Abstract Lung cancer remains the leading cause of cancer-related mortality worldwide. Approximately 85% of cases are classified as non-small cell lung cancer (NSCLC) and activating mutations in the epidermal growth factor receptor (EGFR) are present in about 16% of metastatic NSCLC tumors. The development of EGFR tyrosine kinase inhibitors (EGFR-TKIs) including the third-generation agent Osimertinib, have significantly improved patient outcomes. Nonetheless, its efficacy is limited by both on-target and off-target resistance mechanisms, including the C797S mutation, MET amplification, and histological transformation. Emerging evidence has shown that EGFR signaling influences DNA repair, and that persistent proliferative signaling in EGFR-driven tumors elevates replication stress (RS), making them uniquely vulnerable to inhibition of the DNA damage response (DDR). While prior approaches have primarily targeted DDR kinases such as ATR, CHK1, and WEE1, the role of DNA damage sensors (DDS), including replication protein A (RPA) and the Ku70/80 complex, remains largely unexplored. In this study, we focus on this strategy by targeting RPA and Ku70/80, which recognize DNA structures generated by direct DNA damage or RS and initiate the DDR. Using small molecule inhibitors that target RPA and Ku-DNA binding in combination with clinically relevant EGFR-TKIs, we aimed to dissect the mechanistic interactions between DDR pathways and EGFR signaling to elucidate how DDR modulation impact therapeutic response and resistance in EGFR-driven NSCLCs. Our results show that the DDS inhibitors NERx-329 (RPAi) and Ku-DBi 3392 (Ku70/80) in combination with Osimertinib further enhances antiproliferative and cytotoxic effects in TKI-sensitive, EGFR-mutant NSCLC cells in an additive manner. Our data demonstrate that DDR inhibition is effective in TKI-sensitive EGFR-driven models potentiating Osimertinib activity. Importantly, this is independent of a DNA damaging agent, suggesting that inhibition of DDR itself is enough to increase the therapeutic effect of Osimertinib. This effect appears to be EGFR-specific, as no potentiation of Alectinib activity was observed in an EML4-ALK driven NSCLC model. These preliminary findings support a functional crosstalk between DDS-mediated DDR pathways and EGFR signaling highlighting a potential therapeutic strategy to overcome, delay, or prevent the recurrence of resistance in EGFR-driven NSCLC cancers. Citation Format: Pamela L. Mendoza-Munoz, Madison E. Gerbig, John J. Turchi, Shadia I. Jalal. Targeting DNA damage sensors (DDS) to enhance Osimertinib response in EGFR-driven cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 236.
Mendoza-Munoz et al. (Fri,) studied this question.