PROJECT SUMMARY/ABSTRACT: Constitutively active NRF2 signaling is associated with radiation resistance and poor patient outcome in several cancer types, including oral squamous cell carcinoma (OSCC). NRF2 is a transcription factor which drives the phase II antioxidant gene expression program. In cancer, NRF2 is frequently mutated to become constitutively active, resulting in protection from oxidative and metabolic stress, thereby driving radioresistance. The established role of NRF2 in promoting radioresistance makes it an attractive drug target to treat cancer patients. Our group generated the first clinically relevant NRF2 mutant (Nrf2E79Q) GEMM to evaluate the role of NRF2 in cancer initiation, progression and therapeutic resistance. My preliminary data show that mouse embryonic fibroblasts (MEFs) derived from our Nrf2E79Q GEMM have increased cell viability and proliferation following radiation treatment compared to those with wild-type NRF2. These findings are consistent with the literature and demonstrate that NRF2 protects cells from oxidative stress and radiation. Through genetic crossing, we have isolated NRF2 expression to the oral cavity and will induce OSCC by treating the mice with a chemical carcinogen. For my thesis project, I will study the role of NRF2 in OSCC in vivo and in vitro using tumor cells derived from our model, analyzing cell viability, cell proliferation, and gene expression differences between Nrf2E79Q and wild-type upon radiation treatment. I hypothesize that NRF2 promotes resistance to radiation in oral squamous cell carcinoma and inhibiting NRF2 will mitigate this resistance. Despite extensive evidence of a negative impact of NRF2 activity on clinical outcome, there remains no FDA approved NRF2 inhibitors. As a transcription factor, NRF2 is inherently difficult to directly target. Therefore, we sought to identify druggable targets—specifically kinases—that regulate NRF2. To this end, we performed a high-throughput live cell imaging chemical screen using the Published Kinase Inhibitor Set (PKIS). I have validated numerous hits from the screen as NRF2 inhibitors, including compounds with strong selectivity to multiple PI3Ks. In addition to studying the role of NRF2 activation in oral keratinocytes and OSCC, my thesis work will also test the effectiveness of these inhibitors in sensitizing Nrf2E79Q OSCC cells to radiation. Further, to empower translational relevance, I will create human isogenic cell lines with a Nrf2E79Q allele using CRISPR to study whether inhibiting NRF2 decreases radioprotection in a human OSCC. Validation of the effectiveness of these kinase inhibitors will bring the field a step closer to targeting NRF2 in cancer and ultimately improve patient outcomes. The work prosed in thi...