Abstract 4450: Targeting KEAP1/NRF2 signaling sensitizes KRAS-driven NSCLC to KRAS inhibitors

The emergence of KRAS G12C inhibitors has paved the way for new therapeutic approaches in NSCLC patients with KRAS mutations. However, the efficacy of these inhibitors has been limited in NSCLC based on clinical trial results. Numerous studies have uncovered that the co-occurrence of additional mutations, particularly in KEAP1 and STK11, is associated with a poorer prognosis for KRAS G12C-mutated NSCLC patients. KEAP1 is a negative regulator of the NRF2 signaling pathway. KEAP1 mutations lead to constitutive activation of NRF2, thus promoting tumor growth and drug resistance. In this study, we employed siRNA technique to specifically target NRF2, and found that targeting NRF2 could be a promising therapeutic approach to improve the efficacies of KRAS inhibitors in NSCLC. Firstly, we compared the basal mRNA and protein levels of NRF2 between cell lines with and without KEAP1 mutation. The results showed that the basal mRNA levels were comparable across 5 cell lines including A549, H2122, HEK293, H292 and LoVo. Only A549 and H2122, two cell lines with KEAP1 mutation, expressed high NRF2 protein levels. This observation aligns with the established mechanism that KEAP1 mutation leads to the accumulation of NRF2 through a protein degradation-repression manner. Furthermore, luciferase reporter assay demonstrated that NRF2 transcription was activated in A549 and H2122 in comparison to HEK293. Accordingly, the expression of NRF2 downstream genes, such as AKR1B10 and AKR1C1, was relatively higher in KEAP1-mutated A549 and H2122 cells than in KEAP1-intact cells. To investigate the impact of NRF2 knockdown on cell proliferation, siRNA was performed in A549 and H2122 cells. The knockdown efficiency was verified through qRT-PCR and western blot analysis. The results revealed that knockdown of NRF2 could significantly inhibit cell growth in both A549 and H2122 cells. To ensure that the inhibitory effects of NRF2 knockdown were not limited to the two cell lines, we conducted an analysis of NRF2 dependency in NSCLC using the Depmap database. The results indicated that KEAP1/NRF2 pathway-activating mutations could predict NRF2 dependency in NSCLC, even in the broader context of lung cancer. Moreover, two downstream genes of NRF2, AKR1B10 and AKR1C1, were observed down-regulated in A549 and H2122 cells post NRF2 knockdown. Finally, we assessed the combined effectiveness of NRF2 knockdown and KRAS inhibitors in the A549 and H2122 cells. The results demonstrated that silencing NRF2 could significantly improve the responsiveness of both the pan-KRAS inhibitor RMC-6236 and the KRAS G12C inhibitor Sotorasib in A549 and H2122 cells. Taken together, our study demonstrates that knockdown of NRF2 can enhance the effectiveness of KRAS inhibitors in treating NSCLC patients with KEAP1 mutations. This finding provides the scientific foundation for developing combination therapy strategies that target both NRF2 and KRAS to improve patient outcomes. Citation Format: Xianwen Yang, Peiling Ren, Guiping Li, Ying Yu, Guangxiu Dai. Targeting KEAP1/NRF2 signaling sensitizes KRAS-driven NSCLC to KRAS inhibitors [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 4450.