add to the current knowledge regarding the safe use of VTX. One of the limitations of the present study was that we did not use knockout models to inhibit the molecular mechanisms involved in VTX-induced cardiotoxicity. Furthermore, we did not measure any in vivo parameters, such as those from echocardiography or blood pressure measurements, which could help to further assess the effects of VTX on heart function. However, the results of the current study shed light on the toxic effects of VTX treatment on the heart, and encourage future studies to further prove the current findings. Further studies are required to fully and comprehensively understand the exact mechanism of VTX-induced cardiotoxicity.",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "The authors are thankful to the Researchers Supporting Project (number RSP-2021/335 ), King Saud University, Riyadh, Saudi Arabia.",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "",
"section_name": "Institutional Review",
"section_num": null
},
{
"section_content": "Funding: This project was funded by the Researchers Supporting Project (number RSP-2021/335 ), King Saud University, Riyadh, Saudi Arabia.",
"section_name": "",
"section_num": ""
},
{
"section_content": "Board Statement: The animal study protocol was approved by the KSU Local Institutional Study Ethics Committee (REC) (protocol code KSU-SE-19-86, 2019 ). Informed Consent Statement: Not applicable.",
"section_name": "Institutional Review",
"section_num": null
},
{
"section_content": "Data Availability Statement: Data are contained within the article.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest.",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.1186/1472-6750-8-6
|
Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail
|
<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>High purity of tumour samples is a necessity for accurate genetic and expression analysis and is usually achieved by positive selection in chronic lymphocytic leukaemia (CLL).</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>We adapted a bifunctional rosette-based antibody cocktail for negative selection of B-cells for isolating CLL cells from peripheral blood (PB). PB samples from CLL patients were split into aliquots. One aliquot of each sample was enriched by density gradient centrifugation (DGC), while the other aliquot of each sample was incubated with an antibody cocktail for B-cell enrichment prior to DGC (RS+DGC). The purity of CLL cells after DGC averaged 74.1% (range: 15.9 – 97.4%). Using RS+DGC, the purity averaged 93.8% (range: 80.4 – 99.4%) with 23 of 29 (79%) samples showing CLL purities above 90%. RNA extracted from enriched CLL cells was of appropriately high quality for microarray analysis.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>This study confirms the use of a bifunctional rosette-based antibody cocktail as an effective method for the purification of CLL cells from peripheral blood.</jats:p> </jats:sec>
|
[
{
"section_content": "Enrichment of tumour cells to a purity of more than 90% is highly desirable for accurate results in many applications, especially for RT-PCR and microarray based expression analysis [1, 2]. In B-cell chronic lymphocytic leukaemia (CLL), such purities have usually been achieved by density gradient centrifugation (DGC) and subsequent fluorescent-activated cell sorting (FACS) or by magnetic cell sorting (MCS) for CD19 positive cells [1]. \n\nStudies focusing on expression analysis in CLL utilising microarrays report median purities of 88 and 90% of CD19 positive cells using DGC [3, 4] though it is likely that selection occurred for samples with high purity. One study applying DGC and FACS of mononuclear cells reported purities of between 90 and 95% of CD5-CD19 co-expressing cells [5]. Three studies [6] [7] [8] reported purities higher than 97% of CD19 positive cells after DGC and MCS. Although high purity is achieved with FACS and MCS, both are time and cost intensive procedures which often are limited in terms of tumour cell yields and applicability, since they require expensive equipment and the processing time depends on the sample volume. Another potential disadvantage is that they are positive selection approaches which might alter gene expression through the activation of cell surface receptors [1]. \n\nOur study focused on adapting a negative selection method that could offer the required purity after the DGC step thereby markedly cutting down the time and cost of sample processing and reducing the risk of altering the gene expression pattern. \n\nWe used a bifunctional antibody cocktail for B-cell enrichment (RosetteSep™ (RS)) that binds erythrocytes (via glycophorin) on one side and white cell populations other than B-cells (via the CD2, CD3, CD16, CD36, CD56 and/ or CD66b antigens) on the other side thus forming dense rosettes of erythrocytes surrounding the unwanted white blood cells when added to whole blood. The increased density of the rosetted cells results in their pelleting by subsequent DGC. This combination of RS incubation and subsequent density gradient centrifugation (RS+DGC) thus results in the depletion of undesired cells and leaves purified B-cells behind that can be harvested from the interface [9]. Here, we investigate whether RS+DGC can also effectively isolate CLL cells at high purity from peripheral blood (PB).",
"section_name": "Background",
"section_num": null
},
{
"section_content": "A preliminary experiment was used to assess the optimal RS concentration that resulted in the best purity. Aliquots of three CLL samples were treated with 50, 60, 70 and 80 μl RS/ml PB to monitor the effect on the resulting purity. \n\nThe experiments indicated that a concentration of 70 μl RS/ml PB resulted in the best purity (see Additional File 1). This was the concentration used to subsequently enrich all CLL samples. Enrichment with RS+DGC was performed in less than 90 minutes and showed higher purities of CD5/CD19 coexpressing cells for every sample compared to the enrichment with DGC alone. The analysed PB samples of CLL patients showed an average CLL cell purity of 74. 1% (ranging from 15. 9 to 97. 4%) after DGC (see Figure 1A and Additional Files 2 and 3). After RS+DGC enrichment, the same samples exhibited an average CLL cell purity of 93. 8% (ranging from 80. 4 to 99. 4%). The average purity of CD5/CD19 co-expressing cells was raised from 74. 1% after DGC to 93. 8% after RS+DGC. The average percentage of CD5 -CD19 + (normal B-cells), CD5 -CD19 -(natural killer cells and monocytes) and CD5 + CD19 -cells (T-cells) was reduced from 1. 4, 10. 1 and 14. 4 to 1. 0, 3. 5 and 1. 6% respectively after RS+DGC (see Additional File 3). \n\nThe purity of the enriched CLL cells (based on CD5/CD19 co-expression) increased with the WBC count of the samples (see Figure 1A ). RS+DGC enrichment resulted in a CLL purity of greater than 90% for all 23 of the 29 samples that showed a WBC count higher than 20 × 106cells/ml PB, while the 6 samples with a WBC count between 7 and 20 × 106cells/ml PB showed CLL purity between 80 and 90% after RS+DGC enrichment. The consistently higher purities achieved with RS+DGC in all 29 samples compared to DGC alone show the efficacy of the rosette based enrichment method, and is comparable with purities achieved by MCS and FACS and superior in terms of time and cost (see Table 1 ). \n\nNot surprisingly, the cell yield also depended on the WBC count (see Figure 1B and Additional File 4). The number of cells harvested can be regulated by increasing (or decreasing) the volume of blood to be processed and by adjusting the volume of added RS antibody cocktail accordingly without any effect on the processing time of 90 minutes. This is another potential advantage over FACS and MCS where increased cell numbers require increased processing time. The RNA extracted from enriched cells using RS+DGC displayed an average RIN of 8. 9 (ranging from 7. 7 to 9. 5), indicating high-quality RNA (see Additional File 3) that subsequently gave excellent results on microarray analysis (data not shown).",
"section_name": "Results and Discussion",
"section_num": null
},
{
"section_content": "This study shows that negative selection using a bifunctional rosette-based antibody cocktail is an effective method to isolate CLL cells of high purity, especially in samples with a WBC count above 20 × 10 6 cells/ml. The short purification time, the independence from expensive and time consuming procedures, such as FACS and MCS (see Table 1 ), and the flexible adjustment of cell yields makes RS+DGC an attractive purification method for a wide spectrum of downstream applications, particularly expression analysis utilising microarrays, in which a CLL purity of >90% is desirable.",
"section_name": "Conclusion",
"section_num": null
},
{
"section_content": "Peripheral blood (PB) samples of CLL patients were obtained as part of a study approved by the Peter MacCallum Ethics of Human Research Committee. The white blood cell (WBC) count ranged from 7. 81 to 437. 08 × 10 6 /ml and averaged 76. 1 × 10 6 /ml. The diagnosis of CLL was based on earlier examination of the patients' blood film and immunophenotyping for CD3, CD4, CD5, CD8, CD10, CD16, CD19, CD20, CD22, CD23, CD38, CD45, CD56, FMC7 and surface immunoglobulin light chain expression. \n\nBlood from CLL patients was incubated with RosetteSep™ (StemCell Technologies Inc., Vancouver, British Columbia, Canada) (RS) at a concentration of 70 μl/ml PB in the dark at room temperature for 20 minutes with gentle manual swirling every 5 minutes. As a control, an aliquot of the same sample was processed the same way except without addition of RS. After incubation, both aliquots of blood were diluted with 4 volumes (rather than 2 volumes as recommended by the manufacturer) of Dulbecco's phosphate buffered saline (PBS) containing 2% foetal bovine serum as otherwise we found that blood samples with high WBC counts were insufficiently diluted for efficient separation. The samples were then underlaid with 3 ml Lymphocyte Separation Medium (MP Biomedicals, Aurora, OH) and centrifuged for 20 minutes at 1,200 g. The enriched cells were subsequently harvested from the interface and washed once in 2 volumes Dulbecco's PBS with 2% foetal bovine serum by centrifuging for 10 minutes at 200 g. \n\nThe purity of the enriched cell population was analysed by staining with a panel of fluorescently labelled antibodies (BD Biosciences, San Jose, CA) in three different tubes. \n\n(Tube 1: CD5-FITC, CD10-PE, CD19-PerCP and CD45-APC. Tube 2: FMC7-FITC, CD23-PE, CD19-PerCP and CD45-APC. Tube 3: CD22-FITC, CD38-PE, CD20-PerCP and CD45-APC). Data acquisition was carried out using a FACScalibur-cytometer (BD Biosciences) and Cell Quest software (BD Biosciences). Ungated data analysis was conducted using Cytomics RXP software (Beckman Coulter, Fullerton, CA). \n\nThe diagnosis of CLL was reconfirmed by assessing positivity for CD5, CD19, CD23 and CD45, weak positivity for CD20; weak positivity or negativity for CD22 and FMC7 and negativity for CD10. After confirming the CLL diagnosis, the CLL purity was assessed by the co-expression of CD5 and CD19. \n\nRNA was extracted from RS+DGC enriched CLL cells by applying a combination of the Trizol-protocol (Invitrogen, Carlsbad, CA, USA) and the RNeasy Micro Kit (Qiagen, Hilden, Germany). The RNA Integrity Number (RIN) of the extracted RNA was determined using the 2100 Bioanalyzer (Agilent, Santa Clara, CA) according to the manufacturer's protocol.",
"section_name": "Methods",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We thank Nancy Messino for assisting with the immunophenotyping and Sophia Halley, Ida Candiloro, Angela Tan, Lasse Kristensen, Michael Krypuy and Chelsee Hewitt for critical reading of the manuscript. Funding for this study was from grants from the CLL Global Research Foundation to AD",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "SE adapted the methodology, carried out the laboratory work, participated in experimental design, and drafted the manuscript. DC participated in the experimental design and supplied clinical specimens. DW participated in the experimental design and supplied clinical specimens. PG assisted with the immunophenotyping. JFS supplied clin-ical specimens. AD participated in experimental design, supervised the laboratory work and brought the manuscript to its final form. All authors read and approved the final manuscript.",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional material",
"section_num": null
},
{
"section_content": "CD5/CD19 immunophenotyping of sample CLL22 (1)",
"section_name": "Additional file 1",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional file 3",
"section_num": null
},
{
"section_content": "The data is sorted by ascending WBC count. RNA Integrity Numbers (RIN) for RNA extracted from CLL cells after RS+DGC enrichment are shown. The figure shows the WBC counts of all CLL peripheral blood samples and the respective purity of the CD5 -CD19 +, CD5 + CD19 +, CD5 - CD19 -and CD5 + CD19 -fractions after DGC and after RS+DGC. This table also displays RNA Integrity Numbers for RNA extracted from purified CLL cells using RS+DGC. Click here for file [http://www. biomedcentral. com/content/supplementary/1472-6750-8-6-S3. pdf]",
"section_name": "White Blood Cell (WBC) counts of fresh CLL peripheral blood samples and their examination for CLL purity after density gradient centrifugation DGC and RosetteSep incubation prior to DGC (RS+DGC) enrichment.",
"section_num": null
},
{
"section_content": "White blood cell (WBC) counts of fresh CLL peripheral blood (PB) samples and yields and purities after RosetteSep incubation prior to density gradient centrifugation (RS+DGC) enrichment. The data is sorted by ascending WBC count. The figure shows the WBC counts of all CLL peripheral blood samples and the respective cell yield and purity of the CD5 + CD19 + fractions after DGC and after RS+DGC. Click here for file [http://www. biomedcentral. com/content/supplementary/1472-6750-8-6-S4. pdf] and JFS and Reece Australia to DW and AD. SE also acknowledges his academic supervisors at the Heinrich Heine University, Prof. Dr. Heinz Mehlhorn and Prof. Dr. Christopher Poremba.",
"section_name": "Additional file 4",
"section_num": null
}
] |
10.3389/fonc.2022.809772
|
Prediction of the Mechanism of Sodium Butyrate against Radiation-Induced Lung Injury in Non-Small Cell Lung Cancer Based on Network Pharmacology and Molecular Dynamic Simulations
|
<jats:sec><jats:title>Background</jats:title><jats:p>Radiation-induced lung injury (RILI) is a severe side effect of radiotherapy for non-small cell lung cancer (NSCLC) ,and one of the major hindrances to improve the efficacy of radiotherapy. Previous studies have confirmed that sodium butyrate (NaB) has potential of anti-radiation toxicity. However, the mechanism of the protective effect of NaB against RILI has not yet been clarified. This study aimed to explore the underlying protective mechanisms of NaB against RILI in NSCLC through network pharmacology, molecular docking, molecular dynamic simulations and <jats:italic>in vivo</jats:italic> experiments.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>The predictive target genes of NaB were obtained from the PharmMapper database and the literature review. The involved genes of RILI and NSCLC were predicted using OMIM and GeneCards database. The intersectional genes of drug and disease were identified using the Venny tool and uploaded to the Cytoscape software to identify 5 core target genes of NaB associated with RILI. The correlations between the 5 core target genes and EGFR, PD-L1, immune infiltrates, chemokines and chemokine receptors were analyzed using TIMER 2.0, TIMER and TISIDB databases. We constructed the mechanism maps of the 3 key signaling pathways using the KEGG database based on the results of GO and KEGG analyses from Metascape database. The 5 core target genes and drug were docked using the AutoDock Vina tool and visualized using PyMOL software. GROMACS software was used to perform 100 ns molecular dynamics simulation. Irradiation-induced lung injury model in mice were established to assess the therapeutic effects of NaB.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>A total of 51 intersectional genes involved in NaB against RILI in NSCLC were identified. The 5 core target genes were AKT1, TP53, NOTCH1, SIRT1, and PTEN. The expressions of the 5 core target genes were significantly associated with EGFR, PD-L1, immune infiltrates, chemokines and chemokine receptors, respectively. The results from GO analysis of the 51 intersectional genes revealed that the biological processes were focused on the regulation of smooth muscle cell proliferation, oxidative stress and cell death, while the three key KEGG pathways were enriched in PI3K-Akt signal pathway, p53 signal pathway, and FOXO signal pathway. The docking of NaB with the 5 core target genes showed affinity and stability, especially AKT1. <jats:italic>In vivo</jats:italic> experiments showed that NaB treatment significantly protected mice from RILI, with reduced lung histological damage. In addition, NaB treatment significantly inhibited the PI3K/Akt signaling pathway.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>NaB may protect patients from RILI in NSCLC through multiple target genes including AKT1, TP53, NOTCH1, SIRT1 and PTEN, with multiple signaling pathways involving, including PI3K-Akt pathway, p53 pathway, and FOXO pathways. Our findings effectively provide a feasible theoretical basis to further elucidate the mechanism of NaB in the treatment of RILI.</jats:p></jats:sec>
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[
{
"section_content": "Background: Radiation-induced lung injury (RILI) is a severe side effect of radiotherapy for non-small cell lung cancer (NSCLC),and one of the major hindrances to improve the efficacy of radiotherapy. Previous studies have confirmed that sodium butyrate (NaB) has potential of anti-radiation toxicity. However, the mechanism of the protective effect of NaB against RILI has not yet been clarified. This study aimed to explore the underlying protective mechanisms of NaB against RILI in NSCLC through network pharmacology, molecular docking, molecular dynamic simulations and in vivo experiments.",
"section_name": "",
"section_num": ""
},
{
"section_content": "The predictive target genes of NaB were obtained from the PharmMapper database and the literature review. The involved genes of RILI and NSCLC were predicted using OMIM and GeneCards database. The intersectional genes of drug and disease were identified using the Venny tool and uploaded to the Cytoscape software to identify 5 core target genes of NaB associated with RILI. The correlations between the 5 core target genes and EGFR, PD-L1, immune infiltrates, chemokines and chemokine receptors were analyzed using TIMER 2. 0, TIMER and TISIDB databases. We constructed the mechanism maps of the 3 key signaling pathways using the KEGG database based on the results of GO and KEGG analyses from Metascape database. The 5 core target genes and drug were docked using the AutoDock Vina tool and visualized using PyMOL software. GROMACS software was used to perform 100 ns molecular dynamics simulation. Irradiation-induced lung injury model in mice were established to assess the therapeutic effects of NaB.",
"section_name": "Methods:",
"section_num": null
},
{
"section_content": "Lung cancer is the most common cause of cancer-related death worldwide (1). Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer, accounting for about 85% (2). The vast majority of NSCLC are diagnosed at an advanced inoperable stage. Concurrent chemoradiotherapy (CHRT) is the standard treatment for locally advanced inoperable NSCLC. CHRT significantly improve the overall survival of advanced NSCLC, with a 5-year survival rate of approximate 30% (3). However, radiation therapy for NSCLC is usually accompanied with the radiation-induced lung injury (RILI) (4). RILI may cause severe dyspnea and chronic pulmonary fibrosis, resulting in poor quality of life and even death (5). Previous studies have reported that inflammatory factors, including transforming growth factor beta (TGF-b) and tumour necrosis factor alpha (TNF-a), and immunological cells such as T helper cells and macrophage played vital roles in the occurrence and progression of RILI (6, 7). However, the exact mechanism of RILI is still unclear. Currently, the main treatment strategy for RILI is the combination of glucocorticoids and antibiotics, but the efficacy is limited. Additionally, the treatment of RILI require a long-term use of glucocorticoids, which may raise severe side effects (7). Therefore, there is an urgent need to explore the underlying mechanism of RILI, and develop effective drugs for RILI treatment. \n\nSodium butyrate (NaB) is a kind of short-chain fatty acid generated from the fermentation of dietary fibers by anaerobic bacterial within the colon (8). In addition, NaB is confirmed as a histone deacetylase inhibitor (HDACi). Many studies have proved that some traditional Chinese medicines can protect against RILI (9, 10), and NaB has also been reported to reduce radiation toxicity. Lee et al. (11) has demonstrated that NaB could alleviate radiation-induced cognitive dysfunction. Previous studies have shown that NaB could improve the efficacy of radiotherapy without damaging normal mucosa (12). Perona et al. (13) has reported that intraperitoneally administration of NaB would optimize the irradiation results. It is worth mentioning that inflammation is the most vital feature of acute lung injury (AIL) and RILI. A large number of previous studies have confirmed that NaB has extensive antiinflammatory and immunomodulatory effects (14) (15) (16). NaB was shown to markedly downregulate the levels of interleukin 1b (IL1b) and TNF-a, and suppress the expression of nuclear factor kB, to attenuate immune response and relieve severe disruption of lung tissue structural (17). Additionally, the antitumor effect of NaB was revealed (18) (19) (20). In our previous study, we found that the combined therapy of NaB and docetaxel can additively inhibit proliferation and promote apoptosis of lung adenocarcinoma cells (21). Although accumulating evidences mentioned above indicated that NaB has both anti-radiological toxicity and anti-inflammatory effects for RILI, the mechanism by which NaB protected NSCLC from RILI has not been clarified. \n\nNetwork pharmacology is a new discipline based on the theory of systems biology and multi-direction pharmacology, which can predict the pharmacological mechanism of drugs in disease through identifying multiple potential targets and signaling pathways (22, 23). Molecular docking and molecular dynamics simulation (MDs) are mainly used to predict the binding capability and stability of drug and target genes, and realize the virtual screening of the binding complex with drug and target gene (24) (25) (26). In this study, we employ network pharmacology, molecular docking, MDs, and in vivo experiments to explore the underlying mechanisms of NaB for the treatment of RILI in NSCLC.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "METHODS",
"section_num": null
},
{
"section_content": "The research procedure of our bioinformatic analysis is shown in the flowchart (Figure 1 ). First, we identified 51 intersectional genes associated with NaB, RILI and NSCLC. Second, based on the protein-protein interaction network (PPI) of the 51 intersectional genes, 10 hub genes were further screened out. Subsequently, 5 core target genes were identified as target genes of NaB against RILI. Also, the 51 intersectional genes were performed GO enrichment analysis and KEGG analysis. The relationships of the 5 core target genes with EGFR, PD-L1, immune cells infiltration, chemokines and chemokine receptors were investigated. In the end, the 5 core target genes were performed molecular docking and MDs analyses.",
"section_name": "Schematic Diagram of the Bioinformatic Analysis",
"section_num": null
},
{
"section_content": "First, the 2D structural information of NaB (CAS:156-54-7) was downloaded from the NCBI PubChem (https://pubchem. ncbi. nlm. nih. gov/), and entered into the PharmMapper database (http://www. lilab-ecust. cn/pharmmapper/) to predict the potential targets. The names of target genes found from the PharmMapper database were converted to the formal gene names using the UniProt database (https://www. uniprot. org/). To optimize the identification of NaB target genes, we also conducted a literature retrieval, and excluded duplicates. The retrieval term was \"(sodium butyrate [Title/Abstract]) AND (gene [Title/Abstract])\", and the retrieval time was limited to 2016-2021. The involved genes of RILI and NSCLC were identified from Online Mendelian Inheritance in Man (OMIM) (http://omim. org/) and GeneCards database (https://www. genecards. org/). Finally, we obtained 51 intersectional genes for NaB, NSCLC and RILI through the intersection of Venny 2. 1 tool (http://bioinfogp. cnb. csic. es/tools/venny/index. html).",
"section_name": "Search and Identification of Common Targets",
"section_num": null
},
{
"section_content": "The 51 intersectional genes were uploaded to the STRING database (https://string-db. org/) to yield an interaction network. The protein type was chosen as \"Homo sapiens\" and the other parameters were set to default values. The protein interaction network files were imported into Cytoscape 3. 8. 2 software. The top 10 hub genes were screened by CytoHubber plug-in and MCC algorithm in Cytoscape 3. 8. 2 software. According to the values of \"Degree\", \"Betweenness Centrality\" and \"Closeness Centrality\", we identified the first 5 core target genes. \n\nCorrelation Analyses Between the Top 5 Core Genes and EGFR, PD-L1, Immune Infiltrates, Chemokines and Chemokine Receptors\n\nBased on the analysis of TIMER 2. 0 (http://timer. cistrome. org/), TIMER (https://cistrome. shinyapps. io/) and TISIDB (http://cis. hku. hk/TISIDB/) databases, we further investigated the correlations between the 5 core target genes and EGFR, PD-L1, immune infiltrates, chemokines and chemokine receptors in lung adenocarcinoma (LUAD) and lung squamous cell carcinomas (LUSC) which are both the most common types of NSCLC.",
"section_name": "Protein-Protein Interaction Network Construction and Hub Genes Screening",
"section_num": null
},
{
"section_content": "The 51 intersectional genes were imported into the Metascape database (https://metascape. org/) to perform GO and KEGG enrichment analyses, and bubble maps were drawn via bioinformatics online tool (http://www. bioinformatics. com. cn/). According to the degree of genes enrichment and p-value, we screened out the top 12 most likely KEGG signaling pathways (p<0. 0001) from the first 20, and identified target genes enriching in these pathways. The identified genes were uploaded to Cytoscape 3. 8. 2 software to construct a \"component-targetpathway\" map. Then, 3 key signaling pathways closely associated with RILI were screened out, and visualized using the KEGG database (https://www. genome. jp/kegg/).",
"section_name": "GO and KEGG Pathway Enrichment Analyses",
"section_num": null
},
{
"section_content": "Subsequently, the best protein crystal structures of the 5 core target genes were downloaded from the RCSB PDB (https:// www. rcsb. org/) database. These proteins were operated to remove water molecules, add polar hydrogen, and build active pockets using the AutoDock Vina tool (27). The 3D structure of NaB was imported into Chem3D 17. 1 for optimization. In the AutoDock module, we ran to dock NaB to the proteins of 5 core target genes for 100 times using the Lamarckian Genetic Algorithm (LGA). Other parameters were set to the default values. The lowest binding energy for molecular docking was taken as the final result and visualized by PyMOL software. AKT1 with the lowest docking scores were used for the subsequent MDs.",
"section_name": "Molecular Docking Analysis",
"section_num": null
},
{
"section_content": "To further verify the reliability of docking results, GROMACS software was used to perform molecular dynamics simulation (MDs) analysis of AKT1 and NaB compounds. Before proceeding with the simulation, the general AMBER force field (GAFF) was used for substrates, while the partial atomic charges were obtained from the RESP method by Multwfn (28, 29). The missing parameters for the ligands were generated by the parmchk utility from AMBER tools. Na + ions were added into the protein surface to neutralize the total charges of the systems. The systems were solvated in a rectangular box of TIP3P waters extending up to minimum cutoff of 15 Å from the protein boundary. The steepest descent and conjugate gradient method were used to optimize the energy of the initial structure. Then under canonical ensemble for 0. 05 ns, the system was gently annealed from 10 to 300 K with a weak restraint of 15 kcal/mol/Å. Under isothermal-isobaric ensemble at target pressure of 1. 0 atm and target temperature of 300K, 1 ns of density equilibration was performed by Langevinthermostat and Berendsen barostat with pressure-relaxation time of 0. 001 ns and collision frequency of 0. 002 ns. After minimizations and equilibrations, MDs run of 100 ns was performed for ATK1-NaB complex systems using GROMACS software (30). Finally, according to the analysis of the GROMACS software, we get the corresponding root mean square deviation (RMSD) and root mean square fluctuation (RMSF), which can be used to evaluate the stability of ATK1-NaB complex system.",
"section_name": "Molecular Dynamic Simulations Analysis",
"section_num": null
},
{
