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Principal Investigator: YVES POMMIER
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2024
Award: $1,196,333
Funding agency: National Cancer Institute
We are testing chemical candidate compounds in our TOP3B cellular and biochemical assays. We have engineered cloned cell lines with reporters ("green-GFP" and "red-mCherry") allowing concurrent cultures of TOP3B wild-type (TOP3B+/+) and knockout (TOP3B-KO = TOP3B-/-) cells and used them for high throughput screening. The 2,500 compound NCATS Library has been screened in our lab. A transfer of our TOP3B-engineered cells to the CCR Molecular Target Laboratory has been set up with Barry O'Keefe at the NCI-Frederick. We are also testing the NCI libraries including natural products. Identified drugs are further tested in our biochemical assays with recombinant TOP3B as we have the assays running to study the biochemical and structural determinants of nucleic acid cleavage by TOP3B. Anti-viral activity is tested by our collaborators in Weidong Wang and Mariano Garcia-Blanco's groups. Identified drug leads, which we have patented and published in the Proceedings of the National Academy of Sciences are being optimized with medicinal chemist colleagues. We have successfully obtained and published the structure of TOP3B with its single-stranded DNA and RNA substrates, discovered new catalytic residues involved in metal binding to the enzyme catalytic site, and developed new biochemical assays to study the catalytic steps of the catalytic reaction. We are currently developing additional substrate and have obtained the first structures of the C-terminal domain of the enzyme with its cofactor TDRD3. Using our TOP3B knockout cell lines and our self-poisoning (R338W) TOP3B construct as well as wild-type and catalytic-dead constructs (Y336F), we have shown the importance of TOP3B in resolving R-loops and avoiding genomic damage. We observed elevated R-loops in TOP3B knockout cells (TOP3BKO), which are suppressed by TOP3B transfection. R-loop-inducing agents, the topoisomerase I inhibitor camptothecin, and the splicing inhibitor pladienolide-B also induce higher R-Loops in TOP3BKO cells. Camptothecin- and pladienolide-B-induced R-loops are concurrent with the induction of TOP3B cleavage complexes (TOP3Bccs). RNA/ DNA Hybrid IP-Western blotting show that TOP3B is physically associated with R-loops. Biochemical assays using recombinant TOP3B and oligonucleotides mimicking R-loops show that TOP3B cleaves the single-stranded DNA displaced by the R-loop RNA-DNA duplex. IP-Mass Spectrometry and IP-Western experiments reveal that TOP3B interacts with the R-loop helicase DDX5 independently of TDRD3. Finally, we demonstrate that DDX5 and TOP3B are epistatic in resolving R-loops in a pathway parallel with Senataxin. We proposed a decatenation model for R-loop resolution by TOP3B-DDX5 protecting cells from R-loop-induced damage. We are also engaged in a genomic screen using CRISPR-Cas9 and our isogenic TOP3B knockout cell lines to discover the synthetic lethal genes in cells lacking TOP3B. Because TOP3B knockout mice are viable and their phenotypes has not been fully characterized, we are determining the defects of TOP3B knockout mice compared to their wild-type counterparts. We have also successfully generated dominant-negative TOP3B knockin mice based on our R338W mutant. Our preliminary results show that the TOP3B knockout mice have immune defects with splenomegaly and altered immune cells with inflammatory phenotype. The TOP3B knockout mice are also tumor-prone as their lymphoid cells have increased R-loops and genomic damage in their immune cells. The knockin mice appear to have a different phenotype with growth defect and sterility.
