The nucleoside analog cytarabine (ara-C) has been the mainstay of acute myeloid leukemia (AML) chemotherapy for more than 40 years and is one of the most important drugs used to induce remission in patients with AML. A number of studies suggest that the intracellular concentration of its active metabolite, ara-CTP, varies widely among patients and in turn is associated with variability in clinical response. The objective of the current proposal is to determine the molecular basis of this variation in intracellular ara-CTP levels by identifying and evaluating the pharmacogenomic effect of genetic polymorphisms in key genes in the ara-C metabolic pathway, with the long-term goal of explaining the variability observed in treatment response and toxicity profile among AML patients receiving ara-C chemotherapy. The hypothesis of this research is that genetic polymorphisms in key genes in the ara-C metabolic pathway -specifically: deoxycytidine kinase (DCK, a rate limiting activating enzyme); 5'nucleotidase (NT5C2), deoxycytidine deaminase (CDA), and deoxycytidylate deaminase (DCTD) (3 main inactivating enzymes); human equilibrative nucleoside transporter (hENT1/ SLC29A1, ara-C uptake transporter); and ribonucleotide reductase (enzyme regulating intracellular dCTP pools, and consisting of RRM1 and RRM2 subunits) -form the molecular basis of the inter-patient variability observed in intracellular ara-CTP concentration and sensitivity to ara-C. This hypothesis is based on the observations that the above mentioned candidate genes have been shown by various in vitro and in vivo studies to be associated with the clinical response and/or sensitivity to ara-C and demonstrate a wide inter- patient variability in their expression.
The specific aims of the proposed research are: 1) To identify and functionally characterize the coding genetic polymorphisms in key ara-C metabolic pathway genes in lymphoblast samples with European (CEPH) or African (YRI) ancestry; 2) To identify and characterize regulatory cis-genetic polymorphisms associated with mRNA expression of the candidate genes; and 3) To determine the association of functionally significant genetic polymorphisms in the candidate genes with clinical phenotypes such as candidate gene expression and ara-C sensitivity in diagnostic blasts, and intracellular ara- CTP levels and the extent of DNA synthesis inhibition in post ara-C treatment leukemic blasts from pediatric AML patients enrolled in the St. Jude AML2002 protocol. The use of CEPH and YRI samples will allow us to use HAPMAP genotype data and to study any ethnic differences in genotype frequencies and in candidate gene expression. The results from this study would also be applicable to other nucleoside analogs such as gemcitabine, decitabine, clofarabine, etc. that are metabolized by the same pathway. Understanding genetic variation in the key candidate genes involved in the metabolism of ara-C will provide us an opportunity to identify patients at increased risk of adverse reactions or decreased likelihood of response, based upon their genetic profile, which in future could help in dose optimization to reduce drug toxicity without compromising on efficacy. Relevance Statement: Acute myeloid leukemia (AML) is the second most common childhood leukemia and has the worst outcome of all major childhood cancers. Ara-C is the main drug used in AML chemotherapy but the cellular levels of its active form, ara-CTP, vary widely among patients and in turn are associated with variability in clinical response to ara-C treatment. The proposed research seeks to explain this variability by studying the inherited genetic variation in key genes involved in ara-C metabolism and may, in future, help to optimize ara-C dosing based on patient genetics. ? ? ? ?
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