Internal Tandem Duplication of Flt3 (ITD-Flt3) Bypasses Growth Factor Mediated Survivin Expression, while Disruption of Survivin Accelerates Cell Death in Hematopoietic Cells Expressing ITD-Flt3.

The inhibitor of apoptosis protein Survivin is barely detectable in normal adult tissues but is over-expressed in almost all cancers and hematopoietic malignancies and confers resistance to apoptosis and aberrant cell proliferation. We have shown that hematopoietic growth factors induce Survivin expression in normal CD34⁺ cells in both cell cycle dependent and independent fashion and that oncogenic Ras can enhance Survivin expression independent of growth factor stimulation. In addition, over-expression of Survivin blocks apoptosis of hematopoietic progenitor cells through a p21^WAF1/Cip1 (p21) dependent mechanism. However the mechanisms responsible for Survivin overexpression in malignant hematopoietic cells is poorly understood. Flt3 is a type III tyrosine kinase receptor expressed mainly in the primitive hematopoietic compartment. Internal tandem duplication (ITD) mutations of the Flt3 gene that activate the Flt3 tyrosine kinase are frequently found in patients with AML and associated with poor prognosis. Furthermore, their ectopic expression in IL-3 dependent Ba/F3 cells results in IL-3-independent proliferation and a myeloproliferative disease in mice. In order to understand the mechanism by which ITD-Flt3 enhances cell survival, we examined whether ITD-Flt3 can bypass Survivin up-regulation independent of hematopoietic growth factors and if Survivin lies down stream of ITD-Flt3 in prolonging cell survival. Incubation of primary umbilical cord blood CD34⁺ cells with Flt3 ligand induced Survivin expression, indicating that Survivin lies down stream of Flt3 signaling pathways in CD34⁺ cells. While Survivin expression was comparable in Ba/F3 cells expressing ITD-Flt3 (W51, W73 and W78 mutants, kindly provided by Dr. DG Gilliland) and wild-type Flt3 when maintained in the presence of IL-3, ITD-Flt3 prevented down-modulation of Survivin expression induced by IL-3 withdrawal even during G₀/G₁ phase of cell cycle, suggesting that up-regulation of Survivin is not a consequence of cell cycle progression associated with ITD-Flt3 expression. The increase in Survivin by ITD-Flt3 was associated with reduced active caspase-3 and prolonged cell survival coincident with induction of p21 expression and Tyrosine 15 phosphorylation of CDK1 (Cdc2), which have been implicated in anti-apoptosis. These results are consistent with a p21 dependent anti-apoptosis effect of Survivin in primary hematopoietic progenitor cells and suggest the involvement of Survivin-p21 and CDK1 in ITD-Flt3 mediated cell survival. Overexpression of dominant negative T34A Survivin inhibits the enhanced survival induced by ITD-Flt3 in Ba/F3 cells following IL-3 withdrawal, suggesting that the survival effect of ITD-Flt3 is mediated by Survivin. Similar to ITD-Flt3, overexpression of ectopic wild-type Survivin results in enhanced cell survival in Ba/F3 cells but failed to induce cell cycle progression and IL-3-independent proliferation, indicating that Survivin alone is not sufficient for induction of growth factor independent proliferation and the cell cycle enhancing effect of ITD-Flt3. In summary, our findings indicate that ITD-Flt3 up-regulates Survivin expression independent of hematopoietic growth factors and that the ITD-Flt3/Survivin axis provides a survival advantage in transformed hematopoietic cells. Disruption of these pathways may increase apoptosis of malignant hematopoietic cells and provide therapeutic benefit.