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. Author manuscript; available in PMC: 2012 Feb 1.
Published in final edited form as: Mol Cancer Ther. 2011 Jun 9;10(8):1407–1418. doi: 10.1158/1535-7163.MCT-11-0205

Enhanced efficacy of IGF1R inhibition in paediatric glioblastoma by combinatorial targeting of PDGFRα/β

Aleksandra Bielen 1, Lara Perryman 1, Gary M Box 2, Melanie Valenti 2, Alexis de Haven Brandon 2, Vanessa Martins 2, Alexa Jury 1, Sergey Popov 1, Sharon Gowan 2, Sebastien Jeay 3, Florence I Raynaud 2, Francesco Hofmann 3, Darren Hargrave 4, Suzanne A Eccles 2, Chris Jones 1,4
PMCID: PMC3160488  EMSID: UKMS35725  PMID: 21659463

Abstract

Paediatric glioblastoma (pGBM), although rare, is one of the leading causes of cancer-related deaths in children, with tumours essentially refractory to existing treatments. We have identified IGF1R to be a potential therapeutic target in pGBM due to gene amplification and high levels of IGF2 expression in some tumour samples, as well as constitutive receptor activation in pGBM cell lines. In order to evaluate the therapeutic potential of strategies targeting the receptor, we have carried out in vitro and in vivo preclinical studies using the specific IGF1R inhibitor NVP-AEW541. A modest inhibitory effect was seen in vitro, with GI50 values of 5-6μM, and concurrent inhibition of receptor phosphorylation. Specific targeting of IGF1R with siRNA decreased cell viability, diminished downstream signalling through PI3-kinase and induced G1 arrest, effects mimicked by NVP-AEW541, both in the absence and presence of IGF2. Hallmarks of PI3-kinase inhibition were observed after treatment with NVP-AEW541 by expression profiling and Western blot analysis. Phospho-RTK arrays demonstrated phosphorylation of PDGFRα/β in pGBM cells suggesting co-activation of an alternative RTK pathway. Treatment of KNS42 with the PDGFR inhibitor imatinib showed additional effects targeting the MAP-kinase pathway, and co-treatment of the PDGFR inhibitor imatinib with NVP-AEW541 resulted in a highly synergistic interaction in vitro, and increased efficacy after 14 days therapy in vivo compared with either agent alone. These data provide evidence that inhibition of IGF1R, in combination with other targeted agents, may be a useful and novel therapeutic strategy in pGBM.

Keywords: IGF1R, PDGFR, PI3-kinase, MAP-kinase, paediatric glioblastoma

Introduction

High grade glioma in children continues to have a dismal clinical outcome, with a median overall survival for patients with supratentorial WHO grade IV glioblastoma multiforme (pGBM) of 11 months (1). Current standard of care is still questionable, with the Stupp protocol (2) for adult cases of radiotherapy plus temozolomide commonly used despite an absence of published data demonstrating a clearly beneficial therapeutic effect (3). Novel, molecularly targeted agents have demonstrated little efficacy in early phase clinical trials (4-6), a problem exacerbated by concerns over drug delivery coupled with an incomplete knowledge of the importance of the specific target in the childhood setting.

Recent molecular profiling data has demonstrated that there is a spectrum of genomic aberrations in high grade glioma covering patients of all ages, with paediatric cases tending to have an overlapping, but distinct biology from their adult counterparts (7-11). Such differences can be demonstrated at the levels of both large scale and highly focal DNA copy number alterations, as well as gene expression signatures defining molecular subclassifications. In particular, paediatric high grade glioma appear to be both preferentially and differentially driven by PDGFR signalling (10), with only a minority of cases demonstrating the EGFR-driven subtype largely present in adults (12-14).

In adults, high grade glioma with EGFR amplification/overexpression were reported to be mutually exclusive with a smaller subset of tumours which had extremely high levels of the ligand IGF2 (15). IGF2 was found to convey a strong growth-promoting effect in tumour-derived neurospheres, signalling through PI3-kinase (15). Given the distinctions between EGFR and PDGFR in adult and paediatric high grade glioma, we reasoned that dysregulation of the IGF signalling system was likely to co-exist with alterations in PDGFR, and thus may be a useful target in a significant proportion of childhood cases.

