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Background Supratentorial RELA fusion (ST-RELA) ependymomas (EPNs) are resistant tumors without an approved chemotherapeutic treatment. Unfortunately, the molecular mechanisms that lead to chemoresistance traits of ST-RELA remain elusive. The aim of this study was to assess RELA fusion-dependent signaling modules, specifically the role of the Hedgehog (Hh) pathway as a novel targetable vulnerability in ST-RELA. Methods Gene expression was analyzed in EPN from patient cohorts, by microarray, RNA-seq, qRT-PCR, and scRNA-seq. Inhibitors against Smoothened (SMO) (Sonidegib) and Aurora kinase A (AURKA) (Alisertib) were evaluated. Protein expression, primary cilia formation, and drug effects were assessed by immunoblot, immunofluorescence, and immunohistochemistry. Results Hh components were selectively overexpressed in EPNs induced by the RELA fusion. Single-cell analysis showed that the Hh signature was primarily confined to undifferentiated, stem-like cell subpopulations. Sonidegib exhibited potent growth-inhibitory effects on ST-RELA cells, suggesting a key role in active Hh signaling; importantly, the effect of Sonidegib was reversed by primary cilia loss. We, thus, tested the effect of AURKA inhibition by Alisertib, to induce cilia stabilization/reassembly. Strikingly, Alisertib rescued ciliogenesis and synergized with Sonidegib in killing ST-RELA cells. Using a xenograft model, we show that cilia loss is a mechanism for acquiring resistance to the inhibitory effect of Sonidegib. However, Alisertib fails to rescue cilia and highlights the need for other strategies to promote cilia reassembly, for treating ST-RELA tumors. Conclusion Our study reveals a crucial role for the Hh pathway in ST-RELA tumor growth, and suggests that rescue of primary cilia represents a vulnerability of the ST-RELA EPNs.

Background Medulloblastoma (MB) is the most common malignant paediatric brain tumour and a leading cause of cancer-related mortality and morbidity. Existing treatment protocols are aggressive in nature resulting in significant neurological, intellectual and physical disabilities for the children undergoing treatment. Thus, there is an urgent need for improved, targeted therapies that minimize these harmful side effects. Methods We identified candidate drugs for MB using a network-based systems-pharmacogenomics approach: based on results from a functional genomics screen, we identified a network of interactions implicated in human MB growth regulation. We then integrated drugs and their known mechanisms of action, along with gene expression data from a large collection of medulloblastoma patients to identify drugs with potential to treat MB. Results Our analyses identified drugs targeting CDK4, CDK6 and AURKA as strong candidates for MB; all of these genes are well validated as drug targets in other tumour types. We also identified non-WNT MB as a novel indication for drugs targeting TUBB, CAD, SNRPA, SLC1A5, PTPRS, P4HB and CHEK2. Based upon these analyses, we subsequently demonstrated that one of these drugs, the new microtubule stabilizing agent, ixabepilone, blocked tumour growth in vivo in mice bearing patient-derived xenograft tumours of the Sonic Hedgehog and Group 3 subtype, providing the first demonstration of its efficacy in MB. Conclusions Our findings confirm that this data-driven systems pharmacogenomics strategy is a powerful approach for the discovery and validation of novel therapeutic candidates relevant to MB treatment, and along with data validating ixabepilone in PDX models of the two most aggressive subtypes of medulloblastoma, we present the network analysis framework as a resource for the field.

