PROJECT SUMMARY Head and neck cancer (HNC) is devastating. Even in the setting of curable disease, 1 of every 2 patients diagnosed ultimately succumb. Additionally, survivors are left with debilitating toxicity due in large part to the toxicity of radiation, leading to poor quality of life increased rates of depression and suicide. Therefore, there is a critical need to develop precision oncology approaches which match radiation treatment approaches to HNC biology. HPV-associated (HPV+) HNC most commonly arises in the oropharynx (OPC) and is associated with much greater sensitivity to radiation. However, up to 20% of patients with HPV+ tumors will also be failed by radiation. Conversely, HPV- HNC that express p16 exhibit favorable outcomes compared to truly p16 negative tumors. This proposal seeks to understand why this is so. Specifically, we have identified the protein p16, a clinical surrogate for HPV, to be driving response to radiation and PARP inhibition outside of its usual cell cycle regulatory function, by inhibiting DNA damage repair (DDR). Although p16 is generally thought to be functionally inactive in HPV+ HNC, and absent in HPV- HNC, our data suggests that p16 is functioning via non-canonical signaling in HNC to induce a state of BRCAness and is key to understanding the differential radiation response in HPV+ and HPV- tumors. In this proposal we build upon the substantial data we have generated for a non-canonical p16-mediated pathway active in HNC – leading to upregulation of HUWE1, downregulation of USP7 and a shift in DDR – by first characterizing the relationship between p16 and Sp1. Although, we have linked Sp1 to the activation of our non-canonical p16 signaling pathway, as well as outcome in HNC following the combination of cisplatin and radiation, the nature of this interaction is unclear. We will both characterize this interaction as well as utilize state-of-the-art methodology to globally characterize the effects of p16 and Sp1 on mRNA and protein levels within HNC pre-clinical models and patient tumors. We will then comprehensively examine the relationship between p16 and Sp1 on DDR and radiation or Olaparib response using a combination of immunodeficient and -competent murine orthotopic models as well as 400 HPV+ and HPV- OPC tumors treated uniformly with cisplatin and radiation in the largest cohort of its kind to date. Finally, we will utilize USP7 inhibitors in clinical development in combination with either radiation or PARP inhibition in pre-clinical models. Completion of this project will establish biomarkers of outcome in both HPV+ and HPV- HNSCC downstream of p16, evaluate USP7 as a viable target for sensitization to radiation and Olaparib, and identify novel targets for precision medicine in HNC.