MDR1 and TNF are biomarkers of birinapant response in murine AMLs. (A) Whole-cell lysates from HoxA9/Meis1, MLL-AF9-Nras^G12D, and MLL-ENL AML cells were probed with the indicated antibodies, with actin used as the loading control. M# indicates the different membranes. Tumor numbers indicate leukemias originating from individual mice. HoxA9/Meis1 (n = 5; tumors 87, 236, and 11) (B); MLL-AF9-Nras^G12D (n = 4; tumors 11, 12, 350, and 374) (C); and MLL-ENL (n = 4; tumors 457, 126, 106, and 123) (D). AML cells were treated with Tari (0.3, 0.6, 1.2, 2.5, and 5 μM), with or without Bir, for 24 hours. (E) Western blot analysis of MDR1 in independent MLL-AF9 AML cells. (F) MDR1^L, birinapant-sensitive (Bir^S), combination-resistant (Combo^R) (blue bars, n = 3; tumor 393); MDR1^H, birinapant-resistant (Bir^R), combination-sensitive (Combo^S) (red bars, n = 4; tumors 145 and 647); MDR1^L, Bir^R, Combo^S+TNF (purple bars, n = 6; tumors 515, 516, and 518); and MDR1^H, Bir^R, and Combo^S+TNF (green bars, n = 3; tumors 394 and 396) AML cells were treated for 24 hours with Bir (500 nM), with or without Tari (500 nM) and with or without TNF (100 ng/mL). (G) TNF production in 4 MLL-AF9 AML subgroups was determined by ELISA. Cells were treated with Bir (500 nM), with or without Tari (500 nM), for 6 hours (1-2 individual tumors for each subgroup; 2-3 repeats). Data are the mean ± SEM throughout. Cell death was measured by flow cytometry analysis of PI uptake and decrease in cell volume. P values were obtained by comparison of control and Bir/Tari treatment. *P < .05.