Combined Targeting of the Proteasome and Bcl-2 Sensitizes Human B-Cell Lymphoma and Multiple Myeloma to Cyclophosphamide in In Vitro and In Vivo Models of These Diseases.

The balance of apoptotic proteins is a critical mechanism used by the cell to maintain the mitochondrial transmembrane potential, which plays a critical role in governing apoptosis. Bortezomib (B) is a proteasome inhibitor that selectively inhibits the 26S proteasome, leading to an increase in many intracellular proteins, including proteins that lead to inhibition of NF-kB, as well as proapoptotic proteins like bik and Bax. Oblimersen (Obl) is a 18 mer oligonucleotide results in decreased transcription of the antiapoptotic protein bcl-2. We hypothesized that the mitochondrial membrane potential could be influenced by the co-administration of these agents to favor aopotosis. We report results of pre-clinical in vitro and in vivo experiments using these agents in combination in various lymphoma (NHL) & myeloma (MM) models. NHL & MM cell lines were exposed to Obl in the presence of cationic lipid and incubated for 48 hours. DNA synthesis & cell growth arrest was quantified by 3H-thymidine incorporation assays at 72 hrs, & revealed a concentration dependent cell killing. Induction of apoptosis in the presence or absence of different concentrations of B was detected by multiparameter flow cytometric analysis. Data reveals addition of bortezomib, even at relatively low concentrations (10 nM), additively improved the effects of Obl in all the cell lines studied. A SCID beige model of aggressive large B-cell lymphoma was used by injecting 1x10e7 cells (SKI-DLCL-1 or RL, a cell line over expressing bcl-2 through the 14:18 translocation) as a subcutaneous flank tumor. In over 6 in vivo experiments, the combination of B (0.5 mg/kg days 3 & 7) + Obl (3 mg/kg days 1,3,5, &7) consistently resulted in about 50% inhibition of growth of both SKI-DLCL-1 & RL compared to that in the control. Surprisingly, results were evident even using a low dose schedule of bortezomib (about one quarter of the MTD), suggesting the possibility of a small sensitizing dose of this drug. In all experiments, growth delay seen with the doublet exceeded the results noted with any singlet (B or Obl alone). Integration of cyclophosphamide at a dose of 50 mg/kg produced marked tumor shrinkage that appeared synergistic in several circumstances. Single agent CTX produced excellent growth delay (similar to any doublet), but in all these cases, tumor re-growth was evident by 21–25 days post tumor transplant. CTX dose & schedule was also important. Same day ‘lump and dump’ of all drugs failed to effect cured, while CTX preceding B consistently produced best results, with cures found only in this triplet cohort. Results suggest that optimal scheduling of agents requires a ‘lead in’ exposure of Obl, followed by CTX preceding all doses of bortezomib. These studies support the efficacy of this regimen.