Combination of Farnesyl Transferase Inhibition (Lonafarnib) and Proteasome Inhibition (Bortezomib) Results in Rapid Caspase Activation and down Regulation of p-AKT in Myeloma cell Lines and Primary Myeloma Cells.

Introduction: Treatment for multiple myeloma (MM) has developed from alkylator based therapies to the use of novel targeted agents. Single agent studies demonstrate activity for agents such as bortezomib and thalidomide, but in order for them to be maximally effective, improved combinations of targeted agents are needed. We experimented with 2 such targeted agents, bortezomib and lonafarnib (SCH66366), with the intent of blocking 2 key signaling pathways and inducing MM cell apoptosis.

Methods: MM.1S, MM.1R, RPMI 8226 and U266 cell lines were used in addition to fresh unmanipulated human cells from patients with relapsed MM. Cell death was assayed using the MTT assay as well as flow cytometry for Annexin V and PI. Western blots and immunoblots were performed to interrogate the effects of therapy on AKT, Bcl-2, Bcl-XL, caspase 3, 8, 9, and other cellular markers of proliferation and death. siRNA was transfected to inhibit AKT1 and AKT2, while overexpression of AKT1 was achieved using cDNA transfection with membrane bound AKT1.

Results: Dose escalation in vitro demonstrated that 8nM was a subtheraputic dose of bortezomib, and 20nM bortezomib was an effective dose, while doses of lonafarnib up to 6uM had modest effects on MM cell death. When both 8nM and 20nM of bortezomib is combined with lonafarnib doses of 6uM, cell death increases significantly. Order of addition may be critical as cell kill is enhanced when bortezomib is administered first followed (3 and 12 hrs later) by lonafarnib. The converse, lonafarnib followed (3 and 12 hrs later) by bortezomib, results in less cell death than when both agents given simultaneously. AnnexinV staining demonstrated that 8nM bortezomib or lonafarnib (5uM) separately resulted in 20% and 10% apoptosis respectively, compared with 88% apoptosis when both agents are given simultaneously. Combination therapy significantly reduces p-AKT (ser 473) levels and STAT 3 as early as 15 hours with no change in total AKT levels, a finding not seen when either agent is given individually. Transfection of 1ug of AKT1 cDNA (constitutively activated) into MM.1R cells did not change the AKT(ser473) phosphorylation, however; transfection of 75ug of siRNA, specific for the AKT1 and AKT2, significantly reduced the AKT(ser473) phosphorylation. There was no attenuation of cell death induced by the combination when 1ug of cDNA was used to overexpress AKT1. The AKT inhibitor LY294002 alone induced apoptosis to a comparable level to bortezomib combined with lonafarnib further supporting p-AKT suppression as the mechanism of action. Downstream targets of AKT show inhibition as well such as a reduction in the phosphorylation of BAD. We have further demonstrated that caspase activation, PARP activation and cleavage, disruption of MDM2 complex, cell-cycle profile, and bcl-2 cleavage are significantly impacted when combination therapy is administered, and not seen with either agent alone. Similar effects on apoptosis induction, caspase activation, and p-AKT down regulation, are seen when human myeloma cells are treated with the combination of bortezomib and lonafarnib in vitro.

Conclusion: The use of combined FTI and proteasome inhibition represents a novel and potentially worthwhile approach to MM therapy and supports the use of these agents together in clinical trials. Further studies are planned to further elucidate the mechanisms of cell death and potential resistance pathways to this combination approach.