Potential mechanisms for the anti-myeloma properties of bisphosphonates

6586 Background: For cell membrane association, small GTPases such as Ras, Rap1A, Rab, and Rho must undergo post-translational isoprenylation modification. Isoprenoid intermediates of the mevalonate pathway, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), are substrates for farnesylation or geranylgeranylation modifications, respectively. It is reported that nitrogen-containing bisphosphonates (BPs), such as zoledronic acid (ZOL), inhibit isoprenylation through inhibition of FPP synthase, the enzyme that catalyzes the synthesis of FPP. A more significant target could be GGPP synthase, the enzyme that catalyzes the synthesis of GGPP. This study was designed to more precisely identify mevalonate pathway targets of BPs. Methods: Human-derived myeloma cell lines, RPMI-8226 and ARH-77 were incubated for 24 hours with either 50μM of ZOL, or 50μM LWS-138, a novel non-nitrogen containing BP found to be a selective GGPP synthase inhibitor. Levels of FPP and GGPP were measured using methods recently reported (Tong, H. et al. Analytical Biochemistry, 336:51, 2005). Isoprenylation status of Rap1A and Rab6 were analyzed by Western blot. Results: In RPMI-8226 cells, baseline levels for FPP and GGPP were 0.115+/- 0.009 pmol/ 106 cells and 0.270 +/- 0.010 pmol/106 cells, respectively. In ARH-77 cells these values were 0.144 +/- 0.009 pmol FPP/ 106 cells and 0.250 pmol GGPP/ 106 cells. When treated with ZOL, GGPP levels decreased by 54%, while FPP levels increased by 297%. When treated with LWS-138 GGPP levels decreased by 95%, while FPP levels increased by 121%. Both ZOL and LWS-138 impaired isoprenylation of Rap1A and Rab6. The addition of 10μM GGPP, but not 10μM FPP restored isoprenylation of these proteins. Conclusions: GGPP synthase is a more likely target of inhibition by BPs in human myeloma cell lines than FPP synthase. Increased FPP levels after treatment with ZOL suggest that ZOL may have multiple sites of activity on the isoprenoid pathway, while LWS-138 is specific for GGPP synthase. No significant financial relationships to disclose.