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

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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 3 year period as part of the Early Surgeon Scientist Program 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.