Pathogenic role of human papillomavirus (HPV) DNA Integration and clonal expansion in cervical tumorigenesis

摘要/摘要 高危型人乳头瘤病毒（hrHPV）感染是宫颈癌的一个必要的关键事件， carcinoma.了解宫颈癌的一个主要进展是认识到HPV DNA是整合在 几乎所有晚期宫颈肿瘤的基因组中。HPV DNA的人类基因组整合1） 病毒癌基因与宿主细胞稳定地结合，2）潜在地驱动宿主癌基因的表达， 侧翼的HPV DNA插入位点，和3）也导致人类基因组重排。HPV整合是 一般认为HPV DNA在人类基因组中是随机发生的，但肿瘤中整合的HPV DNA通常插入在 在独立的肿瘤中有几十个共同的基因，这表明克隆增殖和生存优势 在这些位置含有插入物的细胞。在我的研究实验室培训的最初阶段，我 开发了一个广泛的，新生的研究计划，以1）检测和映射HPV-人DNA连接在单一的 核苷酸分辨率（杂交捕获+下一代测序，HC+NGS），2）彻底 表征整合的病毒DNA结构，以及3）描述两个宫颈中的转录组景观 异型增生和癌临床样品。我还将HC+NGS检测应用于最近开发的细胞 培养模型，其中HPV环状DNA附加体在早期传代中复制，人原代角质形成细胞。我 发现HPV DNA整合在该模型中有效地发生，从而提供了一个有效的系统来研究HPV DNA整合及其功能后果。据我所知，这是第一个执行此操作的系统 研究的类型。我检测到HPV整合到人类基因组的多个位点，其中一些被检测到 多次表明含有它们的细胞的克隆扩增。我假设HPV DNA整合是一种 促进细胞克隆扩增的关键分子事件，并促进发育异常的发展， 浸润性宫颈癌在这个应用程序中，我试图通过生成和使用来发展我的实验室技能。 研究HPV整合对患者克隆扩增的时空后果的新工具 使用多色间期DNA FISH检测衍生的进行性宫颈不典型增生病变（目的1）;评估 培养的角质形成细胞的致瘤特性（Aim 2），并最终测试转录激活的作用 在扩增的克隆群体中侧翼的人类基因（AIM 3）。我会用一个新的条形码系统 基于与定制重组HPV准病毒组合的感兴趣克隆中GFP的CRISPR活化 谱系追踪扩增的HPV DNA整合克隆的方法。我将进行拟议的研究， 5年作为K 08职业发展综合奖的一部分。沿着我的导师们， Cristina Montagna和Jack伦茨博士，我们已经制定了一个培训计划，重点是实验室科学， 方法来实现我的职业目标，成为一个完全独立和资助的临床科学家。

Summary/Abstract Infection with high-risk Human Papillomavirus (hrHPV) is a necessary, key event in cervical carcinoma. A major advance in understanding cervical cancer was the recognition that HPV DNA is integrated into the human genome in almost all advanced cervical tumors. Human genome integration of HPV DNA 1) stably associates the viral oncogenes with a host cell, 2) potentially drives expression of host oncogenes that flank the sites of HPV DNA insertions, and 3) also causes human genome rearrangements. HPV integration is thought to occur randomly in the human genome, but integrated HPV DNA in tumors is often inserted at one of dozens of common genes in independent tumors, which suggests a clonal proliferation and survival advantage of cells containing insertions at these positions. During the initial phase of my research laboratory training, I developed an extensive, nascent research program to 1) detect and map HPV-human DNA junctions at single nucleotide resolution (hybridization capture + next generation sequencing, HC+NGS), 2) thoroughly characterize integrated viral DNA structure, and 3) describe the transcriptomic landscape in both cervical dysplasia and carcinoma clinical samples. I also applied the HC+NGS assay to a recently developed cell culture model where HPV circular DNA episomes replicate in early passage, human primary keratinocytes. I found that HPV DNA integration occurs efficiently in this model, thus providing a potent system to study HPV DNA integration and its functional consequences. To my knowledge, this is the first system for performing this type of study. I detected HPV integrated into multiple sites in the human genome, some of which were detected multiple times indicating clonal expansion of cells containing them. I hypothesize that HPV DNA integration is a key molecular event that promotes clonal expansion of cells, and promotes the development of dysplasia and invasive cervical carcinoma. In this application, I seek to develop my laboratory skills by generating and using new tools to study the spatio-temporal consequences of HPV integration on clonal expansion in patient derived, progressive cervical dysplasia lesions using multi-color, interphase DNA FISH assays (Aim 1); assess tumorigenic properties of cultured keratinocytes (Aim 2) and finally to test the role of transcriptional activation of flanking human genes in the expanded clonal populations (AIM 3). I will utilize a novel barcoding system based on CRISPR activation of GFP in clones of interest combined with a custom recombinant HPV quasivirus approach to lineage trace expanded HPV DNA integrated clones. I will carry out the proposed research over a 5 year period as part of a comprehensive K08 Career Development Award. Along with my co-mentors, Dr. Cristina Montagna and Dr. Jack Lenz, we have developed a training plan focusing on laboratory science and methodology to achieve my career goal of becoming a fully independent and funded clinician scientist.