IGF2 signals through its receptor IGF1R, which plays important roles in development, with constitutive abnormalities in the IGF system causative in foetal and postnatal growth syndromes (16, 17). In the CNS, IGF1R promotes cell growth and survival as well as co-operating with growth and morphogenetic factors that induce cell fate specification and selective expansion of specified neural cell subsets (18). In cancer, there is widespread overexpression and occasional DNA copy number gains of IGF2/IGF1R present in a number of disparate paediatric tumour types (19-25), with amplifications reported in pGBM (8, 11). Considerable promise targeting the receptor in childhood cancers has been shown in the preclinical setting by small molecules, and in early phase clinical studies by humanised monoclonal antibodies, particularly in Ewings sarcoma (26-29).

In the present study we have sought to investigate the potential usefulness of targeting IGF1R in paediatric high grade glioma. By utilising a small panel of well-characterised glioma cell lines derived from children (30), we have screened the small molecule inhibitor NVP-AEW541, which as a tool compound has demonstrated efficacy in a range of tumour types (31-33). We have further explored the mechanisms by which its effectiveness may be enhanced, alighting on a clinically relevant combinatorial strategy with anti-PDGFR therapy which produces a synergistic interaction in vitro and in vivo due to differential inhibition of PI3- and MAP-kinase pathways by the two compounds in pGBM cells.

Materials and Methods

Cell lines and reagents

Glioblastoma cell lines were obtained and cultured as previously described (30), and identity re-authenticated by DNA copy number and gene expression profiling. R− (IGF1R-null) and R+ (IGF1R-overexpressing) mouse fibroblasts were provided by Renato Baserga, Thomas Jefferson University, Philadelphia. NVP-AEW541 and imatinib mesylate were synthesized by Novartis. Temozolomide was obtained from Sigma, Poole, UK. Chemical structures are provided below:

graphic file with name ukmss-35725-f0001.jpg

Growth inhibition

Growth inhibition was determined using the MTS assay as previously described (34). For the assessment of combination effects, cells were treated with increasing concentrations of drugs either alone or concurrently at their equipotent molar ratio and combination indices calculated by the method of Chou&Talalay (35). All values are given as mean±standard deviation of at least three independent experiments.

siRNA

Pre-designed siRNA duplexes directed against IGF1R were purchased from Qiagen (Crawley, UK). Cells were transfected with 100nM IGF1R siRNAs as well as with a scrambled sequence control duplex using HyPerFect (Qiagen) transfection reagent.

Western blot analysis

Immunodetection was performed as previously described (30) using antibodies against IGF1R (#3027), phospho-Aktser473 (#9271), Akt (#9271), phospo-Erk1/2 (#9101), Erk1/2 (#9102), PARP (#9542), caspase-3 (#9662), LC3B (#2775) and β-actin (#9315), all at 1:1000 dilution and purchased from Cell Signaling Technology (Danvers, MA, USA).

Electrochemiluminescent immunoassay

MesoScale Discovery system (MSD, Gaithersburg, MD, USA) 96-well multispot assays for total/phospho-IGF1R/IR/IRS-1, total/phospho-Akt, and total/phospho-Erk1/2 were carried out as per the manufacturer’s protocol. Plates were washed four times, read buffer was added, and the plates were analyzed on a SECTOR6000 instrument (MSD).

Detection of phosphorylated receptor tyrosine kinases

A phospho-RTK assay (R&D Systems, Minneapolis, MN, USA) was used to screen multiple receptor tyrosine kinases, according to manufacturer’s instructions. Phosphorylated PDGFRα/β were measured in a sandwich ELISA assay (Cell Signaling) as previously described (36).

Assessment of autophagy

KNS42 cells were grown on a glass cover-slip and after 24hr treated with 5×GI50 of NVP-AEW541 or equivalent DMSO concentration for 1hr. Cells were fixed with 2.5% glutaraldehyde fixative (pH7.3) at 4°C, postfixed in osmium tetroxide, dehydrated and embedded in epoxy resin. The resin blocks were cut on Leica ultra-cut machines and picked on to 150 mesh Guilder Grids with a support film of Pioloform. Grids were stained with uranyl acetate and lead citrate and viewed in the Hitachi H7600 transmission electron microscope. Autophagy was also assessed in cells cultured in the presence or absence of NVP-AEW541 by measuring levels of cytosolic LC3-I and autophagosome-associated LC3-II by Western blot.

