EVI1 drives leukemogenesis through aberrant activation of ERG. EVI1 drives leukemogenesis through aberrant activation of ERG

Chromosomal rearrangements involving EVI1 (MECOM) define a subtype of acute myeloid leukemia (AML) that is associated with a two-year survival rate of <10%. Gene regulatory functions of EVI1 are largely elusive and no targeted therapeutics exist. We developed experimentally tractable murine and human leukemia models that recapitulate phenotypic and transcriptional features of EVI1-rearranged AML and enable large-scale loss-of-function screens. We characterize EVI1-controlled transcriptional programs in cell culture and in vivo, perform CRISPR screens and identify the ETS-related transcription factor ERG as the only gene that is specifically required for EVI1-driven AML. ERG is transcriptionally activated by EVI1 and overexpressed in EVI1-rearranged AML patients. ERG suppression selectively induces terminal differentiation of leukemia cells. EVI1 becomes dispensable for leukemia cells upon ectopic expression of ERG, indicating that key functions of EVI1 are mediated through aberrant activation of ERG. Interfering with this regulatory axis may therefore provide new entry points for rational therapies. Overall design: RNA-seq of leukemia cells derived from mouse models driven by DOX-regulatable or constitutive RUNX1/EVI1, and NRAS(G12D) upon DOX treatment of mice (31848-31861). Quant-seq of in-vitro-cultured murine leukemia cells driven by constitutive RUNX1/EVI1 and NRAS(G12D) upon knockdown of RUNX1/EVI1 compared to ctrl (160665-160670). Quant-seq of in-vitro-cultured human AML cells (HNT-34) driven by EVI1 upon knockdown of EVI1 compared to ctrl (61648-61653). Quant-seq of in-vitro-cultured murine leukemia cells driven by constitutive RUNX1/EVI1, NRAS(G12D) upon knockdown of EVI1 in the presence or absence of ectopic ERG expression (160677-160688). ATAC-seq of murine leukemia cells driven by RUNX1/EVI1, NRAS(G12D) upon knockdown of RUNX1/EVI1 compared to ctrl (162167, 162169)

涉及EVI1（MECOM）的染色体重排，定义了急性髓系白血病（acute myeloid leukemia, AML）的一个亚型，该亚型的两年生存率低于10%。目前EVI1的基因调控功能在很大程度上仍不明确，且尚无针对该亚型的靶向治疗药物。本研究构建了实验上易于操作的小鼠及人源白血病模型，该模型可重现EVI1重排型AML的表型与转录组特征，并支持大规模功能缺失筛选实验。我们在细胞培养与活体水平上解析了EVI1调控的转录程序，通过CRISPR筛选鉴定出ETS家族转录因子ERG是EVI1驱动型AML所特异性必需的唯一基因。ERG可被EVI1转录激活，并在EVI1重排型AML患者体内呈高表达状态。抑制ERG可选择性诱导白血病细胞终末分化。当在白血病细胞中异位表达ERG时，EVI1便不再是细胞存活所必需的，这表明EVI1的核心功能是通过异常激活ERG来实现的。因此，靶向干预这一调控轴可为基于机制的精准治疗提供全新的切入点。实验整体设计：对经多西环素（DOX）处理的小鼠来源的白血病细胞进行RNA测序（RNA-seq），这些小鼠模型由多西环素可调控型或组成型RUNX1/EVI1及NRAS(G12D)驱动（样本编号：31848-31861）；对由组成型RUNX1/EVI1及NRAS(G12D)驱动的体外培养小鼠白血病细胞，在敲低RUNX1/EVI1后进行定量测序（Quant-seq）并以对照细胞作为参照（样本编号：160665-160670）；对由EVI1驱动的体外培养人源AML细胞（HNT-34），在敲低EVI1后进行定量测序并以对照细胞作为参照（样本编号：61648-61653）；对由组成型RUNX1/EVI1及NRAS(G12D)驱动的体外培养小鼠白血病细胞，在存在或不存在异位ERG表达的情况下敲低EVI1后进行定量测序（样本编号：160677-160688）；对由RUNX1/EVI1及NRAS(G12D)驱动的小鼠白血病细胞，在敲低RUNX1/EVI1后进行转座酶可及性测序（ATAC-seq）并以对照细胞作为参照（样本编号：162167、162169）。