Microtubule Inhibitors Induce Cross‐Resistance to Osimertinib Through <scp>CaMKII</scp> Activation in <scp>EGFR</scp> ‐Mutated <scp>NSCLC</scp>

The current standard postoperative adjuvant therapy for patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) includes chemotherapy, including microtubule inhibitors prior to the administration of osimertinib, an EGFR-tyrosine kinase inhibitor (TKI). However, multidrug resistance following treatment with microtubule inhibitors has been reported, and the optimal sequence of drug administration for EGFR-mutated NSCLC remains undefined. In this study, we investigated whether prior treatment with microtubule inhibitors induces acquired cross-resistance to osimertinib in EGFR-mutated NSCLC cells in vitro. To model acquired resistance, PC-9 cells were exposed to vinorelbine or paclitaxel for 18 weeks-approximating the clinical duration of four adjuvant chemotherapy cycles-and subsequent drug sensitivity and signaling pathway alterations were assessed using cell viability assays, RNA sequencing, and immunoblotting. We found that two human NSCLC cell lines derived from PC-9 exhibited reduced sensitivity to osimertinib after 18 weeks of in vitro treatment with tubulin inhibitors: vinorelbine (PC-9/VNR) and paclitaxel (PC-9/PTX). Furthermore, PC-9/VNR and PC-9/PTX cells showed activation of FZD7 and calcium/calmodulin-dependent protein kinase II (CaMKII), along with increased sensitivity to the CaMKII inhibitor KN-93, which exerted additive or synergistic effects. These findings suggest that CaMKII plays a critical role in EGFR-TKI resistance. This study underscores the importance of optimizing the timing of EGFR-TKI administration in the therapeutic sequence for EGFR-mutated NSCLC.