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.

Independent biological triplicate preparations of LCL-57 cells were treated with increasing doses of IR (1, 2, 5, 10 Gy) and harvested at 1 h; the control cells were mock-irradiated and harvested at 1 h. 500 µg aliquots of lysates were analyzed using the immuno-MRM panel targeting DDR-2 analytes. For testing the effects of kinase inhibition, LCL-57 (Atm+) cells were treated with or without ATM-kinase inhibitor (KU-55933) prior to 5 GY IR induction; control samples were treated with DMSO vehicle. Cells were prepared in biological triplicate using separate aliquots in the presence/absence of the inhibitor and harvested over a time-course of 0.25, 1, 6, and 24 h post-irradiation; controls were harvested 1 h following mock-irradiation. Lysates were analyzed by the DDR-2 multiplexed immuno-MRM assay. To evaluate the utility of the DDR-2 assay in pharmacodynamic profiling in primary human PBMCs, PBMCs were isolated from 3 donors and split into two aliquots; one aliquot was expanded in culture with Staphylococcal Enterotoxin B (SEB+) to induce cell cycling, and the other aliquot had no stimulation (SEB-). For each set of cells, DNA damage was induced by 5 Gy IR, controls were mock-irradiated, and cells were harvested 1 h post-IR.