Egfrviii-Mediated Transactivation of Receptor Tyrosine Kinases in Glioma: Mechanism and Therapeutic Implications

Home , Panitumumab, Rilotumumab

ORIGINAL ARTICLE EGFRvIII-mediated transactivation of receptor tyrosine kinases in glioma: mechanism and therapeutic implications

SA Greenall1,2,3,8, JF Donoghue1,2,8, M Van Sinderen1,2,9, V Dubljevic1,2,10, S Budiman1,2, M Devlin4, I Street5,6,7, TE Adams3,11 and TG Johns1,2,11

A truncation mutant of the epidermal growth factor receptor, EGFRvIII, is commonly expressed in glioma, an incurable brain cancer. EGFRvIII is tumorigenic, in part, through its transactivation of other receptor tyrosine kinases (RTKs). Preventing the effects of this transactivation could form part of an effective therapy for glioma; however, the mechanism by which the transactivation occurs is unknown. Focusing on the RTK MET, we show that MET transactivation in U87MG human glioma cells in vitro is proportional to EGFRvIII activity and involves MET heterodimerization associated with a focal adhesion kinase (FAK) scaffold. The transactivation of certain other RTKs was, however, independent of FAK. Simultaneously targeting EGFRvIII (with panitumumab) and the transactivated RTKs themselves (with motesanib) in an intracranial mouse model of glioma resulted in signiﬁcantly greater survival than with either agent alone, indicating that cotargeting these RTKs has potent antitumor efﬁcacy and providing a strategy for treating EGFRvIII-expressing gliomas, which are usually refractory to treatment.

Oncogene (2015) 34, 5277–5287; doi:10.1038/onc.2014.448; published online 9 February 2015

INTRODUCTION In the U87MG glioma cell line in vitro, EGFRvIII enhances its High-grade glioma (HGG), often simply called glioma, is a antitumor activity by transactivating the hepatocyte growth factor malignant brain tumor that is incurable because of its relatively (HGF) receptor (MET), AXL, platelet-derived growth factor recep- β β refractory nature to the standard of care, which involves surgical tor- (PDGFR- ), vascular endothelial growth factor receptor fi 10,11 resection, radiotherapy and the chemotherapeutic temozolomide. 3 (VEGFR3) and broblast growth factor receptor 3. Further- Glioma has been classified into four distinct molecular subtypes, more, transactivation of MET by EGFRvIII leads to resistance to 1,2 the clinical candidate antibody rilotumumab, which binds to and each characterized by a set of molecular traits. Nearly all gliomas 11 of the classical subtype show amplification and overexpression of neutralizes the MET ligand, HGF. Importantly, EGFRvIII-mediated MET transactivation is present in primary gliomaspheres derived the epidermal growth factor receptor (EGFR) gene. Furthermore, from cells isolated from patient tissue and can be inhibited by the up to 50% of gliomas harboring EGFR amplification also contain EGFR-specific TKI erlotinib.12 Hence, this EGFRvIII-mediated RTK EGFRvIII (also known as de2–7EGFR or ΔEGFR),3 a truncated transactivation has widespread implications for the success of receptor that lacks exons 2–7. EGFRvIII is present as an autoactive, 4 glioma therapy. The tyrosine kinase activity of EGFRvIII is clearly disulfide-bonded homodimer and has been identified as an early- 11 5 required for RTK transactivation; however, the degree of onset event in gliomagenesis. In vitro, EGFRvIII expression in participation of these transactivated RTKs in maintaining the glioma cells has been associated with a phenotype that is more tumorigenic state in vivo, or the mechanism by which they invasive, angiogenic and proliferative6 and makes cells more 7,8 become transactivated, has not been elucidated. refractory to chemotherapeutics and radiotherapy than glioma In this study, we investigated the mechanism of EGFRvIII- cells lacking EGFRvIII. Furthermore, EGFRvIII can also mediate the mediated MET transactivation in U87MG glioma cells and in vitro phosphorylation of AKT, mitogen-activated protein kinase conducted a biochemical and biological assessment of and S6, which are downstream effectors of receptor tyrosine the contribution of several known transactivated RTKs to kinases (RTKs), negating the antitumor activity of single tyrosine the tumorigenic state. Based on these findings, we evaluated kinase inhibitors (TKIs) that target other coexpressed, activated therapeutic strategies for targeting EGFRvIII-positive glioma- RTKs in glioma cells and maintaining clonogenic survival in vitro.9 spheres by using a U87MG xenograft model.

