Functional analysis of somatic cancer mutations in human DNA methyltransferases

DNA测序技术的快速发展为癌症生物学提供了许多重要的新见解，其中包括在癌症组织中鉴定许多体细胞突变。有趣的是，许多受影响的基因直接或间接地介导表观遗传修饰，如IHD、DNMT3A、TET2或EZH2，它们是体细胞癌突变的热点。DNA甲基转移酶(DNMT)中的许多体细胞癌突变可能具有功能获得效应，因为它们是杂合发生的，突变谱显示许多错义突变，但只有少数插入、缺失或无义突变。DNMT的功能获得突变可以激活酶的活性，改变其侧翼序列的偏好，亚核定位或破坏其调控，并通过这些机制之一导致肿瘤细胞中选择基因的高甲基化。我的团队在所有DNA甲基转移酶的表达和纯化方面都有丰富的经验，我们开发了独特的检测系统来研究DNMT的活性、特异性和酶性质。基于我们的专业知识，我们计划进行一项假设驱动的分析，分析DNMT中的癌症突变对酶的催化活性、侧翼序列偏好、加工性、亚核定位、多聚体、调控以及与其他蛋白质如Dnmt3L、PCNA或NP95的相互作用的影响。我们将集中精力研究突变，对其功能机制提出可实验验证的假说。我们工作的重点之一将是研究DNMT3A R882突变的机制，这种突变在癌症样本中非常普遍。将在人类细胞的全基因组DNA甲基化实验中研究显示其酶或生化特性发生有趣变化的变体，并将结果与体外数据相关联。此外，我们还将研究我们实验室开发的DNMT3A抑制剂对表达DNMT3A癌症变体的癌细胞株的影响。我们的结果将有助于确定体细胞癌DNMT突变的致癌作用，并了解DNA甲基化变化是如何导致癌症的。这将支持开发针对含有突变DNMT的肿瘤的更有针对性的治疗方法。

The rapid development of DNA sequencing techniques has provided many important new insights into cancer biology, among them the identification of many somatic mutations in cancer tissues. Interestingly, many of the affected genes mediate epigenetic modifications either directly or indirectly, like IHD, Dnmt3a, TET2 or EZH2 which are hotspots of somatic cancer mutations. Many of the somatic cancer mutations in DNA Methyltransferases (Dnmts) are likely to have a gain-of-function effect, because they occur heterozygously and the mutational spectrum shows many missense mutations but only few insertion, deletions or nonsense mutations. Gain-of-function mutations in Dnmts could activate the enzymatic activity, change its flanking sequence preferences, sub-nuclear localization or compromise its regulation and by one of these mechanisms cause hypermethylation of select loci in cancer cells. My group has great experience in the expression and purification of all DNA methyltransferases and we have developed unique assay systems to study the activity, specificity and enzymatic properties of Dnmts. Based on our expertise, we plan a hypothesis driven analysis of the effects of the cancer mutations in Dnmts on the enzymes' catalytic activity, flanking sequence preferences, processivity, sub-nuclear localization, multimerization, regulation and interaction with other proteins like Dnmt3L, PCNA or NP95. We will concentrate our efforts on mutations, for which experimentally testable hypotheses for their functional mechanism could be proposed. One focus of our work will be the investigation of the mechanism of the Dnmt3a R882 mutations which are very prevalent in cancer samples. Variants showing interesting changes of their enzymatic or biochemical properties will be studied in genome wide DNA methylation experiments in human cells and the results correlated with the in vitro data. In addition, we will study the effect of Dnmt3a inhibitors developed in our lab on cancer cell lines expressing the Dnmt3a cancer variants. Our results will help to define the cancerogenic effect of somatic cancer Dnmt mutations and to understand how changes in DNA methylation lead to cancer. This will support the development of more targeted therapies for tumors containing mutated Dnmts.