Project Summary
Contact
Selected Publications

Project Summary

Aim of the collaborative activities of the Department of Bioinformatics at EMBL and the Department of Applied Tumor Biology at the University of Heidelberg are to use comprehensive data sets obtained in the frame of the human genome project to delineate potential novel diagnostic markers and therapeutic targets of tumors using state of the art bioinformatics technology. Candidate markers are being validated by the Department of Applied Tumor Biology and novel diagnostic as well as preventive / and or therapeutic strategies are designed and validated in clinical trials. These activities were in the past already part of several successful joint grant projects funded by the BMBF, the Deutsche Krebshilfe, and the European Union [6th framework]. The results of these projects have been published in several publications and led to the design of a phase I / II immunotherapy trial for colorectal cancer. As a representative example for our partnership and collaboration, we outline here our research programme on microsatellite unstable tumours with focus on colorectal cancer.

Background
Genetic instability and accumulation of mutations are a major characteristics of malignant tumors. In consequence of a defective mismatch repair system, microsatellite unstable [MSI] tumors accumulate mutations in multiple genes, particularly in regions harboring repetitive DNA sequences. If such mutations affect the coding region of microsatellites the frameshifts lead to truncated proteins or proteins with altered functions 1. In addition, the frameshifts result in expression of c-terminal neo-peptides that might induce an immune response 2-4.

Research Focus 1 [identification of coding microsatellites and frequency of frameshift mutations in MSI tumours]
In cooperation with the Bioinformatics group of P. Bork at the EMBL we have established a comprehensive database comprising all coding, untranslated and intronic mononucleotide repeats [MNRs] of the human genome. This combined effort for the first time permitted to use the complete information gathered by the human genome sequencing projects to systematically delineate potential candidate genes that may be mutated during MSI-driven carcinogenesis. We have determined the mutation frequency of particularly long coding and untranslated MNRs 5-7. Based on all available MNR mutation information we established a statistical model for the prediction of selective MSI target genes in an organ specific manner 8. Experimental analysis of predicted target genes in colorectal cancer extended to colorectal adenomas revealed that these genetic alterations are not restricted to carcinomas but already occurred early during tumorigenesis 9. We now seek to translate these findings to shorter coding MNRs. Finally, we will extend these analyses to the genome of other species with emphasis on the mouse, in order to identify common or different coding microsatellite target genes contributing to the MSI tumor pathway.

Research Focus 2 [functional analysis of selective target genes of MSI]
It is generally believed that only few of the affected genes are targets relevant to carcinogenesis. To differentiate selective MSI target genes [containing pathogenic mutations] from bystander genes [containing mutations without contribution to carcinogenesis] we recently proposed a statistical model as a tool for comprehensive analysis of mutations in MSI tumors 8. We actually prepare an online database that soon will become available as an online public database. Currently, more than 20 candidate genes, harboring potentially pathogenic mutations in MNRs 10 have been predicted by this model. These genes include TGFR2, ACVR2, BAX, EPHB2 and others. Meanwhile, EPHB2 was object of a more detailed analysis in MSI tumors where the functional inactivation of this gene was shown to promote MSI colon cancer 11. The pathophysiologic significance of frameshift mutations in other candidate genes and their role in transformation and tumor progression, however, is largely unknown. Accordingly, our current and future projects aim to functionally characterize gene products that appear to be selected for frameshift mutations in MSI tumours. In particular, we focus our research on the putative tumor suppressor and interferon inducible “absent in melanoma 2 [AIM2]” 12 and on a putative member of the nonsense mediated RNA decay [NMD] pathway [in cooperation with Prof. Dr. A. Kulozik]

Research Focus 3 [immune response against frameshift induced neopeptides]
Expression of highly mutated genes leads to the synthesis of truncated proteins with c-terminal frameshift neopeptides which can be presented in the MHC context on MSI tumor cells. Therefore, genes with coding MNRs with high mutation frequency represent ideal targets for immunological response. In immunological studies we identified several frameshift derived immunogenic peptides and subsequently showed that MSI tumor cells can be lysed in vitro by cytotoxic T-cells in a peptide specific manner 2-4. Based on these preclinical data a clinical protocol for a Phase I/II study has been established for testing dose and toxicity of a frameshift peptide vaccine in MSI tumor patients. We expect the clinical trial to be initiated in 2006.

Research Focus 4 [alternative splicing in colorectal cancer]
Production of mature mRNA transcripts in vertebrates is regulated at three stages: transcription initiation, splicing and polyadenylation. The combinatorial arrangement of variations at each stage generates, from a single gene, a variety of mRNA isoforms with different start sites, exons or 3' UTRs. Alternatively spliced mRNA isoforms and resulting truncated proteins or neo-peptides are associated with numerous diseases, including multiple sclerosis, heart failure, neurodegenerative disorders and cancer. Both MMPU partners are currently part of an EU funded multi-disciplinary collaboration project to identify and characterize alternative transcripts throughout the human genome and assess the differential expression of these forms in normal and disease related tissues with focus on colorectal and lung cancer [manuscript in preparation]. Mutations in microsatellites close to splice junctions can lead to alternative transcripts and as a consequence to tumor specific neo-peptides. The identification, the frequency of MSI and of resulting alternative transcripts as well as the diagnostic and/ or therapeutic value of those target genes will be analyzed in the MSI setting.

