Combining the Ras Inhibitor Salirasib and Proteasome Inhibitors: A Potential Treatment for Multiple Myeloma
Authors: Yaari-Stark Shira , Nevo-Caspi Yael , Jacob-Hircsh Jasmine , Rechavi Gideon , Nagler Arnon , Kloog Yoel 
Published: October 2019

Abstract
Abstract 1810
Multiple Myeloma (MM) is characterized by clonal proliferation of malignant plasma cells that eventually develop resistance to chemotherapy. Novel agents such as Thalidomide, Bortezomib (Velcade) and Lenalidomide improve response rates and prolong progression free and overall survival. Drug resistance, differentiation block and increased survival of the MM tumor cells result from genomic alterations, including high cyclin D and fibroblast growth factor receptor 3 (FGFR3) over-expression as well as mutations in NRas. Interactions between myeloma cells and stromal cells in the tumor microenvironment play a major role in MM resistance. Particularly, activation of NF-κB-mediated upregulation of IL-6 secretion by stromal cells is in connection with signal transduction of the Ras oncogene pathway. Activating mutations of Ras have been reported in 30–50% of MM patients. KRas and NRas are the most frequent mutated, suggesting that active Ras is an appropriate target in MM. Development of oncogenic Ras isoforms can be inhibited by Ras inhibitor, farnesylthiosalicylic acid (FTS, salirasib) which also inhibits fibroblast growth factor (FGF)-stimulated Ras activation. We, therefore, compared the effects of FTS on proliferation of NCIH929 (harboring oncogenic NRas) and of two other MM cell lines, MM1.S and U266, which do not harbor oncogenic NRas. Inhibition of cell proliferation was evident by the reduction in BrdU incorporation into the DNA of cells treated for 24 h with FTS (50, 75, or 100 μM) and by counting cells stained with alamarBlue. NCIH929 responded better than the others cell lines to FTS-induced growth inhibition (P<0.05). The IC50 values were 64μM, 82μM and 82–100μM for NCIH929, MM.1S and U266 MM tumor cell lines, respectively (n=3). Treatment with FTS also significantly reduced total Ras and NRas-GTP in NCIH929 but not in the two other cell types, which was accompanied by a significant decrease in the amount of c-Myc (62±2%), p-ERK (38±5%) and p-Akt (52±1.6%) (n=3). All of these proteins are essential for the proliferation, growth, and survival of myelomas. Gene-expression patterns of control and of FTS-treated NCIH929 cells demonstrated down-regulation of FGFR3 (by 2.44 fold) and FGFR3 protein expression declined significantly (36±5%) in these cells after FTS treatment. FTS also inhibited FGF-stimulated GTP loading of wild-type NRas, and hence ERK activation, in MM-NCIH929. These findings suggested that FGFR3 acts together with NRas to activate the MAPK pathway, and also pointing to the possibility that treatment with FTS affected both early Ras-dependent signaling and long-term Ras-dependent control of gene expression and protein translation. Proteasome inhibitors have emerged as powerful tools for inhibiting NFκB activity in myelomas. We therefore examined the combined effect of the proteasome inhibitor MG132 (0.5-2.5 μM) or bortezomib (2.5μM) and the Ras inhibitor FTS (50 or 75 μM) on the growth of NCIH929 cells. Combination of FTS with the proteasome inhibitor MG132 or bortezomib yielded synergistic inhibitory effect (up to 86±6.4%) of NCIH929 MM cell growth (P<0.001; P<0.05, respectively) (n=3). Lastly, we tested the potential inhibitory capabilities of new FTS derivatives including FTS-esters and amides. The FTS-amides exhibited substantially higher activity (50% higher) than FTS itself, while the FTS-esters were completely inactive. In conclusion, the dependence of MM on FGF3R and Ras pathways make them sensitive to Ras inhibitors such as FTS. The synergistic effects of bortezomib and FTS in NCIH929 cells and presumably in MM might be explained by the two distinct pathways that they affect. Based on these results, we suggest that salirasib (FTS) may be considered, both alone and moreover in combination with proteasome inhibitors, as a potential treatment for MM.

Disclosures:
No relevant conflicts of interest to declare.