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. 2009 Jan;27(1):77-83.
doi: 10.1038/nbt.1513. Epub 2008 Dec 21.

Bead-based profiling of tyrosine kinase phosphorylation identifies SRC as a potential target for glioblastoma therapy

Jinyan Du  1 , Paula Bernasconi, Karl R Clauser, D R Mani, Stephen P Finn, Rameen Beroukhim, Melissa Burns, Bina Julian, Xiao P Peng, Haley Hieronymus, Rebecca L Maglathlin, Timothy A Lewis, Linda M Liau, Phioanh Nghiemphu, Ingo K Mellinghoff, David N Louis, Massimo Loda, Steven A Carr, Andrew L Kung, Todd R Golub
Affiliations

Bead-based profiling of tyrosine kinase phosphorylation identifies SRC as a potential target for glioblastoma therapy

Jinyan Du et al. Nat Biotechnol. 2009 Jan.

Abstract

The aberrant activation of tyrosine kinases represents an important oncogenic mechanism, and yet the majority of such events remain undiscovered. Here we describe a bead-based method for detecting phosphorylation of both wild-type and mutant tyrosine kinases in a multiplexed, high-throughput and low-cost manner. With the aim of establishing a tyrosine kinase-activation catalog, we used this method to profile 130 human cancer lines. Follow-up experiments on the finding that SRC is frequently phosphorylated in glioblastoma cell lines showed that SRC is also activated in primary glioblastoma patient samples and that the SRC inhibitor dasatinib (Sprycel) inhibits viability and cell migration in vitro and tumor growth in vivo. Testing of dasatinib-resistant tyrosine kinase alleles confirmed that SRC is indeed the relevant target of dasatinib, which inhibits many tyrosine kinases. These studies establish the feasibility of tyrosine kinome-wide phosphorylation profiling and point to SRC as a possible therapeutic target in glioblastoma.

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Conflict of interest statement

The authors report no conflicts of interest relevant to this work.

Figures

Figure 1
Figure 1. Luminex immunosandwich assay and IP-MS approach
(a) The left panel shows the Luminex immunosandwich assay. Antibody-coupled beads were incubated with cellular protein lysates to capture tyrosine kinases. A biotinylated phospho-tyrosine antibody and streptavidin R-phycoerythrin conjugate (SAPE) were added sequentially to bind to the phospho-tyrosine residues. The mixture was analyzed with a Luminex 100 instrument, whereby each microsphere is analyzed with two lasers -- one to detect the bead color and thereby the identity of the tyrosine kinase, and the other to detect the R-phycoerythrin signal reflecting the tyrosine phosphorylation levels on the bead. The right panel presents the IP-MS approach. Total protein lysates were subjected to enzymatic digestion. Subsequently, the digested lysates were immunoprecipitated with a cocktail of phospho-tyrosine antibodies to enrich for phospho-tyrosine containing peptides. Upon elution, the peptide mixture was analyzed with mass spectrometry to determine their sequences and phosphorylation sites. (b) Comparison between IP-MS and Luminex data on five cell lines. The tyrosine phosphorylations on tyrosine kinases were assayed on biological replicates with either Luminex immunosandwich assay or IP-MS approach as described. Van diagrams of pY-TKs are shown. Numbers of pY-TKs identified with each method alone or both methods are indicated in parentheses. (c) Luminex fluorescence intensities on selected positive control samples. Lysates were prepared from 293FT cells infected with either a control lentivirus (Ctrl) or virus expressing the tyrosine kinases specified in the figure. The samples were assayed on at least three independent days and the representative raw values are shown. Western analysis on the same samples used in the left panels with either a phospho-specific antibody against pY419SRC or pY530SRC. Due to the high sequence homology, pY419SRC antibody also recognizes the corresponding phosphorylation site in other family members. (d) Technical and biological variations of the Luminex assay. For technical variations (blue bars), protein lysates from 6 cell lines (one sample per line) were analyzed at least 8 times with Luminex assay. For biological variations (red bars), protein lysates from each cell line were harvested 12 times sequentially and analyzed with Luminex assay. Variations of active (dark blue or red) and inactive beads (light blue or red) were determined as described in Materials and Methods.
Figure 2
Figure 2. Luminex screen identifies activated tyrosine kinases in human cancer cell lines and primary glioblastomas
(a) Luminex screen on human cancer cell lines. Total protein lysates were prepared from human cancer cell lines and assayed as described in the Experimental Procedures. The raw data were processed and positive signals were normalized to fold-over-backgrounds. The normalized readings were log-transformed and converted into a gct file and the heat map was generated using the GenePattern 3.0 software. (b) Luminex screen on human primary glioblastomas. Total protein lysates were prepared from freshly frozen patient samples as described in the Experimental Procedures. The data was acquired and processed same as in a). (c) Representative positive and negative pY419SRC IHC staining in primary GBM samples. Primary GBM tissue microarrays were staining as described in the Experimental Procedures. Normal brain samples on the same TMA consistently stained negative for pY419SRC (data not shown).
Figure 3
Figure 3. Dasatinib effectively blocked tumor progression in vitro and in vivo, and resistant mutants of SRC and FYN rescued dasatinib effects in glioma cells
(a) Dasatinib diminished T98G glioma cell viability and migration in vitro. T98G cells were plated onto 96-well plates and treatment with either DMSO or dasatinib at concentrations specified in the figure. The cell viability was assayed with CellTiterGlo. Proliferation rates were assessed with BrdU incorporation at 24h. Apoptosis levels were analyzed with a Caspase3/7 activity assay at 48h. Cell migration through 0.45um membrane was measured at 24h post-treatment. Triplicate samples were assayed for each condition. The averages and standard deviations are presented. (b) Dasatinib effectively inhibited tumor growth in vivo. Mice with established intracranial U87 tumors were treated with imatinib at 75 mg/kg twice a day by oral gavage (n=6) or vehicle (n=5). Tumor burden was assessed by bioluminescence imaging and expressed relative to the start of treatment (Treatment Day 1). Data expressed as mean ± SD. A significant suppression of tumor growth was observed in mice treated with dasatinib at 10 mg/kg twice a day by oral gavage (n=5) by comparison to vehicle treated controls (n=5). Day 14 of treatment was the last imaging point with all vehicle treated animals. Student’s t-test p<0.05. Representative images of dasatinib and vehicle treated animals. Bioluminescence intensity is represented by the indicated false-color scale. (c) T98G cells were plated onto 96-well plates and infected with lentiviruses expressing the tyrosine kinases specified in the figure. 24 hours after infection, cells were treated with dasatinib ranging from 50nM to 5uM. Cell viability was assessed at 96h post-treatment. Triplicate samples were analyzed for each condition. The averages and standard deviations are shown. (d) Total protein lysates were prepared from T98G cells treated with dasatinib at concentrations specified in the figure. The lysates were immunoprecipitated with a total SRC antibody and blotted with either the pY419SRC antibody or a total SRC antibody. (e) Dasatinib-resistant mutant SRC but not other alleles confer resistance in glioma cells, indicating increased SRC activity alone does not confer dasatinib resistance. T98G cells were plated, infected and treated same as in figure 3. Besides the wild-type and drug resistant T341I mutant, cells were also infected with viruses expressing an activating allele (Y530F), a kinase dead allele (K598R) and a double mutant of K598R/T341I. Uninfected cells and lacZ virus infected cells were used as controls. Each condition was assayed in triplicates and the means and standard deviations are presented.
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