Investigating how chemotherapeutic thiopurines inhibit telomerase elongation of telomeres

项目总结/摘要 染色体末端的端粒维持对于癌细胞的生长是必不可少的。在人类中， 末端是由形成端粒酶底物的GGTTAG重复序列组成的单链突出端。 端粒酶是一种逆转录酶，由两个亚基组成：蛋白组分（TERT）和RNA 一种包含用于逆转录多个端粒重复序列的完整模板的组分（TR 在一个单一的端粒结合事件。端粒酶是癌症治疗的有吸引力的靶点，因为它 端粒酶在超过85%的癌细胞中表达，而大多数成年体细胞缺乏端粒酶。硫嘌呤是一类 用于治疗白血病和一些儿科癌症的核苷类似物，但毒性很高。前药6- 开发了硫代-2 ′-脱氧鸟苷（6-硫代-dG）以降低非特异性硫嘌呤毒性。这种药物是 显示出成功地减少了多种癌症类型的小鼠肿瘤异种移植物的生长。6-硫代-dg 治疗也可导致端粒缩短和功能障碍，然而，其机制尚不清楚， 最近在生物化学实验中，我发现人类端粒酶可以很容易地添加6-硫代-dG 这种插入会将端粒的代谢产物6-硫代-dGTP插入到不断增长的端粒链中，但这种插入会强烈抑制端粒酶的活性。 添加额外端粒重复的能力。我发现POT 1-TPP 1通常会增强端粒酶活性， 结合和添加多个重复，不能恢复端粒延长的6-硫代- dGTP。此外，由于端粒酶不能抑制端粒酶活性，6-硫代-dGTP对人端粒酶具有低的微摩尔IC 50。 区分dGTP和6-硫代-dGTP，但不抑制DNA聚合酶的进展，降低其 脱靶撞击我对这个项目的初步数据表明，6-硫代-dG可以破坏端粒酶 当修饰的碱基位于端粒突出端的3'末端时结合。我假设6-硫代脱氧葡萄糖 添加会削弱端粒酶与端粒的相互作用，从而破坏重复添加的循环 合成和抑制细胞中端粒的端粒酶延伸。我会用生化补充剂 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。目的2将确定端粒酶是否可以合成 用6-硫代-dG处理的癌细胞中的DNA和延伸端粒。目的3将确定6-硫代-dG在 通过抑制硫嘌呤生酶NUDT 15增强癌细胞的活性。 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的理解， 端粒在癌细胞中的维持。这也将加强我们对6-硫代-dGTP核苷酸 影响端粒酶催化及其作为停止癌细胞增殖的治疗剂的潜在效用。这 奖学金将使我获得与项目目标相关的新技能，并概述了具体的导师 每个.职业发展也是该奖学金的重点，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有关，并参加国家和国际 会议上介绍了这个研究项目的结果。这个奖学金将帮助我获得培训 成为一名独立调查员。

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.