Pediatric Glioblastoma Cell Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Functional diversity and cooperativity between subclonal populations of pediatric glioblastoma and diffuse intrinsic pontine glioma cells.

The failure to develop effective therapies for paediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively map assess the extent to which this was present in these tumours through subclonal genomic analyses, and to determine whether distinct tumour subpopulations may interact to promote tumorigenesis by generating novel subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced cases revealed multiple tumour subclones, spatially and temporally co-existing in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations which we propose co-operate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, confer an abrogated DNA repair pathway and increased invasion/migration of neighbouring cells, in vitro and in vivo, through chemokine signalling and modulation of integrins. These data indicate that even rare tumour subpopulations may exert profound effects on tumorigenesis as a whole, and may represent a novel avenue for therapeutic development. Unravelling the mechanisms of subclonal diversity and communication in pGBM/DIPG will be an important step to overcoming barriers to effective treatments.

Abstract 3461: PID1, a novel brain tumor growth suppressor, is a target for phosphorylation by a newly identified binding partner

Abstract Background: Our group were first to establish a link between PID1 (Phosphotyrosine Interaction Domain containing 1) and cancer, showing PID1's tumor inhibitory effect in medulloblastoma, glioblastoma and atypical teratoid rhabdoid tumor (ATRT) brain tumor cell lines and correlation of higher PID1 mRNA with longer patient survival. PID1 was first identified in 2006 and is associated with obesity-related insulin resistance and mitochondrial dysfunction. Here we present a novel interacting partner of PID1 which phosphorylates and modulates its growth suppressive functions. Methods: In silico predictions, in vitro kinase assay, phosphoproteomic analysis, co-immunoprecipitation, SDS-PAGE, western blotting, flow cytometry. Cells: CHLA-07-BSGBM pediatric glioblastoma, U87 glioblastoma and D283 medulloblastoma cell lines, HEK293FT, primary pooled mouse embryo fibroblasts (MEFs). Results: Using prediction algorithms we found that PID1 was a likely phosphorylation target of a member of the G protein-coupled receptor kinase (GRK) family. In vitro kinase assays coupled with phosphoproteomic analysis confirmed this phosphorylation. Overexpressed HA-PID1 and this GRK could be reciprocally co-immunoprecipitated in 293FT cells and endogenous GRK was co-IPd by endogenous PID1 in CHLA-07-BSGBM. A kinase-dead GRK mutant co-IP'd with PID1 similar to the wildtype GRK indicating that the GRK-PID1 interaction did not require the kinase function of this GRK. Deletion mutants of PID1 differentially bound the GRK in co-IPs in a manner that corresponded to the differential growth inhibition these PID1 deletion mutants exerted in glioblastoma and medulloblastoma cell lines. Functionally, siRNA silencing of this GRK caused apoptosis (annexin-PI positive cells and PARP-1 cleavage) in Pid1wt MEFs suggesting that this GRK mediates survival in MEFs. Apoptosis by the GRK siRNA was mitigated in Pid1ko compared to Pid1wt MEFs, suggesting that PID1 functions downstream of the GRK and negatively regulates this GRK's pro-survival signaling. Importantly, we found similar results in glioblastoma cells using gain/loss of function of the two proteins. Conclusions: We found that PID1 is phosphorylated by a member of the GRK family and interacts with it, affecting this GRK's pro-survival activity. Experiments are now uncovering the molecular ordering of PID1 and this GRK in mediating the growth-suppressive effect of PID1 in brain tumors. Citation Format: Anup S. Pathania, Xiuhai Ren, Min Mahdi, Eslam Nouri Nigjeh, Yang Fu, Ebrahim Zandi, Xiaojiang Chen, Gregory M. Shackleford, Anat Erdreich-Epstein. PID1, a novel brain tumor growth suppressor, is a target for phosphorylation by a newly identified binding partner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3461.

MutT homolog 1 counteracts the effect of anti-neoplastic treatments in adult and pediatric glioblastoma cells

Glioblastoma, a fatal disease in both adult and pediatric patients, currently has limited treatment options that offer no more than temporary relief. Our experiments with adult and pediatric glioblastoma cell lines showed that radiation induces a dose-dependent increase in the level of MutT homolog 1 (MTH1) - an enzyme that hydrolyzes oxidized purine nucleoside triphosphates. Similarly, the combination of vorinostat, which is a histone deacetylase inhibitor, and ABT-888, which is a PARP-1 inhibitor, enhanced clonogenic death and increased the MTH1 level, relative to each treatment alone. This result suggests that the MTH1 level is directly related to the damage that is inflicted upon the cells, and its activity protects them against anti-neoplastic therapy. Indeed, the MTH1 inhibitor TH588 and MTH1 siRNA increased glioblastoma's response to both radiation and the combination of vorinostat and ABT-888. TH588 also inhibited glioblastoma's capacity for migration and invasion. In normal fibroblasts, low radiation doses and the combination of vorinostat and ABT-888 decreased the level of the enzyme. TH588 did not alter the fibroblasts’ response to radiation and only mildly affected their response to the combination of vorinostat and ABT-888.In summary, the inhibition of MTH1 is required to better realize the therapeutic potential of anti-neoplastic treatments in glioblastoma.

