Targeting the Protein Phosphatase, Shp2, Reduces FLT3-ITD-Induced Hyperproliferation of Murine Hematopoietic Progenitors.

Abstract 827

Internal tandem duplications in the FMS-like receptor tyrosine kinase receptor (FLT3-ITDs) are present in approximately 25% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. FLT3-ITDs induce FLT3 ligand (FL)-independent hyperproliferative cellular growth and promiscuous activation of STAT5; however, the molecular mechanism underlying aberrant activation of these signaling molecules is largely unknown. Upon FLT3 ligand (FL) stimulation, tyrosine 599 (Y599) of WT-FLT3 recruits the protein tyrosine phosphatase, Shp2 and induces cell proliferation and Erk activation. In several FLT3-ITDs, including N51-FLT3 and N73-FLT3, Y599 is duplicated. These findings led us to hypothesize that increased recruitment of Shp2 to N51-FLT3 or N73-FLT3, via Y599, contributes to N51-FLT3- and N73-FLT3-induced cellular hyperproliferation and promiscuous STAT5 activation. Using Baf3 cells stably expressing WT-FLT3, N51-FLT3, or N73-FLT3, co-immunoprecipitation assays demonstrated that Shp2 associates with WT-FLT3 in a FL-dependent manner, while Shp2 is constitutively associated with N51-FLT3 and N73-FLT3 independent of FL-stimulation. To investigate the functional role of Shp2 in FLT3-ITD-induced leukemogenesis, Baf3 cells expressing WT-FLT3, N51-FLT3, or N73-FLT3 were transfected with a mammalian expression vector encoding a U6 polymerase III–directed Shp2-specific short-hairpin RNA (shRNA) or a scrambled shRNA and selected in puromycin. Immunoblot analysis revealed significant reduction of Shp2 expression by the Shp2-specific shRNA and no change in Shp2 expression by the scrambled shRNA in all cell lines. Upon knock-down of Shp2 in Baf3/WT-FLT3 cells, proliferation was minimally reduced based on thymidine incorporation assays; however, knock-down of Shp2 in Baf3/N51-FLT3 and Baf3/N73-FLT3 cells significantly reduced proliferation, both at baseline and in response to FL stimulation. To further test the role of Shp2 in FLT3-ITD-induced transformation, Shp2^flox/flox mice were crossed with Mx1Cre transgenic mice to produce Shp2^flox/flox Mx1Cre+ mice. Shp2^flox/floxMx1Cre+ and Shp2^flox/floxMx1Cre- pairs were treated with 3 intraperitoneal injections of polyI/polyC to induce recombination of the Shp2 allele and generate a null allele in the Mx1Cre+ animals. PCR and immunoblot analysis revealed ablation of the flox allele and reduction of Shp2 protein, respectively, in Shp2^flox/floxMx1Cre+ bone marrow as soon as 2 weeks post injection. Low density mononuclear cells (LDMNCs) isolated from polyI/polyC-treated Shp2^flox/floxMx1Cre- and Shp2^flox/floxMx1Cre+ mice were transduced with either pMSCV-WT-FLT3 or pMSCV-N51-FLT3. Transduced cells were sorted for EGFP (enhanced green fluorescent protein) expression and subjected to thymidine incorporation assays. Shp2^flox/floxMx1Cre+ cells transduced with N51-FLT3 demonstrated significantly reduced proliferation at baseline and in response to FL stimulation compared to Shp2^flox/floxMx1Cre- cells expressing N51-FLT3. To pharmacologically examine the role of Shp2 in FLT3-ITD-induced-hyperproliferation and constitutive activation of Stat5, we utilized a Shp2 inhibitor, II-B08, identified from a library of indole salicylic acid derivatives. II-B08 exhibits an IC₅₀ of 5.5 μM for Shp2 and demonstrates excellent cellular activity. Bone marrow LDMNCs retrovirally transduced with WT-FLT3 or N51-FLT3 were incubated in the absence of or in the presence of increasing concentrations of II-B08 and plated into thymidine incorporation assays. A minimal and non-significant reduction in proliferation was observed in WT-FLT3 expressing cells in the presence of II-B08; however N51-FLT3 expressing cells demonstrated a highly significant reduction in cellular proliferation in a concentration-dependent fashion. In addition to these functional analyses, biochemical studies revealed a reduction in Stat5 activation in N51-FLT3 expressing cells treated with II-B08 compared N51-FLT3 expressing cells treated with DMSO in two independent experiments. Collectively, these data suggest that constitutive recruitment of Shp2 to N51-FLT3 and N73-FLT3 contributes to the FLT3-ITD-induced hyperproliferative phenotype and imply that inhibition of Shp2 function may provide a novel therapeutic approach to FLT3-ITD-bearing leukemias.