mRNA expression profiling analysis

Expression profiling after treatment of SF188 and KNS42 cells with NVP-AEW541 at 5×GI50 by Illumina HT-12 BeadChips was carried out according to the manufacturer’s instructions and normalised using the lumi package in R2.11 (www.r-project.org). All data have been deposited in the MIAME-compliant ArrayExpress database (accession number E-TABM-889). Time-course analysis was carried out using a Pearson correlation coefficient metric. Co-ordinate gene regulation was identified using Gene Set Enrichment Analysis (GSEA, www.broad.mit.edu/gsea/), with a nominal p value cut-off of 0.001. Gene expression signatures were compared to other drug induced profiles using the Connectivity Map (http://www.broad.mit.edu/camp/). Set of differentially expressed were clustered into biological modules on the basis of gene ontology using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) online software (http://david.abcc.ncifcrf.gov).

In vivo antitumour activity of NVP-AEW541 and imatinib

Six to eight week-old female athymic nude mice [CrTac:NCr-Foxn1nu (www.taconic.com)] were utilized to establish subcutaneous pGBM xenograft models. Experiments were conducted in accordance with UK Home Office regulations under the Animals (Scientific Procedures) Act 1986 and UKCCCR guidelines for animal experimentation (37). A subline of KNS42 was generated using serial xenografting and the addition of matrigel and growth factors, as the parental cells developed palpable tumours with a low take rate over 235 days. KNS42_A4 cells (growth rate=36 days, take rate=100%) were implanted subcutaneously, bilaterally, at 5×106 per site. Mice were randomly assigned to three groups (n=6), and treated with NVP-AEW541 (50mg/kg, p.o.b.d., with dose reduced from 75mg/kg on day 11 due to weight loss) and imatinib mesylate (150mg/kg, p.o.b.d.) either alone or in combination, as well as vehicle comprising of 25mM L(+) tartaric acid. Sample extracts were analysed by for drug concentration by LC/MS/MS.

Results

Paediatric high grade glioma cell lines show a moderate sensitivity to small molecule inhibition of IGF1R

We re-analysed our recently compiled molecular profiling studies of paediatric high grade glioma patient samples (GSE19578) and cell lines (E-TABM-579) to explore the mRNA expression of various components of the IGF signalling network. Specific up-regulation of IGF1R was observed in paediatric high grade versus paediatric low-grade and adult high grade cell lines (Figure 1A), and high levels of IGF2, IGF1R, and IRS1 were variously noted in subsets of paediatric high grade glioma patient samples, including cases with PDGFRA amplification (Figure 1B). In our cell line panel, mRNA expression corresponded with wild-type receptor expression by Western blot (Figure 1C) and high levels of phosphorylation were observed in pGBM KNS42 and SF188 cells by an electrochemiluminescent assay (MSD) (Figure 1D). Examining the in vitro growth inhibitory effects of the small molecule inhibitor NVP-AEW541 revealed a relatively modest sensitivity of the paediatric high grade glioma cell lines (Figure 1E), with GI50 values of 5.85–6.36 μM, compared to those of 1.10 and 13.86 μM for the IGF1R-overexpressing R+ and null R− cells, respectively (Figure 1F). Cell survival was diminished in a concentration-dependent manner by NVP-AEW541 both in the absence and presence of the biologically-relevant ligand IGF2 (Supplementary Figure S1).

Figure 1. IGF1R as a therapeutic target in paediatric high grade glioma.

Figure 1

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(A) Affymetrix gene expression data for numerous probesets corresponding to IGF1R in a panel of adult high grade (darkorange), paediatric high grade (darkgreen) and paediatric low grade (lightgreen) cell lines. Data taken from Array Express E-TABM-579. (B) Affymetrix gene expression data for IGF1R (blue), IRS1 (green) and IGF2 (red) in paediatric high grade glioma patient samples. Asterisks represent cases with known PDGFRA amplification. Data taken from Gene Expression Omnibus GSE19578. (C) Western blot for IGF1R protein expression in high grade glioma cells and the IGF1R-null (R−) and overexpressing (R+) fibroblast lines. (D) Quantitative measure of phosphorylated IGF1R in high grade glioma cell lines assessed by the MSD assay. (E) Effects on cell survival of treatment with high grade glioma cells with the IGF1R inhibitor NVP-AEW541. (F) GI50 values for cell lines treated with NVP-AEW541 as assessed by the MTS assay.

