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: Rozenn Jossé, Scott E Martin, Rajarshi Guha, Pinar Ormanoglu, Thomas D Pfister, Philip M Reaper, Christopher S Barnes, Julie Jones, Peter Charlton, John R Pollard, Joel Morris, James H Doroshow, Yves Pommier
• Journal: Cancer research (Cancer Res) Vol. 74 Issue 23 Pg. 6968-79 (Dec 01 2014) ISSN: 1538-7445 [Electronic] United States
• PMID: 25269479 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: ©2014 American Association for Cancer Research.

Chemical References

• 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

Topics

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