1Oncogenic Signalling Laboratory and Brain Cancer Discovery Collaborative, Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, VIC, Australia; 2Monash University, Clayton, VIC, Australia; 3Division of Materials Science and Engineering, Commonwealth Scientific and Industrial Research Organisation, Parkville, VIC, Australia; 4Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, VIC, Australia; 5CRC for Cancer Therapeutics, Bundoora, VIC, Australia; 6The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia and 7Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia. Correspondence: Professor TG Johns, Oncogenic Signalling Laboratory and Brain Cancer Discovery Collaborative, Centre for Cancer Research, MIMR-PHI Institute of Medical Research, 27-31 Wright Street, Clayton, VIC 3168, Australia. E-mail: [email protected] 8These authors contributed equally to this study. 9Current address: Centre for Reproductive Health, MIMR-PHI Institute of Medical Research, Clayton, VIC, Australia and Monash University, Clayton, VIC, Australia. 10Current address: Patrys, Level 6, Equitable House, Melbourne, VIC, Australia. 11These laboratories contributed equally to this study. Received 12 August 2014; revised 19 October 2014; accepted 8 November 2014; published online 9 February 2015 RTK transactivation by EGFRvIII in glioma SA Greenall et al 5278 RESULTS We then assessed whether other RTKs are also transactivated Efficient MET transactivation by EGFRvIII requires full kinase by EGFRvIII. After stimulation of U87MG.vIII cells with EGF, the activity endogenous wtEGFR had substantially higher activation than in Our previous studies demonstrated that EGFRvIII forms a cysteine- the parent U87MG cells (Figure 1d), as did AXL after stimulation of linked homodimer and that this is the main phosphorylated U87MG.vIII cells with the AXL ligand, GAS-6 (Figure 1e). In both 4 cases, total receptor downregulation after ligand stimulation was (active) form of the receptor. We also previously showed that the retarded only in the presence of EGFRvIII. These data show that transactivation of MET by EGFRvIII does not involve secondary active EGFRvIII augments the response of RTKs other than MET to signaling mediators but requires the kinase activity of EGFRvIII.11 ligand stimulation by inhibiting receptor downregulation. However, whether EGFRvIII forms heterodimers with other RTKs is unknown. To better understand how EGFRvIII transactivates MET, we expressed three modified forms of EGFRvIII in U87MG glioma Neutralization of HGF increases cell-surface MET and thereby cells that express low levels of the wild-type (wt) EGFR—a enhances transactivation dimerization-deficient but kinase-active mutant (U87MG.C16S), a To investigate whether the endogenous HGF autocrine loop has a carboxy-terminal signaling-deficient and kinase-reduced mutant role in EGFRvIII-mediated MET transactivation, we blocked (U87MG.DY5) and a kinase-dead mutant (U87MG.DK)—and autocrine ligand-mediated activation of MET by treating U87MG. assessed MET transactivation in these cells, by using a modified vIII cells with the anti-HGF antibody rilotumumab. After rilotumu- non-reducing western blot analysis of whole-cell lysates. The mab treatment, MET phosphorylation at tyrosine residue 1234 (Y1234) and Y1235 was ~ 50% lower in U87MG.vIII cells than in dimerization-deficient (C16S) form of EGFRvIII transactivated MET untreated cells (Figure 1f). This partial reduction in phosphoryla- (Figure 1b) despite not being dimerized and displaying less tion is consistent with rilotumumab’s blocking the autocrine loop autophosphorylation than unaltered EGFRvIII (Figure 1a), indicat- but not the transactivation. Furthermore, the consequent increase ing that EGFRvIII does not need to dimerize to transactivate MET. in total MET (Figure 1f, top) levels correlated with a reduction in The extent of MET transactivation on the western blots was the phosphorylation of Y1003 (Figure 1f), the binding site for CBL, proportional to the degree of EGFRvIII activation, descending in which is involved in the ubiquitination and downregulation of 4 4 the order EGFRvIII C16S (kinase-active) DY5 (kinase-reduced) MET. Thus, transactivation of MET can occur in the absence of HGF. 4DK (kinase-dead). These results show that the degree of EGFRvIII We then investigated the mechanism by which EGFRvIII activation determines the level of MET transactivation. contributes to MET transactivation, by treating U87MG.C16S, U87MG.DY5 and U87MG.DK cells with rilotumumab. After EGFRvIII augments the activation of RTKs in response to ligand rilotumumab treatment, all tested cell lines showed an increase stimulation in total MET compared with untreated cells, as determined by fl U87MG-derived cells have an intrinsic autocrine HGF loop that western blotting and ow cytometry (Figures 1g and h). The levels sustains low-level MET phosphorylation. Surprisingly, treating of p-MET also increased in U87MG.C16S cells and, to a lesser U87MG cells expressing EGFRvIII (U87MG.vIII) with exogenous extent, in U87MG.DY5 cells. By contrast, no increase was observed HGF led to substantially higher MET activation than in parent in U87MG.DK cells, which cannot transactivate MET (Figure 1g). U87MG cells treated with HGF (Figure 1c), suggesting that EGFRvIII Therefore, the observed increases in MET activation do not require HGF, MET downstream signaling (as an increase was mediated by also enhances MET activation in response to ligand. This higher DY5) or EGFRvIII homodimerization but do require an EGFRvIII with activation status was associated with less ligand-induced down- kinase activity. Furthermore, removing endogenous HGF increases regulation of total MET in the EGFRvIII-bearing cells than in the MET levels, leading to more transactivated MET. parent U87MG cells (Figure 1c), suggesting that active EGFRvIII is Administering rilotumumab in the presence of an EGFRvIII aiding the retention of MET on the cell surface. In support of these variant with reduced activity (such as C16S) may be similar to fi ndings, U87MG.DK cells stimulated with HGF did not exhibit giving rilotumumab in the presence of partially inhibited EGFRvIII, increased phosphorylation of MET compared with parental a situation that occurs during glioma therapy with EGFR TKIs.13 U87MG cells, whereas treatment of U87MG.vIII cells overnight Indeed, in U87MG.vIII cells, titrating EGFRvIII with the EGFR TKI with the EGFRvIII inhibitory antibody panitumumab before HGF erlotinib showed that rilotumumab enhanced MET activation stimulation prevented HGF-induced hyperphosphorylation of MET when EGFRvIII activity was partially inhibited (Figure 1i). Thus, (Figure 1c) and restored ligand-induced MET downregulation. cotreatment with rilotumumab and EGFR TKIs may cause an These results demonstrate that the observed augmentation of increase in MET activation in patients. MET stimulation in response to HGF is due to active EGFRvIII Clinical trials in glioma patients have used lapatinib and receptors increasing the retention of MET on the cell surface. erlotinib, which are reversible EGFR TKIs. After treatment cessation,

Figure 1. The kinase activity of EGFRvIII, not its dimerization potential, is crucial for MET transactivation. (a–g, i and j) Reducing western blotting (immunoblotting, IB) analyses (non-reducing in (a)) for total or phosphorylated RTKs (labeled right) in immunoprecipitates (IP) or whole-cell lysates (WCLs) from a U87MG cell line panel (bottom)—parental U87MG (U87MG), U87MG.wtEGFR (EGFR), U87MG.EGFRvIII (vIII), U87MG.C16S (C16S; dimerization-deficient EGFRvIII), U87MG.DY5 (DY5; secondary-signaling-deficient EGFRvIII) and U87MG.DK (DK; kinase- dead EGFRvIII)—treated in vitro as indicated (top). p-MET, p-Y1234/1235; p-EGFR, p-Y1173; p-Y, pan-phosphotyrosine. Pan-actin loading controls are shown where appropriate. (a) Basal EGFR status across the U87MG cell line panel. The locations of the EGFRvIII monomer (M) and dimer (D) are indicated. (b) Transactivated MET status across the U87MG cell line panel. (c) MET and EGFR status after exposure to MET ligand (HGF). Cells were treated for 7 min with phosphate-buffered saline (PBS) (vehicle) or 100 ng/ml HGF. For panitumumab (pani’mab) tests, cells were pretreated overnight with 100 μg/ml antibody before ligand addition. (d) Endogenous wtEGFR status after exposure to EGFR ligand (EGF). Cells were treated for 10 min with PBS or 100 ng/ml EGF. (e) AXL status after exposure to AXL ligand (GAS-6). Cells were treated for 10 min with PBS or 100 ng/ml GAS-6. (f and g) MET status after exposure to anti-MET-ligand antibody (rilotumumab). Cells were treated overnight with PBS or 10 μg/ml rilotumumab (rilo). (h) Cell-surface MET levels across the cell line panel, as measured by flow cytometry after overnight treatment with PBS (blue) or 10 μg/ml rilotumumab (green). Data are presented as the percentage of the total cell population (y axis) versus log10(fluorescence staining intensity) (x axis). (i) MET and EGFR status after exposure to various concentrations of erlotinib alone or in combination with 10 μg/ml rilotumumab. (j) MET and EGFR status upon EGFR inhibition. U87MG.vIII cells were treated for 2 h with 2 μM erlotinib or 1 μM dacomitinib (acute), and then extensively washed and incubated in drug-free medium overnight (washout).

