Combination of Bortezomib with Tipifarnib (R115777) Induces Synergistic Myeloma Cell Apoptosis Via Down-Regulation of HDAC6 and Inhibition of Aggresome Formation.

Background: We have previously demonstrated that the combination of bortezomib and a farnesyl transferase inhibitor (FTI) induce synergistic myeloma cell apoptosis, and that maximal cell death is sequence dependent (AACR meeting-2004, ASH meeting -2005, David, E. et.al., Blood, (2005)106: 4322–4329,). The mechanism responsible for this synergy has been poorly understood. As FTI’s are known to downregulate HDAC6 expression, we further evaluated alternative mechanisms responsible for the observed synergy between bortezomib and tipifarnib.

Methods: Myeloma cell lines and primary human myeloma cells were used for the analysis. Confocal microscopy was used to assess ubiquitination in RPMI8226 cells exposed to each drug combination. Western blot analysis and flow cytometry were used to assess phospho-protein expression and apoptosis in MM.1S cell line.

Results: The use of tipifarnib and bortezomib resulted in a time dependent reduction in HDAC6 expression as measured by western blot. Confocal images revealed that bortezomib treatment, both alone and in combination with tipifarnib resulted in an accumulation of ubiquitinated proteins, suggesting a disruption of the aggresome complex. Moreover, a potential sequence-dependent antagonistic affect was observed when tipifarnib was given prior to bortezomib resulting in the reduction of ubiquitination. Additional experiments also demonstrated that apoptosis induced by the combination was only partially inhibited by the pan-caspase inhibitor ZVAD-FMK. This led us to explore caspase independent causes of apoptosis. Inhibition of cathepsins demonstrated overall apoptosis was significantly inhibited, suggesting that both caspase independent and dependent pathways are important in myeloma cell apoptosis induced by this potent combination. Although FTI alone inhibited GO/G1/S phases to some level, only the combination of bortezomib and tipifarnib resulted in the enhanced level of apoptosis which was confirmed by different biochemical methods.

Conclusion: The use of a FTI represents an important and alternative mechanism by which to inhibit HDAC6 and thus the aggresome pathway of protein degradation. Given that combinations of bortezomib and aggresome inhibition have been shown to be very active in vitro and in animal models, this approach represents an attractive method by which to target both critical pathways of protein degradation within malignant cells.