In order to assess the effects on downstream signalling of IGF1R abrogation in pGBM, we compared genetic knockdown by specific siRNA with pharmacological inhibition by NVP-AEW541. siRNA directed against IGF1R in the pGBM cell lines (exemplified by KNS42, Figure 2A) resulted in efficient reduction of IGF1R, along with inhibition of phospho-Akt (Ser473) with a small increase in phospho-Erk1/2 detected by Western blot (Figure 2B); induction of apoptosis as measured by PARP cleavage (Figure 2B); and a profound cell cycle arrest at G1 determined by FACS (Figure 2C). These observations were replicated in both the absence and presence of IGF2 (Figure 2D). These effects on protein expression (Figure 2E), cell cycle (Figure 2F) were mimicked by NVP-AEW541 in SF188 and KNS42 cells in both a concentration- and time-dependent manner. NVP-AEW541 blocked IGF2-induced PI3-kinase activation via inhibition of phospho-Akt after 1hr of treatment and induced apoptosis as seen by PARP and caspase 3 cleavage after 1hr and 3hr in KNS42 and SF188 cells respectively. As described for the genetic inhibition of IGF1R above, a dose-dependent increase in MAP-kinase activation via phospho-Erk1/2 in a dose-dependent manner was observed. A time- and dose-dependent reduction in phospho-IGF1R and phospho-IRS were also observed by MSD (Supplementary Figure S2).

Figure 2. Effects of downstream signalling of genetic and pharmacological targeting of IGF1R in paediatric glioblastoma cells.

Figure 2

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(A) Relative IGF1R mRNA expression in KNS42 cells transfected with siRNA targeting the gene in KNS42 cells, as determined by quantitative RT-PCR. **p<0.01, t-test (B) Western blots demonstrating efficient knock-down of IGF1R protein in association with diminished phospho-Akt and PARP cleavage in KNS42 cells transfected with IGF1R siRNA. A slight increase in phosphorylated versus total Erk1/2 is also seen. (C) FACS analysis of siRNA-transfected KNS2 cells versus scrambled control oligos demonstrating an accumulation of cells in G1 and sub-G1 phases in contrast to a reduction of S and G2 phases. (D) Western blots confirming the knock-down of IGF1R, reduction in phospho-Akt, induction of PARP cleavage and increase in phospho-Erk1/2 after treatment of KNS42 cells with IGF1R siRNA in the presence of the ligand IGF2. (E) Effects on downstream signalling in KNS42 and SF188 cells after treatment with NVP-AEW541 in the presence of IGF2. Cells were treated for 1,3,6,24,48 hours with 1×,3×,5×GI50 compound (triangle). Treatment with IGF1R inhibitor decreased phospho-Akt, induced PARP and caspase-3 cleavage, and increased phospho-Erk1/2 levels in a time- and concentration-dependent manner. At 48hrs, the highest concentration of compound results in a significant cell death with little protein recoverable. (F) Effects on cell cycle in KNS42 and SF188 cells treated with NVP-AEW541. An accumulation of cells in G1 and sub-G1 phases is induced by NVP-AEW541 in a time- and concentration-dependent manner. At 48hrs, the highest concentration of compound results in a significant cell death.

NVP-AEW541 acts via down-regulation of the PI3-kinase pathway and induces autophagy in paediatric glioblastoma cells

In order to further explore the mechanism of action of NVP-AEW541 in our pGBM models, we carried out Illumina HT-12 expression profiling for both SF188 and KNS42 cells treated with 5×GI50 NVP-AEW541 for 1, 6 and 24 hours. In KNS42, genes commonly dysregulated by IGF1R inhibition in a time-dependent manner relative to vehicle-treated control included numerous cyclins (e.g. CCND2/CCNE1), cyclin-dependent kinases (e.g. CDK2/CDK4), cyclin-dependent kinase inhibitors (e.g. CDKN1A/CDKN2A/B) and other cell cycle regulators (e.g. E2F2/CDC25A) associated with transition through the G1 checkpoint (Figure 3A). Looking for co-ordinately regulated transcripts using GSEA highlighted significant effects on numerous groups of genes associated with cell cycle, and in particular the G1-S phase transition (SERUM_FIBROBLAST_CELLCYCLE, ES=0.696, p<0.00001, FDR q<0.00001, rank=2; CELL_CYCLE, ES=0.619, p<0.00001, FDR q<0.00001, rank=18; CELL_CYCLE_KEGG, ES=0.610, p<0.00001, FDR q<0.00001, rank=24; G1_TO_S_CELL_CYCLE_REACTOME, ES=0.520, p<0.00001, FDR q=0.00685, rank=51) (Figure 3B). Ontology analysis using DAVID further revealed a clustering of genes associated with cell cycle progression (e.g. GO:0007409:Cell_cycle, enrichment=5.71, p=0.00094; GO:) and DNA replication (e.g. GO:0006260:DNA_replication, enrichment=4.06, p=0.0000051) (Figure 3C) in pGBM cells treated with NVP-AEW541. Probing our specific gene signatures using the Connectivity Map resulted in significant hits for the classical inhibitors of PI3-kinase LY294002 (enrichment=0.352, p<0.00001) and wortmannin (enrichment=0.512, p=0.00006) (Figure 3D). Similar, though slightly less statistically significant, results were obtained with SF188 (Supplementary Figure S3).

