Pyrimethamine inhibits mutant NRF2 as a molecular glue

Esophageal squamous cell carcinoma (ESCC) is a deadly disease and one of the most aggressive cancers of the gastrointestinal tract. As a master transcription factor regulating the stress response, nuclear factor erythroid 2-related factor 2 (NRF2) is often mutated, loses negative regulation by Kelch-like ECH-associated protein 1 (KEAP1), becomes hyperactive, and thus causes chemo-radioresistance and poor survival in human ESCC. Our previous research identified pyrimethamine (PYR) as an NRF2 inhibitor and demonstrated that inhibition of dihydrofolate reductase (DHFR) is the major mechanism of its action. Based on these findings, PYR has advanced into a Phase I window-of-opportunity clinical trial. In this study, using isogenic ESCC cell lines, we aimed to investigate the phenotypic consequences of NRF2 activation in ESCC cells and further elucidate the mechanism of action of PYR. While overexpression of wild-type NRF2 (NRF2 ^WT ) promoted squamous differentiation and suppressed proliferation, expression of mutant NRF2 (NRF2 ^Mut ) produced mixed effects on differentiation markers and promoted proliferation. NRF2 activation reduced sensitivity to chemotherapy and radiation, whereas PYR treatment restored chemosensitivity and radiosensitivity in NRF2 ^Mut ESCC cells. Co-immunoprecipitation and proximity ligation assays revealed that PYR selectively enhanced the interaction between NRF2 ^W24C and KEAP1, but not between NRF2 ^WT and KEAP1. Isothermal titration calorimetry (ITC) confirmed the direct binding of PYR to KEAP1 and surface plasmon resonance (SPR) showed that PYR modestly facilitated the interaction between a DLG ^W24C peptide and the Kelch fragment. Molecular docking suggested that PYR bound to a pocket within the Kelch domain near Arg ⁴¹⁵ . In summary, NRF2 activation promotes cell proliferation and therapy resistance in ESCC in a context-dependent manner. PYR functions as a modest molecular glue that selectively restores KEAP1 binding to NRF2 ^W24C , providing a potential therapeutic strategy for NRF2 ^Mut ESCC.