Cell Based Assays Completed with the Mantle Cell Lymphoma Cell Lines Z138 and NCEB-1 Indicate That Combinations of Bortezomid and Flavopiridol Interact To Achieve Synergistic Activity.

Mantle cell lymphoma (MCL) has the poorest long-term survival of all the lymphoma subtypes. CHOP-like regimens currently represent the standard of care for individuals with MCL, but traditional chemotherapeutic regimens do not elicit disease free survival times that are comparable to other sub classes of B-cell lymphomas. Median survival rate of a MCL patient still is approximately 3 years. Therefore new, targeted therapeutics are needed to improve the clinical outcome of MCL. The aim of the study was to identify and characterize combination effects achieved when flavopiridol, a Cyclin D1 inhibitor, and bortezomib, a proteosome inhibitor, are used in a combination setting together or with other agents contained in the CHOP regimen. Two MCL cell lines, Z138 and NCEB-1, were tested for cytotoxicity of drugs alone and in combinations, where alamar blue was used to assess cell viability. The resulting data were analyzed by using the median effect principle introduced by Chou and Talalay. In addition, mechanisms governing measured cytotoxic effects and combination effects were studied by DNA staining (propidium iodine), Caspase 3/7 activation and by western analysis of cyclinD1, NfkB, Bcl-xl, Bax and Bcl-2. The combination of bortezomib and flavopiridol was found to be synergistic in both cell lines studied. Synergistic interactions were dependent on drug-to-drug ratio. In NCEB-1, a ratio 1:12000 (bortezomib:flavopiridol) determined based on the IC₉₀ of each of the individual agents showed synergy over a broad range of fa values (representing the fraction of affected cells). At ratios based on the IC₁₀ and IC₅₀ of the agents when used alone produced effects that were estimated to be antagonistic by the median effect principle. In the Z138 cell line, drug to drug ratios based on the individual agent IC₅₀ and IC₉₀ resulted in synergistic interactions. Consistent with the objective of identifying synergistic combinations, a clear dose reduction was observed to achieve a given therapeutic goal (e.g. 90% cell kill). 10-fold less bortezomib and 55-fold less flavopiridol would be required to achieve 90% cell death/cytostasis based on addition of the agents alone. Mechanistic studies showed that bortezomib in the combination and alone activated the caspase-dependent apoptotic pathway. The combination of bortezomib and flavopiridol for treatment of MCL would appear to an appropriate choice for clinical evaluation, however novel strategies must be developed in order to insure that combination effects observed in cell based screening assays are capture in patients. In this regard it is important to consider drug-drug ratio effects as well as combination engendered toxicities, both parameters that can be evaluated through careful pharmacodynamic assessments in well defined animal models of MCL.