Survivin in Multiple Myeloma: Prognostic and Therapeutic Implications

Abstract 137

The IAP (inhibitor-of-apoptosis) family member, survivin, is one of the most significantly over-expressed genes in malignant cells. Survivin has been reported to inhibit apoptosis and regulate mitosis as well as cytokinesis. We therefore first determined the expression of survivin in CD138-purified multiple myeloma (MM) cells from previously untreated patients at our centers' trial group (TG) (n=246) and the UAMS Arkansas (n=345) validation group (VG). Using the PANP algorithm, survivin is aberrantly expressed in 27% (TG) of MM cell samples. It is not expressed in normal bone marrow plasma cell samples (n=7) while expression increases significantly from MM stage I-III (P<.001). Survivin expression correlates with proliferation as assessed by gene expression- (r=.8, P<.001) or propidium iodide (r=.7, P<.001). Presence of survivin expression correlates with inferior event-free and overall survival in patients undergoing high-dose chemotherapy in the TG (22.6 vs. 35.4 months, P<.001, 52.9 vs. n.r., P=.002) as well as in the VG (12.3 vs. 54.1 months, P<.001, and 17.4 vs. n.r., respectively). These results support the further evaluation of survivin as a therapeutic target in MM. We next assessed the effects of siRNA-mediated knock-down of survivin in vitro. Suppression of survivin by siRNA induced cell cycle arrest and apoptosis in MM cell lines. The small molecule suppressant of survivin, YM155, is currently in clinical development for the treatment of solid tumors. Here, we investigated YM155 for its anti-MM activity. YM155 abrogated proliferation and induced apoptosis in a panel of 10 human MM cell lines and MM cells isolated from multidrug-resistant patients at an IC50 of 4–50nM while the IC50 was not reached in primary bone marrow stromal cells up to 500nM. YM155 was also able to overcome the protective effect of IL-6, IGF-1 and the presence of bone marrow stromal cells, respectively. The induction of apoptosis by YM155 closely correlated with down-regulation of intracellular survivin protein expression within 24 to 36h of treatment with 50–100nM of YM155. However, inhibition of cell proliferation is already detectable at 12h at 5–10nM, suggesting two different dose- and time-dependent mechanisms of action. We therefore performed gene expression and protein profiling on YM155-treated MM cells. Strikingly, these data revealed early up-regulation of the ER stress response (PERK, phospho-eIF2a, ATF4, ATF3) followed by increased CHOP expression and a profound abrogation of proliferation. This appeared to be independent of cellular survivin levels, indicating that the early proliferation arrest at very low nanomolar concentrations is mediated primarily by the ER stress response. Moreover, gene signatures regulated by the IL-6/STAT pathway (CCND1, BCL2L1, MCL1, BIRC5A) were markedly altered upon YM155 treatment. Importantly, IL-6 profoundly sensitized IL-6 responsive MM cell lines to treatment with YM155. We therefore hypothesized that YM155 might abrogate upstream regulatory signaling pathways of survivin expression. Indeed, YM155 abrogated constitutive as well as IL-6 induced phosphorylation of STAT3, an important transcription factor for survivin expression in MM cells. In contrast, phosphorylation of ERK1/2 and AKT remained unchanged. Dephosphorylation of STAT3 closely correlated with the loss of intracellular survivin. In conclusion, we have demonstrated the prognostic significance of survivin expression and a potential therapeutic role for the small molecule suppressant of survivin YM155 in MM.