MDR1 inhibition increases the intracellular concentration of birinapant in MDR1^HAML. (A) The proposed mechanism of action of SM/MDR1i combination therapy in MDR1^H cells. Bir was pumped out of cells by MDR1, and inhibition of MDR1 pumps increased the intracellular levels of birinapant. (B) KG1 AML cells were treated for the indicated times with Bir (500 nM), with or without the MDR1i Tari (1 μM). Whole-cell lysates were probed with the indicated antibodies, with actin used as the loading control. M# indicates the different membranes. (C) KG1 AML cells were treated for up to 48 hours with Bir (500 nM), with or without Tari (1 μM; n = 4). Cell survival was measured by flow cytometry analysis of PI exclusion. (D) Intracellular levels of birinapant in KG1 AML cells treated with Bir (500 nM), with or without Tari (1 μM), were quantified using MS and deuterium-labeled birinapant as a standard (n = 3 independent experiments). Data are the means ± SEM throughout. P values were obtained by comparison of Bir alone and Bir/Tari treatment at the indicated time points. *P < .05; **P < .01; ***P < .001; ****P < .0001.