Interaction Between Heat Shock Protein Inhibitors and Isoprenoid Biosynthetic Pathway Inhibitors In Multiple Myeloma Cells

Abstract 2895

Multiple myeloma (MM) is characterized by the production of substantial quantities of monoclonal protein (MP) by malignant plasma cells. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of MM cells via inhibition of Rab geranylgeranylation, leading to disruption of MP trafficking and induction of the unfolded protein response (UPR) pathway. Inhibitors of the molecular chaperone heat shock protein 90 (HSP90) have also been shown to induce components of the UPR and apoptosis in MM cells and are currently under clinical investigation. Given the sensitivity of MM cells to stressors which activate the UPR, we hypothesized that combining agents that disrupt protein folding (HSP90 inhibitors) with agents that disrupt protein trafficking (IBP inhibitors) would yield enhanced cytotoxicity via their effects on MP levels and the UPR. We therefore evaluated the effects of combining IPB and HSP90 inhibitors in MM, Waldenstrom macroglobulinemia (WM), and IgM-secreting cells. Studies were performed in 2 lambda light chain-secreting human MM cell lines (RPMI-8226, U266), a WM cell line (WM-WSU), and the IgM-secreting RL lymphoblastoid cell line. IBP inhibitors included lovastatin (Lov) (HMG-CoA reductase inhibitor), digeranyl bisphosphonate (DGBP) (geranylgeranyl synthase inhibitor), and 3-PEHPC (geranylgeranyl transferase II inhibitor), while 17-AAG and 17-DMAG were used as HSP90 inhibitors. The combination of Lov and 17-AAG resulted in enhanced cytotoxicity in all tested cell lines at both 24 & 48 h as determined by MTT assays. Isobologram analysis revealed a synergistic interaction for the RPMI-8226, RL, and WM cells and an additive interaction for the U266 cells. Pre-treatment with Lov for 24 h prior to the addition of 17-AAG resulted in a synergistic interaction in all tested cell lines. The enhanced cytotoxicity was prevented by co-incubation with either mevalonate or geranylgeranyl pyrophosphate. Pre-treatment with DGBP yielded a synergistic interaction between DGBP and 17-AAG in the RPMI-8226 cell line. The addition of 3-PEHPC to either 17-AAG or 17-DMAG resulted in a synergistic interaction in the RPMI-8226 cells. Treatment with 17-AAG and 17-DMAG led to a concentration-dependent decrease in secreted lambda light chain (RPMI-8226, U266) or IgM (RL) levels after 24 h as measured with ELISA. As we have previously demonstrated, Lov decreased secreted MP levels. The combination of Lov and the HSP90 inhibitors resulted in a further decrease in secreted MP. Interestingly, 17-AAG and 17-DMAG decreased intracellular lambda light chain levels in the RPMI-8226 & U266 lines while increasing intracellular IgM levels in the RL line. The increase in l light chain induced by Lov in the MM lines was partially prevented by co-incubation with either of the HSP90 inhibitors. The increase in HSP70 levels induced by HSP90 inhibition was not significantly altered by co-incubation with Lov; however, the accumulation of unmodified Rap1a (a marker for inhibition of geranylgeranylation) induced by Lov was diminished in the presence of the HSP90 inhibitors. Pre-treatment with Lov prior to the addition of 17-AAG resulted in enhanced apoptosis, as determined by PARP and calnexin cleavage, in the MM cells while significant changes were not observed following 24 h concurrent treatment. As a measure of induction of the UPR, levels of phosphorylated eIF2a (p-eIF2a) were assessed. While no significant changes were noted following 24 h concurrent treatment, pre-treatment with Lov did yield an increase in p-eIF2a levels compared with 17-AAG alone. In conclusion, these studies are the first to demonstrate synergistic cytotoxic interactions between select IBP inhibitors and HSP90 inhibitors in MM cells and to evaluate the effects of these agents on MP levels. That the effects of these agents on components of the UPR and apoptosis are most evident following pre-incubation with Lov suggests that inhibition of prenylation is required for the interaction. Further studies will be required to determine whether the observed decrease in intracellular and secreted light chain levels induced by the HSP90 inhibitors is a consequence of increased protein degradation or decreased protein synthesis. These studies form the basis for further pre-clinical and clinical investigation into the use of this novel combination in MM and related MP-secreting disorders.