Abstract 1247: Characterizing the evolution of osimertinib resistance in EGFR mutated non-small cell lung cancer

Abstract Osimertinib is a 3rd generation EGFR inhibitor and the newest FDA approved adjuvant therapy for non-small cell lung cancer (NSCLC) with EGFR exon 19 deletions or exon 21 L858R mutation. Although the drug can yield impressive initial responses, the majority of patients eventually progress due to the evolution of resistance. While the molecular mechanisms underlying resistance have been well studied, little is known about the ecological and evolutionary laws that govern the creation and subsequent expansion of these mutant populations. How frequently are different evolutionary routes taken and to what degree is evolution repeatable? To study the repeatability of osimertinib resistance evolution, we conducted a large-scale evolution experiment aimed at characterizing the distribution of resistant states that emerge, along with the pathways and vulnerabilities associated with their development. In our experimental design, we evolve PC-9, an EGFR-mutated NSCLC cell line to osimertinib independently 10 times (evolutionary replicates) for 10 cycles (serial passages). We also evolved 6 control replicates that undergo no treatment. At the end of the experiment, we will employ a multi-faceted approach to studying the resistance phenotype: 1) IC50 against osimertinib; 2) bulk RNA-seq; 3) DNA/mutational profiling; 4) a collateral sensitivity screening against 20 FDA approved drugs; and 5) a radiation sensitivity assay against 6 doses. The initial pretreatment dose response curve (DRC) against osimertinib showed a double plateau, suggesting the pre-existence of tolerant or even resistant subpopulation. In our control arms, we see consistent persistence of our double plateau throughout all 10 cycles, indicating this population is maintained in the absence of treatment but cannot expand. Conversely, across all 8 treated replicates we see a large shift in IC50 within the 1st treatment cycle, consistent with rapid selection and outgrowth of this population. More detailed analysis using time-lapse microscopy shows while these cells have a higher IC50, they also have a reduced growth rate, even if treatment is completely removed again (a “resistance cost”). This suggests potential evolutionary vulnerabilities which could be leveraged through strategic treatment breaks or drug sequences. Citation Format: Mina N. Dinh, Jinling Wu, Masahiro Hitomi, Maximilian Strobl, Jacob G. Scott. Characterizing the evolution of osimertinib resistance in EGFR mutated non-small cell lung cancer [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 1247.