Attenuation of ABCG2-dependent drug efflux by befotertinib restores chemosensitivity in multidrug-resistant non-small cell lung cancer cells

Chemotherapy failure remains a major challenge in cancer treatment, with multidrug resistance (MDR) representing one of the most significant contributing factors. A key mechanism of MDR involves the over-activity of ATP-binding cassette (ABC) transporters, which utilize ATP hydrolysis to actively export a broad range of anticancer agents from tumor cells. This process reduces intracellular drug accumulation and consequently diminishes therapeutic efficacy. Despite extensive efforts to develop novel inhibitors targeting these transporters, few clinically effective agents have emerged, underscoring the need for alternative strategies such as repurposing targeted therapies with favorable safety profiles to modulate drug efflux. In this study, we evaluated befotertinib (D-0316), a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor approved by the National Medical Products Administration, for its ability to overcome ABCG2-mediated drug resistance. At non-cytotoxic concentrations, befotertinib significantly resensitized ABCG2-overexpressing non-small cell lung cancer (NSCLC) cells to chemotherapeutic agents that are ABCG2 substrates and enhanced drug-induced apoptosis in a concentration-dependent manner. Functional assays demonstrated that befotertinib suppresses ABCG2-mediated drug efflux without altering transporter protein expression. Mechanistic analyses further showed that befotertinib stimulates ABCG2 ATPase activity, while molecular docking predicts stable binding within the ABCG2 substrate-binding pocket, suggesting direct interaction with key residues involved in transporter function. Through this mechanism, befotertinib acts as a functional inhibitor of ABCG2, restoring intracellular drug accumulation and enhancing cytotoxic responses. Collectively, these findings indicate that befotertinib may serve as a chemosensitizing agent to overcome MDR in NSCLC with high ABCG2 expression, supporting further evaluation in combination therapeutic strategies.