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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 -
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 -
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 -
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 -
Establishment and Characterization of Multi-Drug Resistant p53-Negative Osteosarcoma SaOS-2 Subline.Diagnostics (Basel, Switzerland) Aug 2023To establish a p53-negative osteosarcoma (OS) SaOS-2 cellular subline exhibiting resistance to specific chemotherapeutic agents, including topoisomerase II inhibitors,...
AIM
To establish a p53-negative osteosarcoma (OS) SaOS-2 cellular subline exhibiting resistance to specific chemotherapeutic agents, including topoisomerase II inhibitors, taxanes, and vinca alkaloids.
METHODS
The OS subline exhibiting resistance to the chemotherapeutic agents indicated above was generated by the stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of doxorubicin (Dox) for 5 months. Half-inhibitory concentrations (IC) for Dox, vinblastine (Vin), and paclitaxel (PTX) were calculated by a colorimetric MTS-based assay. Crystal violet staining was used to assess cellular viability, whereas the proliferation capacities of cancer cells were monitored in real-time by the i-Celligence system. Expression of apoptotic markers (e.g., cleaved PARP and caspase-3), DNA repair proteins (e.g., ATM, DNA-PK, Nbs1, Rad51, MSH2, etc.), and certain ABC transporters (P-glycoprotein, MRP1, ABCG2, etc.) was assessed by western blotting and real-time PCR. Flow cytometry was used to examine the fluorescence intensity of Dox and ABC-transporter substrates (e.g., Calcein AM and CMFDA) and to assess their excretion to define the activity of specific ABC-transporters. To confirm OS resistance to Dox in vivo, xenograft experiments were performed.
RESULTS
An OS subline generated by a stepwise treatment of the parental SaOS-2 cell line with increasing concentrations of Dox resulted in an increase in the IC for Dox, Vin, and PTX (~6-, 4-, and 30-fold, respectively). The acquisition of chemoresistance in vitro was also evidenced by the lack of apoptotic markers (e.g., cleaved PARP and caspase-3) in resistant OS cells treated with the chemotherapeutic agents indicated above. The development of the multidrug resistance (MDR) phenotype in this OS subline was due to the overexpression of ABCB1 (i.e., P-glycoprotein) and ABCC1 (i.e., multidrug resistance protein-1, MRP-1), which was evidenced on both mRNA and protein levels. Due to increased expression of MDR-related proteins, resistant OS exhibited an excessive efflux of Dox. Moreover, decreased accumulation of calcein AM, a well-known fluorescent substrate for both ABCB1 and ABCC1, was observed for resistant OS cells compared to their parental SaOS-2 cell line. Importantly, tariquidar and cyclosporin, well-known ABC inhibitors, retained the intensity of Dox-induced fluorescence in resistant SAOS-2 cells. Furthermore, in addition to the increased efflux of the chemotherapeutic agents from Dox-resistant OS cells, we found higher expression of several DNA repair proteins (e.g., Rad51 recombinase, Mre11, and Nbs1, activated forms of ATM, DNA-PK, Chk1, and Chk2, etc.), contributing to the chemoresistance due to the excessive DNA repair. Lastly, the in vivo study indicated that Dox has no impact on the SaOS-2 Dox-R xenograft tumor growth in a nude mouse model.
CONCLUSIONS
An acquired resistance of OS to the chemotherapeutic agents might be due to the several mechanisms undergoing simultaneously on the single-cell level. This reveals the complexity of the mechanisms involved in the secondary resistance of OS to chemotherapies.
PubMed: 37627905
DOI: 10.3390/diagnostics13162646 -
Science Advances May 2024DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage...
DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage complex (Top1cc) to allow strand rotation and duplex relaxation, which can lead to elevated levels of DNA-RNA hybrids and micronuclei. To comprehend the underlying mechanisms, we have integrated genomic data of Top1cc-triggered hybrids and DNA double-strand breaks (DSBs) shortly after Top1cc induction, revealing that Top1ccs increase hybrid levels with different mechanisms. DSBs are at highly transcribed genes in early replicating initiation zones and overlap with hybrids downstream of accumulated RNA polymerase II (RNAPII) at gene 5'-ends. A transcription factor IIS mutant impairing transcription elongation further increased RNAPII accumulation likely due to backtracking. Moreover, Top1ccs can trigger micronuclei when occurring during late G or early/mid S, but not during late S. As micronuclei and transcription-replication conflicts are attenuated by transcription factor IIS, our results support a role of RNAPII arrest in Top1cc-induced transcription-replication conflicts leading to DSBs and micronuclei.
