-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

88 DNA Replication Stress Due to Loss of R-Loops in Myelodysplastic Syndromes with SF3B1 Mutation

Program: Oral and Poster Abstracts
Type: Oral
Session: 636. Myelodysplastic Syndromes—Basic and Translational: Clonal Trajectories and Novel Therapies
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Translational Research, hematopoiesis, Biological Processes, molecular biology, pathogenesis
Saturday, December 10, 2022: 10:15 AM

David Rombaut1,2,3*, Carine Lefevre, PhD1,4*, Batoul Farhat, PhD1,2*, Sabrina Bondu, PharmD, PhD1*, Anne Letessier, PhD1*, Auriane Lesieur-Pasquier, PharmD1,2*, Daisy Castillo-Guzman, PhD5*, Marjorie Leduc, PhD6*, Emilie-Fleur Gautier, PhD6*, Virginie Chesnais, PhD1*, Alice Rousseau1*, Ismael Boussaid, PharmD, PhD1,2*, Sarah Battault1,2*, Alexandre Houy7*, Didier Bouscary, MD, PhD1,8*, Lise Willems, MD, PhD1,8*, Nicolas Chapuis, PharmD, PhD1,9*, Sophie Park, MD, PhD10, Sophie Raynaud, MD, PhD11*, Thomas Cluzeau, MD, PhD12, Emmanuelle Clappier, PharmD, PhD13*, Pierre Fenaux14, Lionel Ades15, Eric Solary, MD PhD16, Raphael Margueron, PhD17*, Michel Wassef, PhD17*, Olivier Kosmider, MD, PhD1,9*, Samar Alsafadi, PhD7*, Nathalie Droin, PhD18*, Angelos Constantinou, PhD19*, Marc-Henri Stern, MD, PhD7*, Benoit Miotto, PhD1*, Frederic Chedin, PhD5* and Michaela Fontenay, MD, PhD1,2,3,9

1Université Paris Cité, CNRS, INSERM U1016, Institut Cochin, Paris, France
2Equipe Labellisée Fondation pour la Recherche Médicale, Paris, France
3Laboratoire d'Excellence du Globule Rouge, GR-Ex, Paris, France
4Laboratoire d'excellence du Globule Rouge, GR-Ex, Université Paris Cité, Paris, France
5Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, CA
6Platform Proteom'IC, Université Paris Cité, CNRS, INSERM U1016, Institut Cochin, Paris, France
7Institut Curie, PSL Research University, Sorbonne University, INSERM U830, DNA repair and uveal melanoma, Equipe labellisée par la Ligue Nationale contre le Cancer, Paris, France
8Assistance Publique-Hôpitaux de Paris.Centre-Université Paris Cité, Hôpital Cochin, Department of Hematology, Paris, France
9Assistance Publique-Hôpitaux de Paris.Centre-Université Paris Cité, Hôpital Cochin, Laboratory of Hematology, Paris, France
10Department of Clinical Hematology, Centre Hospitalo-Universitaire Grenoble Alpes, Grenoble, France
11Université Côte d'Azur, Centre Hospitalier Universitaire, Laboratory of Hematology, Nice, France
12Département d'Hématologie Clinique, Université Côte d'Azur, CHU Nice, Nice, Provence Alpes Cote d'Azur, France
13Hematology Laboratory, AP-HP, Hôpital Saint-Louis, Paris, France
14Service d'Hématologie Séniors, Hôpital Saint-Louis, Université Paris 7, Paris, France
15Department of Hematology, Université de Paris, Saint-Louis Hospital, Paris, France
16Université Paris-Saclay, INSERM U1287, Gustave Roussy Cancer Campus, Villejuif, France
17Institut Curie, Paris Sciences Lettres Research University, Sorbonne University, INSERM U934, UMR3215, Paris, France
18Université Paris Saclay, INSERM U1287, Gustave Roussy Cancer Campus, Villejuif, France
19Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Université de Montpellier, Montpellier, France

Introduction: Myelodysplastic syndromes (MDS) with SF3B1 mutation represent a subtype of hematopoietic stem cell disorders with bone marrow (BM) erythroid dysplasia and ring sideroblasts. This mutation in a splicing factor gene causes multiple alterations of mRNA maturation. Other mutated splicing factors SRSF2 or U2AF1, produce splicing alterations and formation of unscheduled R-loops, which trigger DNA damage response and genetic instability. Mechanistically, mutant SRSF2 impairs the transcription pause release downstream of TSS allowing invasion of relaxed DNA double helix by nascent RNA. Although previous studies suggested that R-loops inappropriately form in SF3B1 mutant cells, investigations are required to elucidate why MDS with SF3B1 mutation are less prone to genetic instability.

