Dataset Information

Identification of candidate circulating glucocorticoid response biomarkers in humans using a proteomic approach

ABSTRACT: Glucocorticoids used in pharmacological doses for the treatment of a variety of medical conditions, and endogenous glucocorticoid excess – Cushing’s syndrome, may result in several adverse effects, but currently there is no clinically useful biomarker of glucocorticoid activity. A three-phase protein biomarker discovery strategy was used. Proteomic biomarker discovery and qualification was conducted on the secretome of ex vivo-stimulated peripheral blood mononuclear cells (PBMC) isolated from 6 volunteers, incubated ± dexamethasone 100 ng/mL for 4h and 24h. Untargeted proteomics with label-free quantification (LFQ) was conducted to discover candidate proteins which were quantified using targeted proteomics by a custom multiple reaction monitoring mass spectrometry (MRM-MS) assay. Five proteins were selected for serum measurement by immunoassay in 20 healthy volunteers, with blood drawn at baseline and 12h after 4 mg oral dexamethasone. Paired analysis of the discovery proteomics data (576 and 280 proteins for the 4h and 24h secretomes, respectively) generated a shortlist of candidates which were qualified using MRM-MS to obtain protein level intensity data for 39 proteins. In the validation cohort, 3/5 proteins were dexamethasone-responsive, two significantly decreased: lysozyme C (mean±SEM) – 101±5.5 vs 67±4.4 ng/mL, (P<0.0001); nucleophosmin-1 (median (interquartile range)) – 16.6 (14.4-18.4) vs 14.2 (11.1-17.4) ng/mL, (P<0.01), while high mobility group box 2 (mean±SEM) significantly increased – 819±34 vs 984±60 pg/mL (P<0.01). Using an ex vivo proteomic approach in PBMC, we have identified and verified circulating glucocorticoid-responsive proteins which may have potential as serum biomarkers.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Blood

SUBMITTER: Thomas Stoll

LAB HEAD: Michelle M. Hill

PROVIDER: PXD019945 | Pride | 2021-03-14

REPOSITORIES: Pride

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Publications

Ex vivo glucocorticoid-induced secreted proteome approach for discovery of glucocorticoid-responsive proteins in human serum.

Inder Warrick J WJ Mohamed Ahmed A Keshvari Sahar S Barclay Johanna L JL Ruelcke Jayde E JE Stoll Thomas T Nolan Brendan J BJ Cesana-Nigro Nicole N Hill Michelle M MM

Proteomics. Clinical applications 20210312 2-3

<h4>Purpose</h4>To identify glucocorticoid-responsive proteins measurable in human serum that may have clinical utility in therapeutic drug monitoring and the diagnosis of cortisol excess or deficiency.<h4>Experimental design</h4>A phased biomarker discovery strategy was conducted in two cohorts. Secretome from peripheral blood mononuclear cells (PBMC) isolated from six volunteers after ex vivo incubation ± dexamethasone (DEX) 100 ng/mL for 4 h and 24 h was used for candidate discovery and quali ...[more]

PMID: 33641263

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Project description:Glucocorticoids are an essential component of the treatment of lymphoid malignancies and resistance to glucocorticoid therapy constitutes a prominent clinical problem in relapsed and refractory lymphoblastic leukemias. Constitutively active NOTCH signaling is involved in the pathogenesis of over 50% of T-cell lymphoblastic leukemia (T-ALL) which harbor activating mutations in the NOTCH1 gene. Aberrant NOTCH1 signaling has been shown to protect normal thymocytes from glucocorticoid induced cell death. Here we analyzed the interaction of glucocorticoid therapy with inhibition of NOTCH signaling in the treatment of T-ALL. Gamma-secretase inhibitors (GSI), which block the activation of NOTCH receptors, amplified the transcriptional changes induced by glucocorticoid treatment, including glucocorticoid receptor autoinduction and restored sensitivity to dexamethasone in glucocorticoid-resistant T-ALL cells. Apoptosis induction upon inhibition of NOTCH signaling and activation of the glucocorticoid receptor was dependent on transcriptional upregulation of BIM and subsequent activation of the mitochondrial/intrinsic cell death pathway. Finally, we used a mouse xenograft model of T-ALL to demonstrate that combined treatment with dexamethasone and a GSI results in improved antileukemic effects in vivo. These studies provide insight in the mechanisms of glucocorticoid resistance and serve as rationale for the use of glucocorticoid and GSIs in combination in the treatment of T-ALL. Experiment Overall Design: Duplicate samples (biologic replicas) from CUTLL1 cells were treated for 24 hours with vehicle only (DMSO), dexamethasone (1microM), a gamma-secretase inhibitor (CompE 100nM) and a combination of dexamethasome plus gamma secretase inhibitor at the same concentrations indicated before. Gene expression profiling was analyzed to identify gene expression signatures assocuated with glucocorticoid treatment (dexamethasone), inhibition of NOTCH1 by gamma secretase inhibitor (CompE) or the combination of both treatments.

Dataset Information

Identification of candidate circulating glucocorticoid response biomarkers in humans using a proteomic approach

Publications

Ex vivo glucocorticoid-induced secreted proteome approach for discovery of glucocorticoid-responsive proteins in human serum.

Similar Datasets

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