P709: h2ak119ub in the transcriptional regulation of patients with asxl1-mutant chronic myelomonocytic leukemia

Background: Heterozygous truncating mutations in the epigenetic modifier ASXL1 are common in chronic myelomonocytic leukemia (prevalence ~40%) and are independently associated with a poor prognosis. In cell lines (EML, HEK293T), truncating ASXL1 mutations lead to an increase in the PR-DUB activity and a widespread depletion of H2AK119ub. This deubiquitination involves the promoter regions of up-regulated genes and is thought to play an important role in the overexpression of key leukemogenic driver genes. Aims: To assess the association of H2AK119ub occupancy and gene expression in bone marrow mononuclear cells of patients with ASXL1-mutant and -wildtype chronic myelomonocytic leukemia. Methods: We performed RNA-seq and H2AK119ub ChIP-seq on bone marrow mononuclear cells from patients with chronic myelomonocytic leukemia (8 ASXL1-mutant, 8 -wildtype). All libraries were sequenced on an Illumina HiSeq instrument and further processed for data analysis after quality control. Differential gene expression (n=16) and H2AK119ub occupancy (n=14, 2 samples removed during quality control) between ASXL1-mutant and -wildtype patients were assessed. Differential gene expression was performed to define the up-regulated genes in ASXL1-mutant samples. The H2AK119ub occupancy in promoter regions (defined as transcription start site ±3kb) of these up-regulated genes were compared. Results: Median age at diagnosis was 69 years (48-77), 63% of the patients were male. Half of the patients had proliferative chronic myelomonocytic leukemia and half of them had truncating ASXL1 mutations. All ASXL1 variant allele frequencies were compatible with heterozygosity (31-48%). The distribution of co-mutations was similar between ASXL1-mutant and -wildtype patients (p=0.684). The co-mutations involved the spliceosome, epigenetic regulators, chromatin regulators, and cell signaling molecules and was typical for chronic myelomonocytic leukemia. There was a predominant up-regulation of gene expression in ASXL1-mutant patients: 236 genes up- and 35 down-regulated (Figure A, FDR<0.05 for all genes). The up-regulated genes included known leukemogenic driver genes (e.g. several HOXA cluster genes and MEIS1), mitotic kinases (e.g. RET), and other potential drug targets (e.g. EGF, SOCS2, MDK). Functional annotation of these overexpressed genes demonstrated cell differentiation and hematopoietic development to be enriched processes (FDR<0.05 for all terms). We did not observe a global depletion of H2AK119ub between ASXL1-mutant and -wildtype patients (Figure B). Likewise, there was no depletion of H2AK119ub in the promoter regions of the up-regulated genes (Figure B). Differential H2AK119ub occupancy between ASXL1-mutant and -wildtype patients was mostly observed in intronic regions of genes not differentially expressed (n=544) and in intergenic regions (n=470). Conclusion: In patients with ASXL1-mutant chronic myelomonocytic leukemia, we observed an overexpression of key leukemogenic driver genes, consistent with the findings in model systems. However, we did not observe a concomitant depletion of H2AK119ub in the promoter regions of these genes. This might be due to the greater epigenetic heterogeneity in patient samples, the effect of co-mutations on H2AK119ub occupancy, and/ or technical differences. Understanding the regulatory mechanisms determining gene expression in ASXL1-mutant chronic myelomonocytic leukemia remains of great interest for the development of individualized therapeutic approaches for this patient population.Keywords: Epigenetic, Transcriptional regulation, Chronic myelomonocytic leukemia, ASXL1