PROJECT SUMMARY/ABSTRACT Maintaining redox homeostasis is required for cancer growth. Historically, redox stress has been studied in the context of oxidative stress, where high levels of reactive oxygen species (ROS) damage macromolecules including nucleic acids, lipids, and proteins, resulting in a disruption of cellular function. However, the impact of an overly reductive cell state on tumorigenesis and metastasis is largely unknown. This proposal builds on our findings that the precise induction of reductive stress is a specific vulnerability within a subset of non-small cell lung cancers (NSCLCs). Redox homeostasis within tumors is regulated by NRF2, the central transcriptional regulator of antioxidant, which promotes the expression of key metabolic and detoxification genes resulting in a reductive environment. While NRF2 is aberrantly activated in many cancers through mutation of its negative regulator and tumor suppressor KEAP1, its role in KEAP1-WT cancers is poorly understood. To address this question, we conducted preliminary studies by activating NRF2 in over 50 NSCLC cell lines. Unexpectedly, we found that ~15% of NSCLC cell lines are sensitive to NRF2 activation. A genome-wide CRISPR screen identified that genes involved in mitochondrial metabolism and the electron transport chain (ETC) are major sensitizers to NRF2 activation. Mechanistically, we discovered that NRF2 activation disrupts the equilibrium of NADH/NAD+ through ALDH3A1, inducing NADH-reductive stress and leading to blockage of growth and metastasis, which exhibit elevated sensitivity to ETC complex I inhibition. In line with the generation of reductive stress following NRF2 activation, key cysteines on proteins involved in mitochondrial metabolism are reduced as determined by chemical proteomics platforms. Based on these exciting findings, I propose that NRF2 activation leads to thiol reductive stress, resulting in hyper-reduction and inactivation of key cysteines localized to integral proteins required for mitochondrial function and cancer metastasis. In addition to disrupting the NADH/NAD+ balance, NRF2 triggers diverse oxidoreductases and antioxidants that scavenge oxidizing molecules and reduce oxidized proteins from their functional state, through redox modification on cysteines that are essential to mitochondrial metabolism. This will expand our prior research on NRF2 sensitization and provide a mechanistic characterization of how thiol reductive stress contributes to the progression and metastasis of NSCLC, with therapeutic potential.

PROJECT NARRATIVE NRF2 is hyperactivated by oncogenic mutation in ~30% of non-small cell lung cancers (NSCLCs), a destructive cancer with high rates of metastasis. NRF2 coordinates cellular antioxidant responses and promotes an environment that supports cell growth and metastasis, but its overactivation leads to reductive stress in a subset of NSCLCs. The goal of this project is to use cutting-edge functional genomics and chemical proteomics, innovatively uncover how NRF2 activity blocks tumor growth and metastasis through thiol reductive stress leading to a blockage in mitochondrial respiration.