Histone H3.3 G34 Mutations Alter Histone H3K36 and H3K27 Methylation In Cis

Histone H3 encoding genes, particularly H3F3A and H3F3B, the genes encoding the variant histone H3.3, are mutated at high frequency in pediatric brain and bone malignancies. Compared to the extensive studies on K27M and K36M mutations, little is known about the mechanism of G34 mutations found in pediatric glioblastoma or giant cell tumors of the bone. Here we report that unlike the K27M or K36M that affect global histone methylation, the giant cell tumors of the bone G34 mutations (G34L/W) only affect histone H3K36 and H3K27 methylation on the same mutated histone tails (in cis), a mechanism distinct from known histone mutations.

Pediatric glioblastoma cells inhibit neurogenesis and promote astrogenesis, phenotypic transformation and migration of human neural progenitor cells within cocultures

Neural progenitor cell (NPC) fate is influenced by a variety of biological cues elicited from the surrounding microenvironment and recent studies suggest their possible role in pediatric glioblastoma multiforme (GBM) development. Since a few GBM cells also display NPC characteristics, it is not clear whether NPCs transform to tumor cell phenotype leading to the onset of GBM formation, or NPCs migrate to developing tumor sites in response to paracrine signaling from GBM cells. Elucidating the paracrine interactions between GBM cells and NPCs in vivo is challenging due to the inherent complexity of the CNS. Here, we investigated the interactions between human NPCs (ReNcell) and human pediatric GBM-derived cells (SJ-GBM2) using a Transwell® coculture setup to assess the effects of GBM cells on ReNcells (cytokine and chemokine release, viability, phenotype, differentiation, migration). Standalone ReNcell or GBM cultures served as controls. Qualitative and quantitative results from ELISA®, Live/Dead® and BrdU assays, immunofluorescence labeling, western blot analysis, and scratch test suggests that although ReNcell viability remained unaffected in the presence of pediatric GBM cells, their morphology, phenotype, differentiation patterns, neurite outgrowth, migration patterns (average speed, distance, number of cells) and GSK-3β expression were significantly influenced. The cumulative distance migrated by the cells in each condition was fit to Furth's formula, derived formally from Ornstein-Uhlenbeck process. ReNcell differentiation into neural lineage was compromised and astrogenesis promoted within cocultures. Such coculture platform could be extended to identify the specific molecules contributing to the observed phenomena, to investigate whether NPCs could be transplanted to replace lesions of excised tumor sites, and to elucidate the underlying molecular pathways involved in GBM-NPC interactions within the tumor microenvironment.

Selective BCL-XL inhibition promotes apoptosis in combination with MLN8237 in medulloblastoma and pediatric glioblastoma cells

CNS tumors, including medulloblastoma and pediatric glioblastoma (pGBM) account for the majority of solid pediatric malignancies. There remains an unmet need to identify novel treatment approaches in poor prognosis and relapsed pediatric brain tumors, where therapeutic options are limited. Small-molecule B-cell lymphoma 2 (BCL-2) family inhibitors may enhance tumor cell killing when combined with conventional and targeted chemotherapeutic agents. We investigated the effect of disrupting BCL-2 and B cell lymphoma-extra large (BCL-XL) protein function using ABT-263, ABT-199 and WEHI-539 in medulloblastoma and pGBM cells following treatment with MLN8237, an Aurora kinase inhibitor under investigation as a novel agent for the treatment of malignant brain tumors. Tumor cell growth and viability were determined by MTT/WST-1 assays and flow cytometry. Effects on cell phenotype, cell cycle progression, and ploidy were determined by live cell imaging and DNA content analysis. Apoptosis was determined by annexin V/propidium iodide staining and time-lapse microscopy and confirmed by measuring caspase-3/7 activity and western blotting and by short interfering RNA (siRNA) knockdown of BCL-2 associated X protein/BCL-2 antagonist killer (BAX/BAK). ABT-263, in combination with MLN8237, reduced mitotic slippage and polyploidy and promoted the elimination of mitotically defective cells via a BAX/BAK-dependent, caspase-mediated apoptotic pathway. The BCL-XL antagonist, WEHI-539, significantly augmented tumor cell killing when used in combination with MLN8237, as well as sensitized resistant brain tumor cells to a novel BAX activator, SMBA1. In addition, siRNA-mediated knockdown of BCL-XL sensitized pGBM and medulloblastoma cells to MLN8237 and mimicked the effect of combination drug treatment. Selective small-molecule inhibitors of BCL-XL may enhance the efficacy of MLN8237 and other targeted chemotherapeutic agents.

