Investigating how chemotherapeutic thiopurines inhibit telomerase elongation of telomeres

研究化疗硫嘌呤如何抑制端粒酶延长端粒

• 批准号: 10676638
• 负责人: Samantha Sanford
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676638
• 关键词: Adult; Animals; Basic Science; Binding; Biochemical; Biochemistry; Biological Assay; Cancer Cell Growth; Cancer cell line; Catalysis; Cell Line; Cell Proliferation; Cell division; Cells; Chromosomes; Clinical Research; DNA; DNA Damage; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Data Analyses; Deoxyguanosine; Dependence; Dissociation; Dot Immunoblotting; Educational workshop; Effectiveness; Elongation by Telomerase; Event; Fellowship; Fluorescent in Situ Hybridization; Functional disorder; Future; Growth; Human; Impairment; International; Kinetics; LOX gene; Length; Malignant Childhood Neoplasm; Malignant Neoplasms; Measures; Melanoma Cell; Mentors; Metaphase; Monitor; Mus; Myeloproliferative disease; Nucleotides; Oligonucleotides; Outcome; Pharmaceutical Preparations; Pharmacotherapy; Prodrugs; Property; Proteins; RNA; RNA-Directed DNA Polymerase; Research Personnel; Research Project Grants; Reverse Transcription; Series; Site; Somatic Cell; Techniques; Telomerase; Telomerase Inhibitor; Telomerase RNA Component; Telomerase inhibition; Telomere Maintenance; Telomere Shortening; Testing; Therapeutic; Time; Toxic effect; Training; Variant; Visualization; Writing; base; cancer cell; cancer therapy; cancer type; career development; cell growth; clinical efficacy; deoxyguanosine triphosphate; design; experimental study; improved; in vivo; inhibitor; insight; knock-down; leukemia; molecular imaging; nucleoside analog; p53-binding protein 1; prevent; research study; response; single molecule; skills; small hairpin RNA; symposium; targeted cancer therapy; telomere; telomere loss; therapeutic target; thiopurine; tumor; tumor xenograft

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.

项目概要/摘要 染色体末端端粒的维持对于癌细胞的生长至关重要。在人类中，端粒 末端是由 GGTTAG 重复组成的单链突出端，形成端粒酶的底物。 端粒酶是一种逆转录酶，由两个亚基组成：蛋白质成分 (TERT) 和 RNA 组件（TR），包含用于反转录多个端粒重复的完整模板 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标，因为它 在超过 85% 的癌细胞中表达，而大多数成体细胞缺乏端粒酶。硫嘌呤是一类 用于治疗白血病和一些儿童癌症的核苷类似物，但具有剧毒。前药6- 硫代-2'-脱氧鸟苷 (6-thio-dG) 的开发是为了降低非特异性硫嘌呤毒性。这种药是 显示可成功减少多种癌症类型的小鼠肿瘤异种移植物的生长。 6-硫代-dG 治疗也会导致端粒缩短和功能障碍，但其机制直到现在仍不清楚 最近。在生物化学实验中，我发现人端粒酶可以很容易地添加6-硫代-dG 代谢物 6-硫代-dGTP 进入不断增长的端粒链，但这种插入会强烈抑制端粒酶的活性 添加额外端粒重复序列的能力。我发现 POT1-TPP1，通常会增强端粒酶 结合和添加多个重复序列，在 6-thio- 存在的情况下不能恢复端粒伸长 dGTP。此外，由于端粒酶无能力，6-硫代-dGTP 对人端粒酶的 IC50 较低 区分 dGTP 和 6-硫代-dGTP，但不会抑制 DNA 聚合酶的进展，从而减少其 脱靶影响。我的这个项目的初步数据表明 6-thio-dG 可以破坏端粒酶 当修饰碱基位于端粒突出端的 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 合成并抑制细胞内端粒酶的延长。我会使用补充生化， 单分子和细胞方法来检验我的假设。目标 1 将阐明其机制 6-硫代-dG 会破坏端粒酶催化循环。目标2将确定端粒酶是否可以合成 用 6-thio-dG 处理的癌细胞中的 DNA 和延长端粒。目标 3 将确定 6-thio-dG 是否在 通过抑制硫代嘌呤消毒酶 NUDT15 可以增强癌细胞的作用。 完成该项目将提高我们对 6-thio-dG 如何影响端粒酶活性的理解 癌细胞中端粒的维持。它还将增强我们对 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的治疗方法的潜在用途。这 研究金将使我能够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相关，并参加国内和国际会议 会议介绍该研究项目的结果。该奖学金将帮助我获得培训 成为一名独立调查员所必需的。