THE COMBINATION OF MEK INHIBITORS AND ARSENIC TRIOXIDE TRIGGERS IN VITRO AND IN VIVO SYNERGISTIC CYTOTOXICITY IN MULTIPLE MYELOMA THROUGH MULTIPLE SIGNALING PATHWAYS

We here demonstrate that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD0184352 or PD0325901 (PD) (Pfizer), strikingly enhances arsenic trioxide (ATO)-induced cytotoxicity in multiple myeloma (MM) cells through a caspase-dependent mechanism. In vitro, the combination PD/ATO shows a moderate inhibitory effect on normal hematopoiesis and has a minimal effect on normal bone marrow B cells. Co-treatment with PD greatly enhances the ATOinduced p53 accumulation and p73, a p53 paralogue, cooperates with p53 in caspase activation and apoptosis induction in MM cells that retain a functional p53 pathway. Co-treatment with PD strikingly elevates the (DR4+DR5)/(DcR1+DcR2) tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors ratio and caspase-8 activation of ATO-treated MM cells that do not have a functional p53 pathway. Bim interacts with DR4 and DR5 TRAIL receptors and contributes to the PD/ATO-mediated activation of the extrinsic pathway. Furthermore, in MM cells carrying or not a functional p53 the combined treatment increases the level of the pro-apoptotic Bim (PD-mediated) and decreases its neutralizing anti-apoptotic protein Mcl-1 (ATO-mediated) and loss of Bim interferes with both the extrinsic and intrinsic apoptotic pathways activation of PD/ATO-treated cells. Studies in a human plasmocitoma xenograft model show that combination of PD (10 mg/kg) and ATO (2,5 mg/kg) is well tolerated, triggers a dramatic reduction (95- 98%) in tumor growth relative to untreated mice. Immunohistochemistry and western blot analysis of tumors from treated mice demonstrate that the potent anti-MM activity of PD plus ATO in vivo is due to either decreased proliferation (decrement in the number of Ki-67 positive plasma cells and ERK inhibition) and increased apoptosis (Caspase3 activation). Survival is significantly prolonged in PD/ATO-treated animals versus either treatment alone (mean survival time of PD/ATO 2.5 mg/kg: 74 days; PD: 41 days, p=0.003 for PD/ATO versus PD; ATO 5 mg/kg: 36 days, p=0.001 for PD/ATO versus ATO 5mg/kg; ATO 2.5mg/kg: 27 days, p=0.001 for PD/ATO versus ATO 2.5 mg/kg; untreated: 28 days, p=0.001 for PD/ATO versus untreated). Notably, after 21 days of PD/ATO treatment mice show only moderate bone marrow hypoplasia with respect to untreated animals. These preclinical in vitro and in vivo studies provide the framework for clinical trials of PD0325901 with ATO to improve patients outcome in MM.