The Combination of the mTOR Inhibitor Rapamycin and Proteasome Inhibitor Bortezomib Is Synergistic In Vitro in Multiple Myeloma.
Authors: O’Sullivan Garrett , Leleu Xavier , Jia Xiaoying , Hatjiharisi Evdoxia , Ngo Hai , Moreau Anne-Sophie , Tai Yu-Tsu , McMillin Douglas , Mitsiades Constantine , Raje Noopur , Hideshima Teru , Schlossman Robert , Richardson Paul , Munshi Nikhil , Anderson Kenneth , Ghobrial Irene 
Published: October 2019

Abstract
Background: The PI3K and NFk-B/proteasome pathways are major regulators of survival in Multiple Myeloma (MM). Previous studies have demonstrated clinical efficacy of bortezomib in MM; however, not all patients responded to this agent. mTOR inhibitors have demonstrated significant in vitro and in vivo activity in MM, specifically clinical trials with the mTOR inhibitor CCI-779 (Wyeth) in MM. Therefore, we examined whether inhibition of the PI3K pathway by mTOR and proteasome inhibitors may lead to synergistic activity in MM.
Methods: MM cell lines (MM.1S, RPMI, U266, OPM2) were treated with rapamycin 1–5nM (Sigma Aldrich), bortezomib 2.5–10nM (Millenium, MA), or the combination. Cytotoxicity was measured by the MTT assay at 48 hrs; DNA synthesis was measured using thymidine uptake assay; apoptosis was studied using Apo2.7 by flow cytometry, and cell cycle regulation was determined using flow cytometry. To determine whether these agents can overcome the growth advantage conferred by bone marrow stromal cells (BMSCs), we co-cultured cell lines with stromal cells. Normal peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers. Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method, with synergistic activity determined as a combination index (CI) of <1.0.
Results: Rapamycin induced dose-dependent cytotoxicity from 0.1nM to 1nM, with an IC50 of 5nM in MM.1S and OPM2. Interestingly, higher doses did not induce further cytotoxicity, confirming that low doses of rapamycin are as effective as higher doses. RPMI and U266 MM cell lines were less sensitive to rapamycin, with 5nM inducing 40% and 20% decrease in survival, respectively. Bortezomib induced significant inhibition of survival in all MM cell lines with an IC50 of 2.5nM, as previously reported. The combination of agents induced significant inhibition of proliferation as compared to each agent alone, specifically with the combination of 5nM rapamycin with 5nM of bortezomib. In the DNA synthesis assay, the combination of bortezomib and rapamycin was significantly cytotoxic compared to each agent alone, specifically at the dose of 5nM rapamycin and bortezomib 2.5nM. The combination of rapamycin 1 to 5 nM and bortezomib 5 to 10 nM were synergistic with a CI index less than 1.0, as in RPMI (CI=0.4) and U266 (CI=0.2) cell lines. The combination of rapamycin and bortezomib at serial concentrations did not trigger cytotoxicity in PBMCs from normal volunteers, indicating significant cytotoxicity in malignant cells, with lack of toxicity in normal PBMCs and suggesting a therapeutic index. The combination of bortezomib and rapamycin demonstrated a significant inhibitory effect on the growth of MM cell lines even in coculture with stromal cells. Cell cycle analysis demonstrated G1 arrest at 24 and 48 hrs in MM.1S cells. Similar results were obtained using primary CD138+ myeloma cells from patients.
Conclusion: The combination of rapamycin and bortezomib resulted in synergistic in vitro cytotoxicity in MM cells. These results provide the framework for clinical trials evaluating the combination of CCI-779 and bortezomib in MM.