Targeting Rab Geranylgeranylation Disrupts Myeloma-Stromal Cell Interactions

Abstract 1354

Multiple myeloma is highly dependent on the interaction of the malignant plasma cells with the bone marrow environment. In particular, adhesion to bone marrow stromal cells stimulates myeloma cell proliferation and contributes to drug resistance. New therapeutic strategies which target the microenvironment that harbors myeloma are needed. We have previously demonstrated that isoprenoid biosynthetic pathway (IBP) and prenyltransferase inhibitors cause inhibitory effects in myeloma cells, particularly those agents which disrupt Rab geranylgeranylation. Our prior studies focused on the effects of these agents in disrupting monoclonal protein secretion and inducing the unfolded protein response. Whether disruption of Rab geranylgeranylation affects other key cellular processes in myeloma cells or the bone marrow microenvironment remains to be determined. In this study, we examined the effects of IBP and prenyltransferase inhibitors on several key aspects of myeloma-stromal cell interactions, including adhesion, proliferation, and cytokine secretion. The specific inhibitors utilized included lovastatin (HMG-CoA reductase inhibitor), zoledronic acid (farnesyl pyrophosphate (FPP) synthase inhibitor), digeranyl bisphosphonate (DGBP) (geranylgeranyl pyrophosphate (GGPP) synthase inhibitor), FTI-277 (farnesyl transferase (FTase) inhibitor), GGTI-2133 (geranylgeranyl transferase (GGTase) I inhibitor) and 3-PEHPC (GGTase II inhibitor). Studies were performed with the HS-5 human bone marrow stromal cell line as well as the RPMI-8226 and U266 human myeloma cell lines. MTT cytotoxicity assays demonstrated that the stromal cells are more sensitive to agents which deplete cells of isoprenoids than to direct prenyltransferase inhibitors. Co-incubation studies utilizing the isoprenoid intermediates mevalonate, FPP, and GGGP, revealed that depletion of GGPP, but not FPP, is responsible for IBP inhibitor-induced cytotoxicity in HS-5 stromal cells. Disruption of protein prenylation at the relevant drug concentrations was confirmed via immunoblot analysis of cytosolic and membranous fractions obtained from Triton X-114 lysis of HS-5 cells. Pre-treatment of U266 cells with lovastatin or 3-PEHPC for 24 hours significantly decreased the adhesion of those cells to either the HS-5 stromal cell layer or to fibronectin. To determine whether IBP or prenyltransferase inhibitors disrupt secretion of cytokines from the HS-5 cells, IL-6 ELISA studies were performed. Treatment of HS-5 cells for 24 hours with concentrations of IBP inhibitors that induce less than a 20% decrease in cell viability resulted in a decrease in IL-6 secretion by 20–40%. Of note, 3-PEHPC, but not the FTase or GGTase I inhibitors, significantly decreased IL-6 secretion. The sensitivity of RPMI-8226 and U266 cells to lovastatin or 3-PEHPC was not significantly altered when co-cultured with HS-5 cells as determined by a BrdU incorporation assay. In conclusion, these studies are the first to provide evidence for the role of Rab proteins in regulating myeloma cell adhesion and to demonstrate that disruption of Rab geranylgeranylation results in diminished stromal cell cytokine secretion. Further studies are required to determine the identity of the specific Rabs mediating these effects and the underlying mechanisms. This work supports the further investigation into agents which inhibit Rab geranylgeranylation as a novel strategy with which to target myeloma cells and the bone marrow microenvironment.