Modeling ASXL1 Mutation Revealed Myelodysplasia Caused By Derepression of p16Ink4a through Aberrant PRC1-Mediated Histone Modification

Abstract

Clonal hematopoiesis of indeterminate potential (CHIP) is a premalignant condition in patients with blood cells that possess somatic mutations of genes known to be recurrently mutated in hematologic malignancies. Among them is additional sex combs like 1 (ASXL1), whose mutation is frequent in various myeloid malignancies and associated with poor prognosis, and ASXL1 is known to act as an epigenetic modulator. Despite the clinical impact of ASXL1 mutations in myeloid malignancies, lack of animal models expressing mutated Asxl1 at endogenous levels mimicking human ASXL1-mutated diseases hinders from dissecting precise molecular mechanisms of mutated Asxl1. Here in this study, with newly generated knock-in mice carrying a frameshift mutation at the exon 13 of the Asxl1 gene (Asxl1G643WfsX12), which is identical to the most frequent human ASXL1 G646WfsX12 mutation, we extensively investigated the biological effects of the mutant.Asxl1G643fs/+ mice showed decreased body size and weight, resembling hypomorphic features in patients with Bohring-Opitz syndrome carrying de novo constitutive ASXL1 mutation.Asxl1G643fs/+ mice developed slowly progressive peripheral leukopenia, and Wright-Giemsa stain of bone marrow (BM) cells from Asxl1G643fs/+ mice showed dysplastic myeloid cells represented by hypersegmented neutrophils, apoptotic neutrophils and hyposegmented neutrophils with fine nuclear bridging consistent with pseudo-Pelger-Huët anomaly. Flow cytometric analyses of Asxl1G643fs/+ BM cells confirmed aberrant myeloid differentiation with significant increase of granulocytic and monocytic cell fraction, significant decrease of hematopoietic stem cell (HSC) pool (c-kit+ Sca-1+ Lin- (LSK) cells and CD34- CD135- LSK cells), increased apoptosis in hematopoietic stem/progenitor cells (HSPC) and significant accumulation of G1-phase HSC in the cell cycle analysis, all of which were reminiscent of human myelodysplastic syndromes (MDS). Interestingly, Asxl1G643fs/+ mice became moribund about 600 days after birth when compared to Asxl1+/+ littermates with anemia, leukocytosis, neutrophilia/monocytosis and hepatosplenomegaly, suggesting the development of MDS/myeloproliferative neoplasm (MPN)-like disease, most relevant to human cases with chronic myelomonocytic leukemia.RNA sequencing and gene set enrichment analysis in Asxl1G643fs/+ mice identified derepression of sets of genes that are normally repressed by BMI1, a critical regulator of histone modification in polycomb repressive comprex 1 (PRC1). Among these genes were p16Ink4a, p21, and p15Ink4b all of which were significantly upregulated in Asxl1G643fs/+ LSK cells. We performed chromatin immunoprecipitation (ChIP) assay with an anti-H2AK119ub1 antibody using LSK cells from Asxl1G643fs/+ mice and found that H2AK119ub1 level around the promoter region of p16Ink4a was significantly decreased in Asxl1G643fs/+ LSK cells, suggesting perturbation of Bmi1-driven H2AK119ub1 histone modification by mutated Asxl1. Co-immunoprecipitation assay and in vitro pull-down assay revealed that the mutant form of ASXL1 had no ability to interact with BMI1 as opposed to wild type ASXL1 protein. In addition, we performed ChIP assay with an anti-BMI1 and anti-FLAG antibody using a cell line stably expressed FLAG-tagged wild-type (WT) ASXL1 or mutant form (MT), and found that BMI1 level around the promoter region of p16Ink4a was significantly decreased in the ASXL1 MT-expressed cell line, whereas no significant change in the FLAG level between WT and MT. Furthermore, we intercrossed Asxl1G643fs/+ mice with p16Ink4a heterozygous knock out mice to elucidate the effect of p16Ink4a up-regulation in Asxl1G643fs/+ mice, and found restoration of HSC pool and amelioration of increased apoptosis in HSPC obtained from Asxl1G643fs/+p16Ink4a+/- mice. These results indicated that loss of protein interaction between Asxl1 mutant and Bmi1 affected the activity of PRC1, and subsequent derepression of p16Ink4a by aberrant histone ubiquitination could induce cellular senescence, resulting in MDS-like phenotypes represented by decreased HSC pool and aberrant myeloid differentiation in Asxl1G643fs/+ mice.Taken together, our Asxl1 G643fs mutation knock-in mouse line is useful as a model of CHIP or early stage MDS, and our detailed analysis highlights the functional role of ASXL1 mutation on MDS and MDS /MPN development. DisclosuresMasamoto:Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Pfizer Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Kurokawa:Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Celgene Corporation: Speakers Bureau; Pfizer Inc.: Research Funding; Bristol-Myers Squibb: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau.