Abstract
BackgroundAdditional sex combs-like 1 (ASXL1) mutations have been described in all forms of myeloid neoplasms including chronic myelomonocytic leukemia (CMML) and associated with inferior outcomes, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) remains poorly understood. Transformation of CMML to secondary AML (sAML) is one of the leading causes of death in CMML patients. Previously, we observed that transcription factor RUNX1 mutations (RUNX1-MT) coexisted with ASXL1-MT in CMML and at myeloid blast phase of chronic myeloid leukemia. The contribution of RUNX1 mutations in the pathogenesis of myeloid transformation in ASXL1-mutated leukemia, however, remains unclear.MethodsTo evaluate the leukemogenic role of RUNX1-MT in ASXL1-mutated cells, we co-expressed RUNX1-MT (R135T) and ASXL1-MT (R693X) in different cell lines and performed immunoblot, co-immunoprecipitation, gene expression microarray, quantitative RT-PCR, cell proliferation, differentiation, and clonogenic assays for in vitro functional analyses. The in vivo effect was investigated using the C57BL/6 mouse bone marrow transplantation (BMT) model.ResultsCo-expression of two mutant genes increased myeloid stem cells in animal model, suggesting that cooperation of RUNX1 and ASXL1 mutations played a critical role in leukemia transformation. The expression of RUNX1 mutant in ASXL1-mutated myeloid cells augmented proliferation, blocked differentiation, and increased self-renewal activity. At 9 months post-BMT, mice harboring combined RUNX1 and ASXL1 mutations developed disease characterized by marked splenomegaly, hepatomegaly, and leukocytosis with a shorter latency. Mice transduced with both ASXL1 and RUNX1 mutations enhanced inhibitor of DNA binding 1 (ID1) expression in the spleen, liver, and bone marrow cells. Bone marrow samples from CMML showed that ID1 overexpressed in coexisted mutations of RUNX1 and ASXL1 compared to normal control and either RUNX1-MT or ASXL1-MT samples. Moreover, the RUNX1 mutant protein was more stable than WT and increased HIF1-α and its target ID1 gene expression in ASXL1 mutant cells.ConclusionThe present study demonstrated the biological and functional evidence for the critical role of RUNX1-MT in ASXL1-mutated leukemia in the pathogenesis of myeloid malignancies.
Highlights
Additional sex combs-like 1 (ASXL1) mutations have been described in all forms of myeloid neoplasms including chronic myelomonocytic leukemia (CMML) and associated with inferior outcomes, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) remains poorly understood
Expression of Runt-related transcription factor 1 (RUNX1) mutant augmented cell proliferation and impaired differentiation of ASXL1-mutated cells To understand the biological significance of RUNX1MTs with ASXL1-MTs in CMML patients, we first examined the effect of RUNX1 mutant on cell proliferation and differentiation in ASXL1-mutated cells
(See figure on previous page.) Fig. 1 The collaboration of ASXL1 and RUNX1 mutants augmented proliferation and impair differentiation in vitro. a, b IL-3-dependent murine myeloid 32D cells stably transduced with ASXL1-R693X, RUNX1-WT/R135T, and combination of ASXL1-R693X and RUNX1-R135T were cultured in the absence (a) or in the presence (b) of IL-3 for 96 h
Summary
Additional sex combs-like 1 (ASXL1) mutations have been described in all forms of myeloid neoplasms including chronic myelomonocytic leukemia (CMML) and associated with inferior outcomes, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) remains poorly understood. Transformation of CMML to secondary AML (sAML) is one of the leading causes of death in CMML patients. The contribution of RUNX1 mutations in the pathogenesis of myeloid transformation in ASXL1-mutated leukemia, remains unclear. Somatic mutations of additional sex comb-like protein 1 (ASXL1) gene have been described in patients with various types of myeloid malignancies, including myelodysplastic syndromes (MDS) (15–25%), myeloproliferative neoplasms (MPN) (10–15%), 40% of chronic myelomonocytic leukemia (CMML), a few patients with chronic myeloid leukemia (CML), and 15–20% of acute myeloid leukemia (AML) [1,2,3,4,5]. Zhao et al found that RUNX1 mutants exhibited decreased transactivation activity as well as had a dominant-negative function on the WT-RUNX1 as a result of AML transformation in a subset of CML patients [20]
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