Genome Instability induced in cancer cells carrying mutations in Type II topoisomerases

携带 II 型拓扑异构酶突变的癌细胞诱导基因组不稳定性

基本信息

批准号：
10542782
负责人：
JOHN L NITISS
金额：
$ 8万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
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：
2021-12-20 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10542782
关键词：
Adenocarcinoma Cell Alleles Antineoplastic Agents Binding Biochemical Biological Assay Biological Process CRISPR/Cas technology Cell Death Cell Line Cells Characteristics Chromosome Segregation Collaborations Complex DNA DNA Damage DNA Double Strand Break DNA Repair DNA Repair Disorder DNA Sequence DNA Sequence Databases DNA Structure DNA Topoisomerases DNA metabolism DNA strand break Databases Drug Targeting Ectopic Expression Enzyme Induction Enzyme Inhibitor Drugs Enzymes Equilibrium Etoposide Eukaryotic DNA Topoisomerases II Exhibits Failure Gastric Adenocarcinoma Gene Deletion Gene Duplication Generations Genes Genetic Transcription Genome Genomic Instability Heterozygote Human Human Cell Line Hypersensitivity In Vitro Induced Mutation Isoenzymes Laboratories Lesion Ligation Malignant Neoplasms Mammalian Cell Mediating Modeling Molecular Conformation Mutate Mutation Nonhomologous DNA End Joining Nucleotides Oncogenic Pathway interactions Patients Pattern Pharmaceutical Preparations Phenotype Plasmids Play Positioning Attribute Property Protein Isoforms Proteins RAD52 gene Reaction Recurrence Role Second Primary Cancers Site System Testing Topoisomerase Topoisomerase II Transcription Initiation Work Yeasts cBioPortal cancer cell cancer initiation cell killing cytotoxic cytotoxicity drug action homologous recombination in vivo mutant novel prevent promoter protein purification repaired response screening targeted agent targeted treatment tool tumor tumor DNA tumor progression

Adenocarcinoma Cell Alleles Antineoplastic Agents Binding Biochemical Biological Assay Biological Process CRISPR/Cas technology Cell Death Cell Line Cells Characteristics Chromosome Segregation Collaborations Complex DNA DNA Damage DNA Double Strand Break DNA Repair DNA Repair Disorder DNA Sequence DNA Sequence Databases DNA Structure DNA Topoisomerases DNA metabolism DNA strand break Databases Drug Targeting Ectopic Expression Enzyme Induction Enzyme Inhibitor Drugs Enzymes Equilibrium Etoposide Eukaryotic DNA Topoisomerases II Exhibits Failure Gastric Adenocarcinoma Gene Deletion Gene Duplication Generations Genes Genetic Transcription Genome Genomic Instability Heterozygote Human Human Cell Line Hypersensitivity In Vitro Induced Mutation Isoenzymes Laboratories Lesion Ligation Malignant Neoplasms Mammalian Cell Mediating Modeling Molecular Conformation Mutate Mutation Nonhomologous DNA End Joining Nucleotides Oncogenic Pathway interactions Patients Pattern Pharmaceutical Preparations Phenotype Plasmids Play Positioning Attribute Property Protein Isoforms Proteins RAD52 gene Reaction Recurrence Role Second Primary Cancers Site System Testing Topoisomerase Topoisomerase II Transcription Initiation Work Yeasts cBioPortal cancer cell cancer initiation cell killing cytotoxic cytotoxicity drug action homologous recombination in vivo mutant novel prevent promoter protein purification repaired response screening targeted agent targeted treatment tool tumor tumor DNA tumor progression

