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Development of pharmacodynamic biomarkers for ATR inhibitors

Lookup NU author(s): Dr Tao Chen, Fiona Middleton, Professor Nicola CurtinORCiD

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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.Conclusion: 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. (C) 2014 Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies.

Publication metadata

Author(s): Chen T, Middleton FK, Falcon S, Reaper PM, Pollard JR, Curtin NJ

Publication type: Article

Publication status: Published

Journal: Molecular Oncology

Year: 2015

Volume: 9

Issue: 2

Pages: 463-472

Print publication date: 01/02/2015

Online publication date: 13/10/2014

Acceptance date: 30/09/2014

ISSN (print): 1574-7891

ISSN (electronic): 1878-0261

Publisher: Elsevier

URL: http://dx.doi.org/10.1016/j.molonc.2014.09.012

DOI: 10.1016/j.molonc.2014.09.012

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