"section_content": "Female wild-type C57BL/6 mice (8 weeks; 20-22 g) were purchased from the Experimental Animal Center of Guangxi Medical University (Nanning, China) and raised under specific pathogen-free condition. All procedures involving animals were approved by the Guangxi Medical University Experimental Animal Committee, and were performed in accordance with local and International Animal Welfare Guidelines. \n\nIn conducting an experiment, mice were randomly divided into three groups: Group I (Control group) received saline intraperitoneal administration but without irradiation treatment; Group II (IR group) received radiotherapy combined with saline treatment at each time point as NaB. Group III (NaB+IR group) received an intraperitoneal administration of NaB (Sigma-Aldrich, Shanghai, China) half an hour before irradiation at a dose of 500 mg/kg/day dissolved in saline and consolidating for seven consecutive days. The dosage of NaB was referred to previous publication (31). The single irradiation dose in lung was 15 Gy and dose rate was 1 Gy/ min using 60 Co g-rays, referring to previous research (32). Before radiation treatment, mice were anesthetized by isoflurane inhalation and then shielded with lead bricks to protect their head, abdomen, and extremities from radiation. The mice were euthanized after seven days and their lung tissues excised and harvested for further study.",
"section_name": "In Vivo RILI Model and Experimental Design",
"section_num": null
},
{
"section_content": "Lung tissues of sacrificed mice were fixed with formalin, embedded in paraffin, then sliced into 5 mm section. Subsequently, the sections were subjected to standard hematoxylin and eosin staining to assess the histopathologic changes in lung tissue under a light microscope.",
"section_name": "Histological Examination",
"section_num": null
},
{
"section_content": "Lung tissues were homogenized in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitor cocktail (Beyotime Biotechnology, Shanghai, China). BCA protein assay kit (Beyotime Biotechnology, Shanghai, China) was utilized to measure the protein concentration. An aliquot of protein was separated by SDS-PAGE, and transferred onto polyvinylidene difluoride (PVDF) membranes. Blocked with QuickBlock ™ Western Blot Blocking Buffer (Beyotime Biotechnology, Shanghai, China) at room temperature for 30 min, the membranes were incubated with specific antibodies at 4°C overnight, including anti-p-PI3K (1:1000), anti-PI3K (1:1000), anti-p-AKT (1:2000), anti-AKT (1:1000) (All from Cell Signaling Technology, Danvers, MA, USA) and anti-GAPDH (1:10000) (Abcam, Cambridge, MA, USA). After washing 3 times with tris-buffered saline containing Tween 20 (TBST), the membranes were incubated with the corresponding HRP-conjugated secondary antibody (EarthOx Life Sciences, Millbrae, CA, USA) at room temperature for 1 h. After washing 3 times with TBST, the immunoreactive protein bands were determined by luminescent visualization using an enhanced chemiluminescence reagent ECL kit (Beyotime Biotechnology, Shanghai, China). The signal intensity was measured using enhanced chemiluminescence detection system (BioRad, Hercules, CA, USA).",
"section_name": "Western Blot",
"section_num": null
},
{
"section_content": "The Search and Identification of Common Genes for Sodium Butyrate, RILI, and NSCLC\n\nThe 2D structural information of NaB was obtained from the NCBI PubChem database (Figure 2A ). A total of 196 NaB target genes were predicted, while 4839 genes involved in NSCLC, and 5681 genes in RILI were identified. A total of 51 intersectional genes are thought to be involved in the mechanism of NaB against RILI (Figure 2B ). Then, the 51 intersectional genes were used for subsequent research.",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "As shown in Figure 3A, the PPI network displayed the interaction of the 51 intersectional genes. Hub genes network diagram further showed the interaction degree of the 51 intersectional genes, and identified the top 10 hub genes, including AKT1, TP53, NOTCH1, SIRT1, PTEN, CCND1, CDH1, EGFR, HDAC1 and TNF (Figure 3B ). The larger the node and the redder the color represent the stronger the interaction degree. Then, 5 core target genes were screened out based on the \"Degree\" algorithm, including AKT1, TP53, NOTCH1, SIRT1 and PTEN. These 5 core genes were considered to be the most likely target genes for the protective effect of NaB against RILI.",
"section_name": "Protein-Protein Interaction Network and Hub Genes",
"section_num": null
},
{
"section_content": "Given that previous studies have reported that epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) combined with radiotherapy were more inclined to develop RILI (33, 34), we further explored the correlation between the 5 core target genes and EGFR using the TIMER 2. 0 database. We found that the expression of EGFR was closely associated with AKT1, NOTCH1, SIRT1 and PTEN (P<0. 05), but not TP53 (P>0. 05) (Figure 4 ). It has been confirmed that the use of PD-L1 inhibitors increase the risk of RILI (35). We further investigated the correlation between PD-L1 and the 5 core target genes. The result based on the TIMER2. 0 database showed that the expression of PD-L1 was significantly associated with the expressions of TP53 (P<0. 05) in LUAD and LUSC, NOTCH1 (P<0. 05) in LUAD, and PTEN (P<0. 05) in LUAD (Figure 5 ). \n\nSince RILI is closely correlated to immune and inflammatory reaction (6), further analyses using TIMER and TISIDB databases showed that the expressions of the 5 core target genes were associated with immune infiltrations in LUAD (Figure 6 ) and LUSC (Figure 7 ), to a certain extent. For example, the expression of NOTCH1 was closely associated with B cell, CD4 + T cell, macrophage, neutrophil and dendritic cell in LUAD (P<0. 05) (Figure 6C ); PTEN was significantly associated with CD8 + T cell, CD4 + T cell, macrophage, neutrophil and dendritic cell in LUAD (P<0. 05) (Figure 6E ); and SIRT1 was B A FIGURE 3 | Protein-protein interaction (PPI) network of the 51 intersectional genes was analyzed by STRING database (A) and the hub targets network including the top 10 targets were constructed by cytoHubba plug-in (B). In the hub targets network, the larger the node and the redder the color represent the stronger the interaction degree. associated with B cell, CD8 + T cell, CD4 + T cell, macrophage, neutrophil and dendritic cell in LUSC (P<0. 05) (Figure 7D ). In addition, the expressions of AKT1, TP53, NOTCH1 and SIRT1 were mainly negatively correlated with most chemokines and chemokine receptors in either LUAD or LUSC (Figures 8, 9 ); while the expression of PTEN was mainly positively associated with chemokine receptors (Figure 9E ).",
"section_name": "Correlations Between the Top 5 Core Genes and EGFR, PD-L1, Immune Infiltrates, Chemokines and Chemokine Receptors",
"section_num": null
},
{
"section_content": "To further explore the underlying function and mechanism of the 51 intersectional genes, we performed GO and KEGG pathway enrichment analysis. As shown in Figure 10A, the results showed that the top 20 biological processes (BPs) were mainly focused on the regulation of smooth muscle cell proliferation, oxidative stress and cell death, etc. The top 20 KEGG pathways mainly included several pathways involved in cancer, the PI3K-Akt signaling pathway, p53 signaling pathway, FOXO signaling pathway, viral carcinogenesis, Epstein-Barr virus infection, cell cycle, microRNAs in cancer, etc (Figure 10B ). We created a \"component-targetpathway\" map to exhibit the effect of NaB on the 51 intersectional genes and signaling pathways against RILI using the Cytoscape software (Figure 11 ). The most likely signaling pathways that NaB improved RILI were PI3K-Akt signaling pathway, p53 signaling pathway, and FOXO signaling pathway, based on genes enrichment degree and p-value. The p-values of all these 3 pathways are less than 0. 0001. The maps of these 3 key pathways were utilized to explain the mechanism of NaB against RILI (Figure 12 ).",
"section_name": "GO and KEGG Pathway Enrichment Analysis",
"section_num": null
},
{
"section_content": "To evaluate the binding ability of NaB to the 5 core target genes, we performed molecular docking and plotted images. Lower score of binding energy indicates stronger binding affinity of the docked complex; while binding energy < 0 kcal/mol indicates that ligand molecules can spontaneously bind to the receptor proteins (25, 27, 36). Our results revealed that the binding energies of NaB binding to the 5 core target genes were all less than 0 kcal/mol. Specifically, the scores of binding energies are -5. 7 kcal/mol (AKT1-NaB), -3. 42 kcal/mol (TP53-NaB), -3. 9 kcal/mol (NOTCH1-NaB), -5. 28 kcal/mol (SIRT1-NaB), and -4. 2 kcal/mol (PTEN-NaB), respectively, indicating a high affinity between NaB and these 5 core target genes, in particular AKT1-NaB (Figures 13A-E ).",
"section_name": "Molecular Docking",
"section_num": null
},
{
"section_content": "Since the flexibility of the protein and the solvent environment are not considered in the molecular docking process (Figures 14A, B ), we further verify the reliability of the docking results through molecular dynamics simulations (MDs). Considering that AKT1 has the lowest docking energy with NaB, AKT1 was selected for 100 ns MDs. The RMSD was used to judge whether the AKT1 complex system reaches equilibrium during the simulation process. Generally, a smaller RMSD value indicates a more stable system (37). As shown in Figure 14C, the RMSD curve fluctuated around 2. 7Å and the amplitude remained within 3. 5 Å, indicating that the AKT1-NaB complex system was stable and the bond was firm. We used RMSF to evaluate the stability of each amino acid of AKT1 protein in complex system. Except for the loop region at both ends of the AKT1 protein, the RMSF curve fluctuated within 2. 5Å, confirming the strong stability of AKT1-NaB complex (Figure 14D ). In brief, MDs further verified that AKT1-NaB compound was stable and tightly combined.",
"section_name": "MDs",
"section_num": null
},
{
"section_content": "To determine the effect of NaB on RILI, the mice were intraperitoneal injected with 500 mg/kg NaB pre-and postradiotherapy. After 1 week of a single dose of 15Gy local irradiation inducing lung local radiation model, we collected lung tissues for histological evaluation. As shown in Figure 15A, mice in control group showed no significantly destruction in lung tissues; while lung tissues from mice in IR group demonstrated obviously interstitial congestion and edema, with thickened alveolar walls and collapsed alveolar. With NaB (500 mg/kg) treatment, these pathological changes were significantly reversed. Taken together, these results suggested that NaB treatment alleviates RILI.",
"section_name": "NaB Treatment Alleviates Radiation-Induced Lung Injury",
"section_num": null
},
{
"section_content": "Our bioinformatic results reveal that PI3K/AKT pathway maybe a key signaling pathway involved in NaB against RILI. Then, we further investigated the modulatory effect of NaB on PI3K/AKT pathway. As seen in Figures 15B-D, the phosphorylation levels of PI3K and AKT in IR group were increased compared to control group. NaB treatment significantly reversed the radiation-induced phosphorylation of PI3K and AKT. Our results revealed that NaB treatment may protect against RILI by inhibiting the activation of PI3K/AKT signaling pathway.",
"section_name": "NaB Treatment Inhibits PI3K/AKT Signaling Pathway in Radiation-Induced Lung Injury",
"section_num": null
},
{
"section_content": "Radiotherapy for thoracic malignancies is a standard therapeutic strategy in advance NSCLC (38). However, RILI is a severe side effect of radiotherapy with great impact on the curative effect of NSCLC, since it limits the radiation dose, a crucial factor involving in effective tumor killing (39, 40). Although growing evidence suggests that inflammation (41, 42), immune regulation (43, 44), and reactive oxygen species are involved in the occurrence and development of RILI (39), the exact mechanism is still unclarified, and there is no specific drug for treatment. It is extremely necessary to investigate the mechanism of RILI, and develop new drugs. NaB has been reported, by a large number of studies, that exerts potential activities of antiradiation toxicity (11, 12), anti-inflammation (45-47), immunomodulation (48, 49), and anti-tumor (50) (51) (52). The anti-lung cancer effect of NaB has been proved in our preliminary study (21). However, the mechanism of the protective effect of NaB against RILI needs further exploration. In this study, among the 51 intersectional genes of NaB, RILI and NSCLC, 5 core target genes were identified and thought to be involved in the mechanism of NaB against RILI, including AKT1, TP53, NOTCH1, SIRT1 and PTEN. We revealed the close relationship between the 5 core target genes and immune cell infiltration, and most of the chemokines and chemokine receptors. GO analysis of the 51 intersectional genes exhibited that BPs were focused on the regulation of smooth muscle cell proliferation, oxidative stress and cell death. Whereas the three key KEGG pathways were enriched in the PI3K-Akt pathway, p53 pathway, and FOXO pathway. We further constructed a compound-target-pathway network, depicting the relationship between NaB and the involved genes and pathway of RILI. Finally, we demonstrated that the 5 core target genes have good affinity and stability with NaB. Our experimental in vivo also verified that NaB can improve RILI. Collectively, all the findings above showed that NaB may act on multi-target genes, multi-pathway, and multi-function to protect against RILI. Through the PPI network and hub genes analyses of the 51 intersectional genes, AKT1, TP53, NOTCH1, SIRT1 and PTEN were identified as the most likely potential targets of NaB treatment for RILI. All these target genes have been confirmed to correlate to radiation toxicity. A study reported that phosphorylation of AKT increased in the lung of irradiated mice, and myrtol inhibited the phosphorylation of AKT to protect against RILI (53). The AKT-mediated pathway was significantly associated with RILI grade 3 (54). TP53 is a tumor suppressor gene associated with RILI. A study of Yang et al. (55) has shown that the polymorphisms of TP53 and ATM were associated with the risk of RILI in lung cancer patients treated with radiotherapy. Mathew et al. (10) reported that simvastatin could reverse RILI-associated dysregulated gene expression including TP53. Genetic alterations in NOTCH1 were associated with a high mean grade of radiation-induced toxicity in head and neck squamous cell carcinoma (56). SIRT1 has a protective effect against radiation injury (57). Additionally, Zhang et al. (58) reported that active PTEN signaling after radiation is closely related to RILI. Our result in vivo also confirmed the increased phosphorylation levels of AKT with radiotherapy, and NaB protect against radiation-induced lung injury with decreased phosphorylation of AKT. Collectively, all these genes, including AKT1, TP53, NOTCH1, SIRT1 and PTEN, may play a vital role in radiotoxicity, and we speculated that NaB treatment against RILI is associated with the regulation of these target genes. \n\nNumerous studies have found that RILI is closely related to inflammatory cytokine and immune cells (6, 7). In our findings, the expressions of the 5 core target genes showed some relationship with either immune infiltrations or chemokines and chemokine receptors. Based on these findings, we further performed GO function and KEGG pathway analysis of target genes. The results from GO analysis revealed that the major biological processes were enriched in the regulation of smooth colleagues founded the up-regulation of PTEN induced by oxidative stress in damaged vascular VSMCs (63). The biological processes related to the 5 core target genes further support the results of our GO analysis. Noteworthy, these biological processes are also closely related to the occurrence and development of RILI (64, 65). For example, hydrogen therapy was confirmed to attenuate irradiation-induced lung damage by reducing oxidative stress (64). Isoflavone have showed radioprotective effects in irradiated lungs by limiting excessive immune cell homing via vascular endothelium into damaged lung tissue (65). Taken together, NaB may act on these target genes to modulate biological processes including smooth muscle cell proliferation, oxidative stress, and cell death, thereby alleviating RILI. \n\nThe results from KEGG analysis indicated that multiple target genes of NaB were mainly enriched in the PI3K-Akt pathway, p53 pathway, and FOXO pathway, which were shown to play an essential role in radiotherapy. Our RILI model also showed significantly increased phosphorylation of PI3K and AKT with radiotherapy. Notably, NaB treatment suppress the activities of PI3K and AKT, and protect against RILI. Research has shown that severe RILI in lung cancer patients was associated with genetic variants in the PI3K-Akt signaling pathway (54). Repeated radon exposure induced lung injury by activating the PI3K/AKT/mTOR pathway (66). RILI can lead to chronic pulmonary fibrosis, which can be reduced by inhibition of the PI3K/AKT/mTOR pathway (67). Alleviation of radiationinduced DNA damage were associated with downregulating p53 mediated signaling pathway (68). Increased MMP-2 expression mediated by p53 is involved in RILI (69). Additionally, research showed that the baicalein inhibited radiation-induced inflammatory response through upregulating FOXO activation, and down-regulating NF-kB (70). Moskalev et al. (71) demonstrated an essential role of FOXO in hormesis and radiation adaptive response which had a protective effect on the body. Overall, NaB may alleviate RILI by modulating the pathways mentioned above. \n\nThe limitations of this study include the following aspects. First, our results of involved genes and signaling pathways of RILI need a really world cohort to verify. Second, we still need further biological experiments to verify the pharmacological mechanisms of sodium butyrate.",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "In conclusion, NaB may alleviate RILI through multiple target genes including AKT1, TP53, NOTCH1, SIRT1 and PTEN. In addition, multiple signaling pathways involved in the protective effect of NaB against RILI, including PI3K-Akt pathway, p53 pathway, and FOXO pathways. Hence, the mechanism of NaB against RILI is multi-target and multi-pathway. Our findings effectively provide a feasible theoretical basis for further elucidation of NaB in the treatment of RILI.",
"section_name": "CONCLUSION",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "The work was funded by Beijing Xisike Clinical Oncology Research Foundation ( Y-2019AZQN-04532 ), and the Key Program of Science and Technology of Guangxi, China ( AB20159024 ).",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "XZ: Methodology, Writing -original draft, Writing -review and editing. MC: Writing -review and editing. PF: Search information, review. TS: review and editing. SL: Writing -review and editing. WJ: Project administration, Funding acquisition, Resources, Writing -review and editing. All authors contributed to the article and approved the submitted version.",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. \n\nPublisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.",
"section_name": "Conflict of Interest:",
"section_num": null
}
] |
10.1371/journal.pone.0034347
|
Epigenetic Silencing of the Circadian Clock Gene CRY1 is Associated with an Indolent Clinical Course in Chronic Lymphocytic Leukemia
|
Disruption of circadian rhythm is believed to play a critical role in cancer development. Cryptochrome 1 (CRY1) is a core component of the mammalian circadian clock and we have previously shown its deregulated expression in a subgroup of patients with chronic lymphocytic leukemia (CLL). Using real-time RT-PCR in a cohort of 76 CLL patients and 35 normal blood donors we now demonstrate that differential CRY1 mRNA expression in high-risk (HR) CD38+/immunoglobulin variable heavy chain gene (IgVH) unmutated patients as compared to low-risk (LR) CD38-/IgVH mutated patients can be attributed to down-modulation of CRY1 in LR CLL cases. Analysis of the DNA methylation profile of the CRY1 promoter in a subgroup of 57 patients revealed that CRY1 expression in LR CLL cells is silenced by aberrant promoter CpG island hypermethylation. The methylation pattern of the CRY1 promoter proved to have high prognostic impact in CLL where aberrant promoter methylation predicted a favourable outcome. CRY1 mRNA transcript levels did not change over time in the majority of patients where sequential samples were available for analysis. We also compared the CRY1 expression in CLL with other lymphoid malignancies and observed epigenetic silencing of CRY1 in a patient with B cell acute lymphoblastic leukemia (B-ALL).
|
[
{
"section_content": "Accumulating epidemiological and genetic evidence indicates that disruption of circadian rhythms may increase the susceptibility for developing cancer including non-Hodgkin lymphoma (NHL) and also adversely affects tumor progression [1] [2] [3] [4] [5]. At the molecular level, several genes constituting the clock machinery have been found to establish functional interplays with regulators of the cell cycle, and disrupted expression of these genes has been shown to result in aberrant cell proliferation [2, 3, 6, 7]. In a previous study we first described disturbances in the molecular circadian machinery of CLL cells and hypothesized that these alterations may play a role in the molecular pathogenesis of the disease [8]. In particular, we found that the core circadian gene CRY1 is up-regulated in samples from high-risk ZAP-70+/CD38+ CLL patients as compared to their ZAP-702/CD382 counterparts which are characterized by a more benign clinical course [8, 9]. Therefore, based on these data we and others have proposed that CRY1 may serve as a novel prognostic factor which could be useful for the clinical management of CLL patients [8] [9] [10] [11]. Functionally, CRY1 has been shown to be essential to the maintenance of circadian rhythm because of its role in the negative arm of the circadian feedback loop [12]. However, independent of its circadian function it may have an additional role as a transcriptional regulator of a number of genes involved in cell metabolism and proliferation [4, 6, 12, 13]. In the current study we further investigated the role of CRY1 by comparing its expression pattern in molecularly defined CLL subgroups to that of B cells obtained from the peripheral blood of normal donors. Furthermore, we aimed to determine the molecular mechanism(s) underlying deregulated CRY1 expression in CLL.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Peripheral blood samples from 76 CLL patients and 35 normal donors (ND) were analyzed after obtaining written consent according to our institutional guidelines, approved by the Ethics Commission of the University of Essen-Duisburg. The diagnosis of CLL required a persistent lymphocytosis of more than 5. 0610 9 /l and a typical CD19 +, CD20 +, CD5 +, CD23 +, Ig light chain (k or l light chain) restricted immunophenotype as revealed by flow cytometry of peripheral blood cells [14]. Blood samples from CLL patients and ND were drawn in the morning hours and immediately processed. Peripheral blood mononuclear cells (PBMC) were isolated by Lymphoprep density gradient centrifugation (Invitrogen, Karlsruhe; Germany) and cryopreserved until further analysis. Patient selection for this study was based on the availability of viably frozen DMSO preserved PBMC and/or freshly isolated total RNA stored in our CLL cell bank. Clinical and laboratory data of the study population are shown in Table 1. \n\nFive healthy donors and a subgroup of 57 CLL patients were subjected to DNA methylation analysis. Patient characteristics of this subgroup are shown separately in Table S1. For this set of experiments CD19+ cells were positively selected from PBMCs employing the EasySep Human CD19 Selection Kit (StemCell Technologies, Canada) according to the manufacturer's instruction resulting in a. 90% purity of CD19+ B cells. DNA was isolated from the immunomagnetically purified CD19+ cell fraction using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany).",
"section_name": "Patients and samples",
"section_num": null
},
{
"section_content": "Cell surface expression of CD38 was examined by flow cytometry using a previously described panel of fluorochromelabeled monoclonal antibodies (CD38 PE, clone HB7) in a standard three-color flow cytometry approach using a 20% cut-off to define CD38 positivity [15, 16].",
"section_name": "Flow cytometry",
"section_num": null
},
{
"section_content": "Prognostically relevant anomalies of chromosomal regions 11q, 13q, and 17p, and of chromosome 12 were assessed by fluorescence in situ hybridization, as described previously [16].",
"section_name": "Fluorescence in situ hybridization (FISH)",
"section_num": null
},
{
"section_content": "Total RNA from PBMC was extracted with RNeasy Midi Kit (Qiagen, Hilden, Germany) and spectrophotometrically quantified as previously described [8]. First strand DNA was synthesized from 1 mg of RNA using oligo(dT) primers employing a commercially available kit (RT-PCR Amplimers, Becton Dickinson, Heidelberg, Germany) according to the manufacturer's instructions. Real-Time PCR was performed with the ABI Prism 7900HT Sequence Detector (Applied Biosystems, Foster City, CA, USA) using the TaqMan Universal PCR Master Mix protocol (Applied Biosystems). Specific assays for CRY1 (Hs00172734_m1, Assays-on-Demand Gene Expression System, Applied Biosystems), BMAL1 (Hs00154147_m1, Assays-on-Demand Gene Expression System, Applied Biosystems), CLOCK (Hs00231857_m1, Assayson-Demand Gene Expression System, Applied Biosystems), PER1 (Hs00242988_m1, Assays-on-Demand Gene Expression System, Applied Biosystems), PER2 (Hs00256144_m1, Assays-on-Demand Gene Expression System, Applied Biosystems) and GAPDH (Hs99999905_m1, Assays-on-Demand Gene Expression System, Applied Biosystems) were used. All reactions were carried out in a 10 ml final volume containing 5 ml Master Mix, 0. 5 ml of the specific assay and 4. 5 ml of 1:2 diluted cDNA. The amplification was performed under following conditions: 95uC for 10 min followed by 40 cycles of denaturation at 95uC for 15 s and annealing/elongation at 60uC for 1 min. Standard curves for all assays show similar gradients (coefficient of variation 6. 8%, data not shown). CRY1 mRNA expression was normalized against the housekeeping gene glyceralaldehyde-3-phosphate dehydrogenase (GAPDH) as endogenous reference by computing the difference between the respective Ct values (DCt = C t [gene]2C t [GAPDH] ). All PCR reactions were performed in duplicate (mean coefficient of variation for all target genes was below 1%). The mean threshold cycle number (C t ) for each tested mRNA was used to quantify the relative expression of each gene: 2 2DCt. DNA methylation analysis of the CRY1 gene by bisulphite genomic sequencing. Bisulphite treatment of genomic DNA was carried out using the EpiTect Bisulphite Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. The primers for amplifying bisulphite-modified DNA were: CRY1forward: 59-TTTGTGAGGGAAGGTTTAGTTT-39, CRY1reverse: 59-AACAATTTCCAAACCCTCC-39. For possible sequencing of the PCR product we attached a tag (forward 59-CTTGCTTCCTGGCACGAG-39, reverse 59-CAGGAAACAG-CTATGAC-39). The PCR was carried out as follows: denaturation at 95uC for 10 minutes followed by 35 cycles comprising a second denaturation of 30 seconds at 94uC, annealing at 61uC for 30 seconds and extension at 72uC for 45 seconds, followed by 7 minutes elongation at 72uC. The sequence of the PCR product is depicted in Figure S1. PCR products were separated by agarose gel electrophoresis, excised and purified by gel extraction with MinElute Gel Extraction Kit (Qiagen, Hilden, Germany). PCR products were ligated into pGEMH-T Easy Vector (Promega, Madison, USA) and transformed in XL1-Blue Competent Cells (Stratagene, La Jolla, CA, USA). Plasmid DNA isolated from multiple colonies derived from each PCR product were sequenced using the CRY1 reverse primer on an ABI 3130 Genetic Analyzer.",
"section_name": "Real-time reverse-transcriptase-PCR (qRT-PCR)",
"section_num": null
},
{
"section_content": "MassArray assays. Bisulphite treatment of genomic DNA was carried out using the EpiTect Bisulphite Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. The reverse primer contained a T7-promoter tag for in vitro transcription (59-cagtaatacgactcactatagggagaaggct-39) and the forward primer was tagged with a 10 mer (59-aggaagagag-39). Bisulphite-treated DNA was PCR amplified (denaturation at 95uC for 10 minutes followed by 40 cycles comprising a second denaturation of 30 seconds at 95uC, annealing at 61uC for 30 seconds and extension at 74uC for 30 seconds, followed by 10 minutes elongation at 74uC), PCR products were purified by gel extraction with MinElute Gel Extraction Kit (Qiagen, Hilden, Germany). 5 mL of the PCR product were used for the assay. Bisulphite MassArray assays were performed by the Genomics Core Facility, Albert Einstein College of Medicine. The data were analyzed using the analytical pipeline previously published [17]. DNA quality and no-template controls, 0%, and 100% methylated DNA were included in all assays. \n\n50 K SNP array analysis of CRY1 copy number at chromosome 12q23. For SNP array studies, genomic DNA was extracted from CD19 positively selected PBMCs using the QIAamp blood kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Array experiments were performed according to the standard protocol for Affymetrix GeneChip Mapping 50 K arrays (Affymetrix). Briefly, a 250 ng sample of DNA was digested with XbaI, ligated to adaptors, amplified by PCR, fragmented with DNAse I, and biotin-labeled. The labeled samples were hybridized to the Affymetrix 50 K SNP XbaI mapping array (Affymetrix Inc., Santa Clara, CA) followed by washing, staining and scanning. The acquired signal data was normalized with the dChip [18] program, using model-based expression, perfect match (PM)-only background subtraction and quantiles as probe-selection method. The normalized signal was then used as raw copy number per SNP and further analyzed by the GLAD algorithm [19] included in the GenePattern [20] suite, which segmented the data and assigned aggregated copy numbers to segments. A segment was defined as aberrant if its copy number was below 1. 7 (loss) or above 2. 3 (gain). The resulting list of aberrant regions was in addition filtered for regions consisting of more than 10 SNPs to exclude regions resulting from random noise in the copy number signal.",
