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

Abstract

10Persistent expression of high-risk HPV oncogenes is necessary for the development of anogenital and 11 oropharyngeal cancers. Here, we show that E6/E7 expressing cells are hypersensitive to DNA 12 crosslinking agent cisplatin and have defects in repairing DNA interstrand crosslinks (ICL). Importantly, 13 we elucidate how E6/E7 attenuate the Fanconi anemia (FA) DNA crosslink repair pathway. Though 14 E6/E7 activated the pathway by increasing FancD2 monoubiquitination and foci formation, they inhibited 15 the completion of the repair by multiple mechanisms. E6/E7 impaired FancD2 colocalization with double-16 strand breaks (DSB), which subsequently hindered the recruitment of downstream protein Rad51 to DSB 17 in E6 cells. Further, E6 expression caused delayed FancD2 de-ubiquitination, an important process for 18 effective ICL repair. Delayed FancD2 de-ubiquitination was attributed to the increased chromatin 19 retention of FancD2 hindering USP1 de-ubiquitinating activity, and persistently activated ATR/CHK-20 1/pS565 FancI signaling. E6 mediated p53 degradation did not hamper the cell cycle specific process of 21 FancD2 modifications but abrogated repair by disrupting FancD2 de-ubiquitination. Further, E6 reduced 22 the expression and foci formation of Palb2, which is a repair protein downstream of FancD2. These 23 findings uncover unique mechanisms by which HPV oncogenes contribute to genomic instability and the 24 response to cisplatin therapies. 25 42 High-risk human papillomavirus (HR-HPV) E6/E7 oncoproteins are essential for the development of 43 malignancies of the anogenital tract and oropharynx, with HPV16 being the predominant type (1). 44 Cervical and oropharyngeal cancers are the most common HPV-associated malignancies among females 45 and males, respectively (2). Persistent HPV infection destabilizes the cellular genome which can lead to 46 cancer. Genomic instability is likely the result of the numerous interactions of HPV oncoproteins with 47 host tumor suppressors and DNA damage repair (DDR) proteins. Recently, we demonstrated that high-48 risk HPV oncogenes attenuate double-strand break (DSB) repair by impairing the homologous 49 recombination pathway (3). To further elucidate the mechanisms by which HPV oncogenes impair DDR, 50 the present study focuses on the impact of HPV16 oncogenes on the Fanconi anemia-BRCA (FA or FA-51 BRCA) pathway. 52The FA-BRCA pathway is involved in the repair of intra or interstrand crosslinks (ICL), thereby 53 maintaining genomic stability (4, 5) ( Fig S1). ICLs block DNA replication and transcription and, thus, 54 are highly cytotoxic to cells if not repaired. The FA pathway is composed of 22 FA proteins (identified to 55 date) (6). When any one of the FA genes of the FA-BRCA pathway is mutated, individuals have a 56 spectrum of disorders, called Fanconi Anemia, characterized by bone marrow failure, congenital 57 malformations, cancer predisposition, and cellular sensitivity to ICL-inducing agents (7). Upon exposure 58 to DNA crosslinking agents, and during S-phase o...