Pan-AKIs potentiate the TRAIL-induced cell death in MM cells. (Ai) HMCLs seeded at 2.0 × 10⁵ cells/mL were treated sequentially with escalating doses of MK-0457 (15-2000 ng/mL; 0.032-4.3 μM) or PHA-680632 (15-2000 ng/mL; 0.03-4 μM) for 3 hours and subsequently with TRAIL (0.5-50 ng/mL) alone or in combination with pan-AKIs at a fixed ratio of 25:1 for MK-0457/TRAIL and 50:1 for PHA-680632/TRAIL (in combination with pan-AKIs, TRAIL was used at 0.6, 1.2, 2.4, 4.8, 9.6, and 19.2 ng/mL). After 24 hours, cell death was measured by annexin-V binding. Combination index plots were then generated using the Calcusyn software. Combination index values <1.0 indicate synergism, 1.0 indicates additive effect, and >1.0 indicates an antagonistic effect. (Aii) JJN3 were seeded at 2.0 × 10⁵ cells/mL in the presence of dimethyl sulfoxide (vehicle), MK-0457 (0.4 μM), or PHA-680632 (0.8 μM) for 3 hours and then incubated with TRAIL at the concentration of 300 ng/mL. After 24 hours, apoptosis was measured by annexin-V labeling. Values represent means ± SD of 4 independent experiments (*P < .01 vs either treatment alone; Dunnet test). (B) HMCLs were treated with pan-AKIs for 3 hours and then incubated with TRAIL at the indicated doses (ng/mL) in the presence or absence of IGF-1 (50 ng/mL), IL-6 (20 ng/mL) or BMSCs (HMCLs in direct contact to BMSC monolayer in a 5:1 ratio). After 24 hours, HMCLs were harvested and apoptotic HMCLs cells were determined by flow cytometry as CD38/CD138⁺ annexin-V⁺ cells; data represent means ± SD of quadruplicate experiments. Dexamethasone (2 μM) was used as control to monitor the protective effects of growth factors and stromal cells (*P < .05; **P < .005; Tukey-Kramer Honestly Significant Difference test). (Ci-ii) CD138-purified plasma cells from 5 patients with MM were isolated from BM and then seeded at 2.5 × 10⁵ cells/mL. Samples from 1 to 3 were pretreated with MK-0457 (0.4 μM), and samples 4 and 5 were pretreated with PHA-680632 (0.8 μM) and then incubated with TRAIL (9.6 ng/mL) for 24 hours. Sample 4 was cultured in presence or absence of BMSCs. The cell death was measured by annexin-V staining. (i) Results are expressed as the net apoptosis induction [percentage of apoptosis in treated cells minus percentage of apoptosis in dimethylsulfoxide (vehicle-treated cells)] of all 5 primary samples tested. (ii) Histogram represents the mean percentage of apoptosis ± SD (expressed as net difference between the percentages of apoptosis in treated cells minus percentage of apoptosis in vehicle-treated cells) of the results obtained in the 5 different patient samples (*P < .05 vs either treatment alone; Dunnet test). (iii) Peripheral blood mononuclear cells from 3 healthy volunteers were treated with dimethylsulfoxide (vehicle), MK-0457 (0.4 μM), or PHA-680632 (0.8 μM) for 3 hours and then incubated with TRAIL (9.6 ng/mL). After 24 hours of treatment, cell death was then measured by annexin-V staining. (D) Transfection of Aurora A and/or Aurora B, but not the unrelated nonspecific control siRNA, led to a decrease in Aurora kinases protein expression in HMCLs without affecting the levels of the unrelated protein actin. At 48 hours after siRNA transfection, HMCLs were treated with TRAIL at the indicated doses (ng/mL) for 48 hours and the percentages of annexin-V⁺ apoptotic cells were then measured. Values are mean ± SD of 3 independent experiments (*P < .001 vs TRAIL nonspecific si-RNA [CONT]; Dunnet test).