Cytoskeletal remodeling via CAMSAP3 downregulation drives resistance to osimertinib in NSCLC cells.

Osimertinib, also known as AZD9291, is a highly potent and selective EGFR mutants (including exon 19 deletion, L858R/T790M) inhibitor that significantly inhibits EGFR phosphorylation signaling. However, acquired resistance to osimertinib is inevitable in the treatment of non-small cell lung cancer (NSCLC). Microtubules, key cytoskeletal components involved in intracellular cargo transport, mediate EGFR-endosomal recycling, yet their specific role in osimertinib resistance remains to be elucidated. In this study, we found that centrosomal microtubule formation was increased in osimertinib-resistant NSCLC cells, and calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) was identified as the key molecule responsible for the change of microtubule morphology. Genetic modulation via CAMSAP3 silencing in both osimertinib-sensitive cells (in vitro) and xenograft models (in vivo) enhanced microtubule clustering and resistance to osimertinib, whereas CAMSAP3 overexpression in resistant cells partially restored microtubule organization and drug sensitivity. Furthermore, we demonstrated that full-length CAMSAP3 is essential for proper localization of the microtubule-dependent endosomal-lysosomal system. CAMSAP3 depletion caused EGFR translocation to the perinuclear microtubule organizing center (MTOC), thereby blocking plasma membrane recycling and promoting lysosomal degradation. These findings establish CAMSAP3 as a key regulator of EGFR signaling and osimertinib response in NSCLC, suggesting its therapeutic potential for overcoming drug resistance in lung cancer.