High-Risk Human Papillomavirus Oncogenes Disrupt the Fanconi Anemia DNA repair Pathway by Impairing Localization and De-ubiquitination of FancD2

Abstract

Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and oropharyngeal cancers. Here, we show that E6/E7 expressing cells are hypersensitive to DNA crosslinking agent cisplatin and have defects in repairing DNA interstrand crosslinks (ICL). Importantly, we elucidate how E6/E7 attenuate the Fanconi anemia (FA) DNA crosslink repair pathway. Though E6/E7 activated the pathway by increasing FancD2 monoubiquitination and foci formation, they inhibited the completion of the repair by multiple mechanisms. E6/E7 impaired FancD2 colocalization with double-strand breaks (DSB), which subsequently hindered the recruitment of downstream protein Rad51 to DSB in E6 cells. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for effective ICL repair. Delayed FancD2 de-ubiquitination was attributed to the increased chromatin retention of FancD2 hindering USP1 de-ubiquitinating activity, and persistently activated ATR/CHK-1/pS565 FancI signaling. E6 mediated p53 degradation did not hamper the cell cycle specific process of FancD2 modifications but abrogated repair by disrupting FancD2 de-ubiquitination. Further, E6 reduced the expression and foci formation of Palb2, which is a repair protein downstream of FancD2. These findings uncover unique mechanisms by which HPV oncogenes contribute to genomic instability and the response to cisplatin therapies.\n\nAUTHOR SUMMARYHigh-risk human papillomavirus (HPV) causes nearly all cervical and many other anogenital cancers, and oropharyngeal cancers. As cisplatin is the most commonly used drug for cervical and HPV-associated oropharyngeal cancers, it is important to understand how HPV oncogenes disrupt the Fanconi anemia (FA) pathway involved primarily in the repair of cisplatin-induced DNA crosslinks. However, the mechanism by which HPV E6 and E7 attenuate the FA pathway is poorly understood. We demonstrate that E6/E7 expression disrupts crosslink repair and increase cisplatin sensitivity, and attenuate the FA pathway through multiple unique mechanisms. First, E6/E7 causes accumulation of FancD2, a central component of the FA pathway, at the sites away from DNA damage. This results in reduced recruitment of Rad51, another repair protein involved in the pathway. Second, E6 causes delayed FancD2 de-ubiquitination, an important process for effective repair. Third, E6 expressing cells decreases the expression and foci formation of Palb2 repair protein. Together, this work elucidates the mechanisms by which HPV attenuates the repair of DNA crosslinks increasing cisplatin cytotoxicity and efficacy in treating HPV-positive cancers.