Neratinib (HIK-272),an irreversible inhibitor of EGFR and Her-2,is in phase Ⅲ clinical trials for patients with Her-2-positive locally advanced or metastatic breast cancer.The objective of this study was to explore the ability of neratinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABC transporters.Our results showed that neratinib remarkably enhanced the sensitivity of ABCB1-overexpressing cells to ABCBI substrates.Importantly,neratinib augmented the effect of chemotherapeutic agents on inhibiting the growth of ABCBl-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice.Furthermore,neratinib increased the accumulation of doxorubicin in ABCB1-overexpressing cell lines and rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemic blasts.Intriguingly,neratinib stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations.Similarly,neratinib in a concentration-dependent manner inhibited photolabeling of ABCB1 with 125I-lodoarylazidoprazosin (IC50=0.25 μmol/L).However,neither the expression of ABCB1 at mRNA and protein levels nor the phosphorylation of Akt was affected by neratinib at reversal concentrations.Docking simulations are consistent with the binding conformation of neratinib within the large cavity of the transmembrane region of ABCB1,providing computational support for the cross-reactivity of TKIs with human ABCB1,In conclusion,neratinib can reverse ABCBI-mediated multidrug resistance in vitro,ex vivo and in vivo by inhibiting its transport function.