"section_name": "DNA methylation analysis of the CRY1 gene by Bisulphite",
"section_num": null
},
{
"section_content": "Comparisons of clinical and biological parameters between subgroups were carried out using the Mann-Whitney-U test for continuous variables and Fisher's exact test for categorical data. Correlation between CRY1 delta CT and percentage of promoter methylation was tested using Spearman correlation. The Wilcoxon test for paired samples was used to compare CRY1 delta CT analysed in B and T cells from ND. Survival analysis was carried out with the Kaplan-Meier method and differences in treatment free survival between risk groups were tested with the log-rank test. All analyses were performed using R statistical software version 2. 10. 1 (R Development Core Team, Vienna, Austria, http:// www. r-project. org) and GraphPad Prism Version 5. 04 (GraphPad Software).",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We measured the mRNA expression of the circadian gene CRY1 in peripheral blood mononuclear cells (PBMC) containing more than 80% CD19+CD5+ leukemic B cells as determined by multiparameter flow cytometry in a cohort of 76 CLL patients (Table 1 ). In line with our previously published work [8, 9] we observed significantly higher CRY1 mRNA levels in high-risk(HR) patients defined by the expression of CD38 and/or unmutated IgVH genes as compared to their CD38 negative and/or IgVH mutated low risk (LR) counterparts (Figure 1A, 1B ). We could also confirm the prognostic value of CRY1 expression in CLL by comparing the clinical outcome of CLL patients with high vs. low CRY1 expression using the median DCt value as a cut-off. Significant differences in treatment free survival (TFS) were observed between the two groups (Figure S2 ). Of note, comparative analysis with PBMC derived from age matched normal blood donors revealed that these expression differences can be attributed to an under-expression of CRY1 in LR CLL cases rather than over-expression in the HR group (Figure 1A, 1B ). As the cellular composition of normal PBMC and PBMC isolated from CLL patients is known to vastly differ with regard to the content of B and T cells we compared mRNA expression levels of CRY1 in immunomagnetically purified CD19+ B cells and CD3+ T cells from normal donors (Figure S3 ). CRY1 expression was very similar in these normal lymphocyte subsets suggesting that under-expression in LR CLL samples relative to ND controls reflected true down-regulation of the gene in the leukemic cells rather than differences in the cellular composition of the PBMC samples.",
"section_name": "Analysis of CRY1 expression in normal donor derived vs. CLL PBMC",
"section_num": null
},
{
"section_content": "Aiming at investigating the molecular mechanism(s) underlying disrupted CRY1 expression in CLL cells we then speculated that this phenomenon may be explained by aberration of this gene at chromosome 12q23-q24. 1. Exploiting a panel of CLL cases which had been previously investigated for the presence of structural chromosomal abnormalities [21] by high resolution SNP array profiling we could not detect loss of chromosomal material at the CRY1 locus in any of the 55 individual CLL samples analyzed (data not shown). Moreover, an elevated copy number at this position is only detected for samples harbouring a trisomy 12, all other samples exhibit the regular two copies of this gene. The CLL patient cohort of this study (n = 76) comprises 12 cases with a trisomy 12. Among the CD38+ population 10 patients had a trisomy 12 whereas 14 were negative. Among the CD382 patients 2 had a trisomy 12 whereas 28 subjects proved to be negative. No differences in CRY1 mRNA expression were noted neither in the CD38+ nor the CD382 subgroups defined by the presence or absence of trisomy 12 (p = 0. 54 and 0. 49, respectively).",
"section_name": "Dysregulation of CRY1 in CLL cannot be explained by chromosomal aberrations",
"section_num": null
},
{
"section_content": "As CRY1 has been previously shown to undergo aberrant DNA methylation events in various human cancers [7, 22], we then examined whether epigenetic silencing could explain CRY1 mRNA expression differences in CLL. DNA methylation was studied within the promoter region of the CRY1 gene (Figure S1 ) employing sequencing of cloned PCR products generated from bisulphite-modified DNA extracted from immunomagnetically purified CD19+ B cells from the peripheral blood of CLL (N = 14) and normal donors (N = 4). We observed that patients with low CRY1 expression show hypermethylation of the analyzed region in comparison to patients with a high CRY1 expression and normal donors (Figure 2A ). Using the Sequenom MassArray Compact System for methylation analysis in the same CRY1 promoter region we then confirmed these results in a cohort of 58 CLL samples and 5 normal donors (Figures 3A and S4, Table S3 ). LR CLL patients either defined by CD38 or IgVH mutational status showed a significantly higher degree of methylation of the CRY1 promoter in comparison to the HR group. In all, there was a statistically highly significant inverse correlation between the percentage of methylated CpGs and CRY1 mRNA expression as detected by qRT-PCR (Figure 2B and 3B ). Comparing both methods for methylation analysis a high correlation (r = 0. 89, mRNA expression values and percentage of methylated CpG were found to be highly correlated (r = 20. 62, p = 0. 006, Spearman correlation). The regression line in the plot was produced by linear regression analysis using promoter methylation as dependent and CRY1 mRNA expression as independent variable (open circles indicate ND). doi:10. 1371/journal. pone. 0034347. g002 p,0. 0001) proves the consistency and robustness of each of the applied methods (Figure 3C ).",
"section_name": "DNA methylation analysis of the CRY1 gene",
"section_num": null
},
{
"section_content": "To assess the prognostic value of the methylation profile of the CRY1 promoter we defined two groups, i. e. patients with lowly methylated and highly methylated promoter region. The threshold value of 9% was derived from the median value of the CLL samples subjected to MassArray methylation analysis. On survival analysis patients with a highly methylated CRY1 promoter region exhibited a significantly longer treatment-free survival as compared to the hypomethylated patient subgroup (Figure 4 ).",
"section_name": "Prognostic value of CRY1 methylation pattern in CLL",
"section_num": null
},
{
"section_content": "Twenty-seven patients were studied at two or more time points using the qRT-PCR assay. As illustrated in Figure S5, CRY1 mRNA expression was relatively stable over time in the majority of patients with stable disease (10/13 (77%), Figure S5, left panel) as well as in patients with progressive disease (12/14 (86%), right panel). The fact that three of the five patients with increasing CRY1 mRNA levels had a comparably benign course of disease may suggest that changes in CRY1 expression over time are not stringently related to disease progression. Moreover, in 6 of the 14 patients (marked by arrows in Figure S5 ) with a progressive course of the disease first treatment was initiated after collection of the baseline sample in this longitudinal analysis. Although all these patients responded to therapy, no reduction in CRY1 mRNA expression could be documented. It is noteworthy that we did not observe a single case with decreasing CRY1 mRNA levels during follow-up in the entire cohort of patients with samples available for sequential analysis. \n\nInterestingly, one patient with a low baseline CRY1 expression and a correspondingly high degree of CpG island methylation experienced disease progression as evidenced by rising lymphocyte counts and transition from Binet stage A to stage C disease. Increasing CRY1 expression in this particular patient was shown to be associated with loss of CRY1 promoter hypermethylation (Figure S6 ).",
"section_name": "Analysis of CRY1 expression over time",
"section_num": null
},
{
"section_content": "At the molecular level, circadian rhythms are encoded by an autoregulatory loop composed of a set of transcription activators (CLOCK/BMAL1) that induce expression of PER and CRY. Accumulated PER and CRY proteins in turn inhibit BMAL1/ CLOCK transcriptional activity and repress their expression. Thus, aberrant silencing of CRY1 expression in LR CLL may be associated with dysregulated expression of other circadian genes. Indeed, we could observe increased PER2 and CLOCK mRNA levels in CLL as compared to ND derived PBMC, whereas mRNA levels of BMAL1 and PER1 were similar in both groups (Figure Unpaired two-tailed t-test was used to compute p-values. B Samples from CLL patients and ND were subjected to both CRY1 mRNA expression and DNA methylation analysis with the bisulphite MassArray assay. mRNA expression values and percentage of methylated CpG were found to be highly correlated (r = 20. 63, p,0. 0001, Spearman correlation). The regression line in the plot was produced by linear regression analysis using promoter methylation as dependent and CRY1 mRNA expression as independent variable. C Correlation between the methylation data resulting from bisulphite genomic sequencing and the MassArray method showed high consistency (r = 0. 86, p,0. 0001, Spearman correlation). doi:10. 1371/journal. pone. 0034347. g003 S7). However, changes in PER2 and CLOCK expression were noted in both LR and HR CLL samples and could not be related to alterations in CRY1 expression (Figure S7 ) suggesting that these abnormalities may occur independently of each other.",
"section_name": "Analysis of the role of CRY1 within the circadian clock's transcription/translation-based feedback loop in CLL",
"section_num": null
},
{
"section_content": "From a cancer biology standpoint we next wanted to test whether deregulated CRY1 expression may also be observed in other lymphoid malignancies than CLL. To this end we employed the CRY1 qRT-PCR assay to screen RNA samples isolated from a wide range of lymphoproliferative disorders including T prolymphocytic leukemia (T-PLL, n = 10), mantle cell lymphoma (MCL, n = 6), hairy cell leukemia (HCL, n = 3), multiple myeloma (MM, n = 8), plasma cell leukemia (PCL, n = 2) and B and T lineage acute lymphoid leukemia (n = 29 resp. n = 19). In disease entities with more than 5 observations, statistical comparisons with ND derived PBMC did not reveal significant expression differences (Figure 5A ). However, this finding needs to be interpreted with caution because of the small numbers of samples tested in this series. The availability of a comparably large number of ALL samples (n = 48) allowed for a more detailed analysis of this disease group. The clinical characteristics of this patient cohort are given in Table S2. Similar to its distribution in CLL, CRY1 expression in ALL was found to be very heterogeneous where one patient with a mature B-ALL exhibited a markedly low CRY1 transcript level (Figure 5A ). This particular sample demonstrated a high degree of CRY1 promoter methylation while two other ALL samples with a higher CRY1 expression were nearly completely demethylated (Figure 5B ). \n\nNext we investigated the prognostic value of CRY1 expression in ALL. To this end we compared the clinical outcome of ALL patients with high vs. low CRY1 expression using the median DCt value as a cut-off. No significant differences in overall survival (OS) were observed between the two groups (Figure S8 ).",
"section_name": "Analysis of CRY1 expression and CpG island hypermethylation in other hematologic malignancies",
"section_num": null
},
{
"section_content": "In this study, we analyzed the expression of CRY1 mRNA, which encodes a key component of the central and peripheral circadian oscillator, in the PBMC from normal blood donors and patients with CLL [8, 11, 23, 24]. In line with our previous work [8, 9] and that of others [10, 11] we detected elevated CRY1 transcript levels in patients with high risk disease defined by the expression of CD38 and/or unmutated IgVH (UM) genes as compared to their CD38 negative and/or IgVH mutated (M) low risk (LR) counterparts. Lewintre et al. confirmed these results in a recently published microarray-based gene expression profiling study including 36 patients with early-stage CLL [11]. Therefore, determination of CRY1 may have potential as a novel prognostic marker in CLL and should be tested in comparison to other established molecular risk factors in the setting of prospective randomized trials. \n\nWe now uncover that HR CLL cases and ND derived B cells exhibit comparable levels of CRY1 mRNA expression. Thus, disrupted CRY1 expression in CLL can be attributed to downmodulation of CRY1 in LR CLL cases rather than over- expression in the HR group. We then aimed to investigate the molecular mechanisms underlying down-regulation of CRY1 in LR CLL. As CRY1 had been previously shown to undergo aberrant DNA methylation events in various solid human malignancies including breast and ovarian cancers [3, 7, 22], we examined whether epigenetic silencing could also explain the observed CRY1 mRNA expression differences in CLL subgroups. To this end we performed comparative DNA methylation analysis of highly purified CD19+ B cells from the peripheral blood of 57 CLL patients and normal donors. Indeed, our results show that CRY1 is transcriptionally silenced by promoter hypermethylation in LR CLL cases while HR cases and ND derived B cells exhibit hypomethylated CRY1 promoter regions. Importantly, DNA methylation analysis was complemented by qRT-PCR performed on the same samples revealing a statistically highly significant inverse correlation between the percentage of methylated CpGs and CRY1 mRNA expression in individual cases, suggesting a direct regulation of CRY1 expression through methylation of its promoter. We could show that the methylation status of the CRY1 promoter predicts clinical outcome in CLL patients; where aberrant hypermethylation was associated with a more benign course of the disease. To date, only few reports describe an aberrant methylation phenotype as a predictor of outcome in haematological diseases. Recently, Irving et al reported a panel of methylation markers (CD38, HOXA4, BTG4) in which an overall methylation score was significantly associated with time to first treatment in CLL [25]. Olk-Batz et al [26] described that a high methylation profile is associated with an aggressive biological variant of juvenile myelomonocytic leukemia. \n\nAs stability of CRY1 expression by the leukemic cell clone over time is an important prerequisite for its reliable use as a prognostic marker [27], we sequentially analyzed CRY1 expression in 27 patients. CRY1 transcript levels were remarkably stable in the majority of patients and no consistent changes were noted in relation to alterations in disease activity and treatment history of individual patients. Significant expression changes during followup occurred in 19% of the patients, which could limit the value of this marker under routine clinical conditions. However, these findings are limited by the comparably small number of CLL cases analyzed in this study and thus need to be validated in a larger patient cohort. In one patient with a low baseline CRY1 expression and a correspondingly high degree of CpG island methylation disease progression was linked to increasing CRY1 expression and hypomethylation of the CRY1 promoter region. In aggregate, these findings raise the possibility that epigenetic silencing of CRY1 occurs early in the disease and may be lost during disease progression at least in rare cases. Conversely, it appears unlikely that the leukemic cells acquire epigenetic silencing of CRY1 during the course of the disease as none of the 27 patients in the longitudinal analysis showed a decrease of CRY1 expression. It would be interesting to further investigate this hypothesis by systematically comparing the prevalence of CRY1 methylation events in individuals with monoclonal B cell lymphocytosis with Binet stage A, B and C CLL patients. \n\nWhile differential CRY1 expression in HR vs. LR CLL subgroups is now well established [8] [9] [10] [11] 28], the functional consequences of CRY1 down-modulation in the leukemic cells are currently unknown. It is tempting to speculate that epigenetic silencing of CRY1 may contribute to the benign clinical behaviour of LR CLL cases. At first sight this notion appears counterintuitive to the general conception that the core components of the molecular clock machinery may function as tumor suppressor genes [29] [30] [31] [32]. This view is mainly based on epidemiological and genetic evidence indicating that disruption of circadian rhythms might be directly linked to cancer development including non-Hodgkin-lymphoma [2, 5, [33] [34] [35]. While the circadian genes PER1 and PER2 have been clearly shown to function as tumor suppressors in the mouse model [30], a recent study showed that epidermal deletion of BMAL1 in a transgenic mouse model which spontaneously develops squamous tumours leads to significantly fewer neoplastic lesions [36]. Along the same line transgenic CRY deficient mice do not show a predisposition to cancer [6]. Furthermore and somewhat unexpectedly, ablation in the mouse of both CRY genes in a TP53 2/2 background delays the onset of cancer [13]. This latter observation supports our hypothesis that epigenetic silencing of CRY1 may functionally contribute to the indolent clinical behaviour of LR CLL. It is currently unclear whether abrogation of one CRY gene, i. e. CRY1 or CRY2 suffices to fully block the circadian rhythmicity of an individual cell. Studies comparing the biologic characteristics of transgenic mice lacking either one or both CRY genes indicate a certain degree of functional redundancy [37]. \n\nAnother matter of controversy is whether circadian rhythmicity per se or only certain core clock components are involved in tumorigenesis [4]. To address this issue we correlated CRY1 mRNA expression with other components of the circadian clock's transcription/translation-based feedback loop in individual CLL samples. Recently, a number of in vitro and animal studies have further elucidated the important functional role of CRY proteins for the regulation of the molecular clock. For example, work by Ye et al [38] demonstrated that CRY directly interacts with the BMAL1:CLOCK:E-box complex independent of PER resulting in inhibition of the transactivator function of CLOCK:BMAL1. Furthermore, Busino et al. [39] showed in an in vitro model that Cry1 2/2 Cry2 2/2 mouse embryonic fibroblasts are characterized by a loss of oscillation in Per1 and Per2 and exhibit increased Per2 but not Per1 mRNA transcript levels in comparison to control fibroblasts isolated from CRY wild type animals. \n\nHere, we found that the expression of PER2 and CLOCK but not BMAL1 and PER1 are also disrupted in CLL as compared to ND derived control samples. However, contrary to what could be expected from above described findings [38, 39] these abnormalities could neither be correlated with changes in CRY1 expression nor the clinical course of the disease in individual CLL cases. In aggregate, these latter results clearly suggest that defects in the circadian molecular machinery may be a common phenomenon in CLL cells and are not restricted to CRY1. Ongoing work in our laboratory is aimed at determining the molecular and cellular consequences of silencing different circadian genes in CLL cells in vitro using siRNA oligonucleotide technology. \n\nFinally, we wanted to determine whether CRY1 expression may also be disturbed in other lymphoid malignancies than CLL. To this end we measured CRY1 transcript levels in PBMC samples from a range of different disease entities including T-PLL, MCL, HCL, MM, PCL and B and T cell ALL. In a subgroup analysis focussing on ALL we found a heterogeneous expression pattern comparable to that observed in CLL. One patient with a mature B-ALL exhibited a particularly low CRY1 expression which correlated with a high degree of promoter methylation, suggesting that aberrant epigenetic silencing of this gene may also occur in other lymphoid disease entities. However, contrary to CLL survival analysis did not show a significant difference between ALL patients with high and low expression of CRY1, respectively. \n\nIn conclusion, our data indicate that the previously reported CRY1 gene expression differences in LR vs. HR CLL patients [8, 11] are caused by aberrant methylation of the CRY1 promoter in the LR patient subgroup. To our knowledge this is the first report in CLL research linking epigenetic silencing of a specific gene to an indolent clinical course of the disease.",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "Supporting Information",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "We thank Sima Vagaie, Michael Mo ¨llmann, Sana Mohamad and Olga Rempel for excellent technical assistance, Anja Fu ¨hrer and Sabrina Kieruzel for maintaining the CLL biobank and Ute Schmu ¨cker for FACS analyses.",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "indicates a uniquely assayable site. Fragmentation patterns are shown in corresponding colors, yellow highlights primer sequences. Methylation data are shown as an average from duplicates. Bar height denotes percent methylation on a scale from 0% (low) to 100% (high), error bars indicate median absolute deviation. CG sites that are putatively outside the usable mass window are indicated as boxes with gray background. (TIF)",
"section_name": "",
"section_num": ""
}
] |
10.3390/cancers12103041
|
Fc-Engineered Antibodies with Enhanced Fc-Effector Function for the Treatment of B-Cell Malignancies
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<jats:p>Monoclonal antibody (mAb) therapy has rapidly changed the field of cancer therapy. In 1997, the CD20-targeting mAb rituximab was the first mAb to be approved by the U.S. Food and Drug Administration (FDA) for treatment of cancer. Within two decades, dozens of mAbs entered the clinic for treatment of several hematological cancers and solid tumors, and numerous more are under clinical investigation. The success of mAbs as cancer therapeutics lies in their ability to induce various cytotoxic machineries against specific targets. These cytotoxic machineries include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), which are all mediated via the fragment crystallizable (Fc) domain of mAbs. In this review article, we will outline the novel approaches of engineering these Fc domains of mAbs to enhance their Fc-effector function and thereby their anti-tumor potency, with specific focus to summarize their (pre-) clinical status for the treatment of B-cell malignancies, including chronic lymphocytic leukemia (CLL), B-cell non-Hodgkin lymphoma (B-NHL), and multiple myeloma (MM).</jats:p>
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{
"section_content": "Naturally, antibodies (Abs) are produced by B-cells as a polyclonal population, with high specificity for their distinct target antigen and epitope. Antibodies thereby play various important roles in our immune system. The field of therapeutic Abs commenced in 1975, when the development of the mouse hybridoma technology enabled the production of large amounts of murine monoclonal (m) Abs [1]. However, murine mAbs elicited an immunogenic response in human patients. To reduce this immunogenicity, chimeric mAbs, consisting of a constant human domain fused to a variable mouse domain, were developed [2]. The chimeric mAb rituximab targeting cluster of differentiation Cancers 2020, 12, 3041 2 of 24 (CD) 20 was the first FDA-approved mAb for cancer therapy in 1997. The development of advanced design technologies such as human antibody gene expression libraries and transgenic animals allowed the engineering of humanized (the hypervariable region of a murine antibody grafted in a human antibody) and fully human mAbs [3]. To be successfully applied in the clinic, mAbs generally require additional engineering to improve their affinity, limit any biophysical liabilities, and to increase their half-life. Currently, 30 mAbs are clinically approved for treatment of cancer, and this number is rapidly increasing: in the last decade, the number of mAbs that have entered late-stage clinical studies has been tripled [4]. The therapeutic potential of mAbs has been exploited by the development of antibody fusion products, such as bispecific antibodies or antibody drug conjugates, which take advantage of specific antigen binding properties of antibodies to precisely target cytotoxic cells or toxic agents to cancerous cells. A novel development in antibody engineering is the modification of the antibody fragment crystallizable (Fc) region in order to increase the Fc tail-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), to induce tumor cytotoxicity more effectively. Numerous Fc-engineered antibodies have demonstrated clinical activity or are under preclinical investigation. \n\nIn this review, we will outline the novel approaches of engineering Fc domains of mAbs to enhance their Fc-effector function and anti-tumor potency, with specific focus to their (pre-) clinical status for the treatment of B-cell malignancies, including chronic lymphocytic leukemia (CLL), B-cell non-Hodgkin lymphoma (B-NHL), and multiple myeloma (MM). Not described in this review is the application of Fc engineering in order to improve antibody half-life, to silence mAb effector functions in case of antibodies used as receptor agonists or antagonists or as drug delivery vehicles, and to increase the direct, not Fc-effector function-mediated, anti-tumor potency of mAbs.",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "Antibodies are mono-or polymers of immunoglobulins (Ig) consisting of two identical pairs of heavy (H) and light (L) chains, which are linked through non-covalent interactions and disulfide bonds to form a Y-shaped structure [5]. All H and L light chains contain a single variable domain (V L ), which also consists of hypervariable regions. The combination of the (hyper) variable regions of the H and L chains determines the antigen specificity and affinity of an antibody. The L chains contain a single constant (C L ) domain to make a stable link with the H chain. The number of constant domains of the H chain (C H ) is dependent on the isotype of the antibody: IgA, IgD, and IgG contain three (C H 1-3), and IgE and IgM contain four constant domains (C H [1] [2] [3] [4]. The first C H is linked with C L to the variable regions, which together form the fragment antigen binding (Fab) region. The heavily glycosylated C H 2-3 or C H 2-4 domains are linked to C H 1 via a flexible hinge region and constitute the Fc region (Figure 1A ). The engineering of this region, which is responsible for the isotype-and subclass-dependent Fc-mediated effector functions of antibodies, will be the main focus of this review.",
"section_name": "Antibody Structure",
"section_num": "2."
},
{
"section_content": "Upon antigen engagement, IgG antibodies can induce direct anti-tumor effects via triggering the cell death signaling pathways and via blockade of essential receptor systems, as well as indirect antitumor effects via their Fc-mediated effector functions, by engaging other immune cells or killer mechanisms. The Fc-mediated effector functions of antibodies include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complementdependent cytotoxicity (CDC), and have been shown to be crucial for the therapeutic efficacy of most clinically approved antibodies (Figure 1B ). Among the four IgG subclasses, IgG1 and IgG3 induce the strongest Fc-effector functions [6]. However, since IgG1 has the longest half-life and is more stable than IgG3 [7], most therapeutic antibodies with Fc-mediated functions are of IgG1 isotype.",
"section_name": "Fc-Effector Functions",
"section_num": "3."
},
{
"section_content": "The IgG1-induced ADCC and ADCP response is mediated via binding to Fc gamma receptors (FcγR), which are expressed on innate effector cells, including monocytes, monocyte-derived cells, basophils, mast cells, and natural killer (NK) cells. The FcγR family consists of the activating FcγRI (CD64), FcγRIIa (CD32a), FcγRIIIa (CD16a), and FcγRIIIb (CD16b), and the inhibitory FcγRIIb (CD32b). Of all FcγRs, only FcγRI, which plays a major role in myeloid cell activation, is classified as a high-affinity Fc receptor [8]. All other FcγRs require binding of multivalent IgG-antigen immune complexes in order to provide sufficient avidity to activate downstream signaling and induce antibody-mediated ADCC or ADCP [9]. \n\nNatural killer (NK) cells are considered the most potent inducers of ADCC. NK cells as well as monocytes and macrophages express FcγRIIIa, however, only NK cells exclusively express FcγRIIIa ADCC and ADCP are mediated via binding to FcγR and cell death occurs via release of cytotoxic granules and via internalization and degradation of the target, respectively. CDC is mediated via binding to complement protein C1q and cell death occurs via formation of the membrane attack complex (MAC), which consists of complement proteins C5b, C6, C7, and C8 and various copies of C9, and generates pores in the membrane.",
"section_name": "ADCC/ADCP",
"section_num": "3.1."
},
{
"section_content": "Upon antigen engagement, IgG antibodies can induce direct anti-tumor effects via triggering the cell death signaling pathways and via blockade of essential receptor systems, as well as indirect anti-tumor effects via their Fc-mediated effector functions, by engaging other immune cells or killer mechanisms. The Fc-mediated effector functions of antibodies include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), and have been shown to be crucial for the therapeutic efficacy of most clinically approved antibodies (Figure 1B ). Among the four IgG subclasses, IgG1 and IgG3 induce the strongest Fc-effector functions [6]. However, since IgG1 has the longest half-life and is more stable than IgG3 [7], most therapeutic antibodies with Fc-mediated functions are of IgG1 isotype.",
"section_name": "Fc-Effector Functions",
"section_num": "3."
