Abstract 5599: Allosteric regulation of Abl and Bcr-Abl kinases by enhanced SH3:linker interaction

Abstract

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 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 nine High Affinity Linker (HAL) variants of Abl 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. Using cell based assays, we identified Abl-HAL core proteins that remain locked in an inactive 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 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 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5599. doi:1538-7445.AM2012-5599