Role of Promotor Polymorphisms of the MGMT Gene in Alkylation Chemotherapy

描述(申请人提供)：胶质母细胞瘤(GB)是最常见的脑肿瘤类型，通常迅速致死。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，迫切需要了解导致患者不同反应的机制。TMZ通过烷基化肿瘤DNA形成O6-烷基鸟嘌呤(O6-AG)，从而引起细胞凋亡，从而发挥其治疗作用。O6-AG是由DNA修复蛋白O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)修复的，因此，MGMT水平是决定肿瘤对TMZ治疗耐药性的关键因素。由于遗传和表观遗传因素，不同个体的MGMT水平不同。基因启动子/增强子(P/E)区域的单核苷酸多态(SNPs)可以通过改变MGMT转录调控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 SNPs和MGMT转录之间的关系。我们的研究将解决这一重要的未知问题，最终的长期目标是开发敏感和特异的标记物，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。我们将验证MGMT基因启动子/增强子(P/E)区域的SNPs改变MGMT转录调控的假设。MGMT转录的改变将显著影响对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，我们将首先确定存在于MGMT基因P/E区的SNPs，并确定它们在普通人群中的频率。然后，我们将确定这些SNP包含的单倍型，并将使用重组DNA和克隆技术来生成与这些单倍型对应的MGMT构建体。这些构建体将被瞬时地导入靶细胞环境(即培养的人胶质母细胞瘤细胞)，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启动子多态作为癌症患者对烷基化化疗反应的潜在修饰物的作用。获得的新知识最终将有助于改进治疗方式，这将导致提高存活率和改善GB患者的功能恢复。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瘤患者对烷基化化疗的反应比其他患者更好。产生的这些信息将具有重要的临床意义，因为它可能有助于为脑瘤患者开发治疗方案，这些方案根据患者的遗传特征进行个性化定制或改进，以最大限度地提高治疗反应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.