Oncogene (2015) 5277 – 5287 © 2015 Macmillan Publishers Limited RTK transactivation by EGFRvIII in glioma SA Greenall et al 5279 these TKIs might be expunged from cells, and MET transactivation washout, led to the reactivation of EGFRvIII-mediated MET might reoccur. Indeed, treating cells with erlotinib or the transactivation in erlotinib-treated cells but not in dacomitinib- irreversible EGFR inhibitor dacomitinib, followed by a 24 h treated cells (Figure 1j), suggesting that irreversible EGFR

© 2015 Macmillan Publishers Limited Oncogene (2015) 5277 – 5287 RTK transactivation by EGFRvIII in glioma SA Greenall et al 5280 inhibitors may be more effective in vivo at preventing the We further investigated the role of FAK in MET transactivation reactivation of MET transactivation. by assessing its involvement in EGFRvIII•MET heterodimerization. Significantly fewer EGFRvIII•MET heterodimers formed after FAK EGFRvIII and MET form a heterodimer in glioma cells kinase inhibition, as demonstrated by PLA (Figures 3e and f), suggesting that activated FAK facilitates heterodimerization by Our previous study suggested that EGFRvIII and MET directly acting as a scaffold. Dacomitinib did not significantly decrease the interact,11 but this was not formally demonstrated. Therefore, amount of EGFRvIII•MET heterodimer (Figure 3f). Taken together, to determine whether EGFRvIII transactivation of MET was • these data show that a FAK scaffold is required for the mediated by heterodimerization, we examined EGFRvIII MET transactivation of MET by EGFRvIII. heterodimerization using the proximity ligation assay (PLA) (Figure 2a), which detects direct nanoscale interactions between molecules at the cell surface. Optimization of the assay to detect FAK inhibition results in caspase-independent cell death cell-surface EGFRvIII•MET heterodimers was performed in U87MG. Given that FAK is important for EGFRvIII•MET heterodimer vIII cells with labeled antibodies specific for EGFRvIII and MET. A formation, and therefore EGFRvIII signaling, we studied the effects strong fluorescent signal, indicating heterodimer formation, was of the FAK inhibitor CTX-945 on cell viability. In U87MG.vIII cells, observed in U87MG.vIII cells but not in U87MG parental cells, CTX-945 had a half-maximal inhibitory concentration (IC50)of which lack EGFRvIII (Supplementary Figure S1a). Labeling of the 2.4 μM (Supplementary Figure S3a); therefore, we investigated the antibodies did not affect their binding activity compared with mechanism of CTX-945-mediated cell death using a cytotoxic dose unlabeled antibody (Supplementary Figure S1b). The PLA also (5 μM). We found no significant induction of cleaved caspase-3/7 showed a robust EGFRvIII•MET heterodimer signal for U87MG.C16S over 48 h compared with time-matched vehicle controls but significantly less signal in U87MG.DY5 and U87MG.DK cells (Figure 4a), suggesting that FAK inhibition did not induce fi than in U87MG.vIII cells (Figures 2a and b). This finding agrees caspase-dependent apoptosis. Supporting this nding, the pan- with our MET transactivation data, with the weaker MET caspase inhibitor Z-VAD-FMK was unable to rescue cells from fi transactivation in U87MG.DY5 cells than in U87MG.vIII cells CTX-945-induced death (Figure 4b), con rming that caspase explained by the presence of less heterodimer. Furthermore, the activity was not involved in the induction of cell death. We then investigated caspase-independent mechanisms of cell death, homodimeric form of EGFRvIII is not required for interaction with 16 fi namely autophagy and mitochondrial damage. CTX-945 induced MET, as cells expressing dimerization-de cient EGFRvIII (U87MG. fl C16S) contained EGFRvIII•MET heterodimer. Finally, we confirmed an autophagic ux over 24 h, with a progressive increase in LC3B that EGFRvIII and MET bind each other by using co- protein observed in U87MG.vIII and the EGFRvIII-positive primary immunoprecipitation (Figure 2c). Taken together, these results gliomasphere cell line GBM-6, indicating active autophagy confirm that EGFRvIII and MET form a heterodimer at the cell (Figure 4c). Inhibiting autophagy with chloroquine increased the efficacy of a sub-IC dose of CTX-945 at killing glioma cells surface. 50 compared with vehicle and single treatment controls (Figure 4d; Po0.001), indicating that autophagy induced by CTX-945 is The EGFRvIII•MET heterodimer uses FAK as a scaffold cytoprotective. We then assessed mitochondrial damage, using Interestingly, discrete clusters of PLA signal were present in multiplex flow cytometry to track apoptosis and mitochondrial membrane-ruffle-like structures reminiscent of focal adhesions permeability, and found that CTX-945 induced mitochondrial (Figure 2a). Staining U87MG.vIII cells for actin and focal adhesion damage within 48 h, leading to the onset of apoptosis (Figure 4e). kinase (FAK) revealed discrete FAK puncta that were colocalized These data demonstrate that inhibiting FAK in glioma cells with actin-rich membrane ruffles and distinct from the intracellular induces an initial cytoprotective autophagy response that is pools of FAK (Figure 3a). This pattern also coincided with the most eventually overcome by caspase-independent mitochondrial intense areas of PLA signal for the EGFRvIII•MET heterodimer damage, leading to cell death. (Figure 2a) and with areas of intense staining for total EGFRvIII and total MET in U87MG.vIII cells, as determined by immunofluores- Therapeutic cotargeting of EGFRvIII and transactivated RTKs has cence (Supplementary Figures S2a and b). enhanced antitumor activity We therefore assessed the possible role of FAK in EGFRvIII- The above data suggested that targeting FAK may be a promising mediated MET transactivation. We inhibited FAK activity in approach for the treatment of glioma. Indeed, CTX-945 treatment U87MG.vIII cells by using a highly specific FAK TKI, CTX-0294945 14 or FAK knockdown reduced cell migration, inhibited cellular (denoted CTX-945), as shown by western blotting for activated proliferation and decreased cellular adhesion in U87MG.vIII cells (p-Y397) FAK (Figure 3c). CTX-945 or a FAK/PYK2 TKI (CTX- (Supplementary Figures S3b–e). By contrast, in vivo, U87MG.vIII- 0097900; denoted CTX-009) completely inhibited MET transactiva- FAK kd xenografts grew at a rate comparable to U87MG.vIII-scram tion, reducing it to the level observed in U87MG parental cells or controls in nude mice (Supplementary Figure S4), despite a partial U87MG.vIII cells treated with the TKI dacomitinib (which blocks the knockdown of total FAK and significant inhibition of p-Y397 FAK. activity of EGFR) (Figure 3b). By contrast, TKI inhibition of FAK did Investigation of other RTKs that are transactivated by EGFRvIII, not affect EGFRvIII activity (Figure 3b) and did not inhibit the such as AXL, revealed that their transactivation was unaffected by HGF-mediated autocrine activation of MET (Supplementary treating U87MG.vIII cells with FAK inhibitors (CTX-945 or CTX-009) Figure S2d) or EGF-stimulated wtEGFR activation (Supplementary but was abolished by the EGFR inhibitor dacomitinib in vitro Figure S2e). Moreover, no inhibition of MET transactivation was (Figure 5a). This finding suggests that RTKs other than MET are observed after treatment of U87MG.vIII cells with TKIs of another transactivated independently of FAK, reducing the therapeutic focal adhesion protein (integrin-linked kinase) or of Src-family value of targeting FAK. Therefore, we explored an alternative kinases (Supplementary Figure S2c), indicating the specific therapeutic approach to inhibit the growth of EGFRvIII-positive involvement of FAK. Supporting these findings, genetic ablation glioma cells and xenografts. of FAK with a specific short hairpin RNA (shRNA) (U87MG.vIII- First, instead of combining a TKI with a narrow specificity and an FAKkd) resulted in the loss of MET transactivation compared with anti-EGFR therapeutic, we assessed the antitumorigenic potential scrambled shRNA controls (U87MG.vIII-scram), together with of the multi-RTK TKI motesanib (AMG-706), which inhibits VEGFR1, reductions in p-Y397 FAK. Genetic ablation of FAK, in contrast to VEFGR2, VEGFR3, PDGFR-α, PDGFR-β, c-KIT and RET,17 alone or in simply kinase inhibition of FAK, decreased EGFRvIII phospho- combination with the anti-EGFR monoclonal antibody (mAb) rylation as reported previously15 (Figure 3d). panitumumab. Motesanib inhibited PDGFR-β phosphorylation in

