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

Area of Science:

• Biochemistry
• Molecular Biology
• Oncology

Background:

• ATM serine/threonine kinase (ATM) is crucial for DNA double-strand break repair.
• ATM inhibitors are investigated as anti-cancer agents in clinical trials.
• Pharmacodynamic (PD) assays are essential for guiding ATM inhibitor dosage and understanding their mechanism of action.

Purpose of the Study:

• To develop and validate a multiplex assay for quantifying PD biomarkers of ATM inhibition.
• To identify and measure protein expression and DNA damage-responsive phosphorylation related to ATM activity.

Main Methods:

• Developed and analytically validated a 51-plex assay (DDR-2) for protein and phosphoprotein quantification.
• Assessed assay performance, including lower limit of quantification, linear range, inter-assay variability, and peptide stability.
• Applied the assay to quantify signaling in response to ionizing radiation-induced DNA damage.

Main Results:

• The DDR-2 assay demonstrated high sensitivity (median LLOQ 1.28 fmol) and a wide linear range (>3 orders of magnitude).
• Median inter-assay variability was 11% CV, with 86% of peptides showing stability for storage.
• The assay successfully quantified DNA damage signaling in lymphoblast cell lines and primary human peripheral blood mononuclear cells.

Conclusions:

• The validated DDR-2 assay is a robust tool for quantifying PD biomarkers of ATM inhibition.
• This assay can support mechanism of action studies and inform dose selection for ATM inhibitors in anti-cancer therapy.
• The assay facilitates the assessment of DNA damage response in both cell lines and primary cells for preclinical and clinical applications.