Drugging NRF2 to improve radiation therapy in head and neck squamous cell carcinoma - The NRF2 transcription factor is active in more than 30% of head & neck squamous cell carcinomas (HNSCC), owing to pathway activating mutations and mutation-independent mechanisms. NRF2 drives a gene expression program that mitigates oxidative and electrophilic stress, reprograms and enables cancer cell metabolism, and suppresses immune cell infiltration. In HNSCC patients, NRF2 activity portends a poor prognosis; its mutational activation is predictive for resistance to radiation and local regional failure. Though frequently active in human cancers, mouse models suggest that constitutive NRF2 activity is not sufficient for oncogenesis, but rather NRF2 synergizes with oncogenes and tumor suppressors to drive tumor progression. Despite great need, proven and efficacious NRF2 therapeutic inhibitors do not yet exist in the clinic. This revised application is a response to PAR-22-198, the Precision Approaches in Radiation Synthetic Combinations (PAIRS) program. We hypothesize that chemical suppression of NRF2 activity will sensitize HNSCC to RT. Our previous functional genomic, proteomic, and small molecule screens revealed therapeutic targets and pharmacological agents that suppress NRF2 activation in HNSCC. Here we focus on two in vivo efficacious, potent and mechanistically distinct NRF2 chemical inhibitors. First, we previously reported the ANCHOR class of KEAP1 mutants which maintain enzymatic activity for NRF2 ubiquitylation but fail to degrade the NRF2 protein. Working with Vividion Therapeutics, we sought cysteine-reactive molecules to reverse the ANCHOR phenotype. This resulted in VVD065, a highly selective and potent small molecule that induces NRF2 ubiquitylation, degradation and radiosensitivity. Second, our drug screens discovered pyrimethamine (PYR), a FDA approved medicine, as an efficacious inhibitor of NRF2 signaling. When administered to our genetically engineered mouse model (GEMM) carrying an inducible activating NRF2E79Q mutation, PYR reversed esophageal and oral cavity hyperplasia with no observed toxicity. Subsequent structure-activity-relationship optimization resulted in 30-fold more potent small molecule we call WCDD115. Because NRF2 activity drives resistance to radiation therapy (RT), our primary goal is to leverage these two novel NRF2 inhibitors as synthetic combination agents for RT in human and mouse models of HNSCC. Importantly for this work, we recently reported the first NRF2-driven GEMM of spontaneous oral SCC. Our proposed experiments use human HNSCC xenografts, mouse syngeneic tumor grafts, our NRF2- driven oral cavity SCC GEMM, and human and mouse 3-D spheroid cultures to test if VVD065 or PYR/WCDD115 synthetically combine with RT to suppress tumor growth while minimizing toxicity to normal tissues. We will also identify patient stratifying genotypes predictive of susceptibility to NRF2 chemical inhibition and RT sensitization. Together, these studies will produce foundational data for the dosing, scheduling, efficacy and toxicity of NRF2 inhibitors as synthetic combination agents with RT in pre-clinical HNSCC models. The impact of this research will be an improved response of HNSCC to radiation therapy.
