Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. EGFRvIII is a truncated extracellular mutant of the EGF receptor (EGFR) commonly found in GBMs that confers enhanced tumorigenic behavior. To gain a molecular understanding of the mechanisms by which EGFRvIII acts, we have performed a large-scale analysis of EGFRvIII-activated phosphotyrosinemediated signaling pathways and thereby have identified and quantified 99 phosphorylation sites on 69 proteins. Distinct signaling responses were observed as a function of titrated EGFRvIII receptor levels with the phosphatidylinositol 3-kinase pathway being dominant over the MAPK and STAT3 pathways at a high level of EGFRvIII expression. Within this data set, the activating phosphorylation site on the c-Met receptor was found to be highly responsive to EGFRvIII levels, indicating cross-activation of the c-Met receptor tyrosine kinase by EGFRvIII. To determine the significance of this finding, we devised a combined treatment regimen that used a c-Met kinase inhibitor and either an EGFR kinase inhibitor or cisplatin. This regimen resulted in enhanced cytotoxicity of EGFRvIII-expressing cells compared with treatment with either compound alone. These results suggest that the clinical use of c-Met kinase inhibitors in combination with either EGFR inhibitors or standard chemotherapeutics might represent a previously undescribed therapeutic approach to overcome the observed chemoresistance in patients with GBMs expressing EGFRvIII. mass spectrometry ͉ mutant EGF receptor ͉ signal transduction ͉ tyrosine phosphorylation G lioblastoma multiforme (GBM) is the most aggressive form of adult human brain tumor, with median survival of Ͻ12 months (1). This dismal prognosis is due in part to the lack of therapeutic agents available to eliminate the diffuse glioma infiltrate that remains in the brain after surgical resection. Molecular profiling of genetic lesions in these tumors holds the promise of stratifying tumors into categories amenable to targeted treatment modalities. One such example is the application of EGF receptor (EGFR)-targeted therapeutic agents to treat GBMs that overexpress EGFR. Of these tumors, about half express the deletion mutant EGFRvIII, a truncated extracellular mutant of EGFR lacking exons 2-7, including the extracellular ligand-binding domain. Although EGFRvIII is incapable of binding the EGF family of ligands, it has been shown to be constitutively tyrosine-phosphorylated at Ϸ10% relative to ligand-stimulated wild-type (WT) EGFR (2). This truncated mutant receptor has been exclusively found in tumors, suggesting selection for EGFRvIII during the process of tumorigenesis. Clinical studies have demonstrated a correlation between EGFRvIII expression and poor prognosis for patients with GBM, indicating that it may be important in driving tumorigenic behavior in GBMs.Although much work has been done over the past decade to elucidate pathways involved in EGFRvIII receptor s...

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