Therapeutic targeting of Src kinase signal transduction pathways in AML

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DESCRIPTION (provided by applicant): Acute myeloid leukemia (AML) is a malignant disease of the blood with a poor prognosis. The pathogenesis of AML is incompletely understood but current models propose that pathologic, uncontrolled activation of signal transduction pathways is necessary for development of AML. However, the mechanism(s) of activation of signaling pathways in the majority of AML patient samples is not clear. A subset of AML cells contain mutations in the cell surface tyrosine kinase, Flt3, and recent results demonstrate that targeting Flt3 clinically with a Flt3 kinase inhibitor leads to therapeutic benefit. These important findings demonstrate that inhibition of activated signaling pathways in AML is an important approach to therapy of the disease. We undertook an unbiased approach to identify activated signaling molecules in AML. We performed a phospho-protein analysis of tyrosine phosphorylated proteins from 6 AML patient samples. Interestingly, this analysis demonstrated phosphorylation of both lyn and lck tyrosine kinases in AML cells suggesting that Lyn and Lck, two members of the Src family kinases (SFKs) may contribute to activation of signaling pathways in AML. Kinase assays on primary AML cells demonstrate that lck is activated in 80% of AML samples studied and previous work has demonstrated activation of Lyn in the majority of AML cells. To initially determine if SFK's are necessary for AML growth and survival we performed three experiments. "Knock-down" of either Lyn or Lck using siRNA approaches leads to decreased AML colony forming activity. Additionally, in vitro, the Src inhibitor, Dasatinib inhibited the growth of 2/3 samples tested. In vivo, using a xenotransplantation model fo AML, Dasatinib had cytostatic effects over a 14 day treatment period. These initial results demonstrate that SFKs are an attractive target for pathologic signaling in AML cells and a target for therapy. Aims 1 and 2 below will explore these ideas in more detail. Furthermore, biochemical analysis shows that Lyn and Lck each co-immunoprecipitate with STAT5. Based on this data, we hypothesize that there is aberrant signaling in AML cells through a Src family kinase (SFK)-STAT5 signaling pathway. We have initiated functional studies of this pathway. Curiously, decreased expression of STAT5 has only a modest effect on AML colony forming assay. Despite this, decreased expression of STAT5 leads to altered expression of a number of genes including a subunit of PI3 kinase not normally expressed in hematopoietic cells designated PIK3R3, and the cyclin inhibitor p21. Specific Aims 3 and 4 will determine if STAT5 is a direct target of SFK's and what the important targets of STAT5 are in primary AML cells. These complementary approaches will lead to a better understanding of the pathogenesis of AML and improved therapies. PUBLIC HEALTH RELEVANCE: Acute myeloid leukemia (AML) is a life threatening disease that kills thousands of Americans each year including Veterans. One of the features required for development of AML is a dysregulation of the growth promoting signals inside leukemic cells. Whereas normal cells can turn these pathways on and off, in leukemic cells, they are always on leading to altered cell growth. In this application, we propose to study the pathways that are dysregulated in AML and test drugs that may turn them off. If we are successful, such drugs can be used for the therapy of AML in the future.