MECHANISMS OF G-CSF RECEPTOR MEDIATED DIFFERENTIATION

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DESCRIPTION: (Adapted from investigator's abstract) G-CSF is one of the most critical cytokines driving neutrophilic granulocyte differentiation. The majority of AML cases arise from this lineage. However, AML cells respond aberrantly to G-CSF by proliferating without differentiating. The basis for this aberrant response is unknown. Initial attempts to characterize the G-CSF signal transduction pathway in normal and leukemic myeloid cell lines using traditional biochemical studies yielded little information useful as a basis for comparison between normal and leukemic myeloid cells. The investigators have identified three phosphoproteins - pp55, pp70, and pp80 - that are activated and associate with the G-CSFR. The investigators have determined that pp55 is the Src-related protein tyrosine kinase (PTK), Lyn, while pp70 is the non-Src-related PTK, Syk. The investigators have identified a potential tyrosine-based activation motif (TAM) that may serve as a Syk docking site within the distal half of the cytosolic domain region of the receptor shown to be essential and specific for neutrophilic differentiation. The investigators' preliminary studies support the hypothesis that pp80 may be a novel member of the signal transducers and activators of transcription (STAT) protein family, designated StatG. The investigators demonstrated that activation of StatG did not correlate with proliferation; rather, optimal activation requires the differentiation-specific domain of the G-CSFR that contains the putative Syk docking site. In all normal human myeloid cells tested (including CD34+ bone marrow cells), G-CSF activated a DNA-binding complex, G-SIF-A, composed solely of StatG. In contrast to normal human myeloid cells, G-SIF-A in six of seven human AML cell lines and five of twelve AML patient samples contained both StatG and Stat3. These findings suggests that activation of alternative STAT proteins such as Stat3 by G-CSF in some AML cells may contribute to their failure to follow the normal G-CSF-activated differentiation program. This effect may be mediated through competition by Stat3 for StatG binding to promoter elements critical for transactivating differentiation-specific genes. The Specific Aims of this proposal are: AIM I. To purify and clone StatG and to molecularly characterize its interaction with the G-CSFR in normal myeloid cells. AIM II. To characterize Syk's interaction with the G-CSFR in normal myeloid cells and to determine its role in StatG activation, differentiation, and proliferation. AIM III. To characterize G-SIF-A in AML patient samples and to correlate the composition of G-SIF-A (StatG alone vs. StatG + Stat3) with the biologic response of cells to G-CSF, cytogenetic features, surface immunophenotype, FAB subclass, and prognosis. The long range goal of this proposal is to identify the mechanisms that account for the aberrant response to G-CSF in AML cells in order to design rational differentiation therapies targeted to overcome these abnormalities.