title: Development of pharmacodynamic biomarkers for ATR inhibitors.
abstract: BACKGROUND
  ATR, which signals DNA damage to S/G2 cell cycle checkpoints and for repair, is an attractive target in cancer therapy. ATR inhibitors are being developed and a pharmacodynamic assay is needed to support clinical studies.
  
  
  METHODS
  Phosphorylation of ATR targets, Chk1 and H2AX, was evaluated in MCF7 and K562 cells, human volunteer PBMCs and whole blood by Western blot, immunofluorescence microscopy and flow cytometry after DNA damage. The effect of cell cycle phase, ATR knockdown and inhibition on these phosphorylation events was determined.
  
  
  RESULTS
  Hydroxyurea, UV and 4NQO induced Chk1 and H2AX phosphorylation in MCF7 and K562 cells. UV/4NQO activation of ATR was detectable in non-cycling cells. Chk1 phosphorylation was reduced by ATR knockdown and reflects ATR activity for 3 h, H2AX phosphorylation after UV/4NQO is ATR-dependent for 1 h but increasingly ATM and DNA-PK-dependent at later time points. In isolated PBMCs both phospho-targets were detectable after UV/4NQO but in PBMCs from whole blood treated with 4NQO only H2AX was detectable.
  
  
  CONCLUSIONS
  PhosphoChk1 and H2AX are useful biomarkers for ATR inhibition using a variety of immuno-detection methods, but timing may be critical. Importantly, ATR activity is detectable in non-cycling PBMCs allowing them to be used as a surrogate tissue for biomarker measurement. In PBMCs from whole blood treated with 4NQO phosphoH2AX was the most useful biomarker of ATR activity and a clinically viable pharmacodynamic assay for ATR inhibitors has been developed.
authors.affiliation: Newcastle University, Northern Institute for Cancer Research, Paul O'Gorman Building, Medical School, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
authors.affiliation: Newcastle University, Northern Institute for Cancer Research, Paul O'Gorman Building, Medical School, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Fiona K
authors.initials: FK
authors.lastname: Middleton
authors.affiliation: Vertex Pharmaceuticals (Europe) Limited, 86-88 Jubilee Avenue, Milton Park, Abingdon, Oxfordshire, OX14 4RW, UK.
authors.forename: Susanna
authors.initials: S
authors.lastname: Falcon
authors.affiliation: Vertex Pharmaceuticals (Europe) Limited, 86-88 Jubilee Avenue, Milton Park, Abingdon, Oxfordshire, OX14 4RW, UK.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Reaper
authors.affiliation: Vertex Pharmaceuticals (Europe) Limited, 86-88 Jubilee Avenue, Milton Park, Abingdon, Oxfordshire, OX14 4RW, UK.
authors.forename: John R
authors.initials: JR
authors.lastname: Pollard
authors.affiliation: Newcastle University, Northern Institute for Cancer Research, Paul O'Gorman Building, Medical School, Newcastle upon Tyne, NE2 4HH, UK. Electronic address: nicola.curtin@ncl.ac.uk.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Curtin
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D005260:Female; D006657:Histones / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D008297:Male; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000493:pharmacokinetics / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; C468783:H2AX protein, human; D006657:Histones; D009363:Neoplasm Proteins; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1
keywords: 4NQO; ATR; Biomarker; PBMC; UV; pChk1; γH2AX