Topics: Humans; DNA Breaks, Double-Stranded; DNA Replication; DNA Topoisomerases, Type I; Genomic Instability; R-Loop Structures; RNA Polymerase II; Transcription, Genetic
PubMed: 38787953
DOI: 10.1126/sciadv.adm8196 -
Cell Systems Jul 2023Genotoxic stress in mammalian cells, including those caused by anti-cancer chemotherapy, can induce temporary cell-cycle arrest, DNA damage-induced senescence (DDIS), or...
Genotoxic stress in mammalian cells, including those caused by anti-cancer chemotherapy, can induce temporary cell-cycle arrest, DNA damage-induced senescence (DDIS), or apoptotic cell death. Despite obvious clinical importance, it is unclear how the signals emerging from DNA damage are integrated together with other cellular signaling pathways monitoring the cell's environment and/or internal state to control different cell fates. Using single-cell-based signaling measurements combined with tensor partial least square regression (t-PLSR)/principal component analysis (PCA) analysis, we show that JNK and Erk MAPK signaling regulates the initiation of cell senescence through the transcription factor AP-1 at early times after doxorubicin-induced DNA damage and the senescence-associated secretory phenotype (SASP) at late times after damage. These results identify temporally distinct roles for signaling pathways beyond the classic DNA damage response (DDR) that control the cell senescence decision and modulate the tumor microenvironment and reveal fundamental similarities between signaling pathways responsible for oncogene-induced senescence (OIS) and senescence caused by topoisomerase II inhibition. A record of this paper's transparent peer review process is included in the supplemental information.
Topics: Animals; DNA Topoisomerases, Type II; Cellular Senescence; Signal Transduction; MAP Kinase Signaling System; DNA Damage; Mammals
PubMed: 37473730
DOI: 10.1016/j.cels.2023.06.005 -
Science Advances Dec 2023Type II topoisomerases (TOP2) form transient TOP2 cleavage complexes (TOP2ccs) during their catalytic cycle to relieve topological stress. TOP2ccs are covalently linked...
Type II topoisomerases (TOP2) form transient TOP2 cleavage complexes (TOP2ccs) during their catalytic cycle to relieve topological stress. TOP2ccs are covalently linked TOP2-DNA intermediates that are reversible but can be trapped by TOP2 poisons. Trapped TOP2ccs block transactions on DNA and generate genotoxic stress, which are the mechanisms of action of TOP2 poisons. How cells avoid TOP2cc accumulation remains largely unknown. In this study, we uncovered RAD54 like 2 (RAD54L2) as a key factor that mediates a TOP2-specific DNA damage avoidance pathway. RAD54L2 deficiency conferred unique sensitivity to treatment with TOP2 poisons. RAD54L2 interacted with TOP2A/TOP2B and ZATT/ZNF451 and promoted the turnover of TOP2 from DNA with or without TOP2 poisons. Additionally, inhibition of proteasome activity enhanced the chromatin binding of RAD54L2, which in turn led to the removal of TOP2 from chromatin. In conclusion, we propose that RAD54L2-mediated TOP2 turnover is critically important for the avoidance of potential TOP2-linked DNA damage under physiological conditions and in response to TOP2 poisons.
Topics: Poisons; DNA Topoisomerases, Type II; DNA Damage; DNA Repair; DNA; Chromatin
PubMed: 38055811
DOI: 10.1126/sciadv.adi6681 -
Nucleic Acids Research May 2024In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much...
In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.
Topics: Humans; Antigens, Neoplasm; Carbazoles; Chromatin; Diketopiperazines; DNA; DNA Damage; DNA Topoisomerases, Type II; DNA-Binding Proteins; High Mobility Group Proteins; Histones; Intercalating Agents; Poly-ADP-Ribose Binding Proteins; RNA Polymerase I; RNA Polymerase II; RNA Polymerase III; Topoisomerase II Inhibitors; Transcription, Genetic; Transcriptional Elongation Factors; Aclarubicin
PubMed: 38340348
DOI: 10.1093/nar/gkae069 -
Lancet (London, England) Feb 2024Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and a... (Randomized Controlled Trial)
Randomized Controlled Trial
Oral gepotidacin versus nitrofurantoin in patients with uncomplicated urinary tract infection (EAGLE-2 and EAGLE-3): two randomised, controlled, double-blind, double-dummy, phase 3, non-inferiority trials.
