ABSTRACTProtein kinases have emerged as promising therapeutic agents

resulting from the discovery of their role in numerous human

diseases including cancer. While there has been considerable

interest in the therapeutic potential of kinase inhibitors, many have

not been tested by unbiased strategies to validate both inhibitor

specificity and target engagement within living cells. To overcome

these limitations, we have developed a label free targeted LC-MS

workflow for identification and validation of biomarkers for kinase

inhibition that we have applied to characterize inhibitors of protein

kinase CK2. Although our methods are optimized for CK2, they are

easily adaptable for any substrate-kinase or inhibitor-kinase relation

characterization. This workflow is free of phospho-peptide

enrichment which provided a robust platform to measure kinase

inhibition through quantification of both phospho- and non-phospho-

species of the same peptide phosphorylated by the kinase. Since

non-phosphorylated peptides are detected easier in any MS

instrument, our method provided an increased sensitivity to monitor

CK2 inhibition with elongation factor 1-delta (EF1D) and

demonstrated that eukaryotic translation initiation factor 2 (IF2B) is a

superior biomarker of CK2 inhibition. Our comparison of eight

commercially available CK2 inhibitors (TBB, TBBz, DMAT, Ellagic

Acid, Quinalirazin, Resorufin, CX-4945 and inhibitor VIII) showed

that CX-4945 and inhibitor VIII were most effective at the inhibition

of CK2 in human osteosarcoma U2OS and adenocarcinoma HeLa

cells. Overall, our methods have yielded implementation of

systematic platforms for studying CK2 inhibitors and to further

characterize the biological functions of CK2.

Figure 2. Chemical structures of ATP, GTP and the evaluated CK2

inhibitors with their previously reported IC50 values.

ACKNOWLEDGEMENTThe work was supported by Canadian Institutes of

Health Research (CIHR) and Canadian Cancer Society

Research Institute (CCSRI).

Robust label free targeted proteomic workflow to identify and validate biomarkers of kinase

inhibition: comparative evaluation of CK2 inhibitors for their inhibition of CK2 in cells

Laszlo Gyenis1, Sam R. Fess1, Stephanie A. Zukowski1, Jacob P. Turowec1, Paula Pittock1, Gilles Lajoie1 and David W. Litchfield1,2

1Department of Biochemistry and 2Department of Oncology, Schulich School of Medicine and Dentistry, University of Western Ontario, London,

Ontario, N6A 5C1, Canada

Figure 1. Overview of systematic kinase inhibitor validation and biomarker

identification strategies. The identified and validated biomarkers are used

for characterization of kinase dependent cell signaling events and for

monitoring kinase inhibition at clinical settings.

Figure 4. Monitoring CK2 inhibition after 24h of 50μM CX-4945 inhibitor

treatment using U2OS cells with a label free targeted LC-MS (A-F) or

immunoblotting methods (G-H). Pie charts are showing the IF2B (E) or

EF1D (F) targeted CK2 phosphorylated or non-phosphorylated peptide

amounts measured by targeted LC-MS or by the immunoblotting method

(H) quantifying immunoblots with LiCor Odyssey v. 3.0 software.

Figure 3. Monitoring CK2 inhibition by immunoblotting with the indicated phospho-specific antibodies after 24 hours of inhibitor treatment. Lysates marked by

* did not contain phosphatase inhibitors. DMSO treated lysates +/- λ-phosphatase were used as immunoblotting controls of phospho-specific antibodies.

CONCLUSIONS

All kinase inhibitors should be evaluated by unbiased

strategies before conclusions are made about their

specificity, effectiveness and/or are used for

characterization of cell signaling events

Unbiased chemical proteomics strategies developed

for characterization of CK2 inhibitors can be readily

adapted for evaluation of other than CK2 kinase

inhibitors

Inhibitor characterization methods can be combined

with different mass spectrometry strategies (e.g.

immobilised metal affinity chromatography (IMAC),

phosphoprotein and phosphopeptide enrichment

strategies prior to MS analysis, etc.)

Three independent methods (LC-MS, immunoblots &

immunofluorescence) demonstrated that IF2B has

superior ability to monitor cellular CK2 activity

REFERENCES1. Gyenis, L.; Turowec, J. P.; Bretner, M.; Litchfield, D. W., Chemical proteomics and functional proteomics strategies for protein kinase

inhibitor validation and protein kinase substrate identification: Applications to protein kinase CK2. Biochimica et biophysica acta

2013, 1834(7):1352-8.

2. Gyenis, L.; Kus, A.; Bretner, M.; Litchfield, D. W., Functional proteomics strategy for validation of protein kinase inhibitors reveals

new targets for a TBB-derived inhibitor of protein kinase CK2. Journal of proteomics 2013, 81, 70-9.

3. Duncan, J. S.; Haystead, T. A.; Litchfield, D. W., Chemoproteomic characterization of protein kinase inhibitors using immobilized

ATP. Methods Mol Biol 2012, 795, 119-34

4. Gyenis, L.; Duncan, J. S.; Turowec, J. P.; Bretner, M.; Litchfield, D. W., Unbiased functional proteomics strategy for protein kinase

inhibitor validation and identification of bona fide protein kinase substrates: application to identification of EEF1D as a substrate for

CK2. Journal of proteome research 2011, 10, (11), 4887-901.

5. Duncan, J. S.; Gyenis, L.; Lenehan, J.; Bretner, M.; Graves, L. M.; Haystead, T. A.; Litchfield, D. W., An unbiased evaluation of CK2

inhibitors by chemoproteomics: characterization of inhibitor effects on CK2 and identification of novel inhibitor targets. Molecular &

cellular proteomics : MCP 2008, 7, (6), 1077-88.

Figure 5. Monitoring CK2 inhibition in U2OS cells after 24h of 30 or

50μM CX-4945 inhibitor treatments with immunofluorescence using a

phospho-antibody raised against the ac-pS-GDEMIFDPTMSKC-amide

IF2B peptide (A). Results of three independent experiments showing the

phosho-IF2B (FITC)/DAPI intensity signal reduction after CX-4945

treatments. FITC/DAPI intensity mean ratios of three independent

experiments with standard deviation (B). Pie chart are demonstrating the

residual amounts of phosphor-IF2B signal (C) after inhibitor treatments

when DMSO control was considered as 100% signal level.

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Figure 6. Validation of CK2 dependent phosphorylation in U2OS cell line

expressing the inhibitor resistant mutant of CK2 alpha (V66A/I174A) with

Tetracycline regulation. The expression of CK2 alpha (V66A/I174)-HA

and Myc-CK2beta are turned on in the absence of Tetracycline. Bar

charts are showing mean signal intensities measured by the indicated

phospho-antibodies normalized to GAPDH in the immunoblots. The

means with standard deviation of three independent experiments are

showing in each chart.