title: ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase i inhibitors by disabling DNA replication initiation and fork elongation responses.
abstract: Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials.
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland.
authors.forename: Rozenn
authors.initials: R
authors.lastname: Jossé
authors.affiliation: Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences (NCATS), NIH, Rockville, Maryland.
authors.forename: Scott E
authors.initials: SE
authors.lastname: Martin
authors.affiliation: Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences (NCATS), NIH, Rockville, Maryland.
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Guha
authors.affiliation: Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences (NCATS), NIH, Rockville, Maryland.
authors.forename: Pinar
authors.initials: P
authors.lastname: Ormanoglu
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc, National Laboratory for Cancer Research, Frederick, Maryland.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Pfister
authors.affiliation: Vertex Pharmaceuticals (Europe) Ltd, Abingdon, Oxfordshire, United Kingdom.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Reaper
authors.affiliation: Vertex Pharmaceuticals (Europe) Ltd, Abingdon, Oxfordshire, United Kingdom.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Barnes
authors.affiliation: Vertex Pharmaceuticals (Europe) Ltd, Abingdon, Oxfordshire, United Kingdom.
authors.forename: Julie
authors.initials: J
authors.lastname: Jones
authors.affiliation: Vertex Pharmaceuticals (Europe) Ltd, Abingdon, Oxfordshire, United Kingdom.
authors.forename: Peter
authors.initials: P
authors.lastname: Charlton
authors.affiliation: Vertex Pharmaceuticals (Europe) Ltd, Abingdon, Oxfordshire, United Kingdom.
authors.forename: John R
authors.initials: JR
authors.lastname: Pollard
authors.affiliation: Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, Division of Cancer Treatment and Diagnosis (DTP-DCTD), NCI-NIH, Bethesda, Maryland.
authors.forename: Joel
authors.initials: J
authors.lastname: Morris
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland. Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, Division of Cancer Treatment and Diagnosis (DTP-DCTD), NCI-NIH, Bethesda, Maryland.
authors.forename: James H
authors.initials: JH
authors.lastname: Doroshow
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland. pommier@nih.gov.
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D006801:Humans; D064007:Ataxia Telangiectasia Mutated Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002166:Camptothecin / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D019073:HT29 Cells; D000077146:Irinotecan; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011719:Pyrazines / Q000494:pharmacology; D018741:Replication Origin / Q000187:drug effects; D059010:Single-Cell Analysis / Q000379:methods; D013450:Sulfones / Q000494:pharmacology; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology; D019772:Topotecan / Q000494:pharmacology; D007555:Isoxazoles / Q000737:chemistry / Q000494:pharmacology
chemical_list: C560580:3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide; D064007:Ataxia Telangiectasia Mutated Proteins; C576597:ATR protein, human; D002166:Camptothecin; D000071877:Checkpoint Kinase 1; C000605867:CHEK1 protein, human; D004264:DNA Topoisomerases, Type I; C468783:H2AX protein, human; D006657:Histones; D000077146:Irinotecan; D011494:Protein Kinases; D011719:Pyrazines; D013450:Sulfones; C528909:TOP1 protein, human; D059004:Topoisomerase I Inhibitors; D019772:Topotecan; C000598331:berzosertib; D007555:Isoxazoles