Abstract 5419: HDAC1 regulates RUNX1 activity in inv(16) acute myeloid leukemia

Abstract

Abstract RUNX1 and CBFβ form a transcription factor dimer that regulates normal hematopoiesis and leukemogenesis. Inversion of chromosome 16 (inv(16)) is one of the most common mutations in acute myeloid leukemia (AML), fusing CBFβ with the gene encoding smooth muscle myosin heavy chain (MYH11). The fusion protein encoded by CBFB-MYH11 (CM), retains the ability to bind to RUNX1, and together they cause changes in gene expression leading to leukemogenesis. Recently, we found that Histone Deacetylase 1 (HDAC1) is part of the RUNX1:CM complex, that all three proteins co-localize on the promoters of target genes, and that HDAC1 is required for target gene expression. By deacetylating histones, HDAC1 can act as a transcriptional repressor. However, HDAC1 can also deacetylate non-histone proteins, which may explain its unexpected role in gene activation. We hypothesized that HDAC1 acts by deacetylating proteins in the RUNX1:CM complex. To test this, we immunoprecipitated the RUNX1:CM complex and probed for acetyl-lysine. The only visible band was at the expected size for RUNX1, suggesting that RUNX1 is the target of HDAC1 activity. Indeed, cells transfected with RUNX1 and HDAC1 showed significantly less acetylation than cells with RUNX1 alone. To test if HDAC1 deacetylates endogenous RUNX1, we treated leukemia cells from CM-expressing knockin mice with the HDAC1 selective inhibitor entinostat. The level of RUNX1 acetylation was significantly increased in treated cells compared to control, providing further support that HDAC1 deacetylates RUNX1. Acetylation at lysines 24 and 43 of RUNX1 was previously shown to increase its activity. To determine the effect of deacetylation on RUNX1 activity, we used the M-CSFR promoter fused to luciferase. Cells transfected with RUNX1, CBFβ, and HDAC1 showed significantly decreased promoter activity compared to cells with RUNX1 and CBFβ. Importantly, in cells expressing RUNX1 acetylation and deacetylation mimetics, promoter activity was unchanged with the addition of HDAC1, indicating that lysines 24 and 43 are the relevant HDAC1 targets. To test if RUNX1 acetylation status affects the activity of the CM complex, we transfected a CM+ cell line with WT or mutated RUNX1 and examined target gene expression. In CM+ cells over-expressing WT RUNX1, we found similar target gene expression as untransfected CM+ cells. However, expression of either of RUNX1 mutant blocked the CM-induced changes in gene expression. This implies that regulation of RUNX1 acetylation is important for the activity of the RUNX1:CM complex. The observation that both RUNX1 mimetics had the same effect on CM activity may imply that alternate rounds of RUNX1 acetylation and deacetylation is required for continuous RUNX1:CM transcriptional activity. This work provides a possible explanation for HDAC1's role in gene activation in inv(16) AML, and may have implications for other RUNX1 dependent leukemias as well. Citation Format: Lisa Richter, Yiqian Wang, Michelle Becker, Jake Williams, R. Katherine Hyde. HDAC1 regulates RUNX1 activity in inv(16) acute myeloid leukemia [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 5419.