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

摘要/摘要 感染高危人乳头瘤病毒(HrHPV)是宫颈疾病中必要的关键事件 癌症。了解宫颈癌的一个重大进展是认识到HPV DNA是整合的 几乎所有晚期宫颈肿瘤的人类基因组。人乳头瘤病毒DNA 1的人类基因组整合 稳定地将病毒癌基因与宿主细胞关联，2)潜在地驱动宿主癌基因的表达，该宿主癌基因 HPV DNA插入位点的侧翼，以及3)也会导致人类基因组重排。HPV整合是 被认为是随机出现在人类基因组中，但肿瘤中整合的HPV DNA经常插入到 在独立肿瘤中有数十个共同基因，这表明克隆增殖和生存优势 在这些位置包含插入内容的单元格。在我研究实验室培训的最初阶段，我 开发了一项广泛的、新生的研究计划，以1)单次检测和绘制HPV-人类DNA连接 核苷酸拆分(杂交捕获下一代测序，HC NGS)，2)彻底 描述完整的病毒DNA结构，以及3)描述两个宫颈的转录情况 异型增生和癌的临床标本。我还将HC NGS分析应用于一个最近开发的细胞 人原代角质形成细胞HPV环状DNA异构体早期复制的培养模型。我 发现在该模型中HPV DNA的整合是有效的，从而为研究HPV提供了一个有效的系统 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 研究类型。我检测到HPV整合到人类基因组的多个位置，其中一些被检测到 多次表明含有它们的细胞克隆性扩增。我假设HPV DNA整合是一种 关键的分子事件，促进细胞的克隆性扩张，并促进异型增生和 浸润性宫颈癌。在这个应用程序中，我试图通过生成和使用 研究HPV整合对患者克隆扩张的时空影响的新工具 使用多色间期DNA FISH分析衍生的进行性宫颈不典型增生病变(目标1)；评估 培养的角质形成细胞的致瘤特性(目标2)，并最终测试转录激活的作用 人类基因侧翼在扩展克隆种群中的分布(AIM 3)。我将使用一种新的条形码系统 基于CRISPR激活目的克隆中GFP与定制重组HPV准病毒的结合 人乳头瘤病毒DNA扩增克隆的谱系追踪方法。我将在一年多的时间内进行拟议的研究 作为综合K08职业发展奖的一部分，为期5年。和我的导师们一起，戴维斯博士。 克里斯蒂娜·蒙塔尼亚和杰克·伦茨博士，我们制定了一项培训计划，重点是实验室科学和 实现我的职业目标，成为一名完全独立和有资金支持的临床医生科学家。

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.