G-CSF is the cytokine most critical for driving neutrophil differentiation The majority of acute myeloid leukemia (AML) cases arise from this lineage. However, AML cells, unlike their normal myeloid counterparts, respond aberrantly to G-CSF by proliferating without differentiating. The basis for this aberrant response is unknown. The hypo as pursued during the previous funding period was that signaling downstream of the G-CSFR was aberrant in AML cells. G-CSFR was immunoprecipitated in human neutrophils (PMN) after ligand activation. Three phosphoproteins were identified that were activated and associated with the G-CSFR, Lyn, Syk and Stat3. In contrast to Lyn and Syk, Stat3 proved to be required for G-CSF-mediated neutrophilic differentiation in the murine myeloblast cell line model, 32DcI3. However, which Stat3 isoform is responsible for G-CSF-mediated neutrophil differentiation, Stat3a or Stat3beta (or both) has not been determined. Human CD34+cord blood mononuclear cells express predominantly Stat3a while mature PMN express predominantly Stat3beta. Stat3a but not Stat3beta is down regulated midway through the process of G-CSF-induced neutrophilic differentiation of CD34+ cord blood cells and 32Dcl3 cells resulting in a ratio similar to that observed in mature PMN. In contrast, the ratio of Stat3a to Stat3beta protein in AML cell lines is high and remains unchanged following prolonged G-CSF exposure (14 days). Most intriguingly, constitutive overexpression of Stat3a in 32Dc13 cells was shown to inhibit G-CSF-induced neutrophilic differentiation. In this competitive renewal application we will pursue the central hypothesis that the balance of Stat3a to Stat3bet activated by G-CSF in myeloid cells dictates the gene expression profile and, as a consequence, the ability of these cells to differentiate in response to this critical granulopoietin.
The Specific Aims of this proposal are: 1) to determine the effects of genetically altered Stat3a to Stat3B ratios on neutrophil differentiation and cell proliferation in 32Dcl3 cells using stable transfection of Stat3 isoforms in an inducible promoter system, 2) to determine the effects of genetically altered Stat3a to Stat3B ratios on neutrophil differentiation and myeloid progenitor cell expansion in vivo by generating transgenic mice deficient in each isoform and 3) to determine the effects of altered Stat3a and Stat3B ratios on gene expression in 32Dcl3 and in hematopoietic cells from Stat3 isoform deficient mice by directed examination of 13 known Stat3 gene targets and using cDNA microarray analysis. The goal of this proposal is to identify gene targets downstream of Stat3B and Stat3a that promote or delay G-CSF-mediated neutrophil differentiation, respectively. Identification of these target genes and how their regulation is altered in AML cells may account for the aberrant response of these cells to G-CSF.
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