Pediatric brain cancer patients treated with fractionated radiotherapy commonly develop long-term late side-effects including cognitive deficits. Many preclinical models of late effects have been developed that use a single, high dose of radiotherapy, which does not mimic the fractionated schedule children receive clinically. This study aimed to create a mouse model of late effects using clinically-relevant fractionated radiotherapy, and to measure the effects on the developing brain. Juvenile mice were treated at postnatal day 16 with a single dose of 8Gy whole brain radiation, or a mathematically-equivalent fractionated dose of 18Gy (9 x 2Gy daily fractions). Sham control mice received a CT scan, or 9 x sham CT scans. Mice were allowed to grow to young adulthood (63 days). Ex vivo anatomical MRI scans were performed along with diffusion tensor imaging (DTI) and histology. Mice receiving a single 8Gy radiation dose exhibited significantly decreased volumes in areas including the olfactory bulbs (-19%), hippocampus (-7%), corpus callosum (-9%) and motor cortex (-9%). In contrast, mice receiving fractionated radiotherapy showed fewer significantly decreased regions, although olfactory bulbs were reduced (-12%). Furthermore, doublecortin-positive cells were significantly reduced in the dentate gyrus indicating profound effects of radiotherapy on murine neural stem cells. Few radiotherapy-induced differences were observed by DTI, and immunohistochemistry revealed no changes in myelin basic protein, suggesting that white matter is minimally altered in mice. These results show that preclinical models exhibit treatment-induced late effects, and that commonly-used experimental approaches of single dose radiotherapy induce more neurological changes than an equivalent fractionated dose, thus may over-estimate radiotherapy-induced late effects. We have developed a clinically-relevant fractionated dosing protocol in mice, which replicates late effects experienced by children, and can be used to measure long-term effects of novel chemo/radiotherapy treatment combinations, ensuring children with brain cancer receive treatment both effective and safe treatment.

Background Although epidermal growth factor receptor (EGFR) and its truncated, autoactive mutant EGFR variant (v)III are bona fide drivers of tumorigenesis in some gliomas, therapeutic antibodies developed to neutralize this axis have not improved patient survival in a limited number of trials. Previous studies using cells transduced to exogenously express EGFRvIII may have compromised mechanistic studies of anti-EGFR therapeutics. Therefore, we re-assessed the activity of clinical EGFR antibodies in patient-derived gliomaspheres that endogenously express EGFRvIII. Methods The antitumor efficacy of antibodies was assessed using in vitro proliferation assays and intracranial orthografts. Receptor activation status, antibody engagement, oncogenic signaling, and mechanism of action after antibody treatment were analyzed by immunoprecipitation and western blotting. Tracking of antibody receptor complexes was conducted using immunofluorescence. Results The EGFR domain III–targeting antibodies cetuximab, necitumumab, nimotuzumab, and matuzumab did not neutralize EGFRvIII activation. Chimeric monoclonal antibody 806 (ch806) neutralized EGFRvIII, but not wild-type (wt)EGFR activation. Panitumumab was the only antibody that neutralized both EGFRvIII and wtEGFR, leading to reduction of p-S6 signaling and superior in vitro and in vivo antitumor activity. Mechanistically, panitumumab induced recycling of receptor but not degradation as previously described. Panitumumab, via its unique avidity, stably cross-linked EGFRvIII to prevent its activation, while ch806 induced a marked reduction in the active EGFRvIII disulphide-bonded dimer. Conclusions We discovered a previously unknown major resistance mechanism in glioma in that most EGFR domain III–targeting antibodies do not neutralize EGFRvIII. The superior in vitro and in vivo antitumor activity of panitumumab supports further clinical testing of this antibody against EGFRvIII-stratified glioma.

Despite treatment intensification, survival for certain high-risk subgroups of medulloblastoma (MB) such as p53 mutant SHH (SHHa) and MYC amplified Group 3 (Gr3) have remained dismal. Recently, the use of Carboplatin as radiosensitiser with craniospinal irradiation (CSI) has shown survival benefit in children with Gr3 MB. However, there is an ongoing need to discover newer, non-chemo radiosensitisers which have minimal toxicity and unequivocal benefit in high-risk MB. Radiation is a potent inducer of DNA damage in cancer cells. CHK1/2, ATR, and WEE1 kinases are critical regulators of the DNA damage response (DDR). We hypothesised that inhibition of these kinases may enhance radiation-induced cytotoxicity in MB. We utilised small-animal radiotherapy platform to deliver CSI to MB-tumour-bearing mice that closely mimic clinical settings. Here we demonstrate systematic preclinical evaluation of DDR inhibitors (DDRi) as radiosensitiser in MB models. In vitro treatment of Gr3 MB cell lines with either CHK1/2 inhibitor (CHKi, Prexasertib, AZD7762), ATR inhibitor (ATRi, AZD6738), or WEE1 inhibitor (WEE1i, AZD1775) along with radiation ceased post-radiation cell cycle arrest, prohibiting DNA repair, and increasing apoptosis. The combination also reduced the colony-forming ability of MB cells post-irradiation. In vivo, CSI and DDRi, each induced modest apoptosis in orthotopically-implanted high-risk MB mice. When these models were treated with CHKi+CSI, ATRi+CSI or WEE1i+CSI, the effect was cumulative. Importantly, addition of DDRi to CSI significantly prolonged the survival of MB-bearing mice compared to CSI alone. Here we also explored strategy of “vertical blockade” by simultaneous use of CHKi and WEE1i which when combined with CSI also prolonged survival in MB-bearing mice. Additionally, we evaluated various regimens to best deliver these novel agents with CSI, maximising effect and minimising toxicity. Our study opens up new realms of radiosensitisation with the use of DDRi in paediatric medulloblastoma and provides robust preclinical evidence for clinical translation.

