Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

Background: Esophageal squamous cell carcinoma (ESCC) remains a highly lethal malignancy with limited therapeutic options, in part due to frequent activation of nuclear factor erythroid 2-related factor 2 (NFE2L2 or NRF2). Gain-of-function mutations in NRF2 disrupt its negative regulation by Kelch-like ECH-associated protein 1 (KEAP1), resulting in sustained NRF2 signaling that promotes tumor growth and resistance to chemotherapy and radiation. We previously identified the FDA-approved drug pyrimethamine (PYR) as an NRF2 inhibitor and demonstrated that inhibition of dihydrofolate reductase (DHFR) represents the primary mechanism underlying its NRF2-suppressive activity, supporting its advancement into a Phase I window-of-opportunity clinical trial (NCT 05678348). Meanwhile, in NRF2W24C-KYSE70 and NRF2D77V-KYSE180 cells, PYR promoted NRF2Mut ubiquitination and proteasomal degradation and shortened its half-life. This study aims to explore additional modes of action by which PYR inhibits NRF2. Methods: Cell cycle analysis was performed by flow cytometry. Cell proliferation, apoptosis and chemosensitivity were assessed by Live-Cell Analysis System, while radiosensitivity was evaluated using X-ray irradiation and the CellTiter-Glo assay. Molecular interactions between NRF2 and KEAP1 were examined through Co-IP and PLA, and the direct binding of PYR to KEAP1 was quantified using ITC and SPR. Molecular docking and dynamic simulations were employed to predict potential PYR-binding pockets within the Kelch domain. Results: Using genetically defined isogenic ESCC cell models, we show that activation of mutant NRF2 (NRF2Mut) or wild-type NRF2 (NRF2WT) produces distinct, context-dependent effects on squamous differentiation, proliferation, and therapeutic response. We further demonstrate that PYR restores sensitivity to chemotherapy and ionizing radiation in NRF2Mut ESCC cells. Mechanistically, short-term PYR treatment promotes KEAP1-dependent proteasome-mediated degradation of NRF2W24C. Biochemical and biophysical assays indicate that PYR enhances the interaction between KEAP1 and NRF2W24C in a manner associated with KEAP1-dependent proteasomal degradation. Computational modeling further suggests that PYR may engage a pocket within the Kelch domain to facilitate the NRF2W24C-KEAP1 interaction. Conclusions: These findings show that PYR functionally restores KEAP1-mediated NRF2 degradation of select NRF2Mut through a glue-like effect and overcomes therapy resistance in ESCC. Although the proposed glue-like mechanism remains hypothetical, this work supports further investigation into the NRF2–KEAP1 interaction and may inform the development of KEAP1-targeted strategies for NRF2Mut cancers, including ESCC.