Preclinical osimertinib-resistant mouse models for advancing third-generation EGFR-TKI drug development

The efficacy of Osimertinib, a third-generation EGFR tyrosine kinase inhibitor used in non-small cell lung cancer (NSCLC), is well established through clinical trials showing prolonged patient survival with EGFR-TKI therapy. However, a key clinical challenge arises from acquired resistance, frequently mediated by EGFR C797 mutations-notably the triple mutations Del19/T790M/C797S and L858R/T790M/C797S. To systematically investigate Osimertinib resistance and enable the testing of new therapeutic candidates, we have developed a panel of resistant models in both in vitro and in vivo settings. Using the human lung adenocarcinoma NCI-H1975 cell line-which carries the dual EGFR L858R/T790M mutations, we employed precise gene editing to introduce the C797S mutation, generating an isogenic triple-mutant subline (H1975 L858R/T790M/C797S). This model recapitulates the genetic profile of patients who have developed resistance following Osimertinib treatment. In addition to C797S-mediated resistance, we applied prolonged in vitro exposure of multiple cell lines to escalating Osimertinib concentrations, inducing acquired resistance independent of C797 mutations. Resistance was quantitatively confirmed through IC50 assays, which revealed a marked increase in Osimertinib tolerance. These models were further validated in vivo for tumor growth and drug response, confirming their functional resistance. We now possess well-characterized Osimertinib-resistant cell lines, including triple-mutant NCI-H1975 and PC-14 variants, providing essential platforms for dissecting resistance mechanisms and accelerating the development of next-generation treatments.