TAIII Suppresses the Growth of T790M-Mutant Non-Small-Cell Lung Cancer by Targeting the EGFR/ERK Signaling Pathway

Background/Objectives: First-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) are standard first-line treatments for advanced non-small-cell lung cancer (NSCLC). However, acquired resistance often develops via secondary T790M mutations, necessitating new therapies. Timosaponin AIII (TAIII) shows antitumor activity and has been found to suppress EGFR phosphorylation. This study aimed to evaluate the therapeutic potential of TAIII in overcoming T790M-mediated resistance in NSCLC and elucidate its underlying molecular mechanisms. Methods: We evaluated the inhibitory effects of TAIII on proliferation (EdU assay) and migration (Transwell assay) in T790M-mutated H1975 cells. EGFR phosphorylation and downstream signaling were analyzed by Western blotting. Molecular docking was employed to predict the binding of TAIII to EGFR, while CETSA (cellular thermal shift assay) and SIP (Stability of Interaction Partners) assays were used to validate TAIII-EGFR interaction stability. The in vivo antitumor efficacy was further confirmed in nude mouse xenograft models. Results: TAIII inhibited H1975 cell proliferation and migration by downregulating p-EGFR (Y1068) and ERK signaling. Docking showed stable TAIII binding in the EGFR kinase domain via hydrogen bonds at THR-776 and PRO-770, confirmed by CETSA and SIP. At high concentrations, TAIII induced EGFR degradation through autophagy–lysosome pathways. TAIII monotherapy outperformed combinations with gefitinib (CI > 1). Xenograft models confirmed its potent antitumor effect via EGFR phosphorylation inhibition. Conclusions: TAIII demonstrates substantial therapeutic potential for overcoming T790M-mediated resistance in NSCLC by its dual mechanism of EGFR signaling inhibition and receptor degradation, supporting further preclinical and clinical development.