Abstract 4643: Mutant ASXL1 collaborates with HHEX to promote myeloid leukemogenesis

Abstract

Abstract An epigenetic modulator Additional sex combs-like 1 (ASXL1) is recurrently mutated in myeloid neoplasms and its mutations are associated with poor prognosis. Recently, we generated mutant Asxl1 conditional knock-in (Asxl1-MT KI) mice mimicking human ASXL1 E635RfsX15 mutation, one of the most common mutations in myeloid neoplasms (Nagase et al. JEM 2018). Retrovirus-mediated insertional mutagenesis study exhibited susceptibility of Asxl1-MT KI bone marrow cells to myeloid leukemia, and we identified Hematopoietically expressed homeobox (Hhex) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between the mutant ASXL1 and HHEX in myeloid leukemogenesis. We first performed colony-forming assay and found that forced expression of HHEX enhanced colony replating activity and blocked myeloid differentiation in bone marrow hematopoietic stem progenitor cells (HSPCs) derived from ASXL1-MT KI mice, while it showed only modest effect in normal HSPCs. The synergistic effect between the mutant ASXL1 and HHEX in blocking myeloid differentiation was also observed in human HL-60 cells. We next evaluated the role of endogenous Hhex in the mutant ASXL1-expressing cells. Depletion of endogenous Hhex using CRISPR-Cas9 system ameliorated mutant ASXL1-induced differentiation block in 32Dcl3 cells. Depletion of endogenous Hhex in murine mutant ASXL1-expressing leukemia cells [cSAM cells: cells with combined expression of SETBP1 and ASXL1 mutations (Inoue et al. Leukemia 2015), cRAM cells: cells with combined expression of RUNX1 and ASXL1 mutations (Nagase et al. JEM 2018)] also promoted differentiation and increased apoptosis. Furthermore, Hhex deletion profoundly attenuated the colonogenicity of cSAM and cRAM cells and leukemogenicity of cSAM cells. We then investigated target genes of the mutant ASXL1 and HHEX in myeloid neoplasms using public database and our previous RNA-Seq data. Among the potential target genes of the mutant ASXL1 and HHEX, we found that Myb, Etv5 and Oraov1 genes were upregulated by the mutant ASXL1 and HHEX in murine HSPCs. Conversely, Hhex depletion resulted in downregulation of these genes both in cSAM and cRAM leukemic cells. In addition, depletion of Myb, Etv5 or Oraov1 genes significantly abrogated the colonogenicity of cSAM cells. These data suggest that mutant ASXL1 and HHEX cooperatively induce myeloid leukemogenesis via dysregulating Myb, Etv5 and Oraov1. Citation Format: Shuhei Asada, Reina Takeda, Daichi Inoue, Susumu Goyama, Toshio Kitamura. Mutant ASXL1 collaborates with HHEX to promote myeloid leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4643.