American Association for Cancer Research
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Supplementary Methods, Legends for Figures 1-3 and Tables 1-5 from Phosphoproteomics Identifies Driver Tyrosine Kinases in Sarcoma Cell Lines and Tumors

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posted on 2023-03-30, 21:13 authored by Yun Bai, Jiannong Li, Bin Fang, Arthur Edwards, Guolin Zhang, Marilyn Bui, Steven Eschrich, Soner Altiok, John Koomen, Eric B. Haura

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ARTICLE ABSTRACT

Driver tyrosine kinase mutations are rare in sarcomas, and patterns of tyrosine phosphorylation are poorly understood. To better understand the signaling pathways active in sarcoma, we examined global tyrosine phosphorylation in sarcoma cell lines and human tumor samples. Anti-phosphotyrosine antibodies were used to purify tyrosine phosphorylated peptides, which were then identified by liquid chromatography and tandem mass spectrometry. The findings were validated with RNA interference, rescue, and small-molecule tyrosine kinase inhibitors. We identified 1,936 unique tyrosine phosphorylated peptides, corresponding to 844 unique phosphotyrosine proteins. In sarcoma cells alone, peptides corresponding to 39 tyrosine kinases were found. Four of 10 cell lines showed dependence on tyrosine kinases for growth and/or survival, including platelet-derived growth factor receptor (PDGFR)α, MET, insulin receptor/insulin-like growth factor receptor signaling, and SRC family kinase signaling. Rhabdomyosarcoma samples showed overexpression of PDGFRα in 13% of examined cases, and sarcomas showed abundant tyrosine phosphorylation and expression of a number of tyrosine phosphorylated tyrosine kinases, including DDR2, EphB4, TYR2, AXL, SRC, LYN, and FAK. Together, our findings suggest that integrating global phosphoproteomics with functional analyses with kinase inhibitors can identify drivers of sarcoma growth and survival. Cancer Res; 72(10); 2501–11. ©2012 AACR.

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