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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 Article has an altmetric score of 11

Myelodysplastic syndromes are induced by histone methylation–altering ASXL1 mutations

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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 1

ASXL1 mutations inhibit G-CSF–induced myeloid differentiation of 32Dcl3 cells.

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ASXL1 mutations inhibit G-CSF–induced myeloid differentiation of 32Dcl3...
(A) Schematic diagram of ASXL1-WT and 2 mutants, ASXL1-MT1 and ASXL1-MT2. ASXH, ASX homology region; NLS, nuclear localization signal; NR, putative nuclear receptor coregulator binding motifs; PHD, plant homeodomain. (B) Expression of ASXL1-WT and its mutants in 293T cells or 32Dcl3 cells transiently transfected or stably transduced with a FLAG-ASXL1-MT1, ASXL1-MT2, ASXL1-WT, or an empty vector (pMYs-IG). (C) HEK293T cells were transiently transfected with FLAG-ASXL1-MT, FLAG-ASXL1-WT, and Myc-EZH2 cDNA, followed by IP of FLAG epitope and Western blotting for EZH2. Cell lysates were also subject to immunoblotting with anti-FLAG Ab or anti-Myc Ab. (D) ASXL1 protein expression using C-terminus anti-ASXL1 antibodies in leukemia cell lines. Harboring mutations are as follows: K562, heterozygous ASXL1 Y591Y/X; KU812, heterozygous ASXL1 R693R/X; Mono-Mac-6, heterozygous ASXL1 L1393RfsX30; MEG-01, homozygous ASXL1 G646WfsX12; TS9:22, homozygous ASXL1 R693X; KBM5, homozygous ASXL1 G710X. (E) Nuclear localization of ASXL1-MTs. The 293T cells transiently transfected with pMYs-IG, pMYs-FLAG-ASXL1-MT1-IG, pMYs-FLAG-ASXL1-MT2-IG, or pMYs-FLAG-ASXL1-WT-IG were immunostained with anti-FLAG Ab (red) and DAPI (blue). Original magnification, ×600. (F) Surface expression of CD11b in 32Dcl3 cells transduced with indicated plasmid after incubation with 1 ng/ml IL-3 (blue) or 50 ng/ml G-CSF for 6 days (red) was analyzed by flow cytometry (top). Filled histograms show control (IgG). Proportions of segmented cells on days 0, 3, and 6 of G-CSF stimulation are shown (middle). Morphology of the cells 6 days after G-CSF stimulation was assessed by Wright-Giemsa staining (bottom). Original magnification, ×400; scale bars: 20 μm.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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