Terms: <20-(S)-camptothecine><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><22-secocamptothecin-21-oic acid lactone 21><Anti-Cancer Agents><Antibiotic Agents><Antibiotic Drugs><Antibiotics><Antimorphic mutation><Antineoplastic Agents><Antineoplastic Drugs><Antineoplastics><Artificial beta Cells><Artificial β Cells><Assay><Autoregulation><Bacteria><Binding><Bioassay><Biochemical><Biological Assay><Biological Function><Biological Process><C-terminal><CCR><COVID-19 virus><COVID19 virus><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Camptothecin><Cancer Drug><Cas nuclease technology><Catalytic Core><Catalytic Domain><Catalytic Region><Catalytic Site><Catalytic Subunit><Cell Body><Cell Line><Cell Nucleus><Cell Protection><CellLine><Cells><Chemicals><Clinical><Clone Cells><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><CoV-2><CoV2><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><Cytoplasm><Cytoprotection><DNA><DNA Helicases><DNA Topoisomerase II><DNA Topoisomerase III><DNA Topoisomerase IV><DNA Type 2 Topoisomerase><DNA Unwinding Proteins><DNA unwinding enzyme><Defect><Deoxyribonucleic Acid><Dominant Negative><Dominant-Negative Mutant><Dominant-Negative Mutation><Drug Targeting><Drugs><Electron Cryomicroscopy><Engineering><Enlarged Spleen><Enzyme Gene><Enzymes><Family><Gene Transcription><Generalized Growth><Genetic Transcription><Genome Stability><Genomic Stability><Genomics><Growth><High Throughput Assay><Homeostasis><Host Factor><Host Factor Protein><Human><Human Cell Line><Hybrids><Immune><Immunes><Immunoblotting><Inflammatory><Integration Host Factors><KI mice><KO mice><Knock-in Mouse><Knock-out><Knock-out Mice><Knockout><Knockout Mice><Laboratories><Legal patent><Lethal Genes><Libraries><Lymphoid Cell><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Medication><Medicine><Metabolic><Metals><Minor><Miscellaneous Antibiotic><Modeling><Modern Man><Molecular Interaction><Molecular Target><NCATS><National Academy of Sciences><National Center for Advancing Translational Sciences><Natural Products><Neoplastic Disease Chemotherapeutic Agents><Non-Polyadenylated RNA><Nucleic Acid Cleavage><Nucleus><Null Mouse><Oligo><Oligonucleotides><Patents><Pathway interactions><Pharmaceutical Preparations><Phenotype><Physiological Homeostasis><Poisoning><Publishing><RNA><RNA Expression><RNA Folding><RNA Gene Products><RNA Splicing><RNA Viruses><Reaction><Recombinants><Reporter><Reporting><Resolution><Ribonucleic Acid><Role><Running><SARS><SARS corona virus 2><SARS coronavirus disease><SARS-CO-V2><SARS-COVID-2><SARS-CoV disease><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome coronavirus disease><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Single-Stranded DNA><Splenomegaly><Splicing><Sterility><Strains Cell Lines><Structure><System><TOP1><TOP1 gene><TOP2><TOP2A><TOP2A gene><TOP3><TOP3 topoisomerase><TOP3A><TOP3A gene><TP2A><Testing><Tissue Growth><Topo II><Topo III><Topo III-Alpha><Topo IV><Topoisomerase><Topoisomerase II><Topoisomerase III><Topoisomerase IV><Topoisomerase-I Inhibitor><Transcription><Transfection><Translations><Tumor-Specific Treatment Agents><Type II DNA Topoisomerases><United States National Academy of Sciences><Viral Activity><Viral Function><Viral Physiology><Western Blotting><Western Immunoblotting><Wuhan coronavirus><Yeasts><anti-cancer drug><atomic interactions><cancer predisposition><cofactor><coronavirus disease 2019 virus><coronavirus disease-19 virus><cryo-EM><cryoEM><cryogenic electron microscopy><cultured cell line><cytoprotective><drug/agent><engineered beta cell><experiment><experimental research><experimental study><experiments><hCoV19><helicase><high throughput screening><inhibitor><knockin mice><mutant><nCoV2><naturally occurring product><novel><oligos><ontogeny><pathway><poisoned><protein blotting><resolutions><social role><ssDNA><sterile><structural determinants><structural factors><translation><tumor>