Prenylation Inhibitors Block IL-6 and IGF-1 Dependent Activation of RAS Signaling in Multiple Myeloma Cells.

Abstract

Multiple myeloma (MM) is a fatal hematologic malignancy associated with disruption of RAS-to MAP kinase (ERK) signaling. The IL-6 cytokine family and growth factors such as IGF-1 have been demonstrated to promote malignant plasma cell proliferation through stimulation of ERK and PI-3 kinase/AKT signaling. Prenylation inhibitors such as farnesyltransferase inhibitors (FTIs), geranylgeranyl transferase inhibitors (GGTIs) and the competitive HMG-CoA reductase inhibitor lovastatin have been shown to block RAS post-translational modification and disrupt RAS signaling. To assess efficacy of prenylation inhibitors (e.g. FTI L-744,832, GGTI-2147 and lovastatin) to attenuate MM cell responses to IL-6 and IGF-1, inhibitor-treated cells were analyzed by proliferation/viability assays (MTS) and Western blotting to determine phosphorylated MEK-1/2 and ERK-1/2. FTI L-744,832 was found to inhibit growth of MM cells cultured with IL-6 or IGF-1 even more potently than in cytokine/growth factor-free medium (IC50s 1.3 μM, 1.8 μM and 4.2 μM, respectively). IL-6 moderately protected MM cells from inhibitory effects of GGTI-2147, while IGF-1 had no effect (IC50s 1.1 μM and 0.5 μM vs. 0.5 μM, respectively). IL-6 and IGF-1 protected MM cells from lovastatin-induced growth inhibition (IC50s 4.7 μM and 5.0 μM vs. 1.4 μM, respectively). Furthermore, co-treating MM cells with FTI L-744,832 and GGTI-2147 or lovastatin synergistically inhibited proliferation of MM cells regardless of the presence or absence of IL-6 or IGF-1. Western blotting demonstrated that FTI/GGTI or FTI/lovastatin co-treatment more completely blocked activation of MEK-1/2 and ERK-1/2 in NCI-H929 cells than treatment with any of the compounds alone. Co-treatment also elicited greater inhibition of IL-6 and IGF-1 induced MEK-1/2 and ERK-1/2 activation in NCI-H929 cells. IL-6, IGF-1 and prenylation inhibitors had no effects on AKT phosphorylation status in NCI-H929 cells. To evaluate the clinical relevance of these observations, primary MM cells were obtained from bone marrow aspirates (n=6) or from peripheral blood of a patient with plasma cell leukemia/MM (PCL/MM). Primary MM cells were isolated by magnetic cell sorting using CD138-coupled microbeads, titrated with prenylation inhibitors in the presence of IL-6 and synergy was calculated using the CalcSyn program. Activating RAS mutations (4 K-RAS, 1 N-RAS) were found in 4/7 (57%) MM patient samples, with one sample harboring both K- and N-RAS mutations. FTI L-744,832 elicited anti-myeloma effects only at concentrations much higher than those found to inhibit healthy donor CD34+ cells (IC50's 51–396 μM vs. 8.2μM) or MM cell lines (1.1–23.8μM) and thus may be ineffective or cause non-specific toxicity when used as a single agent. However, IC50's calculated for GGTI-2147 and lovastatin were generally higher for CD34+ cells as compared to primary MM cells, supporting specificity of these compounds. Furthermore, combination of FTI with GGTI or lovastatin synergistically inhibited primary MM cell proliferation (IC50's 0.6–23.1 μM). Our results support that inhibition of RAS down-stream signaling is a major mechanism through which FTI/GGTI and FTI/lovastatin co-treatment synergistically inhibit MM cell proliferation, even in the presence of IL-6 and IGF-1. Incomplete response to FTI treatment may be explained by alternative prenylation of K- and N-RAS by GGTase I in the presence of FTIs. As the majority of RAS mutations in MM occur in K- and N-RAS, FTI-resistance due to alternative geranylgeranylation may have therapeutic consequences.