In vitro nuclear magnetic resonance spectroscopy metabolic biomarkers for the combination of temozolomide with PI3K inhibition in paediatric glioblastoma cells

Recent experimental data showed that the PI3K pathway contributes to resistance to temozolomide (TMZ) in paediatric glioblastoma and that this effect is reversed by combination treatment of TMZ with a PI3K inhibitor. Our aim is to assess whether this combination results in metabolic changes that are detectable by nuclear magnetic resonance (NMR) spectroscopy, potentially providing metabolic biomarkers for PI3K inhibition and TMZ combination treatment. Using two genetically distinct paediatric glioblastoma cell lines, SF188 and KNS42, in vitro 1H-NMR analysis following treatment with the dual pan-Class I PI3K/mTOR inhibitor PI-103 resulted in a decrease in lactate and phosphocholine (PC) levels (P<0.02) relative to control. In contrast, treatment with TMZ caused an increase in glycerolphosphocholine (GPC) levels (P≤0.05). Combination of PI-103 with TMZ showed metabolic effects of both agents including a decrease in the levels of lactate and PC (P<0.02) while an increase in GPC (P<0.05). We also report a decrease in the protein expression levels of HK2, LDHA and CHKA providing likely mechanisms for the depletion of lactate and PC, respectively. Our results show that our in vitro NMR-detected changes in lactate and choline metabolites may have potential as non-invasive biomarkers for monitoring response to combination of PI3K/mTOR inhibitors with TMZ during clinical trials in children with glioblastoma, subject to further in vivo validation.

Abstract 1932: Mutations in ATRX increase genetic instability and sensitivity to PARP inhibitors in paediatric glioblastoma cells

Abstract Paediatric glioblastomas (pGBM) are amongst the most common causes of cancer-related deaths in children, and are defined by highly recurrent mutations in H3 histones. Mutations affecting the chromatin remodeling protein ATRX have been reported in 30% of pGBM cases, and are strongly associated with the alternative lengthening of telomeres (ALT) phenotype, but their precise interaction with histone mutations and their role in tumorigenesis remain unclear. We collected sequence data from 262 published and 64 unpublished cases of pGBM and identified somatic ATRX mutations in 54/326 (17%) of cases. ATRX mutations are mainly loss of function mutations, with the majority of frameshift mutations (37/54, 68,5%) found upstream of the helicase domain resulting in truncation of the main functional domain of ATRX. Missense mutations (16/54, 29,6%) reside almost exclusively in the helicase domain (11/54, 20,4%), whereas nonsense mutations are a less common event (7/54, 13%) but present in both the helicase (4/7, 57,1%) and ADD domains (3/7, 42,9%). ATRX mutations commonly co-segregate with H3.3 G34 (16/54) and TP53 (42/54) mutations, and define a subgroup of patients with a longer overall survival (16 months median overall survival in mutant ATRX cases versus 11 months in wild-type ATRX cases, COXPH p = 0.079), though with a greater number of somatic mutations (MWU p = 0.023) and copy number alterations (MWU p = 0.0011) than wild-type cases. We screened a series of 21 primary patient-derived pGBM cell cultures for histone and ATRX mutation status in addition to ATRX protein expression and ALT, and subjected the panel to a high-throughput in vitro cell viability screen of &amp;gt;400 chemotherapeutics and small molecules. We identified a specific genetic dependency for ATRX mutation and sensitivity to distinct PARP inhibitor chemotypes, including olaparib and rucaparib (PARP catalytic inhibitors), and talazoparib (PARP trapper inhibitor). These data were validated using CRISPR-Cas9-engineered ATRX knockout, targeting either the ADD or helicase domain, in SF188 pGBM cells. Gene editing was confirmed by IonTorrent sequencing and Western blot. ATRX mutant clones were also more sensitive to drugs targeting DNA damage response pathways such as bleomycin and sapacitabine. Gene expression analysis of ATRX mutant pGBM samples confirmed an intact homologous recombination pathway and overexpression of PARP1, suggesting an underlying mechanism distinct from that observed in BRCA-mutant breast and ovarian cancers. Ongoing work is aimed at unravelling the specific pathways involved, and evaluating the utility of PARP inhibition in orthotopic pGBM xenografts in vivo. These data suggest a synthetic lethality for PARP inhibitors in ATRX-deficient pGBM cells, and may represent a novel therapeutic strategy for these highly aggressive tumours in children. Citation Format: Janat Fazal-Salom, Mara Vinci, Diana Carvalho, Helen Pemberton, Stephen J. Pettitt, Christopher J. Lord, Alan Mackay, Lynn Bjerke, Chris Jones. Mutations in ATRX increase genetic instability and sensitivity to PARP inhibitors in paediatric glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1932. doi:10.1158/1538-7445.AM2017-1932

