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by Antje Lazarovic last modified 2009-03-31 11:52

Sub-projects of the IG MUTANOM:

Project coordination (TP 1), Prof. Dr. Hans Lehrach, PD Dr. Bodo Lange

It is the goal of this subproject to coordinate and integrate the overall research effort, available infrastructures and resources by employing and combining existing management and research structures with expertise in the field of functional genomics and health research. Communication, reporting and exchange of data are central components in this subproject. We will organise regular project meetings (among the members of the IG) as well as local meetings (on a national level) in order to disseminate knowledge and open the consortium to other research groups and other IGs e.g. as part of the NGFN Plus and NGFN transfer research funding programs „Integrierte Verbünde der medizinischen Genomforschung“. It is the goal of the IG-Mutanom to apply a Systems Biology approach to analyse complex diseases using cancer as a prototypical problem.

Translational Health Research
(TP 2), Prof. Dr. Angela Brand

The subproject aims to advance the translational research through various fields of science and the humanities (biomedicine, biotechnology, bioinformatics, biology, philosophy/social ethics & bioethics, social and political sciences, economics, public health law, epidemiology/biostatistics, public health). So far, innovative genome-based technologies are struggling to reach patients as the basic research sciences are not conceptually linked to health systems research and Public Health. For the benefit of patients the subproject aims to facilitate a process which is using the methodology of health technology assessment (HTA) to translate the results of Mutanom into valuable diagnostic and therapeutic options. In cooperation with the other subprojects the European Centre for Public Health Genomics (ECPHG) at the Faculty of Health, Medicine and Life Sciences of the University of Maastricht / Netherlands will ensure the effective and responsible use of the upcoming knowledge. The subproject will also communicate the results with various stakeholders from politics, ethics, funding bodies the clinical sector and Public Health. The outcomes will include a generic methodology for the assessment of innovative technologies in genomics and a HTA report for the Mutanom applications.

Mutational analysis (TP 3), Dr. Michal Schweiger

For the mutational profiling approach we are interested in fundamental cancer relevant pathways. Once matured, the developed technologies and models will be easily transferable and applicable to other genetic diseases. However, in this project we will focus on sequencing cancer relevant genes and regulatory sequences of a broad spectrum of different tumors and cell lines to detect common aberrations. The sequencing of established cancer cell lines will help to choose optimal cell lines for further proteomic and functional analyses. Different tumor tissues will be provided from our clinical partners. For a comprehensive characterization of the genetic background of the cancer cell lines and tumor tissues we will apply a new, highly sensitive massively parallel sequencing-by-synthesis approach, expected to allow the analysis of up to 8 different samples generating a total of up to 40 million reads at an estimated cost of 3000 $. Since the sequence analysis is based on the sequencing of the amplification products of single DNA molecules, this approach in a sense provides ‘digital’ information. It is therefore considerably less error-prone to the ‘contamination’ of tumor tissue with normal tissue, since this will reduce the frequency of detecting the mutant molecule, rather than reduce the signal of the mutant form, like in normal sequencing or array based analysis procedures

Recombinant cancer cell libraries & drug target recovery (TP 4), Prof. Dr. Jan Mollenhauer

In previous work, we developed a system utilizing site-specific recombination for the serial construction of isogenic cancer cell lines. Starting from an acceptor cell line that is correspondingly modified for site-specific recombination, stable recombinants with either constitutive or inducible over-expression or knock-down of cellular genes of interest can be constructed. Within IG-Mutanom, we pursue four major goals. Firstly, in tight cooperation with the other partners, the system will be modified to expand its use for the serial analysis of protein-protein interaction networks of cancer genes and its mutated variants. Secondly, we aim at constructing acceptor cell lines for the cancer backgrounds relevant to IG-Mutanom, i. e. breast, prostate, and gastrointestinal cancer. At third, these will be used to construct libraries of recombinant isogenic cancer cells, which will subsequently be used to quantify the effects of cancer genes and their mutated variants on cell viability with the goal to identify effectors in different cancer backgrounds. Finally, the most promising configurations resulting from these screens will be used to design synthetic lethal screens, which aim at recovering drug targets, whose inactivation would kill cancer cells but not normal cells. This subproject provides and develops core technologies facilitating the systems biological approach and aims at converting systems biological into translational data via the recovery of novel drug targets.

Quantification of cancer pathways (TP 5), Dr. Ulrike Korf, PD Dr. Holger Sültmann

The goal of this subproject is to systematically determine the consequence of frequent mutations in cancer on the abundances and activation states of key proteins. The effects of the mutation will be determined at the level of the modulation of the activity states of important signalling pathways. To this end, the technology of protein arrays will be utilized. The resulting resources and data sets will be funneled into the analyses and modelling efforts, respectively, of the other subprojects of the IG-MUTANOM

Protein interaction networks (TP 6), Prof. Erich Wanker

Protein-tyrosine kinases (PTKs) are important regulators of intracellular signal-transduction pathways and their activity is usually tightly controlled and regulated. Perturbation of PTK signalling by mutations and other genetic alterations results in deregulated kinase activity and malignant transformation. In this sub-project, we aim at the generation of a comprehensive protein interaction network for human cytoplasmic protein-tyrosine kinases and their mutant counterparts that were identified through bioinformatics and sequencing approaches. For this we will use high-throughput yeast two-hybrid protein-protein interaction (PPI) screening, mass spectrometry-based proteomics and bioinformatics. The protein complex information will then be utilized for the systematic identification of modulators of Ras-Raf-ERK, JNK-STAT and PI(3)K signalling using cell-based reporter assays. This includes systematic RNAi knock-down as well as overexpression experiments with established cell model systems in order to identify proteins that influence signalling in human malignancies.
The results of this sub-project are expected to help our partners within the MUTANOM consortium to identify new tumor suppressors or oncogenes in patient genotyping and phenotypic analyses. Furthermore, this sub-project may contribute to the elucidation of new pathways in oncogenic kinase signalling and the identification of new drug targets for therapy development.

