Abstract Non-small cell lung cancer (NSCLC) accounts for 85-90% of lung cancers. NRF2 is a master transcription factor that orchestrates antioxidant defense. Under basal conditions, KEAP1 binds NRF2, promoting its ubiquitination and degradation. Oxidative stress alters KEAP1 conformation, preventing NRF2 binding and enabling NRF2 nuclear accumulation and activation of cytoprotective genes. NSCLC frequently harbors KEAP1 or NRF2 mutations—often within the KEAP1 Kelch domain or the NRF2 Neh2 domain—that impair KEAP1-mediated turnover and drive constitutive NRF2 activation. Such tumors are resistant to chemo-, radio-, and immunotherapy, yet direct NRF2 inhibitors remain elusive due to the absence of catalytic pockets and concerns about systemic toxicity. To identify synthetic-lethal targets that heighten ROS-induced death in NRF2-active NSCLC, we performed negative-selection CRISPR/Cas9 screens using a focused antioxidant-enzyme sgRNA library. Cells were treated with a sublethal dose of the NQO1-bioactivatable ROS-generating prodrug β-lapachone. As expected, NQO1 was the top sensitizing hit, and TXNRD1/TXN were enriched among resistant hits, consistent with the protective thioredoxin pathway. Unexpectedly, genes required for de novo glutathione synthesis (GCLM, GCLC, GSS) showed minimal dropout, indicating limited contribution of GSH biosynthesis to β-Lap sensitivity. Instead, the NRF2 target gene GSR—responsible for reducing GSSG to GSH—emerged prominently, suggesting that GSR loss uniquely impairs survival under oxidative stress. Across multiple ROS-inducing agents, GSR knockout strongly sensitized KEAP1/NRF2-mutant cells but not KEAP1/NRF2-intact cells. Importantly, toxicity was not driven by global GSH depletion. Rather, we found that mitochondrial GSR is essential: cytosolic-only GSR could not rescue GSR-deficient cells, whereas forced mitochondrial localization fully restored viability. Mechanistically, GSR loss caused severe mitochondrial fragmentation, compromised electron transport chain (ETC) integrity, and reduced ETC activities. GSR-deficient cells accumulated mitochondrial ROS and showed decreased expression of iron-sulfur cluster-containing proteins, collectively driving mitochondrial dysfunction and cell death. In vivo, GSR deletion triggered redox collapse and dramatically sensitized KEAP1-mutant tumors to chemotherapy. Together, these findings reveal an unrecognized mitochondrial redox dependency in KEAP1/NRF2-mutant NSCLC and identify GSR as a promising therapeutic vulnerability for overcoming NRF2-mediated ROS resistance. Citation Format: Yun Huang, Chao Ting, Jing-Chun Lin, Ryan Martin, Duy T. Nguyen, Jose Serrano-Velez, Yun Pyo Kang, Nathan P. Ward, Ana P. Da Silva Gomes, Mingxiang Teng, Chang Jiang. Mitochondrial glutathione reductase as a redox vulnerability in KEAP1/NRF2-mutant non-small cell lung cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6009.
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