},
{
"section_content": "The IgG1-induced ADCC and ADCP response is mediated via binding to Fc gamma receptors (FcγR), which are expressed on innate effector cells, including monocytes, monocyte-derived cells, basophils, mast cells, and natural killer (NK) cells. The FcγR family consists of the activating FcγRI (CD64), FcγRIIa (CD32a), FcγRIIIa (CD16a), and FcγRIIIb (CD16b), and the inhibitory FcγRIIb (CD32b). Of all FcγRs, only FcγRI, which plays a major role in myeloid cell activation, is classified as a high-affinity Fc receptor [8]. All other FcγRs require binding of multivalent IgG-antigen immune complexes in order to provide sufficient avidity to activate downstream signaling and induce antibody-mediated ADCC or ADCP [9]. \n\nNatural killer (NK) cells are considered the most potent inducers of ADCC. NK cells as well as monocytes and macrophages express FcγRIIIa, however, only NK cells exclusively express FcγRIIIa [10]. \n\nTriggering of FcγRIIIa induces phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs), which activates a downstream signaling cascade resulting in the release of cytotoxic granules containing perforin and granzyme in the immune synapse formed between the NK cell and the target cell, leading to target cell death [11]. \n\nIn contrast to NK cells, the phagocytic monocytes, neutrophils, and macrophages co-express activating and inhibitory FcγRs. ADCP can be induced by various activating FcγRs including FcγRIIIa (CD16a), FcγRIIa (CD32A), and FcγRIIIb (CD16b), which all signal via ITAMs, and may be inhibited by FcγRIIb (CD32b), which signals via immunoreceptor tyrosine-based inhibitory motifs (ITIMs). The balance of activating and inhibitory signaling dictates ADCP induction, which occurs via internalization and degradation of the antibody-opsonized target by the phagocyte [12]. \n\nFcγRs are highly polymorphic, their genes are known to have several single-nucleotide polymorphisms (SNPs). Such polymorphisms can affect the affinity of the FcγR for Ig molecules [13]. Both FcγRIIa and FcγRIIIa are known to exist as two allotypic variants, which are associated with clinical response for therapeutic mAbs. In the extracellular domain of FcγRIIa, a C > T substitution at amino acid position 131 results in a histidine (131R) to arginine (131R) replacement [14]. FcγRIIa binds with high affinity to IgG1 and IgG3. The amino acid position 131 is polymorphic for IgG2 binding: the 131H variant can bind to IgG2 with high affinity, while 131R barely binds to IgG2 [13]. For FcγRIIa, a T > G substitution can occur at amino acid position 158 resulting in valine (158V) to phenylalanine (158F) replacement. The FcγRIIIa-158V variant was shown to bind to IgG1 and IgG3 with a higher affinity compared to FcγRIIIa-158F [15]. \n\nThe FcyRIIa (131H/R) and FcyRIIIa (158F/V) polymorphisms are associated with clinical response for several clinical-approved mAbs including rituximab, trastuzumab, and cetuximab [16] [17] [18].",
"section_name": "ADCC/ADCP",
"section_num": "3.1."
},
{
"section_content": "Therapeutic IgG1 antibodies can activate the classical pathway of the complement system by binding of the Fc region to the complement protein C1q, which initiates a cascade of proteolytic cleavage steps. This results in formation of the membrane attack complex (MAC), consisting of the complement products C5b to C9. The MAC generates pores in the cell membrane which initiates target cell lysis, termed complement-dependent cytotoxicity (CDC) [19]. Similar to FcγR, the affinity of C1q for IgG-Fc is low and binding is dependent on multivalent IgG-antigen immune complexes to provide sufficient avidity [20] [21] [22]. The ability of IgG1 antibodies to activate the complement pathway is highly dependent on antigen density, size, and fluidity [23] [24] [25]. High-resolution crystallography studies have recently revealed that, dependent on such factors, specific non-covalent interactions between IgG Fc domains induce ordered hexamer formation (mAb hexamerization) on the cell surface that provides a docking platform for the six-globular-headed C1q molecule and thereby efficiently activates the complement pathway [26].",
"section_name": "CDC",
"section_num": "3.2."
},
{
"section_content": "The hinge and the proximal C H 2 regions of the Fc tail are considered critical for Fc interaction with FcγRs and C1q. The interface of these regions contains the binding sites, while the structural conformation of the C H 2 domain allows engagement of C1q or FcγR. In addition, the C H 2 domains are post-translationally modified by asparagine(N)297-linked glycosylation, and glycosylation and the specific glycan composition contribute to the stability and the dynamics of the C H 2 domains [27] [28] [29]. Glycan components include core units of N-acetylglucosamine (GlcNAc) and mannose, with additional variations in galactose, bisecting GlcNAc, fucose, and sialic acid. \n\nDetailed understanding of Fc interactions with C1q and FcγR opened up opportunities to modulate C1q and FcγR binding by Fc engineering. In order to enhance ADCC, ADCP, and CDC, studies have employed site-directed mutagenesis (sequence variations), Fc glycosylation modification (glycoengineering), and avidity modulation, which will be outlined below. \n\nCancers 2020, 12, 3041 5 of 24",
"section_name": "Fc Engineering to Enhance Fc-Effector Functions",
"section_num": "4."
},
{
"section_content": "",
"section_name": "Enhancing ADCC",
"section_num": "4.1."
},
{
"section_content": "Fc glycosylation is required for binding the low-affinity FcγR [30, 31]. Generally, aglycosylation is thought to completely abrogate FcγR effector functions [32, 33], but several aglycosylated Fc variants with intact FcγR effector function have been reported [34] [35] [36]. Altering the specific composition of the Fc glycan can increase the affinity for FcγR. The removal of core fucose (afucosylation) has been shown to highly increase FcγRIIIa binding affinity and consequently increase ADCC [37, 38] (Figure 2A ). This effect has been attributed to an interaction between the Fc-glycan and the N-glycan attached to Asn 162 of the FcγRIIIa [39], however, the exact nature of the interaction is still debated. It has been suggested that core fucose restricts the number of conformations recognized by the FcγRIIIa N-glycan [40], while others suggest that core fucose inhibits direct carbohydrate-carbohydrate interactions with the receptor glycan [41, 42]. Nevertheless, afucosylation is widely accepted as an effective approach to increase the potency of IgG1 antibodies to induce ADCC.",
"section_name": "Glycoengineering to Enhance FcγR Affinity",
"section_num": "4.1.1."
},
{
"section_content": "Fc glycosylation is required for binding the low-affinity FcγR [30, 31]. Generally, aglycosylation is thought to completely abrogate FcγR effector functions [32, 33], but several aglycosylated Fc variants with intact FcγR effector function have been reported [34] [35] [36]. Altering the specific composition of the Fc glycan can increase the affinity for FcγR. The removal of core fucose (afucosylation) has been shown to highly increase FcγRIIIa binding affinity and consequently increase ADCC [37, 38] (Figure 2A ). This effect has been attributed to an interaction between the Fcglycan and the N-glycan attached to Asn 162 of the FcγRIIIa [39], however, the exact nature of the interaction is still debated. It has been suggested that core fucose restricts the number of conformations recognized by the FcγRIIIa N-glycan [40], while others suggest that core fucose inhibits direct carbohydrate-carbohydrate interactions with the receptor glycan [41, 42]. Nevertheless, afucosylation is widely accepted as an effective approach to increase the potency of IgG1 antibodies to induce ADCC. \n\nTo a lesser extent, Fc galactosylation is also suggested to modulate FcγRIIIa binding. The reported effects of hypergalactosylation on ADCC, however, range from completely absent to positive without addition of afucosylation [38, 43, 44] and positive with addition of afucosylation [45, 46]. These large variations in study results might be explained by differential interactions of the galactose on the different N-glycan arms with FcγR [47]. The effects of Fc sialylation on ADCC have been described to be minimal, and completely absent in addition to afucosylation [43, 46, 48]. To a lesser extent, Fc galactosylation is also suggested to modulate FcγRIIIa binding. The reported effects of hypergalactosylation on ADCC, however, range from completely absent to positive without addition of afucosylation [38, 43, 44] and positive with addition of afucosylation [45, 46]. These large variations in study results might be explained by differential interactions of the galactose on the different N-glycan arms with FcγR [47]. The effects of Fc sialylation on ADCC have been described to be minimal, and completely absent in addition to afucosylation [43, 46, 48].",
"section_name": "Glycoengineering to Enhance FcγR Affinity",
"section_num": "4.1.1."
},
{
"section_content": "High-resolution structural Fc analysis revealed the specific FcγR-binding sites, which has laid the foundation for structure-guided identification of affinity-enhancing mutations. FcγRs interact with residues Leu234-Ser239 on the lower hinge and residues Asp265-Glu269, and Asn297-Thr299 on the C H 2 domain [49, 50]. Since then, it has become clear that numerous positions within or in close proximity to this region can be mutated to improve FcγR binding affinity. Alanine screening in the C H 2 and C H 3 domains revealed that several mutations could enhance binding to FcγRIIIa, with the most potent mutations combined in S298A/E333A/K334A for enhanced ADCC [51]. A study using computational design algorithms and high-throughput screening demonstrated that S239D/I332E mutations could also enhance FcγRIIIa binding and ADCC [52]. Both S298A/E333A/K334A and S239D/I332E highly enhanced binding for the lower-affinity polymorphic variant (F158) of FcγRIIIa [51, 52]. The P247I/A339Q mutations, which were applied in the anti-CD20 mAb ocaratuzumab, have also been shown to enhance binding to the lower-affinity FcγRIIIa [53] (Figure 2B ). \n\nStructural analyses of the Fc-FcγR interaction have revealed that the Fc binding to FcγR is asymmetrical: the receptor binds to different residues on each Fc domain. Hence, it seemed likely that applying mutations to Fc regions asymmetrically could maximize the FcγR binding affinity. Indeed, Fc heterodimeric antibodies improved C H 2 domain stability and the consequent FcγRIIIa binding as compared to a symmetrically mutated Fc variant [54, 55]. In addition, afucosylation of the heterodimeric antibodies further improved the FcγRIIIa binding [55].",
"section_name": "Site-Directed Mutagenesis to Enhance FcγR Affinity",
"section_num": "4.1.2."
},
{
"section_content": "In comparison to affinity modulation, avidity modulation is a less established but more straightforward approach. Fc duplication (or tandem-Fc) or multiplication, whereby multiple Fcs are linked within one IgG1 molecule, has been shown to augment FcγR binding avidity and increase ADCC and ADCP [56] [57] [58] [59] (Figure 2C ). A theoretical safety concern for Fc multiplication strategies is the fact that natural antibody oligomerization may result in unwanted immune activation [60]. However, studies have reported minimal in vitro aggregation and no in vivo adverse events so far [57, 59 ].",
"section_name": "Fc Multimerization",
"section_num": "4.1.3."
},
{
"section_content": "",
"section_name": "Enhancing ADCP",
"section_num": "4.2."
},
{
"section_content": "Strategies enhancing ADCC via increased affinity for FcγRIIIa on NK cells can also enhance ADCP via increased antibody binding to monocytes and macrophages, since these cells also express FcγRIIIa. ADCP induced by neutrophils can be improved as well since neutrophils express FcγRIIIb, which shares 97% sequence homology with FcγRIIIa. It has indeed been demonstrated that afucosylated mAbs can induce higher levels of ADCP [61, 62].",
"section_name": "Glycoengineering to Enhance FcγR Affinity",
"section_num": "4.2.1."
},
{
"section_content": "Different than ADCC, ADCP induction is highly dependent on the balance of binding to the activating receptors versus the inhibitory receptor FcγRIIb. The activating FcγRIIa shares 90% similarities with the inhibitory FcγRIIb [63]. Hence, selectively increasing FcγRIIa binding without influencing or while even decreasing the inhibitory FcγRIIb binding remains a great challenge in enhancing ADCP and requires more careful engineering. Increasing FcγRIIa binding while simultaneously decreasing FcγRIIb was achieved by mutations F243L/R292P/Y300L/V305I/P396L [64]. In another study, in which ADCC could be enhanced by S239D/I332E mutations, a third mutation (A330L) was necessary to improve ADCP because the sole S239D/I332E mutation also resulted in increased binding to FcγRIIb [52]. Another study identified the G236A mutation to selectively enhance FcγRIIa binding. This study demonstrated that the addition of G236A to S239D/I332E and S239D/A330L/I332E resulted in enhanced ADCP, in addition to the improvement of ADCC [65, 66] (Figure 2B ).",
"section_name": "Site-Directed Mutagenesis to Enhance FcγR Affinity",
"section_num": "4.2.2."
},
{
"section_content": "Fc multimerization strategies are not FcγR-specific. Therefore, such strategies will enhance the binding of mAbs to other low-affinity FcγRs, including to the inhibitory FcγRIIb. Nonetheless, it appeared possible to increase FcγRIIa binding and ADCP by Fc multimers [57, 67]. However, since binding to the inhibitory FcγRIIb was also increased [57], Fc multimerization strategies might require further Fc engineering to optimally enhance the ADCP.",
"section_name": "Fc Multimerization",
"section_num": "4.2.3."
},
{
"section_content": "",
"section_name": "Enhancing CDC",
"section_num": "4.3."
},
{
"section_content": "While afucosylation significantly enhances ADCC and ADCP by facilitating the interaction with the FcγRIIIa glycan, it minimally affects CDC [46]. Sialyation seems to have moderate effects on C1q binding. Some studies reported increased and some others reported decreased C1q binding by sialyation [46, 68, 69]. Instead, galactose is the key glycan for C1q binding. Numerous studies demonstrated enhanced C1q binding and CDC by Fc galactosylation [46, [69] [70] [71] (Figure 2A ). Molecular interactions between galactose and amino acid residues on the C H 2 domains possibly increase C1q binding affinity [72]. It has also been suggested that Fc glycosylation modulates Fc/Fc interactions and thereby affects not the affinity but the avidity of C1q binding [73].",
"section_name": "Glycoengineering to Enhance C1q Binding Affinity",
"section_num": "4.3.1."
},
{
"section_content": "The first structural analysis studies revealed that the residues D270, K322, P329, and P331 of the C H 2 domain were critical for the interaction with C1q [74, 75]. More recently, it has been shown that there are two main interaction sites: residues 266-272 and 294-300 on one C H 2 domain and residues 325-331 on the other [76]. Mutations in residues located on or in proximity to these binding sites significantly affected C1q binding: the double mutant K326W/E333S and triple mutant S267E/H268E/S324T enhanced C1q binding and CDC [77, 78] (Figure 2B ). The hinge region also plays a role in complement activation, because this region affects the flexibility of the Fc tail, thereby determining the ability to fix C1q. Indeed, certain mutations in the upper hinge region could enhance C1q binding and CDC [79].",
"section_name": "Site-Directed Mutagenesis to Enhance C1q Binding Affinity",
"section_num": "4.3.2."
},
{
"section_content": "In addition to affinity modulation, site mutagenesis can also be performed in order to modulate avidity. Proceeding from the finding that antibody hexamers facilitate C1q binding, the essential first step in CDC, a novel strategy was developed in order to improve the hexamer forming of antibodies upon target antigen binding. Introducing the specific point mutations E345R and E430G at the Fc and C H 2-C H 3 interface could indeed stimulate the Fc/Fc interactions between antibodies and facilitate the natural concept of antibody hexamerization, leading to superior C1q binding and enhanced CDC [26, 80] (Figure 2B, E ). Since Fc hexamerization by these specific point mutations only occurs upon antigen binding on the cell surface, antibodies generated by this so called \"HexaBody\" technology retain the pharmacokinetics of conventional IgG1 antibodies.",
"section_name": "Antibody Hexamerization to Facilitate C1q Binding",
"section_num": "4.3.3."
},
{
"section_content": "Although both IgG1 and IgG3 can effectively activate complement, IgG3 antibodies can bind C1q more effectively [81]. Therefore, cross-isotype antibodies have been generated by replacing the C H 2 and C H 3 domains of an IgG1 antibody with the corresponding regions of an IgG3 antibody, which increases the CDC response [82] (Figure 2D ).",
"section_name": "Cross-Isotype Antibodies",
"section_num": "4.3.4."
},
{
"section_content": "Although the hybridoma technology revolutionized the field of therapeutic antibodies, most mAbs that are currently approved for therapeutic use are generated by mammalian expression systems, which allow higher antibody yields and preserve post-translational modifications, generating a higher-quality mAb product. Mammalian expression systems often use the variable regions derived from the hybridoma or phage display technologies. The sequence of the desired region is cloned into the appropriate expression vector and subsequently transfected into the expression system. Currently available mammalian expression systems include various Chinese hamster ovary (CHO) cell lines, mouse myeloma (NS0), and mouse hybridoma (Sp2/0) cell lines. In addition, several human expression systems are available, including embryonic kidney (HEK293), amniotic (CAP), a hybrid of HEK293 and lymphoma (HKB-11), and embryonic retina (PER. C6). The human expression systems, however, provide transient expression and are therefore only suitable for preclinical purposes. \n\nThe current approaches of antibody sequence engineering at the Fc site apply site-directed mutagenesis either directly once heavy and light chain sequences are available (for structure-based sequence engineering) or to generate large phage or yeast display libraries to screen for the most optimal Fc variant (empirical-based sequence engineering). Glycoengineered antibodies require more complex adaptations in the manufacturing protocol, which will be outlined below.",
"section_name": "Generation of Fc-Engineered mAbs",
"section_num": "5."
},
{
"section_content": "Mammalian expression systems allow conventional post-translational modifications and can also be modified to alter specific post-translational modifications, such as Fc glycosylation. However, glycosylation is a complex process and cannot be controlled completely as cell culture conditions can alter the glycosylation pattern [83, 84]. To create glycoengineered antibodies in order to develop antibodies with improved ADCC activity, several modified mammalian expression systems were developed. Double knockout of the enzyme α1,6-fucosyltransferase 8 (FUT8), which catalyzes the transfer of fucose from GDP-fucose to N-acetylglucosamine (GlcNAc), in CHO cell lines resulted in the production of afucosylated antibodies [85]. Alternatively, CHO cells were engineered to express β(1,4)-N-acetylglucosaminyltransferase III (GnTIII). IgGs produced by mammalian cells have very low or no bisecting GlcNAc, in contrast to IgGs present in human serum, and increasing the number of bisecting GlcNAc improved ADCC levels [86, 87] ).",
"section_name": "Glycoengineered mAbs",
"section_num": null
},
{
"section_content": "Although various strategies can enhance ADCC, ADCP, or CDC effector function, they do not uniformly increase these effector functions when applied to different antigens, since antigen binding also affects the C1q and FcγR binding via structural allostery [88, 89]. In addition, there is a partial overlap in the Fc-binding sites for C1q and FcγR [6]. Therefore, modulating the Fc tail to enhance ADCC/ADCP can negatively influence CDC, and vice versa. It is thus recommended to evaluate Fc-effector function-enhancing strategies for each target individually. \n\nIn B-cell malignancies, a wide variety of disease-associated targets are available, including various lineage-specific surface molecules. The Fc-engineered mAbs for these target antigens are discussed below for each relevant disease subtype (Table 1 ). 4 ClinicalTrials. gov Identifier: NCT03582033. \n\n6. 1. B-CLL and B-NHL\n\nCD20 is expressed on almost all healthy and malignant B-cells, but is not expressed by precursor B-cells and plasma cells, making it the ideal therapeutic target for B-cell malignancies [90]. The CD20-targeting chimeric mAb rituximab was the first mAb to be approved by the FDA for cancer therapy in 1997 and is currently still part of the first line of immune-chemotherapy regimens for patients with B-NHL and CLL. Although rituximab is capable of both ADCC/ADCP and CDC induction, multiple Fc engineering strategies have been explored to enhance the effector functions of CD20-targeting mAbs, either type I or type II. The CD20 mAbs are classified as type I and II based on their ability to reorganize the CD20 molecules into lipid rafts. Type I CD20 mAbs, such as rituximab, can induce CD20 reorganization and efficiently activate the complement pathway, whereas type II CD20 mAbs are poor complement activators but instead induce direct cell death. Both type I and II mAbs can induce ADCC [91, 92]. \n\nGlycoengineered CD20-targeting mAbs include obinutuzumab (GA101) and ublituximab (TG-1101). Obinutuzumab is a type II glycoengineered (non-fucosylated) humanized anti-CD20 IgG2 mAb which targets a different but overlapping epitope on CD20 compared to rituximab [93]. In comparison to rituximab, a significant clinical benefit of obinutuzumab was observed for FL and CLL in combination with chemotherapy [94] [95] [96], and obinutuzumab has received FDA approval for FL and CLL. Obinutuzumab in combination with CHOP (G-CHOP) did not show a PFS benefit compared to R-CHOP for treatment-naïve DLBCL [97, 98]. Ublituximab is a type I glycoengineered (low-fucose content) chimeric anti-CD20 IgG1 which targets a unique epitope on CD20 and is currently under clinical investigation. Ublituximab increased the ADCC of CLL cells in vitro and ex vivo compared to rituximab [99,100], and induced ADCC in rituximab-resistant B-NHL in in vitro and in vivo models [101]. Ublituximab has shown promising phase 2 and 3 clinical efficacy either as a single agent or in combination with ibrutinib and umbralisib, the first BTK inhibitor and a next-generation PI3K inhibitor, in high-risk CLL and B-NHL patients [102] [103] [104]. In addition, several clinical trials are ongoing, including trials investigating the efficacy in treatment-naïve FL and in progressive CLL (ClinicalTrials. gov Identifier: NCT03828448 and NCT04149821, respectively) and a trial investigating the combination of ublituximab with an anti-PDL1 mAb (TG-1501) (ClinicalTrials. gov Identifier: NCT02535286). \n\nThe relevance of FcγRIIIa polymorphisms for antibodies targeting CD20 has been demonstrated by the higher response rates of rituximab in patients with the 158V variant [16, 105, 106]. Fc-mutated CD20-targeting mAbs that were clinically evaluated and designed to enhance affinity for the low-affinity variant FcγRIIIa-158F include the humanized mAbs ocaratuzumab (AME-133v; LY2469298), PRO131921 (RhuMAb v114), and ocrelizumab. Ocaratuzumab was generated by screening for Fc modifications that enhance ADCC, which led to the identification of the P247I/A339Q mutations that enhanced binding to both allelic variants of FcγRIIIa, in addition to Fab modifications that enhance antigen binding [107]. In vitro, ocaratuzumab induced ADCC in CLL cells at higher levels than rituximab, and similar levels to obinutuzumab [53]. A phase 1/2 clinical trial demonstrated the activity and tolerability of ocaratuzumab in previously treated FL patients with low-affinity FcγRIIIa [108, 109]. PRO131921 is Fc-modified (unspecified) to enhance C1q binding in addition to enhanced FcγRIIIa binding, and was demonstrated to enhance ADCC and CDC in vitro compared to rituximab. A phase 1 trial of PRO131921 in relapsed and/or refractory follicular lymphoma patients who previously received rituximab showed tolerability [110]. However, the clinical development of both ocaratuzumab and PRO131921 has been discontinued [111], no information regarding the reason has been disclosed. Ocrelizumab demonstrated activity in a phase 1-2 trial in patients with relapsed/refractory follicular lymphoma [112], but is, at the moment, only registered for the treatment of patients with multiple sclerosis. \n\nSeveral other Fc-engineered CD20-targeting antibodies have been explored in preclinical studies. The strong CDC induction of IgG3 antibodies targeting CD20 [113, 114] favors the development of IgG1/IgG3 isotype variants, and a CD20-targeting afucosylated IgG1/IgG3 isotype variant, with increased CDC and ADCC levels in vitro [115]. Applying multiple Fc-enhancing strategies simultaneously also proved beneficial for a nonfucosylated rituximab variant containing the S267E/H268F/S324T/G236A/I332E mutations, which enhanced both ADCC and CDC in vitro [116].",
"section_name": "Clinical Experience with Fc-Engineered mAbs for B-Cell Malignancies",
"section_num": "6."
},
{
"section_content": "Similar to CD20, CD37 is expressed on all mature B-cells, but absent or expressed at very low levels on stem cells, precursor B-cells, and plasma cells [117, 118]. Several CD37-targeting therapeutics have been clinically evaluated, including several immunoconjugates but also two Fc-engineered antibodies. BI 836,826 (MAb 37. 1) is an Fc-mutated (S239D/I332E) chimeric IgG1 with enhanced ADCC in addition to pro-apoptotic activity. BI 836,826 demonstrated potent cytotoxicity in CLL cells ex vivo, especially in combination with the PI3K inhibitor idelalisib in relapsed CLL [119, 120]. In phase 1 clinical trials in relapsed/refractory CLL and relapsed/refractory B-NHL, acceptable tolerability and preliminary efficacy was observed [121, 122]. However, a phase 1b/2 trial of BI 836,826 in combination with gemcitabine and oxaliplatin in DLBCL was halted prematurely due to dose-limiting toxicities (DLTs) (ClinicalTrials. gov Identifier: NCT02624492). BI 836,826 has been discontinued from further clinical development. The Fc-engineered DuoHexaBody-CD37 is a biparatopic (dual-epitope-targeting) CD37-targeting IgG1 antibody with the E430G hexamerization-enhancing mutation that induces potent CDC, in contrast to native CD37-targeting antibodies [123]. DuoHexaBody-CD37 showed ex vivo efficacy in B-CLL and various B-NHL (van der Horst et al. [124] ), and a first-in-human clinical trial has recently been initiated (ClinicalTrials. gov Identifier: NCT04358458).",
"section_name": "CD37",
"section_num": "6.1.2."
},
{
"section_content": "B cell-activating factor (BAFF) is an immunomodulatory cytokine which regulates B-cell survival and activation. BAFF can bind to three receptors although only one of them binds BAFF with high specificity: the BAFF receptor (BAFF-R) [125]. BAFF-R is expressed on almost all normal and malignant B-cells, but not on pre-B-cells, and is therefore considered an appropriate target for B-CLL and B-NHL. Ianalumab (VAY736; B-1239) is a fully human BAFF-R-targeting glycoengineered (afucosylated) IgG1 antibody. Although ianalumab also blocks receptor signaling and proliferation, ADCC induction mediated by the afucosylated Fc domain was demonstrated to be crucial for potent cytotoxicity. Furthermore, ianalumab induced higher levels of ADCC in CLL cells than rituximab and the Fc-engineered obinutuzumab, and combining ianalumab with ibrutinib could further enhance efficacy in vivo [126, 127]. A phase 1 clinical trial is currently active to evaluate ianalumab in combination with ibrutinib for CLL patients (ClinicalTrials. gov Identifier: NCT03400176).",
"section_name": "BAFF-R",
"section_num": "6.1.3."
},
{
"section_content": "CD19 expression is restricted to the B-cell lineage but is in contrast to CD20 also expressed on precursor B-cells. CD19 is highly expressed in B-NHL and several leukemias, including CLL and ALL. In addition, although CD19 is generally considered to be absent on plasma cells, it has been shown that some multiple myeloma (MM) cells express CD19 at extremely low density which might suffice for targeted therapy [128]. Unmodified CD19-targeting antibodies induce limited ADCC/ADCP and CDC, partly because they are rapidly internalized [129]. CD19 is therefore mostly used as a target for T-cell engagers, such as bispecific antibodies or chimeric antigen receptors (CARs), but some Fc-engineered CD19-targeting antibodies are also clinically evaluated. The CD19-targeting afucosylated mAbs inebilizumab (MEDI-551) and MDX-1342 and the Fc-mutated (S239D/I332E) mAb tafasitamab (MOR208; XmAb5575) all enhance ADCC levels in vitro relative to native CD19 mAbs [130] [131] [132]. Inebilizumab was tested in phase 1 trials and showed tolerability and preliminary efficacy in CLL, FL, DLBCL, and MM [133]. However, phase 2 trials of inebilizumab in combination with chemotherapy in CLL and DLBCL did not show any significant differences in outcome compared to rituximab in combination with chemotherapy (ClinicalTrials. gov Identifier: NCT01466153; ClinicalTrials. gov Identifier: NCT01453205). A phase 1 study of inebilizumab in relapsed or refractory advanced B-cell malignancies has recently been completed (ClinicalTrials. gov Identifier: NCT00983619). MDX-1342 has been tested in a phase 1 study in in CLL patients (ClinicalTrials. gov Identifier: NCT00593944), but the study was halted prematurely and the program has been discontinued without further disclosure. Tafasitamab was demonstrated safe and efficacious in a phase 1 trial in relapsed CLL and a phase 2 trial in relapsed and refractory B-NHL [134, 135]. Moreover, in vitro studies suggested that lenalidomide can further enhance the ADCC effects of tafasitamab, and the combination with lenalidomide resulted in a high response rate of relapsed and refractory DLBCL patients. [136]. Tafasitamab has been granted accelerated FDA approval in combination with lenalidomide for patients with relapsed DLBCL.",
"section_name": "CD19",
"section_num": "6.1.4."
