Survivin Regulates Aberrant Proliferation of Hematopoietic Progenitor Cells with Self Renewal Capability and Development of Acute Leukemia Induced by Internal-Tandem-Duplication of Flt3.

Internal Tandem Duplication mutations in the Flt3 tyrosine kinase gene (ITD-Flt3) and over-expression of the inhibitor of apoptosis protein Survivin are frequently found in patients with acute myeloid leukemia (AML) and are associated with poor prognosis. We have shown that ITD-Flt3 increases expression of Survivin in Ba/F3 cells and that antagonizing Survivin inhibits growth factor independent proliferation of primary mouse hematopoietic stem and progenitor cells (HSPC) expressing ITD-Flt3 (Fukuda et al. ASH, 2005). This suggests that Survivin may regulate proliferation of AML cells expressing ITD-Flt3. Herein we investigated the functional role of Survivin in self-renewal and proliferation of transformed HSPC and development of acute leukemia induced by ITD-Flt3 in mice. Treatment of ITD-Flt3 positive human AML MV4-11 cells with the ITD-Flt3 kinase inhibitor AG1296 reduced their proliferation by 33±4% coincident with 50% reduction in Survivin protein, suggesting a functional link between ITD-Flt3 and Survivin in AML cell proliferation. Conditional Survivin deletion significantly reduced growth factor independent proliferation of primary mouse marrow c-kit⁺, Sca-1⁺, lin⁻ cells that express ITD-Flt3 by 46±7%, which was further accentuated by AG1296 (76±8% reduction). The effect of Survivin deletion was associated with increased apoptosis measured by Annexin V, sub G1 content and G₀/G₁ arrest (101±38, 169±44 and14±5% increase, respectively). Overexpressing ITD-Flt3 reduced the proportion of lin⁺ cells derived from lin⁻ marrow cells cultured with GM-CSF plus Stem Cell Factor compared to wild-type Flt3, while Survivin deletion in ITD-Flt3 expressing cells partially abrogated the effect (53±6% increase in lin⁺ cells, P<0.05). Furthermore, Survivin deletion dramatically diminished secondary CFU formation induced by ITD-Flt3 in the absence of hematopoietic growth factors (74±5% reduction, P<0.005). These results suggest that ITD-Flt3 promotes self renewal and inhibits differentiation of primary HSPC that is mediated through Survivin. However, over-expressing Survivin alone failed to transform these cells, suggesting that Survivin is required but not sufficient for ITD-Flt3 signaling in primary HSPC. BALB/c mice transplanted with Ba/F3 cells overexpressing ITD-Flt3 (Ba/F3-ITD) died of acute leukemia within 5 weeks (32±1 days), whereas significantly delayed leukemia progression and prolonged survival was observed in mice transplanted with Ba/F3-ITD cells expressing T34A dominant-negative Survivin (70±13 days, P<0.05, N=10), suggesting that Survivin is involved in the acute leukemia induced by ITD-Flt3. These results suggest that Survivin regulates expansion of HSPC with self-renewal capability transformed by ITD-Flt3 and development of ITD-Flt3 positive acute leukemia and that antagonizing Survivin may provide therapeutic benefit for acute leukemias expressing ITD-Flt3. However, since we previously showed that Survivin regulates normal hematopoiesis (Fukuda et al. ASH 2006), development of strategies that disrupt Survivin specifically in malignant hematopoietic cells expressing ITD-Flt3 without affecting normal HSC are warranted.