Myelodysplastic syndromes are induced by histone methylation–altering ASXL1 mutations
Daichi Inoue, … , Omar Abdel-Wahab, Toshio Kitamura
Daichi Inoue, … , Omar Abdel-Wahab, Toshio Kitamura
Published October 8, 2013
Citation Information: J Clin Invest. 2013;123(11):4627-4640. https://doi.org/10.1172/JCI70739.
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Research Article Hematology

Myelodysplastic syndromes are induced by histone methylation–altering ASXL1 mutations

Abstract

Recurrent mutations in the gene encoding additional sex combs-like 1 (ASXL1) are found in various hematologic malignancies and associated with poor prognosis. In particular, ASXL1 mutations are common in patients with hematologic malignancies associated with myelodysplasia, including myelodysplastic syndromes (MDSs), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal–truncating Asxl1 mutations (ASXL1-MTs) inhibited myeloid differentiation and induced MDS-like disease in mice. ASXL1-MT mice displayed features of human-associated MDS, including multi-lineage myelodysplasia, pancytopenia, and occasional progression to overt leukemia. ASXL1-MT resulted in derepression of homeobox A9 (Hoxa9) and microRNA-125a (miR-125a) expression through inhibition of polycomb repressive complex 2–mediated (PRC2-mediated) methylation of histone H3K27. miR-125a reduced expression of C-type lectin domain family 5, member a (Clec5a), which is involved in myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1-MT, while CLEC5A expression was generally low. Thus, ASXL1-MT–induced MDS-like disease in mice is associated with derepression of Hoxa9 and miR-125a and with Clec5a dysregulation. Our data provide evidence for an axis of MDS pathogenesis that implicates both ASXL1 mutations and miR-125a as therapeutic targets in MDS.

Authors

Daichi Inoue, Jiro Kitaura, Katsuhiro Togami, Koutarou Nishimura, Yutaka Enomoto, Tomoyuki Uchida, Yuki Kagiyama, Kimihito Cojin Kawabata, Fumio Nakahara, Kumi Izawa, Toshihiko Oki, Akie Maehara, Masamichi Isobe, Akiho Tsuchiya, Yuka Harada, Hironori Harada, Takahiro Ochiya, Hiroyuki Aburatani, Hiroshi Kimura, Felicitas Thol, Michael Heuser, Ross L. Levine, Omar Abdel-Wahab, Toshio Kitamura

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Figure 7

ASXL1 mutations caused upregulation of miR-125a, leading to the repression of Clec5a.

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ASXL1 mutations caused upregulation of miR-125a, leading to the repress...
(A) Schematic presentation of predicted miR-125a–binding sites in the mouse Clec5a (top) and human CLEC5A 3′UTR (bottom). (B) To confirm that Clec5a is a direct target gene of miR-125a, the WT 3′UTR of Clec5a or the mutated 3′UTR was cloned to downstream of the Renilla luciferase (Rluc) open reading frame. Schematic diagrams of predicted miR-125a–binding sites in the Clec5a-3′UTR and the alignment between miR-125a and either Clec5a-3′UTR (top) or a mutated 3′UTR (bottom) are shown. Three bases in the 3′UTR, corresponding to seed sequences, were replaced with the indicated bases in the mutant form. (C) 293T cells were cotransfected with an internal control vector (pGL3-control) plus either pGL4.74[hRluc/TK]-Clec5a3′UTR-WT or pGL4.74[hRluc/TK]-Clec5a3′UTR-MT plus either pMXs-EF1-miR-125a-Puro or mock (pMXs-EF1-Puro). Luciferase assays were performed with a triplicate set. (D) Relative expression levels of Clec5a by qRT-PCR in 32Dcl3 cells transduced with pMXs-EF1-Puro (mock), pMXs-EF1-miR-125a-Puro, and pMXs-EF1-miR-125b-Puro. (E) Positive rate of Clec5a expression in 32Dcl3 cells transduced with mock, miR-125a, or miR-125b after incubation with 1 ng IL-3 (0 hours) or 50 ng/ml G-CSF (12 hours) was analyzed by flow cytometry at indicated time points. (F) The proportion of mature cells (left) and cytospin preparations (right) of the 32Dcl3 cells expressing mock, miR-125a, or miR-125b cultured in the presence of 50 ng/ml G-CSF for 6 days. Scale bars: 20 μm. *P < 0.05.