},
{
"section_content": "",
"section_name": "Multiple Myeloma (MM)",
"section_num": "6.2."
},
{
"section_content": "CD38 is an attractive target for multiple myeloma due to its high and uniform expression on MM cells, while its expression on myeloid and lymphoid cells and in non-hematopoietic tissue is relatively low. The unmodified CD38-targeting antibody daratumumab received FDA approval in 2019 and induces MM cell cytotoxicity via ADCC, ADCP, and CDC in addition to direct cell death [137]. To further increase the CDC potential of CD38-targeting antibodies, the Fc-engineered antibody HexaBody-CD38 (GEN3014) carrying the E430G hexamerization-enhancing mutation has been developed. HexaBody-CD38 demonstrated superior CDC activity in vitro compared to daratumumab and showed promising anti-tumor activity in vivo [138]. In addition, the Fc multimerization technology has been employed to generate the anti-CD38 selective immunomodulator of the Fc receptor antibody (SIFbody), with enhanced binding to the Fcγ receptors and C1q resulting in CDC activity and NK-and macrophage-mediated killing in vitro. The anti-CD38 SIFbody also demonstrated increased efficacy ex vivo compared to daratumumab [139]. 6. 2. 2. HM1. 24 HM1. 24 was first described to be preferentially overexpressed on normal and malignant plasma cells [140, 141], although more recent studies also demonstrated HM1. 24 expression on B-CLL and lymphoma and several solid tumors [142] [143] [144] [145] [146]. Antibodies targeting HM1. 24 for MM exhibited in vitro and in vivo anti-tumor activity. However, a phase 1 study of the humanized anti-HM1. 24 unmodified antibody AHM in relapsed/refractory MM could not demonstrate significant efficacy. Glycoengineered (afucosylated) variants of AHM and the Fc-mutated (S239D/I332E) anti-HM1. 24 antibody XmAb5592 could enhance ADCC as well as ADCP compared to AHM in preclinical studies, and warrant further clinical testing [147] [148] [149].",
"section_name": "CD38",
"section_num": "6.2.1."
},
{
"section_content": "The intercellular adhesion molecule-1 (ICAM-1/CD54) mediates adhesion of MM cells to bone marrow stromal cells (BMSCs). CD54 is highly expressed on MM cells and associated with advanced disease stage and resistance to chemotherapy, which makes ICAM-1 an interesting target for MM [150, 151]. The unmodified ICAM-1-targeting antibody BI-505 induced potent anti-myeloma activity in vitro and in vivo, which was predominantly macrophage-mediated [152]. BI-505 progressed to clinical trials, and although a phase I trial demonstrated good tolerability, BI-505 lacked significant efficacy in a phase II trial in MM [153, 154]. To potentially enhance efficacy in vivo, Fc engineering (S239D/I332E) has been applied to the anti-ICAM-1 fully human IgG1 antibody MSH-TP15, which binds to a distinct but overlapping epitope compared to BI-505, with enhanced ADCC and ADCP activity in vitro and improved tumor control in vivo compared to its unmodified counterpart [155, 156]. 6. 2. 4. BCMA B-cell maturation antigen (BCMA; CD269; TNFRSF17) plays a significant role in the differentiation of B-cells to plasma cells and is required for plasma cell longevity [157]. BCMA expression is specific for plasma cells and MM cells even overexpress BCMA [158]. Multiple T-cell engagers targeting BCMA are currently being clinically evaluated and expected to receive approval for clinical application soon. SEA-BCMA is a glycoengineered (afucosylated) humanized BCMA-targeting IgG1 antibody and showed promising preclinical activity via induction of ADCC and ADCP as well as a block in proliferation [159]. SEA-BCMA is currently evaluated in a phase 1 safety study in relapsed/refractory MM patients (ClinicalTrials. gov Identifier: NCT03582033).",
"section_name": "ICAM-1",
"section_num": "6.2.3."
},
{
"section_content": "In this review, we have illustrated various strategies to enhance Fc-mediated effector functions and we have summarized their clinical application in chronic lymphocytic leukemia (CLL), B-cell non-Hodgkin lymphoma (B-NHL), and multiple myeloma (MM). Fc-mediated effector functions are being enhanced to increase their anti-tumor potency or when a specific effector function is beneficial, i. e., improving ADCC induction for combination therapy with lenalidomide or in patients with low-affinity FcγR polymorphisms. In addition, Fc engineering can be applied to antibodies which depend on antibody clustering or FcγR-mediated antibody crosslinking for agonism of receptors, such as antibodies targeting the costimulatory protein CD40 or antibodies targeting death receptors 4 or 5, and thus benefit from similar strategies as discussed here [160] [161] [162]. \n\nGenerally, mAbs that are Fc-engineered to improve their effector functions are capable of enhancing in vitro and in vivo anti-tumor potency compared to their parental unmodified mAb. Various Fc-engineered mAbs also demonstrated clinical efficacy, and are already approved for clinical use. However, other Fc-engineered mAbs demonstrated toxicity in clinical trials or failed to induce significant clinical efficacy, and were discontinued for development. Increasing the clinical success of Fc-engineered mAbs requires more empirical in vitro/in vivo screening to determine the most favorable Fc engineering strategy or combination of strategies. In addition, understanding (i) the specific contribution of ADCC, ADCP, and CDC to the clinical efficacy of mAbs in hematological malignancies and (ii) the exact clinical effect of the different Fc engineering strategies could allow for developing Fc engineering strategies customized to a specific target and disease. A step forward in understanding the specific contribution of mAb effector functions is the recent development of Fc engineering strategies that enhance CDC specifically, such as the HexaBody technology. Until recently, clinically evaluated mAbs were mostly Fc-engineered to enhance ADCC function. Hence, evaluating Fc-engineered mAbs with enhanced CDC and potentially comparing them to mAbs with enhanced ADCC could provide crucial information regarding the contribution of mAb effector functions to clinical efficacy and toxicity, and the results of such clinical trials are highly anticipated. \n\nTo conclude, our advanced knowledge of Fc structure and Fc-mediated effector function has enabled the clinical development of Fc-engineered mAbs. Expanding our clinical experience with these Fc-engineered mAbs will provide valuable information that could allow the development of antibodies with tailor-made effector functions.",
"section_name": "Conclusions and Future Perspective",
"section_num": "7."
}
] |
[
{
"section_content": "This research received no external funding.",
"section_name": "",
"section_num": ""
}
] |
10.1038/s41375-018-0342-3
|
Recurrent activating STAT5B N642H mutation in myeloid neoplasms with eosinophilia
|
Determining the underlying cause of persistent eosinophilia is important for effective clinical management but remains a diagnostic challenge in many cases. We identified STAT5B N642H, an established oncogenic mutation, in 27/1715 (1.6%) cases referred for investigation of eosinophilia. Of the 27 mutated cases, a working diagnosis of hypereosinophilic syndrome (HES; n = 7) or a myeloid neoplasm with eosinophilia (n = 20) had been made prior to the detection of STAT5B N642H. Myeloid panel analysis identified a median of 2 additional mutated genes (range 0-4) with 4 cases having STAT5B N642H as a sole abnormality. STAT5B N642H was absent in cultured T cells of 4/4 positive cases. Individuals with SF3B1 mutations (9/27; 33%) or STAT5B N642H as a sole abnormality had a markedly better overall survival compared to cases with other additional mutations (median 65 months vs. 14 months; hazard ratio = 8.1; P < 0.001). The overall survival of STAT5B-mutated HES cases was only 30 months, suggesting that these cases should be reclassified as chronic eosinophilic leukemia, not otherwise specified (CEL-NOS). The finding of STAT5B N642H as a recurrent mutation in myeloid neoplasia with eosinophilia provides a new diagnostic and prognostic marker as well as a potential target for therapy.
|
[
{
"section_content": "Eosinophilia, defined as an elevation of the peripheral blood (PB) eosinophil count above 0. 5 × 10 9 /L, is conventionally divided into three main categories: primary, secondary (reactive) and idiopathic. Primary eosinophilia is a clonal hematologic disorder in which the eosinophils form part of the neoplastic clone. Secondary, non-clonal eosinophilia may be driven by a wide range of underlying conditions including allergic disorders, autoimmunity, infectious diseases, lymphoproliferative disorders, solid tumours, drug reactions and other conditions. Idiopathic eosinophilia is a diagnosis of exclusion when no primary or secondary cause can be identified [1]. \n\nClonal eosinophilia is seen in the context of a myeloid neoplasm and particularly the World Health Organisation (WHO)-defined entities 'chronic eosinophilic leukemia, not otherwise specified' (CEL-NOS) and 'myeloid and lymphoid neoplasms with rearrangement of PDGFRA, PDGFRB or FGFR1 or with PCM1-JAK2, ETV6-JAK2 or BCR-JAK2' (MLN-eo). Clonal eosinophilia may also be associated with other WHO subtypes of myeloproliferative neoplasm (MPN-eo) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN-eo) [1] [2] [3]. \n\nIdentifying the underlying cause of eosinophilia is important for patient management but can be challenging in the absence of an overt myeloid neoplasm or discernible secondary cause. Primary eosinophilia is strongly associated with constitutively activated tyrosine kinase (TK) signalling, and to date more than 70 TK fusion genes have been identified in myeloid neoplasms as a consequence of reciprocal translocations or other genomic rearrangements [3]. Identification of these fusions usually confirms a specific diagnosis and is an indication for targeted therapy. Notably, imatinib induces rapid and durable complete clinical, hematologic and molecular remissions in >90% of patients with a PDGFRA or PDGFRB fusion gene, conferring excellent progressionfree and overall survival (OS) [4, 5]. Fusions involving JAK2, FGFR1 or FLT3 are associated with a more aggressive clinical course and may be responsive to other small molecule inhibitors [6] [7] [8]. Some TK-fusion negative eosinophilia cases test positive for KIT D816V or JAK2 V617F, whereas others have mutations in a range of genes associated with myeloid neoplasms such as TET2, ASXL1, EZH2 or SETBP1 [9] [10] [11]. It has been suggested that a rapid and durable response to corticosteroids is uncommon in cases with primary eosinophilia and instead points towards a diagnosis of secondary eosinophilia, if that is not already apparent [12]. \n\nIn this study, we have used genomic approaches to focus on the identification of novel somatic abnormalities in patients with suspected primary eosinophilia. We have identified a recurrent somatically acquired point mutation in STAT5B leading to an N642H substitution in several cases. This mutation is known to activate STAT5B but was previously thought to be restricted to lymphoproliferative disorders.",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Our study included samples from individuals referred for routine diagnostic analysis of persistent unexplained eosinophilia and/or patients diagnosed with MPN or MDS/MPN with eosinophilia according to standard morphologic, hematologic and laboratory criteria [2]. Cases that tested positive for KIT D816V, FIP1L1-PDGFRA or other recognised TK fusion genes were excluded. The study design adhered to the tenets of the Declaration of Helsinki and was approved by the National Research Ethics Service (UK) Committee South West and the institutional review board of the Medical Faculty of Mannheim, Heidelberg University as part of the 'German Registry on Disorders of Eosinophils and Mast Cells'. DNA or RNA was extracted from total PB or bone marrow (BM) leukocytes using standard procedures. T-cells were selected using CD3 microbeads (Miltenyi Biotech, Cologne, Germany) and stimulated to divide by co-cultivation with CD2/CD3/CD28 in the presence of interleukin-2 (T-cell activation/expansion kit; Miltenyi Biotech).",
"section_name": "Patients and samples",
"section_num": null
},
{
"section_content": "RNAseq data for the initial cohort of 14 cases is available at ArrayExpress (www. ebi. ac. uk/arrayexpress; accession number E-MTAB-7492). RNAseq data was analysed for point mutations using an in-house pipeline. Briefly, raw RNAseq data in fastq format were aligned to the reference genome (human_g1k_v37) using STAR aligner and a twostep process. In the first step, splice junctions were detected by an initial alignment of the fastq files. In the second step, final alignments were determined using the splice junctions as a guide [13]. After alignment the bam files were sorted, de-duplicated and read groups were added using Picard (http://broadinstitute. github. io/picard). In preparation for variant calling, GATK was used to hard-clip intronic sequences (SplitNCigarReads), reassign mapping qualities (ReassignOneMappingQuality) and recalibrate base quality scores (BaseRecalibrator). Variants were called using GATK HaplotypeCaller and ignoring soft clipped bases, which minimises false positive and false negative calls. A final set of high-quality variants in variant call format (VCF) was produced by selecting variants with a phred-scaled confidence threshold of 20 and excluding variants with low depth corrected quality scores (QD < 2), significant strand bias (FS > 30) or SNP clusters of 3 or more SNPs within 35 base pairs. ANNOVAR [14] was used to annotate variants with respect to genes, databases of known polymorphisms (Exome Aggregation Consortium, 1000 genomes, exome sequencing project, dbSNP), and pathogenicity estimates (avsift, polyphen, etc. ). A shortlist of potentially relevant variants was created by applying additional filters, which excluded non-coding variants, synonymous SNVs and variants with an alternate allele frequency greater than 1% in public databases of known variation: 1000 genomes (1000G; www. ncbi. nlm. nih. gov/variation/tools/1000genomes/), exome sequencing project (ESP; http://evs. gs. washington. edu/EVS/) and exome aggregation consortium (ExAC; http://exac. broadinstitute. org/). Variants passing these filters were further highlighted if they were located in a manually curated list of candidate genes (n = 581 genes), annotated as pathogenic in Clinvar, overlapped with a somatic mutation in hematopoietic and lymphoid tissue in COSMIC (https://ca ncer. sanger. ac. uk/cosmic) or were recurrently mutated.",
"section_name": "RNAseq analysis",
"section_num": null
},
{
"section_content": "A tetra-primer ARMS assay [15] was designed using http:// primer1. soton. ac. uk/primer1. html with inner primers to specifically amplify the normal and mutant STAT5B alleles and outer primers to produce a positive control STAT5B band for each reaction. PCR primers were: forward outer (FO), 5′-CGATCAGGAAACACGTAGATAAGGTAATT-3; reverse outer (RO), 5′-AAATGGAGATTTCTATTGGAG CCATTAT-3; forward wild-type specific (Fwt), 5′-TCT CTGGTGGTAAAAGGCATCAGGTT-3; reverse mutantspecific (Rmt), 5′-TTATTGATCTAGAGGAAAGAATGT TTTAGC-3. The FO and RO were used at a final concentration of 0. 5 μM whereas the inner primers Fwt and Rmt were used at 2. 5 μM. Amplification reactions were performed using AmpliTaq Gold DNA polymerase, 25 ng genomic DNA at an annealing temperature of 60 °C for 35 cycles. Cases showing mutant bands after agarose gel electrophoresis were confirmed by independent amplification followed by Sanger sequencing.",
"section_name": "Amplification-refractory mutation system (ARMS) test for STAT5B N642H",
"section_num": null
},
{
"section_content": "Sanger sequencing was used to screen for other STAT gene mutations using primers designed to amplify from cDNA: STAT3 exons 20-22 (5′-GGGCCATCTTGAGCACTAAG-3′ and 5′-CACAGATAAACTTGGTCTTCAGG-3′); STAT5A exons 16-17 (5′-GGACCTTCTTGTTGCGCTTT-3′ and 5′-GGCGGTCAGGAAACACATAG-3′) and STAT5B exons 16-17 (5′-AGTGACTCAGAAATTGGCGG-3′ and 5′-GGCC TGGTCCATGTACGT-3′). ARMS assays were designed for JAK3 V722I and SOCS1 Q201H. For JAK3 V722I, a standard tetra primer assay was designed using PCR primers: forward outer (FO), 5′-CAATAGACCCACCCCAATCTCCCCA-GAC-3′; reverse outer (RO), 5′-GCAAGGAAGTGGATCC CTGATCCCACTT-3′; reverse wild-type-specific (Rwt), 5′-GCCACGGTCTGGGAAGTGTTTAGTGtCG-3′; forward mutant-specific (Fmt), 5′-ATCCAGGGCACTGATGGGCAT GGTTAT-3′. FO, RO, Rwt and Fmt primers were all used at a final concentration of 0. 5 μM in PCR reactions using Ampli-Taq Gold DNA polymerase, 25 ng genomic DNA and an annealing temperature of 68 °C for 35 cycles. Design of a tetra primer assay for SOCS1 Q201H proved to be difficult and therefore we used a simplified mutation-specific assay with primers forward (F) 5′-CCAGGAGGGGGAGGACCCCCT-CAAGAGG-3′ and reverse mutant-specific (Rmt), 5′-CGC GACTACCTGAGCTCCTTCCCCTTCGAC-3′ at an annealing temperature of 70 °C for 35 cycles. All ARMS assays were validated with control samples, and positive/negative controls were included on each run. The Illumina Trusight Myeloid Sequencing Panel (Illumina, San Diego, CA) was used to screen STAT5B-mutated samples for additional somatic mutations. Samples were processed according to the manufacturer's protocol, run on an Illumina Miseq and results interpreted using Alissa Interpret (Agilent, Cheadle, UK) using a variant allele frequency (vaf) cut off of ≥5%.",
"section_name": "Other mutational analysis",
"section_num": null
},
{
"section_content": "Mononuclear cells from cryopreserved or fresh primary cells were grown in methylcellulose with cytokines, without erythropoietin (Methocult H4035 Optimum), no EPO, Stemcell Technologies (Vancouver, BC, Canada). DNA was prepared for sequencing using the PicoPlex whole genome amplification kit (Rubicon Genomics Inc., Ann Arbor, MI). Colonies were first plucked into 80 µl phosphate buffered saline, spun down and resuspended in 2. 5 µl of PicoPlex cell extraction buffer followed by DNA extraction and amplification according to the manufacturer's instructions. The DNA was then cleaned up using the QIAquick PCR purification kit (Qiagen, Hilden Germany) prior to amplification and Sanger sequencing using primers designed to target specific mutations.",
"section_name": "Colony growth and sequencing",
"section_num": null
},
{
"section_content": "OS probabilities were estimated using the Kaplan-Meier method and compared by the log-rank (Mantel-Cox) test using SPSS v25 (IBM Corporation, Armonk, NY, USA). OS was defined as the time between diagnosis and the date of death or last contact.",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We previously described RNA-seq analysis to search for cryptic fusion genes in 14 patients with MPN-eo or idiopathic hypereosinophilia with a normal karyotype [6]. Reanalysis of these data for possible point mutations identified 10 candidate variants in 6 cases (Table 1 ). No variants were seen in 8 cases, including the 2 previously reported to have DIAPH1-PDGFRB or ZMYM2-FLT3 fusions [6]. Two of the 6 cases had known myeloid driver mutations: JAK2 V617F in case E166 and SF3B1 K666N in case E11076. Of the other variants, STAT5B N642H (NM_012448: c. A1924C) was seen in two cases (E11076 and E11493) and stood out as this is a known driver mutation in lymphoproliferative disorders [16]. Furthermore, this mutation was recently reported in the T-cells and other cell lineages of two young children with eosinophilia [17].",
"section_name": "STAT5B N642H mutation identified by RNAseq",
"section_num": null
},
{
"section_content": "We used an ARMS assay to rapidly screen additional retrospective cases for STAT5B N642H (Fig. 1a ). In an initial screen, we found that 2/30 cases with suspected MPN-eo tested positive compared to 0/74 cases with other chronic myeloid neoplasms. Subsequent screening focused only on cases referred for investigation of eosinophilia: of 1671 cases screened, 23 further cases tested positive. Overall, therefore, we found that 27/1715 (1. 6%) eosinophilia patients harboured STAT5B N642H. For 25/26 cases confirmed by Sanger sequencing, the mutation-specific peak was at a similar height or somewhat lower than the wild type peak, suggesting that the majority of leukocytes were heterozygous for STAT5B N642H. In one sample (E492) only the STAT5B mutant peak was seen suggesting the dominance of a homozygous or hemizygous mutant clone (Supplementary Figure 1 ). In 4/4 cases tested, the STAT5B mutant clone was not detected in cultured T-cells confirming that it was acquired somatically (Supplementary Figure 2 ).",
"section_name": "Identification of additional cases with STAT5B mutations",
"section_num": null
},
{
"section_content": "Other somatic STAT5B and STAT3 mutations have been identified in lymphoproliferative disorders. To test if these mutations might also be associated with persistent eosinophilia, we sequenced known mutation hotspot regions of STAT3 and STAT5B in 153 cases but no variants were seen apart from STAT5B N642H. No human mutations have been reported in STAT5A but due to the high homology with STAT5B we also sequenced part of this gene, but again no variants were found (149 cases).",
"section_name": "Other STAT mutations",
"section_num": null
},
{
"section_content": "We screened all 27 STAT5B N642H cases for mutations in other genes associated with myeloid neoplasms (Fig. 1b ). Overall, mutations were seen in a median of two additional genes (range 0-4), with 4 cases showing no additional variants (Group 1). Of note, 9 cases had SF3B1 mutations (Group 2) of which 4 were detected as sole additional abnormalities, 3 were seen in combination with single mutations in TET2 or DNMT3A and 2 had additional mutations in genes encoding signalling or splicing components. Fourteen cases did not have SF3B1 mutations but instead had a diverse range of epigenetic, signalling, transcription factor and other splicing mutations with no clear patterns emerging in terms of co-mutated genes (Group 3). All additional mutations are detailed in Supplementary Table 1.",
"section_name": "Additional mutations in STAT5B-mutated cases",
"section_num": null
},
{
"section_content": "The clinical features for all cases are summarised in Table 2. There was a preponderance of males (19 males, 8 females) and the median age was 70 (range 7-89; n = 27) with a median eosinophil count at the presentation of 6 × 10 9 /L (range 0. 5-27; n = 26). In most cases eosinophilia was apparent at diagnosis but in 3 cases this was acquired during the course of myelodysplastic syndrome (MDS; cases E2594 and E11837) or MPN (case E13661). Basophilia was noted in several cases. A working diagnosis of idiopathic hypereosinophilic syndrome (HES) had been made in 7 cases prior to the finding of STAT5B N642H and CEL-NOS in 2 cases, supported by the finding of an additional chromosome 8 by cytogenetic analysis in both cases. The Kaplan-Meier estimate for OS of STAT5B-mutated HES cases was only 30 months, which is very short compared to published series [11], and suggests that these cases should be reclassified as CEL-NOS [10]. The remaining 18 cases had been diagnosed with another myeloid neoplasm, most commonly a subtype of MDS/MPN (n = 11). The PB and BM from a representative case with CEL-NOS and STAT5B N642H as a sole abnormality are shown in Fig. 2. \n\nThere was no obvious correlation between the clinical diagnosis and the molecular classification described above. For example, the 7 cases initially diagnosed with HES were split between molecular group 1 (n = 1), group 2 (n = 2) and group 3 (n = 4) and the 9 cases with SF3B1 mutations had been diagnosed with 7 different entities, including HES (n = 2) and 6 WHO-defined subtypes of myeloid neoplasms. There was, however, a clear correlation between molecular features and outcome. Focusing on patients with eosinophilia at diagnosis, the OS for cases in group 3 was markedly inferior to cases in groups 1 and 2 [median 14 months vs. 65 months; hazard ratio (HR) = 8. 1 (95% CI: 1. 9-23); P < 0. 0004, Fig. 3a ]. By contrast age, gender and white cell count were not significantly associated with OS in this relatively small group, whereas eosinophil count approached significance (≤6 × 10 9 /L, 49 months vs. >6 × 10 9 /L, 17 months; P = 0. 06). No significant effect of mutation number was seen when all mutations were considered but, interestingly, when genes strongly associated with age-related clonal hematopoiesis (i. e. DNMT3A and TET2) were excluded from the analysis, the OS for cases with mutations in 2 or more additional genes was significantly worse than that of cases with 0 or 1 additional mutations [median 18 months vs. 50 months; HR = 6. 5 (95% CI: 2. 1-30) P = 0. 001, Fig. 3b]. As for therapy, 5 patients were treated imatinib for at least 2 months but none responded. One patient with KIT D816V-positive systemic mastocytosis with associated hematologic neoplasm (SM-AHN) responded to midostaurin but the response was lost after 9 months. Some patients showed clinical improvement and reduction in eosinophil counts with corticosteroids but in most cases this was partial and/or transient.",
"section_name": "Clinical features associated with STAT5B N642H and other mutations",
"section_num": null
},
{
"section_content": "To understand if the acquisition of STAT5B N642H is an early or late event in the development of myeloid neoplasia, we genotyped myeloid colonies grown from 4 cases with multiple mutations. For case E10926, 6 mutations in 5 genes had been identified by bulk analysis. By colony analysis, we found a major clone with 5 mutations that included STAT5B N642H and KRAS V14I but not KIT D816V, and a minor clone that included KIT D816V but not STAT5B N642H or KRAS V14I. All colonies tested positive for SF3B1 and two independent TET2 mutations and thus branching evolution can be inferred with STAT5B N642H acquired in a later subclone. For 2 of the other cases, linear evolution was apparent with STAT5B and ASXL1 mutations acquired as late events. The fourth case was uninformative with all colonies positive for the 3 mutations detected on bulk analysis (Fig. 4a ). For two cases with increasing eosinophil counts, the STAT5B N642H vaf, as estimated by Sanger sequencing, increased over time (Fig. 4b ).",
"section_name": "Clonal hierarchy",
"section_num": null
},
{
"section_content": "Other recurrent mutations identified by RNAseq were also considered. Both PRF1 N252S and SOCS1 Q210H were identified in two individuals (Table 1 ). PRF1 encodes perforin 1, a gene associated with familial hemophagocytic lymphohistiocytosis. However, the N252S variant has been shown to be a non-functional rare single nucleotide polymorphism (SNP) and was therefore not considered further [18]. SOCS1 encodes suppressor of cytokine signalling 1, which takes part in a negative feedback loop to attenuate cytokine signalling and therefore has obvious potential relevance to myeloid neoplasia. The status of the Q210H variant is unclear: it is seen at a frequency of 0. 2-0. 6% in the 1000G, ESP and ExAC control datasets, as are some known somatic driver mutations such as JAK2 V617F. For one case, we tested buccal cells and found the variant was constitutional (data not shown). To test the possibility that Q210H might be more widespread in myeloid neoplasia, we tested cases referred for investigation of eosinophilia ( V722I was seen in a single case and has been reported previously as an activating mutation [19] but is also present at 0. 4-1% in control datasets and its pathogenicity has been disputed. We found that 4/307 cases with suspected myeloid neoplasia tested positive for JAK3 V722I (one of which also had the mutation in cultured T-cells) as well as 3/88 normal controls (P = 0. 19), again suggesting it is an irrelevant polymorphism. Similarly, RPS19 T55M is also believed to be a polymorphism [20].",
"section_name": "Assessment of other variants identified by RNAseq",
"section_num": null
},
{
