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. 2021 Jul 30;13(15):3843.
doi: 10.3390/cancers13153843.

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

Jeffrey R Whiteaker  1 , Tao Wang  1 , Lei Zhao  1 , Regine M Schoenherr  1 , Jacob J Kennedy  1 , Ulianna Voytovich  1 , Richard G Ivey  1 , Dongqing Huang  1 , Chenwei Lin  1 , Simona Colantonio  2 , Tessa W Caceres  2 , Rhonda R Roberts  2 , Joseph G Knotts  2 , Jan A Kaczmarczyk  2 , Josip Blonder  2 , Joshua J Reading  2 , Christopher W Richardson  2 , Stephen M Hewitt  3 , Sandra S Garcia-Buntley  2 , William Bocik  2 , Tara Hiltke  4 , Henry Rodriguez  4 , Elizabeth A Harrington  5 , J Carl Barrett  5 , Benedetta Lombardi  5 , Paola Marco-Casanova  5 , Andrew J Pierce  5 , Amanda G Paulovich  1
Affiliations

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

Jeffrey R Whiteaker et al. Cancers (Basel). .

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.

Keywords: DNA damage response; immuno-MRM; multiple reaction monitoring; targeted proteomics.

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Conflict of interest statement

E.A.H., J.C.B., P.M.-C. and A.J.P. are or were employees of Astra-Zeneca. A.J.P. current affiliation: Crescendo Biologics Ltd.

Figures

Figure 1
Figure 1
Immuno-MRM enables highly multiplexed protein quantification. (a) Assay workflow consists of generation of a protein lysate followed by enzymatic digestion (e.g., trypsin). Stable isotope labeled standards unique to each targeted peptide sequence are spiked to the sample at a known concentration. Custom monoclonal antibodies coupled to magnetic beads are used to enrich the endogenous peptides and labeled standards. The eluate is analyzed by multiple reaction monitoring mass spectrometry, where analyte peptides and internal standards coelute with equivalent relative areas of monitored transitions. High specificity is maintained through optimal selection of fragment ion transitions to monitor. (b) Characterization of performance figures of merit of the assay. A representative response curve for the heavy peptide DSGSQEVLSELR spiked into cell lysate shows a typical linear range. Repeatability is characterized by the distribution of CV values for intra- (within day) and inter- (between day) assay repeatability. Each point corresponds to the average CV for a peptide measured at three concentrations, Low (Lo), Medium (Med), and High (Hi) in triplicate over five days (n = 15 at each concentration for a peptide). Stability shows the distribution of percent difference for samples stored at 24 h at 4 °C and after two freeze-thaw cycles relative to immediate analysis. Sequential enrichment shows the distribution of percent difference and CV values following enrichment using the flow-through of a sample from another immuno-MRM assay compared to direct enrichment. For box plots, the white line shows the median value, boxes show the inner quartiles, and the whiskers show 5–95% of data.
Figure 2
Figure 2
The DDR-2 immuno-MRM assay detects changes in phosphorylation in response to ionizing radiation. (a) Heatmap showing unsupervised clustering of analytes detected in LCL-57 exposed to increasing levels of irradiation (1,2,5,10 Gy); control samples were mock-irradiated. All samples were harvested at 1 h. Peak area ratios (light:heavy) were normalized for each peptide analyte. Peptide analyte labels indicated gene symbol, followed by modification site and the first four letters of the peptide sequence. Nonmodified peptides are indicated by “pan”. (b) Bar plots showing peak area ratio (light:heavy) of analytes with greater than 2-fold change in concentration detected by immuno-MRM. Error bars show the standard deviation of biological triplicate analysis. In t-test between irradiated and mock results, one asterisk (*) indicates p value smaller than 0.05 (p < 0.05); two asterisks (**) indicate p value smaller than 0.01 (p < 0.01); three asterisks (***) indicate p value smaller than 0.001 (p < 0.001).
Figure 3
Figure 3
The DDR-2 immuno-MRM assay detects changes in phosphorylation dynamics due to ATM kinase inhibition. (a) Heatmap showing unsupervised clustering of analytes in LCL-57 exposed to ionizing radiation in the presence of ATM inhibitor (or control vehicle, DMSO). Peak area ratios (light:heavy) were normalized for each peptide analyte. (b) Bar plots showing peak area ratios (light:heavy) of analytes with greater than 2-fold change quantified by immuno-MRM. Blue lines show control (DMSO vehicle) and red lines show samples treated in the presence of ATM inhibitor. Error bars the standard deviation of biological triplicate analysis.
Figure 4
Figure 4
Pharmacodynamic profiling of cell signaling dynamics. (a) Heatmap showing unsupervised clustering of analytes and samples for PBMCs exposed to ionizing radiation in the presence of ATM, ATR, or DNA-PK kinase inhibitors. Peak area ratios (light:heavy) were normalized for each peptide analyte. (b) Bar plots showing peak area ratios (light:heavy) for selected analytes. For each kinase inhibitor, the pair of mock-treated (green) and irradiated (orange) cells are plotted. Error bars show the standard deviation of triplicate analysis (SEB+, ATMi and DMSO samples) or the range of duplicate analysis (SEB+, DNAPKi samples). To meet the assay sample requirements, two individuals (i.e., biological duplicate reps) were used for DNAPKi samples, and a single individual sample was used (i.e., singlicate analysis) for the ATRi samples. For the unstimulated cells (SEB−), lysates from two individuals were pooled to obtain sufficient material (i.e., singlicate technical analysis).
Figure 5
Figure 5
Pharmacodynamic markers of kinase inhibition identified by comparing protein and phosphorylation levels in PBMCs. The ratio of concentration levels in PBMCs (SEB+) determined by the immuno-MRM assay is plotted for four comparisons: (a) 5 Gy versus mock-irradiation, (b) irradiated in the presence of ATM inhibitor versus vehicle (DMSO), (c) irradiated in the presence of ATR inhibitor versus vehicle (DMSO), and (d) irradiated in the presence of DNAPK inhibitor versus vehicle (DMSO). The log(2) ratio is plotted for each comparison. Large changes are labeled with the analyte and phosphorylation site (if applicable).
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