The Protein Tyrosine Phosphatase, Shp2, Positively Contributes to FLT3-ITD-Induced Malignant Disease in Vivo and Co-Localizes with Nuclear Phospho-STAT5 in FLT3-ITD-Expressing Leukemic Cells.

Abstract 2420

Internal tandem duplications in the fms-like tyrosine kinase receptor (FLT3-ITDs) confer a poor prognosis in individuals with acute myeloid leukemia (AML). Based on the finding that the protein tyrosine phosphatase, Shp2, interacts with WT FLT3 tyrosine (Y) 599, which is commonly duplicated in FLT3-ITDs, we hypothesized that increased recruitment of Shp2 to FLT3-ITDs contributes to FLT3 ligand (FL)-independent hyperproliferation and aberrant STAT5 activation. Co-immunoprecipitation studies demonstrated constitutive association of Shp2 with the FLT3-ITD, N51-FLT3, as well as with STAT5. Additionally, we found that genetic disruption of Ptpn11, the gene encoding Shp2, significantly reduced N51-FLT3-induced hematopoietic cell hyperproliferation and STAT5 hyperphosphorylation in vitro. To investigate these findings further, Lin- bone marrow cells from Shp2^flox/flox;Mx1Cre⁺ animals were retrovirally transduced with N51-FLT3, sorted to homogeneity, and transplanted into lethally irradiated congenic recipients. Transplanted animals were treated with polyI:polyC to delete Shp2 or with phosphate buffered saline (PBS control) 4 – 6 weeks following transplantation, and animals were followed temporally. The majority of PBS-treated animals (16/18) died of hematologic malignancy. In contrast, animals with Shp2 deletion (polyI:polyC-treated, n=16) succumbed to malignant disease less frequently (10/16), demonstrated a significantly prolonged survival (p=0.024 by log-rank test), and had smaller spleen sizes compared to the PBS-treated animals. Notably, Y599 has been shown to recruit Shp2 to WT FLT3 and mutation of Y599 to phenylalanine (F) within WT FLT3 causes a reduction in FL-stimulated cell proliferation. Thus, we generated point mutants including N51-Y599F1 bearing the Y to F mutation at the first Y599 and N51-Y599F1/2 bearing Y to F mutation at both the first and duplicated Y599. Murine bone marrow low density mononuclear cells were transduced with each construct and subjected to ³H-thymidine incorporation and immunoblot for proliferation and STAT5 activation, respectively. While mutation of the first Y599 alone failed to reduce proliferation or STAT5 phosphorylation, mutation of both the first and duplicated Y599 significantly reduced cellular proliferation and phospho-STAT5 levels. To investigate molecular mechanisms underlying how constitutive association of Shp2 with STAT5 may promote FLT3-ITD-induced leukemogenesis, we utilized the human FLT3-ITD positive AML-derived cell line, MV411. While previous studies have demonstrated nuclear localization of Shp2 in AML samples, the role of nuclear Shp2 in leukemia has never been investigated. We utilized in situ immunofluorescence to examine nuclear distribution of Shp2 and potential co-localization with phospho-STAT5. Strong nuclear expression of Shp2 was observed in MV411 cells, and upon merging of images, nuclear Shp2 co-localized strongly with nuclear phospho-STAT5, suggesting that Shp2 may work with STAT5 within the nucleus to enhance gene expression promoting leukemogenesis. We chose to examine the BCL2L1 promoter, a STAT5-responsive promoter which regulates expression of the prosurvival protein, Bcl-X_L. Using chromatin immunoprecipitation assays, we found Shp2 is present at functional interferon-g activation sites (GAS) within the BCL2L1 promoter. Furthermore, knockdown of Shp2 in MV411 cells resulted in reduced phospho-STAT5 levels and reduced BCL2L1 promoter-directed luciferase expression. Moreover, using a novel small molecule Shp2 inhibitor, the proliferation of N51-FLT3-expressing bone marrow progenitors and primary AML samples was significantly reduced in a dose-dependent manner. Our findings suggest that constitutive association of Shp2 with N51-FLT3 promotes hyperproliferation and that either genetic disruption of Shp2 expression or mutation of the Shp2 binding sites on N51-FLT3 significantly abrogates N51-FLT3-induced hyperproliferation, STAT5 hyperactivation, and N51-FLT3-induced hematologic malignancy in vivo. Furthermore, Shp2 and STAT5 appear to work functionally in the nucleus to promote STAT5-responsive, pro-leukemogenic gene expression. Collectively, these studies demonstrate that Shp2 positively contributes to FLT3-ITD-induced leukemia and suggest that Shp2 inhibition may provide a novel therapeutic approach to AML.