Project Summary Ferroptosis is an iron-dependent form of nonapoptotic cell death that is induced by excessive lipid peroxidation. Previous studies by us and others identified ferroptosis as a natural tumor suppression mechanism and showed that ferroptosis inactivation, like apoptosis inactivation, contributes to tumor development. Recently, we and others also showed that radiotherapy (RT) can potently induce ferroptosis and suggested that ferroptosis inducers (FINs) can be used in RT to overcome radioresistance. However, the underlying mechanisms of ferroptosis in radioresistance and the exact cancer or genetic contexts in which to target ferroptosis in RT still remain largely unexplored. This application aims to determine the mechanisms by which ferroptosis inactivation contributes to radioresistance in KEAP1-mutant lung cancer cells and to assess the combination of RT and FINs in treating KEAP1-mutant lung cancers. KEAP1 is commonly mutated in lung cancer, and KEAP1-mutant lung cancers are resistant to RT. KEAP1 mutation or deficiency in lung cancer stabilizes NRF2 and promotes an NRF2-mediated antioxidant response. Our recent publication and new preliminary data support our central hypotheses that (i) KEAP1 deficiency promotes radioresistance largely through inhibiting ferroptosis, and KEAP1 regulates ferroptosis through NRF2 transcriptional targets SLC7A11 and other unidentified downstream targets; and (ii) combining RT and FINs that inactivate SLC7A11 (or other potential ferroptosis inhibitors identified from our studies) is an effective therapeutic strategy to overcome radioresistance in KEAP1-mutant lung cancers without causing significant damage in normal tissues. To test our hypotheses, we will pursue the following specific aims: Specific Aim 1: To determine the mechanisms by which KEAP1 regulates ferroptosis and radioresistance in lung cancer cells. Specific Aim 2. To determine the effectiveness of combining FINs with RT for treating KEAP1-mutant lung cancer. Our proposed studies are expected to identify novel mechanisms of ferroptosis and radioresistance and to identify effective new therapeutic strategies to overcome radioresistance in lung cancer treatment. Our proposed studies will have a significant impact on both our understanding of the fundamental mechanisms of ferroptosis and radiation biology and our ability to target ferroptosis-related radioresistance in cancer treatment.