Cellular signalling networks (TP 7), Prof. Reinhold Schäfer

The task of subproject 7 is to investigate the roles of candidate genes in controlling proliferation, cellular survival and various neoplastic properties. The standard approach for testing putative oncogenes is to express the candidate gene and its mutated derivative under the control of heterologous promoters in appropriate recipient cells and to assess their impact on cellular parameters, typically associated with the transformed state. Proliferation without anchorage is a stringent feature of cells with tumorigenic potential and will be used as a convenient and reliable read-out. Putative tumor suppressing activity of candidate genes will be assayed in tumorigenic cell lines by RNA interference. A typical experiment involves silencing of the gene in question in a transient manner, followed by analysis of growth characteristics in standard culture and on poly-hema-coated vessels that abolish contact of cells to their substratum and selectively permit proliferation of transformed cells. Several alternatives to this approach will be tested. Gene silencing can be achieved in a stable fashion to monitor the long-term effects of gene ablation. The candidate transformation suppressor can also be assayed in normal recipient cells by co-expression with an established oncogene such as mutated Ras. This read-out is based on the capacity of the candidate to directly counteract oncogenic signal transduction. At the molecular level, we will assess candidate gene effects on receptor tyrosine kinase/Ras/ MAP-kinase signal transduction and related pathways. To begin to understand candidate gene effects at the systems level, we will investigate their impact on the genetic program of cells expressing the candidate cancer or anti-cancer gene by expression profiling. To this end, we will use a previously characterized customized DNA microarray, representing validated and functionally annotated Ras pathway targets.

Mouse disease models (TP 8), Prof. Bernhard Herrmann, Dr. Markus Morkel

The aim of our subproject is to determine the effect of selected disease-related mutations in-vivo, using advanced mouse tumor models. We employ Recombination-Mediated & Cassette
Exchange(RMCE) to integrate mutant versions of oncogenes, tumor suppressors & and other disease-related genes into the genome in a controlled manner. Importantly, our mouse models provide (i) uniform genetic background (C57Bl/6), (ii) fast integration of transgenes into a defined locus (ROSA26), (iii) tissue-specific transgene expression, and (iv) conditional transgene activation and repression via Tet-ON/OFF. Mice containing & these novel inducible transgenes can be crossed to existing tumor models in C57Bl/6 background, for instance to APCmin mice. The possibility of systematic in-vivo analysis of the roles of selected mutations in the mouse will likely unravel important aspects of tumor biology that cannot be assessed in cell culture or in retrospective analysis of human tumor samples. Our studies thus provide an essential link between the cell-based assays and future clinical studies.

Protein complex composition and function in disease (TP 9), PD Dr. Bodo Lange

This subproject aims to analyse the effect of disease relevant mutations on the composition and function of protein complexes. The project will deliver biochemical and functional data on the effect of genetic changes identified in cancer tissue or cancer cell lines.
The goals of this subproject are:
(1) to characterize protein-protein interactions identified under “wild type” versus cancer-relevant “mutant” conditions, pertinent to cell proliferation, cell transformation and metastasis;
(2) to define the biochemical and functional consequences of “mutant” protein-protein interactions, using tissue culture systems of isogenic cancer cell lines;
(3) to define the consequence of physiological (cancer-relevant) cues (chemicals, stress) on protein-protein interactions and their posttranslational modifications.

The contribution of this project to the overall goal of the IG-Mutanom is the provision of bicochemical and functional data for the modelling of disease related signalling pathways. This, together with data from the other subprojects, will be used in the development of models predicting the consequences of genetic somatic aberrations in cells.

Data integration and modelling (TP 10), Christoph Wierling, Dr. Ralf Herwig

Delivers the computational framework for the IG, in particular an integrated framework for data analysis and correlation, comprehensive network annotations of reactions affected by mutated cancer genes and the kinetic modelling of relevant network modules. In the course of this IG we plan 1.) to integrate the heterogeneous project data types in an existing, XML-based data integration system by implementing new XML schemas and digesters for the indexing process, 2.) to extend the pathway database integration system CPDB with information on mutated genes and 3.) to develop a predictive cancer model based on intensive parameter fitting with experimental data.

Quantitative Proteomics (TP 11), PD Dr. Gerard Drewes, Dr. Gerard Jobety

The goal of this sub-project is to elucidate the mechanisms by which mutations in proteins in tumors cause a de-regulation of their associated protein complexes. To this goal, a selection of the mutant proteins studied by the IG-Mutanom consortium will be subjected to quantitative interactome analysis. In the first step, protein tagging and affinity chromatography will be employed to isolate native protein complexes formed by the mutated and wild-type proteins from cultured tumor cell lines. In the next step, we will then use state-of-the-art quantitative mass spectrometry to map all proteins in the purified protein complex and to quantify their relative amounts. By directly comparing the data obtained for wildtype and mutated proteins, differences in the stoichiometry of the complexes will be calculated. From these data, the molecular basis of the abnormal signal transduction in the tumor can be deduced. Together with additional orthogonal data generated by IG-Mutanom, this should enable the establishment of immunoaffinity-based analysis methods for tumor biopsies with the final goal of developing assays for diagnostic and compound screening purposes.


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