Figure 3. Expression profiling of KNS42 cells treated with NVP-AEW541 highlights gene signatures associated with a PI3-kinase mediated cell cycle arrest.

Figure 3

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(A) Heatmap demonstrating expression of genes associated with the G1 to S phase transition of the cell cycle in KNS42 cells treated with 5×GI50 NVP-AEW541 for 1,6,24hrs. Genes are coloured according to global, not relative, expression values. (B) Gene set enrichment analysis of NVP-AEW541-treated KNS42 cells identified numerous groups of genes associated with the cell cycle. Enrichment score plots demonstrating strong negative correlation (down-regulation) with treatment with inhibitor are given. (C) Gene ontology analysis using the DAVID bioinformatic tool. A decrease in expression of numerous genes associated with processes such as cell cycle control and DNA replication are seen in KNS42 cells treated with NVP-AEW541. Bars represent fold enrichment of the most significant gene ontology groups, coloured by Bonferroni-corrected p values. (D) Connectivity Map analysis of NVP-AEW541-induced gene signatures in KNS42 cells. The barview is constructed from 6100 horizontal lines representing individual treatment instances ordered by their corresponding enrichment with the NVP-AEW541 signature (positive–green, negative–red, no enrichment–grey). All instances involving the PI3-kinase inhibitors LY-294002 and wortmannin are coloured black, with corresponding highest scoring treatment instances given in the table.

Given the strong evidence to suggest that NVP-AEW541 was acting predominantly via the PI3-kinase pathway in pGBM cells, we tested whether we could replicate our previously published observations regarding a differential response to combination with TMZ in KNS42 and SF188 (38). Both cell lines are resistant to TMZ (GI50>100μM), SF188 due to an unmethylated MGMT promoter, whilst KNS42 is MGMT-independent (38). Combination of TMZ and NVP-AEW541 resulted in a highly synergistic interaction with KNS42 cells (CI=0.61), though it was antagonistic in SF188 (CI=2.16), as determined by median effects analysis (Figure 4A). This mirrored the results seen with the dual PI3-kinase/mTOR inhibitor PI-103, an effect we hypothesise is at least in part a PI3-kinase dependent process leading to an upregulation of a HOX/stem cell signature (38). PI3-kinase inhibition is known to induce autophagy in glioblastoma cells (39), and we demonstrated similar effects in KNS42 cells with inhibition of IGF1R by both Western blot for increased LCB3-II (Figure 4B) and electron microscopy, demonstrating the formation of autophagic structures including autolysosomes and amphisomes upon treatment with NVP-AEW541 (Figure 4C,D).

Figure 4. Treatment with NVP-AEW541 overcomes resistance to temozolomide and induces autophagy in paediatric glioblastoma KNS42 cells.

