Sequential senescence-escape cycles drive genomic heterogeneity and osimertinib resistance in EGFR-mutant NSCLC

Acquired resistance to osimertinib remains a critical challenge in the management of EGFR-mutant (EGFR⁺) NSCLC. Although most patients initially respond, relapse is universal, even after prolonged remissions, suggesting that this interval reflects a durable drug-induced proliferative arrest consistent with cellular senescence. We demonstrate Osimertinib-Induced Senescence (OsIS) in EGFR⁺ NSCLC cells and show that, several weeks after drug withdrawal, senescent cells resume proliferation. To determine how repeated senescence and escape influence therapeutic response and genomic evolution, we generated isogenic cell lines expanded through four sequential rounds of OsIS over approximately six months. Four distinct evolutionary trajectories were selected, each exhibiting varying degrees of resistance to EGFR inhibitors. Although OsIS-derived lines retained sensitivity to cisplatin, they displayed heterogeneous responses to pemetrexed, while sensitivity to tubulin-interacting drugs and navitoclax was preserved. Genomic analyses confirmed that resistance did not arise from de novo single nucleotide mutations or copy number amplification in EGFR or MET. Instead, all OsIS-derived lines had increased tumor mutational burden and acquired mutational signatures associated with base-excision repair defects, replication stress, and, in the most resistant line, oxidative stress. Despite these signatures, therapies directed at DNA repair or replication stress were uniformly ineffective, indicating that resistance was not driven by discrete genomic lesions but rather by age-associated mutational drift. These findings support a model in which OsIS functions as an evolutionary bottleneck whose escape promotes genomic heterogeneity and promotes resistance to EGFR inhibition.