Pharmacogenomics and Mechanisms of Cytidine Analogues

描述（由申请人提供）：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有希望的结果。吉西他滨的作用是由于包括药物转运蛋白、催化药物活化和失活的酶以及药物靶点的"途径"。然而，关于吉西他滨反应变化的决定因素知之甚少，特别是我们目前对这种药物的了解所描述的途径之外的基因中的单核苷酸多态性（SNP）。为了鉴定对吉西他滨应答变异具有重要意义的其他基因，我们使用300 Human Variation Panel淋巴母细胞样细胞系作为常见遗传变异的模型系统，进行全基因组表达相关性研究，以鉴定表达水平与吉西他滨细胞毒性变异（IC50值）显著相关的基因。一个最重要的候选基因FKBP5，一个编码51 kDa亲免蛋白的基因，被证明会影响对吉西他滨应答的凋亡途径。具体而言，FKBP5的较低表达与吉西他滨诱导的细胞毒性抗性相关。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活化影响吉西他滨反应，并且与FKBP5基因表达和蛋白质功能相关的遗传变异可能显著促进吉西他滨反应的变异。在本申请中，我们提出确定FKBP 5调节AKT活化的机制，然后使用小鼠模型和来自用吉西他滨治疗的胰腺癌患者的肿瘤样品测试FKBP 5在吉西他滨应答中的作用。此外，我们还将使用300个淋巴母细胞系确定与FKBP5基因表达和吉西他滨反应相关的基因序列变异，然后使用这些SNP进行功能基因组研究。最后，我们将对胰腺癌患者的DNA进行基因型-表型相关性研究，以确定影响FKBP5表达和/或蛋白质功能的SNP是否可能影响吉西他滨治疗胰腺癌的反应。总之，这一综合性系列实验将增强我们对吉西他滨耐药机制的理解，并可能确定有助于预测吉西他滨治疗胰腺癌反应的生物标志物。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化的决定因素知之甚少，特别是我们目前对该药物代谢和"靶点"的知识所描述的"途径"之外的基因中的单核苷酸多态性（SNP）。为了鉴定对吉西他滨应答变异重要的其他基因，我们使用了300个人类变异组淋巴母细胞样细胞系作为模型系统，结合全基因组方法来鉴定一个最佳候选基因FKBP 5，其表达与吉西他滨敏感性显著相关。在本申请中，基于广泛的初步数据，我们建议研究FKBP5调节吉西他滨反应的机制，并鉴定FKBP5中的遗传变异，其可用作生物标志物以帮助预测吉西他滨在胰腺癌治疗中的反应。

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.