Oncogene (2015) 5277 – 5287 © 2015 Macmillan Publishers Limited RTK transactivation by EGFRvIII in glioma SA Greenall et al 5281 U87MG cells (Figure 5b), showing that it inhibits its target in transactivation in this study, which is in agreement with the glioma cells. Motesanib slightly inhibited the growth of three finding that active EGFR and AXL directly interact in triple- primary gliomasphere cell lines compared with vehicle (Figure 5c) negative breast cancer.23 However, as PDGFR-β and the RTK IGF-IR and showed additive antitumor activity on the GBM-L1 cell line in can interact with FAK in cells in vitro,22 it is possible that a FAK combination with panitumumab (Po0.0001). scaffold may be used in the EGFRvIII-mediated transactivation of Given this finding, and our published findings that the these RTKs. This process might also occur in other cancer types combination of panitumumab and the anti-HGF antibody and could be particularly important in non-small-cell lung rilotumumab is synergistic in vivo against glioma xenografts,11 carcinoma, in which activating mutations in the tyrosine kinase we compared the antitumor efficacy of motesanib, panitumumab domain of the EGFR drive MET transactivation.24 and rilotumumab, alone and in various combinations, against We also demonstrated that active EGFRvIII reduces the down- subcutaneous U87MG.vIII glioma xenografts (Figure 6a and regulation of HGF-stimulated MET, leading to increased activation Table 1). We confirmed our previous findings11 that EGFRvIII of MET by its ligand. This enhancement was also seen with other mediates significant resistance to rilotumumab. Panitumumab by RTKs transactivated by EGFRvIII, including AXL and the endo- itself significantly reduced U87MG.vIII xenograft growth compared genous wtEGFR when stimulated using GAS-6 and EGF, respectively. with vehicle-treated xenografts. The combination of motesanib The expected downregulation of both total wtEGFR and total AXL and rilotumumab significantly reduced the tumor volume was also reduced in the presence of EGFRvIII compared with compared with vehicle (P = 0.0001), rilotumumab alone parental cells, suggesting a similar mechanism to that observed (P = 0.0002) or motesanib alone (P = 0.02). These data demonstrate for MET. Because EGFRvIII forms a heterodimer with MET, the that, despite not directly targeting EGFRvIII, significant tumor mechanism underlying this increased response of RTKs to ligand suppression can be achieved by cotargeting EGFRvIII- stimulation may be a heterodimer-induced reduction in RTK transactivated RTKs and autocrine MET signaling. The combination downregulation after stimulation with ligand. Supporting this of motesanib and panitumumab demonstrated significant tumor hypothesis is the observation that EGFRvIII downregulation is inhibition compared with vehicle (P = 0.0001) or motesanib alone significantly impaired in glioma cells.25 The observed increase in (P = 0.0001) but not panitumumab alone (P = 0.07), and the RTK response to ligand stimulation offers another mechanism by rilotumumab and panitumumab combination also significantly which EGFRvIII bestows such a substantial tumorigenic advantage reduced tumor volumes compared with rilotumumab alone in glioma. Our findings are in contrast to those of a recent study (P = 0.0001) and panitumumab alone (P = 0.02). Once treatment demonstrating that EGF-mediated stimulation results in a ceased, tumor regrowth was markedly delayed in mice that had unidirectional increase in EGFRvIII activity, with wtEGFR increasing received panitumumab-containing combination treatments com- the activity of EGFRvIII,26 suggesting that the EGFRvIII and wtEGFR pared with single agent groups and the motesanib and interaction differs depending on the cellular background with rilotumumab combination group. respect to ligand or other scaffold molecules. EGFRvIII-mediated We then assessed whether the key drug combinations were as MET transactivation still occurred upon complete neutralization of efficacious in the native tumor environment, by using an the HGF autocrine loop by rilotumumab, in agreement with intracranial U87MG.vIII model. Unlike in the subcutaneous model, studies of the HGF-negative cell line U373.EGFRvIII,10 demonstra- the combination of motesanib and rilotumumab did not increase ting that ligand is not required for MET transactivation. Hence, we survival compared with vehicle controls (Figure 6b). Similarly to suggest a model by which basal transactivation of MET is the subcutaneous model, panitumumab as a single agent augmented by HGF. First, EGFRvIII recruits FAK to the inner provided a significant benefit, with mice surviving 40 days longer membrane, where it also associates with MET to form the than vehicle controls. The addition of motesanib to panitumumab EGFRvIII•MET heterodimer, allowing direct transactivation to occur. provided an additional survival benefit, to 62 days longer than Second, paracrine or autocrine HGF stimulation enhances MET vehicle controls (the end of the experimental period), with no activation further as a result of the EGFRvIII•MET heterodimer mortality recorded in this period. Histological analysis revealed reducing the downregulation of MET. that all treatment groups eventually developed tumors, although One major impediment to EGFR TKI therapy for patients with in the motesanib plus panitumumab group only a single tumor glioma is poor tumoral penetration and accumulation of regrew (Figure 6c). These data demonstrate that a combination of drugs.13,27,28 Thus, EGFR is inhibited suboptimally, below the agents targeting both EGFRvIII and the RTKs that are commonly threshold required to induce apoptotic cell death.13 Our findings transactivated by EGFRvIII provides additional survival advantages show that rilotumumab, when used in combination with a in an orthotopic model of glioma. suboptimal concentration of erlotinib, results in increased cell- surface MET and subsequently enhanced EGFRvIII-mediated MET transactivation compared with cells not expressing EGFRvIII. We DISCUSSION showed that rilotumumab treatment decreased the phosphoryla- In this study, we show that the EGFRvIII-mediated transactivation tion of MET at Y1003, which is the CBL engagement site.29,30 Thus, of MET in HGG involves the formation of an EGFRvIII•MET the inherently low level of EGFRvIII degradation,25 especially in the heterodimer and that FAK is required to sustain the MET presence of erlotinib, and the rilotumumab-induced decrease in transactivation. FAK is a ubiquitously expressed intracellular CBL-mediated degradation of MET leads to an increase in MET, tyrosine kinase that is linked to integrin signaling, cellular and hence the EGFRvIII•MET complex, on the cell surface. Given migration, cytoskeletal rearrangements and invasion and can that HGF probably contributes to the observed resistance to EGFR directly bind activated RTKs in HGG.18 Furthermore, negative TKIs in non-small-cell lung carcinoma,31 it is reasonable to regulatory microRNAs that target FAK and EGFR, such as miR-7, are postulate that therapeutics such as rilotumumab may increase commonly downregulated in HGG,19,20 and the consequent high the efficacy of these agents. However, because MET transactiva- expression of the FAK protein is an indicator of poor prognosis in tion by autoactivated kinase domain EGFR mutants is also patients with glioma.21 observed in non-small-cell lung carcinoma,24 it would be prudent Previous studies have identified that FAK interacts with to assess whether the combination of rilotumumab and an EGFR activated MET22 and EGFRvIII15 via FAK’s FERM domain. In TKI stimulates transactivation of MET in these tumors compared normal brain tissue, FAK associates with integrins; however, in with those expressing wtEGFR. HGG, FAK switches to associating with activated RTKs such as After partial knockdown of FAK by RNA interference, we EGFR and PDGFR, possibly in part facilitating RTK cross-talk,18 as observed no effect on U87MG.vIII xenograft growth. Further described in this study. FAK was not associated with AXL examination showed that other EGFRvIII-transactivated RTKs were

