TOP3B as host factor and drug target

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Topoisomerase III-beta (TOP3B) is the only known human RNA topoisomerase. It belongs to the type IA topoisomerase family, which is conserved across species (Topo I and Topo III in bacteria, Top3 in yeast). Unlike other topoisomerases, TOP3B is not only present in the nucleus but is also abundant in the cytoplasm TOP3B has also been reported as positive host factor for SARS-CoV-2. We hypothesize that TOP3B acts by removing topological barriers associated with cellular processing of the long (30 kb) and folded RNA of SARS-CoV-2 (transcription - translation - replication - packaging), in addition to its fundamental roles in cellular homeostasis (transcription and translation). Although inhibitors of topoisomerases I and II are established anticancer drugs (targeting TOP1 and TOP2) and antibiotics (targeting both bacterial TOP2 enzymes: gyrase and Topo IV), they do not affect TOP3B and there is no clinical inhibitor for TOP3 or bacterial TOP1A. As we have established TOP3B knockout human cells and found them viable with only minor baseline growth reduction, we have screened for selective TOP3B inhibitors using our isogenic human cell lines with and without TOP3B target. We have been the first to report novel inhibitors and we have patented them. They are being tested by our collaborators in SARS-CoV-2-infected and other RNA virus-infected cells. Our second aim is to define the biological functions of TOP3B and particularly how it regulates R-loops and genome stability. We are studying cell-based systems including isogenic TOP3B knockout cell lines and TOP3B knockout mice to determine their phenotypes including R-loop accumulations, genomic instability, immune and metabolic deficiency, synthetic lethality and cancer predisposition. We have successfully set up cryo-Electron-Microscopy (CryoEM) to study the molecular and atomic interactions of TOP3B with its DNA and RNA substrates and set the stage to elucidate the binding of TOP3B inhibitors. Project Summary Aim 1: Discovery of TOP3B poisons and analysis of their molecular mechanism of action: We have tested 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 have patented and published in the Proceedings of the National Academy of Sciences are being optimized with medicinal chemist colleagues. Aim 2: Elucidation of the structure of human TOP3B with and without its DNA and RNA substrates, protein cofactors and inhibitors: 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. Aim 3: Elucidation of the roles of TOP3B in DNA and RNA metabolism: 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 have demonstrated elevated R-loops in TOP3B knockout cells (TOP3BKO), which are suppressed by TOP3B transfection, and extended this finding to Top3B knockout mice. 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 have demonstrated 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 have performed ia genomic screen using CRISPR-Cas9 and isogenic TOP3B knockout cell lines to discover the synthetic lethal genes in cells lacking TOP3B. The most prominent pathways involve RNA splicing, translation and proteasomal degradation. We have further established the importance of TOP3B for splicing using IP-mass-spectrometry and RNA sequencing. The study is being written up for submission. Aim 4: In vivo studies in TOP3B knockout mice: 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 knock-in mice appear to have a different phenotype with growth defect and sterility. The study has been submitted for publication and is being revised.