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

Paradigms and Technologies


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.


Jossé, Rozenn; Martin, Scott E; Guha, Rajarshi; Ormanoglu, Pinar; Pfister, Thomas D; Reaper, Philip M; Barnes, Christopher S; Jones, Julie; Charlton, Peter; Pollard, John R; Morris, Joel; Doroshow, James H; Pommier, Yves;


  • Ataxia Telangiectasia Mutated Proteins/ antagonists & inhibitors
  • Ataxia Telangiectasia Mutated Proteins/ metabolism
  • Camptothecin/ analogs & derivatives
  • Camptothecin/ pharmacology
  • Cell Line, Tumor
  • DNA Damage
  • DNA Replication/ drug effects
  • DNA Topoisomerases, Type I/ metabolism
  • HT29 Cells
  • Histones/ genetics
  • Histones/ metabolism
  • Humans
  • 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

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