Combination of NVP-BEZ235 and Enzastaurin on B-Cell Lymphoma Cell Lines: An Effective Therapeutic Strategy

Abstract 2719

Non-Hodgkin's lymphoma is the most common hematologic neoplasm in adults. Chemotherapy combined with CD-20 monoclonal antibodies has improved survival in both indolent and aggressive B-NHL. However, a substantial subset of patients does not achieve a cure or long disease remission. This has promoted the identification of new targeted treatments and new agents that have shown promising efficacy for future B-NHL therapies. The phosphatidylinositol 3-kinase (PI3K) mammalian target of rapamicin (mTOR) pathway mediates proliferation, survival and drug resistance in lymphoma cells. NVP-BEZ235 (BEZ235) is a new, orally bio available inhibitor of PI3K and mTOR and a representative of a new class of anti-tumour agents. In the current study, the efficacy of the combination of two orally available inhibitor to PI3K/mTOR (BEZ235) and PKCbeta/AKT (enzastaurin) was evaluated in B-cell lymphoma cell lines (RL, WSH-NHL, Jeko and Granta). First, we tested the anti-lymphoma activity of BEZ235 alone and in combination with enzastaurin, everolimus and perifosine. Results using MTT assay were expressed as fraction of cells killed by the individual drug or the combination in the drug-treated versus untreated cells. The interaction between drugs was analyzed by isobologram analysis using the STACorp8.2 software program based upon the Chou-Talalay method to determine if the combination were additive or synergistic. We found that enzastaurin, everolimus and perifosine enhanced the cytotoxicity triggered by BEZ235; a clear synergistic interaction (CI<1) appeared after 48 hours using low concentrations of the all compounds. We examined the functional effects of BEZ235 alone and in combination on apoptosis in lymphoma cells. We demonstrated that BEZ235 (20nM) alone after 24 hours induces an increase of 8–10% of apoptotic cells versus untreated, instead BEZ235 (20nM) in combination with enzastaurin (5microM) after 24 hours induces an increase of 25%. We next defined mechanisms whereby BEZ235 alone and in combination induce apoptosis in lymphoid cells. In particular, BEZ235 combined with enzastaurin induces both intrinsic and extrinsic apoptosis pathways with caspase 3, caspase 9, caspase 8 cleavage. We also showed that the combination of BEZ235 and enzastaurin decreases viability and induce apoptosis in B-cell lymphoma cell lines and peripheral blood mononuclear cells (PBMCs) from lymphoma patients. The combination has no effect on normal PBMCs and suppresses cell prolipheration of B-cell lymphoma cell lines (RL and Jeko) when co-cultured with bone marrow stromal cells in a system that mimics the bone marrow microenvironment. BEZ235, enzastaurin, everolimus and perifosine are inhibitors of intracellular pathways, thought we investigated effects of BEZ235 alone and in combinations with the other compounds in targeting p-AKT, p-mTOR, p-GSK3beta, p-p70, p-p90, p-MAPK, p-4EBP1 and cyclin D1 pathways by Western Blot. In addition, we demonstrated that BEZ235 plus enzastaurin resulted in increased expression of pro-apoptotic Bim, and in decrease expression of anti-apoptotic Bcl-2, which could not be abrogated by BEZ235 alone. In conclusion, our data suggest that in B cell lymphoma cell lines, BEZ235 in combination with enzastaurin elicits its antitumor effect better that combinated with perifosine and everolimus. Our data reveals that the drug combination targets phosphorilation of PI3K/Akt/mTOR pathways and induces both intrinsic and extrinsic apoptosis pathways. Furthermore, inhibition of Bcl-2 anti-apoptosis family members may, in part, explain the efficacy of signalling blockade in lymphoma cells and suggests an additional therapeutic targeting strategy. Therefore, these preclinical data support the potential use of BEZ235 in patients with NHL, and in particular provide rationale for combination with enzastaurin.