MLN4924 interacts synergistically with belinostat to induce apoptosis in both p53 wt and deficient leukemia cells. (A-B) p53^wt OCI-AML-3 and MOLM-13 (FLT3-ITD), as well as U937 (p53-null) and MV-4-11 (p53^mut, FLT-ITD), cells were exposed to the indicated concentrations of MLN4924 ± belinostat (PXD-101, nM) for 24 hours. (C) Murine Ba/F3 cells were stably transfected with constructs encoding either wt-FLT3 or FLT3-ITD. Cells were exposed to the indicated concentrations of PXD-101 ± MLN4924 for 24 hours. (D) wt-p53 OCI-AML-3 cells were stably transfected with shRNA constructs targeting human TP53 (shTP53) or scrambled sequence as a negative control (shNC), and then treated with 100 to 300 nM PXD-101 (P) ± 300 nM MLN4924 for 24 hours. For panels A-D, the percentage of Annexin V⁺ apoptotic cells was determined by FCM (*P < .05, **P < .01). (E) U937 cells were incubated with 300 nM PXD-101 ± 100 nM MLN4924 for 24 hours, after which cleavage of caspase 3, 7, 9, and PARP was monitored by western blot analysis (top panels). Alternatively, U937 cells were exposed (24 hours) to varying concentrations of MLN4924 ± PXD-101 at a fixed ratio (1:1), after which the percentage of Annexin V⁺ cells was determined, and median dose-effect analysis was then used to characterize the nature of the interaction between these agents. Combination index (CI) values <1.0 denote a synergistic interaction. The results are representative of 3 separate experiments. β-act, β-actin; c-Casp, cleaved caspase; CF, cleaved fragment; PXD, PXD-101; Veh, vehicle.