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4116 Multiomic Characterization of Myelodysplastic Neoplasms (MDS) with Micromegakaryocytes Highlights the Role of EZH2-RUNX1 deregulation in Disease Physiopathology and Response to Targeted Therapies

Program: Oral and Poster Abstracts
Session: 602. Myeloid Oncogenesis: Basic: Poster III
Hematology Disease Topics & Pathways:
Research, MDS, Translational Research, Chronic Myeloid Malignancies, hematopoiesis, Diseases, Myeloid Malignancies, Biological Processes, Technology and Procedures, pathogenesis, omics technologies
Monday, December 11, 2023, 6:00 PM-8:00 PM

Miranda Fernandez-Serrano, MSc1,2*, Alba Mesa, MD3*, Ignacio Campillo-Marcos, PhD4,5*, Marta Casado-Pelaez4*, Shubhra Bhattacharya6*, Laura Palomo, PhD7*, Wencke Walter, PhD8*, Pau Marin-Escudero, MSc1*, Mar Mallo9*, Julia Montoro10*, Francesc Sole, PhD11, Elisabetta Mereu, PhD12*, Caterina Mata13*, David Corujo, PhD6*, Torsten Haferlach, MD, PhD8, Marcus Buschbeck, PhD14*, Manel Esteller, MD15,16,17,18, David Valcarcel, MD, PhD10, Silvia Saumell, MD, PhD19,20* and Gael Roue, PhD21

1Lymphoma Tranlastional group, Josep Carreras Leukaemia Research Institute, Badalona, Spain
2Department of Biochemistry and Molecular Biology, Autonomous University of Barcelona (UAB), Barcelona, Spain
3Hematology Department, ICO-Hospital Universitari Germans Trias i Pujol, Badalona, Spain
4Cancer Epigenetics group, Josep Carreras Leukaemia Research Institute, Badalona, Spain
5Centro de Investigación Biomédica en Red Cáncer (CIBERONC), Madrid, Spain
6Chromatin, Metabolism and Cell Fate group, Josep Carreras Leukaemia Research Institute, Badalona, Spain
7Experimental Hematology, Vall d’Hebron Institute of Oncology (VHIO), Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain
8MLL Munich Leukemia Laboratory, Munich, Germany
9Myelodysplastic Syndromes Research Group, Josep Carreras Leukaemia Research Institute, ICO-Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Barcelona, Spain
10Service of Hematology, Hospital Universitari Vall d’Hebron, Barcelona; Vall d’Hebron Instituto de Oncología (VHIO), Barcelona, Spain
11MDS Group, Josep Carreras Leukaemia Research Institut, ICO-Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, Barcelona, Spain
12Cellular Systems Genomics group, Josep Carreras Leukaemia Research Institute, Badalona, Spain
13Single Cell Unit, Josep Carreras Leukaemia Research Institute, Badalona, Spain
14Chromatin, Metabolism and Cell Fate group, Josep Carreras Leukaemia Research Institute (IJC), Badalona, ESP
15Josep Carreras Leukaemia Research Institute, Badalona, Spain
16School of Medicine, Physiological Sciences Department, University of Barcelona, Badalona, Spain
17Centro de Investigacion Biomedica en Red Cancer, Madrid, Spain
18Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain
19Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, Barcelona, Spain
20Department of Hematology, University Hospital Vall d’Hebron, University Autònoma of Barcelona (UAB). Experimental Hematology Unit, Vall d’Hebron Institute of Oncology (VHIO), Barcelona, ESP
21Lymphoma translational group, Josep Carreras Leukemia Research Institute, Badalona, Spain

Micromegakaryocytes (micro-MKs), an unequivocal myeloid dysplastic feature in adults, are present in 15-20% of myelodysplastic neoplasms (MDS). We recently associated the detection of these small megakaryocytes with a mono- or bi-lobated nucleus in bone marrow (BM) smears of MDS patients with an unfavorable prognosis. The aim of the present study was to elucidate the molecular bases of micro-MK formation and its impact on MDS response to targeted therapies.

