Bcr-Abl is the oncogenic tyrosine kinase expressed as a result of the Philadelphia translocation in chronic myelogenous leukemia (CML). Src family kinase inhibitors and dominant-negative mutants block Bcr- Abl-induced transformation of myeloid progenitor cells, strongly suggesting that Bcr-Abl cooperates with Src in CML pathogenesis. Experiments proposed here will investigate the molecular mechanisms that regulate the interaction of Bcr-Abl with Src kinases, determine whether Src kinases affect Bcr-Abl kinase activity and sensitivity to the anti-CML drug STI-571 (Gleevec), and look at the genetic requirement for Src kinases in a model system for CML:
Aim 1 : Test the hypothesis that association with Bcr-Abl is sufficient to induce Src kinase activation. Recent data from our laboratory show that interaction with Bcr-Abl involves Src family kinase SH2 and SH3 domains, suggesting that association with Bcr-Abl may be sufficient to induce sustained Src kinase activation in vivo. Using fibroblast and myeloid cell transformation assays, we will test this idea by co-expressing Hck and Lyn with regions of Bcr-Abl previously shown to bind to these myeloid Src family members in vitro. Identification of the Bcr-Abl regions essential for Src family kinase activation will define a novel molecular surface for anti-CML drug design.
Aim 2 : Test the hypothesis that Src kinases phosphorylate Bcr-Abl in vivo and modulate its kinase activity and sensitivity to inhibition by STI-571. Preliminary data show that Src kinases directly phosphorylate the c-Abl kinase domain on the activation loop tyrosine in vitro. Bcr-Abl mutants lacking Src family kinase phosphorylation sites and selective inhibitors will be used to determine whether Src family kinases affect Bcr-Abl activity and sensitivity to STI-571, which selectively inhibits the inactive conformer of Abl.
Aim 3 : Test the genetic requirement for Src family kinases in Bcr-Abl signal transduction and STI-571 sensitivity. The transforming activity of Bcr-Abl will be assessed in hematopoietic colony-forming assays of bone marrow cells from mice lacking the three major myeloid Src family members (Hck/Lyn/Fgr), all of which interact with Bcr-Abl. Triple-knockout cells will also be used to address the requirement for Src family members in coupling Bcr-Abl to Stats and other downstream effectors as well as sensitivity to STI-571. Successful completion of these studies will provide strong validation of myeloid-specific Src kinases as targets for second-generation anti-CML drug development.
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