Exp Mol Med.  2016 Dec;48(12):e279. 10.1038/emm.2016.114.

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

Affiliations
  • 1Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands. h.verheul@vumc.nl

Abstract

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"‰Î¼g of lysate protein and up to 400"‰Î¼M 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"‰Î¼g 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.


MeSH Terms

Adenosine Triphosphate
Cell Line
Cell Line, Tumor
Dasatinib
Erlotinib Hydrochloride
Humans
Peptides
Phosphorylation
Phosphotransferases
Protein-Tyrosine Kinases*
Substrate Specificity
Tyrosine*
Adenosine Triphosphate
Dasatinib
Erlotinib Hydrochloride
Peptides
Phosphotransferases
Protein-Tyrosine Kinases
Tyrosine
Full Text Links
  • EMM
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr