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.

Figure 1

Conditions influencing optimal basal profile signal intensity. Average signal intensity ±s.d. of the 143 peptide spots is represented unless stated otherwise. (a) Basal profile signal intensities obtained with increasing lysate protein input. Optimal signal intensity was obtained using 7.5 and 10 μg of lysate protein input per array for HCT116 and MDA-MB-231, respectively. (b) Basal profile signal intensities obtained with different ATP concentrations. ATP concentrations up to 400 μM strongly increased signal intensity, while at higher concentrations the curve deviated from linearity. (c) Comparison of signal intensities with different lysis buffers. Compared to T-PER and RIPA lysis buffers, M-PER resulted in more efficient and consistent lysis of HCT116 (P<0.001 compared to both buffers, Student's t-test) and 786-O cells (P<0.001 relative to RIPA; T-PER P=0.807). Average signal intensity relative to M-PER is shown. (d) Comparison of trypsin-based cell lysis and scraping-based lysis. Trypsin-based lysis of 786-O cells enhanced signal intensity when compared to the standard scraping procedure. Incubation with 2 μM sunitinib prior to trypsin-based lysis and ex-vivo spiking of the same concentration in a scraping-based lysate resulted in an approximately 25% decrease of average signal intensity compared to the control sample (P<0.001 in both comparisons). (e) Freeze-thaw cycles. Relative to the first freeze-thaw cycle after lysate storage, average signal intensity of HCT116 lysate was not affected by additional cycles (P=0.98, ANOVA). (f) Sample conservation on ice. Conservation of sample constituents of four patient-derived tumor lysates on ice for three consecutive measurements resulted in a non-significant decline of average sample signal intensity (P=0.25, ANOVA).

Figure 2

Recombinant Src and Axl kinase substrate specificity. Microarrays were incubated with 125 ng ml⁻¹ of recombinant Src or 500 ng ml⁻¹ Axl kinase. (a) Raw images of array spot phosphorylation after 1 h demonstrate significant overlap in peptide phosphorylation. Some peptides were predominantly phosphorylated by either Src (row 3-column 11 and 5–6) or Axl (for example, spots 9–11 and 10–5). (b) Correlation plot of 143 peptide signal intensities showing relative substrate preference for Src and Axl kinase, indicated by the red and blue circle, respectively.

Figure 3

Microarray reproducibility and linearity. Inter- and intra-chip correlations between peptide phosphorylation levels as assessed by Pearson's correlations (r) using 12 technical replicates of HCT116 lysate (7.5 μg) measured on 3 chips in a single run. (a) Linearity plot of two representative technical replicates. (b) CV plot of the technical replicas shown in (a). The threshold was set at 15% as recommended by the microarray manufacturer. (c) Linearity plot of biological replicates of 786-O cells in three independent experiments. (d) CV plot of the biological replicas shown in (c). Threshold at CV 15%. CV, coefficient of variation as determined by the ratio of the standard deviation to the mean signal intensity.

Figure 4

Basal profile comparison of 10 tumor-related samples. Samples were measured using triplicate samples of 7.5 μg of lysate protein per array. (a) Heatmap of log2-transformed normalized signal intensities of the top 65 phosphorylated peptides in 10 different tumor cell or tissue-derived samples, sorted from high (red) to low (blue) signal intensity. The top 10 substrates included CD79A, ENOG, EFS, SRC8, PLCG1, FRK, P85A and PAXI. (b) ‘Phosphorylation landscape' indicating marginal absolute signal intensity differences between the tumor tissues (colorectal cancer, HCC, melanoma, lung cancer, pNET, MDA-MB-231 human breast cancer xenograft, HT29 chorioallantoic membrane model) and cell lines (HT29 colorectal cancer, NIH/3T3 mouse fibroblast, 786-O renal cell cancer). CAM chick chorioallantoic membrane; HCC hepatocellular carcinoma, pNET pancreatic neuroendocrine tumor.

Figure 5

Inhibition of kinase activity. (a) TKI incubation versus spiking. Prior to lysis, HCT116 cells were incubated in vitro ±2 μM sunitinib, and subsequent lysates were spiked ±2 μM sunitinib (drug incubation/drug spiking). Signal intensity of the top 15 substrates is represented from high to low. Although inhibition patterns were similar, inhibition potency increased for sunitinib-incubated versus spiked samples, while additional spiking of previously incubated samples did not increase the degree of inhibition. (b) Competition with ATP. The HCT116 lysate was spiked with 4 μM sunitinib, 25 μM sorafenib or 10 nM dasatinib in the presence of increasing ATP concentrations. Despite average phosphorylation inhibition of 65–85% at 100 μM ATP, increasing ATP concentrations induced signal intensity and attenuated the inhibitory effects of sunitinib and sorafenib. With dasatinib, signal intensity increased to a lesser extent, suggesting partial ATP-independent inhibition of kinase activity. (c and d) Substrate-specific inhibition. Microarrays were incubated with 125 ng ml of recombinant Src or 500 ng ml Axl kinase±85 nM and 2 μM of the Src-inhibitor dasatinib and Axl-inhibitor R428, respectively. Both drugs resulted in near-complete inhibition of phosphorylation. The results of the top 25 phosphorylated substrates are shown.

Figure 6

TKI inhibition profiles of four patient samples. (a) Inhibition heatmap of four patient-derived tumor lysates (two breast, two lung cancer) demonstrating inhibition ratios of the top 80 peptides achieved by spiking with five TKIs. Peptides are sorted from high inhibition ratio (dark blue) to low, and green indicates absence of inhibition. (b) Representative patient example of top 30 highest intensity substrates sorted from high to low based on control samples. The control samples revealed the absolute phosphorylation intensities without inhibitor for each sample. Subsequently, absolute phosphorylation inhibition achieved by spiking with sorafenib, erlotinib, sunitinib and dasatinib are shown. Inhibition potency varies between the drugs, while no differential inhibition of individual peptides is observed.