Combined Treatment with the Mek Inhibitor PD0325901 and Arsenic Trioxide Has Potent Antitumor Activity in Vivo against Human Multiple Myeloma Xenograft Model.

Despite recent advances in therapy, Multiple myeloma (MM) remains incurable because of the high resistance to apoptosis and both intrinsic and acquired drug resistance. Therefore, new therapeutic strategies are needed to improve patient outcome. We recently demonstrated that blockade of the MEK/ERK signaling module, using the small-molecule inhibitors PD184352 or PD0325901 (PD), strikingly enhances arsenic trioxide (ATO)-induced cytotoxicity in MM cells through a multiple modulation of apoptotic regulatory proteins, including p53 family proteins, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, several Bcl-2 family proteins and caspases, that depend on the functionality of the p53 pathway (Blood prebublished on line june 26, 2008). Furthermore, we also demonstrated that PD plus ATO treatment induces early tumor (volume approximately 200 mm³) regression, prolongs survival and is well tolerated in vivo in a human plasmacytoma xenograft model. The aim of this study was to investigate whether the combined treatment with PD and ATO is effective in animals with more advanced tumors; thus we used a murine model in which MM RPMI 8226 cells were injected subcutaneously into NOD-SCID mice and when the tumors reached approximately 1000mm³, mice were randomized (n=6/group) to receive vehicle or PD0325901 at 10 mg/kg administered by oral gavage or ATO (3.75 mg/kg) injected intraperitoneally or PD/ATO on a 5-days-a-week schedule for 3 consecutive weeks. Treatment of RPMI 8226 MM-advanced tumor-bearing mice with PD0325901 (10mg/kg) significantly reduced MM-tumor growth as compared to control (P <.01 Tukey-Kramer test), ATO (3.75 mg/kg) had minimal effect on the growth of tumors, which increased as in control mice. Importantly, when PD (10mg/kg) was combined with ATO (3.75mg/Kg), there was a significant reduction in tumor size and growth rate relative to untreated or PD treated mice (P<.001 for PD/ATO versus control, and P<.01 for PD/ATO versus PD Tukey-Kramer test). The combination of PD and ATO (3.75mg/Kg) significantly prolonged survival compared with treatment with either drug alone and was well tolerated in vivo because no differences in body weight and general appearance was noted in mice during the treatment. We next investigated the in vivo effects of the drug combination on proliferation and apoptosis; whole tumor-cell tissues and tumor lysates from mice treated for five days (n=2/group) were subjected to immunohistochemical staining and immunoblotting to assess in vivo phosphorylation of ERK, the proliferative antigen, Ki-67, and cleaved caspase-3. Tumor tissues from PD0325901 (10mg/kg) treatments resulted in profound p-ERK inhibition compared with tumor tissues from vehicle control or ATO-treated animals. In agreement with these data, a significant decrement in the number of Ki-67 positive plasma cells was noted in tumor sections from PD-treated mice relative to tumors from mice receiving either vehicle control or ATO (3.75mg/Kg) treatment alone thereby confirming the tumors growth retardation observed in PD-treated mice. Either PD (10mg/kg) or ATO (3.75mg/Kg) alone did not increase caspase activation compared with tumors from control cohorts. However, the combination PD/ATO dramatically activated caspase-3 in advanced tumors. Notably, consistent with our previous in vitro study demonstrating the involvement of the Bim pathway in MM PD/ATO-induced apoptosis, immunoblotting of MM tumors form PD plus ATO-treated mice showed an elevated ratio of proapoptotic Bim to antiapoptotic Mcl-1 compared with treatment with either drug alone. Collectively, our previous and present findings suggest that combining PD with ATO induces both cytostatic and cytotoxic responses in vivo, resulting in regression of early or advanced tumors, prolongs survival in vivo, and is well tolerated in vivo. In conclusion, our preclinical in vivo studies provide the framework for testing PD0325901 and ATO combination therapy in clinical trials aimed to improve patient outcome in MM.