High-throughput Epigenomic Mapping of Regulatory Elements in Ovarian Cancer at Basepair Resolution

项目摘要/摘要 卵巢上皮癌(OC)是美国最致命的妇科癌症。它由几个 组织类型，每种生物类型都有不同的临床挑战。透明细胞OC(CCOC)代表了 研究不足的组织类型，以对标准化疗的低应答率和缺乏有效性为特征 治疗选择。子宫内膜样癌(ENOC)是一种密切相关的组织类型。CCOC和ENOC有一个共同点 组织起源(子宫内膜异位症)和关键基因突变，但在临床行为上明显不同。我们的 最近发表的研究表明，这些突变中的大多数在未转化的子宫内膜异位症中都可以检测到， 进一步表明了恶变的不同机制。我们试图描绘出以下贡献 在这个过程中改变了表观遗传学和转录控制。最常见的组织类型，高级浆液性 OC(HGSOC)中存在关键抑癌基因BRCA1和RAD51C的表观遗传失活 同源DNA修复(HR)途径，一个强调其治疗意义的途径。这些肿瘤通常 尽管最初对铂类药物治疗反应良好，但五年内复发。原发性卵巢癌的表观遗传学研究进展 通常没有对组织类型进行分层，或者严重偏向最常见的 组织类型为HGSOC。即使对于HGSOC，表观遗传学分析也只在有限的基因组覆盖范围内进行， 主要集中在基因启动子上。促进剂在最近的文献中引起了极大的兴趣，因为它是 动态使用的基因组隔间，但原始人类OC样本的增强子研究，特别是 那些具有不同组织类型的，通常是缺乏的。这在一定程度上要归因于现有技术的局限性。 为了解决这一知识差距，我们建议实施一种创新的成本效益工具，与 主要的人体样本，以使用靶向技术分析增强剂和其他监管元素 联合分析DNA甲基化和核小体占有率(Target-Nome Seq)。我们还将开发 应用这项技术所需的相关生物信息学管道(目标1)。我们将使用Target-Nome序列和 转录组图谱分析300例原发OC肿瘤标本，以及显微解剖的肿瘤和支持物 样本子集上的间质隔间(目标2)。我们希望通过这个丰富的数据集来解决这项研究 和上述临床问题(目标3)。我们将使用增强子和启动子表观遗传状态，以及 新的非编码RNA-增强子RNA(ERNA)类别以及转录水平的表达 确定转录调控网络的因素和候选目标基因。通过分析哪些网络 在不同的组织类型中发生变化，我们将更好地了解不同的分子组成 这些组织类型中的每一个。我们的Focus Target-Nome Seq技术相对较高的测序深度将 也使我们能够评估肿瘤间和亚克隆的异质性，以阐明肿瘤的潜在机制 复发。

PROJECT SUMMARY / ABSTRACT Epithelial ovarian cancer (OC) is the deadliest gynecological cancer in the US. It consists of several histotypes, each biologically distinct with different clinical challenges. Clear cell OC (CCOC) represents a much understudied histotype marked by low response rates to standard chemotherapies and a lack of effective therapeutic options. Endometrioid OC (ENOC) is a closely related histotype. CCOC and ENOC share a common tissue of origin (endometriosis) and key genetic mutations but differ markedly in their clinical behavior. Our recently published study showed that most of these mutations were detectable in untransformed endometriosis, suggesting further distinct mechanisms of malignant transformation. We seek to delineate the contributions of altered epigenetics and transcriptional control in this process. The most prevalent histotype, high grade serous OC (HGSOC) harbors epigenetic inactivation of key tumor suppressor genes BRCA1 and RAD51C in the homologous DNA repair (HR) pathway, a pathway highlighted for its therapeutic relevance. These tumors often recur within five years despite initial good response to platinum therapy. Past epigenetic studies of primary OC have usually been conducted without stratification of histotypes, or heavily biased towards the most common histotype HGSOC. Even for HGSOC, epigenetic profiling has been performed with only limited genome coverage, focused primarily on gene promoters. Enhancers have incurred much interest in recent literature as the most dynamically used compartment of the genome, but enhancer studies of primary human OC samples, especially those of distinct histotypes, are generally lacking. This is attributable in part to limitations of existing technology. To address this knowledge gap, we propose to implement an innovative cost-effective tool, compatible with primary human samples, to profile enhancers and other regulatory elements using a targeted technology that jointly profiles DNA methylation and nucleosome occupancy (Target-NOMe Seq). We will also develop the associated bioinformatic pipeline needed to apply this technology (Aim 1). We will use Target-NOMe Seq and transcriptome profiling to analyze 300 bulk OC tumor samples, as well as microdissected tumor and supportive stromal compartments on a subset of samples (Aim 2). With this rich dataset we hope to address the research and clinical questions described above (Aim 3). We will use enhancer and promoter epigenetic states, and a new category of non-coding RNA - enhancer RNA (eRNA), as well as the expression levels of transcription factors and candidate target genes to define transcriptional regulatory networks. By analyzing which networks are altered in the different histotypes, we will gain a better understanding of the distinct molecular makeup of each of these histotypes. The relatively high sequencing depth of our focused Target-NOMe Seq technology will also allow us to assess intertumor and subclonal heterogeneity to shed light on potential mechanisms of tumor recurrence.