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Molecular Oncology I – Hematooncology

Mgr. Martin Trbušek, Dr.
Research Group Leader

THEMATIC RESEARCH FOCUS

RESEARCH AREAS

• Role of tumour suppressor p53 in cancer
• Pathogenesis and therapy of chronic lymphocytic leukemia
• Drug testing on cancer cells

MAIN OBJECTIVES

• Mapping of key genetic defects in cancer cells. Genetic analyses of cancer cells in relation to the prediction of therapy response and resistance to modern anti-cancer drugs. Stratification of cancer patients for DNA-damaging and/or biological therapy. Detailed mapping of TP53 and ATM defects in cancer cells, including thorough functional, mutational, and cytogenetic analysis.
• Drug testing on selected high-risk cancer cells.

CONTENT OF RESEARCH

Development of novel therapeutic strategies for high-risk cancer patients

Genetic abnormalities of cancer cells that confer drug resistance have been attracting intensive interest for decades. Alterations of key tumour-suppressor gene, i.e. TP53 (coding for p53 protein) is the most common type of defect, which substantially reduces therapeutic options. For the study of novel approaches aiming at the elimination of high-risk cancer cells with p53 mutation, we intend to use chronic lymphocytic leukemia (CLL) as a model system. This leukemia, the most frequent type of all leukemias in the Western world, is still an incurable disease. Clinical heterogeneity is its characteristic feature, with the worst outcome being clearly associated with defects in TP53. Its inactivation (monoallelic or biallelic) almost inevitably leads to requirement of therapy, which, however, almost uniformly fails. Bad prognosis and a somewhat delayed poor therapeutic response are also associated with abnormalities (mutation, complete inactivation) of the ATM gene. This gene codes for a kinase cooperating with p53 after the induction of ds-DNA damage.

This work package will consist of the following complementary steps:

(1) detailed mapping of TP53 and ATM defects in CLL cells, including thorough functional, mutational and cytogenetic analysis; this continuous characterisation will provide a stable source of suitable cell cultures for testing, because virtually no permanent CLL cell lines exist;

(2) expression profiling (protein-coding genes, miRNAs) of CLL cells after drug administration, with the aim of identifying molecular determinants of resistance, in addition to TP53 and ATM defects;

(3) in vitro (cell culture) and in vivo (mouse model) testing of characterised CLL cells for their sensitivity to drugs and experimental compounds.

The following types of agents will be tested: (a) current modern drugs, which are used in clinical practice including B-cell-directed monoclonal antibodies combined with DNA-damaging agents; (b) commercially available preclinical chemical compounds, e.g. agents for wt-p53 stabilisation, reactivation of p53-mutated protein, drug transport, CDK inhibitors and possibly others; (c) chemical inhibitors of DNA damage repair, which have been shown recently to be enhanced in the resistant CLL cells, e.g. inhibitors of DNA-PK, ATM, Chk1, Chk2 and potentially other proteins involved; (d) agents (siRNAs possibly followed in selected cases by specifically designed chemical compounds) targeting newly identified (in this project) targets in CLL cells conferring resistence to DNA damage or to other selected therapeutic agents.

The outcome of the project is expected in several areas: (a) improvement of the current therapeutic protocols used for the treatment of high-risk CLL patients with the aim of improving their disease-free interval and overall survival; (b) suggestions for certain novel therapeutic strategies to overcome a block in the ATM/p53 functioning in CLL cells and possibly other cancer cells; (c) the identification of novel markers of drug resistance and testing of their utility for therapeutic purposes. Not only publications, but also patent applications are expected in the last two areas.

Technologies used: functional assay for TP53 gene (FASAY), functional assay for ATM activity towards p53 protein (phospho-sites specific Western-blotting), mutation and deletion analyses of TP53 and ATM genes (direkt sequencing, resequencing microarrays), expression microarrays for genes and microRNAs, tests of cellular viability, xenograft mouse model of CLL (NOD/SCID mice, sub-lethaly irradiated), bioinformatic processing, molecular modelling.

KEY RESEARCH EQUIPMENT

PLANNED RESEARCH INFRASTRUCTURE

Core Facility

The research group will be one of the principal users of the equipment available within CEITEC Genomics Core Facility.

Technology Units

Molecular oncology

CURRENT RESEARCH INFRASTRUCTURE

Clean laboratories (class II) for work with tissue culture cell lines and primary cells, which are fully equipped (e.g. for work with viral constructs), facility for cellular transfections (Lonza), clean laboratories for work with immunodeficient mice and their treatment, equipment for a complete work with proteins, including 2-D electrophoresis, 7300 Real-Time PCR System, VICTOR (multiplate reader), basic (common) equipment for a complete work with DNA and RNA.

MAIN PROJECTS

• In vitro sensitivity/resistance of CLL cells with inactivated p53 on modern therapy (NR8445), Ministry of Health, 2005-2007, Martin Trbušek, University Hospital Brno.
• Sensitisation of B-CLL cells to cytostatics by monoclonal antibody rituximab –identification of responsible proteins (NR9301), Ministry of Health, 2007-2009, Martin Trbušek, University Hospital Brno.
• Prognostic stratification of CLL patients according to genetic alterations of TP53 gene – detection of mutations using functional analysis in combination with modern approaches (NS9858), Ministry of Health, 2009-2011, Martin Trbušek, the University Hospital Brno.

SELECTED PUBLICATIONS

• TRBUSEK, M., SMARDOVA, J., MALCIKOVA, J., et all. Missense mutations located in structural p53 DNA-binding motifs are associated with extremely poor survival in chronic lymphocytic leukemia. J. Clin. Oncol. 2011, 29, p. 2703-2708.
• ZENZ, T., VOLLMER, D., TRBUSEK, M., et all. TP53 mutation profile in chronic lymphocytic leukemia: Evidence for a disease specific profile from a comprehensive analysis of 268 mutations. Leukemia. 2010, 24, p. 2072-2079.
• CEJKOVA, S., ROCNOVA, L., POTESIL, D., SMARDOVA, J., NOVAKOVA, V., CHUMCHALOVA, J., ZEZULKOVA, D., BORSKY, M., DOUBEK, M., BRYCHTOVA, Y., POSPISILOVA, S., KLABUSAY, M., MAYER, K., TRBUSEK, M. Presence of heterozygous ATM deletion may not be critical in the primary response of chronic lymphocytic leukemia cells to fludarabine. Eur. J. Haematol. 2009, 82, p. 133-142.
• MALCIKOVA, J., SMARDOVA, J., ROCNOVA, L., TICHY, B., KUGLIK, P., VRANOVA, V., CEJKOVA, S., SVITAKOVA, M., SKUHROVA FRANCOVA, H., BRYCHTOVA, Y., DOUBEK, M., BREJCHA, M., KLABUSAY, M., MAYER, J., POSPISILOVA, S., TRBUSEK, M. Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection, impact on survival and response to DNA damage. Blood. 2009, 114, p. 5307-5314.
• MALCIKOVA, J., SMARDOVA, J., PEKOVA, S., CEJKOVA, S., KOTASKOVA, J., TICHY, B., FRANCOVA, H., DOUBEK, M., BRYCHTOVA, Y., JANEK, D., POSPISILOVA, S., MAYER, J., DVORAKOVA, D., TRBUSEK, M. Identification of somatic hypermutations in the TP53 gene in B-cell chronic lymphocytic leukemia. Mol. Immunol. 2008, 45, p. 1525-1529.