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Updated: Apr 20, 2026
Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis
Published on: March 20, 2021
Development of pharmacodynamic biomarkers for ATR inhibitors
Tao Chen1, Fiona K Middleton1, Susanna Falcon2
1Newcastle University, Northern Institute for Cancer Research, Paul O'Gorman Building, Medical School, Newcastle upon Tyne, NE2 4HH, UK.
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.
Insights
Developing a pharmacodynamic assay for ATR inhibitors is crucial for cancer therapy. This study found that phospho-Chk1 and phospho-H2AX are reliable biomarkers for ATR activity, even in non-cycling cells.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- The ATM and Rad3-related (ATR) kinase is a key regulator of DNA damage response pathways, making it an attractive target for cancer therapies.
- Development of ATR inhibitors necessitates a robust pharmacodynamic assay to support clinical trials.
Purpose of the Study:
- To evaluate phospho-Chk1 and phospho-H2AX as biomarkers for ATR activity.
- To assess the utility of peripheral blood mononuclear cells (PBMCs) as a surrogate tissue for measuring ATR activity.
- To develop a clinically viable pharmacodynamic assay for ATR inhibitors.
Main Methods:
- Western blot, immunofluorescence microscopy, and flow cytometry were used to assess Chk1 and H2AX phosphorylation in various cell lines and human blood samples.
- DNA-damaging agents (Hydroxyurea, UV, 4NQO) were employed to induce ATR activation.
- The impact of ATR knockdown and inhibition on phosphorylation events was investigated.
Main Results:
- Hydroxyurea, UV, and 4NQO induced Chk1 and H2AX phosphorylation in cancer cell lines.
- ATR activation was detectable in non-cycling cells, and phospho-Chk1 reflected ATR activity for up to 3 hours.
- Phospho-H2AX was a more persistent marker of ATR activity in PBMCs from whole blood, becoming increasingly ATM and DNA-PK dependent over time.
Conclusions:
- PhosphoChk1 and H2AX serve as valuable biomarkers for assessing ATR inhibition across different detection methods, with timing being a critical factor.
- Non-cycling PBMCs are suitable surrogate tissues for ATR biomarker measurement.
- PhosphoH2AX in PBMCs from whole blood represents a clinically viable biomarker for ATR activity, enabling the development of a pharmacodynamic assay for ATR inhibitors.
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