Abstract B75: Allosteric regulation of Abl and Bcr-Abl kinases by enhanced SH3:linker interaction.
Authors: Panjarian Shoghag B. , Chen Shugui , Iacob Roxana , Wales Thomas E. , Engen John R. , Smithgall Thomas E. 
Published: June 2012

Abstract
Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinical management of CML has been revolutionized by imatinib, a selective ATP-competitive inhibitor of Bcr-Abl kinase activity. Despite the clinical success of imatinib in the chronic stage of CML, it is less effective in advanced disease due to the emergence of drug resistance often caused by point mutations in the Bcr-Abl kinase domain. Recently, we demonstrated that introduction of the “gatekeeper” resistance mutation T315I into a recombinant c-Abl “core” protein (consisting of the myristoylated N-Cap, SH3, SH2 and kinase domains) enhanced basal kinase activity. Imatinib selectivity is achieved in part by binding and stabilizing the unique inactive conformation of the Abl kinase domain. This conformation is stabilized allosterically by intramolecular binding of the SH3 domain to the SH2-kinase linker, which in turn docks against the back of the kinase domain. The goal of this project, therefore, was to determine whether kinase activation induced by T315I, as well as a mutation in a recently described allosteric inhibitor binding site in the kinase domain C-lobe (A356N mutant), could be overcome by enhancing the SH3:linker interaction. For these experiments, we engineered ten recombinant High Affinity Linker (HAL) proteins based on the c-Abl regulatory region (SH3-SH2-Linker) in which SH3:linker interaction was tightened through sequential addition of proline residues to the linker. Enhanced SH3:linker interaction was confirmed by analysis of SH3 dynamics using hydrogen-deuterium exchange mass spectrometry. We then incorporated the HAL coding sequences into the c-Abl core, and assessed kinase activation following transfection of 293T cells. Immunoblot analysis with autophosphorylation site-specific antibodies showed that five of the Abl-HAL core proteins remained inactive, suggesting that linker proline substitution did not alter the downregulated conformation. To determine the impact of the HAL sequences on activating mutations in the kinase domain, the strongest HAL sequence was combined with the T315I and A356N mutations. Remarkably, the HAL substitution completely reversed Abl core activation by the A356N mutation, and partially reversed the effect of T315I. We next introduced the HAL sequence into p210 Bcr-Abl coding region (wild-type and T315I), and expressed these forms of Bcr-Abl along with matched controls in the human myeloid leukemia cell line, TF-1. The Bcr-Abl HAL mutants transformed TF-1 cells to a cytokine-independent phenotype as effectively as wild-type Bcr-Abl but were much more sensitive to induction of apoptosis by the allosteric inhibitor, GNF-2. Furthermore, TF-1 cells transformed with Bcr-Abl-HAL bearing a wild-type kinase domain also showed greater sensitivity to imatinib and the related inhibitor nilotinib, suggesting that enhanced SH3:linker interaction structures the kinase domain in a favorable conformation for drug action. Our results provide new evidence that SH3:linker interaction not only has a strong allosteric effect on the regulation of the c-Abl kinase core, but also on Bcr-Abl sensitivity to both ATP-competitive and allosteric inhibitors as well.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B75.