Projects / Programmes
Pharmacogenetic approaches to research, diagnosis and therapy of leukemia
| Code |
Science |
Field |
Subfield |
| 3.04.00 |
Medical sciences |
Oncology |
|
| Code |
Science |
Field |
| B200 |
Biomedical sciences |
Cytology, oncology, cancerology |
| Code |
Science |
Field |
| 3.01 |
Medical and Health Sciences |
Basic medicine |
Pharmacogenetics, Therapy individualization, 6-mercaptopurine, acute lymphoblastic leukemia, drug toxicity, thiopurine S-methyltransferase, S-adenosylmethionine
Researchers (15)
Organisations (2)
Abstract
The proposed project is motivated by the requirement of translation of genomic, transcriptomic and metabolomic methodologies to clinical medicine with the goal to improve both diagnosis and treatment of pediatric leukemia patients.
State-of-the art and Objectives
Despite many advances in the understanding of cancer biology, therapeutic treatment of tumors remains essentially empiric. The main problem of chemotherapy is the narrow therapeutic index of antitumor agents that results in severe toxicity and low treatment outcome. The use of pharmacogenomics to individualize drug therapy offers the potential to improve drug effectiveness, reduce adverse side effects, and provide cost-effective pharmaceutical care.
Thiopurine drugs are used in the treatment of acute lymphoblast leukaemia (ALL), the most common childhood cancer. Our previous studies have delineated the genotype-to-phenotype correlation for the thiopurine S-methyltransferase (TPMT), which catalyzes methylation and thus deactivation of 6-MP, and further linked the low enzyme activity to the onset of severe toxicity of 6-MP. Beside genotype other factors also influence the activity of TPMT. Identification and understanding of factors influencing TPMT activity is crucial for the improvement of the efficacy and safety of ALL treatment. We therefore intend to further investigate the competitive metabolic routes for thiopurines. By dddressing the molecular mechanisms of thiopurine action and selected enzyme function we intend to search for a novel potential targets for new-generation of therapeutic agents.
Aims of the project
1.Identification of biological markers and their implementation in clinical practice
We will try to identify additional factors influencing TPMT activity., by testing the correlation between levels of methionine cytotoxic metabolites of 6-MP and TPMT activity.
2. Identification of novel therapeutically relevant biological targets
Delineation of the impact of TPMT on intracellular metabolism will allow the characterization of relevant enzymes, metabolites and differentially expressed genes as candidates for clinically relevant targets with potential roles in the modulation of cell- and tissue-specific processes (oxidative stress, methionine metabolism, DNA methylation).
3. Elucidation of molecular mechanism of thiopurine therapy response
The differential genome, transcriptome and metabolome analysis will be preformed on thiopurines resistant and non-resistant cells. The selected candidate genes, metabolites and enzymes will be implemented in clinical practice as diagnostic test for optimization of thiopurine therapy in ALL patients.
4. Development of 6-MP dosing algorithm. The dosing algorithm will enable the calculation of the optimal individual dose for each patient, according to his or her demographic, phenotypic and genotypic characteristics.
Relevance of the proposed project
The project will be conducted by an international, interdisciplinary team including partners from University of Ljubljana, Faculty of Pharmacy, Department of Pediatrics, University Clinical Center Ljubljana (dr. Janez Jazbec), Pediatric hospital St. Anna Kinderspital in Vienna, Austria (dr. Leo Kager), Estonian Genome Project and Institute for molecular and cell biology, University of Tartu (dr. Andres Metspalu), and Sackler Faculty of Medicine, University of Tel Aviv (dr. David Gurwitz). Translation of genomic, transcriptomic and metabolomic methodologies in the field of pharmacogenetics shall improve both diagnosis and treatment of pediatric leukemia patients. All of this indicates a high scientific and socio-economic relevance. Proposed approach and expertise of the group shall assure high-scientific relevance and accomplishments of project’s ambitious goals.
Significance for science
The relation between 6-mercaptopurine (6-MP) toxicities and genotype of its crucial metabolizing enzyme, thiopurine S-methyltransferase (TPMT), represents one of the best pharmacogenetic examples. Although there is a strong evidence of good phenotype-to-genotype correlation, some patients respond unexpectedly on therapy. This indicates that metabolism of thiopurines depends, not only on particular enzyme, but rather on a complex metabolic network including 5-methyltetrahydrofolate (5-Me-THF) (Karas-Kuzelicki N., Pharmacogenomics. 2009). Previously, we confirmed the synergy of TPMT and methyltetrahydrofolate reductase (MTHFR) low activity alleles with elevated risk of 6-MP related side effects in ALL patients (Karas-Kuzelicki N., Leukemia. 2009). Based on our latest findings, we examined the toxicity of 6-MP using multifactorial pharmacogenomic approach. By means of TaqMan technology, 15 EBV-transformed human lymphoblastoid cell lines (LCLs) from healthy donors were genotyped for polymorphic alleles of enzymes that could influence 5-Me-THF and methionine metabolism: TPMT*2, TPMT*3B, TPMT*3C, MTHFR 677C)T, MTHFR 1298A)C, MTRR 66A)G, MTHFD 1958G)A, BHMT 742G)A, GNMT 1298C)T. Cytotoxicity of 6-MP was determined by MTS test after 72 h exposure to various drug concentrations (1,0–100 µM). All cell lines demonstrated logarithmic cytotoxic response with increasing concentrations of 6-MP. Different genotype patterns revealed distinct cell sensitivity on 6-MP; IC50 extensively ranged between 3.8 and 20.9 µM. The sensitivity towards 6-MP did not associate with TPMT alleles heterozygosity. However, the net effect of above mentioned polymorphic alleles on cell survival showed statistical significance: LCLs with less than four alleles that could lead to decreased 5-Me-THF concentration had lower sensitivity on 6-MP compared with LCLs with higher number of such alleles (Mann–Whitney test, p(0,05). Current outcomes are consistent with the hypothesized assumption that the effect of 6-MP on cell survival relies on combination of polymorphisms in genes for enzymes in a complex folate and methionine metabolic pathways. Nevertheless, extended investigation should be performed using higher number of distinct LCLs. Furthermore, the path through which 5-Me-THF influences the sensitivity on 6-MP should be elucidated. Since S-adenosylmethionine (SAM) stabilizes wild type and heterozygous TPMT (Milek M., Biochem Pharmacol. 2009) 5-Me-THF could act via altered SAM concentrations.
Significance for the country
Contribution to the of individualized therapy of Acute Llymphoblastic Leukemia (ALL) patients. The research on pharmacogentetics has the direct impact on the economy and society and is demonstrated by an improved quality of life of patients undergoing the therapy and by favorable pharmacoeconomic indicators. Severe side effects are observed in patients due to the differential response of individuals to therapy and due to the conventional drugs prescribing philosophy that all individuals of the same age group need the same dose of the drug, i.e. »one size fits all«. Pharmacogenetic testing enables individualized approach leading to the rationalization of therapy and improved life quality of patients. Based on the pharmacogenetic findings we are able to explain the inter-individual differences to 6-mercaptopurine treatment and the onset of toxicity. We have successfully transferred the basic research practice to clinical environment. By genotyping the pediatric patients with life-threatening toxicity we have enabled the basis for individualization of therapy, and therefore eliminated toxicity, and overall contributed to quality of their life.
Most important scientific results
Annual report
2010,
2011,
2012,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2010,
2011,
2012,
final report,
complete report on dLib.si