© 2015 Macmillan Publishers Limited Oncogene (2015) 5277 – 5287 RTK transactivation by EGFRvIII in glioma SA Greenall et al 5282 active, as a result of FAK-independent transactivation by EGFRvIII. invasion. Another FAK inhibitor, TAE226, has shown initial This mode of activation probably negates the loss of FAK activity antitumor efﬁcacy against multiple glioma cell lines, including with respect to tumor growth, although we cannot exclude the U87MG.vIII cells, in several in vitro assays, but this activity did not possibility that FAK is involved in other tumor processes such as translate into in vivo efﬁcacy in an orthotopic xenograft model.32

Figure 3. The EGFRvIII•MET heterodimer is concentrated in actin-rich membrane ruffles and uses FAK as a scaffold. (a) U87MG.vIII cells were stained for total FAK (green), total actin (phalloidin; blue) and DNA (red) and visualized by immunofluorescence. Overlay, fluorescence images superimposed on phase-contrast images. Arrows, discrete concentrations of FAK and phalloidin staining localized to peripheral membrane ruffle structures. Bar, 50 μm. (b–d) Reducing western blotting (immunoblotting, IB) analyses for total or phosphorylated RTKs (labeled right) in immunoprecipitates (IP) or whole-cell lysates (WCLs) from parental U87MG (U87MG) and U87MG.EGFRvIII (vIII) treated as indicated (top). p-MET, p-Y1234/1235; p-EGFR, p-Y1173; and p-FAK, p-Y397. Pan-actin loading controls are shown where appropriate. (b) MET and EGFR status upon FAK inhibition (2 h treatment with the FAK inhibitors CTX-945 (5 μM) or CTX-009 (10 μM) or the irreversible pan-HER inhibitor dacomitinib (1 μM)). A U87MG parental control was included for basal MET and EGFR status. A nonspecific mouse IgG was used as an immunoprecipitation control on U87MG.vIII lysates. (c)FAKstatus following a 2 h treatment of U87MG.vIII cells with vehicle or CTX-945. (d) FAK, MET and EGFR status in U87MG.vIII cells stably expressing a scrambled shRNA (scram) or a FAK-targeting shRNA (FAK kd). (e and f) PLA analysis of EGFRvIII•MET heterodimer in U87MG.vIII cells treated for 2 h as indicated. (e) Representative confocal images. PLA signal, green; nucleus, blue. Bar, 30 μm. (f)Quantification of PLA signal after drug treatment. Data are the mean PLA signal per cell ± s.e.m.; n = 50 cells per treatment; *Po0.05, PLA signal compared with vehicle.

Figure 2. EGFRvIII and MET associate by heterodimerization. (a) Representative images for PLA conducted on the U87MG cell line panel. Phase- contrast images show the cells in situ,andfluorescent images depict the PLA signal (green) and the nucleus (blue). Overlays demonstrate the cellular localization of the PLA signal. Bar, 30 μm for magnified images (zoom) and 50 μm for all other images. Arrows indicate distinct clusters of PLA signal. (b)Quantification of PLA signals per cell indicating the amount of heterodimer being formed. Data are the mean ± s.e.m. n = 25 cells per cell line. N.S., not significant. (c) Co-immunoprecipitation of MET and EGFRvIII demonstrating the heterodimeric complex. The indicated U87MG-based cell lines (labeled bottom) were lyzed, MET was immunoprecipitated (IP) and then western analyses (IB) were conducted for total MET (top) and EGFR (bottom). A nonspecific mouse IgG was used as an immunoprecipitation control on each cell lysate.

Figure 4. Speciﬁc FAK inhibition using CTX-945 results in caspase-independent cell death caused by mitochondrial damage. (a) Cleaved caspase-3/7 assay on U87MG.vIII cells treated with the FAK inhibitor CTX-945 (5 μM). Data are the mean percentage cleaved caspase-3/7 signal normalized to the mean time-matched vehicle controls ± s.e.m. (b) MTS cell viability assays for U87MG.vIII cells treated for 72 h with 5 μM CTX-945, a pan-caspase inhibitor (25 μM Z-VAD-FMK) or both. Data are the mean cell viability normalized to the mean vehicle control results ± s.e.m. (c) Western blot analyses (IB) of LC3B status of U87MG.vIII or GBM-6 cells treated with vehicle (V) or 5 μM CTX-945 (C). Whole-cell lysates (WCLs) were probed for total LC3B (top) or pan-actin (loading control; bottom). (d) MTS cell viability assays for U87MG.vIII cells treated for 72 h with 2 μM CTX-945, an autophagy inhibitor (20 μM chloroquine (CQ)) or both. Data are the mean cell viability normalized to the mean vehicle control results ± s.e.m. (e) Flow cytometry analysis of mitochondrial permeability and apoptosis in U87MG.vIII cells treated with 5 μM CTX-945 for 48 h. Data are Annexin V staining versus ﬂuorescent signal from a dye indicating mitochondrial membrane potential.

