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

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 μg of lysate protein and up to 400 μM 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.

Axitinib: A Photoswitchable Approved Tyrosine Kinase Inhibitor

“…We therefore aimed to use a kinase activity profiling technique that can be set up under controlled light conditions. In this context, we investigated the kinase activity of HUVECs after treatment with ( E )‐ or ( Z )‐axitinib by using the PamGene Pamstation® and PamChip® technology . The advantage of this method, with regard to photosensitive compounds, is that the experiments can be performed in the dark or under controlled light conditions.…”

“…For nonirradiated ( Z )‐axitinib (Figure b), one of three data sets was neglected due to clear outliers. Data analysis was performed as described by Labots et al . For signal evaluation, Bionavigator 6 software (PamGene) was used according to the manufacturer's protocol.…”

“…In this context,w ei nvestigated the kinase activity of HUVECs after treatment with (E)-or (Z)-axitinib by using the PamGene Pamstation and PamChip technology. [54][55][56][57] The advantage of this method, with regard to photosensitive compounds, is that the experimentsc an be performed in the dark or under controlled light conditions. Furthermore, different excitation wavelengths, ranging from l = 460 to 650 nm, are availablefor readout.…”

“…In the irradiation experiment, the reaction mixture containing (Z)-axitinib was irradiated at l = 385 nm for 30 s prior to addition to the PamChip.F or nonirradiated (Z)-axitinib (Figure 6b), one of three data sets was neglected due to clear outliers. Data analysis was performed as described by Labots et al [55] For signal evaluation, Bionavigator 6s oftware (PamGene) was used according to the manufacturer's protocol.…”

Analysis of contribution of protein phosphorylation in the development of the diseases

“…Новым подходом в использовании микрочипов для анализа онкогенных сигнальных путей стала разработка пептидных чипов с субстратной специфичностью к тирозиновым киназам [51,60]. Пептиды на чипе фосфорилируются киназами, присутствующими в аналите, и связываются с флуоресцентно-меченными антителами к фосфотирозину пептидных субстратов.…”