"section_content": "STAT5 is a key component of cytokine-induced signal transduction cascades, and a critical downstream mediator of transformation by oncogenic TKs such as BCR- Sex: male (M) or female (F); Age in years (y) at presentation; Status: dead (D) or alive (A) at specified number of months (m) after presentation or first detection of eosinophilia (E2594, E11837, E13661); white cell count (wcc), platelets (plt) and eosinophils (eos) ×10 9 /L at diagnosis or first detection of eosinophilia; haemoglobin (Hb) in g/L; diagnosis: atypical chronic myeloid leukemia (aCML), hypereosinophilic syndrome (HES), myeloproliferative neoplasm, unclassifiable (MPN-U); myelodysplastic/myeloproliferative neoplasm (MDS/MPN), myelodysplastic syndrome with single lineage dysplasia (MDS-SLD), chronic myelomonocytic leukemia (CMML), multiple myeloma (MM), systemic mastocytosis with associated hematological neoplasm (SM-AHN), MDS/MPN, unclassifiable (MDS/MPN-U), MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), MDS with multilineage dysplasia (MDS-MLD), chronic eosinophilic leukemia, not otherwise specified (CEL-NOS), polycythemia vera (PV); molecular group (MG): 1 = no additional mutations; 2 = SF3B1 mutated; 3 = additional mutations but SF3B1 unmutated; not available (n/a) a Indicates the 4 cases for whom STAT5B N642H was absent in cultured T-cells ABL1, JAK2 V617F, FLT3-ITD and ZNF198-FGFR1 [21] [22] [23] [24] [25]. STAT5 is encoded by two different genes, STAT5A STAT5B, located closely together at chromosome 17q11. 2 that encode proteins with >90% amino acid identity and largely redundant functions. Nevertheless, targeted disruption of STAT5A and STAT5B gives rise to distinct phenotypes in mice and only STAT5B has been reported to be a target of mutations in cancer [26]. STAT5A N642H was initially identified as a constitutively activating mutation in a random ex vivo mutagenesis screen [27]. The corresponding mutation in STAT5B was subsequently identified as a somatically acquired driver mutation in 1-37% of patients with various lymphoid neoplasms including large granular lymphocytic leukemia [16], paediatric T-cell acute lymphoblastic leukemia [28], T-cell prolymphocytic leukemia [29], γδ-T-cell lymphoma [30] and two cases of lymphocyte-driven early onset nonclonal eosinophilia with urticaria, dermatitis and other features [17]. The most frequent mutation seen in these T-cell disorders is STAT5B N642H, but other mutations are also seen, specifically STAT5B Y665F and STAT3 Y640F, N647I and D661V/Y [31]. Isolated reports have identified single cases of myeloid neoplasms, specifically chronic neutrophilic leukemia [32] and MLN-eo [33], that tested positive for STAT5B N642H as well as 2 cases that developed clonal hematopoiesis following aplastic anaemia [34]. \n\nOur findings are the first to identify STAT5B N642H as a recurrent mutation in myeloid neoplasms with eosinophilia. \n\nWe demonstrated the absence of STAT5B N642H in cultured T-cells from 4 cases, one of whom (case E12614) was in molecular group 1, i. e. had no additional mutations. All 4 group 1 cases, and indeed most cases in this study, had STAT5B vafs which suggested the great majority of leukocytes were heterozygously mutated (Supplementary Figure 1 ). Although we cannot exclude the possibility that some T-cell subsets might be part of the mutant clone, our data strongly suggest that STAT5B N642H drives primary eosinophilia irrespective of the presence or absence of additional mutations. \n\nCytokine stimulation results in phosphorylation of STAT5B by receptor or non-receptor TKs. Dimerization of tyrosine phosphorylated (pY) STAT5B is mediated by trans-SH2 domain/phosphotyrosine binding, and the dimers then translocate to the nucleus and activate transcription of target genes [26]. N642 is located within the SH2 domain of STAT5B, close to the phosphotyrosine-binding loop. Rather than being constitutively active, the N642H mutant shows prolonged pY-STAT5 levels upon cytokine stimulation due to greatly enhanced stability of N642H homodimers [30]. Transgenic expression of STAT5B N642H under the control of the Vav1 promoter (which is believed to be active in all hematopoietic cell types, including stem cells) resulted in transplantable CD8+ T-cell neoplasia. \n\nFig. 2 Peripheral blood film (a), bone marrow smear (b) and bone marrow trephine biopsy section at ×20 (c) and ×100 (d). The blood film showed 40% neutrophils, 28% eosinophils, 4% basophils, 12% lymphocytes and 7% monocytes. The eosinophils were morphologically close to normal with only a very minor degree of vacuolation and hypogranularity but there was an increase in non-lobated forms. Bone marrow cellularity and megakaryocyte numbers were increased. The increased cellularity was due to an increase in all three granulocyte lineages (neutrophils, basophils and eosinophils). The trephine biopsy sections show hypercellularity, disorganisation and an increase in cells of neutrophil and eosinophil lineages. Reticulin was not increased Although eosinophilia was not noted, there was a mild expansion of mature and immature myeloid lineage cells [35]. The apparent absence myeloid neoplasia with eosinophilia in this mouse model may be the result of a number of factors, including the precise expression of STAT5B, the effect of other somatic mutations, the cell type in which STAT5B N642H arises and the rapid onset of Tcell neoplasia. \n\nWe observed a striking influence of additional mutations on patients with STAT5B N642H that mirrors established associations in related myeloid disorders, particularly the relatively good prognostic influence of SF3B1 mutations and the adverse influence of multiple mutations, both of which have been described in MDS [36, 37]. In addition, only one case with an SF3B1 mutation tested positive for one or more mutations in SRSF2, ASXL1 and RUNX1 (S/A/R), known to be an adverse prognostic factor in advanced SM [38], whereas 10/14 cases in group 3 were S/A/R positive (P = 0. 009; Fisher's exact test). \n\nThe combination of JAK2 V617F and SF3B1 mutations is associated with MDS/MPN with ring sideroblasts and thrombocytosis [39]. In our series, the working diagnosis of patients with STAT5B N642H and SF3B1 mutations ranged from HES to MDS/MPN (Table 2 ). Stored material from most cases was not available for central morphological review but we envisage that prospective analysis of new cases will help to define more accurately the features b OS for cases with mutations in 2 or more additional genes (excluding DNMT3A and TET2) was significantly worse than that for cases with 0 or 1 additional mutations (median 18 months vs. 50 months; P = 0. 001). Of the 9 cases with mutations in ≥2 additional genes, 8 were in group 3 Previous studies have shown that STAT5A is required for eosinophil differentiation of cord blood-derived CD34+ [40]. Our findings, however, suggest that STAT5B N642H may be a driver of eosinophilia. First, in most of our cases eosinophilia was apparent at diagnosis and the level of STAT5B N642H as assessed by Sanger sequencing indicated that the mutation was present in the majority of cells. In 4 of these cases STAT5B N642H was detected as a sole abnormality, although we cannot exclude the possibility of mutations in genes not covered by the myeloid panel. Second, analysis of 2 cases with increasing eosinophil counts showed an increase in the STAT5B N642H vaf. Three additional cases acquired eosinophilia during the course of their disease but unfortunately samples were not available for analysis from the pre-eosinophilia phase. \n\nThe finding that STAT5B N642H shows prolonged activation following cytokine signalling suggests that targeting upstream TKs may ameliorate the activity of mutant STAT5B. Indeed, the T-cell neoplasms induced by transgenic STAT5B N642H was markedly suppressed by JAK1/2 inhibition [35]. Furthermore, a small molecule inhibitor of STAT5B dimerization has been shown to inhibit the growth of FLT3-ITD positive AML cells [41]. Further studies will be required to determine if myeloid disorders associated with STAT5B N642H are targetable with small molecule inhibitors.",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Compliance with ethical standards",
"section_num": null
},
{
"section_content": "Acknowledgements This study was funded by Bloodwise Specialist Programme Grant no. 13002 to NCPC, WJT and AC. We thank Professor Satu Mustjoki, Helsinki for providing control samples with STAT5B mutations.",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Conflict of interest",
"section_num": null
},
{
"section_content": "The authors declare that they have no conflict of interest. \n\nOpen Access This article is licensed under a Creative Commons Attribution 4. 0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4. 0/.",
"section_name": "Conflict of interest",
"section_num": null
}
] |
10.1371/journal.pone.0116791
|
A Comparison of Assays for Accurate Copy Number Measurement of the Low-Affinity Fc Gamma Receptor Genes FCGR3A and FCGR3B
|
Le locus FCGR3 codant pour le récepteur d'activation de faible affinité FcγRIII, joue un rôle vital dans l'immunité déclenchée par les fonctions effectrices et régulatrices cellulaires. Il a déjà été rapporté que le numéro de copie des gènes FCGR3A et FCGR3B affecte la sensibilité à plusieurs maladies auto-immunes et affections inflammatoires chroniques. Cependant, de telles études d'association génétique donnent souvent des résultats incohérents ; elles nécessitent donc des tests robustes avec un faible taux d'erreur. Nous avons étudié la précision et l'efficacité de l'estimation de FCGR3 CNV en comparant Sequenom MassARRAY et le rapport paralogue test-restriction enzyme digest variant ratio (PRT-REDVR). De plus, étant donné que de nombreuses études d'association génétique de FCGR3B CNV ont été réalisées en utilisant la PCR quantitative en temps réel, nous avons également inclus l'évaluation de la performance de cette méthode dans l'estimation de la CNV multi-allélique de FCGR3B. Le test qPCR a montré une distribution considérablement plus large de l'intensité du signal, introduisant potentiellement une erreur dans l'estimation du nombre de copies et des taux de faux positifs plus élevés. Sequenom et PRT-REDVR ont tous deux montré un biais systématique moindre, mais Sequenom a biaisé vers un nombre de copies normal (CN = 2). L'écart entre Sequenom et PRT-REDVR pourrait être attribué soit au bruit des effets de lot dans les mesures individuelles. Notre étude suggère que PRT-REDVR est plus robuste et précis dans le génotypage du CNV de FCGR3, mais met en évidence les besoins de plusieurs tests indépendants pour une validation approfondie lors de la réalisation d'une étude d'association génétique avec des CNV multi-alléliques.
|
[
{
"section_content": "Copy number variation (CNV) is defined as genetic variation involving a loss or gain of large segments of DNA (typically over 1 kb), a definition that includes simple deletions and duplications [1]. CNV can affect phenotype by altering gene dosage, disrupting coding sequences, or perturbing long-range gene regulation [2], and has been associated with susceptibility to various autoimmune and infectious diseases [3] [4] [5] [6] [7]. \n\nFcγ receptors function as cell surface receptors for the Fc region of IgG, are found on the surface of natural killer cells, monocytes, neutrophils, and mast cells, and play a critical role in immunity. In humans, there are three main types of Fcγ receptors, high-affinity FcγRI and low affinity FcγRII and FcγRIII [8]. They bind to IgG-antigen immune complex and initiate either inhibitory or activating responses within the cell [9]. FcγRII receptors are encoded by FCGR2A, 2B, 2C and FcγRIII receptors are encoded by FCGR3A and FCGR3B respectively, and these genes are located as a cluster at 1q23. 3. FCGR3A and FCGR3B encode FcγRIIIA and FcγRIIIB respectively, which are different from each other both in their attachment to the cell surface and in their expression pattern: FcγRIIIA has a transmembrane region and is expressed on natural killer cells, and FcγRIIIB is attached to the cell membrane by a glycophosphoinositol anchor and is expressed primarily on neutrophils [10]. FCGR3A and FCGR3B are paralogous genes each on an 82 kb segmental duplication which is ~98% identical at the DNA level. FCGR3B carries the two common alleles of the human neutrophil antigen HNA1 that differ by four amino acid substitutions: HNA1a and HNA1b [11]. Recurrent non-allelic homologous recombination between the two segmental duplications generates CNV within populations affecting both FCGR3A and FCGR3B [12] [13] [14]. \n\nThe CNV of FCGR3B has been reported to be associated with susceptibility to a number of autoimmune diseases including systemic lupus erythematosus (SLE), organ-specific autoimmunity, and rheumatoid arthritis [14] [15] [16]. However, genetic association studies for multiallelic CNV remains problematic as spurious copy number calls could lead to false association thus yield inconsistent replications [17] [18] [19]. This is predominantly due to lack of robust and accurate methods in assaying such genetic variation. Therefore in this paper, we compare three typing assays: paralogue ratio test-restriction enzyme digest variant ratio (PRT-REDVR) [13], real-time qPCR [16] and Sequenom MassARRAY (Sequenom Inc. Brisbane, Australia).",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "",
"section_name": "Ethical approval and subject recruitment",
"section_num": null
},
{
"section_content": "The QiaAMP Blood Mini Kit (Qiagen, Germany) was used to extract DNA from 200 μl of peripheral blood sample according to the manufacturer's protocol. Concentration and purity of the extracted DNA were determined by spectrophotometer Nanodrop ND-1000. The quality of DNA was confirmed by electrophoresis on a 1% agarose gel, followed by SyBr safe staining to ensure that the DNA samples were not degraded based on the clear single band generated.",
"section_name": "DNA extraction",
"section_num": null
},
{
"section_content": "The primer sequences for the target gene FCGR3B and the reference gene FOXP2 were obtained from Fanciulli et al. [16]. A total of 5 μl (10 ng/µl) genomic DNA was amplified in a reaction mixture containing 12. 5 μl iQ SyBr Green Supermix (BioRad), 1 μl (7 μM/μl) of each of forward and reverse primers, and made up to total volume of 25 μl with ddH 2 O. Cycling conditions were 95˚C for 3 min, and then 40 cycles of 95˚C for 30 s, followed by 60˚C for 15 s and 72˚C for 30 s. The FCGR3B primer sequences were: Forward: 5 0 -CACCTTGAATCTCATCCC-CAGGGTCTTG-3 0 and Reverse: 5 0 -CCATCTCTGTCACCTGCCAG-3 0. The amplification was carried out using BioRad CFX96 Touch Real-Time PCR Detection System. \n\nThe efficiency of the assay was determined by the generation of a standard curve: a series of five-fold dilutions of a single genomic DNA sample from 50 ng/μl to 0. 08 ng/μl (10 ng to 0. 016 ng in each qPCR). All reactions were run in triplicate. For a high quality assay, amplification should be linear across the entire dilution series with amplification efficiencies between 90-110%. Normalization to the control gene Forkhead Box P2 (FOXP2) (Forward: 5 0 -TGA-CATGCCAGCTTATCTGTTT-3 0 and Reverse: 5 0 -GAGAAAAGCAATTTTCACAGTCC-3 0 ) [16] was used to give an estimate of copy number. Copy number of the target sequence in each test sample was determined by using the comparative CT (2-CT) approach. The relative copy number of the target gene in each test sample in relation to the reference gene was determined by relative quantification through the comparative CT (ΔΔCT) calculation method as previously mentioned by Livak and Schmittgen [20]. Assumption of exact doubling of the target sequence was made in this method. After normalisation to the median value of all samples in the assay, which was assumed to represent a diploid copy number of 2, deletions of FCGR3B were called for samples with a value <1. 5, and duplications of FCGR3B were called for samples with a value >2. 6. These values were chosen based on clustering of raw copy number values of the entire sample set.",
"section_name": "Real-time quantitative PCR (qPCR)",
"section_num": null
},
{
"section_content": "Sequenom MassARRAY uses the primer extension approach for both relative, and absolute determination of CNV calls [21]. For both FCGR3A and FCGR3B three assays were designed at the 3-prime, 5-prime and centre of each gene, targeting a single nucleotide variant (SNV) that distinguished the two paralogues [22] (Table 1 ). In effect, these SNV sites were being used for paralogue-specific quantification, hence the relative signal from each paralogue was investigated. All test samples were assayed with one multiple primer master mix, and primer extension PCR was performed. The extended products were analysed by SEQUENOM MALDI-TOF mass spectrometry. \n\nTable 1. Primer sequences and single nucleotide variation (SNV) identified for FCGR3A/B in Sequenom MassARRAY.",
"section_name": "Sequenom MassARRAY genotyping",
"section_num": null
},
{
"section_content": "Sequence SNV identified There are two steps in Sequenom MassARRAY allowing the inference of genomic copy number. The first stage relies on comparison of an amplicon for the region of interest with a known amount of a competitor DNA amplicon of known genotype, using primer extension and mass spectrometry to quantify the amount of each variant at a particular SNV. Comparison of the ratio of the genomic amplicon with the competitor DNA provides information about the copy number of the region of interest. The second stage relies on a comparison of the amount of amplicon for the region of interest and the amount of a control amplicon where diploid copy number is assumed to be two, co-amplified from the same genomic DNA. Three control regions were included in the study: i) chr11: 31,700,000-31,880,000; ii) chr6: 43,864,065-43,904,064; iii) chr7: 113,800,000-114,150,000, reference coordinates based on genome assembly build hg18. Approximately 100-120 bp of oligonucleotide sequence corresponding to the control and test regions was synthesized and titrated by serial dilution to optimise the iPLEX experiment (Sequenom) for equal peak sizes for the control regions and test region. The 2N controls were used to normalize intra-assay sample to sample loading variation, and the CNV of interest against a normal control. \n\nThe details of analysis of copy number calls for MassARRAY are described in the Sequenom Quickguide [21]. In brief, absolute copy number was calculated from the logEC50 value generated via the titration series in Sequenom QGE software provided by Sequenom Inc. The EC50 value is the point at which the peak areas of the test DNA and the competitor DNA are equal, representing a 1:1 concentration of the molecules in the reaction (ratio 1:1). This point is defined as the effective concentration required for obtaining 50% of the maximal effect (EC50). Subsequently the absolute copy numbers obtained were further normalized as was done in the qPCR mentioned earlier.",
"section_name": "Primer",
"section_num": null
},
{
"section_content": "The PRT-REDVR assays were carried out as described according to Hollox et al. [13]. Briefly the amplification of 10 ng of DNA was performed in a final volume of 10 μl, with 0. 5 μM forward primer and 0. 5 μM FAM-or HEX-labelled reverse primer, in a reaction buffer. The primer sequences used are shown in Table 2. The two amplifications were carried out (with FAM-or HEX-labelled primer respectively), to allow detection and co-electrophoresis of the amplicons on the capillary electrophoresis. This allows internal calibration of each experiment. Products were amplified using 30 cycles of: 95˚C for 30 s, 56˚C for 30 s and 70˚C for 30 s followed by single chase of 56˚C for 1 min then 70˚C for 20 min to reduce levels of single-stranded DNA products. After the PCR cycle, 1 µl of a 10% to 20% dilution of each PCR product was added to 10 µl deionized formamide, and analysed by electrophoresis on an ABI 3100 Genetic Analyzer (Applied Biosystems, Warrington, UK), with an injection time of 30 s. \n\nThe respective PCR amplicons sized 67 bp corresponding to chromosome 1, and 72 bp corresponding to chromosome 18, were recorded for both FAM-and HEX-labelled products. The ratio of the areas under the 67 bp peak and the 72 bp peak was compared, and the results were accepted if the coefficient of variation (standard deviation divided by the mean) was <0. 15. The mean of the FAM and HEX ratio was used in further analysis. Both the mean ratios and 9 reference standards with known copy numbers obtained from Human Random Control DNA (European Collection of Cell Cultures) were used as the calibration for each experiment, and the resulting linear regression was used to estimate the copy number of FCGR3 for the samples studied, as described in [13].",
"section_name": "Paralogue Ratio Test (PRT)",
"section_num": null
},
{
"section_content": "Two REDVR assays were used in this study: one distinguishes variant from FCGR3A and FCGR3B (c. 733C>T, corresponding to the arginine to stop codon change that defines FCGR3B) and the other distinguishes neutrophil antigens HNA1a and HNA1b (g. 147C>T) [13]. Amplification of two regions in duplex was carried out using primer sequences as shown in Table 3 with concentration of 0. 5 μM and the conditions described above, except with an annealing temperature of 53˚C: 2 μl of PCR product was digested with 10 units of Taq α I restriction enzyme (New England Biolabs) in 50 mM Tris-Cl (pH 7. 9 at 25˚C), 100 mM NaCl, 10 mM MgCl 2,1 mM dithiothreitol in a final volume of 10 μl for 4 hours at 65˚C. Digested products were analysed with capillary electrophoresis on an ABI 3100 Genetic Analyzer (Applied Biosystems) and analysed with GeneScan software (Applied Biosystems). Mean ratios of the product were used along with the reference standard for experimental calibration and the result was used to estimates copy number calls. \n\nThe PRT analysis for copy number call was performed in combination with the REDVR analysis, using a maximum likelihood approach described previously [13]. PRT produces the sum of the copy number calls for FCGR3A and FCGR3B genes whilst the REDVR estimates the copy numbers of FCGR3A and FCGR3B based on the ratio determined [13]. Analyses were performed using Microsoft Excel unless otherwise stated.",
"section_name": "Restriction Enzyme Digest Variant Ratio (REDVR)",
"section_num": null
},
{
"section_content": "We first assessed the distribution of the raw copy number calls for FCGR3A from Sequenom (Fig. 1A ). Distinct clustering was observed around the integers, indicating relatively high consistency of this assay in FCGR3A. 81% (101/161) of the absolute copy number from Sequenom were in agreement with PRT-REDVR, but the Sequenom data tended to be skewed towards a diploid copy number of two (Fig. 1B ; S1 Table ). A higher rate of inconsistency between these two assays was observed with higher copy number (Fig. 2 ). \n\nNext, we analysed the copy number calls for FCGR3B with the sample genotyped with all three assays namely, qPCR, Sequenom and PRT-REDVR (N = 80). In general, copy number called by qPCR showed a greater variability (gains or losses) (Fig. 3 ), and relatively broader distribution compared to Sequenom (Fig. 4 ), suggesting lower specificity of qPCR, in line with the previous report [23]. The performance of the assays was assessed by matching the data for PRT, qPCR and Sequenom (Fig. 5 ; S2 Table ). qPCR revealed a lower consistency with the two assays (Fig. 5A-C ); whereas the raw copy number call from Sequenom was in higher agreement with PRT-REDVR (Fig. 5D ). We observed that 78. 2% of the copy number calls for FCGR3B were in agreement between PRT-REDVR and Sequenom; while qPCR showed a lower concordance rate with PRT-REDVR (60. 1%) and Sequenom (75. 4%) (Table 4 ; S2 Table ). \n\nThe discordance increased with the increase of copy number calls. The concordance rate for all the three assays though, has been relatively poor (57. 3%). This observation agrees with the report from the previous study [23]. CNV typing was repeated on the 10 of the discrepant samples by qPCR and PRT-REDVR and showed that six of these samples the discrepant measurements are reproducible. We speculate that this could be due to small repeat specific deletions, or perhaps reflect single nucleotide variation underneath oligonucleotide primers that reduce or abolish amplification from one repeat [13]. \n\nCollectively, our analyses strongly suggest that, i) the higher copy number, the greater discrepancy in CNV estimation between assays; and ii) neither Sequenom nor qPCR assay is suitable for multi-allelic copy number variation genotyping. \n\nTo assess the quality of the copy number calls from PRT, we showed that both FAM-and HEX-labelled PRT duplicates were highly correlated, except for one outlier (Fig. 6 ). Although the clusters overlap, final copy number calls are made using information both from the PRT data and the two REDVR assays. The raw Sequenom data (logEC50) generated from the three distinct probes did correlate well with each other, but was slightly affected by the distance between probes especially with the highly copy number variable FCGR3B (Fig. 7 ). However the advantages of having three probes outweighs the disadvantages. Another potential drawback of the Sequenom assay is the batch effect observed (Fig. 7 ), which indicates that large changes in absolute copy number can be generated by batch effect alone, although comparison with the control probes may reduce this effect in the relative copy number estimates. \n\nPRT is a PCR-based assay using a single pair of primers to simultaneously amplify two specific products in a single reaction: one from a single-copy reference locus and the other from the copy variable test locus of interest-in this case, the FCGR3 region. The copy number of the test locus is then estimated from the ratio of test to reference PCR products. \n\nPRT relies on co-amplification of the test and reference locus using the same primer pair, with test and reference distinguished by a small deletion, subsequently differentiated and quantified using capillary electrophoresis. The primer pairs were designed in such a way that that it amplifies a unique reference locus on chromosome 18 [13]. The test and reference PCR products can be distinguished by a small difference in size [13]. One limitation of this PRT assay is that its development requires the presence of paralogue DNA sequence in the CNV regions to allow comparison of ratio between reference and test locus, which at times not applicable to many CNV loci. In addition, it only amplifies copies of a generic FCGR3, owing to the fact that this region shows up to 98. 3% sequence homology, posing a challenge in designing primers specific for FCGR3A and FCGR3B CNV genotyping. Therefore, a multiplex REDVR assay was used to amplify across the nucleotide which changed an arginine in FCGR3A to a stop codon in FCGR3B, and a nucleotide that distinguished HNA1a from HNA1b. The a/b variant can be distinguished by digestion using Taq α I. The ratio of 3A:3B and HNA1a:HNA1b also provides extra information on copy number helping distinguish copy numbers. \n\nIn both PRT and REDVR, inter-experiment calibration was corrected by running known controls showing high agreement for total copy number between methods. This highlights the advantage of this assay whereby it generates more reliable integer copy number calls with inter-validation whilst easing the interpretation. Our study suggests that qPCR could potentially introduce false-positive calls, therefore CNV association studies based on qPCR should be counter validated. Indeed, many studies showed contradictory findings on copy number and association with disease development, for instance CCL3L1 in HIV [24], and DEFB4 in Crohn's disease [19, 25]. The disadvantage of qPCR though is common across copy number assays and this is expected in view of the principle of CNV and the chemistry of qPCR amplification. The absolute values also vary usually upon repetition since qPCR is technically demanding. This again is a common phenomenon observed in most qPCR assays [26]. \n\nReports of CNV genotyping based on Sequenom have been relatively lacking. However, more conservative calls in this assay potentially dilute true positive calls of CNV. \n\nIn summary, we suggest that qPCR assay is not suitable for the use in large scale case-control association studies for multi-allelic genes like FCGR3. On the other hand, PRT-REDVR presents a relatively more reliable result as compared to both qPCR and Sequenom MassAR-RAY [24]. The sequence requirements for PRT assay development mean that it is limited to certain sequences, and an assay cannot necessarily be designed for a small sequence region, for example, one specific exon. However, the precision and accuracy of PRT-REDVR are equivalent to those of MLPA and MAPH, and it has the advantage of potential high-throughput analysis with the small amount of DNA required as of PCR-based methods [27]. Advantages further include the robustness of assay, small amount of DNA, though potentially introducing false negative result, running cost, and feasibility of equipment. The DNA integrity though is a major determining factor for the specificity and robustness of an assay. Independent assay such as microsatellite therefore, should warrant the informativeness of this technology [13].",
"section_name": "Results and Discussion",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "The authors thank all medical officers and staff nurses from Hospital Kota Bharu, Hospital USM and Hospital Sungai Buloh for assisting in sample recruitment.",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This study is supported by UiTM DANA Kecemerlangan ( 600-RMI/ STDANA 5/3/Fst (281/ 2009 )), FRGS 2010 ( 600-RMI/ ST/FRGS 5/3/Fst (69/ 2010 )), and Long-term Research Grant Scheme (LRGS) for Infectious Disease 2011 ( 600-RMI/ LRGS 5/3 (3/2011) ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.",
"section_name": "",
"section_num": ""
}
] |
10.3389/fimmu.2021.784691
|
Bone Marrow Lymphoid Niche Adaptation to Mature B Cell Neoplasms
|
<jats:p>B-cell non-Hodgkin lymphoma (B-NHL) evolution and treatment are complicated by a high prevalence of relapses primarily due to the ability of malignant B cells to interact with tumor-supportive lymph node (LN) and bone marrow (BM) microenvironments. In particular, progressive alterations of BM stromal cells sustain the survival, proliferation, and drug resistance of tumor B cells during diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL). The current review describes how the crosstalk between BM stromal cells and lymphoma tumor cells triggers the establishment of the tumor supportive niche. DLBCL, FL, and CLL display distinct patterns of BM involvement, but in each case tumor-infiltrating stromal cells, corresponding to cancer-associated fibroblasts, exhibit specific phenotypic and functional features promoting the recruitment, adhesion, and survival of tumor cells. Tumor cell-derived extracellular vesicles have been recently proposed as playing a central role in triggering initial induction of tumor-supportive niches, notably within the BM. Finally, the disruption of the BM stroma reprogramming emerges as a promising therapeutic option in B-cell lymphomas. Targeting the crosstalk between BM stromal cells and malignant B cells, either through the inhibition of stroma-derived B-cell growth factors or through the mobilization of clonal B cells outside their supportive BM niche, should in particular be further evaluated as a way to avoid relapses by abrogating resistance niches.</jats:p>