The concept that EGFRvIII mediates RTK transactivation is which are scaffolded by FAK in a manner that sustains MET supported by our previous study showing that the anti-EGFR transactivation in glioma. We also demonstrate that transactivated antibody panitumumab reduces both MET and PDGFR-β phos- RTKs can be targeted independently of the main oncogenic driver, phorylation in U87MG.vIII cells following short-term treatment EGFRvIII, and show that the RTKs make a crucial contribution to in vitro.11 Furthermore, this study showed that administering tumor recurrence after anti-EGFR therapy. These in vivo data panitumumab in combination with rilotumumab additively indicate a potential new strategy for treating glioma in the clinic: reduced tumor growth in vivo. We demonstrated that a combining anti-EGFR and multi-RTK targeting therapeutics to combination of panitumumab and motesanib reduced tumor induce a more potent antitumorigenic effect in EGFRvIII-bearing growth in a manner similar to panitumumab and rilotumumab, gliomas than targeting EGFRvIII alone. demonstrating that other RTKs apart from MET can be cotargeted with EGFRvIII to enhance antitumorigenic efficacy. Motesanib MATERIALS AND METHODS alone in vivo did not show any efficacy, indicating that sustained Cell lines EGFRvIII signaling mediates substantial resistance to a TKI U87MG and the U87MG-derived cell lines are described elsewhere.4,11 The targeting multiple RTKs simultaneously, including those associated status of all cell lines was routinely tested by western blotting to confirm with angiogenesis. Combining motesanib with rilotumumab expression, activation status and dimerization. The gliomasphere lines (which targets and neutralizes the HGF autocrine loop) resulted GBM-6, HK-301 and GBM-L1 were maintained in StemPro neural stem cell in a significant reduction in tumor growth, demonstrating that medium containing Dulbecco's modified Eagle's medium/F12, StemPro targeting as many transactivated RTKs as possible can be Neural Stem Cell SFM Supplement, 0.02 μg/ml basic fibroblast growth μ efficacious even in the presence of continued direct signaling factor, 0.02 g/ml epidermal growth factor, 2.5 mM GlutaMAX and from EGFRvIII. Thus, both direct signaling from EGFRvIII and its penicillin/streptomycin (Invitrogen, Melbourne, VIC, Australia). transactivation of other RTKs contribute to the tumorigenicity of EGFRvIII. Furthermore, using motesanib in combination with Reagents panitumumab caused complete regression of 80% of orthotopic The anti-EGFRvIII mAb mAb 806 and the anti-MET mAb LMH 85 were xenografts. Interestingly, Akhavan et al.12 recently showed that described previously.33,34 Rilotumumab and motesanib (which need to be transcription of the PDGFR-B gene, a motesanib target, is obtained via an MTA) and panitumumab (Vectibix) were provided by fi derepressed and subsequently activated when EGFRvIII is Amgen (Thousand Oaks, CA, USA); dacomitinib was provided by P zer inhibited by an EGFR TKI in xenograft models and patients. (New York, NY, USA); erlotinib (Tarceva) was purchased from Selleck Chemicals (Houston, TX, USA). The FAK/PYK2 inhibitor CTX-0097900 Therefore, some of motesanib’sefficacy in combination with fi β (PF-562 271) and the speci c FAK inhibitor CTX-0294945 were synthesized panitumumab might be due to its targeting of PDGFR- by the CRC for Cancer Therapeutics (Melbourne, VIC, Australia). Rabbit anti- reactivation caused by long-term panitumumab treatment. MET polyclonal antibody C-28, both alone and conjugated to agarose Collectively, these findings define a mechanism and a cellular beads, and protein A/G agarose beads were purchased from Santa Cruz location (i.e., the cell membrane) for EGFRvIII•MET heterodimers, Biotechnology (Santa Cruz, CA, USA). The following antibodies were

Figure 5. FAK-independent AXL transactivation and activity of motesanib on glioma cells. (a) Western analyses (IB) of AXL status in U87MG.vIII cells treated for 2 h as indicated. AXL was immunoprecipitated (IP) and probed for total AXL (top) and pan-phosphotyrosine (bottom). A U87MG parental control was included for basal AXL status. A nonspecific rabbit IgG was used as an immunoprecipitation control on U87MG. vIII lysates. (b) Western blot analysis of PDGFR-β status in U87MG cells treated with vehicle or 10 μM motesanib. PDGFR-β was immunoprecipitated and probed for total PDGFR-β (top) and pan-phosphotyrosine (bottom). (c) Proliferation assays after GBM-6, HK301 or GBM-L1 gliomaspheres were treated for 72 h with vehicle, 10 μM motesanib, 10 μg/ml panitumumab or both. Data are the mean inhibition of proliferation normalized to the mean vehicle control results ± s.e.m. also used: rabbit monoclonal anti-phosphorylated (Y1234/1235) MET Flow cytometry (3D7), mouse monoclonal anti-MET (L41G3), rabbit monoclonal anti- Cells were stained for 2 h at 4 °C with 10 μg/ml LMH 85 or mAb 806 in ice- phosphorylated (Y1173) EGFR (53A5), rabbit polyclonal anti-FAK, rabbit cold serum-free medium containing 0.1% bovine serum albumin (and then monoclonal anti-LC3B (D11) and rabbit monoclonal anti-AXL (C89E7) 1:300 anti-mouse-AF647 for 1 h at 4 °C). Cells were analyzed on a (Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal FACSCanto II flow cytometer (BD Biosciences). anti-phosphorylated (Y845) EGFR, anti-mouse-AF488, anti-mouse-AF647- phalloidin (Invitrogen); mouse anti-pan-phosphotyrosine (pY20) (Invitro- Proliferation gen); mouse monoclonal anti-FAK (77/FAK) (BD Transduction, San Jose, CA, USA). Other reagents were as follows: human HGF; Z-VAD-FMK, Caspase- Proliferation in response to drug treatment was assessed at 72 h using the MTS assay (Promega) or the ViaLight assay (Lonza) according to the Glo 3/7 Assay (Thermo Scientific, Melbourne, VIC, Australia); human EGF, manufacturer’s instructions. chloroquine hydrochloride, iodoacetamide (Sigma-Aldrich, St Louis, MO, USA); and human GAS-6 (R&D Systems, Minneapolis, MN, USA). Immunofluorescence Proximity ligation assay Immunofluorescence was conducted as described previously.35 PLAs were conducted using the Duolink System (Olink Bioscience, Uppsala, Sweden). Briefly, mAb 806 was labeled with biotin, and LMH 85 was labeled with digoxigenin using a OneShot Labeling Kit (SoluLink, Xenograft models – Kensington, SA, Australia); the manufacturer’s instructions (CUSTOM For subcutaneous xenograft studies, 6 8-week-old BALB/c nu/nu female protocol section) were then followed. Images were captured using a mice (Animal Resources Centre, Perth, WA, Australia) were inoculated once with 100 μl tumor cells in growth medium in both flanks. All experiments DeltaVision deconvolution microscope (GE Healthcare, Little Chalfont, UK). 11 Image preparation and measurement of PLA fluorescent signals per cell were conducted using established tumor models. The treatments was conducted using Imaris (3D and 4D Real-Time Interactive Data administered were 100 mg motesanib per kg body weight (daily), 50 mg panitumumab per kg body weight (on alternate days) or 1.5 mg Visualization software), with 3D Spotfinder (BitPlane Scientific Software, rilotumumab per kg body weight (on alternate days). Treatments Zurich, Switzerland). commenced on day 8 after tumor inoculation and were administered by Immunoprecipitation and western blotting intraperitoneal injection over 11 days. Tumor volumes were measured as described previously.11 Immunoprecipitation and western blotting were performed as previously Intracranial xenograft experiments were performed as published 11,31 described. previously.36 The treatments administered were 100 mg motesanib per kg body weight, 50 mg panitumumab per kg body weight or 5 mg Dimerization analysis of EGFRvIII rilotumumab per kg body weight. Treatments commenced on day 4 after Assays were conducted as described previously,4 except using TXC buffer tumor cell inoculation and were administered by intraperitoneal injection containing iodoacetamide instead of RIPA buffer. on alternate days for 11 days.

