The overexpression of ATP-binding cassette (ABC) transporters has been proved to 
be a major trigger for multidrug resistance (MDR) in certain types of cancer. In 
our study, we investigated whether osimertinib (AZD9291), a third-generation 
irreversible tyrosine kinase inhibitor of both activating EGFR mutations and 
resistance-associated T790M point mutation, could reverse MDR induced by ABCB1 
and ABCG2 in vitro, in vivo, and ex vivo Our results showed that osimertinib 
significantly increased the sensitivity of ABCB1- and ABCG2-overexpressing cells 
to their substrate chemotherapeutic agents in vitro and in the model of 
ABCB1-overexpressing KBv200 cell xenograft in nude mice. Mechanistically, 
osimertinib increased the intracellular accumulations of doxorubicin (DOX) and 
Rhodamine 123 (Rho 123) by inhibiting the efflux function of the transporters in 
ABCB1- or ABCG2-overexpressing cells but not in their parental sensitive cells. 
Furthermore, osimertinib stimulated the ATPase activity of both ABCB1 and ABCG2 
and competed with the [(125)I] iodoarylazidoprazosin photolabeling bound to 
ABCB1 or ABCG2, but did not alter the localization and expression of ABCB1 or 
ABCG2 in mRNA and protein levels nor the phosphorylations of EGFR, AKT, and ERK. 
Importantly, osimertinib also enhanced the cytotoxicity of DOX and intracellular 
accumulation of Rho 123 in ABCB1-overexpressing primary leukemia cells. Overall, 
these findings suggest osimertinib reverses ABCB1- and ABCG2-mediated MDR via 
inhibiting ABCB1 and ABCG2 from pumping out chemotherapeutic agents and provide 
possibility for cancer combinational therapy with osimertinib in the clinic. Mol 
Cancer Ther; 15(8); 1845-58. ©2016 AACR.