Mutations in a variety of genes have been identified in MDS patients. Among them, mutations of additional sex combs-like 1 (ASXL1), found in 15-20% of MDS patients, have been identified as an independent poor prognostic factor. We previously demonstrated that C-terminal–truncating ASXL1 mutations (ASXL1-MT) inhibited myeloid differentiation and induced an MDS-like disease in mice after 1~2 years by inhibiting polycomb repressive complex 2–mediated methylation of histone H3K27 (Inoue et al. J Clin Invest. 2013). Given that ASXL1 mutations have been shown to be related to high-risk MDS or leukemic transformation, it is not clear how ASXL1-mutated MDS clones can transform into advanced MDS or AML.

First, we examined genetic alterations in 368 WHO-defined MDS patients; ASXL1 mutations were detected in 64 of them (17.39%). Intriguingly, the patients with ASXL1 mutations had a significantly higher incidence of the concurrent SET binding protein 1 (SETBP1) mutation than those with the wild-type ASXL1 (6 out of 64, 9.38% vs. 2 out of 304, 0.66%, P=0.0005). Moreover, among ASXL1-mutated MDS patients, those harboring SETBP1 mutations had a higher incidence of leukemic transformation than those without (P=0.042), and MDS patients with both mutations had a significantly shorter overall survival compared to those without SETBP1 mutations (median, 10.5 vs. 22.5 months, P=0.046). In addition, we demonstrated that most SETBP1 mutations, such as D868N, occur in the PEST domain of the SKI homology region, preventing ubiquitination and subsequent proteasomal degradation. These results prompted us to investigate whether SETBP1 mutations play a critical role in the leukemic transformation of ASXL1-mutated MDS cells.

In in vitro experiments, the expression of SETBP1-D868N enhanced myeloid colony formation of ASXL1-MT-transduced LSK cells, augmenting ASXL1-MT-induced differentiation blocking of 32Dcl3 cells. Of note, SETBP1-D868N collaborated with ASXL1-MT to induce AML after a short latency (median survival, 73 days) in a murine BMT model, while all mice expressing either ASXL1-MT or SETBP1-D868N survived for 6 months after transplantation (P<0.0001). Mice with leukemia induced by the combination of ASXL1-MT and SETBP1-D868N exhibited remarkable leukocytosis, anemia, thrombocytopenia, macrocytosis, hematosplenomegaly and hypercellular BM when compared to control mice.

To clarify the molecular mechanism leading to leukemic transformation, we first investigated the Pp2a-Akt pathway because SETBP1 protein has been shown to interact with SET oncoprotein, resulting in Pp2a phosphorylation and subsequent inhibition. Consistent with previous reports using overexpression systems of SETBP1 wild type protein (SETBP1-WT), BM cells of leukemic mice displayed phosphorylated Pp2a and Akt compared to those of the control mice. Administration of FTY720, a Pp2a activator, efficiently repressed the growth rate in vitro and slightly improved the survival of serially transplanted mice. Next, using RNA-seq and GSEA, we demonstrated that SETBP1-D868N enriched hematopoietic stem cell-related genes and posterior Hoxa genes. Chromatin immnoprecipitation assay showed that both SETBP1-WT and SETBP1-D868N interacted with the promoter regions of Hoxa9 and Hoxa10, raising the possibility that a gain-of-function mutant of SETBP1 enhances transcription of these genes, directly or indirectly. Moreover, GSEA indicated global repression of the TGF-β signaling pathway and reciprocal upregulation of the Myc pathway in leukemic mice.

In conclusion, our data provide evidence for the role of SETBP1 mutations in leukemic transformation and suggest the resulting deregulated pathways as potential therapeutic targets to prevent disease progression in MDS.


Harada:Kyowa Hakko Kirin Co., Ltd.: Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.

Sign in via your Institution