|
[
{
"section_content": "B-cell non-Hodgkin lymphomas (B-NHL) are a heterogeneous group of hematological malignancies that emerge from different stages of normal mature B-cell differentiation (1). Lymphoma evolution and treatment are complicated by a high prevalence of relapses (2) primarily due to the ability of malignant B cells to interact with protective lymph node (LN) and bone marrow (BM) microenvironments (3) (4) (5). In agreement, several studies have correlated BM involvement with worsened prognosis and impaired chemotherapeutic response in B-cell lymphomas (6) (7) (8). This review delves into the current knowledge of the BM stromal cell modifications induced by the protumoral niche establishment in B-NHL with a specific focus on diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL). Interestingly, these three B-NHL subtypes displayed various BM involvement, with 11%-34% of DLBCL (9, 10), 70%-80% of FL (11), and virtually all CLL cases showing BM infiltration at diagnosis (Table 1 ). Moreover, this review highlights the newly described role of extracellular vesicles (EVs) in the seeding of the BM niche. EVs are released during homeostasis and cell activation, with pleiotropic effects on signaling between cells. EV cargos are enriched in nucleic acids, proteins, and lipids. Briefly, the International Society of Extracellular Vesicles had classified EVs into three main groups: i) exosomes, the small vesicles with diameters ≤100-150 nm that are formed inside multivesicular bodies; ii) microvesicles, medium-size vesicles of plasma membrane origin with diameters of up to 1000 nm; and iii) apoptotic bodies, the large vesicles with diameters > 1000 nm that are produced by apoptotic cells (12). Excellent reviews on the biomolecular and functional characteristics of EVs as well as on the techniques used for EV isolation and characterization have recently been published (13, 14). \n\nDLBCL is the most common aggressive B-NHL and accounts for approximatively 24% of new NHL cases (15). Gene expression analysis and study of genomic alterations have identified distinct genetic subtypes in DLBCL, reflecting differential pathogenesis, and associated with distinct clinical behavior (16) (17) (18) (19). Interestingly, recent studies have highlighted the impact of tumor microenvironment (TME) heterogeneity on tumor B-cell biological features and on DLBCL patient outcome (20, 21). \n\nFL accounts for about 20% of adult lymphoma and is an indolent disease characterized by prolonged periods of remissions preceding relapses and ultimate transformation into DLBCL in about 30% of cases. The genetic hallmark of FL is the t (14, 18) translocation occurring during the V(D)J recombination of immunoglobulin genes in the BM. The resulting deregulation of BCL2 provides a selective survival advantage to B cells during the germinal center (GC) reaction, triggering illegitimate recirculation of t (14, 18) pos post-GC B cells detectable in most healthy individuals. Iterative (re)entry of these FL precursor cells inside GC favors accumulation of additional genetic alterations sometimes converging towards overt FL (22). Importantly, FL is the paradigm of a neoplasia fully dependent on a complex microenvironment network that coevolves with tumor B cells to create a tumor supportive niche in both LN and BM (23, 24). \n\nCLL is the most common hematologic malignancy in adults in Western countries. CLL is preceded by a stage of monoclonal B-cell lymphocytosis and is characterized by the accumulation of mature clonal B cells resistant to apoptosis in the blood, BM, and lymphoid organs. Patients with CLL have a heterogeneous clinical course with some never needing treatment, while others require treatment immediately after diagnosis or during illness due to a more symptomatic and unfavorable clinical course. In typical CLL cases, the tumor B cell clone exhibits an abnormal expression of markers like CD5, CXCR4, and ZAP-70, that are used to stratify the disease in conjunction with the mutational status of the BCR reflecting different cell of origin (25, 26). Despite fully disseminated presentation, TME provides crucial survival signals to malignant CLL cells within the proliferation centers of LN and BM (27). \n\nIn these three mature B-cell neoplasms, specialized tumor niches support survival, proliferation, and drug resistance of tumor B cells. These highly heterogeneous niches include defective tumor immunity, due to altered recruitment and cell exhaustion of cytotoxic cells, to the amplification of immunosuppressive cells, or to immune escape mechanisms developed by tumor B cell themselves, hampering tumor recognition, immune synapse formation, or anti-tumor cell activation (23, 24, 27). Conversely, fully functional tumor permissive cells, including CD4 pos T cell, myeloid cell, and stromal cell subsets, could be found. The relationship between LN and BM protumoral niches and how the similarities and differences between these microenvironments could impact malignant B-cell features remains elusive. In FL, malignant B cells found in the BM are characterized by a lower cytological grade, a decreased proliferation, and a reduced CD10 expression compared with LN FL B cells (28). Moreover, their gene expression profile reflects their reduced proliferation and active metabolism (29). Finally, somatic hypermutation analysis and targeted deep sequencing demonstrate that different FL B-cell subclones could be detected within LN versus BM, and suggested that FL originates in the LN and infiltrates BM early in the course of the disease, allowing further accumulation of BM-specific (28, 30, 31). Besides the exact cell composition and supportive signals provided by BM niches, a major issue remains to establish how these niches evolve during tumor development, from the pre-tumoral stage to overt lymphoma, during remissions and relapses.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "BM constitutes the primary site for the maintenance and differentiation of hematopoietic stem cells (HSCs) and for Bcell lymphopoiesis. Different stromal cell niches dynamically control these processes. Seminal papers have recently proposed a molecular atlas of the BM stromal cells at the single cell resolution, including osteoblasts, perivascular cells, endothelial cells, and mesenchymal stromal cells, providing clues on how various stromal cell subtypes could interact with HSCs and differentiating B-cell subsets (32) (33) (34). In the context of B-NHL, dynamic interactions between BM stromal cells and tumor B cells have been described to play a key role in converting the BM TME into a tumor supportive niche (34) (35) (36). DLBCL, FL, and CLL display distinct patterns of BM infiltration (Table 1 ). DLBCL show a mixed pattern of BM involvement that can potentially range from localized focal infiltrates to complete disruption of BM by lymphoma cell proliferation (37). In contrast, FL infiltration is primarily localized to the paratrabecular regions as nodular aggregates admixed with lymphoid-like TME (38). In CLL several BM infiltration patterns can be found including mixed nodular-interstitial, interstitial, and diffuse (39). In each cases, stromal cells exhibiting specific functional phenotype support recruitment, survival, and proliferation of tumor B cells, mimicking the cancer-associated fibroblasts (CAFs) described in solid cancers.",
"section_name": "LYMPHOMA BM STROMAL MICROENVIRONMENT",
"section_num": null
},
{
"section_content": "BM DLBCL-CAFs have been poorly explored in situ. In contrast, in FL, BM-CAFs, like their LN counterparts, overexpress CXCL12 involved in the recruitment, adhesion, and activation of FL B cells (40) (Table 1 ). Moreover, they ectopically express CXCL13 and CCL19, the two lymphoid chemokines classically expressed by LN follicular dendritic cells (FDC) and fibroblastic reticular cells (FRC) respectively, thus recreating GC-like structures able to recruit and support CXCR5 pos CCR7 pos FL B cells (41, 42). \n\nCLL B lymphocytes could be attracted in vitro to BM stromal cells whose protective effects require close cell proximity (43) (44) (45). This colocalization of CLL tumor cells with their supportive stromal cell niche relies on the deregulation of several chemokine pathways (Table 1 ). The demonstration that the clinical efficacy of BCR inhibitors in CLL is mediated, at least in part, by the inhibition of chemokine receptor activity and the corresponding mobilization of tumor cells out of their protective niches further highlights the crucial role of stromal cell-derived chemokine in CLL survival (46). First, high expression of CXCR4 on the surface of peripheral blood CLL cells triggers their migration to BM stromal cells producing CXCL12 (45, (47) (48) (49). CXCR4 surface expression is regulated by its ligand, thus explaining the decrease in CXCR4 expression on tissue tumor B cells, while recirculating CLL B cells express high levels of CXCR4. In parallel, blood CLL cells express high amounts of CCR7 (50). Indeed, the recycling of CXCR4 and CCR7 receptors is potentiated in CLL cells and contributes to their stronger expression (51). Recently, it was shown that p66Shc (SHCtransforming protein 1), which limits the recycling of CXCR4 and CCR7 by inhibiting their de-phosphorylation, is deficient in CLL (52). Interestingly, CCR7 could also form heterodimers with CXCR4 thus disrupting the CXCR4/CXCL12 downstream signaling and reducing B-cell retention within BM (53). Furthermore, other proteins expressed by CLL cells, such as ZAP70 or CXCR7 have been shown to regulate the function of CXCR4 (54, 55). Altogether, the modulation of CXCR4 function could regulate the homing capacity of CLL cells within BM. Second, CXCR5, the CXCL13 receptor, is also expressed at high levels by CLL cells (56, 57). However, conversely to the ectopic induction of CXCL13-expressing FDC in FL BM, CXCL13 seems to be only involved in CLL B cell homing into LN and the increase of CXCL13 level in the plasma of CLL patients is correlated with LN size but not BM infiltration (58). Finally, integrin a4b1 (VLA-4) plays a prominent role in the homing of CLL cells to BM niches. VLA-4 major ligands, fibronectin and VCAM-1, are constitutively present on BM stromal cells and endothelial cells and are upregulated by inflammatory signals in a NF-kB-dependent manner (59). In mouse xenograft models, CLL cells from VLA-4 neg patients showed significantly lower BM homing rates than those from VLA-4 pos patients. In contrast, the spleen homing rates did not significantly differ. Clinically, the VLA-4 status directly drives in the extent of human BM infiltration (60).",
"section_name": "BM Stromal Cells Support B-Cell Recruitment",
"section_num": null
},
{
"section_content": "In DLBCL, the upregulation of Notch-3 in tumor cells under close cell-cell contact with BM-derived stromal cells has been implicated in the development of aggressive lymphoma cells (61). In turn, such direct interaction between DLBCL cells and stromal cells mediates an increase in B-cell activating factor (BAFF) expression by stromal thus resulting in a decrease of chemotherapy-induced B-cell apoptosis (62, 63) (Table 1 ). One of the factors involved in the regulation of DLBCL B-cell interaction with the BM stromal niche is the level of Jun expression. Indeed, Jun-regulated genes mediate the interaction of malignant cells with stromal cells and extracellular matrix proteins and impact extranodal localization (64). There is also evidence for tumor permissive effects of BM stromal cells on DLBCL cells through secretion of IL-6 and IL-17A, which promote both cell proliferation and drug resistance (8). Finally, the crosstalk between malignant B cells and stromal cells in DLBCL could also impact metabolic reprogramming in DLBCL. DLBCL have been early considered as metabolically heterogeneous (65, 66). Non-malignant cells from TME including stromal cells have been proposed to contribute to DLBCL metabolism by providing metabolic intermediates (67) but no data specifically address this issue in BM versus LN niches even if the use of specific metabolic inhibitors have been recently explored in some DLBCL subsets (68). \n\nIn FL, tumor B cells are strongly dependent on direct interactions with a microenvironment close to that of normal GC, including in particular follicular helper T cells (Tfh), myeloid cells, and lymphoid stromal cell subsets (23, 24, 69). The protumoral role of infiltrating lymphoid stromal cells has been demonstrated in particular by the identification of ectopicallyinduced FRC-and FDC-like cells within invaded BM (40, 70). To date the origin and heterogeneity of the stromal cells supporting FL B cells within LN and BM are not perfectly understood and it is very likely that several FL CAF subtypes co-exist and organize different cell niches with specific functions (38). Stromal cells supporting FL B cell survival have been initially identified as lymphoid-like stromal cells obtained in vitro by stimulation of BM mesenchymal precursors by TNF-a (TNF) and Lymphotoxin-a1b2 (LT) or by direct contact with malignant B cells (3). Interestingly, BM stromal cells obtained from FL patients display a specific gene expression profile even after in vitro amplification, suggesting an imprinting on these cells by the tumor context (40, 63, 71). VLA-4, which is expressed by FL-CAFs, is involved in the growth of GC lymphomas and their resistance to anti-CD20 treatments (72). In vitro, FL stromal cells decrease tumor B cell apoptosis through a set of partially resolved mechanisms, including the production of hedgehog ligands (Hh), BAFF and TGF-b, over-expression of ABC-type multi-drug transporters, and activation of a c-MYC/HDAC6 loop in tumor cells (24, 73). Moreover, CXCL12 contributes to FL B cell activation and synergize with BCR signaling (40). To date, the metabolism of FL remains broadly unexplored. Gene expression profile of FL B cells obtained from medullary niche reveals a decreased expression of the genes involved in of glycolysis, fatty acid synthesis, and OxPhos pathway compared to LN B cells (29). However, the role of stromal cells from BM versus LN niches in FL B-cell metabolic reprogramming remains to be evaluated. \n\nCLL B cells could interact with stromal cells via different receptor/ligand couples including ICAM-1/LFA-1 (74), VCAM-1/ VLA-4 (75-78), CXCR5/CXCL13 (79), BCMA/BAFF, or TACI/ BAFF (80), or by transpresentation of IL-15 from stromal cells to B cells (81). Amon those, ICAM-1, VCAM-1 and BAFF have been shown to be expressed by BM stromal cells. These interactions could lead to leukemic cell survival via a CD44-dependent mechanism involving up-regulation of MCL-1 in CLL B cells (82), activation of NF-kB pathway (80), and result in migration and proliferation of leukemic cells. In the same way, the interaction between CD100 (on CLL B-cell surface) and Plexin-B1 (present on BM stromal cells) extends CLL B cell viability and enhances proliferation (83). The mutual activation of stromal cells and tumor cells also depends on the CLL-mediated activation of Notch2 in BM stromal cells, leading to C1q overexpression the reciprocal activation of the canonical Wnt pathway in CLL cells (84) Moreover, BM stromal cell derived CXCL12 exhibits a pro-survival effect on CLL tumor cells (44, 85, 86). BM Stromal cells may also induce protective epigenetic modifications in CLL B cells including hypomethylation of the lysine 27 of histone H3 protein subunit (H3K27me3) (87). Finally, BM stromal cells have an important role on CLL metabolism. CLL cells have a net increase of reactive oxygen species (ROS) compared to their normal counterpart and are highly sensitive to cellular antioxydants, such as glutathione, to maintain their redox balance. BM stromal cells trigger glutathione synthesis by CLL cells through cysteine release, thus protecting tumor cells from drug-induced apoptosis (88). Moreover, BM stromal cells contribute to the glycolytic shift in CLL cells, at least in part by the Notch/Myc axis, triggering an increased glycolysis associated with higher lactic acid production, glucose uptake, and glucose transportation (89, 90).",
"section_name": "BM Stromal Cells Support B-Cell Survival",
"section_num": null
},
{
"section_content": "Beyond these functions of direct B-cell support, lymphoma CAFs are thought to be the organizers of the tumor niche. A role for the composition of the stromal-cell derived extracellular matrix in the pathogenesis of DLBCL was recently identified within tumor LN, raising the question of its direct and indirect impact on tumor growth, as an example through the modulation of immune cell infiltration, within invaded BM (21). \n\nFL-CAFs overexpress the chemokine CCL2 within invaded BM, thus triggering the recruitment of monocytes that are then converted into pro-angiogenic and anti-inflammatory macrophages (71). FL tumor-associated macrophages have been shown to play a key role in the growth of FL B cells through the transpresentation of IL-15 and the triggering of BCR-dependent signaling involving DC-SIGN-expressing macrophages and oligomannose residues introduced in FL BCR (91, 92). BM and LN FL-CAFs could also promote the recruitment and survival of pro-tumoral neutrophils through the release of large amounts of IL-8 (63). Of note, in DLBCL, tumor cells have been shown to produce themselves IL-8 involved in the recruitment of APRIL-producing neutrophils (93). Moreover, BM and LN FL-infiltrating stromal cells also overexpress the immunosuppressive molecule PGE2 (94) involved in the recruitment or activation of suppressor cells such as Tregs and MDSCs (95). Finally, CAFs have been shown in solid tumors to physically hamper the recruitment of cytotoxic T cells to the tumor and CD8 pos T cells are retained at the periphery of FL tumor aggregates in both LN and BM, suggesting that FL-CAFs could contribute to tumor exclusion in lymphomas (96) (97) (98). \n\nOverall, it is clear that close interactions of tumor B cells with stromal cells within the BM, together with modulation of chemokines and cytokines directly influence the growth of DLBCL, FL and CLL, providing evidence that the BM niche plays a critical role in both lymphoma survival and drug resistance. Regardless of their cell of origin, the mechanisms underlying the differentiation of lymphoma CAFs are of the utmost importance given their potential as therapeutic targets.",
"section_name": "BM Stromal Cells Organize the Tumor Niche",
"section_num": null
},
{
"section_content": "FL tumor B cells could directly contribute to the commitment of BM stromal precursors into an FRC-like phenotype overexpressing CCL2 and IL-8 through TNF-dependent mechanisms (3, 63, 71). Moreover, even if they produce less LT than normal centrocytes, the large number of GC-like B cells ectopically found in invaded FL BM probably contributes to a local overproduction of LT that synergizes with TNF for the induction of lymphoid stroma commitment. However, surrounding non-malignant cells could also participate in the polarization of FL-CAFs. Neutrophils, recruited by IL-8-producing BM FL stromal cells, could in turn contribute to their differentiation into FRC-like cells through activation of the NFkB pathway (63). In addition, LN FL-Tfh overexpress IL-4 which induces a Transglutaminase hi Podoplanin low CD106 hi CXCL12 hi phenotype on human stromal cell precursors. FL-Tfh also produce high amounts of TNF and LT, which sensitize stromal cell precursors to the effect of IL-4, notably through increased expression of the STAT6 signaling molecule (40). Even iffully mature Tfh have not been detected within FL BM, IL-4 and CXCL12 have been shown to be correlated in invaded FL BM (40). Finally, some of the recurrent genetic alterations in FL regulate the re-education of the tumor niche by tumor B cells. In particular, the gain-of-function mutations of the histone methyltransferase EZH2, which occurred early in 20% to 30% of FL, are proposed to uncouple GC B cells from the critical Tfh checkpoint whereas switching them to FDC dependency (99). EZH2-mutated GC B cells downregulate many genes linked to Tfh signaling, fail to engage Tfh, thus limiting recycling toward the dark zone of GC, and survive in the light zone as proliferating centrocytes overexpressing LT, TNF, and BAFFR, all involved in GC B-cell/FDC crosstalk. HVEM loss-of-function mutations detected in about 40% of patients with FL have been associated, in a murine model of FL and in FL patients, with an amplification of Tfh producing large amounts of IL4, TNF, and LT, and able to activate FL-CAF within LN (100). No study had currently evaluated how these genetic events could impact FL TME coevolution within BM. Even if such data are essentially lacking in the context of DLBCL, some recurrent genetic alterations have been recently associated with a specific TME pattern, with some of them related to overexpression of genes associated with GC-like stroma or extracellular matrix/FRC/CAF genes (21). \n\nFinally, LT produced by CLL cells is involved in the polarization and/or in situ generation of the tumor stromal network and the secretion of CXCL13, IL-6, and IL-8 (74, 79). Moreover, the leukemic clone produces retinoic acid in the stromal microenvironment which contributes, at least in part, to the CXCL13 induction (101). \n\nIn addition to the factors described above, tumor derived EVs seem to be involved in the communication between tumor cells and their TME, in particular CAFs. Such mechanism could play a central role in triggering initial induction of tumor-supportive niche within distant sites, including BM.",
"section_name": "EMERGENCE OF THE BM LYMPHOMA STROMAL MICROENVIRONMENT",
"section_num": null
},
{
"section_content": "To date no study has explored the putative involvement of EVs in the induction of a BM lymphoma stromal niche in the context of DLBCL. Moreover, only few studies have investigated the involvement of EVs in the pathophysiology of FL (Figure 1 ). \n\nRecently, FL-derived EVs were shown to modulate the gene expression profile of BM stromal cells, triggering an upregulation of HSC niche factors including CXCL12, angiopoetin-1, KITLG, or IL-7, and increasing the capacity of stromal cells to interact specifically with BM FL B cells and support their survival and their quiescent phenotype (29). Interestingly, the phenotype of EVtreated stromal cells is quite different from that obtained under treatment by TNF/LT or coculture with FL B cells supporting a role of EVs in the activation of BM stromal cells before BM seeding by malignant B cells. In fact, the level of CXCL12 is increased in noninvolved BM plasma suggesting that FL EVs could shape the BM stromal niche before BM infiltration by tumor cells or at distance from this BM infiltration (unpublished data). In the same way, the analysis of the gene expression profile of BM stromal cells highlights a continuum ranging from healthy donor BM stromal cells, to stromal cells obtained from FL patients without BM involvement, and finally from FL-invaded BM (29). Altogether these data suggest that EVs could contribute to CXCL12 upregulation in the absence of direct contact with malignant B cells and could then synergize with IL-4 produced by infiltrating T cells admixed with FL B cells to further enhance local CXCL12 production. Interestingly, BM stromal cells activation by FLderived EVs was shown to rely on TGF-b dependent pathways something that is reminiscent of the role of TGF-b in the B-cell/ stromal cell crosstalk within FL LN (42). How TGF-b and STAT6 pathways could synergize for the acquisition of FL CAF phenotype within FL BM remains to be explored. \n\nBidirectional crosstalk has also been reported between CLL Bcells and their surrounding stroma via EVs (Figure 1 ). CLL B cells release large amounts of exosomes that show strong expression of CD37, CD9, and CD63. Ibrutinib, a Btk inhibitor, significantly reduces the amount of plasma exosomes in CLL patients. Likewise, in vitro treatment of CLL cells with Idelalisib (a PI3K inhibitor) decreases exosome secretion, something that is not observed during treatment with fludarabine (102). This result highlights the role of the BCR-PI3K pathway in controlling exosome secretion in CLL. Besides BCR itself, CLL supportive TME produces BAFF, APRIL, CD31, and plexin B1 that all protect CLL cells from spontaneous apoptosis by synergizing with BCR signaling (44, 103) and could influence EV secretion. The comparison of the mRNA content of EVs produced by B cells from healthy donors versus patients with CLL, and stimulated or not through the TLR9 pathway, shows enrichment for the kinases of the BCR pathway, LYN, SYK, MAPK1, MAPK2, and the antiapoptotic proteins BCL2 and BCL3 in CLL-derived EVs. These EVs released by tumor B cells transfer their mRNA content to non-malignant cells in the TME (104). Microvesicles derived from malignant CLL cells and detected in peripheral blood also deliver the receptor tyrosine kinase Axl into BM stromal cells leading to the activation of a AKT/mTOR/p70S6K/HIF-1a axis resulting in an increase in VEGF synthesis (105). This increase in VEGF is associated with an increased neovascularization in medullary (106) and extramedullary tissues, as well as a paracrine pro-survival stimulation of tumor B cells (107). The miRNA content of CLL B cell-derived exosomes is strongly enriched in miR-21, miR-155, miR-146a, miR-148a, and let-7g (108). BM stromal cells treated in vitro with these CLL exosomes acquire an inflammatory pro-tumoral phenotype, while endothelial cells increase their capacity for angiogenesis (108). These effects are consistent with what is known about the effect of miR-21 and miR-146a in the transition from normal fibroblast to CAFs (109) (110) (111) (112). Indeed, CLL miR-146a pos exosomes induce the transition of BM stromal precursors into CAFs showing overexpression of a-SMA and FAP (113). In addition, CLL exosomes show specific enrichment in miR-202-3p, able to decrease expression of Sufu (a component of the hedgehog pathway) in stromal cells and to trigger stromal cell proliferation (114). Finally, EVs isolated from cultures of CLL BM stromal cells induce a significant decrease in spontaneous apoptosis of tumor B cells and an increase in their chemoresistance to several drugs, including fludarabine, ibrutinib, idelalisib, and venetoclax. In addition, these EVs induce changes in the gene expression profile of CLL cells mimicking the transcriptomic signatures obtained after BCR stimulation (115).",
"section_name": "ROLES OF EVS IN THE INDUCTION OF BM LYMPHOMA STROMAL NICHE",
"section_num": null
},
{
"section_content": "Analyzing the deregulation of extracellular proteins or miRNAs in the blood and tumor niches of patients during B cell tumorigenesis is a reliable tool for the identification of new tumor-targeted therapies. For example, the detailed mode of action of the CD30 antibody-drug conjugate Brentuximab vedotin in DLBCL is not well understood since the clinical outcome seems to be partially independent of the CD30 expression on the tumor cells. However, as CD30 pos bystander cells are enriched in the tumor tissue in many cases of DLBCL, CD30 might be released within TME-derived EVs. Thus a model was proposed in which even in the absence of CD30 on the tumor cells, EVs can transport the targeting protein from cells of the TME to tumor cells (116). This model would explain the clinical efficacy of Brentuximab vedotin also in cases of lack of the targeting antigen on tumor cells. In the same way, DLBCL EVs carrying miR-125b-5p can reduce tumor sensitivity to rituximab by inhibiting TNFAIP3 expression and reducing CD20 expression (117). Whether the miR-125b-5p/TNFAIP3 axis can be used as a therapeutic approach for increasing DLBCL sensitivity to anti-CD20 antibodies requires further investigations. \n\nEVs released by B cell could carry CD39 and CD73, two surface molecules known to hydrolyze ATP released by dying cancer cells into adenosine that hijacks CD8 T cell immune activity by binding the A2A adenosine receptors (118). One could speculate that B-cell-derived EVs may have a similar effect. The decrease of B-cell-derived EVs bearing CD73 and CD39 can be achieved by deregulating the docking protein RAB27A (118). This could be performed using an inactivated Epstein-Barr virus carrying siRNA, but it is also possible to generate EVs derived from cell lines producing RAB27A siRNA and to specifically deliver it to tumor cells. \n\nUltimately, thanks to their molecular structure mimicking the plasma membrane of the cells and their capability to reverse their cargo into target cells, exosomes could be shaped and filled of drug molecules, acting as drug-delivery systems. In fact, cancer vaccine clinical trials relying on the administration of exosomes produced by dendritic cells (Dexosomes), exploited to shuttle antigenic determinants of immune response, were conducted to immunize patients in the context of solid tumors (119) (120) (121). In the same way, systemic administrations of TNF-Related Apoptosis-Inducing Ligand (TRAIL)-armed exosomes have shown a great anti-tumor effectiveness against FL/DLBCL cell lines both in vitro and in a mouse model (122).",