Figure 4

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(A) Co-treatment of pGBM cells with NVP-AEW541 and temozolomide (TMZ) resulted in a high degree of synergy in KNS42 cells (combination index=0.61) as calculated by the median effect analysis. By contrast, SF188 cells showed an antagonistic interaction (CI=2.16). (B) Western blot of LC3 expression in response to NVP-AEW541 treatment of IGF2-stimulated KNS42 cells. An increased conversion of the cytosolic LC3-I to the autophagosome-associated LC3-II form is observed in a time- and concentration-(1×,3×,5×GI50) dependent manner. (C) Electron microscopy of vehicle-treated KNS42 cells. Little vacuolation is observed, with cytoplasm mostly containing multivesicular bodies (arrow). (i) Original magnificatio×5000. (ii) Original magnification×25000. (D) Treatment of KNS42 cells with NVP-AEW541 at 5×GI50 for 1hr. Electron micrographs reveal the accumulation of large and small cytoplasmic vacuoles. (i) Original magnificatio×5000. (ii) Vacuoles filled with electron-dense inclusions resembling autolysosomes (arrow). Original magnificatio×20000. (iii) Cytoplasm with numerous large empty vacuoles, and smaller ones surrounded by double membrane indicative of autophagosomes (arrow). Original magnification×20000. (iv) Autolysosomes filled with homogeneous digested material (arrow), in some cases fusing together. Original magnificatio×20000.

Paediatric glioblastoma cells have constitutive co-activation of IGF1R and PDGFR, and co-targeting the receptors shows enhanced efficacy in vitro and in vivo

Despite the clear targeting of PI3-kinase signalling after inhibition of IGF1R by NVP-AEW541 in pGBM cells, the growth inhibitory effects in vitro were relatively modest. In order to explore the mechanisms by which the cells may be surviving such targeted drug treatment, we carried out phospho-receptor tyrosine kinase arrays. Both KNS42 and SF188 had constitutively active IGF1R, confirming the MSD data, however in addition were found to have high levels of PDGFRα and PDGFRβ, respectively (Figure 5A). Overexpression of wild-type PDGFRα/β was confirmed in these lines by Western blot, as was activation of both PI3- and MAP-kinase pathways upon stimulation with the ligand PDGF-BB (Figure 5B). Treatment of the cells with the PDGFR (and other RTK) inhibitor imatinib as a single agent resulted in broad insensitivity, with GI50 values in the range 30.6–35.0 μM (not shown). In order to determine the efficacy of dual targeting of both IGF1R and PDGFRα/β, we carried out a median effects analysis in vitro. KNS42 cells (PDGFRα co-activated) showed a highly synergistic interaction (CI=0.44), whilst the effects with SF188 (PDGFRβ activated) were additive (CI=1.0) (Figure 5C). Similar additivity was observed with U87MG (PDGFRβ-activated) and Res259 (c-Kit-activated) (data not shown). Analysis of downstream signalling components demonstrated a time- and concentration-dependent inhibition of both phospho-Akt and phospho-Erk1/2, revealing co-inhibition of the MAP-kinase pathway in contrast to the PI3-kinase selectivity of NVP-AEW541 (Figure 5D). Apoptosis via PARP and caspase 3 cleavage (Figure 5D) and a G1 cell cycle arrest were also observed (Figure 5E) in parallel with inhibition of PDGFRα/β by ELISA (Supplementary Figure S4), albeit at relatively high doses of the compound.

Figure 5. Co-activation of IGF1R and PDGFRα/β in paediatric glioblastoma cells and inhibition of both PI3- and MAP-kinase pathways by imatinib.

Figure 5

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(A) Phosphorylated receptor tyrosine kinase assay shows co-activation of IGF1R with PDGFRα in KNS42 cells, and PDGFRβ in SF188. (B) Western blot confirmation of wild-type protein expression of PDGFRα/β in KNS42 and SF188 cells, respectively, as well as activation of both PI3- and MAP-kinase pathways upon stimulation with ligand PDGF-BB. (C) Median effects analysis of combining NVP-AEW541 and imatinib on high grade glioma cells in vitro. KNS42 cells show a highly synergistic interaction (CI=0.44), whilst an additive effect was observed for SF188 (CI=1.0). (D) Effects on downstream signalling in KNS42 and SF188 cells after treatment with imatinib in the presence of PDGF-BB. Treatment with imatinib decreased phospho-Akt and phospho-Erk1/2, and induced PARP and caspase-3 cleavage in a time- and concentration (1×,3×,5×GI50)-dependent manner. At 48hrs, the highest concentrations of compound results in a significant cell death with little protein recoverable. (E) Effects on cell cycle in KNS42 and SF188 cells treated with imatinib. An accumulation of cells in G1 and sub-G1 phases is induced by imatinib in a time- and concentration-dependent manner. At 48hrs, the highest concentrations of compound results in a significant cell death.