TMOD-09. A PATIENT-DERIVED XENOGRAFT MODEL MIMICKING RECURRENT PEDIATRIC MALIGNANT GLIOMA

Abstract BACKGROUND: Pediatric high grade glioma (pHGG) is a devastating cancer of the central nervous system. The prognosis of this most malignant form of brain tumor remains poor, largely due to the inherent resistance of pHGG to therapy, and inevitably leads to recurrence. Unfortunately, because recurrent glioma, or glioma remaining after conventional therapy, frequently has very different molecular characteristics from the original tumor in addition to a vastly different biological behavior, recurrent pHGG has proven exceedingly difficult to treat, with very few treatment options providing clinically meaningful outcomes. As part of the NCI Pediatric Preclinical Testing Program, several pediatric brain cancer cell lines, including the ones used in this study, were developed and characterized. However, to our knowledge, none have been utilized in development of a rat xenograft model of recurrent pHGG. METHODS: Pediatric glioblastoma cells (SJ-GBM2) were obtained from the Children’s Oncology Group Cell Culture and Xenograft Repository. Irradiated SJGBM2-10gy cells were generated by exposure to a total radiation dose of 10 Gy. Following irradiation, the resultant SJGBM2-10gy cells were tested for cell viability and allowed to reach confluence prior to stereotactical implantation into the right striatum of male athymic rats. RESULTS: Less than 1% of total cells survived radiation. Both irradiated and parental cell lines formed tumors in 100% of the animals following intracranial implantation. Irradiated SJGBM2-10gy cells exhibited a more aggressive tumor growth than the parental SJ-GBM2 cell line, resulting in mortality on average 14 days earlier. Histopathological analysis is underway and more advanced imaging studies are planned. CONCLUSION: To our knowledge this is the first report of an intracranial rat xenograft model mimicking recurrent pHGG using irradiated cell lines. It represents a robust and expedient in vivo model for studying recurrent pHGG biology.

Pediatric and adult glioblastoma radiosensitization induced by PI3K/mTOR inhibition causes early metabolic alterations detected by nuclear magnetic resonance spectroscopy.

Poor outcome for patients with glioblastomas is often associated with radioresistance. PI3K/mTOR pathway deregulation has been correlated with radioresistance; therefore, PI3K/mTOR inhibition could render tumors radiosensitive. In this study, we show that NVP-BEZ235, a dual PI3K/mTOR inhibitor, potentiates the effects of irradiation in both adult and pediatric glioblastoma cell lines, resulting in early metabolic changes detected by nuclear magnetic resonance (NMR) spectroscopy. NVP-BEZ235 radiosensitises cells to X ray exposure, inducing cell death through the inhibition of CDC25A and the activation of p21cip1(CDKN1A). Lactate and phosphocholine levels, increased with radiation, are decreased after NVP-BEZ235 and combination treatment, suggesting that inhibiting the PI3K/mTOR pathway reverses radiation induced metabolic changes. Importantly, NVP-BEZ235 potentiates the effects of irradiation in a xenograft model of adult glioblastoma, where we observed a decrease in lactate and phosphocholine levels after seven days of combination treatment. Although tumor size was not affected due to the short length of the treatment, a significant increase in CASP3 mRNA was observed in the combination group. Taken together, our data suggest that NMR metabolites could be used as biomarkers to detect an early response to combination therapy with PI3K/mTOR inhibitors and radiotherapy in adult and pediatric glioblastoma patients.

Penfluridol suppresses glioblastoma tumor growth by Akt-mediated inhibition of GLI1.

