Role of Survivin in Drug Resistant B-Cell Acute Lymphoblastic Leukemia.

Abstract 10

Survivin/BIRC5, an inhibitor of apoptosis (IAP) protein,is critical for the survival and proliferation of cancerous cells and has become the target of an increasing number of preclinical novel therapies against primarily solid tumors. Survivin is expressed in AML and ALL cells and has been implicted in leukemia relapse. Here we test the hypothesis that Survivin is critical to chemotherapeutic drug resistance in ALL. To test this hypothesis, we initially compared survivin mRNA levels in both patient-derived B-ALL cells and B-ALL cell lines versus normal B-cells in various stages of development using real-time PCR. ALL cells encompassing various cytogenetic subgroups showed significantly greater mRNA levels of Survivin versus normal B cell precursors ranging from 2 to greater than 20-fold versus controls. To determine whether Survivin contributes to drug resistance, we lentivirally overexpressed Survivin in primary B-ALL and B-ALL cell lines. Overexpression of Survivin attenuated the effect of Vincristine on ALL cell proliferation when compared to ALL cells transduced with empty vector controls. Vincristine IC₅₀ value determination for the control was 0.1 nM, whereas Survivin overexpression resulted an IC₅₀ value of 10 nM (p<0.01). Similarly, significantly higher concentrations of L-Asparaginase (>0.01 U/l vs ab >0.1; p<0.05) and Dexamethasone (0.1 nmol/l vs >10 nmol/l; p<0.013) were needed to achieve drug cytotoxicity. We conclude that Survivin overexpression decreases sensitivity of ALL cells to standard chemotherapy. Conversely, lentivirally-mediated survivin shRNA knockdown of the same cell type significantly sensitized a greater percentage of the leukemia to 10nM vincristine versus non-silencing controls. Survivin shRNA targeting resulted in a 30.2% greater affected population than controls, assayed using MTT (p<0.003). Overexpression or knockdown of Survivin was confirmed by Western Blot and real-time PCR. Of note, expression levels of Survivin is heterogeneous within the same ALL sample: Transduction of patient-derived ALL cells with a Survivin-GFP reporter construct revealed that a small subpopulation of Survivin-GFP high ALL exhibiting approximately 7-fold higher levels of endogenous Survivin than bulk leukemia cells. Consistent with cell cycle-dependent regulation of Survivin, Survivin-GFP high ALL were enriched in G2/M phase of the cell cycle. In addition, endogenous variations of Survivin protein levels were in the same range as changes in Survivin protein levels in overexpression and knockdown experiments, which indicates that these experiments tested physiologically occurring levels of Survivin. Next, we determined whether Survivin contributes to self-renewal of drug resistant ALL cells. Survivin overexpression of primary samples yielded three times more colonies, than controls in a primary plating CFU assay (p<0.019). Taken together, our data suggests that targeting Survivin in B-ALL may sensitize to chemotherapy and highlights the role of Survivin in drug resistant B-ALL as a target for novel therapies.