BACKGROUND
Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and a unique binding site, providing well balanced inhibition of two type II topoisomerase enzymes. Oral gepotidacin is under investigation to treat uncomplicated urinary tract infections. We aimed to compare the efficacy and safety of oral gepotidacin with that of nitrofurantoin in adolescent and adult female individuals with uncomplicated urinary tract infections.
METHODS
EAGLE-2 and EAGLE-3 were phase 3, randomised, multicentre, double-blind, double-dummy, non-inferiority (10% margin) trials, in which patients were enrolled at 219 centres worldwide. Patients assigned female at birth, non-pregnant, aged 12 years or older, weighing 40 kg or more, with two or more symptoms of dysuria, frequency, urgency, or lower abdominal pain, and with evidence of urinary nitrite, pyuria, or both were eligible for inclusion. Patients were randomly assigned (1:1) centrally by interactive response technology to receive oral gepotidacin (1500 mg twice daily for 5 days) or oral nitrofurantoin (100 mg twice daily for 5 days), with randomisation stratified by age category and history of recurrent uncomplicated urinary tract infections. Patients, investigators, and the sponsor study team were masked to treatment assignment. The primary endpoint, therapeutic response (success or failure) at test-of-cure (ie, day 10-13), was evaluated in randomly assigned patients with nitrofurantoin-susceptible qualifying uropathogens (≥10 colony-forming units [CFU] per mL) and who received at least one dose of study treatment. Conforming to regulatory guidance, therapeutic success was defined as combined clinical success (ie, complete symptom resolution) and microbiological success (ie, reduction of qualifying uropathogens to <10 CFU/mL) without other systemic antimicrobial use. Safety analyses included patients who were randomly assigned and who received at least one dose of study treatment. The trials are registered with ClinicalTrials.gov, NCT04020341 (EAGLE-2) and NCT04187144 (EAGLE-3), and are completed.
FINDINGS
Studies were undertaken from Oct 17, 2019, to Nov 30, 2022 (EAGLE-2), and from April 23, 2020, to Dec 1, 2022 (EAGLE-3). 1680 patients in EAGLE-2 and 1731 patients in EAGLE-3 were screened for eligibility, of whom 1531 and 1605 were randomly assigned, respectively (767 in the gepotidacin group and 764 in the nitrofurantoin group in EAGLE-2, and 805 in the gepotidacin group and 800 in the nitrofurantoin group in EAGLE-3). After an interim analysis, which was prospectively agreed as a protocol amendment, both studies were stopped for efficacy. Thus, the primary analysis population included only patients who, at the time of the interim analysis data cutoff, had the opportunity to reach the test-of-cure visit or were known to not have attained therapeutic success before the test-of-cure visit. In EAGLE-2, 162 (50·6%) of 320 patients assigned gepotidacin and 135 (47·0%) of 287 patients assigned nitrofurantoin had therapeutic success (adjusted difference 4·3%, 95% CI -3·6 to 12·1). In EAGLE-3, 162 (58·5%) of 277 patients assigned gepotidacin and 115 (43·6%) of 264 patients assigned nitrofurantoin had therapeutic success (adjusted difference 14·6%, 95% CI 6·4 to 22·8). Gepotidacin was non-inferior to nitrofurantoin in both studies and superior to nitrofurantoin in EAGLE-3. The most common adverse event with gepotidacin was diarrhoea (observed in 111 [14%] of 766 patients in EAGLE-2 and in 147 [18%] of 804 patients in EAGLE-3), whereas the most common adverse event with nitrofurantoin was nausea (in 29 [4%] of 760 patients in EAGLE-2 and in 35 [4%] of 798 patients in EAGLE-3). Cases were mostly mild or moderate. No life-threatening or fatal events occurred.
INTERPRETATION
Gepotidacin is an efficacious oral antibiotic with acceptable safety and tolerability profiles. As a first-in-class investigational oral antibiotic with activity against common uropathogens, including clinically important drug-resistant phenotypes, gepotidacin has the potential to offer substantial benefit to patients.
FUNDING
GSK and the US Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority.
Topics: Adult; Adolescent; Infant, Newborn; Humans; Female; Nitrofurantoin; Treatment Outcome; Anti-Bacterial Agents; Urinary Tract Infections; Research; Double-Blind Method; Acenaphthenes; Heterocyclic Compounds, 3-Ring
PubMed: 38342126
DOI: 10.1016/S0140-6736(23)02196-7