Functional analysis of somatic cancer mutations in human DNA methyltransferases

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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.