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Nature Chemical Biology May 2023Etoposide is a broadly employed chemotherapeutic and eukaryotic topoisomerase II poison that stabilizes cleaved DNA intermediates to promote DNA breakage and...
Etoposide is a broadly employed chemotherapeutic and eukaryotic topoisomerase II poison that stabilizes cleaved DNA intermediates to promote DNA breakage and cytotoxicity. How etoposide perturbs topoisomerase dynamics is not known. Here we investigated the action of etoposide on yeast topoisomerase II, human topoisomerase IIα and human topoisomerase IIβ using several sensitive single-molecule detection methods. Unexpectedly, we found that etoposide induces topoisomerase to trap DNA loops, compacting DNA and restructuring DNA topology. Loop trapping occurs after ATP hydrolysis but before strand ejection from the enzyme. Although etoposide decreases the innate stability of topoisomerase dimers, it increases the ability of the enzyme to act as a stable roadblock. Interestingly, the three topoisomerases show similar etoposide-mediated resistance to dimer separation and sliding along DNA but different abilities to compact DNA and chirally relax DNA supercoils. These data provide unique mechanistic insights into the functional consequences of etoposide on topoisomerase II dynamics.
Topics: Humans; Etoposide; Topoisomerase II Inhibitors; DNA Topoisomerases, Type II; DNA
PubMed: 36717711
DOI: 10.1038/s41589-022-01235-9 -
The Journal of Clinical Investigation Mar 2024Development of effective strategies to manage the inevitable acquired resistance to osimertinib, a third-generation EGFR inhibitor for the treatment of EGFR-mutant...
Development of effective strategies to manage the inevitable acquired resistance to osimertinib, a third-generation EGFR inhibitor for the treatment of EGFR-mutant (EGFRm) non-small cell lung cancer (NSCLC), is urgently needed. This study reports that DNA topoisomerase II (Topo II) inhibitors, doxorubicin and etoposide, synergistically decreased cell survival, with enhanced induction of DNA damage and apoptosis in osimertinib-resistant cells; suppressed the growth of osimertinib-resistant tumors; and delayed the emergence of osimertinib-acquired resistance. Mechanistically, osimertinib decreased Topo IIα levels in EGFRm NSCLC cells by facilitating FBXW7-mediated proteasomal degradation, resulting in induction of DNA damage; these effects were lost in osimertinib-resistant cell lines that possess elevated levels of Topo IIα. Increased Topo IIα levels were also detected in the majority of tissue samples from patients with NSCLC after relapse from EGFR tyrosine kinase inhibitor treatment. Enforced expression of an ectopic TOP2A gene in sensitive EGFRm NSCLC cells conferred resistance to osimertinib, whereas knockdown of TOP2A in osimertinib-resistant cell lines restored their susceptibility to osimertinib-induced DNA damage and apoptosis. Together, these results reveal an essential role of Topo IIα inhibition in mediating the therapeutic efficacy of osimertinib against EGFRm NSCLC, providing scientific rationale for targeting Topo II to manage acquired resistance to osimertinib.
Topics: Humans; Acrylamides; Carcinoma, Non-Small-Cell Lung; Aniline Compounds; ErbB Receptors; Lung Neoplasms; DNA Topoisomerases, Type II; Cell Line, Tumor; Topoisomerase II Inhibitors; Drug Resistance, Neoplasm; Animals; Mice; Mutation; Poly-ADP-Ribose Binding Proteins; Drug Synergism; DNA Damage; Piperazines; Etoposide; Xenograft Model Antitumor Assays
PubMed: 38451729
DOI: 10.1172/JCI172716 -
Molecular Cell Apr 2024The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free...
The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.
Topics: DNA Topoisomerases, Type II; Chromosomes; Mitosis; Interphase; Polymers
PubMed: 38521067
DOI: 10.1016/j.molcel.2024.02.025 -
Proceedings of the National Academy of... Jul 2023Type II topoisomerases transiently cleave duplex DNA as part of a strand passage mechanism that helps control chromosomal organization and superstructure. Aberrant DNA...
Type II topoisomerases transiently cleave duplex DNA as part of a strand passage mechanism that helps control chromosomal organization and superstructure. Aberrant DNA cleavage can result in genomic instability, and how topoisomerase activity is controlled to prevent unwanted breaks is poorly understood. Using a genetic screen, we identified mutations in the beta isoform of human topoisomerase II (hTOP2β) that render the enzyme hypersensitive to the chemotherapeutic agent etoposide. Several of these variants were unexpectedly found to display hypercleavage behavior in vitro and to be capable of inducing cell lethality in a DNA repair-deficient background; surprisingly, a subset of these mutations were also observed in sequences from cancer genome databases. Using molecular dynamics simulations and computational network analyses, we found that many of the mutations obtained from the screen map to interfacial points between structurally coupled elements, and that dynamical modeling could be used to identify other damage-inducing alleles present in cancer genome databases. This work establishes that there is an innate link between DNA cleavage predisposition and sensitivity to topoisomerase II poisons, and that certain sequence variants of human type II topoisomerases found in cancer cells can act as DNA-damaging agents. Our findings underscore the potential for hTOP2β to function as a clastogen capable of generating DNA damage that may promote or support cellular transformation.
Topics: Humans; Mutagens; Topoisomerase II Inhibitors; Etoposide; DNA Topoisomerases, Type II; DNA Damage; DNA; Neoplasms
PubMed: 37406101
DOI: 10.1073/pnas.2302064120 -
Scientific Reports Jan 2023Type II topoisomerases (TOP2) poisons represent one class of the most successful and widely prescribed chemotherapeutics, which is frontline therapy for a myriad of...