Contact

Magnus von Knebel Doeberitz, MD
Department of Applied Tumour Biology
Institute of Pathology
University of Heidelberg
Im Neuenheimer Feld 220/222
69120 Heidelberg
Tel: +49-6221-56 2877
Fax:+49-6221-56 5981
E-mail: knebel@med.uni-heidelberg.de
Website

Peer Bork, PhD
Joint Coordinator, Senior Group Leader
Structural and Computational Biology Unit
European Molecular Biology Laboratory
Meyerhofstraße 1
69117 Heidelberg
Tel: +49 6221 387-8526
Fax: +49 6221 387-519
E-mail: peer.bork@embl.de
Group home page

Selected Publications

1. Kloor, M., von Knebel Doeberitz, M., and Gebert, J. F. Molecular testing for microsatellite instability and its value in tumor characterization. Expert.Rev.Mol.Diagn., 5: 599-611, 2005.
2. Schwitalle, Y., Linnebacher, M., Ripberger, E., Gebert, J., and von Knebel Doeberitz, M. Immunogenic peptides generated by frameshift mutations in DNA mismatch repair-deficient cancer cells. Cancer Immun., 4: 14, 2004.
3. Ripberger, E., Linnebacher, M., Schwitalle, Y., Gebert, J., and von Knebel Doeberitz, M. Identification of an HLA-A0201-restricted CTL epitope generated by a tumor-specific frameshift mutation in a coding microsatellite of the OGT gene. J.Clin.Immunol., 23: 415-423, 2003.
4. Linnebacher, M., Gebert, J., Rudy, W., Woerner, S., Yuan, Y. P., Bork, P., and von Knebel Doeberitz, M. Frameshift peptide-derived T-cell epitopes: a source of novel tumor-specific antigens. Int.J.Cancer, 93: 6-11, 2001.
5. Woerner, S. M., Gebert, J., Yuan, Y. P., Sutter, C., Ridder, R., Bork, P., and von Knebel Doeberitz, M. Systematic identification of genes with coding microsatellites mutated in DNA mismatch repair-deficient cancer cells. Int.J.Cancer, 93: 12-19, 2001.
6. Sutter, C., Dallenbach-Hellweg, G., Schmidt, D., Baehring, J., Bielau, S., von Knebel Doeberitz, M., and Gebert, J. Molecular analysis of endometrial hyperplasia in HNPCC-suspicious patients may predict progression to endometrial carcinoma. Int.J.Gynecol.Pathol., 23: 18-25, 2004.
7. Findeisen, P., Kloor, M., Merx, S., Sutter, C., Woerner, S. M., Dostmann, N., Benner, A., Dondog, B., Pawlita, M., Dippold, W., Wagner, R., Gebert, J., and von Knebel Doeberitz, M. T25 repeat in the 3' untranslated region of the CASP2 gene: a sensitive and specific marker for microsatellite instability in colorectal cancer. Cancer Res., 65: 8072-8078, 2005.
8. Woerner, S. M., Benner, A., Sutter, C., Schiller, M., Yuan, Y. P., Keller, G., Bork, P., Doeberitz, M. K., and Gebert, J. F. Pathogenesis of DNA repair-deficient cancers: a statistical meta-analysis of putative Real Common Target genes. Oncogene, 22: 2226-2235, 2003.
9. Woerner, S. M., Kloor, M., Mueller, A., Rueschoff, J., Friedrichs, N., Buettner, R., Buzello, M., Kienle, P., Knaebel, H. P., Kunstmann, E., Pagenstecher, C., Schackert, H. K., Moslein, G., Vogelsang, H., von Knebel Doeberitz, M., and Gebert, J. F. Microsatellite instability of selective target genes in HNPCC-associated colon adenomas. Oncogene, 24: 2525-2535, 2005.
10. Woerner, S. M., Benner, A., Yan, P., Yuan, Y. P., Bork, P., von Knebel Doeberitz, M., and Gebert, J. Improved prediction of selective target genes of mismatch repair deficient tumorigenesis. 2006 [submitted].
11. Alazzouzi, H., Davalos, V., Kokko, A., Domingo, E., Woerner, S. M., Wilson, A. J., Konrad, L., Laiho, P., Espin, E., Armengol, M., Imai, K., Yamamoto, H., Mariadason, J. M., Gebert, J. F., Aaltonen, L. A., Schwartz S Jr, and Arango, D. Mechanisms of inactivation of the receptor tyrosine kinase EPHB2 in colorectal tumors. Cancer Res., 65: 10170-10173, 2005.
12. Dihlmann, S., Kloor, M., Woerner, S., Youmans, H., and von Knebel Doeberitz, M. Inactivation of "Absent in Melanoma 2 [AIM2]" by genetic and epigenetic mechanisms in mismatch repair deficient colon carcinomas. Cancer Res. 2006 [submitted].