MECOM is a master repressor of myeloid differentiation through dose control of CEBPA in acute myeloid leukemia [ChIP-seq]

The transcription factor MECOM, located at 3q26, is essential for hematopoietic stem cells (HSCs) in healthy individuals. Enhancer translocations, due to 3q26 rearrangements, drive out-of-context MECOM expression in an aggressive subtype of acute myeloid leukemia (AML). Aberrantly expressed MECOM is essential for the survival and immature phenotype of these leukemia cells. Direct depletion of MECOM using an endogenous auxin-inducible degron immediately upregulates expression of myeloid differentiation factor CEBPA. MECOM depletion is also accompanied by a severe loss of CD34 expression and gain of mature myeloid cell surface marker CD15. MECOM exerts its inhibitory effect on differentiation by binding to the +42kb CEBPA enhancer, which is required for neutrophil development. This is partially dependent on the interaction between MECOM and its essential co-repressor CTBP2, a potential target in this type of AML. We demonstrate that CEBPA overexpression can bypass the MECOM-mediated block of differentiation. In addition, AML patients with MECOM overexpression through enhancer hijacking show significantly reduced CEBPA. Our study provides insight into the mechanism by which MECOM maintains the stem cell state in AML. Furthermore, CEBPA levels are an important read-out for successful targeting of MECOM in 3q26-rearranged AML. We introduced an auxin-inducible degron (AID) (V5-AID-T2A-eGFP) 3’ of MECOM in inv(3) cell line MUTZ3. Next, chromatin immunoprecipitation followed by sequencing (ChIP-Seq) for EVI1, CTBP2, various transcription factors (p300, RUNX1) and histone modifications (H3K27ac, H3K27me3, H3k9me3) was performed in this cell line, either after auxin-induced MECOM degradation or in the absence of treatment.

位于3q26区域的转录因子MECOM（MECOM），在健康个体的造血干细胞（hematopoietic stem cells, HSCs）中发挥核心功能。因3q26染色体重排引发的增强子易位，会在侵袭性急性髓系白血病（acute myeloid leukemia, AML）亚型中驱动MECOM的异位表达。异常高表达的MECOM对这类白血病细胞的存活及未成熟表型维持至关重要。利用内源性生长素诱导型降解子（auxin-inducible degron, AID）直接敲除MECOM，可即刻上调髓系分化因子CEBPA（CEBPA）的表达。MECOM敲除同时会伴随CD34表达的严重缺失，以及成熟髓系细胞表面标志物CD15的表达上调。MECOM通过结合+42kb位置的CEBPA增强子发挥分化抑制作用，该增强子是中性粒细胞发育所必需的顺式调控元件。这一调控过程部分依赖于MECOM与其必需辅阻遏因子CTBP2（CTBP2）的相互作用，而CTBP2是此类AML的潜在治疗靶点。本研究证实，CEBPA过表达可绕过MECOM介导的分化阻滞。此外，通过增强子劫持实现MECOM过表达的AML患者，其CEBPA表达水平显著降低。本研究为MECOM维持AML干细胞状态的分子机制提供了全新见解。同时，CEBPA的表达水平可作为3q26重排AML中MECOM靶向治疗成功与否的重要检测指标。我们在inv(3)细胞系MUTZ3的MECOM基因3'端引入了生长素诱导型降解子（AID）（V5-AID-T2A-eGFP）。随后，分别在生长素诱导MECOM降解及未处理的该细胞系中，针对EVI1、CTBP2、多种转录因子（p300、RUNX1）以及组蛋白修饰（H3K27ac、H3K27me3、H3K9me3）开展了染色质免疫沉淀测序（ChIP-Seq）实验。