Leukemogenic Functions of Mutant ASXL1 Are Regulated By CDK-Mediated Phosphorylation

Additional sex combs-like 1 (ASXL1) is recurrently mutated in various myeloid neoplasms and its mutations confer poor prognosis. Most of the ASXL1 mutations in myeloid malignancies are frameshift or nonsense mutations, leading to expression of C-terminally truncated form of ASXL1 (ASXL1-MT). Recently, we have demonstrated that monoubiquitination of ASXL1-MT at lysine 351 plays an important role in enhancing the catalytic activity of BAP1, resulting in reduction of H2AK119ub and upregulation of posterior HOXA genes to promote myeloid leukemogenesis (Asada et al. Nat commun. 2018). However, roles of other post-translational modifications of ASXL1 including phosphorylation remain to be elucidated. In this study, we performed nano liquid chromatography tandem mass spectrometry (nano LC-MS/MS) and found that ASXL1 was phosphorylated at multiple serine (S) and threonine (T) residues including S503 residue, the major phosphorylated site of ASXL1. We generated an antibody that specifically recognizes phosphorylated S503 of ASXL1, and showed that CDK1/2 phosphorylated both wildtype ASXL1 and ASXL1-MT. However, phosphorylation status of ASXL1-MT at serine 503 did not affect its oncogenic activity. Thus, we considered the possibility that phosphorylations of other residues would compensate the effects of phosphorylation of ASXL1-MT at serine 503. Using public database and our data from mass spectrometry, we found additional five S/T residues containing a CDK phosphorylation motif (S/T-P). We then established a non-phosphomimetic mutant form of ASXL1-MT (ASXL1-MT-6A) whose six S/T residues were substituted to alanine. To compare the oncogenic functions between ASXL1-MT-6A and ASXL1-MT, we first assessed the impact of these mutants on differentiation of hematopoietic cells. Expression of ASXL1-MT-6A more strongly impaired differentiation of human CD34+ cord blood (CB) cells towards granulocytes and erythrocytes than that of ASXL1-MT. These effects of ASXL1-MT-6A on differentiation block were also observed in HL-60 cells. Next, we assessed the impact of phosphorylation of ASXL1-MT on its leukemogenic functions using RUNX1-ETO and N-Ras G12V-induced leukemia models, because RUNX1-ETO fusion or N-Ras mutations frequently coexist with ASXL1 mutations in patients with myeloid neoplasms. In agreement with our previous report, expression of ASXL1-MT enhanced proliferation of RUNX1-ETO expressing CB cells. ASXL1-MT-6A showed the stronger growth promoting effect than ASXL1-MT i