Pan-Aurora Kinase Inhibitor Mk-0457 Synergistically Potentiates Apo2L/Trail Cytotoxicity in Multiple Mieloma Cells Sensitive and Resistant to Bortezomib.

Abstract 1837

Poster Board I-863

Multiple Myeloma (MM) cells are extremely resistant to apoptosis and currently new potential drug combinations are under investigation. Aurora kinase inhibitors have been shown to abrogate proliferation and induce apoptosis in human myeloma cells lines (HMCLs) and primary myeloma cells. In addition previous studies have shown the antimyeloma activity of Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) as a single agent or in combination with certain chemotherapeutic agents. The aim of this study was to investigate whether the combined treatment with pan-Aurora kinase inhibitor MK-0457 also known as VX-680(Vertex/Merck) and Apo2L/TRAIL has cytotoxic effects on MM cells. Because we found that MK-0457 (0.2–0.5 μM) partially activated the extrinsic caspase-8 mediated pathway both in HMCLs RPMI 8226 (highly sensitive to Apo2L/TRAIL: median lethal dose (LD₅₀) at 48 hours was 4.9 ng/mL), and bortezomib-resistant 8226/R5 (barely sensitive to Apo2L/TRAIL: LD₅₀ at 48 hours was 90.9 ng/mL) we attempted to examine whether MK-0457 potentiates the Apo2L/TRAIL-mediated apoptosis in HMCLs that have differential sensitivity to Apo2L/TRAIL. We first analyzed the pharmacologic interactions between MK-0457 and Apo2L/TRAIL using a fixed-ratio experimental design and found that the combined treatment resulted in the synergistic induction of apoptosis in both HMCLs (Chou-Talalay method): after 48 hours of treatment the averaged Combination Index values calculated from the ED50 (50% effective dose), ED75 and ED90, in MK-0457 plus Apo2L/TRAIL were 0.04 ± 0.05 and 0.03 ± 0.02 in RPMI 8226 and 8226/R5 respectively. Consistent with these results, we found that Aurora-A and -B were expressed at similar levels in RPMI 8226 and 8226/R5 cells and we demonstrated that the functional knock-out of Aurora-A or -B gene expression by small interfering (si)RNAs significantly increased (P< .001 Dunnett test) the TRAIL- induced apoptosis in both HMCLs. To investigate the molecular mechanisms by which MK-0457/Apo2L/TRAIL induced MM cell apoptosis first we compared the effect on caspase activation in RPMI 8226 and 8226/R5. The cells were treated with MK-0457 (0.2–0.5 μM) and/or Apo2L/TRAIL (2.4 ng/mL and 9.6 ng/mL in RPMI 8226 and 8226/R5 respectively) for 24 hours and caspase activation and PARP fragmentation were analyzed by Western blotting: the treatment with MK-0457 strongly potentiated the Apo2L/TRAIL -induced activation of caspase-8, caspase-3 and PARP cleavage in both HMCLs. In addition, a strong activation of caspase-9 was observed in the MK-0457/Apo2L/TRAIL-treated 8226/R5. Using caspase blocking peptides, specific siRNA against caspase-8 or caspase-9 and Western immunoblotting we demonstrated the involvement of primarily caspase-8 and -3 in MK-0457/Apo2L/TRAIL -induced apoptosis in RPMI 8226 and 8226/R5: the inhibition of caspase-8 significantly (P< .001 Dunnett test) reduced the MK-0457/Apo2L/TRAIL -induced apoptosis in both cell lines. Moreover, the pancaspase inhibitor Z-VAD-FMK protected MM cells from MK-0457/Apo2L/TRAIL -induced apoptosis, confirming that caspase activity was indispensable in MK-0457/Apo2L/TRAIL -induced apoptosis. Since antiapoptotic Mcl-1 and proapoptotic Bim play a pivotal role in controlling MM cell survival and apoptosis, and Bim, as previously demonstrated, can interfere with the activation of both intrinsic and extrinsic apoptotic pathways in MM cells, we analyzed their expression in MK-0457/Apo2L/TRAIL treated cells. We found that monotreatment with neither MK-0457 nor Apo2L/TRAIL (or their combination) was able to substantially modulate the expression of Mcl-1 or Bim in RPMI 8226; in contrast in HMCL 8226/R5, that showed low sensitivity to Apo2L/TRAIL, the treatment with Apo2L/TRAIL increased the intracellular amount of the anti-apoptotic protein Mcl-1 and MK-0457 reverted Apo2L/TRAIL-induced up-regulation of Mcl-1, thus correlating with the enhanced cytotoxicity of combined treatment. In conclusion, our data indicate that targeting aurora kinase potentiates the apoptotic effect of Apo2L/TRAIL in MM cells with differential sensitivity to Apo2L/TRAIL through the activation of the extrinsic pathway. More important, MK-0457/Apo2L/TRAIL can induce apoptosis in MM cells displaying resistance to bortezomib. Together, these findings suggest that a strategy combining Aurora kinase inhibitors with Apo2L/TRAIL warrants attention in MM.