Role of Promotor Polymorphisms of the MGMT Gene in Alkylation Chemotherapy

描述（由申请人提供）：胶质母细胞瘤（GB）是最常见的脑肿瘤类型，通常迅速致命。临床试验表明，替莫唑胺（TMZ）联合化疗对患者有益。不幸的是，只有少数患者对这种治疗有反应，因此，迫切需要了解患者之间不同反应的机制。TMZ通过将肿瘤DNA烷基化形成O 6-烷基鸟嘌呤（O 6-AG）引起细胞凋亡而发挥其治疗作用。O 6-AG由DNA修复蛋白O 6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）修复，因此，MGMT水平构成了决定肿瘤对TMZ治疗耐药性的关键因素。MGMT的水平因遗传和表观遗传因素而异。该基因启动子/增强子（P/E）区域的单核苷酸多态性（SNP）可通过改变MGMT转录的调节来影响蛋白质的表达。到目前为止，还没有研究系统地描述P/E SNP和MGMT转录之间的关系。我们的研究将解决这个重要的未知问题，最终的长期目标是开发敏感和特异的标志物，可以区分那些最有可能对化疗有反应的患者和那些没有反应的患者。我们将检验MGMT基因启动子/增强子（P/E）区域的SNP改变MGMT转录调控的假设。MGMT转录的改变将显著影响烷基化化疗的敏感性和GB治疗的结果。为了验证我们的假设，我们将首先确定MGMT基因P/E区域中存在的SNP，并确定它们在普通人群中的频率。然后，我们将确定这些SNPs所包含的单倍型，我们将使用重组DNA和克隆技术来产生对应于这些单倍型的MGMT构建体。将这些构建体瞬时转染到靶细胞环境（即培养的人胶质母细胞瘤细胞）中，以确定每种单倍型对MGMT启动子活性的影响。这项研究产生的信息将具有重要的翻译意义，因为它将阐明MGMT启动子多态性作为癌症患者对烷基化化疗反应的潜在修饰剂的作用。所获得的新知识将最终有助于改善治疗方式，从而提高GB患者的生存率并改善功能恢复。公共卫生相关性：该项目的重点是了解MGMT基因序列中遗传变异的意义。产生的新信息可以解释为什么一些脑肿瘤患者对烷基化化疗的反应比其他人好。所产生的这种信息将具有重要的临床意义，因为它可以帮助开发针对脑肿瘤患者的治疗方案，所述治疗方案基于患者的遗传特征而被单独定制或改进，以使治疗反应最大化并减少与治疗相关的有害副作用。

DESCRIPTION (provided by applicant): Glioblastoma (GB) is the most common type of brain tumor and is usually rapidly fatal. Clinical trials indicate that treatment with temozolomide (TMZ) coupled with chemotherapy is beneficial to patients. Unfortunately, only a small number of patients respond to such treatment and, therefore, there is a critical need to understand the mechanisms responsible for the differing responses among patients. TMZ exerts its therapeutic effect by alkylating tumor DNA to form O6-alkylguanine (O6-AG) which causes apoptosis. O6-AG is repaired by the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), and, therefore, MGMT levels constitute a critical factor in determining tumor resistance to TMZ treatment. Levels of MGMT vary among individuals due to genetic and epigenetic factors. Single nucleotide polymorphisms (SNPs) in the promoter/enhancer (P/E) region of the gene can affect the expression of the protein by altering the regulation of MGMT transcription. To date, no studies have systematically characterized the relationship between P/E SNPs and MGMT transcription. Our research will address this important unknown, with the overarching long-term goal of ultimately developing sensitive and specific markers that can distinguish those patients who would most likely be responsive to chemotherapy from those who are not. We will test the hypothesis that SNPs in the promoter/enhancer (P/E) region of the MGMT gene alter the regulation of MGMT transcription. Alteration in MGMT transcription would significantly influence sensitivity to alkylation chemotherapy and the outcome of GB treatment. To test our hypothesis, we will first identify the SNPs that exist in the P/E region of the MGMT gene and determine their frequency in the general population. We will then determine the haplotypes that these SNPs encompass, and we will use recombinant DNA and cloning techniques to generate MGMT constructs corresponding to these haplotypes. These constructs will be transiently transfected into the target cell environment (i.e. cultured human glioblastoma cells) to establish the effect each haplotype has on MGMT promoter activity. The information generated from this research will have important translational implications, since it will clarify the role of MGMT promoter polymorphisms as potential modifiers of response to alkylation chemotherapy in cancer patients. The new knowledge gained would ultimately help enhance treatment modalities, which would lead to increasing survival rates and improving functional recovery for GB patients. PUBLIC HEALTH RELEVANCE: The focus of this project is on understanding the significance of the inherited variations in the MGMT gene sequence. The new information generated can explain why some patients with brain tumors respond better than others to alkylation chemotherapy. This information generated will have important clinical implications as it could help in developing therapeutic regimens for patients with brain tumors that are individually tailored or refined, based on a patient's genetic profile, to maximize therapeutic response and reduce deleterious side- effects associated with treatments.