Abstract (Project 3) Radiation therapy (RT) plays a critical role in the treatment of non-small cell lung cancer (NSCLC). While advances in tumor imaging and radiation delivery techniques have significantly improved RT, advances in genomic and molecular understanding of tumors have largely failed to impact management of patients treated with RT. Therefore, development of “precision radiation oncology” approaches, defined as the use of molecular biomarkers to personalize RT, remains a major unmet need. Our long-term goal is to develop novel, molecularly-based precision radiation oncology approaches for NSCLC patients treated with RT. We previously demonstrated that NSCLCs with KEAP1 or NFE2L2 mutations are radioresistant and have high rates of local recurrence after RT. In this proposal we will develop a personalized radiosensitization strategy for KEAP1/NFE2L2 mutant tumors based on inhibition of glutaminase, which plays a critical role in biosynthesis of cellular free radical defenses. Prior studies have found conflicting results regarding the ability of glutaminase inhibition to radiosensitize NSCLC. Our central hypothesis is that glutaminase inhibition can radiosensitize KEAP1/NFE2L2 mutant but not wildtype NSCLCs and if correct would help explain the previous conflicting results. We will therefore test if our genotype-specific radiosensitization approach has efficacy in KEAP1/NFE2L2 mutant but not wildtype NSCLC in vivo. We will also test if our approach increases normal tissue toxicity. Additionally, we will identify specific KEAP1 mutations that cause radioresistance in order to enable identifying patients with radioresistant tumors. In collaboration with Project 1, we will also test if the complement inhibitor PMX205 can serve as an additional radiosensitizer for KEAP1/NFE2L2 mutant NSCLC. Finally, in collaboration with Project 2 we will test if glutaminase inhibition can radiosensitize NFE2L2-mutant head and neck cancers. If successful, our project will establish glutaminase inhibition as a precision radiation oncology strategy for personalized radiosensitization of KEAP1/NFE2L2 mutant NSCLC. This approach could widen the therapeutic index of RT since it would only expose patients at highest risk of local recurrence to the potential additional toxicities of adding a radiation sensitizer. Our results will serve as the basis for developing clinical trials to attempt to translate this strategy. These trials will involve genotyping of NSCLC patients undergoing RT in order to select patients with KEAP1/NFE2L2 mutations for concurrent treatment with RT and glutaminase inhibitors. Importantly, our findings will serve as proof-of-principle that can likely also translate to other tumor types with frequent KEAP1/NFE2L2 mutations.