Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma

Abstract

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...

Navigating cancer network attractors for tumor-specific therapy

“…As predicted by R.L. and Pawson 66 several years ago, new pharmaceutical strategies that target networks instead of single proteins are becoming available 47,48 . We predict this trend will not only continue, but also include recent advances that highlight the possibility to 'cure' networks using time-and order-dependent therapies 68 .…”

“…In line with their importance, network-attacking mutations have attracted more attention in recent years [43][44][45][46][47][48] . Moreover, information has been accumulating steadily about how specificity in signaling networks and modular protein domains emerges [49][50][51] , leading to the definition of determinants of specificity in protein domains 52,53 .…”

“…Combination drugs that interfere with disease networks (so-called network medicine 66 ) have been shown to lead to a better response than single-hit therapies by causing secondary perturbations to signaling networks 47,48,67 . Recent work by the Yaffe laboratory represents a clear leap forward within the field of network medicine 68,69 .…”

Network‐based drugs and biomarkers

“…However, most current biomarker discovery is not taking this principle into consideration, which could have potentially dangerous implications. For example, should the phosphorylation site on JNK be used as a biomarker, it could have damaging consequences for the patient, as the context-dependent or multivariate nature of the regulation of this site would not be immediately apparent [13,22,23]. It could very well be that in a given patient or tumour context this site would be anti-apoptotic at a specific stage of disease progression.…”

“…Instead we encourage that the network models themselves should be deployed directly as markers. As we are moving towards multi-dimensional and integrative network models of cell behaviour, we expect that such models will be useful not just for determining network structures that can be targeted but also as predictive markers of disease emergence or progression [4,[22][23][24][25]. This will require robust quantitative network assays to become more widespread and userfriendly [21].…”