Homeobox Transcription Factor Hhex Promotes Myeloid Leukemia in Cooperation with Mutant ASXL1
Authors: Takeda Reina , Asada Shuhei , Park Sung-Joon , Yokoyama Akihiko , Kanai Akinori , Visconte Valeria , Hershberger Courtney , Hayashi Yasutaka , Yonezawa Taishi , Tamura Moe , Fukuyama Tomofusa , Matsumoto Akiko , Yamasaki Satoshi , Nakai Kenta , Inaba Toshiya , Shibata Tatsuhiro , Inoue Daichi , Honda Hiroaki , Goyama Susumu , Maciejewski Jaroslaw P. , Kitamura Toshio 
Published: November 2019

Additional sex combs-like 1 (ASXL1) mutations are frequently found in myeloid malignancies such as AML, MDS and MPNs. Our previous study on mutant Asxl1 expressing knock-in (Asxl1-MT KI) mice revealed that Asxl1-MT impaired normal hematopoiesis (Nagase R et al. J. Exp. Med. 2018). However, Asxl1-MT alone was insufficient to develop myeloid leukemia. Thus, additional factors are required for myeloid transformation in ASXL1-mutated cells. Retrovirus-mediated insertional mutagenesis approaches demonstrated susceptibility of Asxl1-MT KI cells to myeloid leukemia, and helped identifying Hematopoietically expressed homeobox (Hhex) gene as a common retrovirus integration site. Therefore, we here investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis.
We first assessed the effects of ASXL1-MT and HHEX on proliferation and differentiation of murine and human hematopoietic stem progenitor cells (HSPCs). Expression of HHEX enhanced proliferation and blocked differentiation of HSPCs expressing ASXL1-MT, while it showed only a modest effect in normal HSPCs. Some of the mice transplanted with cells expressing ASXL1-MT and HHEX developed myeloid leukemia. Expression of ASXL1-MT and HHEX dramatically promoted the growth of RUNX1-ETO-expressing Cord Blood (CB) cells in vitro by promoting cell cycle and inhibiting apoptosis. Moreover, ASXL1-MT and HHEX synergistically accelerated development of two distinct types of myeloid leukemia driven by RUNX1-ETO9a or FLT3-ITD in vivo. These data indicate that ASXL1-MT and HHEX cooperatively work in leukemic transformation of myeloid cells.
Next, we evaluated the role of endogenous HHEX in ASXL1-MT-expressing leukemia cells. We here used two murine mutant ASXL1-expressing leukemia cells (cSAM cells: cells with combined expression of SETBP1 and ASXL1 mutations, cRAM cells: cells with combined expression of RUNX1 and ASXL1 mutations). Depletion of Hhex using CRISPR-Cas9 system profoundly attenuated the colony-forming ability and leukemogenicity of cSAM and cRAM cells. Similarly, depletion of HHEX attenuated the growth of human leukemia cell lines harboring an ASXL1 mutation including MEG-01 and Kasumi-1 by inducing apoptosis and differentiation. In contrast, the growth of ASXL1 wildtype cell lines such as THP-1 and U937 was unaffected by HHEX depletion. Thus, endogenous HHEX promotes survival of ASXL1-mutated leukemia cells.
To elucidate the underlying molecular mechanisms, we performed RNA-seq using RUNX1-ETO expressing CB cells. Gene set enrichment analysis revealed that genes related to leukemia stem cells were more enriched in cells expressing both ASXL1-MT and HHEX than control cells. In combination with this RNA-seq data and ChIP-seq using 293T cells, we identified Myb and Etv5 as candidate genes upregulated by HHEX expression. We confirmed that ASXL1-MT and HHEX upregulated Myb and Etv5 in murine HSPCs by RT-qPCR and HHEX bound to promoter regions of MYB and ETV5 genes in HL-60 cells by ChIP-qPCR. Conversely, depletion of Hhex reduced expression of Myb and Etv5 in cSAM and cRAM cells. Luciferase reporter assay revealed that co-expression of ASXL1-MT and HHEX cooperatively enhanced promoter activity of MYB. In addition, preliminary expression analyses of primary cells showed that HHEX/ETV5 mRNA expression levels were significantly and MYB mRNA expression tended to be higher in primary AML samples including ASXL1 mutations compared to the ones of healthy controls (Beat AML).
Finally, we assessed the role of MYB or ETV5 in leukemogenesis driven by ASXL1 mutations. Depletion of Myb or Etv5 reduced colony-forming activity in cSAM and cRAM cells by promoting apoptosis or differentiation, respectively. Furthermore, ectopic expression of MYB or ETV5 significantly reversed the reduced colony-forming activity of Hhex-depleted cSAM cells.
Taken together, our study demonstrates that ASXL1-MT and HHEX are cooperative events promoting myeloid leukemogenesis through upregulation of MYB and ETV5.

Disclosures
Maciejewski: Novartis: Consultancy; Alexion: Consultancy.