展开

项目摘要

DNA topoisomerase II (Top2) alters DNA topology by making a double-strand break in DNA and passing an intact duplex through the break. The transient double strand break is generated through a protein/DNA covalent intermediate termed the cleavage complex. Mammalian cells contain two Top2 isoforms termed Top2α and Top2β. The two enzymes have distinct biological functions with Top2α playing a key role in chromosome segregation and Top2β having unique roles in transcription. While the DNA cleavage mechanism of Top2 allows cells to catalyze changes in DNA conformation without the dangers of frank DNA double strand breaks, a variety of mechanisms can interfere with the cleavage/religation equilibrium of the enzyme. For example, anti-cancer drugs like etoposide generate elevated levels of cleavage complexes leading to cytotoxicity and genome instability. Drugs targeting Top2 also cause secondary malignancies that arise from enzyme induced DNA damage. Since Top2 can cause genome instability, a hallmark of cancer, it is plausible that Top2 failure could be a driver of genome instability leading to cancer. We identified unique mutants in yeast Top2 and the two human isoforms that generated high levels of DNA damage in the absence of inhibitors of the enzyme. These mutant enzymes have a unique characteristic: while they can be expressed in yeast cells proficient for repairing DNA damage, their expression in DNA repair deficient cells, such as rad52- cells leads to cell killing. We examined whether one of these mutants, (top2-F1025Y,R1128G) induced mutations in yeast and found that expression of this allele led to a novel mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Recently, it has been shown that human cancer cells that express Top2α-K743N are associated with a mutational signature closely related to that induced by yeast top2-F1025Y,R1128G suggesting that Top2α- K743N is a mutator that gives rise to genome instability. In this application, we will study the biochemical characteristics of Top2α-K743N. In addition, a small number of recurrent mutations have been found in cancer cells in Top2α or Top2β. Notably, Top2β-R656H has been recovered from more than 16 tumors of diverse origin. We will apply the tools we developed for studying top2-F1025Y,R1128G to the recurrent mutations found in both Top2α and Top2β. We will use expression of the mutant enzymes in rad52- cells and induction of etoposide hypersensitivity as a preliminary screen to determine whether the mutant proteins induce cytotoxic DNA damage. Putative DNA damaging Top2 proteins will be will be purified and characterized for the ability to generate DNA damage in vitro. Finally, we will begin to generate mammalian cell lines that express the mutant alleles by either CRISPR/Cas9 editing or ectopic expression to characterize how the mutant enzymes can lead to genome instability. Our results will establish whether Top2 alterations found in tumors can be oncogenic.

DNA拓扑异构酶II（TOP2）通过在DNA中进行双链断裂并通过在休息过程中完整的双工。通过蛋白质/DNA产生瞬态双链断裂共价中间体称为裂解复合物。哺乳动物细胞包含两个称为TOP2的同工型 TOP2α和TOP2β。这两种酶具有不同的生物学功能，top2α在染色体分离和TOP2β在转录中具有独特的作用。而DNA裂解机制 TOP2的允许细胞催化DNA构象的变化，而无需弗兰克DNA双链的危险断裂，多种机制会干扰酶的裂解/宗教。为了例如，抗癌药（例如依托泊苷）产生较高水平的切割复合物，导致细胞毒性和基因组不稳定性。靶向top2的药物也会引起次要恶性肿瘤酶诱导DNA损伤。由于TOP2可能会导致基因组不稳定性，这是癌症的标志，因此是合理的 Top2失败可能是导致癌症的基因组不稳定性的驱动力。我们确定了独特的突变体酵母Top2和两个在没有抑制剂的情况下会产生高水平DNA损伤的两个人类同工型酶。这些突变酶具有独特的特征：虽然它们可以在酵母中表达细胞熟练修复DNA损伤，它们在DNA修复缺陷细胞中的表达，例如RAD52-细胞导致细胞杀死。我们检查了这些突变体之一（TOP2-F1025Y，R1128G）是否诱导突变酵母并发现该等位基因的表达导致了一个新型的突变特征，该签名是从头开始的 DNA序列的重复取决于DSB修复的非同源最终连接途径。最近，已经表明，表达TOP2α-K743N的人类癌细胞与A 突变特征与酵母Top2-F1025Y（R1128G）诱导的密切相关，这表明TOP2α- K743N是导致基因组不稳定性的突变器。在此应用中，我们将研究生化 TOP2α-K743N的特性。另外，在癌症中发现了少量复发突变 TOP2α或TOP2β的细胞。值得注意的是，TOP2β-R656H已从16多个潜水员的肿瘤中回收起源。我们将应用我们开发的工具用于研究Top2-F1025Y，R1128G的复发突变在TOP2α和TOP2β中发现。我们将使用Rad52细胞中突变酶的表达并诱导依托泊苷超敏反应作为初步筛查，以确定突变蛋白是否诱导细胞毒性 DNA损伤。假定的DNA损伤TOP2蛋白将被纯化并以具有的能力为特征体外产生DNA损伤。最后，我们将开始生成表达突变体的哺乳动物细胞系 CRISPR/CAS9编辑或Ecopic表达的等位基因，以表征突变酶如何引导基因组不稳定性。我们的结果将确定在肿瘤中发现的TOP2改变是否可能具有致癌性。