Glioblastoma (GBM) is the most common brain tumor with poor survival rate. Our results show that penfluridol, an antipsychotic drug significantly reduced the survival of ten adult and pediatric glioblastoma cell lines with IC50 ranging 2–5 μM after 72 hours of treatment and induced apoptosis. Penfluridol treatment suppressed the phosphorylation of Akt at Ser473 and reduced the expression of GLI1, OCT4, Nanog and Sox2 in several glioblastoma cell lines in a concentration-dependent manner. Inhibiting Akt with LY294002 and siRNA, or inhibiting GLI1 using GANT61, cyclopamine, siRNA and CRISPR/Cas9 resulted in enhanced cell growth suppressive effects of penfluridol. On the other hand, overexpression of GLI1 significantly attenuated the effects of penfluridol. Our results further demonstrated that penfluridol treatment inhibited the growth of U87MG tumors by 65% and 72% in subcutaneous and intracranial in vivo glioblastoma tumor models respectively. Immunohistochemical and western blot analysis of tumors revealed reduced pAkt (Ser 473), GLI1, OCT4 and increase in caspase-3 cleavage and TUNEL staining, confirming in vitro findings. Taken together, our results indicate that overall glioblastoma tumor growth suppression by penfluridol was associated with Akt-mediated inhibition of GLI1.

EXTH-36. SCREENING COMPOUNDS THAT TARGET THE DNA DAMAGE RESPONSE FOR BLOOD-BRAIN-BARRIER PERMEABILITY

For glioblastoma (GBM), development of strategies that stabilize disease progression and ultimately achieve a cure will require comprehensive approaches towards target selection and design of therapies. Activation of the DNA damage response (DDR) by temozolomide and irradiation induces signaling pathways with actionable therapeutic targets (Akt, Chk1, and Mdm2), providing fertile ground to develop novel anti-GBM combination therapies. Using pediatric and adult GBM cells with clinically relevant molecular profiles, isobologram analyses indicated that combinations of small molecule inhibitors to these DDR-induced targets can synergistically block in vitro cell growth and decrease resistance of GBM cells to temozolomide. Knowing the degree of blood-brain-barrier (BBB) permeability is particularly important in the selection of therapeutic compounds for the treatment of GBM. To this end, we adapted the Mouse Brain Uptake Assay (MBUA), which determines the BBB apparent permeability (Papp, nm/sec) for drugs across an intact BBB with the assumption that uptake rate is an important contributor to brain concentrations. When Papp is plotted as a function of cLogD and compared with calibration compounds, specific mechanistic interpretation is possible. Optimal BBB Papp values fall within the region of brain uptake limited only by cerebral blood flow, and not limited by low passive permeability or active efflux. Inhibitors of interest have been analyzed using the MBUA. BBB permeability, as measured by Papp, was in the order RG7388>RG7112>nutlin3a for MDM2 inhibitors and CHIR124>AZD7762 for Chk1 inhibitors. Survival in an intracranial GBM xenograft model indicated that an intermittent dosing regimen of temozolomide with Akt (GDC0068) and Mdm2 (RG7112) inhibitors exhibiting moderate BBB permeability significantly reduced GBM growth with minimal toxicity. Studies are in progress to determine if the next-generation Mdm2 inhibitor RG7388 with a more favorable BBB Papp can further improve efficacy of combination therapy and offer new therapeutic options for GBM.

The histone deacetylase inhibitor PCI-24781 as a putative radiosensitizer in pediatric glioblastoma cell lines.

BackgroundGlioblastoma (GBM) is considered to be one of the most aggressive tumors of the central nervous system (CNS). Even with the use of modern treatment protocols, the prognosis remains reserved, with children with GBM having a mean survival of 12–15 months.MethodsIn the present study we investigated the potential radiosensitizing effect of PCI-24781, a potent pan-histone deacetylase inhibitor (HDACi), on the SF188 and KNS42 cell lines of pediatric GBM. Cell proliferation rates, clonogenicity and apoptosis were compared in the presence and absence of treatment with PCI-24781. We also compared the clonogenicity rates of the irradiated SF188 and KNS42 cell lines with or without previous treatment with PCI-24781 at the doses of 0.25–16 μM. In addition, we investigated the effects of PCI-24781 on the expression of some of the main proteins responsible for the repair of double-strand DNA breaks caused by irradiation.ResultsThe inhibitor blocked cell proliferation, induced death by apoptosis and reduced the colony forming capacity of the cell lines, both of them showing a significant decrease of colony formation at all irradiation doses. The expression of the Rad51 protein, important for the homologous recombination (HR) repair pathway, and of the DNA-PKcs, Ku70 and Ku86 proteins, important for the non-homologous end joining (NHEJ) repair pathway, was more reduced when the irradiated cell line was previously treated with PCI-24781 than when it was treated exclusively with radiotherapy.ConclusionsThese findings demonstrate that HDACi PCI-24781 has a radiosensitizing profile that compromises the repair of double-strand DNA breaks in cells of pediatric GBM treated with radiotherapy.

