Nucleosome Binding Protein HMGN1 Promotes HSPC Competitive Advantage with Decreased Quiescence and Cooperates with AML Oncogenes to Block Differentiation and Increase Clonogenicity

Abstract

Acute myeloid leukemia (AML) is characterized by rapid growth and differentiation arrest of immature myeloblasts. The AML blast is defined by having “open” chromatin, thus, we hypothesized that alterations of chromatin compaction may promote AML. The nucleosome binding protein HMGN1 is known to decompact chromatin and is located on chr21q22 in one of the most recurrently amplified regions in AML. We previously presented in vitro data showing that HMGN1 overexpression delayed myeloid differentiation in association with an increase in global levels of Histone H3K27 acetylation downstream of the histone acetyltransferases (HATs) CBP and p300. The goal of this study was to define the effect of HMGN1 overexpression on hematopoiesis in vivo and to identify leukemia oncogenes that cooperate with HMGN1 in primary hematopoietic cells.We analyzed native WT and OE-HMGN1 (transgenic overexpressing HMGN1) hematopoiesis at steady state in young and older mice. OE-HMGN1 transgenic mice had a significant increase in lineage-negative cells with age (Fig A, *p=0.02). We next performed competitive bone marrow transplantation with CD45.1 (WT) vs CD45.2 (WT or OE-HMGN1) donors measuring the relative contribution of CD45.1 and 45.2 cells to hematopoiesis over time after 3 consecutive serial transplants, each at 16 weeks. OE-HMGN1 stem and progenitor cells (HSPCs) had a progressive clonal advantage during serial transplantation (Fig B, *p<0.0002). This clonal advantage was observed across multiple HSPC populations and in all mature lineages. Lin-Sca1+Kit+ (LSK) and Lin-Kit+ (LK) HSPCs were sorted 4 months after transplantation for cell cycle analysis, ex vivo differentiation assays, and RNA-sequencing. OE-HMGN1 HSPCs were less quiescent in vivo, with fewer cells in G0 (Fig C, LK p= 0.0007, LSK p=0.01), and had a relative defect in differentiation capacity in vitro compared to WT. Gene set enrichment analysis was consistent with these phenotypes, showing enrichment of senescence and cell cycle regulatory gene expression programs in WT compared to OE-HMGN1 HSPCs.HMGN1 overexpression alone did not cause leukemia in mice. To screen for synergy between HMGN1 and AML oncogenes, we immortalized myeloid progenitors from WT or OE-HMGN1 mice with a HoxB8 transgene and measured growth properties after transduction with retroviruses expressing one of several AML-associated fusion proteins (BCR-ABL, MLL-AF9, PML-RAR, AML-ETO9a, MOZ-TIF2) or mutated oncogenes (FLT3, NPM1, NRAS, GATA2) with GFP. AML-ETO9a (AE9a) and MOZ-TIF2 had the most significant cooperation with HMGN1, most notably by enhancing their ability to block myeloid differentiation in vitro. This is of interest because, like we found for HMGN1, AE9a and MT2 are known promote leukemia via chromatin effects dependent on CBP/p300 and/or acetyltransferase activity. In confirmatory experiments, combination of AE9a and HMGN1 exacerbated the delay in myeloid differentiation induced by either gene alone (p<0.05), and HMGN1 enhanced serial colony formation capacity of AE9a in semi-solid media (p<0.05).In vivo transplantation of WT or OE-HMGN1 c-Kit+ HSPCs transduced with AE9a led to development of AML ~110 days after injection in both groups. However, subpopulation analysis of AE9a-expressing leukemias showed that LSK and MPP-like populations were expanded in the context of OE-HMGN1 compared to WT (p<0.05). Remarkably, global H3K27ac levels were significantly higher in LT-HSC-like HMGN1-OE AE9a leukemia cells using single cell subpopulation analysis by intracellular flow cytometry compared to WT (Fig D p=0.008), similar to what we observed in HSPCs after competitive transplant. Ex vivo clonogenic capacity was increased in AE9a leukemias that overexpressed HMGN1 in an LTC-IC assay (Fig E *p<0.05). Limiting dilution secondary transplants are ongoing to test if these differences correlate with increased leukemia-initiating capacity per cell in vivo in leukemias that overexpress HMGN1.Our study suggests that overexpression of HMGN1 provides a clonal advantage to hematopoietic populations including long-term stem cells. HMGN1 also cooperates with specific AML oncogenes, possibly those associated with H3K27 acetylation/HAT activity, to enhance leukemic phenotypes. Further study is needed to determine if HMGN1 overexpression confers specific therapeutic vulnerabilities, such as to HAT inhibitors or other chromatin-targeting agents. [Display omitted] DisclosuresLane:Stemline Therapeutics: Research Funding; N-of-one: Consultancy.