Understanding Telomere Elongation Mechanisms in Cancer

了解癌症中的端粒延长机制

基本信息

批准号：
10364650
负责人：
Taylor Nicholas Takasugi
金额：
$ 3.16万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10364650
关键词：
ATRX gene Amino Acids Binding Proteins Biological Assay CRISPR/Cas technology Cancer cell line Candidate Disease Gene Carcinoma Cells Characteristics Chromosomal translocation Chromosomes Clinical Complex DNA DNA Damage DNA damage checkpoint Data Dissection Educational Activities Functional disorder Genetic Genetic Heterogeneity Genome Genomic Instability Goals Homeostasis Homologous Gene Human Immunofluorescence Immunologic Invaded Length Malignant - descriptor Malignant Neoplasms Mammals Measures Mediating Mus Mutate Mutation Neoplasm Metastasis Pathway interactions Pharmaceutical Preparations Phenotype Physicians Play Population Process Proteins Quantitative Reverse Transcriptase PCR RNA Interference Regulation Research Research Proposals Role SCID Mice Scientist Signal Transduction Site-Directed Mutagenesis Subcutaneous Injections Telomerase Telomere Length Maintenance Telomere Maintenance Telomere Pathway Testing Tissues Training Variant cancer cell cancer initiation genomic profiles in silico loss of function mouse genome mutant neoplastic cell next generation sequencing novel programs promoter reconstitution recruit repaired response telomere trait transcriptome sequencing tumor tumor initiation tumor progression tumorigenesis tumorigenic

项目摘要

Project Summary Malignant cancers must activate telomere maintenance mechanisms to achieve replicative immortality. Mutations in the human Protection Of Telomeres 1 (hPOT1) protein are frequently detected in cancers with abnormally long telomeres. This result suggests that the loss of hPOT1 function disrupts regulation of telomere length homeostasis to promote telomere elongation. The mouse genome encodes two POT1 proteins, POT1a and POT1b. I have generated Pot1b null tumors that display markedly elongated telomeres mediated by telomerase. In Aim 1, I will measure telomerase expression and its recruitment to telomeres to determine if changes in telomerase dynamics has led to telomere length increase in these tumors. Then I will investigate the mechanism underlying changes in telomerase dynamics by testing the role of DDR observed in Pot1b null tumors and the role of POT1b-TPP1 interactions in telomerase-mediated length maintenance. Pot1b null tumors also display very heterogeneous telomere length with extremely long ss G-overhang that is reminiscent of telomeres generated by Alternative Lengthening of Telomeres (ALT), a telomere length maintenance pathway through homology directed repair and independent of telomerase function. When Pot1b loss is combined with telomerase deletion, telomeres rapidly shorten and potentially select for ALT activation. Aim 2 will test for activation of the ALT maintenance in Pot1b-/-; Tert-/- MEFs. I will also determine if the Pot1b-/-; Tert-/-; Atrx-/- genetic background is sufficient to activate ALT. Furthermore, I will reconstitute POT1b domain mutants in ALT positive cells to investigate the mechanisms utilized by POT1b to repress ALT. Finally, I will perform RNA-seq to identify novel factors contributing to ALT activation in Pot1b-/-; Tert-/- MEFs. Together, these aims explore the central hypothesis that POT1b represses the activation of telomere maintenance mechanisms. Aim 1 will investigate the mechanism leading to telomerase-mediated telomere hyper-elongation in the absence of POT1b and Aim 2 will determine if the combined loss of POT1b and TERT activates ALT. Completion of these aims will contribute to our knowledge of the telomere maintenance mechanisms necessary to promote replicative immortality and cancer initiation.

项目概要恶性肿瘤必须激活端粒维持机制才能实现复制永生。人类端粒保护 1 (hPOT1) 蛋白的突变经常在以下癌症中检测到：异常长的端粒。这一结果表明 hPOT1 功能的丧失扰乱了端粒的调节长度稳态促进端粒伸长。小鼠基因组编码两种 POT1 蛋白，POT1a 和 POT1b。我已经生成了 Pot1b 无效肿瘤，这些肿瘤显示出由以下因素介导的显着拉长的端粒：端粒酶。在目标 1 中，我将测量端粒酶表达及其向端粒的募集，以确定是否端粒酶动力学的变化导致这些肿瘤的端粒长度增加。然后我将调查通过测试 Pot1b 无效肿瘤中观察到的 DDR 的作用，研究端粒酶动力学变化的潜在机制以及 POT1b-TPP1 相互作用在端粒酶介导的长度维持中的作用。 Pot1b 无效肿瘤还表现出非常异质的端粒长度，具有极长的 ss G 突出端这让人想起端粒选择性延长 (ALT) 产生的端粒，一种端粒长度通过同源定向修复且独立于端粒酶功能的维持途径。当 Pot1b 端粒酶缺失与端粒酶缺失相结合，端粒迅速缩短，并可能选择 ALT 激活。目标 2 将测试 Pot1b-/- 中 ALT 维持的激活； Tert-/- MEF。我还将确定 Pot1b-/- 是否；叔-/-; Atrx-/- 遗传背景足以激活 ALT。此外，我将重构 POT1b 域 ALT 阳性细胞中的突变体，以研究 POT1b 抑制 ALT 的机制。最后，我会进行 RNA-seq 以确定 Pot1b-/- 中导致 ALT 激活的新因素； Tert-/- MEF。这些目标共同探讨了 POT1b 抑制端粒激活的中心假设维护机制。目标 1 将研究导致端粒酶介导的端粒的机制在缺乏 POT1b 和 Aim 2 的情况下的超伸长将决定 POT1b 和 TERT 的组合损失是否激活 ALT。完成这些目标将有助于我们对端粒维护的了解促进复制永生和癌症发生所必需的机制。