Abstract 395: Triamterene and Asinex 1 target DLG and ETGE mutants of NRF2 in esophageal squamous cell carcinoma

Abstract Introduction: NRF2 is a master transcription factor that regulates oxidative stress response. Esophageal squamous cell carcinoma (ESCC) frequently harbors gain-of-function NRF2 mutations that cause NRF2 overexpression, drive chemo- and radio-resistance, and lead to poor survival in human patients. NRF2 interacts with KEAP1’s Kelch domain through the DLG (Trp24-Leu30) and ETGE (Leu76-Leu84) motifs, which are mutation hotspots in human ESCC. Hence, there is a need to develop targeted therapies against NRF2Mut-ESCC carrying DLG or ETGE mutations, without inhibiting NRF2WT. Our previous study has validated two FDA-approved drugs (Pyrimethamine and Mitoxantrone) as NRF2Mutinhibitors in human ESCC cells and a genetically modified murine model (Redox Biology 2023; 67:102901). This study aims to further identify inhibitors that target DLG and ETGE mutants of NRF2 in ESCC. Methods: The effects of Triamterene (FDA-approved diuretic) and Asinex 1 (novel compound) on NRF2 expression, half-life, and ubiquitination were examined in NRF2W24C-KYSE70, NRF2D77V-KYSE180, NRF2D29H-TE14, NRF2G81S-OE21, NRF2WT-KYSE450, and KEAP1Null-KYSE450 cells. Proximity ligation assay (PLA) was performed to investigate the KEAP1-NRF2 interaction. Seven Triamterene analogues were synthesized for a structure-activity relationship study. Results: Both Triamterene and Asinex 1 displayed dose- and time-dependent inhibitory effects on NRF2W24C and NRF2D77V expression, respectively, with limited cytotoxicity. Both compounds did not impact NRF2WT expression in NRF2WT-KYSE450 and KEAP1Null-KYSE450 cells, indicating their selective inhibition of NRF2Mut. Triamterene and Asinex 1 significantly reduced the half-life and enhanced ubiquitination of NRF2W24C and NRF2D77V, respectively. In addition, Triamterene and Asinex 1 significantly inhibited the expression of NRF2D29H in TE14 cells and NRF2G81S in OE21 cells, respectively. PLA showed a significant increase in KEAP1-NRF2W24C interaction by Triamterene. The structure-activity relationship study and computer modeling suggested that Triamterene binds KEAP1 (in particular Arg415), thus modifies the conformation of Kelch domain to fit DLGW24C Conclusion: Triamterene and Asinex 1 inhibit NRF2Mut in ESCC cells with specificity for DLG mutants and ETGE mutants, respectively. Both compounds are being further investigated for their mechanisms of action as molecular glues and efficacy in genetically modified mice carrying DLG and ETGE mutations. Citation Format: Zachary A. Ladd, Candice Bui-Linh, Chorlada Paiboonrungruang, Boopathi Subramaniyan, Zhaohui Xiong, Tao Gao, Haining Wang, Francis Spitz, Xiaoxin Chen. Triamterene and Asinex 1 target DLG and ETGE mutants of NRF2 in esophageal squamous cell carcinoma [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 395.