Abstract 392: Pyrimethamine inhibits NRF2Mut in esophageal squamous cell carcinoma as a molecular glue

Abstract 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 and becomes hyperactive, and thus causes chemo-radioresistance and poor survival in human ESCC. In our previous study, we have identified pyrimethamine (PYR) as an NRF2 inhibitor using a small molecule screening, and further validated it as an inhibitor of NRF2 expression in NRF2Mut ESCC cells in vitro and Nrf2E79Q-driven esophageal phenotype in mice. As a result, PYR has moved into a Phase I window of opportunity trial (NCT05678348). This study is aimed to investigate the molecular and phenotypic consequences of NRF2 hyperactivation in ESCC, and elucidate the mechanism of action of PYR. We first generated isogenic human ESCC cells through CRISPR-Cas9 knockout for comparison with its parental cells. RNAseq and Western blotting showed that NRF2Mut overexpression downreulated the expression of squamous differential markers and upregulated the expression of proliferation markers, while NRF2WT overexpression showed different patterns. Flow cytometry and live cell imaging further showed that NRF2 activation modulated cell cycle distribution and proliferation, but not apoptosis of human ESCC cells. PYR increased chemosensitivity and radiosensitivity in NRF2Mut ESCC cells. Co-immunoprecipitation (Co-IP) and proximity ligation assay (PLA) suggested that PYR promoted NRF2-KEAP1 binding as a molecular glue in NRF2Mut ESCC cells, but not in NRF2WT ESCC cells. In summary, NRF2 hyperactivation differentially modulated squamous differentiation and cell proliferation in NRF2Mut and NRF2WT ESCC cells. PYR enhanced chemo-radiosensitivity by acting as a molecular glue specifically in NRF2Mut ESCC cells, supporting PYR as a novel therapy for NRF2Mut ESCC. Citation Format: Zhaohui Xiong, Chorlada Paiboonrungruang, Boopathi Subramaniyan, Candice Bui-Linh, Yahui Li, Huan Li, Francis Spitz, Xiaoxin Chen. Pyrimethamine inhibits NRF2Mut in esophageal squamous cell carcinoma as a molecular glue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 392.