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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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/摘要