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

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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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Figure 1: Luminex immunosandwich assay and IP-MS approach.
Figure 2: Luminex screen identifies activated tyrosine kinases in human cancer cell lines and primary glioblastomas.
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.

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Acknowledgements

We would like to thank Levi Garraway and Johnathan Fletcher for cell lines. We thank Julie Dang for her technical support on the pY419SRC IHC experiment. J.D. is supported by a fellowship from the Leukemia and Lymphoma Society and The Irving Family. I.K.M. is funded by the Brain Tumor Funders' Collaborative. The project has been funded in part with funds from the National Cancer Institute's (NCI) Initiative for Chemical Genetics, National Institutes of Health, under contract no. N01-CO-12400, and through the NCI's Integrative Cancer Biology Program.

Author information

Correspondence to Todd R Golub.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Methods (PDF 288 kb)

Supplementary Table 1

Tyrosine phosphorylated proteins identified with IP-MS method in five cancer cell lines. (XLS 36 kb)

Supplementary Table 2

Capture antibodies and positive control samples used in Luminex immunosandwich assay. (XLS 35 kb)

Supplementary Table 3

Tyrosine phosphorylation levels on antibody validation samples. (XLS 75 kb)

Supplementary Table 4

Tyrosine phosphorylation levels of wild-type and dasatinib-resistant tyrosine kinase mutants measured by Luminex assay. (XLS 48 kb)

Supplementary Table 5

Tyrosine phosphorylation levels in six cell lines used for measuring technical and biological variations. (XLS 194 kb)

Supplementary Table 6

Tyrosine phosphorylation levels on cancer cell lines. (XLS 91 kb)

Supplementary Table 7

Tyrosine phosphorylation levels and genomic data on the primary glioma samples. (XLS 26 kb)

Supplementary Table 8

pY419SRC immunohistochemsitry on primary GBM tissue microarrays. (XLS 22 kb)

Supplementary Table 9

Dasatinib EC50 and SRC phosphorylation levels in cancer cell lines. (XLS 28 kb)

Supplementary Table 10

ORF amplification and mutagenesis primers for generating dasatinib-resistant mutants. (XLS 24 kb)

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