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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

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中制造双链断裂并传递DNA来改变DNA拓扑结构