Methods: A total of 96 patients including 53 MDS with SF3B1 mutation and 43 other low risk MDS was enrolled in this study. RNA-seq of BM mononuclear cells (MNC) was performed in 21 MDS with and 6 MDS w/o SF3B1 mutation. BM CD34+-derived erythroblasts were expanded from 69 MDS and 22 healthy controls to analyze transcriptome, proteome, R-loop landscape, and DNA replication stress. For global proteome analysis, after protein digestion, peptides were fractionated on SCX column and analyzed by mass spectrometry. R-loops were detected by DNA-RNA immunoprecipitation (DRIP-seq) using S9.6 antibody. Immunofluorescence labeling of pRPA32, 53BP1 and γH2AX, DNA combing, BrdU assay and metabolomics were used to investigate DNA replication stress and DNA biosynthesis in human primary erythroblasts or in murine proerythroblastic (proE) G1E-ER4 Crispr-cas9 Sf3b1K700E/+ cell line. expressing GATA1 under the control of estrogen receptor.

Results: In SF3B1-mutated BM MNC samples, DeSeq2 analysis of RNA-seq data revealed 1,058 upregulated genes (log2(FC)>0.6, BH-adj P-value<0.05) involved in DNA replication, cell cycle, nucleotide biosynthesis, DNA repair, and erythroid differentiation. Analysis of splicing identified 3,937 differential splicing events (ΔPSI>0.10, BH-adj P-value<0.05) including 1,256 events of intron retention (IR). Among IR events, 384 loss of IR events correlated with altered expression of 293 genes. In SF3B1-mutated erythroblasts, loss of IR events were more frequent in polychromatophilic erythroblasts (polyE: 383) than in proE/early basophilic erythroblasts (eBasoE: 171) suggesting that gene extinction mediated by IR during normal differentiation was altered. Quantitative proteomics revealed 924 significantly deregulated proteins with little overlap with IR transcripts, consistently involved in DNA damage, DNA repair, and nucleotide biosynthesis pathways.

DRIP-seq was performed in proE/eBasoE of 8 MDS w/o or with SF3B1 mutation to investigate R-loop formation. Stringent peak calling identified 2,208 shared peaks in wild-type samples compared to 159 shared peaks in SF3B1-mutated samples, suggesting a loss of R-loops. Integrative analysis and DRIP-qPCR demonstrated that R-loop losses overlapped IR loss events, consistent with the notion that intron excision prevents R-loop formation. Compared to SRSF2 or U2AF1-mutated erythroblasts, SF3B1-mutated cells exhibited a significant increase of DNA synthesis by BrdU labeling and of replication fork speed by DNA combing. Immunofluorescence analysis detected pRPA32 foci marking single strand DNA but no evidence of double strand breaks in contrast to SRSF2 mutant cells in which 53BP1 or γH2AX foci were detected. Compared to isogenic cell lines, Sf3b1K700E/+ proE G1E-ER4 cells demonstrated higher proliferation rate and fork speed in proliferation and in estradiol-induced differentiation conditions, and significantly lower amounts of deoxynucleotides (dATP, dCTP, TTP) suggesting persistence of DNA synthesis during differentiation. Targeting of DNA polymerase by aphidicolin or ribonucleotide reductase by low doses of hydroxyurea partially rescued erythroid differentiation.

Conclusion: Our data demonstrated a highly specific signature of DNA replication stress at transcriptomic and proteomic level in SF3B1-mutant cells. High replication fork speed reflects the loss of R-loops in these cells, which may reduce the risk of genetic instability at the expense of normal differentiation. Pharmacological targeting of the DNA replication stress could improve erythropoiesis.

Disclosures: Bouscary: Abbvie: Honoraria. Willems: Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Park: Sandoz: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cluzeau: AbbVie, Astellas, Servier: Speakers Bureau; BMS/Celgene, Novartis, Jazz: Consultancy, Speakers Bureau. Fenaux: AbbVie, BMS, Janssen, Jazz, Novartis: Consultancy, Honoraria, Research Funding.

*signifies non-member of ASH