Investigating how chemotherapeutic thiopurines inhibit telomerase elongation of telomeres

项目摘要/摘要 染色体末端端粒的维持对癌细胞的生长至关重要。在人类中，端粒 以形成端粒酶底物的GGTTAG重复序列组成的单链突出物结束。 端粒酶是一种逆转录酶，由两个亚基组成：蛋白质组分(TERT)和RNA 含有用于逆转录多个端粒重复序列的完整模板的成分(Tr) 在单个端粒结合事件期间。端粒酶是癌症治疗的一个有吸引力的靶点，因为它是 在85%以上的癌细胞中表达，而大多数成年体细胞缺乏端粒酶。硫代嘌呤是一类 核苷类似物用于治疗白血病和一些儿科癌症，但毒性很大。前药6- 硫代-2‘-脱氧鸟苷(6-硫代-DG)是为了减少硫代嘌呤的非特异性毒性而开发的。这种药是 显示成功地减少了多种癌症类型的小鼠移植瘤的生长。6-硫代-DG 治疗也可以导致端粒缩短和功能障碍，然而，其机制直到 最近。在生化实验中，我发现人的端粒酶可以很容易地将6-硫代-DG 代谢产物6-硫代-dGTP与不断增长的端粒链结合，但这种插入会强烈抑制端粒酶的 能够添加额外的端粒重复序列。我发现POT1-TPP1，它通常可以增强端粒酶 结合和增加多个重复，不能恢复端粒在6-硫代存在下的伸长 DGTP。此外，6-硫代-dGTP对人端粒酶具有低的微摩尔IC50，这是因为端粒酶不能 区分dGTP和6-thio-dGTP，但不抑制DNA聚合酶的进展，减少其 偏离目标的撞击。我为这个项目提供的初步数据表明，6-硫代-DG可以破坏端粒酶 当修饰的碱基位于端粒突出物的3‘端时结合。我假设6-硫代-DG 添加会损害端粒酶与端粒的相互作用，从而扰乱重复添加的循环 细胞内端粒的合成和抑制端粒酶的延长。我将使用补充生化， 单分子和细胞方法来验证我的假设。目标1将阐明 6-硫代-DG破坏端粒酶催化循环。目标2将确定端粒酶是否可以合成 6-硫代-DG处理的癌细胞的DNA和延伸的端粒。AIM 3将确定6-硫代-DG在 癌细胞通过抑制硫代嘌呤核糖核酸酶NUDT15而得到增强。 完成这个项目将提高我们对6-硫代-DG如何影响端粒酶活性和 癌细胞中的端粒维持。它还将加深我们对6-硫代-dGTP核苷酸如何 影响端粒酶催化及其作为阻止癌细胞增殖治疗的潜在效用。这 奖学金将使我获得与项目目标相关的新技能，并概述具体的导师 每个人。职业发展也是该奖学金的一个重点，其中包括参加研讨会的计划 与实验技术、数据分析和科学写作相关，并参加国内外 会议介绍这一研究项目的成果。这笔奖学金将帮助我获得培训 成为一名独立调查员所必需的。

Project Summary/ Abstract Telomere maintenance at chromosome ends is essential for the growth of cancer cells. In humans, telomeres end in a single strand overhang consisting of GGTTAG repeats that form the substrate for telomerase. Telomerase is a reverse transcriptase composed of two subunits: a protein component (TERT) and an RNA component (TR) which contains an integral template used to reverse transcribe multiple telomeric repeats during a single telomere binding event. Telomerase is an attractive target for cancer therapies because it is expressed in over 85% of cancer cells, while most adult somatic cells lack telomerase. Thiopurines are a class of nucleoside analogs used to treat leukemia and some pediatric cancers but are highly toxic. The prodrug 6- thio-2'-deoxyguanosine (6-thio-dG) was developed to reduce non-specific thiopurine toxicity. This drug was shown to successfully reduce the growth of mouse tumor xenografts for multiple cancer types. 6-thio-dG treatment can also cause telomere shortening and dysfunction, however, the mechanism was unknown until recently. In biochemistry experiments, I discovered that human telomerase can readily add the 6-thio-dG metabolite, 6-thio-dGTP, to a growing telomere chain, but this insertion then strongly inhibits telomerase’s ability to add additional telomeric repeats. I found that POT1-TPP1, which normally enhances telomerase binding and the addition of multiple repeats, cannot restore telomere elongation in the presence of 6-thio- dGTP. In addition, 6-thio-dGTP has a low micromolar IC50 for human telomerase due to telomerase’s inability to discriminate between dGTP and 6-thio-dGTP but does not inhibit DNA polymerase progression, reducing its off-target impact. My preliminary data for this project demonstrate that 6-thio-dG can disrupt telomerase binding when the modified base is located at the 3’ end of the telomeric overhang. I hypothesize that 6-thio-dG addition impairs telomerase’s interaction with the telomere, thereby disrupting cycling for repeat addition synthesis and inhibiting telomerase elongation of telomeres in cells. I will use complementary biochemical, single-molecule, and cellular approaches to test my hypothesis. Aim 1 will elucidate the mechanism by which 6-thio-dG disrupts the telomerase catalytic cycle. Aim 2 will determine whether telomerase can synthesize DNA and extend telomeres in cancer cells treated with 6-thio-dG. Aim 3 will determine if 6-thio-dG efficacy in cancer cells is enhanced by inhibiting the thiopurine sanitase, NUDT15. Completing this project will improve our understanding of how 6-thio-dG impacts telomerase activity and telomere maintenance in cancer cells. It will also enhance our understanding of how 6-thio-dGTP nucleotide impacts telomerase catalysis and its potential utility as a therapeutic for halting cancer cell proliferation. This fellowship will allow me to acquire new skills related to the project’s aims and outlines specific mentors for each. Career development is also a strong focus in this fellowship, which includes plans to attend workshops related to experimental techniques, data analysis, and scientific writing and attend national and international conferences to present the findings of this research project. This fellowship will help me acquire the training necessary to become an independent investigator.