Figure 6. Combined inhibition of multiple RTKs reduces EGFRvIII tumorigenicity and increases survival in vivo.(a) Tumor growth curves of subcutaneous U87MG.vIII xenografts in mice treated with vehicle or RTK inhibitors alone or in combination from day 8 after U87MG.vIII cell inoculation. Data are the mean tumor volume ± s.e.m. over time. The treatment phase (gray, from day 8 after U87MG.vIII cell inoculation) and recovery phase (white, from day 19 after U87MG.vIII cell inoculation) of the experiment are indicated. (b) Kaplan–Meier survival curves for mice with intracranial U87MG.vIII tumors treated with vehicle or RTK inhibitors alone or in combination. Data are the percentage of animals surviving in each group at various time points. The treatment phase (gray, from day 4 after U87MG.vIII cell inoculation) and recovery phase (white, from day 15 after U87MG.vIII cell inoculation) of the experiment are indicated. (c) Shown are whole-mount Aperio ScanScope images (Leica Biosystems, Sydney, NSW, Australia) of hematoxylin- and eosin-stained coronal sections of mouse brains, in which areas of tumor development can be identiﬁed. The number of tumors that formed relative to the number of mice for each treatment group is shown within parentheses. Arrows indicate the tumors that are present in each coronal section.

This study was conducted in accordance with the Australian Code of Table 1. RTK-inhibitory antibodies slow U87MG.vIII xenograft tumor Practice for the Care and Use of Animals for Scientific Purposes (2004) and growth approved by Monash Medical Centre Animal Ethics Committee A (approval 2011/76). Treatment Tumor volume P-value (mean ± s.e.m., mm3)a (versus vehicle) Statistical analysis Vehicle 1057.6 ± 109.9 N/A All results are mean ± s.e.m. An analysis of variance with Dunnett’s post hoc Rilotumumab 1353.7 ± 129.1 0.02 test was conducted to assess whether data sets with a normal distribution ± Motesanib 720 80.7 0.08 were significantly different. Po0.05 was considered significant. In vivo ± Panitumumab 103.4 15.3 0.0001 data are mean tumor volume ± s.e.m. for each treatment group, and ± Motesanib+rilotumumab 393.9 117.4 0.0018 significance differences were analyzed with Student’s t-test. Motesanib+panitumumab 58.4 ± 12.8 0.0001 Rilotumumab+panitumumab 57.7 ± 7.9 0.0001 Abbreviations: N/A, not applicable; RTK, receptor tyrosine kinase. CONFLICT OF INTEREST aTumor volumes were measured on day 16 of the subcutaneous U87MG. MD and IS are inventors listed on patent applications for FAK inhibitors. TGJ has vIII xenograft trial. received project funding from Amgen.

