Mechanisms of De Novo Methylation in Cancer

描述（由申请人提供）：该基金的目标是了解DNA甲基化模式的建立和遗传机制，并开发可以干扰胞嘧啶甲基化并重新激活沉默基因的药物。这项研究导致FDA最近批准了两种DNA去甲基化剂（5-aza-CR和5-aza-CdR）用于治疗骨髓增生异常综合征。在未来五年的项目中，我们希望利用表观基因组分析来了解DNA甲基化模式是如何通过DNA甲基转移酶和特定染色质组分之间的相互作用来建立和维持的。为此，我们开发了一种定制的NimbleGen阵列，可以在正常和转化细胞的1，800个转录起始位点（TSS）以综合方式分析核小体，组蛋白修饰和DNA甲基化。在具体目标1中，我们将利用平铺阵列来映射正常（PrEC）和转化的前列腺癌细胞（PC 3）中的核小体。然后，我们将确定在PC 3细胞中或在HCT 116结肠癌细胞中干扰DNA甲基化修饰如何改变组蛋白标记的分布，所述组蛋白标记集中于由多梳抑制复合物2（PRC 2）施加的组蛋白H3-K27 me 3标记。在具体目标2中，我们将确定在DNA甲基化恢复为HCT 116衍生物（DKO）期间表观基因组如何重组，其中三种DNA甲基转移酶中的两种（DNMT 1和DNMT 3B）已被遗传敲除。在具体目标3中，我们将跟进我们的新结果，这些结果显示从头甲基转移酶DNMT 3A和3B与核小体的强锚定。我们希望确定酶如何与核小体相互作用，以便我们能够理解特定模式是如何建立的。在具体目标4中，我们将继续寻求开发DNA去甲基化药物，这些药物比FDA目前批准用于癌症治疗的药物更稳定，并且能够逆转异常的DNA甲基化，组蛋白修饰和核小体定位。这些目标的实现将对我们理解癌症的表观遗传学产生重大影响，并与治疗和预防癌症的新策略直接相关。公共卫生相关性：在过去的几年中，已经清楚的是，体细胞可遗传的表观遗传过程导致的基因异常沉默可以直接导致人类癌症的形成。虽然我们知道DNA甲基化模式的改变在肿瘤抑制基因的沉默中起着重要作用，但我们不知道这些改变的模式是如何建立的，也不知道正常模式是如何在人类发育过程中建立和遗传的。该领域最近的兴奋集中在涉及多梳抑制复合物（PRCs）的基因沉默机制的潜在作用上，多梳抑制复合物对于正常发育是必需的，并且最近被发现在使肿瘤抑制基因失活中也起作用。这些PRCs可以直接沉默基因本身，也以某种方式建立基因更持久的沉默诱导DNA甲基化。最近，FDA批准了两种DNA去甲基化剂和一种组蛋白去乙酰化酶抑制剂用于治疗特定类型的癌症。

DESCRIPTION (provided by applicant): The objectives of this grant, which has been funded for almost 30 years, have been to understand the mechanisms for the establishment and inheritance of DNA methylation patterns and to develop drugs which can interfere with cytosine methylation and reactivate silenced genes. This research has led to the recent approval by the FDA of two DNA demethylating agents (5-aza-CR and 5-aza-CdR) for the treatment of myeloid dysplastic syndrome. In the next five year period of the project, we hope to take advantage of epigenomic analysis to understand how DNA methylation patterns are established and maintained by an interaction between DNA methyltransferases and specific chromatin components. To do this, we have developed a custom NimbleGen array allowing for the analyses of nucleosomes, histone modifications and DNA methylation in an integrated way at 1,800 transcription start sites (TSS) in normal and transformed cells. In Specific Aim 1, we will utilize the tiling array to map nucleosomes in both normal (PrECs) and transformed prostate cancer cells (PC3). We shall then determine how interfering with DNA methylation pharmacologically in PC3 cells or genetically in HCT116 colon cancer cells alters the distribution of histone marks focusing on the histone H3-K27me3 mark applied by the polycomb repressive complex 2 (PRC2). In Specific Aim 2, we shall determine how the epigenome is reorganized during the restoration of DNA methylation to HCT116 derivatives (DKO) in which two of the three DNA methyltransferases (DNMT1 and DNMT3B) have been genetically knocked down. In Specific Aim 3, we will follow-up on our new results which show the strong anchoring of the de novo methyltransferases DNMT3A and 3B to nucleosomes. We wish to determine how the enzymes interact with nucleosomes so that we can understand how specific patterns are established. In Specific Aim 4, we will continue our quest to develop DNA demethylating drugs which are more stable than those currently approved by the FDA for cancer treatment and which are able to reverse aberrant DNA methylation, histone modifications and nucleosome positioning. Achievement of these aims should have major impact in our understanding of the epigenetics of cancer and have direct relevance to new strategies to treat and prevent cancer. PUBLIC HEALTH RELEVANCE: It has become clear over the last few years that the abnormal silencing of genes by somatically heritable epigenetic processes, can contribute directly to the formation of human cancers. Although we know that altered patterns of DNA methylation play a fundamental role in the silencing of tumor suppressor genes, we do not know how these altered patterns are set up or how normal patterns are established and inherited during human development. Recent excitement in the field has focused on the potential role of gene silencing mechanisms involving the polycomb repressive complexes (PRCs), which are essential for normal development and have recently been found also to play a role in inactivating tumor suppressor genes. These PRCs can directly silence genes by themselves and also somehow set up genes for more permanent silencing induced by DNA methylation. Recently, the FDA has approved two DNA demethylating agents and one histone deacetylase inhibitor for the treatment of particular kinds of cancer.