PO46NANOPARTICLE-ASSOCIATED CELL PENETRATING AND CYTOTOXIC PEPTIDES AS A NOVEL THERAPEUTIC STRATEGY FOR MALIGNANT GLIOMA

INTRODUCTION: Local neurosurgically-applied drug-delivery systems offer an opportunity to target glioblastoma (GBM) residual disease, but consideration must be given to the uptake of delivered agents once diffused from a drug-delivery depot. We assess the glycosaminoglycan-binding enhanced transduction of nanoparticles coated with genetically-engineered peptides, as a direct corollary to biomaterial-based localised drug-delivery systems. METHOD: GBM cells were transduced with biocompatible iron oxide magnetic nanoparticles (dMNP) coated with membrane docking (P21) and cell penetrating (8R) peptides. Protein transduction efficiency was determined by cytochemical staining and metabolic assays. To test the efficacy of the cytotoxic peptide SHEPHERDIN (SHEP), which disrupts the interaction of Survivin with HSP 90, GBM cells were exposed to acute doses of dMNP-P21-8R-SHEP and effects on metabolism measured. RESULTS: Nuclear and cytoplasmic protein transduction of dMNP-8R-P21 was evident in 100% of adult and paediatric GBM cells after 3h exposure, whereas after 16h, only 25% of uncoated nanoparticles traverse the cellular membrane by direct translocation and/or endocytosis. Protein-mediated transduction of dMNP-P21-8R-SHEP results in dose-dependent proliferation impairment of adult and paediatric GBM cells (IC50 25 µM and 20 µM respectively). CONCLUSION: Protein-mediated cellular transduction via membrane docking and cell penetrating peptides has the potential to deliver anti-cancer cargos to brain tumours and diversifies nanoparticle functionality. Early findings characterising SHEP-mediated GBM viability loss and controlled release of dMNP-P21-8R-SHEP from a polymeric microparticle drug-delivery matrix will be discussed.

HG-09 * INFANTILE GLIOBLASTOMA WITH SARCOMATOUS HISTOLOGY DRIVEN BY ETV6:NTRK3 FUSIONS ARE SENSITIVE TO TRK INHIBITION BY PHA-848125

Approximately 1% of all paediatric brain tumours occur at less than one year of age, and glioblastoma (GBM) account for only 2-9% of these. Their clinical presentation, location, histology, behaviour, response to therapy and outcome differ from GBMs of older children and adults, and they do not appear to be associated with known histone H3 or IDH1-driven subgroups. Recent genome and RNA sequencing data highlighted recurrent NTRK1-3 fusions in 4/10 non-brainstem GBM under the age of 3 years, including one case of ETV6-NTRK3 in a cerebral hemispheric tumour diagnosed at 1 month old. These fusions are usually associated with congenital mesoblastic nephroma and congenital infantile fibrosarcoma, and upon review of a series of cases diagnosed as glioblastoma under the age of 1yr, 6/14 were found to present with a predominant spindle cell morphology, a relatively circumscribed interface with adjacent brain, and without prominent infiltration. These tumours were positive for GFAP as well as desmin, CD56 and CD99. One case was diagnosed in utero by ultrasound, and at birth underwent surgical resection from which primary cell cultures were established under stem cell conditions on laminin-coated plates. Exome and RNA sequencing revealed the presence of the ETV6:NTRK3 fusion transcript as the sole somatic alteration, validated by RT-PCR, Sanger sequencing and FISH. 450K methylation profiling clustered the tumour with desmoplastic infantile ganglioglioma rather than subgroups of paediatric GBM. Primary cells treated with the CNS-penetrant small molecule Trk inhibitor PHA-848125, resulted in marked sensitivity (GI50 = 200nM) compared to cerebral hemispheric (H3 wild-type) and thalamic (H3.3K27M) paediatric GBM cells (GI50 = 1.8-4.6mM). A subset of infantile/congenital brain tumours classified as GBM appear to represent a distinct entity more closely related, biologically and histologically, to mesenchymal cancers. Agents targeting ETV:NTRK3 may be a useful novel treatment strategy in these children.

