Determining the Role of DNA Methylation Deregulation in Oncogenesis

项目总结/摘要 DNA（胞嘧啶-5）-甲基转移酶3A（DNMT 3AMut）的体细胞突变发生在>20-30%的 急性髓性 (AML)使其成为这种致命的人类疾病中最常见的突变基因之一。 DNMT 3AMut与AML的不良临床结果相关。然而， DNMT 3AMut对AML发展的贡献仍然远未明确。我们报告说，DNMT 3AMut促进了 细胞转化，并在具有起始RAS突变的小鼠中建立真正的AML表型。 这种新的小鼠AML模型的整合转录组和表观基因组分析显示，DNMT 3AMut 直接与靶基因的增强子结合，在结合位点诱导局灶性DNA低甲基化。这些 DNMT 3AMut介导的事件导致控制干细胞的基因表达程序的异常激活 自我更新（特别是Meis 1结）、抗分化和肿瘤细胞促存活。我们基于发现的 表观遗传抑制剂筛选进一步鉴定了DOT 1 L组蛋白甲基转移酶对于 DNMT 3AMut诱导的异常基因激活。我们假设DNMT 3AMut诱导局灶性DNA低表达， 白血病 促进DOT 1 L依赖性自我更新激活（特别是Meis 1）的基因增强子甲基化， 分化和肿瘤促存活基因，其共同促成AML发病机制。 解剖 分子事件及其机制 DNMT3AMut - 介导的AML进展应提供关键的 新的治疗策略的见解。为了实现这一目标，我们将使用尖端的CRISPR/Cas9技术， 确定DNMT 3AMut引起的增强子DNA低甲基化在诱导异常基因中的因果作用， 激活和促进AML决定了DNMT 3AMut激活的基因通路是什么 在体外和体内对小鼠AML发展至关重要 (aim（1）我们将 通过我们建立的功能丧失研究， 鼠AML模型（目的2）;第三，在转化目的中，我们将使用人AML细胞系和原代AML细胞系。 携带DNMT 3AMut的患者来源的异种移植物（PDX）模型，以确定DNMT 3AMut对表观基因组 人类AML中的基因改变、基因表达失调和恶性生长（目的3）。我们希望定义 DNMT 3AMut诱导的AML表观遗传/基因变化，并期望确定DNMT 3AMut 促进AML进展。因为某些已确定的途径，如DOT 1 L和Bcl 2， 可与现有的化合物 ，完成我们建议的研究不仅可以促进一个新的 机理的认识 DNMT 3AMut相关 AML，但也将产生创新的治疗方法， 治疗受影响的癌症患者。

PROJECT SUMMARY/ABSTRACT Somatic mutation of DNA (cytosine-5)-methyltransferase 3A (DNMT3AMut) occurs in >20-30% of acute myeloid (AML) patients making it one of the most frequently mutated genes in this deadly human disease. DNMT3AMut correlates with poor clinical outcome of AML. However, the molecular mechanism by which DNMT3AMut contributes to AML development remains far from clear. We reported that DNMT3AMut promotes cell transformation and establishes a bona fide AML phenotype in mice possessing the initiating RAS mutation. Integrated transcriptome and epigenomic profiling of this new murine AML model revealed that DNMT3AMut binds directly to enhancers of target genes inducing focal DNA hypo-methylation at binding sites. These DNMT3AMut-mediated events result in aberrant activation of a gene-expression program controlling stem cell self-renewal (notably a Meis1 node), anti-differentiation and tumor cell pro-survival. Our discovery-based epigenetic inhibitor screen further identified the DOT1L histone methyltransferase to be essential for DNMT3AMut-induced aberrant gene activation. We hypothesize that DNMT3AMut induces focal DNA hypo- leukemia methylation at gene enhancers promoting DOT1L-dependent activation of self-renewal (notably Meis1), anti- differentiation and tumor pro-survival genes, which collectively contribute to AML pathogenesis. Dissecting the molecular events and mechanism underlying DNMT3AMut -mediated AML progression should provide critical insights into new treatment strategies. Towards this goal, we will use cutting-edge CRISPR/Cas9 technologies to define the causal role for enhancer DNA hypomethylation due to DNMT3AMut in inducing aberrant gene activation and promoting AML determine what DNMT3AMut-activated gene pathways are essential for AML development in mice in vitro and in vivo (aim 1); we will through loss-of-function studies in our established murine AML models (aim 2); and third, in a translational aim, we will use human AML cell lines and primary patient-derived xenograft (PDX) models bearing DNMT3AMut to define effects of DNMT3AMut on epigenomic alteration, gene expression deregulation and malignant growth in human AML (aim 3). We expect to define the DNMT3AMut-induced epigenetic/gene changes in AML and expect to identify the pathway by which DNMT3AMut promotes AML progression. Because certain identified pathways such as DOT1L and Bcl2 are potentially druggable with the existing compounds , completion of our proposed research should not only promote a new mechanistic understanding of DNMT3AMut-associated AML but will also yield innovative therapeutics for the treatment of affected cancer patients.