Oncogene (2015) 5277 – 5287 © 2015 Macmillan Publishers Limited RTK transactivation by EGFRvIII in glioma SA Greenall et al 5287 ACKNOWLEDGEMENTS 15 Liu M, Yang Y, Wang C, Sun L, Mei C, Yao W et al. The effect of epidermal growth We acknowledge the facilities and scientific and technical assistance of the Histology factor receptor variant III on glioma cell migration by stimulating ERK Facility, Microimaging facility and Monash Animal Services, at MIMR-PHI Institute of Medical phosphorylation through the focal adhesion kinase signaling pathway. Archiv 502 – Research. We thank Dr D Dadley-Moore for editing the manuscript. SAG is funded by a Biochem Biophys 2010; :89 95. Commonwealth Scientific and Industrial Research Organisation (CSIRO) OCE Postdoctoral 16 Martinvalet D, Zhu P, Lieberman J, Granzyme A. Induces caspase-independent fi 22 Fellowship. JFD is funded by a Cure Cancer Australia Foundation postdoctoral fellowship mitochondrial damage, a required rst step for apoptosis. Immunity 2005; : – and a Victorian Cancer Agency Early Career Seed Grant (ECSG1108). TGJ is funded by 355 370. fi National Health and Medical Research Council Project Grants 1028552 and 1012020, the 17 Polverino A, Coxon A, Starnes C, Diaz Z, DeMel T, Wang L et al. AMG 706, an oral, Victorian Government’s Operational and Infrastructure Support Program and the Cure multikinase inhibitor that selectively targets vascular endothelial growth factor, platelet-derived growth factor, and kit receptors, potently inhibits angiogenesis Brain Cancer Foundation. This work was supported by the CRC for Cancer Therapeutics, an and induces regression in tumor xenografts. Cancer Res 2006; 66:8715–8721. initiative of the Australian Government. Rilotumumab and motesanib must be obtained 18 Riemenschneider MJ, Mueller W, Betensky RA, Mohapatra G, Louis DN. In situ through an MTA. Rilotumumab, panitumumab and motesanib were provided by Amgen analysis of integrin and growth factor receptor signaling pathways in human (rilotumumab and motesanib through an MTA); and dacomitinib was provided by Pfizer. glioblastomas suggests overlapping relationships with focal adhesion kinase activation. Am J Pathol 2005; 167: 1379–1387. AUTHOR CONTRIBUTIONS 19 Kefas B, Godlewski J, Comeau L, Li Y, Abounader R, Hawkinson M et al. MicroRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway SAG, JFD, TEA and TGJ conceived the idea and designed the experiments; SAG, and is down-regulated in glioblastoma. Cancer Res 2008; 68: 3566–3572. JFD, MVS, VD and SB performed the experiments; SAG, JFD, MVS, VD, MD, IS, 20 Kong X, Li G, Yuan Y, He Y, Wu X, Zhang W et al. MicroRNA-7 inhibits epithelial- TEA and TGJ analyzed and interpreted the data; SAG, JFD, MD, IS, TEA and TGJ to-mesenchymal transition and metastasis of breast cancer cells via targeting FAK 7 wrote and revised the manuscript; MD and IS provided reagents; and TEA and expression. PLoS One 2012; : e41523. 21 Ding L, Sun X, You Y, Liu N, Fu Z. Expression of focal adhesion kinase and TGJ supervised the study. phosphorylated focal adhesion kinase in human gliomas is associated with unfavorable overall survival. Transl Res 2010; 156:45–52. REFERENCES 22 Chen TH, Chan PC, Chen CL, Chen HC. Phosphorylation of focal adhesion kinase on tyrosine 194 by Met leads to its activation through relief of autoinhibition. 1 Sturm D, Witt H, Hovestadt V, Khuong-Quang DA, Jones DT, Konermann C et al. Oncogene 2011; 30:153–166. fi Hotspot mutations in H3F3A and IDH1 de ne distinct epigenetic and biological 23 Meyer AS, Miller MA, Gertler FB, Lauffenburger DA. The receptor AXL diversifies subgroups of glioblastoma. Cancer Cell 2012; 22:425–437. EGFR signaling and limits the response to EGFR-targeted inhibitors in 2 Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson MD et al. Integrated triple-negative breast cancer cells. Sci Signal 2013; 6: ra66. genomic analysis identifies clinically relevant subtypes of glioblastoma characterized 24 Guo A, Villén J, Kornhauser J, Lee KA, Stokes MP, Rikova K et al. Signaling networks by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 2010; 17:98–110. assembled by oncogenic EGFR and c-Met. Proc Natl Acad Sci USA 2008; 105: 3 Gan HK, Cvrljevic AN, Johns TG. The epidermal growth factor receptor variant III 692–697. (EGFRvIII): where wild things are altered. FEBS J 2013; 280: 5350–5370. 25 Grandal MV, Zandi R, Pedersen MW, Willumsen BM, van Deurs B, Poulsen HS. 4 Ymer SI, Greenall SA, Cvrljevic A, Cao DX, Donoghue JF, Epa VC et al. Glioma EGFRvIII escapes down-regulation due to impaired internalization and sorting to specific extracellular missense mutations in the first cysteine rich region of lysosomes. Carcinogenesis 2007; 28:1408–1417. epidermal growth factor receptor (EGFR) initiate ligand independent activation. 26 Fan QW, Cheng CK, Gustafson WC, Charron E, Zipper P, Wong RA et al. EGFR Cancers 2011; 3: 2032–2049. phosphorylates tumor-derived EGFRvIII driving STAT3/5 and progression in glio- 5 Del Vecchio CA, Giacomini CP, Vogel H, Jensen KC, Florio T, Merlo A et al. EGFRvIII blastoma. Cancer Cell 2013; 24:438–449. gene rearrangement is an early event in glioblastoma tumorigenesis and 27 Mellinghoff IK, Wang MY, Vivanco I, Haas-Kogan DA, Zhu S, Dia EQ et al. Molecular expression defines a hierarchy modulated by epigenetic mechanisms. Oncogene determinants of the response of glioblastomas to EGFR kinase inhibitors. N Engl J 2013; 32: 2670–2681. Med 2005; 353: 2012–2024. 6 Gan HK, Kaye AH, Luwor RB. The EGFRvIII variant in glioblastoma multiforme. 28 Wen PY, Chang SM, Lamborn KR, Kuhn JG, Norden AD, Cloughesy TF et al. Phase I/II J Clin Neurosci 2009; 16:748–754. study of erlotinib and temsirolimus for patients with recurrent malignant 7 Mukherjee B, McEllin B, Camacho CV, Tomimatsu N, Sirasanagandala S, gliomas: North American Brain Tumor Consortium trial 04-02. Neuro-Oncology Nannepaga S et al. EGFRvIII and DNA double-strand break repair: a molecular 2014; 16:567–578. mechanism for radioresistance in glioblastoma. Cancer Res 2009; 69: 4252–4259. 29 Peschard P, Fournier TM, Lamorte L, Naujokas MA, Band H, Langdon WY et al. 8 Nagane M, Levitzki A, Gazit A, Cavenee WK, Huang HJ. Drug resistance of human Mutation of the c-Cbl TKB domain binding site on the Met receptor tyrosine glioblastoma cells conferred by a tumor-specific mutant epidermal growth factor kinase converts it into a transforming protein. Mol cell 2001; 8: 995–1004. receptor through modulation of Bcl-XL and caspase-3-like proteases. Proc Natl 30 Peschard P, Ishiyama N, Lin T, Lipkowitz S, Park M. A conserved DpYR motif in the Acad Sci USA 1998; 95: 5724–5729. juxtamembrane domain of the Met receptor family forms an atypical c-Cbl/Cbl-b 9 Stommel JM, Kimmelman AC, Ying H, Nabioullin R, Ponugoti AH, Wiedemeyer R tyrosine kinase binding domain binding site required for suppression of et al. Coactivation of receptor tyrosine kinases affects the response of tumor cells oncogenic activation. J Biol Chem 2004; 279: 29565–29571. to targeted therapies. Science 2007; 318:287–290. 31 Turke AB, Zejnullahu K, Wu YL, Song Y, Dias-Santagata D, Lifshits E et al. 10 Huang PH, Mukasa A, Bonavia R, Flynn RA, Brewer ZE, Cavenee WK et al. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. Quantitative analysis of EGFRvIII cellular signaling networks reveals a Cancer Cell 2010; 17:77–88. combinatorial therapeutic strategy for glioblastoma. Proc Natl Acad Sci USA 2007; 32 Liu T-J, LaFortune T, Honda T, Ohmori O, Hatakeyama S, Meyer T et al. Inhibition 104: 12867–12872. of both focal adhesion kinase and insulin-like growth factor-I receptor kinase 11 Pillay V, Allaf L, Wilding AL, Donoghue JF, Court NW, Greenall SA et al. suppresses glioma proliferation in vitro and in vivo. Mol Cancer Ther 2007; 6: The plasticity of oncogene addiction: implications for targeted therapies directed 1357–1367. to receptor tyrosine kinases. Neoplasia 2009; 11:448–458, 442–458. 33 Greenall SA, Gherardi E, Liu Z, Donoghue JF, Vitali AA, Li Q et al. Non-agonistic 12 Akhavan D, Pourzia AL, Nourian AA, Williams KJ, Nathanson D, Babic I et al. bivalent antibodies that promote c-MET degradation and inhibit tumor growth De-Repression of PDGFRβ Transcription Promotes Acquired Resistance to EGFR and others specific for tumor related c-MET. PLoS One 2012; 7: e34658. Tyrosine Kinase Inhibitors in Glioblastoma Patients. Cancer Discovery 2013; 3: 34 Johns TG, Stockert E, Ritter G, Jungbluth AA, Huang HJ, Cavenee WK et al. Novel 534–547. monoclonal antibody specific for the de2-7 epidermal growth factor receptor 13 Vivanco I, Robins HI, Rohle D, Campos C, Grommes C, Nghiemphu PL et al. (EGFR) that also recognizes the EGFR expressed in cells containing amplification Differential sensitivity of glioma- versus lung cancer-specific EGFR mutations to of the EGFR gene. Int J Cancer 2002; 98: 398–408. EGFR kinase inhibitors. Cancer Discov 2012; 2:458–471. 35 Johns T, Perera RM, Vernes SC, Vitali AA, Cao DX, Cavenee WK et al. The efficacy 14 Street I, de Sylva M, Lackovic K, Ganame D, Holloway G, Anderson R et al. Abstract of epidermal growth factor receptor-specific antibodies against glioma xenografts LB-308: combination of CTx-0294945 a highly selective inhibitor of focal adhesion is influenced by receptor levels, activation status, and heterodimerization. kinase with bevacizumab in pre-clinical models of breast cancer. In: Proceedings of Clin Cancer Res 2007; 13: 1911–1925. the 103rd Annual Meeting of the American Association for Cancer Research;31 36 Donoghue JF, Bogler O, Johns TG. A simple guide screw method for intracranial March–4 April 2012; AACR: Chicago, IL; Philadelphia, PA; Cancer Res 2012, p 72. xenograft studies in mice. J Vis Exp 2011; 55: e3157.

Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)