Targeting DNA Methylation and the Cancer Epigenome

项目概要 我们的实验室于 1980 年发现 DNA 甲基化（表观遗传修饰）会影响基因表达 和细胞分化。高通量测序和癌症基因组的意外结果 Atlas 和其他项目表明，癌症的许多突变都发生在改变表观基因组的基因中。这个 验证了我们的长期假设，即异常的表观遗传过程是人类的主要贡献者 癌症并提供新的治疗机会。了解表观基因组在癌症中如何变化 需要一种我们在过去五年中制定的综合方法。我们希望确定 DNA 甲基化、核小体定位和组蛋白等关键特征的机制 修改相互影响。然后我们将确定 DNA 甲基化抑制剂 (DNMTi) 的工作原理。 这笔资助旨在使用我们强大的新 NOMe-seq 技术来了解之间的关系 DNA甲基化和核小体定位；使用击倒和其他方法来检查 改变染色质重塑酶和组蛋白修饰酶对整个表观基因组的影响； 并了解表观基因组如何控制内源性逆转录病毒（ERV）。我们将研究如何 表观基因组在人类癌症中发生了改变，不仅表征了基因启动子区域的变化，还表征了 增强子和绝缘子区域以及基因本身。需要解决的主要问题包括 1) 为什么 染色质修饰物有这么多突变，这些突变对染色质结构有什么影响 表观基因组？ 2) 激活癌症/睾丸基因表达的功能后果是什么 5-氮杂核苷？ 3) 哪些双链RNA被5-氮杂核苷激活以及这些双链RNA是如何激活的 与细胞反应有关吗？ 4）我们可以设计表观遗传药物的组合来增加 5-氮杂核苷治疗的效果如何？ 5) 冷冻电镜可以帮助可视化与以下相关的复合物吗？ 染色质的结构和功能？我们还将研究 TET 酶的作用，该酶氧化 5- 甲基胞嘧啶转化为 5-羟甲基胞嘧啶，需要维生素 C 作为辅助因子，酶 G9A 和 SETDB1，使组蛋白 H3K9 甲基化。增加 ERV 表达的组合将是 优先考虑，因为最近的数据强烈表明 ERV 表达可能与细胞变化有关，并且 很可能会影响癌症的临床结果。癌症患者经常缺乏维生素C，这意味着 补充可能会显着增加 TET 活性和患者反应。我们的方法不仅是设计 全面了解表观基因组，同时制定策略以提高患者对表观基因组的反应 药物，也许通过定义未来合理的药物组合，特别是利用 DNMTi。成功的 该项目应该具有快速的机械和转化影响，因为 DNMTi 已获得 FDA 批准或正在 目前正在进行癌症治疗试验。

Project Summary Our laboratory discovered in 1980 that methylation of DNA (epigenetic modification) affected gene expression and cell differentiation. High-throughput sequencing and the unexpected outcomes from The Cancer Genome Atlas and other projects show that many mutations in cancer are in genes that modify the epigenome. This has validated our long-term hypothesis that abnormal epigenetic processes are major contributors to human cancer and offer novel therapeutic opportunities. Understanding how the epigenome is changed in cancer requires an integrated approach, which we have developed over the last five years. We wish to determine the mechanisms by which key features such as DNA methylation, nucleosome positioning, and histone modifications influence each other. We then will determine how DNA methylation inhibitors (DNMTi’s) work. This grant is designed to use our powerful new NOMe-seq technology to understand the relationship between DNA methylation and nucleosomal positioning; to use knock-down and other approaches to examine the effects of altering both chromatin-remodeling and histone-modifying enzymes on the epigenome as a whole; and to understand how the epigenome controls endogenous retroviruses (ERVs). We will study how the epigenome is altered in human cancer, characterizing changes not only in gene promoter regions but also in enhancer and insulator regions and in genes themselves. Major questions to be addressed include 1) Why are there so many mutations in chromatin modifiers, and what are the effects of these mutations on the structure of the epigenome? 2) What are the functional consequences of activating the expression of cancer/testis genes by 5-azanucleoside? 3) What double-stranded RNAs are activated by 5-azanucleosides and how do these relate to cellular responses? 4) Can we design combinations of epigenetic drugs which might increase the effectiveness of 5-azanucleoside treatment? and 5) Can cryo-EM help to visualize complexes relevant to chromatin structure and functions? We will also study the roles of TET enzymes, which oxidize 5- methylcytosine to 5-hydroxymethylcytosine and require vitamin C as a cofactor, and the enzymes G9A and SETDB1, which methylate histone protein H3K9. Combinations that increase the expression of ERVs will be prioritized, because recent data strongly suggests that ERV expression may be linked to cellular changes, and quite possibly to clinical outcomes in cancer. Cancer patients are often deficient in vitamin C, implying that supplementation may markedly increase TET activity and patient response. Our approach is designed not only to understand the epigenome holistically but also to devise strategies which will increase patients' responses to drugs, perhaps by defining future rational drug combinations, in particular making use of DNMTi’s. Success of this project should have rapid mechanistic and translational impact, as DNMTi’s are FDA-approved or are currently in trials for cancer treatment.