Physical and Functional Map of Bcr-Abl Signalling

The postgenomic perspectives and technologies call for a reassessment of familiar cellular processes in a comprehensive, parallel and integrative way. Despite years of studies and considerable insight into the molecular mechanism of action of the oncogenic Bcr-Abl fusion kinase, we still lack a detailed map of its effectors and their corresponding pathways. Knowledge is patchy and is currently reassembled conceptually by individual observations done by different laboratories using different cellular and experimental systems. In light of the importance of the problem, we propose here to elucidate a systematic comprehensive and coherent map of Bcr-Abl signalling using, for the first time, a defined standardized cellular setting that could be exploited to develop strategies for combination therapy or enhance the effectiveness of second-generation Bcr-Abl inhibitors. Constitutive tyrosine kinase activity is central to the capacity of Bcr-Abl to transform haematopoietic cells, leading to chronic myelogenous leukemia (CML). This forms the basis for the unprecedented clinical success of the small- molecule Bcr-Abl inhibitor Imatinib/Gleevec that has become a paradigm for modern targeted cancer therapy. Despite this success, many patients develop resistance to Imatinib due to point mutations in the Abl kinase domain. The outlined project represents a unique opportunity to exploit recent insights and newly developed tools to orchestrate a comprehensive, interdisciplinary and explorative approach to further understand signal transduction of Bcr-Abl at the physical and functional level. Combining protein complex purification with mass-spectrometry identification followed by thorough functional analysis and validation holds the opportunity to identify novel pivotal elements in the pathway that could represent potential targets for alternative or combination therapy with Imatinib. This also offers the possibility to define disease-stage specific differences and to monitor and predict disease progression. The project plan we are proposing here aims at i) the generation of a physical protein-protein interaction map of the Bcr-Abl signalling pathway in one cell type and under standardized conditions. We will tackle this objective by purifying protein complexes by tandem affinity purification coupled with liquid chromatography tandem mass spectrometry (TAP-LC-MS/MS) followed by bioinformatic network analysis and ii) RNAi-perturbation studies on candidate novel and pivotal network elements for functional validation and mapping. iii) Last, the quantitative characterization of selected pivotal pathway components will sharpen the understanding of Bcr-Abl pathway stoichiometry and will be the first step towards a comprehensive mathematical model of Bcr-Abl signalling. These approaches will reconcile genetic and biochemical data on Bcr-Abl signalling, consolidate a unified picture of the diverse existing data points on Bcr-Abl signalling and permit the identification of new therapeutic intervention points of the Bcr-Abl signalling network with pharmacological potential in Imatinib-complementing CML therapies.

The postgenomic perspectives and technologies call for a reassessment of familiar cellular processes in a comprehensive, parallel and integrative way. Despite years of studies and considerable insight into the molecular mechanism of action of the oncogenic Bcr-Abl fusion kinase, we still lack a detailed map of its effectors and their corresponding pathways. Knowledge is patchy and is currently reassembled conceptually by individual observations done by different laboratories using different cellular and experimental systems. In light of the importance of the problem, we propose here to elucidate a systematic comprehensive and coherent map of Bcr-Abl signalling using, for the first time, a defined standardized cellular setting that could be exploited to develop strategies for combination therapy or enhance the effectiveness of second-generation Bcr-Abl inhibitors. Constitutive tyrosine kinase activity is central to the capacity of Bcr-Abl to transform haematopoietic cells, leading to chronic myelogenous leukemia (CML). This forms the basis for the unprecedented clinical success of the small- molecule Bcr-Abl inhibitor Imatinib/Gleevec that has become a paradigm for modern targeted cancer therapy. Despite this success, many patients develop resistance to Imatinib due to point mutations in the Abl kinase domain. The outlined project represents a unique opportunity to exploit recent insights and newly developed tools to orchestrate a comprehensive, interdisciplinary and explorative approach to further understand signal transduction of Bcr-Abl at the physical and functional level. Combining protein complex purification with mass-spectrometry identification followed by thorough functional analysis and validation holds the opportunity to identify novel pivotal elements in the pathway that could represent potential targets for alternative or combination therapy with Imatinib. This also offers the possibility to define disease-stage specific differences and to monitor and predict disease progression. The project plan we are proposing here aims at i) the generation of a physical protein-protein interaction map of the Bcr-Abl signalling pathway in one cell type and under standardized conditions. We will tackle this objective by purifying protein complexes by tandem affinity purification coupled with liquid chromatography tandem mass spectrometry (TAP-LC-MS/MS) followed by bioinformatic network analysis and ii) RNAi-perturbation studies on candidate novel and pivotal network elements for functional validation and mapping. iii) Last, the quantitative characterization of selected pivotal pathway components will sharpen the understanding of Bcr-Abl pathway stoichiometry and will be the first step towards a comprehensive mathematical model of Bcr-Abl signalling. These approaches will reconcile genetic and biochemical data on Bcr-Abl signalling, consolidate a unified picture of the diverse existing data points on Bcr-Abl signalling and permit the identification of new therapeutic intervention points of the Bcr-Abl signalling network with pharmacological potential in Imatinib-complementing CML therapies.