Background: Disruption of Ras-to-MAP kinase (MAPK/ERK) signaling has been implicated in the molecular pathogenesis of multiple myeloma. Inhibitors of Ras posttranslational modification (FTase, REPase and PPMTase inhibitors), Raf and MEK are promising, novel types of nontoxic cancer therapeutics. Recently, it was also reported that lovastatin can inhibit the prenylation and thus activation of Ras proteins. In order to assess the effects of farnesyl transferase inhibitors (FTIs), geranylgeranyl transferase inhibitors (GGTIs), MEK inhibitors and statins in myeloma cells, six multiple myeloma cell lines were subjected to a panel of these inhibitors and assayed for viability, colony formation, cell cycle progression, MAP kinase kinase (MEK-1/2) activation and induction of apoptosis.
a) Activation of the Ras-to-MAPK pathway in multiple myeloma cell lines. Activation of the Ras-to-MAPK pathway was determined by kinase assays and Western blotting. Kinase assays and immunoblotting revealed that myeloma cell lines that harbor Ras mutations (e.g. L-363, NCI-H929, OPM-2) had higher MAPK activity versus cell lines which have wild-type Ras (e.g. LP-1, U266).
b) Activation of the Ras-to-MAPK pathway during cell cycle progression was quantified by a phospho-MEK specific FACS assay. Multiple myeloma cell lines which harbor activating K-Ras (OPM-2, RPMI-8226) and N-Ras (L-363, NCI-H929) mutations were found to have higher amounts of cell-cycle-dependent phospho-MEK in G0/G1 and G2/M as compared to cell lines with wild-type Ras (LP-1, U266). Interestingly, highest amounts of phospho-MEK were observed in OPM-2 and NCI-H929 which also express high levels of mutationally activated (K650M) and wild-type fibroblast growth factor receptor-3, respectively.
c) Inhibition of growth of multiple myeloma cell lines. In colony forming assays, co-treatment of RPMI-8226 cells with FTI L-744,832 and the MEK inhibitor U0126 led to synergistic growth inhibition. FTI alone had an IC50 of 0.27 µM, U0126 had an IC50 of 0.17 µM and the combination of both compounds decreased the IC50s to 0.06 µM. Lovastatin and FTI L-744,832 also synergistically inhibited growth of myeloma cell lines as quantified by cell proliferation assays (MTT). Co-treatment of RPMI-8226 cells for 96 hours with FTI L-744,832 (IC50 29.5 µM) and lovastatin (IC50 3.2 µM) resulted in overall lower IC50s (1.1 µM). Growth inhibition was also synergistic in OPM-2 cells, with IC50s of 1.6 µM for L-744,832, 4.4 µM for lovastatin and 0.6 µM for the combination of the two drugs. Growth inhibition of the multiple myeloma cell line L-363 was also found to be synergistic upon co-treatment with L-744,832 (IC50 29.8 µM) and lovastatin (IC50 11.9 µM) as the combination resulted in a lower IC50 (2.2 µM). Synergistic drug interactions were calculated using the CalcuSyn program.
d) Induction of apoptosis by lovastatin. Co-treatment of the multiple myeloma cell line OPM-2 for 48 hours with FTI L-744,832 and lovastatin led to increased amounts of apoptotic cells as detected by the TUNEL assay (19.1% versus 0.3 % and 3.3 %, respectively). However, at 72 hours, co-treatment elicited no appreciable change in the amount of apoptosis induced by lovastatin alone (73.6 % versus 71.9 %). Incubation of the multiple myeloma cell line NCI-H929 for 72 hours with 5 µM lovastatin also caused considerable apoptosis (>90 %) as measured by the TUNEL assay.
e) Inhibition of Ras processing by FTIs and lovastatin. Ras processing was monitored by Western blotting. Inhibition of H- and N-Ras processing was observed in myeloma cell lines treated with FTI L-744,832 alone and in combination with GGTIs. Treatment of myeloma cell lines with lovastatin inhibited N-Ras processing even more potently than FTIs. Increased amounts of the N-Ras protein were observed in samples treated with lovastatin and FTI L-744,832. Rheb processing was also found to be inhibited by lovastatin, while no change in the prenylation status of RhoA was observed.
Conclusions: These data demonstrate that disruption of Ras-to-MAPK signaling inhibits multiple myeloma cell growth. Lovastatin was particularly effective in inhibiting Ras prenylation and inducing apoptosis in myeloma cell lines. It will be of great interest to investigate the effects of these agents (alone and in combination) on the expression of proteins which are important in the pathogenesis of multiple myeloma.