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

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.

SUBMITTER: Whiteaker JR

PROVIDER: S-EPMC8345163 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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

Whiteaker Jeffrey R JR Wang Tao T Zhao Lei L Schoenherr Regine M RM Kennedy Jacob J JJ Voytovich Ulianna U Ivey Richard G RG Huang Dongqing D Lin Chenwei C Colantonio Simona S Caceres Tessa W TW Roberts Rhonda R RR Knotts Joseph G JG Kaczmarczyk Jan A JA Blonder Josip J Reading Joshua J JJ Richardson Christopher W CW Hewitt Stephen M SM Garcia-Buntley Sandra S SS Bocik William W Hiltke Tara T Rodriguez Henry H Harrington Elizabeth A EA Barrett J Carl JC Lombardi Benedetta B Marco-Casanova Paola P Pierce Andrew J AJ Paulovich Amanda G AG

Cancers 20210730 15

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 p ...[more]

PMID: 34359745

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Project description:BackgroundOvarian cancer is the most lethal gynecologic malignancy in women, and high-grade serous ovarian cancer (HGSOC) is the most common subtype. Currently, no clinical test has been approved by the FDA to screen the general population for ovarian cancer. This underscores the critical need for the development of a robust methodology combined with novel technology to detect diagnostic biomarkers for HGSOC in the sera of women. Targeted mass spectrometry (MS) can be used to identify and quantify specific peptides/proteins in complex biological samples with high accuracy, sensitivity, and reproducibility. In this study, we sought to develop and conduct analytical validation of a multiplexed Tier 2 targeted MS parallel reaction monitoring (PRM) assay for the relative quantification of 23 putative ovarian cancer protein biomarkers in sera.MethodsTo develop a PRM method for our target peptides in sera, we followed nationally recognized consensus guidelines for validating fit-for-purpose Tier 2 targeted MS assays. The endogenous target peptide concentrations were calculated using the calibration curves in serum for each target peptide. Receiver operating characteristic (ROC) curves were analyzed to evaluate the diagnostic performance of the biomarker candidates.ResultsWe describe an effort to develop and analytically validate a multiplexed Tier 2 targeted PRM MS assay to quantify candidate ovarian cancer protein biomarkers in sera. Among the 64 peptides corresponding to 23 proteins in our PRM assay, 24 peptides corresponding to 16 proteins passed the assay validation acceptability criteria. A total of 6 of these peptides from insulin-like growth factor-binding protein 2 (IBP2), sex hormone-binding globulin (SHBG), and TIMP metalloproteinase inhibitor 1 (TIMP1) were quantified in sera from a cohort of 69 patients with early-stage HGSOC, late-stage HGSOC, benign ovarian conditions, and healthy (non-cancer) controls. Confirming the results from previously published studies using orthogonal analytical approaches, IBP2 was identified as a diagnostic biomarker candidate based on its significantly increased abundance in the late-stage HGSOC patient sera compared to the healthy controls and patients with benign ovarian conditions.ConclusionsA multiplexed targeted PRM MS assay was applied to detect candidate diagnostic biomarkers in HGSOC sera. To evaluate the clinical utility of the IBP2 PRM assay for HGSOC detection, further studies need to be performed using a larger patient cohort.

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

Publications

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

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