Project Summary Control of the redox homeostasis is essential to cancer cell proliferation and requires the delicate maintenance of oxidative and reductive metabolic pathways. This equilibrium is controlled by signal transduction pathways and imbalances lead to redox stress that potently blocks cancer growth. Much work has focused on the role of oxidative stress in cancer proliferation, however, the converse– reductive stress and its impact on malignant cells is poorly understood. We have studied the role of redox control in cancer in the context of modification of proteinaceous cysteines by reactive oxygen species and the NRF2 transcription factor pathway. NRF2 functions as the master regulator of the cellular antioxidant response and promotes the expression of key metabolic and detoxification genes to generate a reductive environment and negate oxidative stress. NRF2 is activated in many cancers including ~30% of non small cell lung cancers (NSCLC) through mutation of its negative regulator KEAP1. While NRF2 has been extensively studied in KEAP1-mutant NSCLCs, we wondered what role this pathway plays in the proliferation of NSCLC cell lines which are wildtype (WT) for KEAP1. To this end, we pharmacologically activated NRF2 in 50+ NSCLC cell lines (WT for KEAP1) and measured their proliferation. Unexpectedly, we find that in ~16% of NSCLC cell lines, NRF2 activation results in a severe block in proliferation. A genome wide CRISPR screen identifies that genes involved in mitochondrial metabolism, mitochondrial fusion and the electron transport chain (ETC) are major sensitizers to NRF2 activation when lost and can function as companion biomarkers for NRF2-sensitivity. In line with the generation of reductive stress following NRF2 activation, key cysteines on enzymes involved in mitochondrial metabolism and mitochondrial fusion are reduced as determined by chemical proteomic platforms. To explain these surprising biological characteristics we propose the following hypothesis: NRF2 activation in a subset of NSCLC cell lines promotes an overly reductive environment that decreases the activity of key enzymes in mitochondrial metabolism and mitochondrial respiration and fusion. The inactivation of these pathways synergize to block cell growth. In this grant application, we build on our research surrounding NRF2 sensitization and mechanistically characterize the role of reductive stress in NSCLC proliferation. In this grant application, we will comprehensively define KEAP1-dependence by identifying NRF2 regulation of mitochondrial metabolism/fusion at the protein, cellular and organismal levels. The research proposed herein, takes full advantage of a series of recently conceived methods: chemical proteomics, genome-wide CRISPR screens and untargeted metabolomics, which have previously been deployed in isolation, to be used in an integrated manner to effectively dissect how protein reduction underlies protein malfunction and KEAP1-dependence. These studi...