Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition

SIMPLE SUMMARY: Functionality of the cellular DNA damage response (DDR) network affects risk for developing cancer, and the DDR is also a target of cancer therapies. Thus, it is important that we have reliable laboratory methods for determining the activity of this network. We describe the development and analytical validation of a targeted mass spectrometry-based 51-plex assay (DDR-2) for measuring proteins and post-translational modifications related to the DDR. The findings demonstrate identification of potential novel pharmacodynamic biomarkers. ABSTRACT: The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.