Effects of Isoprenoid Biosynthetic Pathway Inhibition on Glucose Uptake in Myeloma Cells

Abstract

Multiple myeloma (MM) is a plasma cell malignancy characterized by the synthesis and secretion of high levels of monoclonal protein (MP). MM cells are dependent on glucose consumption and GLUT transporter activity and there has been interest in targeting glucose uptake as a therapeutic strategy. Our work has shown that inhibitors of the isoprenoid biosynthetic pathway (IBP) disrupt Rab GTPase geranylgeranylation thus impairing MP trafficking in MM cells, leading to an accumulation of MP in the ER and induction of the unfolded protein response and apoptosis. However, there are over 60 different Rab GTPases in mammalian cells involved in regulating a wide range of membrane trafficking events including exocytic, endocytic and lysosomal pathways, whose disruption may also be contributing to MM cell death. Global gene expression analysis of MM cells treated with the Rab geranylgeranyl transferase (GGTase) inhibitor, 3-PEHPC, revealed novel pathways impacted by Rab GTPase inhibition, including genes involved in glucose metabolism. As previous work with statins have suggested that statin-induced inhibition of glucose uptake may be a consequence of either blocking protein geranylgeranylation or cholesterol depletion, we were interested in determining whether disruption of isoprenoid synthesis and/or Rab geranylgeranylation has an impact on glucose uptake in MM cells. Notably, MM cells are unique in their dependence on additional members of the GLUT family including GLUT4 whose cell surface expression is regulated by Rabs. We therefore examined the effects of IBP inhibitors and direct prenyl transferase inhibitors on glucose uptake in MM cells (RPMI-8226, U266 and MM.1S) using flow cytometric analysis of the uptake of 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose), a fluorescent analog of D-glucose. The IBP inhibitors lovastatin (an inhibitor of HMG-CoA reductase) and digeranyl bisphosphonate (DGBP) (an inhibitor of geranylgeranyl diphosphate synthase) reduced glucose uptake in a concentration- and time-dependent manner in all cell lines (except U266 which we have previously shown to be resistant to DGBP) by 20-50% compared to untreated control cells. To determine whether the effects of these drugs on glucose uptake are a consequence of depletion of a specific isoprenoid species, experiments were performed in which cells were incubated in the presence or absence of the IBP inhibitors and intermediates in the IBP, namely mevalonate, FPP or GGPP. Co-incubation of lovastatin with mevalonate or GGPP completely prevented the effects of lovastatin on glucose uptake in MM.1S and U266 cells while GGPP prevented the block in glucose uptake induced by DGBP in MM.1S cells. However, co-incubation of lovastatin with FPP partially prevented the effects of lovastatin in U266 cells but had no effect in MM.1S cells. Treatment of U266 cells, but not RPMI-8226 or MM.1S cells, with the squalene synthase inhibitor zaragozic acid resulted in a significant decrease in glucose uptake. MM.1S and U266 cell lines were treated with individual prenyltransferase inhibitors (FTI-277, GGTI-2133 or 3-PEHPC) to distinguish between the effects of altering cellular isoprenoid levels versus directly protein prenylation. Whereas none of the prenyltransferase inhibitors had significant effects on the U266 cells, only inhibition of Rab GGTase by 3-PEHPC resulted in a significant decrease in glucose uptake in MM.1S cells. In aggregate these studies demonstrate that Rab inhibition via disruption of geranylgeranylation diminishes glucose uptake in MM cells. In addition, a cholesterol-dependent effect related to IBP inhibition was observed in the U266 cells but not in the other tested MM cells, suggesting that MM cells may vary in their dependence on glucose uptake by distinct GLUT transporters. Future studies will investigate these findings in primary MM cells and identify the specific GLUT family members which are affected by direct Rab GGTase inhibitors as well as by IBP inhibitors. Improved understanding of the mechanisms underlying the effects of these inhibitors will inform further development of therapies targeting glucose uptake in MM. DisclosuresHolstein:Amgen: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.