Overexpression of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) in cancer cells is known to cause multidrug resistance (MDR),which severely limits the clinical efficacy of chemotherapy.Currently,there is no FDA-approved MDR modulator for clinical use.In this study,rociletinib (CO-1686),a mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI),was found to significantly improve the efficacy of ABCG2 substrate chemotherapeutic agents in the transporter-overexpressing cancer cells in vitro and in MDR tumor xenografts in nude mice,without incurring additional toxicity.Mechanistic studies revealed that in ABCG2-overexpressing cancer cells,rociletinib inhibited ABCG2-mediated drug efflux and increased intracellular accumulation of ABCG2 probe substrates.Moreover,rociletinib,inhibited the ATPase activity,and competed with[125I]iodoarylazidoprazosin (IAAP) photolabeling of ABCG2.However,ABCG2 expression at mRNA and protein levels was not altered in the ABCG2-overexpressing cells after treatment with rociletinib.In addition,rociletinib did not inhibit EGFR downstream signaling and phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK).Our results collectively showed that rociletinib reversed ABCG2-mediated MDR by inhibiting ABCG2 efflux function,thus increasing the cellular accumulation of the transporter substrate anticancer drugs.The findings advocated the combination use of rociletinib and other chemotherapeutic drugs in cancer patients with ABCG2-overexpressing MDR tumors.