title: A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells.
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.
authors.affiliation: Departments of Radiation Oncology ; Pharmacology and Chemical Biology.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Bakkenist
authors.affiliation: Medicine, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA 15213-1863.
authors.forename: R Kenneth
authors.initials: RK
authors.lastname: Czambel
authors.affiliation: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263.
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Hershberger
authors.affiliation: Medicine, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA 15213-1863.
authors.forename: Hussein
authors.initials: H
authors.lastname: Tawbi
authors.affiliation: Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, 5117 Centre Avenue, Pittsburgh, PA 15213-1863.
authors.forename: Jan H
authors.initials: JH
authors.lastname: Beumer
authors.affiliation: Medicine, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA 15213-1863.
authors.forename: John C
authors.initials: JC
authors.lastname: Schmitz
keywords: ATM; DNA damage response; H2AX; PBMCs; doxorubicin