Pharmacogenomics and Mechanisms of Cytidine Analogues

描述(申请人提供)：胞苷类似物吉西他滨是治疗胰腺癌的一线化疗药物，在乳腺癌和非小细胞肺癌的治疗中也显示出良好的效果。吉西他滨的作用是通过一条包括药物转运体、催化药物激活和失活的酶以及药物靶点的“途径”实现的。然而，关于吉西他滨反应变异的决定因素，特别是我们目前对该药物所描述的途径之外的基因的单核苷酸多态(SNPs)，我们知之甚少。为了确定吉西他滨反应中其他重要的变异基因，我们使用300个人类变异委员会淋巴母细胞系作为常见遗传变异的模型系统，进行全基因组表达关联研究，以确定与吉西他滨细胞毒性变异显著相关的表达水平的基因(IC50值)。其中一个候选基因FKBP5，一个编码51 kDa免疫亲和素的基因，被证明影响吉西他滨的凋亡途径。具体地说，FKBP5的低表达与对吉西他滨诱导的细胞毒性的抵抗有关。我们还证明了FKBP5在AKT磷酸化中的抑制作用。因此，我们假设FKBP5通过负性调节AKT激活来影响吉西他滨的反应，与FKBP5基因表达和蛋白功能相关的遗传变异可能对吉西他滨的反应有显著的影响。在这项应用中，我们建议确定FKBP5调节AKT激活的机制，然后使用小鼠模型和接受吉西他滨治疗的胰腺癌患者的肿瘤样本来测试FKBP5在吉西他滨反应中的作用。此外，我们还将使用300个淋巴母细胞系来确定与FKBP5基因表达和吉西他滨反应相关的基因序列变异，然后利用这些SNPs进行功能基因组研究。最后，我们将与胰腺癌患者的DNA进行基因-表型相关性研究，以确定影响FKBP5表达和/或蛋白功能的SNPs在用于治疗胰腺癌时是否会影响对吉西他滨的反应。综上所述，这一系列的综合实验将加深我们对吉西他滨耐药机制的理解，并可能识别有助于预测吉西他滨在胰腺癌治疗中的反应的生物标志物。公共卫生相关性：胞苷类似物吉西他滨是治疗胰腺癌的一线化疗药物。然而，关于吉西他滨反应变异的决定因素，特别是我们目前对该药物的代谢和靶点所描述的“途径”之外的基因的单核苷酸多态(SNPs)，知之甚少。为了确定更多与吉西他滨反应变异有关的重要基因，我们使用300个人类变异小组淋巴母细胞系作为模型系统，并结合全基因组方法确定了一个首选候选基因FKBP5，其表达与吉西他滨的敏感性显著相关。在这一应用中，基于大量的初步数据，我们建议研究FKBP5调节吉西他滨疗效的机制，并确定FKBP5的基因变异可能被用作帮助预测吉西他滨治疗胰腺癌疗效的生物标志物。

DESCRIPTION (provided by applicant): The cytidine analogue gemcitabine is first line chemotherapy for the treatment of pancreatic cancer, and it has also shown promising results in the treatment of breast cancer and non-small cell lung cancer. Gemcitabine has its effect as a result of a "pathway" that includes drug transporters, enzymes catalyzing drug activation and inactivation, and drug targets. However, very little is known with regard to determinants of variation in gemcitabine response, especially single nucleotide polymorphisms (SNPs) in genes outside of the pathway described by our current knowledge of this drug. In order to identify additional genes of importance for variation in gemcitabine response, we have used 300 Human Variation Panel lymphoblastoid cell line as a model system for common genetic variation to perform genome-wide expression association studies to identify genes with expression levels that were significantly associated with variation in gemcitabine cytotoxicity (IC50 values). One top candidate gene, FKBP5, a gene encoding a 51 kDa immunophilin, was shown to affect the apoptotic pathway in response to gemcitabine. Specifically, lower expression of FKBP5 was associated with resistance to gemcitabine-induced cytotoxicity. We also demonstrated an inhibitory role for FKBP5 in AKT phosphorylation. As a result, we hypothesize that FKBP5 affects gemcitabine response by negatively regulating AKT activation and that genetic variation associated with FKBP5 gene expression and protein function might contribute significantly to variation in gemcitabine response. In this application, we propose to determine mechanisms by which FKBP5 regulates AKT activation, followed by testing the role of FKBP5 in gemcitabine response using mice models and tumor samples from pancreatic cancer patients treated with gemcitabine. In addition, we will also determine gene sequence variation that is associated with FKBP5 gene expression and response to gemcitabine using 300 lymphoblastoid cell lines, followed by performing functional genomic studies with these SNPs. Finally, we will perform a genotype-phenotype correlation study with DNA from pancreatic cancer patients to determine whether SNPs that affect FKBP5 expression and/or protein function might influence response to gemcitabine when used to treat pancreatic cancer. In summary, this comprehensive series of experiments will enhance our understanding of mechanisms of gemcitabine resistance and may identify biomarkers that might help predict gemcitabine response in the treatment of pancreatic cancer. PUBLIC HEALTH RELEVANCE: The cytidine analogue gemcitabine is first line chemotherapy for the treatment of pancreatic cancer. However, very little is known with regard to determinants of variation in gemcitabine response, especially single nucleotide polymorphisms (SNPs) in genes outside of the "pathway" described by our current knowledge of the metabolism and "targets" for this drug. In order to identify additional genes of importance for variation in gemcitabine response, we have used 300 Human Variation Panel lymphoblastoid cell lines as a model system, together with genome-wide approaches to identify one top candidate gene, FKBP5, for which expression was significantly associated with gemcitabine sensitivity. In this application, based on extensive preliminary data, we propose to investigate mechanisms by which FKBP5 regulates response to gemcitabine and to identify genetic variation in FKBP5 that might be used as a biomarker to help predict gemcitabine response in the treatment of pancreatic cancer.