Chronic myelogenous leukemia is characterized by the presence of the chimeric p210 bcr/abl protein which shows elevated protein tyrosine kinase activity relative to the normal c-abl tyrosine kinase. Hematopoietic progenitors isolated from CML patients in the chronic phase contain constitutively tyrosine phosphorylated protein, GAT62 (GAP-associated with tyrosine phosphorylated p62), that migrates at 62 KD by SDS page and associates with p120 bcr/abl GTPase-activating protein (GAP). The gene encoding for this protein has very recently been identified. The focus of this proposal is to elucidate the role of the GAT62 protein in development and hemopoiesis, and to clarify how GAT62 function relates to leukemogenesis with the following Specific Aims: 1. To define in knock out mice and null ES cell lines, the role of GAT62 in ontogenesis and hemopoiesis. We will characterize GAT62 expression in the developing embryo and in the adult mouse. Using homologous recombination technology, we will disrupt the GAT62 gene in mouse Embryonic Stem (ES) cells and mice or embryos homozygous for the GAT62 inactivating mutation will be generated and studied. We will define the developmental role of the GAT62 gene by characterizing the embryonic or adult phenotype resulting from its inactivation. We will specifically study hemopoiesis in GAT62 mutants. Different experimental approaches will be undertaken depending on whether mice lacking GAT62 re viable or on the stage of embryonic development at which they die. In parallel, we will produce GAT62 null ES cells and study the capacity of these cells to differentiate towards hemopoietic precursors in vitro and in vivo in chimeras and RAG-/- complementation assays. 2) To establish the role of GAT62 in leukemia promotion and progression. We will define whether the tyrosine phosphorylation and the resulting functional activation of GAT62 by the bcr/abl fusion products is a crucial event in CML and ALL (Acute Lymphoblastic Leukemia) leukemogenesis. To this end we will interbreed transgenic mice harboring the p210 bcr/abl and p190 bcr/abl fusion genes in the germ line, that develop leukemias through the activity of the chimeric proteins, with the GAT62 knockout mice in order to generate transgenes for the p210 bcr/abl and p120 bcr/abl fusion molecules in a GAT6 /- and -/- genetic background. Hemopoiesis and leukemogenesis in the resulting transgene combinations will be analyzed.
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