Enhanced Levels of Apoptosis by Combination of Farnesyl Transferase Inhibition (Tipifarnib) and Proteasome Inhibition (Bortezomib) in Myeloma Cell Lines and Primary Myeloma Cells and Its Mechanistic Effects on Akt and Caspase Pathways.

Introduction:

Despite significant advances in the treatment of myeloma, patients invariably become resistant to therapy. Therefore, novel treatment strategies are needed to overcome resistance. Overexpression of the anti-apoptotic protein Akt has been associated with resistance to bortezomib induced apoptosis. We have previously shown that treatment with farnesyl transferase inhibitors (FTI) is associated with synergisitic myeloma cell apoptosis when combined with bortezomib. In this study we explored the mechanism of action of the combination of bortezomib with tipifarnib, a FTI with previously demonstrated clinical activity in patients with hematologic malignancies. Our hypothesis is that the combination of bortezomib and tipifarnib will result in synergistic cell death by overcoming the anti-apoptotic effects of Akt.

Methods:

MM.1S, MM.1R, RPMI8226 and U266 cell lines were used in addition to fresh unmanipulated human myeloma cells from patients with relapsed MM. Cell proliferation was measured using the MTT assay. Cell death was measured by flow cytometric analysis of Annexin V and propidium iodide staining in the presence or absence of both agents and the broad spectrum caspase inhibitor Z-VAD-FMK (ZVAD). Caspase activity was assessed by Western blot ananlysis of cleaved caspases. Transient transfection of cell lines of using activated Akt, wild type Akt and BCL2 was also performed.

Results:

Dose escalation in vitro demonstrated that 8nM was a subtherapeutic dose of bortezomib, and 20nm bortezomib was an effective dose as a single agent. Doses of tipifarnib alone up to 5μM had modest effects on MM cell death. When 8nM or 20nM of bortezomib are combined with tipifarnib at doses of 5μM, cell death increases significantly in MM cell lines. Combination resulted in increased caspase 3, 8, and 9 activities in MM cell lines. Inhibition of caspase activities were confirmed with the broad spectrum caspase inhibitor ZVAD. Individual caspase inhibitor studies after 18 hours of combination treatment suggested that the inhibition of apoptosis is mainly mediated through caspase 8 and caspase 6 as measured by Annexin-V staining in MM.1S cells. Additionally, similar studies with the pan-caspase inhibitor ZVAD also suggested that there are caspase independent pathways resulting in inducing apoptosis of MM.1S cells. Combination therapy significantly reduces phos-Akt as early as 24hrs in MM cells, although, complete inhibition of phos-473-Akt varies between cell lines. Overexpression of activated Akt or wild type Akt and the anti-apoptotic protein Bcl2 in MM.1S did not abrogate the effect of combination on apoptosis. Primary human MM cells also demonstrated synergistic cell death when exposed to the combination at clinically achievable levels.

Conclusion:

The combination of tipifarnib with bortezomib is associated with greater cell death than either agent alone in both myeloma cell lines and patient myeloma cells. Therefore, we propose that the use of combined tipifarnib and bortezomib represents a novel and potentially active approach to MM therapy. The synergistic mechanism involved in the combination warrants further investigation.