A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in
Christopher J Bakkenist1, R Kenneth Czambel2, Pamela A Hershberger3
1Departments of Radiation Oncology ; Pharmacology and Chemical Biology.
Abstract:
Pharmacologic inhibition of DNA repair may increase the efficacy of many cytotoxic cancer agents. Inhibitors of DNA repair enzymes including APE1, ATM, ATR, DNA-PK and PARP have been developed and the PARP inhibitor olaparib is the first-in-class approved in Europe and the USA for the treatment of advanced BRCA-mutated ovarian cancer. Sensitive pharmacodynamic (PD) biomarkers are needed to further evaluate the efficacy of inhibitors of DNA repair enzymes in clinical trials. ATM is a protein kinase that mediates cell-cycle checkpoint activation and DNA double-strand break repair. ATM kinase activation at DNA double-strand breaks (DSBs) is associated with intermolecular autophosphorylation on serine-1981. Exquisite sensitivity and high stoichiometry as well as facile extraction suggest that ATM serine-1981 phosphorylation may be a highly dynamic PD biomarker for both ATM kinase inhibitors and radiation- and chemotherapy-induced DSBs. Here we report the pre-clinical analytical validation and fit-for-purpose biomarker method validation of a quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs). We explore the dynamics of these phosphorylations in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro, and show that ATM serine-1981 phosphorylation is increased in PBMCs in sarcoma patients treated with DNA damaging chemotherapy.
Insights
Measuring ATM serine-1981 phosphorylation in peripheral blood mononuclear cells (PBMCs) can serve as a sensitive biomarker. This method helps evaluate DNA repair inhibitors and DNA damage from chemotherapy in cancer patients.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Pharmacologic inhibition of DNA repair enzymes enhances cytotoxic cancer therapies.
- Approved drugs like olaparib (PARP inhibitor) highlight the potential of DNA repair inhibitors.
- Need for sensitive pharmacodynamic (PD) biomarkers to assess DNA repair inhibitor efficacy in clinical trials.
Purpose of the Study:
- To validate a method for analyzing ATM and H2AX phosphorylation as PD biomarkers.
- To explore the dynamics of these phosphorylations in response to DNA damage and inhibitors.
- To assess the utility of ATM serine-1981 phosphorylation in sarcoma patients undergoing chemotherapy.
Main Methods:
- Developed and validated a quasi-quantitative dual multiplexed immunoblot assay.
- Analyzed ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs).
- Investigated phosphorylation dynamics in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro.
Main Results:
- ATM serine-1981 phosphorylation is a sensitive and dynamic biomarker for DNA double-strand breaks (DSBs).
- The validated immunoblot method can simultaneously analyze ATM and H2AX phosphorylation in PBMCs.
- Increased ATM serine-1981 phosphorylation was observed in PBMCs from sarcoma patients treated with DNA-damaging chemotherapy.
Conclusions:
- ATM serine-1981 phosphorylation is a promising PD biomarker for evaluating DNA repair inhibitors and DNA damage.
- The developed multiplexed immunoblot assay provides a robust method for PD biomarker analysis in clinical settings.
- This approach can aid in optimizing cancer treatment strategies involving DNA repair inhibition and chemotherapy.
