ASXL1 mutations occur frequently in myeloid neoplasms, but the mechanisms by which the mutant ASXL1 induces leukemogenesis remain unclear. In the present study, we found a mutually reinforcing effect between a C-terminally truncating ASXL1 mutant (ASXL1-MT) and the histone H2A deubiquitinase BAP1. Biochemical analyses revealed that BAP1 stabilized ASXL1-MT and induced its monoubiquitination at lysine 351. The monoubiquitinated ASXL1-MT in turn increased the nuclear retention and catalytic function of BAP1. Thus, ASXL1-MT forms a hyperactive complex with BAP1 and reduces global ubiquitination of histone H2A at lysine 119 (H2AK119ub) in cells.

We assessed the role of the ASXL1-MT/BAP1 complex in hematopoietic differentiation using in vitro cell culture assays with cell lines (32Dcl3 and TF1), mouse bone marrow (BM) cells, and human cord blood (CB) CD34+ cells. These assays revealed that ASXL1-MT alone modestly, and coexpression of ASXL1-MT and BAP1 dramatically impaired multi-lineage differentiation of hematopoietic stem/progenitor cells towards granulocytes, macrophages, mast cells, and mature erythroid cells. Interestingly, ASXL1-MT/BAP1 complex did not inhibit, but rather promoted monocytic differentiation, which may account for the frequent detection of ASXL1 mutations in patients with chronic myelomonocytic leukemia (CMML).

We next assessed the role of ASXL1-MT and BAP1 in myeloid leukemogenesis. Coexpression of ASXL1-MT and BAP1 increased the colony forming activity of mouse BM cells, but was not sufficient to transform them. Next, we examined the possible cooperation between ASXL1-MT and RUNX1-ETO in promoting myeloid leukemogenesis because ASXL1 mutations have been frequently detected in RUNX1-ETO leukemia. Forced expression of ASXL1-MT accelerated the development of leukemia driven by RUNX1-ETO9a (a shorter isoform of RUNX1-ETO with a strong leukemogenic activity) in a mouse transplantation model, and also promoted the growth of RUNX1-ETO-expressing human CB cells in vitro . These growth-promoting effects of ASXL1-MT were further enhanced by BAP1 coexpression in RUNX1-ETO leukemia cells. Thus, ASXL1-MT and BAP1 promote myeloid leukemogenesis in cooperation with RUNX1-ETO.

Mechanistically, we identified HOXA cluster genes and IRF8 as direct targets of ASXL1-MT and BAP1. RNA-seq and ChIP-qPCR assays revealed that ASXL1-MT/BAP1 complex bound the promoter regions of Hoxa5, Hoxa7, Hoxa9, and Irf8 genes, and induced upregulation of these genes via profound reduction of H2AK119ub. The upregulation of HOXA genes and IRF8 likely contributes to the ASXL1-MT/BAP1-induced leukemogenesis and monopoiesis, respectively.

Finally, we deleted BAP1 using CRISPR/Cas9 in mouse BM cells transformed by combined expression of ASXL1-MT and a SETBP1 mutant (SETBP1-D868N). Strikingly, BAP1 depletion in the cells led to a significant decrease of leukemogenicity and Hoxa gene expression. We then examined whether Bap1 is also required for leukemogenicity of MLL-fusion leukemia, a well-known leukemia subtype characterized by Hox gene dysregulation. Again, Bap1 depletion reduced colonogenicity and Hoxa gene expression in MLL-AF9-expressing mouse BM cells. Moreover, we confirmed the essential role of endogenous BAP1 to support the growth of human myeloid leukemia cell lines with ASXL1 mutations (Kasumi-1, MEG-01 and TS9;22) or MLL-fusions (MOLM13 and THP1).

In summary, our study demonstrated the critical role of BAP1 in ASXL1-MT-induced aberrant myeloid differentiation, myeloid leukemogenesis, and upregulation of HOXA genes and IRF8 . Targeting enzymatic activity of BAP1 can be a promising therapeutic strategy for myeloid neoplasms with ASXL1 mutations, and potentially for a broad range of myeloid neoplasms with HOX dysregulation.


Kitamura: Daiichi Sankyo: Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.

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