Abstract
Internal tandem duplications of the FMS-like receptor tyrosine kinase (FLT3-ITDs), an in-fame insertion of several amino acids within the juxtamembrane domain, are present in 25% of acute myeloid leukemia (AML) patients and confer a poor prognosis. FLT3-ITDs induce FLT3 ligand (FL)-independent hyperactivation of Erk and promiscuous activation of STAT5; however, the molecular mechanisms underlying aberrant activation of these signaling molecules is largely unknown. Tyrosine 599 (Y599) of WT FLT3 recruits the protein tyrosine phosphatase, Shp2, upon stimulation with FL, resulting 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, results in enhanced Shp2 activation and contributes to N51-FLT3- and N73-FLT3-induced cellular hyperproliferation, Erk hyperactivation, and promiscuous STAT5 activation. Using Baf3 cells stably expressing WT FLT3, N51-FLT3, or N73- FLT3, co-immunoprecipitation assays demonstrated that Shp2 is phosphorylated and associates with WT FLT3 in a FL-dependent manner. However, in contrast, Shp2 is constitutively hyperphosphorylated and associated with FLT3-N51 and FLT3-N73 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. Western blot 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. 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.
Dual Specificity Phosphatases: From Molecular Mechanisms to Biological Function
