Evi1 Governs Kdm6b-Mediated Histone Demethylation to Regulate the Laptm4b-Driven mTOR Pathway in Hematopoietic Progenitor Cells
Authors: Wu Qiong , Yu Chunjie , Yu Fang , Guo Yiran , Sheng Yue , Li Liping , Li Yafang , Zhang Yutao , Hu Chao , Wang Jue , He Tong-Chuan , Huang Yong , Ni Hongyu , Hou Zhigang , Wu Wenshu , Wang Greg G , Lyu Jianxin , Qian Zhijian 
Published: December 2024

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic disorders in which the production of blood cells is disrupted, resulting in a lack of functional blood cells. This clinical phenotypes of MDS are nonspecific and can overlap with various other benign or malignant conditions, such as myeloproliferative neoplasm (MPN). Approximately 30% of MDS patients ultimately develop acute myeloid leukemia (AML). Ecotropic viral integration site 1 (Evi1) was first identified in mice as a common site of ecotropic viral integration, inducing myeloid malignancies. Human EVI1 (MECOM) gene is located on chromosome 3q26, and the rearrangements of chromosome 3q26, which leads to upregulation of EVI1, frequently occurs in myeloid malignant diseases including MDS, AML, and Chronic Myeloid Leukemia (CML). MDS, AML and CML with inv(3)/t(3;3) rearrangements often present similar pathological features with poor prognosis. High expression of EVI1 is also detected in a subgroup of MDS/MPN. However, the molecular mechanisms underlying the role of EVI1 overexpression in leukemogenesis in vivo remain incompletely understood, and targeted therapies for MDS/AML patients with EVI1 overexpression are currently lacking.
In our study, we present a novel transgenic mouse model allowing conditional activation of Evi1 expression at low levels in 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 (MDS/MPN)-like disease. To investigate the mechanism underlying the Evi1-OE mediated development of the MDS/MPN-like disease, we performed integrative analysis of gene expression profiling and EVI1 binding sites in HSPCs by RNA-seq and the cleavage under target and release using nuclease followed by sequencing (CUT&RUN-seq). Lysine demethylase 6b (Kdm6b) was identified as a direct target of Evi1 in primary HSPCs in mice. Kdm6b is a member of JmjC domain-containing histone demethylases that specifically removes methyl groups from H3K27me3 to activate its target genes. We found that Evi1 directly binds to the promoter of Kdm6b, and Evi1-overexpressing HSPCs have a reduced level of H3K27me3 compared to WT HSPCs. Moreover, analysis of the H3K27me3 CUT&RUN-seq results showed a global decrease in H3K27me3 enrichment specifically at the promoter regions of target genes following Evi1 overexpression. This suggests that Kdm6b at least partially mediates the effects of Evi1 overexpression in HSPCs. The demethylation of H3K27me3 induced by the upregulation of Kdm6b in Evi1-overexpressing HSPCs resulted in the upregulation of multiple genes, including Laptm4b. Analysis of public data showed that the expression levels of both KDM6B and LAPTM4B were positively correlated with EVI1 expression in these patients. Further western blot analysis showed that KDM6B and LAPTM4B are upregulated in both EVI1high human leukemia cells and Evi1-overexpressing mouse HSPCs, and LAPTM4B activates the mTOR pathway in these cells. Additionally, both KDM6B inhibitor and LAPTM4B knockdown inhibit growth and induce apoptosis in EVI1high human leukemia cells and Evi1-overexpressing mouse HSPCs, significantly reducing their colony-forming ability. Moreover, LAPTM4B knockdown partially reverses Evi1-induced abnormal hematopoiesis in vivo.
Our findings highlight the presence of EVI1/KDM6B/H3K27me3/LAPTM4B signaling axis in EVI1-overexpressed myeloid malignancies and suggest that pharmacologic approaches to interrupt the Evi1/Kdm6b/H3K27me3/Laptm4b/mTOR signaling axis may serve as important strategies to treat EVI1high hematopoietic malignancies.