137 - Exploiting Dependencies Created By KEAP1/NFE2L2 Mutations in NSCLC to Overcome Radiation Resistance
Presenter(s)
W. X. Mai1, Y. J. Jeon2, L. Guan3, L. A. Soto4, S. Dutt4, N. Kastelowitz1, J. C. Cheng1, B. W. Loo Jr5, and M. Diehn4; 1Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, 2Department of Integrative Biotechnology, Sungkyunkwan University, Seoul, Korea, Republic of (South), 3Radiation Oncology, Stanford University, Stanford, CA, 4Department of Radiation Oncology, Stanford University, Palo Alto, CA, 5Stanford University School of Medicine, Stanford, CA
Purpose/Objective(s): Radiation is a major treatment modality employed in early-stage and locally advanced non-small cell lung cancer (NSCLC). Notably, NSCLC tumors with KEAP1/NFE2L2 mutations are associated with elevated rates of local recurrence post-radiation treatment. Thus, the identification of radiosensitization strategies is paramount. Despite the KEAP1/NFE2L2 axis being established as a driver of radiation resistance, attempts at targeting this pathway have not resulted in clinical success. To address these needs, we performed the first ever genome-wide CRISPR knockout screen in a KEAP1-altered NSCLC cell line under radiation exposure to identify potential radiosensitizing targets.
Materials/Methods: For the CRISPR/Cas9 gene knockout screen, a Trp53-/- Keap1-/- murine NSCLC cell line previously developed by our group was used. We employed a library consisting of sgRNAs against ~20,000 protein-coding genes (10 sgRNA/ gene). Next, cells were either untreated (control) or subjected to 12 Gy over 4 fractions (3 Gy every 4 days). DNA was harvested 16 days following the first treatment and sequenced. CasTLE analysis was utilized to score genes based on effect size in radiated vs. untreated cells. Promising radiosensitizing targets were further verified in isogenic human H1299 KEAP1 wildtype (KEAP1WT) and knockout (KEAP1KO) cell lines using a clonogenic survival assay.
Results: We performed a CRISPR/Cas9 knockout screen to identify radiosensitizing targets in KEAP1KO NSCLC cells. CasTLE analysis using a 5% false discovery rate revealed 1,649 genes essential for survival after radiation (p<0.05). KEGG pathway analysis showed a notable depletion of genes involved in metabolism (n=73, p<0.01). Upon interrogation of individual depleted genes with the greatest effect size, we identified 15 actionable targets with available small molecule inhibitors, including ATM. Given ATM has been demonstrated to support survival in KEAP1-altered tumors and the availability of clinical-grade ATM inhibitors, we prioritized it for validation. Even at low doses (1nM), ATM inhibition preferentially sensitized H1299 KEAP1KO cells to radiation (p<0.05), restoring radiosensitivity to wildtype levels. Moreover, this sensitizing effect appears to stem from ATM’s noncanonical role in redox homeostasis rather than its established function in DNA repair, as ATM inhibition selectively depleted glutathione in KEAP1KO cells (p<0.01) compared to KEAP1WT.
Conclusion: This study presents the first genome-wide knockout screen in KEAP1-altered NSCLC after radiation. Our results reveal a strong dependency on metabolic pathways for radiation resistance and highlight ATM as a radiosensitizing target. While ATM is canonically linked to DNA repair, our data suggest its role in KEAP1-altered NSCLC is tied to redox homeostasis. These findings demonstrate the potential of this approach to uncover clinically relevant dependencies and guide targeted therapeutic strategies in this radioresistant subset of NSCLC.