Finally we determined whether these combination effects observed in vitro would translate to the in vivo setting. We thus dosed female athymic nude mice bearing KNS42_A4 subline subcutaneous xenografts with either 50mg/kg NVP-AEW541 alone (reduced from 75mg/kg after day 11), 150mg/kg imatinib alone, or both compounds in combination, and compared tumour growth with vehicle-treated controls. At these doses, final tumour concentrations of drug exceeded in vitro GI50 values in KNS42_A4 cells for both NVP-AEW541 (11.0μM, 1.5×GI50) and imatinib (38.6μM, 1.5×GI50). KNS42_A4 xenografts treated with both compounds showed significantly reduced tumour volumes starting day 10 post-treatment and maintained until sacrifice compared with either agent alone (p=0.0265–0.0218,ANOVA) (Figure 6A). Final tumour weights showed a similar trend towards enhanced efficacy of the combination treatment (p=0.0842,t-test) (Figure 6B). Pharmacodynamic endpoints assayed by MSD demonstrated specific downregulation of phospho-IGF1R by NVP-AEW541 (p<0.0001,t-test) in contrast to imatinib (Figure 6C), although imatinib did cause a significantly greater reduction in phospho-IRS1 levels (p=0.007,t-test) than the IGF1R inhibitor (p=0.0267,t-test) (Figure 6D). The most profound parallel inhibition of the PI3K and MAPK pathways was achieved in the combination (p=0.0002 and p<0.0001, respectively, t-test), with only modest effects observed on phospho-Akt by either compound alone (p=0.162, NVP-AEW541 and p=0.0496, imatinib, t-test) (Figure 6E), and only imatinib demonstrating inhibition of phospho-Erk1/2 (p<0.0001, t-test) (Figure 6F).

Figure 6. Antitumour activity of combined inhibition of IGF1R and PDGFRα in an in vivo xenograft tumour model of paediatric glioblastoma.

Figure 6

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(A) Combination of NVP-AEW541 and imatinib reduced tumour volume (percentage of day 0) more than either compound alone in the KNS42_A4 subcutaneous xenograft model. **p<0.01, *p<0.05, t-test. (B) A reduced final tumour weight was observed for the NVP-AEW541/imatinib combination compared with control tumours or those treated with either agent alone (p<0.0863,t-test). (C) MSD assay for phosphorylated IGF1R. NVP-AEW541 resulted in a significant inhibition of phospho-IGF1R, alone or in combination, in contrast to imatinib alone. (D) Inhibition of phospho-IRS1 was observed after treatment with NVP-AEW541, and especially imatinib, in KNS42_A4 xenografts. (E) Phospho-Akt levels showed a greater reduction with the combination of NVP-AEW541 and imatinib than either compound alone. (F) Treatment with imatinib, alone or in combination, significantly reduced phospho-Erk1/2 levels, in contrast to NVP-AEW541 alone. ***p<0.001, **p<0.01, *p<0.05, t-test.

Discussion

Paediatric high grade glioma, like the disease in adults, is in clear need of novel therapeutic strategies. Early clinical trials with receptor tyrosine kinase inhibitors directed against EGFR and PDGFR have thus far shown little efficacy (4-6), although it is not known how much of this is due to poor tumour penetration or inappropriate patient selection. What is clear however, from the experience in numerous other tumour types, is that monotherapy using these selective kinase inhibitors is ultimately unlikely to prove effective due to inherent and/or acquired resistance mechanisms (40-42). In glioblastoma, one form of resistance which may be either inherent or acquired is the presence of co-activated receptor tyrosine kinases, such as EGFR and MET (43), and it has been suggested that combination treatments targeting these, amongst other kinases may bring more patient benefit (44-46).

In the childhood setting, in both the primary tumours and cell lines, we have noted the IGF signalling pathway to be a potential therapeutic target, with the presence of gene amplifications, and specific overexpression of the receptor (IGF1R), ligand (IGF2) or adaptor molecule (IRS1). In our in vitro model systems, specifically targeting the receptor conveyed only a modest growth inhibitory effect despite constitutive activation of IGF1R. Identification of those additional RTKs that may also be constitutively activated in these pGBM cell lines has provided clues to design appropriate combination strategies to enhance IGF1R inhibitor treatment.