"section_name": "DISRUPTING THE EV \"REMOTE COMMUNICATION\" TO IMPROVE LYMPHOMA PROGNOSIS",
"section_num": null
},
{
"section_content": "Despite very interesting recent data highlighting BM as a survival niche for lymphoma B cells, numerous controversies remain open on the role of the BM versus LN niches during the early step of lymphomagenesis or at the stage of post-treatment minimal residual disease that could generate relapse. In FL, both pretumoral B cells and early committed precursor cells, that will give rise to overt FL, have been shown to be enriched in BM (22). However, transformation events required iterative passages throughout the GC making it difficult to define precisely whether BM is a primary or a secondary tumor niche. The influence of tumor genetics or patient features on the capacity of tumor B cells to home and develop into BM remains completely unexplored. A major limitation for all BM-dedicated studies is the limited availability of good quality samples to perform phenotypic, transcriptomic, and functional studies and the lack of iterative sampling allowing evaluation of the impact of disease evolution or therapeutic strategies. BM aspirates are scarce and do probably not include the whole diversity of tumor/TME components, in particular stromal cells. Moreover, fixed BM biopsies are very difficult to exploit for spatial transcriptomics and even multiplex immunohistofluorescence approaches. Such technical issue hampers a precise evaluation of spatial heterogeneity in B-cell lymphomas integrating BM as a key tumor site. \n\nAltogether, many evidence support the clinical interest of targeting the crosstalk between BM stromal cells and malignant B cells, through the inhibition of stroma-derived B-cell growth factors, the mobilization of clonal B cells outside their supportive BM niche, or the reprogramming of tumor-supportive stromal cells. Identifying the best therapeutic options, and how to combine them with tumor-targeting drugs or immunotherapy approaches will be the major challenge in the field.",
"section_name": "CONCLUSION",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "This work was supported by research grants from Fondation ARC ( PGA1 RF20170205386 ) and the Institut National du cancer (INCA AAP PNP19-009 ).",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "ED wrote the paper, SM reviewed the paper, and KT supervised and wrote the paper. All authors contributed to the article and approved the submitted version.",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. \n\nPublisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.",
"section_name": "Conflict of Interest:",
"section_num": null
}
] |
10.1038/bcj.2015.39
|
A novel antibody–drug conjugate targeting SAIL for the treatment of hematologic malignancies
|
<jats:title>Abstract</jats:title><jats:p>Although several new therapeutic approaches have improved outcomes in the treatment of hematologic malignancies, unmet need persists in acute myeloid leukemia (AML), multiple myeloma (MM) and non-Hodgkin’s lymphoma. Here we describe the proteomic identification of a novel cancer target, SAIL (Surface Antigen In Leukemia), whose expression is observed in AML, MM, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). While SAIL is widely expressed in CLL, AML, MM, DLBCL and FL patient samples, expression in cancer cell lines is mostly limited to cells of AML origin. We evaluated the antitumor activity of anti-SAIL monoclonal antibodies, 7-1C and 67-7A, conjugated to monomethyl auristatin F. Following internalization, anti-SAIL antibody–drug conjugates (ADCs) exhibited subnanomolar IC<jats:sub>50</jats:sub> values against AML cell lines <jats:italic>in vitro</jats:italic>. In pharmacology studies employing AML cell line xenografts, anti-SAIL ADCs resulted in significant tumor growth inhibition. The restricted expression profile of this target in normal tissues, the high prevalence in different types of hematologic cancers and the observed preclinical activity support the clinical development of SAIL-targeted ADCs.</jats:p>
|
[
{
"section_content": "5] [6] There are currently more than 35 ADCs in clinical development, 7, 8 and even though some promising results have been reported, the available data suggest that developing highly efficacious therapeutics through this modality may be more complex than initially expected. 9 ne of the main challenges in the development of novel ADCs is the identification of a cell surface protein that is selectively expressed in tumors and that allows for efficient internalization of the payload to provide a clinical benefit. 10 Another challenge is to couple a highly specific monoclonal antibody (mAb) to the appropriate linker-toxin combination to achieve the desired safety and efficacy profile. 11 ere we describe the proteomic identification of the novel cell surface antigen SAIL (Surface Antigen In Leukemia) and the preclinical characterization of ADCs with potent in vitro and in vivo activity against SAIL-expressing hematologic tumors.",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "All human cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA), Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) or the Japanese Collection of Research Bioresources Cell Bank (JCRB; Osaka, Japan) and were maintained as recommended.",
"section_name": "Cell lines",
"section_num": null
},
{
"section_content": "Procedures to obtain specimens were conducted under institutional review board approval with all patients signing informed consent. Fresh specimens from acute myeloid leukemia (AML) and multiple myeloma (MM) patients and normal peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) from nondiseased donors were acquired from AllCells (Emeryville, CA, USA). Fresh chronic lymphocytic leukemia (CLL) specimens were from Billings Clinic (Billings, MT, USA) and the University of Florida. Additional frozen AML and CLL patient specimens for flow analysis were from AllCells and the University of California San Diego, respectively. Primary solid tumors and normal adjacent control samples were from CHTN (The Cooperative Human Tissue Network) or the National Disease Research Interchange. CHTN is funded by the National Cancer Institute. \n\nSurface-tagged antigen analysis and liquid chromatographycoupled tandem mass spectrometry Specimens were received within 6-24 h of sample collection. Upon receipt, the specimens were surface labeled using methods similar to those previously described. 12 Before labeling, solid tumor specimens and adjacent tissues were mechanically and enzymatically dissociated and the samples were chromatographically enriched for tagged proteins using a solid-phase affinity resin. \n\nEluted proteins from surface-tagged antigen were identified and quantitated using an LTQ-Orbitrap Velos Pro hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) configured with an EASY-nLC (Thermo Fisher Scientific) instrument for in-line nanoflow liquid chromatography. Resulting data were searched against the Uniprot human FASTA database using the SEQUEST algorithm executed on the Sorcerer 2 platform (SageN Research, San Jose, CA, USA). The relative quantitative levels of identified proteins were determined using the spectral counting method. 13 Spectral counts were tabulated and transformed to Percent Normalized Spectral Abundance Factor (% NSAF) values to account for differences in protein length and variability in sample input 14, 15 using Scaffold software (Proteome Software, Portland, OR, USA). Statistical significance between groups was calculated using the Wilcoxon ranksum test. \n\nAntibody generation and binding assays SAIL-binding mouse mAbs were generated by standard hybridoma methodology after immunization with mouse sarcoma cells stably transfected with the human SAIL antigen. \n\nApparent antigen-binding Kd of anti-SAIL mAb 67-7A and 7-1C was established by peptide enzyme-linked immunosorbent assay (ELISA) or by cell-based flow cytometry methods. 16, 17 For ELISA Kd studies, plates coated with human extracellular domain (ECD) peptide were incubated with increasing concentrations of antibodies. After incubation with an HRPconjugated secondary antibody (Jackson Immunoresearch, WestGrove, PA, USA), luminescence data were obtained and used to calculate an apparent Kd with 95% confidence intervals using Prism software version 6 (GraphPad, San Diego, CA, USA). For the Kd studies using flow cytometry, mouse sarcoma cell lines engineered to express full-length human SAIL were incubated with increasing concentrations of antibodies. Cells were then incubated with an Alexa Fluor 647-conjugated secondary F(ab')2 specific for the mouse IgG Fc and flow cytometry analysis was conducted to calculate an apparent Kd using Prism software.",
"section_name": "Patient samples and normal controls",
"section_num": null
},
{
"section_content": "All cell lines and primary samples were stained at a saturating concentration of 10 μg/ml for 30 min on ice using Alexa Fluor 647conjugated 7-1C antibody. Primary samples were co-stained with multiple tumor markers as described in the figure legend. Antibodies were purchased as follows, Miltenyi Biotec (Bergisch Gladbach, Germany): CD33 (catalog number 130-091-732), CD34 (130-095-393), CD38 (130-099-151); Biolegend (San Diego, CA, USA): CD5 (300622), CD19 (302208), CD56 (318332); eBioscience (San Diego, CA, USA): CD3 (48-0037-42), CD14 (25-0149), Lineage cocktail (22-7778-72). 7-1C and isotype matched control-Alexa Fluor 647 were prepared using the Alexa Fluor 647 Antibody Labeling Kit (Life Technologies, Carlsbad, CA, USA). SAIL copy number was determined by interpolation on a calibration curve generated by Quantum Simply Cellular bead standards (Bangs Laboratories Inc., Fischers, IN, USA). Data acquisition was performed using a MACS-Quant 10 (Miltenyi Biotec) and all data analysis was performed using FlowJo 9. 4. 11 (Flowjo LLC, Ashland, OR, USA). \n\nAntibody internalization studies were performed as previously described. 18 In brief, the cells were incubated with 7-1C-Alexa Fluor 488 antibody at a saturating concentration of 15 μg/ml on ice for 1 h, washed and incubated at 37 °C for the indicated times. Following incubation, cells were immediately chilled and surface quenched for 30 min on ice using anti-Alexa Fluor 488 Rabbit IgG (30 μg/ml, Life Technologies, catalog number A11094) and analyzed by flow cytometry.",
"section_name": "Flow cytometric analysis and internalization assay",
"section_num": null
},
{
"section_content": "All formalin-fixed, paraffin-embedded tissue microarrays for lymphoma and normal tissues were obtained from TriStar Technology Group (Rockville, MD, USA) and US Biomax (Rockville, MD, USA). In situ SAIL RNA analysis on microarrays was performed using the RNAscope technology as previously described (Advanced Cell Diagnostics, Hayward, CA, USA). 19 RNA ISH (in situ hybridization) was typically performed in parallel with positive control peptidylpropyl isomerase B to assess tissue RNA integrity. Staining intensity for ISH was scored as 0 (negative), 1 (weak), 2 (moderate) or 3 (strong). Images were acquired at a digital magnification of × 120 using Nanozoomer slide scanner software (Hamamatsu Photonics, Hamamatsu, Japan).",
"section_name": "RNA in situ hybridization",
"section_num": null
},
{
"section_content": "ADCs of anti-SAIL mouse mAbs (67-7A and 7-1C) were generated by conjugating an average of 3. 5 monomethyl auristatin F (MMAF) molecules per antibody. MMAF was conjugated to the cysteine residues via a maleimidocaproyl (mc) linker as previously described. 20 totoxicity assays with hematologic malignant cell lines and normal PBMC subsets To assess ADC cytotoxicity, cells were plated in 384-well plates (Greiner Bio-One, Monroe, NC, USA) at 4000 cells per well in 40 μl of media. MMAFconjugated anti-SAIL antibodies were serially diluted from 250 nM (except for experiments on sarcoma cells which started at 900 nM) and added to appropriate wells in duplicate. Cell plates were then incubated for 3 days, followed by lysis in CellTiter-Glo (CTG) assay reagent (Promega, Madison, WI, USA). CTG luminescence was quantified on a Synergy HT plate reader (BioTek, Winooski, VT, USA) and graphed. IC 50 and s. e. m. were calculated using Prism's nonlinear curve fitting. \n\nMonocytes and T cells were isolated from PBMCs by using the Pan Monocyte and Pan T-cell Isolation Kits (Miltenyi Biotec). Monocytes (7500) and 1000 T cells were plated in RPMI-1640 with Glutamax (Life Technologies), 10% heat-inactivated human AB serum (MP Biomedicals, Burlingame, CA, USA) and 100 μg/ml primocin (Invivogen, San Diego, CA, USA) in 384-well plates. T-cell proliferation was induced with CD2/CD3/ CD28 activation beads and 20 ng/ml IL-2 (Miltenyi Biotec) and the monocyte phenotype was maintained with 10 ng/ml M-CSF (Peprotech, Rocky Hill, NJ, USA). After a 3-day incubation with 67 nM of ADC, 25 nM free MMAE, or 4. 1 μM cytarabine, viability was assessed with CTG assay reagent. The CTG luminescence data was normalized against a no-treatment control. One representative experiment of multiple is shown. \n\nOCI-AML3 cells stably transduced with MISSION lentiviral particles expressing short hairpin RNA targeting SAIL were generated according to manufacturer's instructions (Sigma-Aldrich, St Louis, MO, USA). Stably transfected populations were selected in 0. 2 μg/ml puromycin for 7 days before the cytotoxicity assay. One representative experiment of two is shown.",
"section_name": "Generation of ADCs",
"section_num": null
},
{
"section_content": "Six-to 8-week-old female CB17 severe combined immunodeficiency mice were obtained from Charles River (Wilmington, MA, USA). Subcutaneous tumors were generated by an injection of 1 × 10 7 cancer cells/mouse in a mixture of phosphate-buffered saline (without magnesium or calcium) and BD Matrigel (BD Biosciences, San Jose, CA, USA) at a 1:1 ratio in the right flank. Mice were randomized when tumors reached a size of 65-200 mm 3 into treatment groups. The animal studies contained the following number of mice per experimental point: Figure 6a, n = 9; Figure 6b, n = 9; Figure 6c, n = 6. ADCs 67-7A-mcMMAF, 7-1C-mcMMAF and isotype-mcMMAF (3 mg/ kg) and cyclophosphamide (150 mg/kg) were administered intravenously (2 weekly doses). Body weights and tumors were measured in a nonblinded manner once and twice weekly, respectively. Tumor volume was calculated as described. 21 Statistical significance between treatment and control groups was calculated using the Student's two-tailed t-test (Prism software). A P-value o0. 05 was considered statistically significant. The animal experiments in this publication were performed in accordance with protocols approved by the Igenica Biotherapeutics Institutional Review Board -Animal Care and Use Committee.",
"section_name": "In vivo studies",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "3] [24] The method, adapted for interrogation of primary tissue specimens, employed freshly isolated primary tumors and normal tissues as source material for cell surface protein profiling. Biotinylation of intact cells from patients and nondiseased donors, followed by isolation of labeled proteins, allowed for the enrichment of cell surface proteins before the analysis by mass spectrometry. \n\nA novel protein (C16orf54, Uniprot Accession Number Q6UWD8), hereafter referred to as SAIL, was first identified based on its high expression in CLL primary samples (Figure 1, Supplementary Table 1 ). Normalized spectral counts (converted to % NSAF) were significantly elevated in 36 out of 40 CLL samples compared with normal PBMC and BMMC controls. SAIL was also detected in primary AML samples (4 out of 14) and MM samples (1 out of 33). The proteomic evaluation was expanded to various types of solid tumors and normal adjacent tissues, including colorectal, lung, ovarian, pancreatic and sarcoma cancer samples, but SAIL was not detected in either these malignant or normal tissues within the limits of assay sensitivity 25, 26 (Supplementary Table 1 ). The restricted expression profile of this novel cell surface antigen in normal tissue and its abundance in CLL provided the rationale for investigating its potential as an antibody target. \n\nSAIL is a 224 amino-acid (aa) protein with one predicted transmembrane domain and a 31 aa ECD (Supplementary Figure 1 ). Before our proteomic identification of SAIL in hematologic cancer samples, it had previously been described as a transcriptional target of RUNX1/AML1 expressed during the development of the mouse hematologic system. 27 neration and characterization of antibodies against SAIL A panel of 277 mouse antibodies against SAIL was generated by immunizing mice with syngeneic cells stably expressing fulllength human SAIL. Antibodies 7-1C and 67-7A were selected as lead therapeutic candidates based on binding properties during screening. More detailed binding studies of the two lead mAbs were conducted in ELISA assays that employed a peptide corresponding to the ECD region of SAIL (Table 1 ). Both 7-1C and 67-7A bound the ECD peptide with apparent Kd values ranging between 1. 2 nM and 1. 6 nM. Binding characteristics were further evaluated in flow cytometry analyses performed with cells stably expressing SAIL. The Kd values for the full-length SAIL antigen were comparable to those measured with the ECD peptide (Table 1 ).",
"section_name": "Proteomic identification of the novel surface antigen SAIL",
"section_num": null
},
{
"section_content": "To corroborate the MS data, SAIL expression in primary AML, CLL and MM tumor samples was evaluated by flow cytometry. Uniform cell surface expression of SAIL was observed in all the CLL samples (n = 20) evaluated by flow cytometry (Figure 2a ), whereas more variable expression was noted in AML (n = 13) and MM (n = 7) primary samples (Figures 2b and c ). These findings were consistent with what was observed during the proteomic analysis and may reflect the more heterogeneous phenotype of AML and MM patient populations, compared with the CLL patient population. SAIL expression was detected in multiple hematologic cell subpopulations in normal PBMC (n = 6) and BMMC (n = 5) specimens by flow cytometry analysis (Figures 2d and e ). Within the limits of assay sensitivity, mass spectrometry detected SAIL expression in at least 10% of normal PBMC and BMMC controls (Supplementary Table 1 ). 25, 26 o assess whether SAIL was present in hematologic malignancies beyond the initial three types evaluated, an expanded analysis of lymphoma samples was performed by RNA ISH analysis (Figure 3 ). Eighty-seven percent prevalence of positive SAIL expression was noted in B-cell lymphomas by ISH analysis. Of particular interest was the fact that in addition to the follicular lymphoma (FL) samples, the majority of samples of both the activated B-cell (ABC) and germinal center B-cell (GCB) subtypes of diffuse large B-cell lymphoma (DLBCL) exhibited SAIL staining. \n\nTo confirm the proteomic data that suggested SAIL expression is minimal in normal tissues, an evaluation of normal tissue microarrays was performed using ISH (Supplementary Table 2 ). SAIL expression was noted in lymphoid tissues (lymph node, spleen, thymus and tonsil), whereas epithelial staining was sparse in five tissues (cervix, esophagus, gallbladder, pancreas and uterus) and high in one tissue (bladder urothelium). SAIL mRNA expression was undetectable by ISH in 13 other normal epithelial tissues.",
"section_name": "SAIL expression in hematologic malignancies and in normal tissues",
"section_num": null
},
{
"section_content": "In contrast to the high prevalence of expression observed in primary B-cell lymphoma samples, SAIL expression in cancer cell lines was found to be mostly restricted to lines of the myeloid lineage. SAIL copy-number enumeration by flow cytometry showed that the antigen is moderately expressed on most cell lines with copy numbers ranging between 5000 and 30 000 (Figure 4 ). \n\nAnti-SAIL ADCs were generated by conjugating mcMMAF to mAbs 7-1C and 67-7A. These ADCs were evaluated for cytotoxicity against sarcoma cells overexpressing the target and against cell lines that had undergone copy-number analysis. The SAIL-specific ADCs exhibited potent in vitro cytotoxic activity against the sarcoma cell line expressing high levels of SAIL, but had no effect on the nontransfected parental sarcoma line, demonstrating target-specific cytotoxicity (Figures 5a and b ). Across a panel of cancer cell lines, copy number generally did not correlate with ADC activity in vitro, a phenomenon observed for ADCs against other targets. 16, 28 For example, KG1 cells were found to express the highest levels of SAIL yet were resistant to cell killing by the ADC, despite their sensitivity to free (unconjugated) monomethyl auristatin E (MMAE) (Figures 4 and 5f ). Conversely, the ADCs demonstrated potent cytotoxic activity against the monocytic NOMO1, THP1 and OCI-AML3 cell lines, which express lower amounts of SAIL (14 000 to 16 000 copies per cell) compared with KG1 (Figures 5c, 5d and 5e versus 5f). Because ADCs generally rely on internalization for cytotoxicity, one sensitive and one nonsensitive AML cell line were evaluated for anti-SAIL mAb internalization. Flow cytometry-based internalization assays showed that anti-SAIL mAb 7-1C was internalized by OCI-AML3 but not by KG1 cells, supporting the hypothesis that sensitivity to anti-SAIL ADCs is driven by internalization (Supplementary Figure 2 ). \n\nImpact of the anti-SAIL ADC on normal hematologic cell populations with the highest degree of antigen expression, including monocytes and proliferating T cells, was evaluated in vitro. These PBMC subpopulations were not found to be sensitive to anti-SAIL ADC (Supplementary Figure 3 ). Whereas free MMAE affected viability by at least 50%, the anti-SAIL ADC did not affect viability at a higher molar concentration, indicating that the anti-SAIL ADC showed selectivity toward a subset of AML models. \n\nTo confirm that the activity observed against the AML cell lines was dependent on SAIL expression, knockdown experiments utilizing SAIL-specific short hairpin RNA constructs were performed in OCI-AML3 cells. In these studies, a 3. 5-fold reduction in SAIL protein expression by flow cytometry was achieved and correlated with decreased anti-SAIL ADC activity (Figure 5g, short hairpin RNA pool 1). \n\nTaken together, these data demonstrated that anti-SAIL ADCs were antigen-specific and exhibited potent cytotoxic activity toward AML cell lines in vitro. \n\nIn vivo efficacy of anti-SAIL ADCs To assess the antitumor activity of the anti-SAIL ADCs in vivo, two subcutaneous cell line xenograft models, chosen based on SAIL expression and ADC sensitivity in vitro, were utilized. In the OCI-AML3 and THP1 AML models, significant tumor growth inhibition was observed for the anti-SAIL ADCs compared with the nontargeting isotype control ADC at a dose level of 3 mg/kg (Figure 6a and b ). In contrast, naked (that is, unconjugated) 67-7A and 7-1C did not exhibit significant activity in the OCI-AML3 model (Figure 6c ), indicating that the main antitumor mechanism of the ADC is through the specific delivery of conjugated cytotoxic payload into tumor cells.",
"section_name": "In vitro evaluation of anti-SAIL ADCs",
"section_num": null
},
{
"section_content": "Antibody-drug conjugates represent a promising new class of anticancer therapeutics that combine the specificity of an antibody with the potent activity of cytotoxic drugs. Many of the ADCs that are in clinical development are based on antigens that had previously been tested as targets for traditional unarmed antibody therapeutics. 0] [31] More recent research has focused on identifying targets whose biological role in cancer may be unknown, but whose expression profile is mostly restricted to malignant tissue. \n\n] [24] SAIL was first identified as part of a target discovery effort utilizing primary CLL samples, where it was found to be overexpressed in most tumors tested relative to normal PBMCs or BMMCs from nondiseased donors (Figure 1, Supplementary Table S1 ). While SAIL expression prevalence was 90% in CLL, only 29% of AML and 3% of MM specimens showed expression. It should be noted that the relatively low abundance of malignant cells in AML and MM samples may lead to an underestimation of protein expression levels of certain targets. Using this proteomics-based method, SAIL expression was not detected in a large set of colorectal, lung and ovarian cancer samples or their matched normal adjacent tissues. An analysis of SAIL expression in other solid tumor indications is currently in progress. \n\nAntibodies against SAIL were developed using cell-based immunization methods in order to ensure that the protein would be recognized in the context of its native conformation at the cell surface. Two lead antibody clones, 67-7A and 7-1C, were selected for further characterization based on binding properties during the initial screening efforts. Binding affinity studies on cells and recombinant SAIL peptide demonstrated that both antibodies bound human SAIL with Kd values in the range of 0. 6-1. 6 nM. \n\nFlow cytometry studies confirmed that SAIL was expressed in all CLL samples evaluated as well as in the majority of AML and MM patient samples (Figure 2 ). An expanded analysis of SAIL expression in hematologic malignancies by ISH showed that the transcript was expressed with very high prevalence (87%) in DLBCL and FL tumor specimens (Figure 3 ). The fact that SAIL was expressed at similar levels in both the ABC and GCB subtypes of DLBCL is of clinical interest, given the low success rate of R-CHOP shown to date in ABC-DLBCL. 32, 33 The selective tumor expression of SAIL mRNA was also confirmed by an ISH analysis of normal tissues, which demonstrated expression predominantly in lymphoid tissues. Studies with immunohistochemical reagents are planned to confirm protein expression in DLBCL, FL and normal tissues. \n\nAn extensive analysis of SAIL expression in cancer cell lines was undertaken using a flow cytometry-based copy enumeration method. Interestingly, the SAIL expression pattern noted in primary lymphoma samples was not replicated in established lymphoma cancer cell lines, but was found to be mostly restricted to cancer cell lines of myeloid origin (Figure 4 ). In those cell lines, the copy number per cell was generally modest, ranging from 5000 to 30 000, similar to what has been reported for CD33, another clinical ADC target. 34 he vast majority of ADCs currently in clinical development employ microtubule inhibitors (auristatins or maytansinoids) as cytotoxic payloads. For this reason, we decided to generate ADCs against SAIL by conjugating the two lead antibodies to the auristatin MMAF. The anti-SAIL ADCs showed cytotoxic activity against a subset of AML cell lines-OCI-AML3, THP1 and NOMO1 (Figure 5 ). \n\nTo understand what other factors may influence ADC response, flow cytometry-based internalization studies were performed on selected cell lines. Results demonstrated that the degree of cellular uptake of ADC correlated with its cytotoxic activity, consistent with findings for other targets like CD22. 28, 35 n two AML cell line xenograft models, anti-SAIL ADCs were found to exhibit strong antitumor activity compared with a nonspecific isotype control ADC or with naked anti-SAIL mAbs (Figure 6 ). For the clinical development of a SAIL-targeted ADC therapeutic, humanized antibody variants are being evaluated in order to minimize potential immunogenicity in humans. In addition, site-specific conjugation approaches have been demonstrated to achieve a superior therapeutic index compared with conventional conjugation approaches, 36 and therapeutic anti-SAIL ADC candidates under development may harness similar sitespecific ADC technology. \n\nIn conclusion, we have characterized the expression of a novel ADC target, SAIL. Expression of SAIL is restricted in normal tissues but is present in different types of hematologic malignancies. Moreover, we have demonstrated that ADCs against SAIL have high in vitro potency and demonstrate high antitumor activity in vivo in multiple xenograft models. Given the body of data generated and the unmet clinical need in indications where SAIL is expressed with high prevalence, clinical development of anti-SAIL ADCs may be warranted.",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "We are grateful to Jason Damiano and Sally Bolmer for critical reading of the manuscript. We thank Joseph Zachwieja, Steven Gomez and John Lippincott for their contribution to antibody generation, Jonathan Melnick and Kristy Venstrom for their contribution to in vitro cytotoxicity assays, and Michelle Lai for the proteomic analysis of primary tumor specimens.",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "",
"section_name": "CONFLICT OF INTEREST",
"section_num": null
},
{
"section_content": "All authors are employees of Igenica Biotherapeutics. Igenica Biotherapeutics is a company funded by venture capital firms -The Column Group, 5AM Ventures, OrbiMed and Third Rock Ventures.",
"section_name": "CONFLICT OF INTEREST",
"section_num": null
},
{
"section_content": "Volume ± SEM, mm3 Time, days This work is licensed under a Creative Commons Attribution 4. 0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons. org/licenses/ by/4. 0/ Supplementary Information accompanies this paper on Blood Cancer Journal website (http://www. nature. com/bcj)",
"section_name": "OCI-AML3 THP1",
"section_num": null
}
] |
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