<h3>Purpose/Objective(s)</h3> HPV-mediated oropharyngeal squamous cell carcinoma (OPSCC) portends improved prognosis under current therapeutic regimens, driving the charge for de-escalation trials. A subgroup of patients with HPV-mediated OPSCC do not respond to traditional therapies, highlighting the need for predictive biomarkers for patient selection. HPV-16 E2 has shown promise as a potential biomarker. The aims of this study are to evaluate E2 status as a predictive marker in a cohort of HPV+ OPSCC and to investigate the role of E2 in treatment response in HPV + head and neck cancer cell line models. <h3>Materials/Methods</h3> Ninety-three patients with HPV+ OPSCC were included in the pilot cohort. Samples from all 93 patients underwent targeted genomic sequencing. E2 reads were quantified utilizing a novel HPV caller pipeline. E2 counts were normalized to E6 reads and the median ratio was used as a cutoff for E2 wild type (WT) or E2 loss. Kaplan-Meier curves were performed for disease free survival. For the <i>in vitro</i> component of this study, we leveraged CRISPR-Cas9 profiling and open reading frame overexpression to create two paired models of E2 loss and E2 overexpression in patient derived HPV+ head and neck cancer cell lines. Clonogenic assays were used to evaluate the effect of E2 on cisplatin sensitivity. RNAseq was used to investigate the mechanism of E2 modulation on cisplatin sensitivity. <h3>Results</h3> In the cohort of 93 patients with OPSCC, loss of E2 was significantly associated with worse disease free survival (p<0.05). In the cell line models, loss of E2 resulted in increased cisplatin resistance while E2 overexpression resulted in increased cisplatin sensitivity. RNAseq of paired cell lines and gene set enrichment analysis of these data identified that E2 status is significantly associated with chromatin assembly, differentiation, cell cycle, DNA-damage repair, and immune-remodeling. Further evaluation with qPCR and western analysis demonstrated increased expression of DNA damage repair genes with loss of E2. <h3>Conclusion</h3> Together these results provide preliminary data promoting E2 as a clinical biomarker in HPV+ OPSCC and suggest that upregulation of DNA damage repair may play a role in the mechanism of treatment resistance with loss of E2. Further studies are warranted to evaluate the role of E2 as a potential biomarker for patient selection in OPSCC.