Type II topoisomerases (TOP2) poisons represent one class of the most successful and widely prescribed chemotherapeutics, which is frontline therapy for a myriad of systemic cancers and solid tumors, including lymphomas, leukemias, and lung cancer. Despite this, treatment with this class of drugs induces unwanted side effects (including cardiovascular morbidity and secondary malignancies). Additionally, the emergence of drug resistance also greatly compromises the clinical use of these drugs. To enhance therapeutic efficiency while lowering unwanted side effects, new insights into effective combination therapy are required. In this study we found that KU60019, a novel, and highly specific ATM kinase inhibitor interferes with the association of ATM with TOP2β and stabilizes TOP2β-DNA cleavage complex, thereby impairing the repair of TOP2 poison-induced DSBs and contributes to genome stability, leading to accelerated cell death. In H1299 as well as in A549 lung cancer cell lines, biologically, KU60019 combined with VP-16 (one of the TOP2 poisons) synergistically suppressed the growth of cells and survival and triggered a much higher apoptosis rate. In summary, we provide a proof-of-concept strategy that ATM inhibitors combined with TOP2 poison would synergistically suppresses lung cancer cell survival as well as reduce DNA damage responses, thus may lowering the possibility of cardiotoxicity and secondary malignancy linked to therapy.
Topics: Humans; DNA Topoisomerases, Type II; Topoisomerase II Inhibitors; Etoposide; Lung Neoplasms; Ataxia Telangiectasia Mutated Proteins
PubMed: 36650267
DOI: 10.1038/s41598-023-28185-z -
EMBO Reports Jul 2023The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for...
The bacterial toxin CcdB (Controller of Cell death or division B) targets DNA Gyrase, an essential bacterial topoisomerase, which is also the molecular target for fluoroquinolones. Here, we present a short cell-penetrating 24-mer peptide, CP1-WT, derived from the Gyrase-binding region of CcdB and examine its effect on growth of Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus and a carbapenem- and tigecycline-resistant strain of Acinetobacter baumannii in both axenic cultures and mouse models of infection. The CP1-WT peptide shows significant improvement over ciprofloxacin in terms of its in vivo therapeutic efficacy in treating established infections of S. Typhimurium, S. aureus and A. baumannii. The molecular mechanism likely involves inhibition of Gyrase or Topoisomerase IV, depending on the strain used. The study validates the CcdB binding site on bacterial DNA Gyrase as a viable and alternative target to the fluoroquinolone binding site.
Topics: Animals; Mice; Staphylococcus aureus; Anti-Bacterial Agents; DNA Gyrase; DNA Topoisomerase IV; Peptides
PubMed: 37166011
DOI: 10.15252/embr.202255338 -
Nature Communications Dec 2023The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell...
The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (-30 to +20) that has the strongest affinity to TOP2B within -423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.
Topics: Humans; DNA; DNA Topoisomerases, Type II; Etoposide; Genes, Immediate-Early; Mitogen-Activated Protein Kinase 1; Phosphorylation; Transcriptional Activation
PubMed: 38097570
DOI: 10.1038/s41467-023-44089-y -
PLoS Genetics Jan 2021Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases...
Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.
Topics: Animals; Cell Cycle Proteins; Chromatids; Chromosomal Proteins, Non-Histone; Chromosome Segregation; DNA Topoisomerases, Type II; Drosophila melanogaster; Kinetochores; Larva; Metaphase; Microtubules; Mitosis; Spindle Apparatus; Cohesins
PubMed: 33513180
DOI: 10.1371/journal.pgen.1009304 -
Science (New York, N.Y.) Jun 2023Cyanotriazole compounds "poison" topoisomerase II of pathogenic trypanosomatids.
Cyanotriazole compounds "poison" topoisomerase II of pathogenic trypanosomatids.
Topics: Antiparasitic Agents; DNA Topoisomerases, Type II; Triazoles; Topoisomerase II Inhibitors; Trypanosomatina; Animals; Humans
PubMed: 37384682
DOI: 10.1126/science.adi5925 -
Developmental Cell Jan 2020GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein...
GCNA proteins are expressed across eukarya in pluripotent cells and have conserved functions in fertility. GCNA homologs Spartan (DVC-1) and Wss1 resolve DNA-protein crosslinks (DPCs), including Topoisomerase-DNA adducts, during DNA replication. Here, we show that GCNA mutants in mouse and C. elegans display defects in genome maintenance including DNA damage, aberrant chromosome condensation, and crossover defects in mouse spermatocytes and spontaneous genomic rearrangements in C. elegans. We show that GCNA and topoisomerase II (TOP2) physically interact in both mice and worms and colocalize on condensed chromosomes during mitosis in C. elegans embryos. Moreover, C. elegans gcna-1 mutants are hypersensitive to TOP2 poison. Together, our findings support a model in which GCNA provides genome maintenance functions in the germline and may do so, in part, by promoting the resolution of TOP2 DPCs.
Topics: Animals; Caenorhabditis elegans; DNA Damage; DNA Repair; DNA Replication; DNA Topoisomerases, Type II; DNA-Binding Proteins; Genome; Genomic Instability; Germ Cells; Male; Mice; Mice, Inbred C57BL; Mitosis; Mutation; Nuclear Proteins; Spermatocytes; Spermatogenesis
PubMed: 31839538
DOI: 10.1016/j.devcel.2019.11.006