Abstract B15: Pediatric glioblastoma cell lines maintain response to developmental growth factors

Abstract Glioblastoma (GBM) is a highly invasive brain tumor arising from an astrocytic cell lineage. In the United States, nearly 500 children each year develop GBMs. Pediatric GBM has a different mutational, methylation, and gene expression spectra than adult GBM, implicating a strong role for developmental processes in disease pathogenesis. The developmental growth factors that define the astrocytic lineage include epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Expression of these growth factors also distinguishes pediatric versus adult GBM. Here, we use EGF or PDGF as a stimulus for malignant behavior in GBM cell lines isolated from either adult or pediatric patients to understand whether the cell line models parallel growth factor receptor signaling preferences observed clinically. In response to these stimuli, pediatric cell lines demonstrate distinct protein signaling and invasion phenotypes from adult cell lines in vitro, and generate invasive, multifocal lesions in an orthotopic xenograft model. Thus, distinguishing features of adult and pediatric GBM are maintained in the cell line models that will be used to test therapeutic regiments preclinically. Both of the receptors activated by EGF and PDGF are known to interact with a promising therapeutic target in adult glioblastoma: signal transducer and activator 3 (STAT3). STAT3 acts as either an oncogene or tumor suppressor depending on cellular context such as the activating receptor complex and phosphatase and tensin homolog (PTEN) mutational status. Our studies will therefore address the differential implications of STAT3 therapy for pediatric versus adult GBM. Citation Format: Marie Renee Mooney. Pediatric glioblastoma cell lines maintain response to developmental growth factors. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B15.

Abstract 2451: Insulin-like growth factor-1 receptor (IGF-1R) inhibitors downregulate p53 expression and upregulate the Warburg effect in paediatric glioblastoma cells

Abstract Introduction: Previously we showed that Picropodophyllin (PPP) - an inhibitor of Insulin-Like Growth Factor-1 Receptor (IGF-1R) promotes the Warburg effect and this effect is associated with the downregulation of p53 after 24h of PPP-treatment. Activation of IKK-NF-κB, by loss of p53, has been reported to increase glycolysis in cancer (1). Aims: To understand the molecular mechanism that links p53 downregulation with increased glycolysis after PPP treatment, we studied the changes in glucose metabolism in 3 different IGF-1R inhibitors - PPP, OSI906 and NVP-AEW541 (NVP) and related these changes to IKK-NF-κB expression and other molecular markers. Methods: KNS42 cells were treated for 24 hours with 5xIC50 of either PPP (2.4µM), OSI906 (220µM) or NVP (40µM). Culture media from each treated group and controls were analyzed by 1H-MRS. Lactate dehydrogenase-A (LDH-A), p53, pAKT, pS6, MCT-1, MCT-4, Glut-1, pIKK and β-actin (loading control) were examined by western blot (WB). LDH enzyme activity was also measured in PPP- and NVP-treated cells. Results: After 24 hours of OSI906, NVP and PPP treatment, decreases of 30% (p&amp;lt;0.01), 30% (p&amp;lt;0.01) and 50% (p&amp;lt;0.001) in cell number were found respectively, when compared with their respective controls. 1H-MRS analysis showed increases in glucose uptake (p&amp;lt;0.01) and lactate excretion (p&amp;lt;0.0001) in cells treated with the 3 IGF-1R inhibitors when compared with their corresponding controls. Larger increases were observed in the PPP-treated group when compared to the other 2 groups. In all 3 treatments, WB showed decreases in p53 and pAkt, an increase in Glut-1 and no change in MCT-1 expression. PPP-treated cells showed an increase in pS6 but a decreased pS6 expression was shown in OSI906- and NVP-treated cells. MCT-4 and pIKK expressions were higher in OSI906- and NVP-treated cells, but unchanged in the PPP-treated cells. LDH enzymatic activities were found to be significantly increased in PPP (p&amp;lt;0.01) and NVP (p&amp;lt;0.0001) treated groups, when compared with controls. Discussions and Conclusion: Our data indicated that the observed increased Warburg effect after IGF-1R inhibition is associated with increased Glut-1 expression, LDH activity and downregulation of p53 but the molecular mechanism is different in PPP when compared to OSI906 and NVP-treated cells. The increased Warburg effect after OSI906 and NVP treatment may be driven by the activation of the IKK-NF-κB pathway following the downregulation of p53, whereas upregulation of pS6 was seen in the PPP-treated cells. The activation of the mTOR-S6K pathway has been linked with the reduction of p53 expression (2). Further investigation is needed to examine why KNS42 cells have a different molecular response to PPP compared with OSI906 and NVP treatment. 1) Kawauchi et al., Nature Cell Biology 30(9): 1792-5, 2007. 2) Lai et al., EMBO 29: 2994-3006, 2010. Citation Format: Anne-Christine Wong Te Fong, Gabriela Andrejeva, Aleksandra Bielen, Chris Jones, John Griffiths, Martin O. Leach, Yuen-Li Chung. Insulin-like growth factor-1 receptor (IGF-1R) inhibitors downregulate p53 expression and upregulate the Warburg effect in paediatric glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2451. doi:10.1158/1538-7445.AM2014-2451

Lactate and choline metabolites detected in vitro by nuclear magnetic resonance spectroscopy are potential metabolic biomarkers for PI3K inhibition in pediatric glioblastoma.

