New treatment strategies for multiple myeloma by targeting Bcl-2 and the mevalonate pathway

Abstract

Multiple myeloma is a B-lineage neoplasia. Enhanced proliferation and defects in the regulation of programmed cell death account for the expansion of the malignant clone. Emergence of drug resistance is the primary cause of treatment failure in myeloma. Various studies have indicated that inhibition of drug-induced apoptosis is an important mechanism of drug resistance in various human cancers including myeloma. Understanding of mechanisms that regulate apoptosis and growth will allow for the development of new biologically-based therapies that overcome drug resistance and reduce myeloma tumor growth. This thesis has focused on the role of Bcl-2 and the mevalonate pathway in the maintenance of myeloma tumor cell survival and resistance to drug-induced apoptosis.

Bcl-2 in myeloma In myeloma, the role of Bcl-2 is unclear. The importance of Bcl-2 in drug-induced apoptosis in myeloma was investigated by using an antisense approach. G3139 is a oligodeoxynucleotide (ODN) complementary to the first 6 codons of the Bcl-2 mRNA. Treatment of ex vivo purified myeloma tumor cells with Bcl-2 antisense ODNs resulted in a sequence-specific reduction of Bcl-2 mRNA, followed by a sequence-specific decrease in Bcl-2 protein levels. Down-regulation of Bcl-2 resulted in enhanced sensitivity of both chemosensitive and chemoresistant myeloma tumor cells to dexamethasone or doxorubicin-induced apoptosis. Based on these in vitro findings a clinical trial was started to evaluate the effect of Bcl-2 antisense in 10 myeloma patients that were heavily pretreated. G3139 was administered for 7 days as a continuous intravenous infusion and VAD chemotherapy was started at day 4. Administration of Bcl-2 antisense resulted in a moderate reduction of Bcl-2 protein levels in peripheral blood circulating myeloma cells. Four (40%) patients achieved a partial response and 3 (30%) a minor response, indicating that 70% of the patients responded to the combination of G3139 and VAD. Five out 7 responding patients were refractory to VAD chemotherapy alone. The responses observed in our trial provide the proof of principle that Bcl-2 protein is involved in drug resistance in vivo and that inhibition of Bcl-2 expression increases the in vivo therapeutic efficacy of cytotoxic agents in myeloma.

Mevalonate pathway in myeloma The mevalonate pathway produces a variety of products including farnesyl and geranylgeranyl isoprenoids and cholesterol. In the second part of this thesis, we focused on the importance of the mevalonate pathway in myeloma. Blocking of the mevalonate pathway through the inhibition of HMG-CoA reductase by lovastatin induced apoptosis and inhibited proliferation of myeloma and lymphoma cells, moreover lovastatin also sensitized these cells to cytotoxic agents. These effects were due to the inhibition of geranylgeranylation through the depletion of geranylgeranylpyrophosphate (GGPP) by lovastatin, whereas depletion of farnesylpyrophosphate (FPP) leading to inhibition of farnesylation had no or only minor effects. The role of protein geranylgeranylation in the regulation of myeloma cell growth and survival was confirmed in experiments with specific inhibitors of geranylgeranyl transferase I (GGTase I) and farnesyl transferase (FTase). Inhibition of GGTase I activity reduced proliferation and induced apoptosis, with only small effects of inhibition of FTase. Inhibition of geranylgeranylation either by depletion of GGPP or inhibition of GGTase I activity reduced expression of the anti-apoptotic protein Mcl-1, which is a key regulator of survival in myeloma. Studies using specific inhibitors of geranylgeranylated proteins suggested that Rac-1 and/or Cdc42 GTPases play a role in myeloma cell growth, but not survival. Introduction of dominant-negative Tat-Rac-1 protein reduced proliferation, whereas constitutively active Tat-Rac-1 stimulated proliferation of myeloma cells. Both dominant-negative and constitutively active Rac-1 had no effect on myeloma cell survival. These data suggest that Rac-1 plays a role in myeloma cell proliferation, and that the suppression of proliferation by lovastatin and GGTase I inhibitors may be, at least in part, due to inhibition of geranylgeranylation of Rac-1. These preclinical studies indicate that inhibition of geranylgeranylation represents a novel therapeutic approach in the treatment of myeloma and lymphoma.