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The MTH1 Inhibitor TH588 Is a Microtubule-Modulating Agent That Eliminates Cancer Cells by Activating the Mitotic Surveillance P

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The MTH1 Inhibitor TH588 Is a Microtubule-Modulating Agent That Eliminates Cancer Cells by Activating the Mitotic Surveillance P www.nature.com/scientificreports OPEN The MTH1 inhibitor TH588 is a microtubule-modulating agent that eliminates cancer cells by activating Received: 28 May 2019 Accepted: 26 September 2019 the mitotic surveillance pathway Published online: 11 October 2019 Nadia Gul1, Joakim Karlsson 2, Carolina Tängemo4, Sanna Linsefors1, Samuel Tuyizere1, Rosie Perkins1, Chandu Ala1, Zhiyuan Zou1, Erik Larsson3, Martin O. Bergö5 & Per Lindahl1,3 The mut-T homolog-1 (MTH1) inhibitor TH588 has shown promise in preclinical cancer studies but its targeting specifcity has been questioned. Alternative mechanisms for the anti-cancer efects of TH588 have been suggested but the question remains unresolved. Here, we performed an unbiased CRISPR screen on human lung cancer cells to identify potential mechanisms behind the cytotoxic efect of TH588. The screen identifed pathways and complexes involved in mitotic spindle regulation. Using immunofuorescence and live cell imaging, we showed that TH588 rapidly reduced microtubule plus-end mobility, disrupted mitotic spindles, and prolonged mitosis in a concentration-dependent but MTH1- independent manner. These efects activated a USP28-p53 pathway – the mitotic surveillance pathway – that blocked cell cycle reentry after prolonged mitosis; USP28 acted upstream of p53 to arrest TH588- treated cells in the G1-phase of the cell cycle. We conclude that TH588 is a microtubule-modulating agent that activates the mitotic surveillance pathway and thus prevents cancer cells from re-entering the cell cycle. Te mut-T homolog 1 (MTH1, also known as NUDT1) inhibitor TH588 has shown promise as an anticancer compound in preclinical studies1,2. It is toxic to a wide range of human cancer cell lines at concentrations that are tolerated by primary or immortalized cells1. In addition, daily injections of TH588 in mice markedly reduced the growth rate of a patient-derived visceral metastasis of a malignant melanoma at concentrations that did not afect body weight, blood cell counts, or liver/heart/kidney parameters1. Te cytotoxic efect of TH588 on human cancer cell lines has been confrmed in other laboratories3–7, supporting further development of this compound. Although independent studies have confrmed that TH588 inhibits MTH1 at nanomolar concentrations4–8, accumulating evidence suggests that its anti-cancer efect is mediated by other mechanisms: First, the concentra- tions of TH588 required for its cytotoxic efect are several fold higher than those required for MTH1 inhibition1,5. Second, a number of alternative MTH1 inhibitors have been developed, none of which are toxic when tested on human cancer cell lines4–7. Tird, CRISPR-mediated knockout of MTH1 failed to reproduce the cytotoxic efect of MTH1 knockdown that was previously reported in human cancer cell lines5,9. And fourth, mice lacking MTH1 show a small but signifcant increase in spontaneous cancers10. A number of alternative mechanisms have been suggested for the anti-cancer efects of TH588 including lipophilicity-related efects, isoform-specifc MTH1 tar- geting, tubulin depolymerization, oxidative damage, and downregulation of the PI3K-Akt-mTOR axis5,6,11–14, but the question remains unresolved12,15. Tere are no reports on the use of unbiased screens to defne genes and pathways that underlie TH588’s efects. 1Wallenberg Laboratory, Department of Molecular and Clinical Medicine, Institute of Medicine, University of Gothenburg, SE-413 45, Gothenburg, Sweden. 2Department of Surgery, Institute of Clinical Sciences, University of Gothenburg, SE-416 85, Gothenburg, Sweden. 3Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, SE-405 30, Gothenburg, Sweden. 4Centre for Cellular Imaging, University of Gothenburg, Box 413, SE-40530, Gothenburg, Sweden. 5Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 83 Huddinge, Sweden. Correspondence and requests for materials should be addressed to P.L. