Evaluation of a tyrosine kinase peptide microarray for tyrosine kinase inhibitor therapy selection in cancer

• Published in: Experimental & molecular medicine Vol. 48; no. 12; p. e279
• Main Authors: Labots, Mariette, Gotink, Kristy J, Dekker, Henk, Azijli, Kaamar, van der Mijn, Johannes C, Huijts, Charlotte M, Piersma, Sander R, Jiménez, Connie R, Verheul, Henk M W
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 16.12.2016; Springer Nature B.V; Nature Publishing Group
• Subjects: 631/61/32; 692/53/2423; 82; Biomedical and Life Sciences; Biomedicine; Cell Line, Tumor; Drug Screening Assays, Antitumor - methods; High-Throughput Screening Assays - methods; Humans; Medical Biochemistry; Molecular Medicine; Neoplasms - drug therapy; Neoplasms - metabolism; Original; original-article; Protein Array Analysis - methods; Protein Kinase Inhibitors - pharmacology; Protein-Tyrosine Kinases - antagonists & inhibitors; Protein-Tyrosine Kinases - metabolism; Stem Cells
• ISSN: 2092-6413; 1226-3613; 2092-6413
• DOI: 10.1038/emm.2016.114

Personalized cancer medicine aims to accurately predict the response of individual patients to targeted therapies, including tyrosine kinase inhibitors (TKIs). Clinical implementation of this concept requires a robust selection tool. Here, using both cancer cell lines and tumor tissue from patients, we evaluated a high-throughput tyrosine kinase peptide substrate array to determine its readiness as a selection tool for TKI therapy. We found linearly increasing phosphorylation signal intensities of peptides representing kinase activity along the kinetic curve of the assay with 7.5–10 μg of lysate protein and up to 400 μ M adenosine triphosphate (ATP). Basal kinase activity profiles were reproducible with intra- and inter-experiment coefficients of variation of <15% and <20%, respectively. Evaluation of 14 tumor cell lines and tissues showed similar consistently high phosphorylated peptides in their basal profiles. Incubation of four patient-derived tumor lysates with the TKIs dasatinib, sunitinib, sorafenib and erlotinib primarily caused inhibition of substrates that were highly phosphorylated in the basal profile analyses. Using recombinant Src and Axl kinase, relative substrate specificity was demonstrated for a subset of peptides, as their phosphorylation was reverted by co-incubation with a specific inhibitor. In conclusion, we demonstrated robust technical specifications of this high-throughput tyrosine kinase peptide microarray. These features required as little as 5–7 μg of protein per sample, facilitating clinical implementation as a TKI selection tool. However, currently available peptide substrates can benefit from an enhancement of the differential potential for complex samples such as tumor lysates. We propose that mass spectrometry-based phosphoproteomics may provide such an enhancement by identifying more discriminative peptides. Cancer: Testing sensitivity of enzymes to drugs A quick test could reveal the sensitivity of tumor cells to drugs targeting the tyrosine kinase enzymes involved in many cancers. These enzymes control the activity of many other proteins by adding phosphate groups to them. Cancer can develop when some enzymes become over-active or are over-produced by underlying genetic mutations. Henk Verheul and colleagues at the VUmc Cancer Center, VU University Medical Center in Amsterdam, The Netherlands, worked with a surface layer coated with small fragments of proteins to which the enzymes can add phosphates. When lysates from cancer cells are passed across this layer the activity of their tyrosine kinase enzymes is revealed by a fluorescent signal generated by antibodies binding to the added phosphate groups. By adding specific anti-cancer drugs in particular tyrosine kinase inhibitors, their ability to inhibit the enzyme activity can be measured. After further refinement the test may offer guidance for anti-cancer therapy in patients.