Abstract 4052: Biological Variations in NRF2 Hotspot Mutants

Abstract Background: NRF2 is a transcription factor that is a master regulator for the cell’s antioxidant response. Under normal conditions, NRF2 is rapidly degraded by cytoplasmic association with KEAP1 and subsequent ubiquitination and proteasomal degradation. Activating mutations in NRF2 contribute to lung cancer transformation. D29H, R34G, and E79Q are the most common mutations found in lung squamous cell carcinoma. Methods: H520 lung cancer cells harbor an intact NRF2/KEAP system. H520 mutant cell lines were generated using lentivirus transduction of either WT or mutant NRF2 and confirmed by Western blot. Immunofluorescence was used to examine the effect of mutations on NRF2 levels and subcellular localization. Cells were tested for ROS levels by DCFFA fluorescence and subcellular NRF2 localization before and after treatment with tert-butyl hydroperoxide. Colony forming ability was tested after radiation with 0, 2, 4, or 6 Gy. Results: D29H and R34G mutations result in increased NRF2 levels and stress-independent translocation to the nucleus compared to wild type NRF2. These mutant cells also showed higher expression of the NRF2 target gene G6PD. E79Q has little difference compared to wild type. All mutants showed decreased DCF fluorescence. With regards to colony formation ability, mutant cell lines were not statistically different from WT cells. Conclusion: Our findings identified D29H and R34G as having increased nuclear levels and transactivation compared to WT cells. Despite the increased activity of NRF2 in these two mutations, there was little evidence for increased radiation resistance. These results indicate differential biological response of common NRF2 mutations. Citation Format: Syed A. Ashraf, Anh Thi Thai, Jinghai Wu, McKenzie Kreamer, Katarina Benejova, Terry M. Williams, Martin Benej, Ioanna Papandreou, Nicholas C. Denko. Biological Variations in NRF2 Hotspot Mutants [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 4052.