Disruption of the Inhibitor-Of-Apoptosis Protein, Survivin, Sensitizes Bcr-abl Positive Cells to STI571 (Gleevec®)-Induced Apoptosis.

Abstract The Bcr-abl oncogene is found in cells of >95% of patients with CML and encodes a cytoplasmic protein with constitutive tyrosine kinase activity. Bcr-abl induces hematopoietic cell transformation and protects cells from apoptosis induced by numerous stimuli. Bcr-abl activates Myc, Ras, raf, Pl3K, and c-jun kinases that are critical for transforming activity, however, the signaling pathways between Bcr-abl and the apoptosis machinery are just beginning to be determined. It is known that Bcr-abl can exert an antiapoptotic effect by blocking mitochondrial release of cytochrome C. Although increased knowledge of Bcr-abl pathways resulted in the design of selective tyrosine kinase inhibitors such as STI571 (Gleevec), the development of drug resistance limits efficacy. Survivin is a member of the highly conserved inhibitor-of-apoptosis (IAP) family of endogenous caspase inhibitors. Similar to other IAPs, Survivin blocks apoptosis by inhibiting caspases 3, 7 and 9. However in contrast to other IAPs, Survivin is not expressed in most adult tissues but aberrantly overexpressed in all cancers and hematopoietic malignancies. Targeting of Survivin by antisense (AS) or dominant-negative (DN) strategies in transformed cell models induces apoptosis. Survivin is expressed in Bcr-abl+ CML cells in blast crisis, but not in cells from patients with Bcr-ablneg CML. Furthermore, high expression of Survivin is found in Adriamycin resistant Bcr-abl+ K562 cells. These findings led us to investigate whether Survivin is involved in the antiapoptotic effects of Bcr-abl and if Survivin disruption can facilitate apoptosis in Bcr-abl+ cells. Transient transfection of human Mo7e and mouse BaF3 hematopoietic cells with the Bcr-abl oncongene results in significantly elevated expression of Survivin mRNA and protein. The mRNA of 2 other Survivin splice variants, Survivin-2B and Survivin-ΔEx3 were also upregulated. In transfected Mo7e cells, Survivin promoter activity was upregulated 2–4 fold compared to parental Mo7e cells, determined using a luciferase reporter construct. In addition to Survivin, the IAP family member ILP was also upregulated by Bcr-abl. Disruption of Survivin expression/function in K562 cells that contain endogenous Bcr-abl and in Bcr-abl transfected Mo7e cells by ectopic expression of AS or DN T34A or C84A mutant Survivin constructs, significantly promoted apoptosis induced by the Bcr-abl tyrosine kinase inhibitor, Gleevec by ≥2-fold, in a time and dose dependent manner. Enhanced apoptosis induced by AS or DN Survivin was accompanied by caspase dependent cleavage of Bcr-abl oncoprotein (>50% decrease in Bcr-abl protein) that was blocked by the caspase inhibitor Z-VAD-fmk; disruption of mitochondria membrane potential (>35% increase in TMRMnegexpression); and enhanced cytochrome C release, quantified by westerns. Similarly, disruption of Survivin mRNA in K562 cells by a 20-mer antisense oligonucleotide resulted in >40% increase in mitochondrial disruption. In contrast, forced expression of wild-type Survivin in K562 cells protected cells from Gleevec-induced apopotosis. In summary, our results demonstrate that the Bcr-abl oncogene regulates Survivin transcription and production, which represents a new signaling pathway downstream of Bcr-abl that may be helpful in understanding the pathophysiology of CML. Targeted Survivin disruption may sensitize Bcr-abl+ CML cells to Gleevec-induced apoptosis and have therapeutic potential, particularly in the development of drug resistance.