Abstract 4996: MLL3 haploinsufficiency preserves self-renewal capacity in HSCs with extensive cumulative division histories

Abstract

Abstract KMT2C/MLL3 is one of the most commonly mutated tumor suppressor genes in human cancer. Haploinsufficiency at the KMT2C/MLL3 locus often occurs in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) as a result of mono-allelic 7q deletions. Mll3 haploinsufficiency has been shown to accelerate AML in mice, but it is not clear how it regulates normal hematopoietic progenitors or why it is required to suppress leukemogenesis. We used a novel loss-of-function mouse allele to characterize hematopoiesis in Mll3-deficient fetal livers and adult bone marrow. Mll3 deletion caused a modest increase in fetal liver hematopoietic stem cells (HSCs) and a modest decrease in lineage restricted progenitors (HPCs). In contrast, Mll3 heterozygosity caused significant expansion of the HSC population in adult bone marrow without affecting more committed progenitor populations. Mll3+/- and Mll3-/- HSCs were able to repopulate lethally irradiated mice, and repopulating activity was enhanced in secondary and tertiary transplants relative to wild type HSCs. Thus, Mll3 haploinsufficiency enhances HSC self-renewal capacity. We next tested whether Mll3 regulates the balance between self-renewal and commitment in non-transplanted HSCs. We used H2B-GFP pulse-chase assays to assess the division histories of HSCs over time. After a 6-week chase, wild type HSCs retained high levels of H2B-GFP expression, consistent with prior studies showing that adult HSCs divide infrequently. In contrast, almost all Mll3+/- HSCs had low to moderate H2B-GFP expression levels, indicative of an extensive division history. This difference was not associated with an increase in the percentage of cycling (S/G2/M) HSCs, and it was not associated with a loss of self-renewal capacity that occurs with cumulative divisions in wild type HSCs. Thus, Mll3 haploinsufficiency allows cumulative HSC divisions to accrue without compromising self-renewal. Mll3 encodes a histone methyltransferase that binds active enhancers and promotes transcription. This raised the question of whether Mll3 deletion results in a loss of commitment-related gene expression, increased self-renewal gene expression or altered epigenetic landscapes. To our surprise, gene expression was indistinguishable between wild type and Mll3+/- HSCs. However, ATAC-seq analysis revealed patterns of nucleosome depletion in Mll3+/- HSCs that closely resembled wild type HPCs. No differences were observed in the ATAC-seq profiles of wild type and Mll3+/- HPCs. These observations suggest that as Mll3+/- HSCs self-renew, they remodel their epigenetic landscapes to resemble committed progenitors, yet they do not activate commitment related genes, and they do exit the stem cell pool. The data suggest that stress conditions that drive HSCs into cycle, such as the chemotherapy regimens that often precede 7q- MDS, may select for Mll3 haploinsufficient HSC clones. Citation Format: Theresa Owuor, Shaina Porter, Cluster Andrew, Riddhi Patel, Jeffrey Magee. MLL3 haploinsufficiency preserves self-renewal capacity in HSCs with extensive cumulative division histories [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4996.