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:14667 | https://doi.org/10.1038/s41598-019-51205-w 1 www.nature.com/scientificreports/ www.nature.com/scientificreports A Lentiviral Lentiviral dox-Cas9 cDNA gRNA library 14 cell divisions in 4 µM TH588 or DMSO + dox Target identification: TH588-treated Deep sequencing of mutant cell pool PCR-amplified lentiviral H460 cell Cas9-expressing Mutant cell pool gRNA inserts H460 cells Untreated mutant cell pool B C Cell cycleKinase FDR 15 3 1.2 0.15 Ctrl ) 0.10 DMSO 0.9 10 2 0.05 4 µM TH588 0.6 Gene score 5 (Log2-ratio 1 0.3 0.0 5 2 1 4 3 1 8 4 1 4 6 F 0 A TK TS Accumulated cell doublings 0510 15 20 RIF1 CINP TP53 A PLK1 ABL1 FZR TPX2 AA USP KIF2 BRD3 MSH GRK5 PNCK MCM6 BIRC MUSK WNK1 IKBKB TERF CAB39 USP28 STYK1 SSNA1 KPNA2 RAB1A CDK1 CENP SPDYC NUMA1 Days AURK TRIM24 SEPT1 YE NUP107 STK17A CGREF1 EIF2AK CSNKE1 MAPRE RACGAP1 ARHGEF10 D E CGREF1 Centrosome:AURKA:TPX2:HMMR CINP MSH4 Aurora A signaling PNCK role of ran in mitotic spindle regulation SPDYC Importin-alpha/Importin-beta/NuMA/Tpx2 GRK5 TERF2ABL1 WNK1 NuMA/Tpx2 TRIM24 pronucleus USP28 STYK1 p-S15,S20-TP53 Tetramer:BIRC5 Gene TP53 Aurora A/RasGap/Survivin USP8 IKBKB ARHGEF10 RasGAP-AURKA/AURKB-survivin complex CAB39 KPNA2 TNF-alpha/NF-kappa B signaling complex 5 CDK16 TPX2 NuMA/Tpx2/Hklp2 KIF23 STK17A p-T288-AURKA:TPX2 AURKA NUMA1 Centraspindlin FZR1 BIRC5 PLK1 YEATS4 GRAS P65/GM130/RAB1/GTP/PLK1 1 1 1 2 3 5 1 1 F A MCM6 B RACGAP1CSNK1E NUP107 BRD3 RIF1 TP53 PLK FZR TPX2 KIF2 IKBK BIRC SSNA RAB1A KPNA CENP AURK RIF1 NUMA CSNK1E CENPF AATK MAPRE1 SSNA1 RACGAP MAPRE1 RAB1A MUSK SEPT11 Gene Ontology Pathways Protein complexes Cellular component BioCarta BioCarta EIF2AK4 PID CORUM Textmining PID Curated databases Reactome Experimentally determined Co-expression Figure 1. CRISPR/Cas9 screening of TH588-treated cells identifed protein complexes and pathways associated with mitotic spindle regulation. (A) Doxycycline-inducible Cas9-expressing cells were infected with lentiviral gRNA libraries to generate complex mutant cell pools (MCPs) for screening. Te MCPs were passaged in TH588 or DMSO for 14 cell divisions before determining the gRNA repertoire (and hence the repertoire of mutations) in the selected cell populations by massive parallel sequencing of PCR-amplifed lentiviral inserts. (B) Growth curves showing accumulated cell doublings of MCPs that were passaged in TH588 or DMSO. (C) Gene scores for cell cycle genes (lef) and kinase genes (right), analogous to average gRNA fold-change (Log2-ratio) in TH588-treated MCPs compared to controls as calculated with the MAGeCK MLE algorithm. Genes with false discovery rates (FDR) < 0.2 are shown. (D) A protein interaction network constructed with candidate genes for both libraries (FDR < 0.2) using the STRING database of known or predicted protein-protein interactions. Te STRING database integrates diverse types of evidence and the color of the edges corresponds to the type of supporting evidence. Te color of the nodes corresponds to the FDR value presented in panel C. (E) Graphic representation of candidate genes and their corresponding functional annotations for gene ontology terms and pathways and protein complexes that were statistically overrepresented among candidate genes with FDR < 0.1 in our screen. Te analysis was performed with ConcensusPathDB and shows annotations with q < 0.05 in the respective database. Here we performed a pooled lentiviral CRISPR screen to identify gene knockouts that rescue H460 human lung cancer cells from the cytotoxic efect of TH588. Data from such screens can identify mechanisms behind drug efects and drug resistance mechanisms. Results CRISPR screen of TH588-treated cells identifed complexes and pathways associated with mitotic spindle regulation. To elucidate mechanisms behind the pharmacological anti-cancer effect of TH588, we performed pooled lentiviral CRISPR screens to identify gene knockouts that rescue prolifera- tion or viability of TH588-treated human H460 lung cancer cells (Fig. 1A). To make H460 cells accessible for screening, we infected cells with lentiviral Cas9 and generated clones expressing doxycycline-inducible Cas9 (Supplementary Fig. S1A). Cas9-expressing cells were infected with two guide RNA (gRNA) libraries targeting SCIENTIFIC REPORTS | (2019) 9:14667 | https://doi.org/10.1038/s41598-019-51205-w 2 www.nature.com/scientificreports/ www.nature.com/scientificreports 1000 cell cycle genes and 500 kinase genes, and treated with blasticidin to produce mutant cell pools16. Each gene was targeted by 10 diferent gRNAs. Massive parallel sequencing of PCR-amplifed lentiviral inserts showed that 9 or 10 gRNAs per gene were detected for more than 95% of the targeted genes, indicating that virus transduction efciency and sequencing depth were sufcient (Supplementary Fig. S1B). To select for mutations that rescue the cytotoxic efect, we passaged the mutant cell pools for 18 days in 4 µM TH588, which markedly reduced cell proliferation (Fig. 1B). In a parallel study, we passaged the same mutant cell pools in 2 µM auranofn, a thioredoxin reductase inhibitor that kills cells by inducing oxidative stress. Control mutant cell pools were passaged with and without DMSO. Te abundance of all gRNAs was determined by massive parallel sequencing of PCR-amplifed lentiviral inserts. Principal component analyses showed that TH588- and auranofn-treated samples separated along the two frst principal components, whereas untreated and DMSO-treated control samples clustered together (Supplementary Fig. S1C). Tese results indicate
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