It is significant that we observed co-activation of PDGFRα/β in the cells. PDGFRA amplification is the most common genomic alteration seen in paediatric high grade glioma (7-11), and we have demonstrated a specific PDGFRA-driven transcriptional program to be active in these tumours even in the presence of normal gene copy number (10). We have also noted less frequent alterations targeting the ligands PDGFA, PDGFB, and the receptor PDGFRB (10, 11). In pGBM, PDGF/IGF dysregulation may co-segregate, demonstrating that the co-RTK activation observed in our models has clinical relevance. In fact this mechanism of PDGFR co-activation with RTKs in paediatric high grade glioma may be a more general mechanism, as we have previously noted in the small subgroup of childhood cases to harbour the EGFRvIII deletion mutation, which notably included anaplastic oligodendroglioma and gliosarcoma samples (36). Treatment with a PDGFR inhibitor alone was ineffective in our models, and only in combination with IGF1R inhibition did we observe efficacy in vitro and in vivo. Of note, greater combinatorial efficacy was observed with activation on PDGFRα than PDGFRβ or c-Kit. By exploring the mechanisms of action of the small molecule inhibitors targeting the specific receptors, we have been able to identify the key roles played by the different components of downstream signalling pathways in this synergistic interaction.

Numerous lines of evidence point to NVP-AEW541 acting highly specifically as an inhibitor of PI3-kinase in the pGBM cells. As well as observing down-regulation of phospho-Akt and G1 arrest, gene expression signatures showed a high degree of overlap with published data on PI3-kinase inhibitors and the Connectivity Map database; we further demonstrated the induction of autophagy, a known consequence of PI3-kinase inhibition in U87MG glioma cells (39). Our previous report of a differential combinatorial response to PI3-kinase/temozolomide in pGBM cells (38) was replicated with NVP-AEW541, presumably at least in part through inhibition of high HOX gene expression in KNS42, but not SF188 cells, a process known to be controlled by a PI3-kinase-mediated epigenetic mechanism (47).

NVP-AEW541 elicited an adaptive up-regulation of MAP-kinase signalling at the highest doses and longest drug exposure times in the presence of the biologically relevant ligand IGF2. This is a potential source of the limited in vitro efficacy of the IGF1R inhibitor, as MAP-kinase has been reported to contribute to a negative regulatory feedback loop promoting IGF-driven Akt activation via Erk/IRS1 (48), which may counterbalance the inhibition through PI3-kinase. Imatinib, on the other hand, inhibited both PI3- and MAP-kinase pathways equally in the presence of PDGF-BB, and it appears that it is this dual inhibition which is of key importance, resulting in a synergistic interaction in vitro and in vivo when the two small molecules were combined in cells with constitutive co-activation of IGF1R and PDGFRα. It is notable that in our subcutaneous xenografts experiments, as well as the specific inhibition of phospho-IGF1R and phospho-Erk with NVP-AEW541 and imatinib alone, there was enhanced down-regulation of both phospho-Akt and phospho-IRS1 in the combination treatments.

As we move toward combinatorial targeted therapies in high grade glioma of all ages, such data provide a preclinical rationale not only for the use of agents targeting co-activated RTKs themselves, but also for the addition of a PI3-kinase or MEK inhibitor to the anti-PDGFR/IGF1R compounds. In the case of adding a PI3-kinase inhibitor to anti-PDGFR therapy, this may obviate any concerns harboured regarding side-effects from co-inhibiting the structurally homologous insulin receptor, whilst the addition of an anti-MEK compound to agents targeting IGF1R may be more clinically effective than the disappointing results of inhibitors of PDGFR itself. It is worth noting that neither of the small molecule signalling inhibitors used in the present study crosses the blood-brain barrier, a further key consideration when developing novel strategies to treat these highly malignant CNS tumours.

Supplementary Material

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Acknowledgements

We would like to thank Dr Daphne Haas-Kogan (UCSF) and Dr Michael Bobola (University of Washington) for provision of the paediatric glioma cell lines. Expression profiling was carried out by UCL Genomics (University College London), and we thank Anita Grigoriadis (Breakthrough Breast Cancer Unit, King’s College London) for bioinformatic assistance. We thank Dr Alice Warley and Ken Brady from the Centre for Ultrastructural Imaging, King’s College London for preparation of samples and assistance with transmission electron microscopy. We also extend our thanks to Dr David Dinsdale (University of Leicester) for the assessment of EM images. This research is supported by Cancer Research UK (C13468/A6718 and C309/A8274). We acknowledge NHS funding to the NIHR Biomedical Research Centre.

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