Evi1 governs Kdm6b-mediated histone demethylation to regulate the Laptm4b-driven mTOR pathway in hematopoietic progenitor cells
Qiong Wu, Chunjie Yu, Fang Yu, Yiran Guo, Yue Sheng, Liping Li, Yafang Li, Yutao Zhang, Chao Hu, Jue Wang, Tong-chuan He, Yong Huang, Hongyu Ni, Zhiguang Huo, Wenshu Wu, Gang Greg Wang, Jianxin Lyu, Zhijian Qian
Qiong Wu, Chunjie Yu, Fang Yu, Yiran Guo, Yue Sheng, Liping Li, Yafang Li, Yutao Zhang, Chao Hu, Jue Wang, Tong-chuan He, Yong Huang, Hongyu Ni, Zhiguang Huo, Wenshu Wu, Gang Greg Wang, Jianxin Lyu, Zhijian Qian
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Research Article Hematology

Evi1 governs Kdm6b-mediated histone demethylation to regulate the Laptm4b-driven mTOR pathway in hematopoietic progenitor cells

Abstract

Ecotropic viral integration site 1 (EVI1/MECOM) is frequently upregulated in myeloid malignancies. Here, we present an Evi1-transgenic mouse model with inducible expression in hematopoietic stem/progenitor cells (HSPCs). Upon induction of Evi1 expression, mice displayed anemia, thrombocytopenia, lymphopenia, and erythroid and megakaryocyte dysplasia with a significant expansion of committed myeloid progenitor cells, resembling human myelodysplastic syndrome/myeloproliferative neoplasm–like (MDS/MPN–like) disease. Evi1 overexpression prompted HSPCs to exit quiescence and accelerated their proliferation, leading to expansion of committed myeloid progenitors while inhibiting lymphopoiesis. Analysis of global gene expression and Evi1 binding site profiling in HSPCs revealed that Evi1 directly upregulated lysine demethylase 6b (Kdm6b). Subsequently, Kdm6b-mediated H3K27me3 demethylation resulted in activation of various genes, including Laptm4b. Interestingly, KDM6B and LAPTM4B are positively correlated with EVI1 expression in patients with MDS. The EVI1/KDM6B/H3K27me3/LAPTM4B signaling pathway was also identified in EVI1hi human leukemia cell lines. We found that hyperactivation of the LAPTM4B-driven mTOR pathway was crucial for the growth of EVI1hi leukemia cells. Knockdown of Laptm4b partially rescued Evi1-induced abnormal hematopoiesis in vivo. Thus, our study establishes a mouse model to investigate EVI1hi myeloid malignancies, demonstrating the significance of the EVI1-mediated KDM6B/H3K27me3/LAPTM4B signaling axis in their maintenance.

Authors

Qiong Wu, Chunjie Yu, Fang Yu, Yiran Guo, Yue Sheng, Liping Li, Yafang Li, Yutao Zhang, Chao Hu, Jue Wang, Tong-chuan He, Yong Huang, Hongyu Ni, Zhiguang Huo, Wenshu Wu, Gang Greg Wang, Jianxin Lyu, Zhijian Qian

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Figure 5

Evi1 regulates H3K27me3 level through Kdm6b.

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Evi1 regulates H3K27me3 level through Kdm6b. (A) Western blot (WB) analy...
(A) Western blot (WB) analysis showing H3 modifications and Kdm6b expression using Lin–c-Kit+ cells from WT and Evi1-overexpressing mice. Actin and H3 were used as loading controls. (B) Box plot showing metagene analysis for the H3K27me3 level at the promoter regions of upregulated genes in Evi1-overexpressing Lin–c-Kit+ cells. (C and D) ChIP-qPCR analyses indicate that Evi1 directly binds to the promoter region of Kdm6b in Lin–c-Kit+ mouse BM cells (C) and erythroid myeloid lymphoid (EML) cells (D). (E and F) Assessment of the expression level of EVI1 and KDM6B at mRNA level in U937, Kasumi-3, and AML1 cells by RT-qPCR. (G) Western blot assessed the protein levels of EVI1, KDM6B, H3K27me3, and H3K4me3 in U937, Kasumi-3, and AML1 cells. (H) WB analysis confirming the protein expression of FLAG-tagged EVI1 in U937 cells. (I) ChIP-qPCR analysis indicates that exogenous EVI1 directly binds to the promoter region of KDM6B in U937 cells. (J) WB analysis showing the EVI1 enrichment for the ChIP-qPCR analysis in AML1 cells. (K) ChIP-qPCR analysis indicates that endogenous EVI1 directly binds to the promoter region of KDM6B in AML1 cells. (L) WB analysis shows the downregulation of KDM6B and upregulation of H3K27me3 in AML1 cells transduced with KDM6B shRNAs. Data are representative of at least 2 independent experiments. In B, median values are indicated by the line within the box plot (minimum to maximum whiskers). P value was calculated by a 2-tailed Mann-Whitney test. In C–F, I, and K, data are presented as mean ± SD, with ordinary 1-way ANOVA with Dunnett’s multiple-comparison test used for C–F and 2-tailed Student’s t test for I and K. *P < 0.05, ***P < 0.001.