YM155 Exerts Potent Cytotoxic Activity Against Quiescent (G0/G1) Multiple Myeloma and Bortezomib Resistant Cells Via Inhibition of Mcl-1 and Survivin

Multiple myeloma (MM) is a molecularly heterogeneous hematologic malignancy and remains mostly incurable despite the recent improvement of treatment strategies by several novel agents. Therefore, it is important to develop more efficacious drug against MM. YM155, a novel small molecule suppressant of survivin, shows anti-proliferative activities against various human cancer cells. YM155 was identified in a survivin gene promoter assay by high throughput screening of chemical libraries. In the present study, we investigated the cytotoxic mechanism of YM155 against human myeloma cells including bortezomib (BTZ) resistant cells (U266/BTZ). Three myeloma cell lines, U266, KMS-11 and KMS-12, were employed. YM155 inhibited the cell growth of these cell lines with the IC50 value of below 5 nM. YM155 suppressed the expression of mRNA and protein of survivin. We also found that YM155 inhibited the protein expression of Mcl-1, as an essential anti-apoptotic protein for survival of myeloma cells. In addition, we observed that YM155 markedly suppressed the phosphorylation of STAT3, which is known as transcription factor of Mcl-1. When KMS-12 cells were incubated with IL-6, phosphorylation of STAT3 and upregulation of Mcl-1 protein were observed. Treatment of KMS-12 with YM155 inhibited these events and eventually induced apoptosis in KMS-12 cells. Interestingly, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. RQ-PCR analysis indicated that the level of Mcl-1 mRNA was not affected after YM155 treatment. Immunoblot analysis showed that proteasome inhibitor MG-132 blocked the inhibition of Mcl-1 expression by YM155, suggesting that proteasome-mediated degradation is involved in YM155-induced Mcl-1 downregulation. MM is a low-growth-fraction disease and low proliferation of MM seems to contribute to resistance to various anticancer drugs. To determine whether YM155 shows cytotoxic effect against quiescent (G0/G1) MM cells, U266 were cultured in low-serum medium to enrich the G0/G1 population. Dual-parametric flow cytometric analysis using Hoechest33342 and the RNA specific dye pyronin Y revealed that YM155 potently induced cell death of quiescent (G0/G1) MM cells. In quiescent MM cells, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. We also examined whether similar effect of YM155 could be observed in primary MM cells. The majority of primary MM cells from patients was found to be in quiescent phase by cell-cycle analysis. YM155 showed similar cytotoxic activity against primary MM cells. In contrast, Ara-C, the S-phase specific anticancer drug, never killed quiescent primary MM cells. We established BTZ-resistant MM cell line (U266/BTZ). The IC50 value was 45-fold higher than its parental cell line. DNA sequencing data indicated that U266/BTZ cells possess a point mutation, G322A, in the gene encoding the proteasome beta-5 subunit. YM155 almost equally exhibited cytotoxic activity against U266/BTZ compared with parental cells. U266/BTZ displayed significantly lowered amounts of bcl-2, survivin and aurora-B kinase proteins. Interestingly, U266/BTZ overexpressed the Mcl-1 protein. Treatment with YM155 rapidly suppressed Mcl-1 protein expression and induced apoptosis. These data suggest that overexpression of Mcl-1 may contribute to bortezomib resistance and downregulation of Mcl-1 by YM155 could overcome it. In conclusion, our data indicate that YM155 may exert robust cytotoxic activity against quiescent (G0/G1) MM and bortezomib resistant cells via inhibition of Mcl-1 and survivin.