ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase i inhibitors by disabling DNA replication initiation and fork elongation responses

Cancer Res. 2014 Dec 1;74(23):6968-79. doi: 10.1158/0008-5472.CAN-13-3369. Epub 2014 Sep 30.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Ataxia Telangiectasia Mutated Proteins / antagonists & inhibitors
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Camptothecin / analogs & derivatives
  • Camptothecin / pharmacology
  • Cell Line, Tumor
  • Checkpoint Kinase 1
  • DNA Damage
  • DNA Replication / drug effects*
  • DNA Topoisomerases, Type I / metabolism*
  • HT29 Cells
  • Histones / genetics
  • Histones / metabolism
  • Humans
  • Irinotecan
  • Organothiophosphorus Compounds / pharmacology*
  • Phosphorylation / drug effects
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Pyrazines / pharmacology*
  • Replication Origin / drug effects*
  • Single-Cell Analysis / methods
  • Sulfones / pharmacology*
  • Topoisomerase I Inhibitors / pharmacology*
  • Topotecan / pharmacology

Substances

  • 3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide
  • H2AX protein, human
  • Histones
  • Organothiophosphorus Compounds
  • Pyrazines
  • Sulfones
  • Topoisomerase I Inhibitors
  • Irinotecan
  • Topotecan
  • VX
  • Protein Kinases
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • CHEK1 protein, human
  • Checkpoint Kinase 1
  • DNA Topoisomerases, Type I
  • TOP1 protein, human
  • Camptothecin