Project Summary Rapidly proliferating cancer cells generate reactive oxygen species (ROS) that if left unchecked inhibit cell growth. To counter this stress, cancer cells and in particular non small cell lung cancers (NSCLC) rely on the activation of the NRF2 transcription factor, leading to the massive upregulation of key metabolic and detoxification proteins needed to restore redox balance. While directly targeting NRF2 with chemical inhibitors is challenging, we hypothesized that hyperactivation of this pathway would lead to an alteration of specific signaling and metabolic pathways required for the proliferation of these cells (co-dependencies), which themselves could be inhibited with small molecules. To identify these co-dependencies in NSCLC, we enriched for proteins containing reactive cysteines, which can be used as a chemical handle to develop inhibitors. This chemical proteomics screen identified hundreds of reactive cysteines regulated by NRF2, including a cryptic cysteine (C274) in the orphan receptor NR0B1. NR0B1 expression is severely restricted to those NCSLC cells and patient tumors with deregulated NRF2 signaling, where it functions as part of multimeric transcriptional complex to support the NRF2 gene expression program. As C274 in NR0B1 is necessary for NR0B1-complex formation, we exploited this residue to develop a small molecule inhibitor that covalently binds to it, subsequently disrupting the protein-protein interactions of NR0B1 and blocking the growth of NRF2-dependent cells, but not NRF2-independent cells. Thus, we have revealed NR0B1 as a druggable co-dependency of the NRF2 pathway. In this grant application, we build on our research on NR0B1 and further identify co-dependent pathways with NRF2 that can be pharmacologically interrogated. Using a powerful chemoproteomic framework, we will comprehensively define NRF2 co-dependencies by: 1) mapping the landscape of cysteine reactivity regulated by NRF2 in lung xenograft models, allowing us to identify cysteines on key proteins in the NRF2 pathway, which may become targetable opportunities in vivo 2) undertaking a small molecule screen to identify compounds that selectively inhibit the proliferation of NRF2-dependent NSCLC cell lines. Importantly, integrating a chemoproteomic platform into this screen, will allow for the rapid identification of co-dependent proteins, offering an unparalleled map of druggable NRF2 co-dependencies. The research proposed herein, takes full advantage of advanced cancer models and chemoproteomic technologies to reveal pharmacologically tractable proteins which are needed for the proliferation of NRF2-addicted cells and may provide a generalizable platform for inhibiting genetically defined cancers. These studies will not only provide a comprehensive understanding of NRF2 biology but might also lay the foundation for translational therapeutics benefiting lung cancer patients with deregulated NRF2 signa...