Pharmacogenetics of Ara-C Metabolic Pathway

核苷类似物阿糖胞苷(Ara-C)40多年来一直是急性髓系白血病(AML)化疗的主要药物，也是诱导AML患者缓解的重要药物之一。许多研究表明，其活性代谢物Ara-CTP的细胞内浓度在不同患者之间差异很大，进而与临床反应的可变性有关。当前建议的目的是通过鉴定和评估Ara-C代谢途径中关键基因的遗传多态的药物基因组效应来确定这种细胞内Ara-CTP水平变化的分子基础，长期目标是解释接受Ara-C化疗的AML患者在治疗反应和毒性方面观察到的差异。这项研究的假设是，Ara-C代谢途径关键基因的遗传多态--具体地说：脱氧胞苷激酶(DCK，一种限速激活酶)；5‘核苷酸酶(NT5C2)、脱氧胞苷脱氨酶(CDA)和脱氧胞苷脱氨酶(DCTD)(三种主要的失活酶)；人类平衡核苷转运体(hENT1/SLC29A1，Ara-C摄取转运体)；以及核苷酸还原酶(调节细胞内dCTP池的酶，由RRM1和RRM2亚单位组成)-构成患者细胞内Ara-CTP浓度和对Arc-C敏感性的分子基础。这一假说是基于这样的观察，即各种体外和体内研究表明，上述候选基因与Ara-C的临床反应和/或敏感性有关，并表明它们的表达在患者之间存在广泛的变异性。这项研究的具体目的是：1)在欧洲(CEPH)或非洲(YRI)血统的淋巴母细胞样本中识别和功能表征关键的Ara-C代谢途径基因的编码遗传多态；2)识别和表征与候选基因的mRNA表达相关的调节性顺式遗传多态；以及3)确定候选基因中具有显著功能的遗传多态与临床表型的关联，如候选基因在诊断细胞中的表达和Ara-C敏感性，以及细胞内Ara-CTP水平和DNA合成抑制的程度。CEPH和YRI样本的使用将使我们能够使用HapMap基因数据，并研究基因频率和候选基因表达的任何种族差异。这项研究的结果也适用于通过相同途径代谢的其他核苷类似物，如吉西他滨、地西他滨、氯法拉滨等。了解参与Ara-C代谢的关键候选基因的遗传变异将为我们提供一个机会，根据他们的基因图谱来识别不良反应风险增加或反应可能性降低的患者，这将有助于未来在不影响疗效的情况下优化剂量以减少药物毒性。相关声明：急性髓系白血病(AML)是第二常见的儿童白血病，是所有主要儿童癌症中预后最差的。Ara-C是AML化疗中使用的主要药物，但其活性形式Ara-CTP的细胞水平在不同患者之间差异很大，进而与Ara-C治疗的临床反应的差异有关。这项拟议的研究试图通过研究涉及Ara-C新陈代谢的关键基因的遗传变异来解释这种变异性，并在未来可能有助于基于患者遗传学优化Ara-C的剂量。

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.