Inhibiting Translation as a Novel Strategy to Target Multiple Myeloma

Multiple myeloma (MM) cell survival is dependent on the ability to produce and secrete immunoglobulins, requiring precise coordination of protein synthesis and degradation. Inhibition of these processes may be responsible for the exquisite sensitivity of MM to drugs such as bortezomib and histone deacetylase inhibitors, which respectively block proteosomal and aggresomal-mediated protein degradation. We hypothesized that MM cells would also be sensitive to inhibition of mRNA translation and have tested silvestrol, a plant-derived natural product that has previously been shown to inhibit ribosome recruitment and to be cytotoxic to B lymphocytes.

Silvestrol was resuspended in DMSO and diluted for use in RPMI medium. A panel of human MM cell lines, representing a range of chromosomal abnormalities observed in this disease, were cultured in RPMI with 10% FCS and appropriate supplementation. Primary mouse embryonic fibroblasts (MEFs) were prepared and passaged 7–10 times until senescence was observed by viability studies. Cytotoxicity was determined using MTT viability assay and apoptosis by flow cytometry using annexin V-PE, CD138-PECy5 conjugated antibodies and 7-AAD. Primary patient samples obtained by bone marrow aspiration were prepared by ficoll isolation and peripheral blood mononuclear cells were cultured in IMDM with 20% FCS with or without silvestrol followed by flow cytometric analysis for apoptosis. Polysome profiling and immunoblotting were performed on extracts of cells treated with silvestrol at various time points in order to determine a mechanism of action as well as to observe cell signaling pathways.

All MM cell lines tested with silvestrol showed profound inhibition of cell growth and exhibited cell death within 48h, with an average IC50 of 10nM. Furthermore, the slow growing cell line, MM1.S and its dexamethasone resistance counterpart, MM1.R, were equally sensitive to silvestrol and at the same IC50 range as more rapidly growing lines. Apoptotis was confirmed by AnnexinV flow cytometric assay in MM cell lines following exposure to 10–20nM silvestrol for 24h. As MM is not usually characterized by a high proliferative index, we tested silvestrol in non-dividing, senescent MEFs and observed cytotoxicity and apoptosis at similar concentrations. Silvestrol was observed to be cytotoxic to the CD138 fraction of primary MM patient bone marrow samples at 10 and 50nM concentrations after 24 and 48h of incubation. Silvestrol was shown to inhibit translation in MM cell lines as evidenced by inhibition of ribosome binding and a decrease in polysome content with a concomitant increase in 80S ribosomes. Western blot analyses suggest that silvestrol significantly decreases NFKB activity in cell lines that constitutively process NFKB2. MCL1, a BCL2 family inhibitor of apoptosis, is also rapidly downregulated in silvestrol treated MM cells, suggesting a possible mechanism for silvestrol-induced apoptosis. In vivo, we have determined that no toxicity was evident at daily 0.2mg/kg injections in the transgenic and immunocompetent vk*MYC mouse model of MM that has shown biologic and therapeutic fidelity to human MM. Silvestrol has already shown tumoricidal activity in xenographic models of other malignancies and its efficacy in vk*MYC MM mice is currently being explored.

These preclinical studies suggest that, because of its unique biology, MM is particularily susceptible to the cytotoxic effects of translation inhibition by silvestrol. These observations warrant clinical evaluation of silvestrol in MM.

No relevant conflicts of interest to declare.