Abstract 2875: Resistance to conventional chemo and radiotherapy associated with increased DNA repair and stem cell-like phenotype in head and neck squamous cell carcinoma expressing HPV E5

Abstract Human papillomavirus-associated head and neck squamous cell carcinoma (HPV+ HNSCC) is one of the fastest-rising cancers in the US. While HPV+ HNSCC patients exhibit better responses to chemoradiation compared to HPV- HNSCC patients, a subset of HPV+ patients do not respond as well and develop recurrent/refractory disease. In this study, we investigated the potential role of HPV E5 in promoting resistance to conventional chemo- and radiotherapy, and the underlying molecular mechanisms. HPV E5 is a small hydrophobic viral oncoprotein with multiple functions including regulation of tumor cell differentiation and apoptosis, modulation of H+ ATPase responsible for acidification of late endosomes, and immune modulation. To test the impact of HPV E5 on therapeutic resistance, we generated HNSCC cell lines and normal keratinocytes expressing HPV16 E5. Upon treatment with radiation (2Gy, 4Gy, 8Gy) and/or cisplatin, HNSCC cells and keratinocytes expressing HPV 16 E5 had increased cell viability, decreased apoptosis, and increased colony-forming capacity compared to empty vector (EV) control lines. Specifically, treatment with 4Gy radiation, 4µM cisplatin, or the combination led to a ~2-fold decrease in % of apoptotic cells in HPV16 E5-expressing HNSCC cells compared to EV and HPV16 E6/E7 HNSCC cells (p <0.05). Examination of DNA damage response and repair pathways indicated that while HPV16 E5 HNSCC cells initially had increased phosphorylation of H2AX 6 hours post treatment, at 24 hours post treatment H2AX phosphorylation in HPV16 E5 cells was significantly decreased compared to EV or HPV16 E6/E7 HNSCC cells. These findings indicate increased DNA repair capacity of HPV16 E5 HNSCC cells. To identify mechanisms underlying this phenotype, we performed RNA-seq and observed enhanced signaling pathways associated with epithelial development and increased expression of stem cell markers in HPV16 E5 HNSCC cells compared to EV control lines. Functionally, we observed a ~2.5-fold increase in sphere-forming capacity and increased expression of markers associated with stem cell phenotype in HPV16 E5 HNSCC cells compared to EV and HPV E6/E7 HNSCC cells. To determine whether these findings translated clinically, 18 HPV+ HNSCC patients from the GSE74927 dataset were grouped according to median expression level of HPV E5 (High vs Low). In support of our hypothesis, HPV E5-High patients exhibited enrichment of pathways associated with extrinsic apoptotic signaling, pluripotency of stem cells, and positive regulation of DNA repair. Taken together these results identify HPV E5 as a potential mediator of resistance to conventional radiation and chemotherapy in HNSCC and suggest that patient tumors expressing HPV E5 may have increased DNA repair capacity and cell survival after cytotoxic therapies. Citation Format: Souvick Roy, Sayuri Miyauchi, Riley Jones, Sangwoo Kim, Andrew B. Sharabi. Resistance to conventional chemo and radiotherapy associated with increased DNA repair and stem cell-like phenotype in head and neck squamous cell carcinoma expressing HPV E5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2875.