Magnoliae flos Overcomes EGFR ‐ TKI Resistance in NSCLC by Enhancing EGFR Ubiquitination

Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) remains a significant obstacle in treating non-small cell lung cancer (NSCLC). There is an urgent need for novel therapeutic approaches to overcome such resistance. This study aimed to investigate the potential of Magnoliae flos (MF) to reverse EGFR-TKI resistance and to elucidate its underlying molecular mechanisms. Network pharmacology was employed to predict the anti-NSCLC potential and molecular targets of MF. Predictions were validated through in vitro and in vivo experiments, including cell viability assays, flow cytometry for apoptosis and cell-cycle analysis, wound-healing assays, hierarchical extraction, high-performance liquid chromatography-mass spectrometry (HPLC-MS), Western blotting, small-molecule inhibitor screening, and patient-derived xenograft (PDX) models. Bioinformatics analyses were conducted to identify signaling pathways associated with EGFR-TKI resistance. Bioinformatics analysis suggested that MF modulates signaling pathways implicated in EGFR-TKI resistance. In the PDX model of EGFR-TKI-resistant NSCLC, the chloroform layer extract of MF (Ext. C) significantly suppressed tumor growth. Ext. C inhibited the EGFR/PI3K/AKT pathway, reduced EGFR protein expression, induced apoptosis, arrested the cell cycle, and impaired cell migration. HPLC-MS analysis identified magnolin (Mag), fargesin (Far), and kobusin (Kob) as bioactive compounds. Functional assays demonstrated these monomers exerted a synergistic anti-tumor effect, with Mag showing the greatest potency. Small-molecule inhibitor screening further revealed that Mag shortened the half-life of EGFR by promoting its ubiquitination and degradation. MF and its active constituents exhibit potent anti-tumor activity against EGFR-TKI-resistant NSCLC by promoting EGFR ubiquitination and downregulation of the EGFR/PI3K/AKT pathway. These findings suggest MF as a promising therapeutic candidate for overcoming EGFR-TKI resistance and provide a basis for further preclinical development.