PI3K SYSTEMS BIOLOGY · In-depth quantification and characterisation of PI 3-kinase signalling networks at a System Biology level

In-depth quantification and characterisation of PI 3-kinase signalling networks at a system biology level

This Marie Curie post-doctoral fellowship was focused on a family of proteins named phosphoinositide 3-kinases (PI3K). It is known that members of this family of enzymes are overactive in a large number of diseases, including different types of cancer, metabolic syndromes (such as diabetes), and inflammation. Enzymes in the PI3K pathway are therefore attractive targets for the development of therapies against a large array of diseases. Advancing these developmental efforts will benefit from a better understanding of how PI3Ks modulate the biochemistry of cells in health and disease. Hence, in our project PI3K SYSTEMS BIOLOGY, we decided to characterise, as comprehensively as possible, the biochemical pathways downstream of these proteins. To this end, we took a completely unbiased approach to the biochemical analysis of cells in which the activity of PI3Ks had been modulated by either pharmacological inhibitors or by genetic means. In-depth analysis of kinase pathways is now possible due to the recent development of techniques based on mass spectrometry. These methods allow the detection and quantification of thousands of phosphorylation sites on proteins in relatively short times. Since, by definition, each phosphorylation site is the result of a kinase activity, these large-scale phosphoproteomics approaches allow investigating kinase signalling without a preconception of the pathways or modules in the biochemical network that may be affected by perturbations to the system. Prior to the start of this project, several groups had reported the development of different techniques for phosphoproteomics. Published methods for phosphoproteomics required labelling of proteins (either metabolically or chemically) with amino acids or tags enriched with heavy isotopes of carbon and nitrogen. These methods, although useful in other contexts, were not completely suitable for this project because we needed to compare large number of samples and we also wished to analyse the role of signalling enzymes in primary tissues. Therefore, the first part of the project involved the refinement of biochemical extraction procedures for enrichment phosphopeptides in a reproducible manner. Such phosphopeptides could then be quantified reliably using label-free techniques based on mass spectrometry (Montoya et al., Methods, 2011). We also modified a computer program, termed Pescal, which the host group had written for the analysis of mass spectrometry data. This improvement consisted in comparing the theoretical isotopic distribution of peptide masses to the calculated ones, thus providing the basis for more accurate analysis. Overall, these developmental efforts contributed to add robustness to a workflow for label-free phosphoproteomics which was then used for the rest of this project. Importantly, in addition to contributing to the work of this project, this refined technology was also used by other members of the host group and by the host department, including other Marie Curie projects. The technology that this project contributed to refine was licensed to a company named Activiomics (see http://www.activiomics.com online for further details). This is a spin-out from the host university which offers phosphoproteomics analyses services to biotechnology and pharmaceutical client companies. Therefore, this project also had a positive impact on European Union (EU)'s competitiveness in the biotechnology sector. As for the biochemical research directly related to this project, we identified several novel phosphorylation sites modulated by different inhibitors that target specific PI3K isoforms in cells derived from mice. These results are currently being collated into a research paper for publication. We also identified novel phosphorylation sites downstream of PI3K inhibitors (and inhibitors of kinases in pathways parallel to PI3K) in leukemia cells. This work (Alcolea et al., MCP, 2012) showed that sensitivity of resistance of cancer cells to kinase inhibitors is not solely dependent on the activity of the pathway being targeted, and that the activity of parallel kinase pathways contributes to how well cell cancer cells respond to therapies that target the kinase network. Measuring the activities of kinase pathways within the network as comprehensively as possible (potentially using techniques similar to those derived from this and other projects in the host laboratory) would be beneficial in predicting the best therapy for each cancer patient. As a continuation of this work, we showed that our refined techniques for large-scale phosphoproteomics could be used to measure the activity of kinase pathways in a highly parallel fashion in primary cancer cells. We thus profiled 20 cases of primary leukaemias and discovered specific phosphoproteomics signatures that predicted the sensitivity of these cells to PI3K inhibitors in clinical development (Casado et al., Science Signalling, in press). We believe that these proof-of-concept studies will have a positive impact in the development of personalised cancer medicine, thus, in turn, impacting on the well-being of society in general. In summary, this Marie Curie project participated in the refinement of techniques for label-free phosphoproteomics. These methods were then used to identify novel proteins downstream of PI3K and to study the contribution of PI3K and related pathways to cancer from a system biology standpoint. In addition to addressing its original objectives, our work resulted in know-how that supported the progression of other projects in the host institution; this refined expertise will also serve as the basis for future projects. Therefore, we are convinced that the impact of the PI3K SYSTEMS BIOLOGY project will reach beyond its original objectives.

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