The phosphoinositide 3-kinase (PI3K) pathway is believed to be of key importance in pediatric glioblastoma. Novel inhibitors of the PI3K pathway are being developed and are entering clinical trials. Our aim is to identify potential non-invasive biomarkers of PI3K signaling pathway inhibition in pediatric glioblastoma using in vitro nuclear magnetic resonance (NMR) spectroscopy, to aid identification of target inhibition and therapeutic response in early phase clinical trials of PI3K inhibitors in childhood cancer. Treatment of SF188 and KNS42 human pediatric glioblastoma cell lines with the dual pan-Class I PI3K/mTOR inhibitor PI-103, inhibited the PI3K signaling pathway and resulted in a decrease in phosphocholine (PC), total choline (tCho) and lactate levels (p<0.02) as detected by phosphorus (31P)- and proton (1H)-NMR. Similar changes were also detected using the pan–Class I PI3K inhibitor GDC-0941 which lacks significant mTOR activity and is entering Phase II clinical trials. In contrast, the DNA damaging agent temozolomide (TMZ), which is used as current frontline therapy in the treatment of glioblastoma postoperatively (in combination with radiotherapy), increased PC, glycerophosphocholine (GPC) and tCho levels (p<0.04). PI-103-induced NMR changes were associated with alterations in protein expression levels of regulatory enzymes involved in glucose and choline metabolism including GLUT1, HK2, LDHA and CHKA. Our results show that by using NMR we can detect distinct biomarkers following PI3K pathway inhibition compared to treatment with the DNA-damaging anti-cancer agent TMZ. This is the first study reporting that lactate and choline metabolites are potential non-invasive biomarkers for monitoring response to PI3K pathway inhibitors in pediatric glioblastoma.

HISTONE DEACETYLASE INHIBITOR PCI-24781 SHOWS ANTIPROLIFERATIVE EFFECTS AGAINST PEDIATRIC GLIOBLASTOMA CELLS

BACKGROUND: Glioblastoma (GBM) is one of the most aggressive tumors affecting the central nervous system (CNS). Despite combined aggressive treatment schedules, the prognosis of children with GBM remains very poor. Epigenetic mechanisms may interfere with the process of carcinogenesis, and the acetylation of DNA can modulate the expression of genes related to the development and progression of malignancies. Clinical studies have shown that histone deacetylases inhibitors (HDACi are clinically active and well tolerated in the treatment of several tumors. Therefore, this study aims to evaluate the therapeutic potential of HDACi PCI-24781 in pediatric GBM lines, SF188 and KNS42 by proliferation assay. METHODS: Pediatric GMB cell-lines were cultured in HAM-F10 supplemented with 10% fetal bovine serum, at 37°C in a humidified 5% CO2 incubator. Cells were seeded in 96-well plates at 2x103 per well. After 24h, cells were treated with PCI-24781 at different concentrations (0.5, 1, 2, 4, 8 and 16 µM for 24, 48, 72 and 96h). Culture medium was removed and replaced with culture medium containing 5 µL of Resazurin dye in each well. Cells were incubated for 6h at 37°C, and the product was measured at 570 nm and 595 nm using a microplate reader. Statistical analysis was performed by One- and Two-way ANOVA and Bonferoni post-hoc. RESULTS: Both cell-lines were sensitive to PCI-24781 treatment, demonstrating an inhibition of proliferation (P < 0.05). For the SF188 cell-line, the strongest effect was observed at the doses of 4 µM, 8 µM and 16 µM at 96h (growth inhibition of 93%). The KNS42 cell-line showed a time-dependent antiproliferative pattern; the best effect was observed at 96h, when growth inhibition was of approximately 84%. It was observed time effect difference of proliferation between 24h and 48h (P < 0.05), but not between 48 h and 72 h. CONCLUSIONS: These primary data demonstrates that PCI-24781 can induce the inhibition of cell proliferation in pediatric GBM cells. Further experiments will be performed to assess the ability of this HDACi in modulating the cellular response in combinatin to ionizing radiation. Financial Support: FAPESP (process no. 2013/15891-8). SECONDARY CATEGORY: Preclinical Experimental Therapeutics.