Everolimus Shows Synergistic Anti-Myeloma Effects with Bortezomib Via the AKT/mTOR Pathway

Introduction: Multiple myeloma (MM) is characterized by the proliferation of malignant plasma cells and a subsequent overabundance of monoclonal paraprotein (M protein). Everolimus ,as an oral mammalian target of rapamycin (mTOR) inhibitor, plays an important role in multiple myeloma. In the phase I trial explored the use of everolimus in relapsed and/or refractory multiple myeloma patients ,the median time to progression was 90 days (range, 13 to 278 days),and no dose-limiting toxicity was observed at daily dose of 10 mg. At the same time, the observed responses are promising in some testing combination strategies. The pre-clinical activity of pan-histone deacetylase inhibitor LBH589 in combination with everolimus caused potent G0/G1 arrest and induced pronounced apoptosis, the combination led to down regulation of pStat3, cyclins, CDKs and XIAP and up regulation of pro-apoptotic Bcl-2 family proteins. Everolimus enhances the cytotoxicity of bendamustine in multiple myeloma cells through a network of pro-apoptotic and cell-cycle-progression regulatory proteins.Down regulation of vascular endothelial cell growth factor and everolimus synergistic antitumor activity in multiple myeloma cells. Furthermore, In a phase I study of everolimus in combination with lenalidomide in relapsed or refractory multiple myeloma patients, the overall response rate was 65% and median progression-free survival was 5.5 months. Bortezomib was the first therapeutic proteasome inhibitor to be tested in humans with MM. However, the combination of bortezomib and everolimus for the treatment of MM is still less reported.

Methods: To evaluate the in vitro and in vivo anti-tumor activity of the combination of everolimus with bortezomib.CCK-8 was used to explore the proliferation of MM cells. Flow cytometry was used to determine the effect on apoptosis of cells.Western blot and Immunohistochemical staining was used to determine the epression of proteins.

Results: In our study, the experimental results demonstrated that everolimus combined with bortezomib suppressed cell proliferation. Treatment with 20 nM everolimus in combination with 10 nM bortezomib for 72h achieved the lowest cell survival rate in both U266 and LP-1 MM cell lines. The combination of everolimus and bortezomib was synergistic in both cell lines indicated by combination index (CI) values of <0.8.Flow cytometry was used to determine the effect on apoptosis of bortezomib in combination with everolimus. everolimus alone and bortezomib alone induced approximately 22.5% and 17.2% apoptosis, combination treatment induced28.8% apoptosis in U266 cells, and the results were 15.6%,10.0% and 16.7% in LP-1 cells. Western blot was used to explore the mechanisms. The expression of AKT1, mTOR, p70S6K1, 4EBP1 and NF-kB was significantly down regulated while the expression of Caspase-3was significantly up regulated in combination group than other alone group. Furthermore, Synergistic antitumor effect of everolimus and bortezomib was observed in the animal model carrying the myeloma xenograft on the 25th day after tumor implantation . The results of immunohistochemical staining further demonstrated that the expression of AKT1, mTOR, p70S6K1, 4EBP1 and NF-kB was significantly down regulated and the expression of Caspase-3 was significantly up regulated in combination group.

Conclusion:Our results shows synergistic antitumor effects of the combination therapy of bortezomib and everolimus induced myeloma cell apoptosis in vitro and in vivo xenograft mouse model ,which mainly through the inhibition of the AKT/mTOR pathway. Our results provided provide a rationale application for the combinatorial treatment of MM with proteasome inhibitor agents and mTOR inhibitors in future clinical practice.