We selected retrospectively a cohort of 40 MDS patient samples subjected to NGS myeloid panel and RNA sequencing and carried out a morphological re-evaluation that included micro-MKs recounting. Cases with isolated del(5q) or SF3B1 mutation were excluded. MDS cases were selected according to the following morphological findings: no dismegakaryopoiesis, dismegakaryopoiesis without micro-MK, low rate of micro-MK (<10%; micro-MKlow) and high rate of micro-MK (>40%; micro-MKhigh). No patient harbored an intermediate (10-40%) rate of micro-MK. A comparative characterization of 8 representative patients was carried out subjecting diagnosis BM samples to Tapestri and Chromium single cell proteogenomic and transcriptomic platforms, gene set enrichment analysis, and deep cytogenetic characterization by optical genome mapping. Functional validation studies were performed using the megakaryoblastic cell line MEG01 differentiated to a mature MK phenotype by exposure to phorbol myristate acetate (PMA), as well as using the CRISPR-Cas9 tool for gene editing. Finally, activity of investigational drugs including 5-azacytidine, lenalidomide, venetoclax, rigosertib and pevonedistat, was evaluated in MEG01-activated cells and in vivo using a chicken embryo chorioallantoic membrane (CAM) model of MDS patient-derived xenograft (PDX).

The most frequently mutated genes across all cohorts were ASXL1 (58%), SRSF2 (38%), RUNX1 (30%), TET2 (30%), STAG2 (20%), EZH2 (18%), DDX41 (10%), U2AF1 (10%), and ZRSR2 (10%). Interestingly, EZH2 and RUNX1 mutations were enriched in the micro-MKhigh group (EZH2: 0%, 10%, 14.3% vs. 66.7%, p 0.006; RUNX1: 0%, 20%, 35.7% vs. 83.3%, p 0.003), and both mutations co-occurred in 85.7% (6/7) of the EZH2 mutated cases. Single cell analyses revealed an enrichment of RUNX1 alterations in three populations within micro-MKhigh BM samples, including hematopoietic stem and progenitor cells, non-classical monocytes and immature erythroid cells. These samples were also characterized by a 5-fold increase in MK progenitor abundance and by the upregulation of a set of 10 EZH2-repressed genes in neutrophil-like cells and myeloid progenitors, with 7q loss presumably affecting the EZH2 locus in 50% of the cases with micro-MK. Using the in vitro MK differentiation model, we further demonstrated that EZH2 downregulation hampered the acquisition of MK surface markers CD41 and CD61, and that venetoclax was the sole agent retaining notable cytotoxic activity in differentiated cells when compared to undifferentiated megakaryoblasts. Accordingly, in a first-in-kind in vivo CAM-MDS transplant model, venetoclax was able to impair BM infiltration by human cells in a PDX model of micro-MK+ MDS.

In summary, supporting the notion that EZH2 and RUNX1 exert a crucial role in the regulation of megakaryopoiesis, our present findings suggest that deregulation of EZH2-RUNX1 activity by loss-of-function mutation and/or genetic deletion affecting myeloid precursors, impairs a proper megakaryocytic differentiation process in MDS. Thus, this phenomenon may be considered as a characteristic feature of MDS cases with an elevated proportion of micro-MK. Of note, preliminary in vitro and in vivo results support the use of venetoclax for the treatment of this subgroup of patients with dismal outcome.

Disclosures: Walter: MLL Munich Leukemia Laboratory: Current Employment. Haferlach: MLL Munich Leukemia Laboratory: Current Employment, Other: Equity Ownership. Esteller: Incyte: Research Funding; Ferrer International: Research Funding; Quimatrix: Consultancy. Roue: Onconova Therapeutics: Research Funding.

*signifies non-member of ASH