Epigenetic basis of MDS/MPN overlap syndrome with compound loss of Kmt2c and Dnmt3a

Abstract Monosomy 7, or loss of 7q (-7/del(7q)), is one the most common chromosomal anomalies in myelodysplastic syndromes (MDS, ~10% of patients), a malignant disorder of the blood system with few treatment options. There are currently no targeted therapies available for -7/del(7q) MDS, and patients progress to a deadly acute myeloid leukemia (AML). The 7q segment is rich in genes that are implicated in MDS pathophysiology, including KMT2C encoding a histone H3 lysine4 methyltransferase (depositing H3K4me1) that marks active gene enhancers during myeloid differentiation. Co-occurring DNA methyltransferase DNMT3A mutations are significantly overrepresented in -7/del(7q) MDS, especially in the absence of other chromosomal aberrations (23%). Complete loss of DNMT3A in hematopoietic stem cells (HSCs) biases them towards self-renewal due to a failure to epigenetically shut off stem-cell-related genes. Loss of KMT2C leads to aberrant myelopoiesis and increases HSC number at baseline by regulating self-renewal. Generally, only one allele of KMT2C and DNMT3A is lost in MDS, suggesting that these haploinsufficient tumor suppressors may need to cooperate to drive disease. We hypothesized that disruption of Dnmt3a cooperated with loss of Kmt2c creating an aberrant epigenetic state that initiates malignant cell signaling and supports myeloid malignancy. We generated mice that lacked one copy of Kmt2c, Dnmt3a, or both specifically in the hematopoietic system with poly(I:C)-inducible Mx1-Cre and subjected them to comprehensive immunophenotypic and functional characterization from early to late age (10 to 80+ weeks). Young (10-12 weeks old) animals of all genotypes showed unperturbed hematopoiesis in both mature and the stem and progenitor cell (HSPC) compartments. Next, serial competitive bone marrow repopulation assays demonstrated increased competitiveness of the Kmt2c/Dnmt3a double-mutant cells in secondary transplants and retained tri-lineage reconstitution ability in tertiary transplants, unlike single gene knockout and WT controls. In tumor watch studies using non-competitively transplanted recipients, Kmt2c/Dnmt3a double-heterozygous BM chimeras developed a MDS/MPN overlap syndrome with long latency characterized by neutrophilia and/or monocytosis accompanied by anemia, occasional thrombocytopenia, and splenomegaly. These experiments indicate that concurrent perturbation of two layers of epigenetic regulation through combined loss of Dnmt3a and Kmt2c is sufficient to prime hematopoiesis to malignant transformation, although hematopoietic aging might also play a role. To address this, we profiled a cohort of aged primary mice (80+ weeks old). Although Kmt2c/Dnmt3a compound heterozygous mice didn't develop overt malignancy, we observed expansion of the stem/progenitor-enriched LSK (Lineage–Sca-1+cKit+) population, with the primitive long-term (LT)-HSCs defined as LSK CD150+CD48– being most significantly perturbed. Consistently, we found persistence of Kmt2c/Dnmt3a double-mutant LT-HSCs after third round of competitive transplantation indicating a robust a gain in self-renewal, even though this cell population was not detectable in other genotypes. To enable mechanistic studies into combined disruption of Dnmt3a and Kmt2c in the development of a myeloid malignancy, we harnessed a well-established HoxB8-ER method to immortalize primary aged HSPCs from wildtype single heterozygous and double heterozygous knockout mouse marrow, creating a scalable and renewable source of cells capable of myeloid differentiation. Pathway analysis of ~1000 differentially expressed genes after granulocytic differentiation found genes involved in JAK-STAT and TGF-beta signaling uniquely enriched in Kmt2c/Dnmt3a double mutants, which are often dysregulated in MPNs. Overall, our studies demonstrate that combined loss of Kmt2c and Dnmt3a results in a perturbed epigenetic state that predisposes to the development of age-related MDS/MPN overlap syndrome by endowing the most primitive HSCs with enhanced self-renewal capacity. We are currently interrogating the epigenetic profiles (histone marks, chromatin accessibility) resulting from combined disruption of Dnmt3a and Kmt2c aiming to identify gene targets and signaling networks for future therapeutic intervention. Better understanding of the molecular pathogenesis of the -7/del(7q) MDS with concurrent DNMT3A mutations is critical to develop more effective treatment approaches and to improve outcomes for MDS patients.