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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA101828-01A1
Application #
6778748
Study Section
Cancer Molecular Pathobiology Study Section (CAMP)
Program Officer
Mufson, R Allan
Project Start
2004-07-01
Project End
2008-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
1
Fiscal Year
2004
Total Cost
$238,868
Indirect Cost
Name
University of Pittsburgh
Department
Genetics
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Panjarian, Shoghag; Iacob, Roxana E; Chen, Shugui et al. (2013) Structure and dynamic regulation of Abl kinases. J Biol Chem 288:5443-50
Pene-Dumitrescu, Teodora; Smithgall, Thomas E (2010) Expression of a Src family kinase in chronic myelogenous leukemia cells induces resistance to imatinib in a kinase-dependent manner. J Biol Chem 285:21446-57
Choi, Yongmun; Seeliger, Markus A; Panjarian, Shoghag B et al. (2009) N-myristoylated c-Abl tyrosine kinase localizes to the endoplasmic reticulum upon binding to an allosteric inhibitor. J Biol Chem 284:29005-14
Iacob, Roxana E; Pene-Dumitrescu, Teodora; Zhang, Jianming et al. (2009) Conformational disturbance in Abl kinase upon mutation and deregulation. Proc Natl Acad Sci U S A 106:1386-91
Engen, J R; Wales, T E; Hochrein, J M et al. (2008) Structure and dynamic regulation of Src-family kinases. Cell Mol Life Sci 65:3058-73
Pene-Dumitrescu, T; Peterson, L F; Donato, N J et al. (2008) An inhibitor-resistant mutant of Hck protects CML cells against the antiproliferative and apoptotic effects of the broad-spectrum Src family kinase inhibitor A-419259. Oncogene 27:7055-69
Chen, Shugui; Dumitrescu, Teodora Pene; Smithgall, Thomas E et al. (2008) Abl N-terminal cap stabilization of SH3 domain dynamics. Biochemistry 47:5795-803
Chen, Shugui; O'Reilly, Linda P; Smithgall, Thomas E et al. (2008) Tyrosine phosphorylation in the SH3 domain disrupts negative regulatory interactions within the c-Abl kinase core. J Mol Biol 383:414-23
Chen, Shugui; Brier, Sebastien; Smithgall, Thomas E et al. (2007) The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding. Protein Sci 16:572-81

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