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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&gt;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 chroni(...TRUNCATED)
"In chronic lymphocytic leukemia (CLL) the occurrence and the impact of antibody responses toward tu(...TRUNCATED)
[{"section_content":"Immune dysfunctions are a key feature of chronic lymphocytic leukemia (CLL) and(...TRUNCATED)
[{"section_content":"","section_name":"CONFLICTS OF INTEREST","section_num":null},{"section_content"(...TRUNCATED)
10.1371/journal.pone.0004434
"Tumorigenic Potential of Olfactory Bulb-Derived Human Adult Neural Stem Cells Associates with Activ(...TRUNCATED)
"Multipotent neural stem cells (NSCs) have been isolated from neurogenic regions of the adult brain.(...TRUNCATED)
[{"section_content":"Due to their ability to self-renew and to differentiate towards the neuronal ph(...TRUNCATED)
[{"section_content":"","section_name":"Acknowledgments","section_num":null},{"section_content":"We t(...TRUNCATED)
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 microenviron(...TRUNCATED)
[{"section_content":"ellular senescence is an autonomous tumour-suppressor mechanism that can be tri(...TRUNCATED)
[{"section_content":"","section_name":"Acknowledgements","section_num":null},{"section_content":"We (...TRUNCATED)
End of preview. Expand in Data Studio

SciLake Fulltext Corpus

The SciLake Fulltext Corpus is a collection of scientific papers parsed and segmented by section, primarily designed for research in the development and evaluation of NLP models. This dataset contains 1,000 full-text papers from various scientific domains, including Neuroscience, Cancer, Transport, and Energy, along with an additional 5,000 random papers from general scientific domains. All papers have been curated with licenses that allow for legal usage, specifically CC-BY and Public Domain.

The dataset provides detailed metadata and full-text sections, offering a robust resource for domain-specific and general scientific research, dataset annotation, model training, and evaluation.

Corpus Overview

  • 1,000 Full-Text Papers Segmented by Section:
    • Domain-specific sections: Neuroscience 🧠, Cancer 🦀, Transport 🛻, Energy 🪫.
    • Each paper is segmented into sections such as Introduction, Methods, Results, etc.
  • 5,000 Random Papers from General Scientific Domains:
    • Mix of stratified sampled by MAG level 0 to ensure diversity across multiple domains and disciplines, and random sample.

Example of Dataset Structure:

{
'doi': DOI,
'title': TITLE,
'description': ABSTRACT,
'fulltext_sections': [
        {
            'section_name': SECTION_NAME_1,
            'section_num': SECTION_NUM_1,
            'section_content': SECTION_CONTENT_1,
        },
        ...
],
'fulltext_additional': [
        {
            'section_name': SECTION_NAME_1,
            'section_num': SECTION_NUM_1,
            'section_content': SECTION_CONTENT_1,
        },
        ...
]

How to use

from datasets import load_dataset

dataset_ds = load_dataset("SIRIS-Lab/scilake-fulltext-corpus")

Licensing Information

The SciLake Fulltext Corpus is released under the following licenses:

CC-BY (Creative Commons Attribution), licenses have been obtained from the publisher’s landing page, PDFs, metadata in OpenAire, and Unpaywall, filtering fro those with license CC-BY or Public Domain.

Dataset Acquisition

The papers included in this dataset were sourced through the OpenAIRE index, with random selection to ensure diverse content. The license information was verified by cross-referencing the publisher’s landing pages, metadata from OpenAire, and the Unpaywall database. Papers were retained if they had a CC-BY license or were in the Public Domain.

Funding

This work was partially funded by a projects under EU’s HORIZON Research and Innovation Programme:

  • SciLake (grant agreement No 101058573).

Contact

For more information, or if you have questions, please contact us at sirislab[at]sirisacademic.com.

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