Abstract PR06: Cooperative HPV and YAP-mediated oncogenic reprogramming of oral progenitors into cancer stem cells at single cell resolution
Authors: Faraji Farhoud , Ramirez Sydney I. , Clubb Lauren M. , Sato Kuniaki , Quiroz Paola Y. Anguiano , Galloway William M. G. , Hoang Thomas S. , Medetgul-Ernar Kate , Marangoni Pauline , Molinolo Alfredo A. , Califano Joseph A. , Smith Quinton , Klein Ophir D. , Tamayo Pablo , Gutkind J. Silvio 
Published: September 2023

Abstract
Human papillomavirus-positive head and neck squamous cell carcinoma (HPV+HNSC) constitutes the most common HPV-driven malignancy in the United States, and yet the molecular and cellular mechanisms underlying carcinogenesis in HPV+HNSC remain poorly understood. The decades long latency period and incomplete penetrance linking oral HPV infection to HPV+HNSC suggest that oncogenic events beyond E6-E7 expression are required for carcinogenesis. YAP is a transcriptional co-activator that coordinates cell stemness, growth, proliferation, and survival. We previously identified alterations resulting in YAP activation as a potential oncogenic event in HNSC. We thus hypothesized YAP activation in the context of E6-E7 expression may represent a key tumor initiating event leading to HPV+HNSC. We developed a genetically engineered murine system to investigate the effects of local induction of YAP activation and E6-E7-expression in Keratin14+ (K14+) oral epithelial progenitor cells (OEPCs). In this system, intralingual administration of tamoxifen and dietary administration of doxycycline enables spatiotemporally controlled expression of E6-E7 and/or YAP. Employing this model, we identify combined E6-E7 and YAP activation (E+Y) as sufficient to induce highly penetrant carcinoma formation as early as 10 days after transgene induction. Immunofluorescent analyses showed E+Y resulted in the ectopic expansion of a mitotically active (Ki67+), stem-like (TP63+, SOX2+) epithelial cell population with invasive potential. Bulk RNAseq of microdissected tongue epithelia showed that E+Y uniquely induced transcriptional programs promoting cell cycle progression, epithelial cell plasticity, and acute inflammatory response, which were not observed upon sole activation of either the E6-E7 or YAP transgene. scRNAseq of transgene-induced tongue epithelial cell suspensions identified 8 distinct clusters, including previously defined physiologic oral epithelial cell states and novel transgene-associated clusters. E+Y resulted in the emergence of a unique epithelial cell cluster with transcriptional characteristics consistent with a cancer stem cell population, with enrichment in YAP, mTORC1, E2F, and MYC-driven programs, and pan-cancer cell states including cell cycle, hypoxia, squamous differentiation, and partial EMT. Implantation of E+Y cells resulted in tumor formation in 100% of NSG mice, indicating that E+Y drives the emergence of a cancer stem cell population. We next defined transcriptionally co-expressed modules driving E+Y tumor initiating capacity. Among TCGA-HNSC subjects, 8 of 12 modules were enriched in tumor compared to matched normal tissue. Interestingly, 3 modules (G1/S, cell migration, and focal adhesion) were associated with overall and disease-free survival. Our findings show YAP activation in the context of E6-E7 expression is sufficient for rapid carcinogenesis. We illuminate cellular processes driving malignant conversion at single cell resolution, and show enrichment of these processes in human HNSC exhibiting poor prognosis.
Citation Format: Farhoud Faraji, Sydney I. Ramirez, Lauren M. Clubb, Kuniaki Sato, Paola Y. Anguiano Quiroz, William M. G. Galloway, Thomas S. Hoang, Kate Medetgul-Ernar, Pauline Marangoni, Alfredo A. Molinolo, Joseph A. Califano, Quinton Smith, Ophir D. Klein, Pablo Tamayo, J. Silvio Gutkind. Cooperative HPV and YAP-mediated oncogenic reprogramming of oral progenitors into cancer stem cells at single cell resolution [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR06.