Medulloblastoma (MB) consists of four core molecular subgroups with distinct clinical features and prognoses. Treatment consists of surgery, followed by radiotherapy and cytotoxic chemotherapy. Despite this intensive approach, outcome remains dismal for patients with certain subtypes of MB, namely MYC-amplified Group 3 and TP53-mutated SHH. Using high-throughput assays, six human MB cell lines were screened against a library of 3208 unique compounds. We identified 45 effective compounds from the screen and found that inhibition cell cycle checkpoint kinases (CHK1 and 2) synergistically enhanced the cytotoxic activity of clinically-used chemotherapeutics cyclophosphamide, cisplatin, and gemcitabine. To identify the best-in-class inhibitor, multiple CHK1/2 inhibitors were assessed in mice bearing intracranial MB. When combined with DNA-damaging chemotherapeutics, CHK1/2 inhibition reduced tumor burden and significantly increased survival of animals with high-risk MB, across multiple different models. In total, we tested 14 different models, representing distinct MB subgroups, and data were validated in three independent laboratories. Pharmacodynamics studies confirmed central nervous system penetration. In mice, combination treatment significantly increased DNA damage and apoptosis compared to chemotherapy alone, and studies with cultured cells showed that CHK inhibition disrupted chemotherapy-induced cell cycle arrest. Our findings indicated CHK1/2 inhibition, specifically with LY2606368 (prexasertib), has strong chemosensitizing activity in MB that warrants further clinical investigation. Moreover, these data demonstrated that we developed a robust and collaborative preclinical assessment platform that can be used to identify potentially effective new therapies for clinical evaluation for pediatric MB.

Simple Summary Radiation-induced glioma (RIG) is a highly aggressive brain cancer arising as a consequence of radiation therapy, for which there is currently no effective treatment. In order to test new drugs in the hope of finding more effective therapies, we need mouse models that faithfully replicate human RIG. Our laboratory collected tumour cells at autopsy from a paediatric patient with RIG following treatment for a different brain tumour. Using these cells, we created a mouse brain tumour model that retains all the characteristics and features of the original patient tumour from which it was derived. This unique model allowed us to study the progression of RIG in the brain, and to identify drugs that may be effective in the treatment of this disease. This mouse model will also allow us to test the efficacy of new treatments, with the hope of improving the prognosis for patients diagnosed with this disease. Abstract Radiation-induced glioma (RIG) is a highly aggressive brain cancer arising as a consequence of radiation therapy. We report a case of RIG that arose in the brain stem following treatment for paediatric medulloblastoma, and the development and characterisation of a matched orthotopic patient-derived xenograft (PDX) model (TK-RIG915). Patient and PDX tumours were analysed using DNA methylation profiling, whole genome sequencing (WGS) and RNA sequencing. While initially thought to be a diffuse intrinsic pontine glioma (DIPG) based on disease location, results from methylation profiling and WGS were not consistent with this diagnosis. Furthermore, clustering analyses based on RNA expression suggested the tumours were distinct from primary DIPG. Additional gene expression analysis demonstrated concordance with a published RIG expression profile. Multiple genetic alterations that enhance PI3K/AKT and Ras/Raf/MEK/ERK signalling were discovered in TK-RIG915 including an activating mutation in PIK3CA, upregulation of PDGFRA and AKT2, inactivating mutations in NF1, and a gain-of-function mutation in PTPN11. Additionally, deletion of CDKN2A/B, increased IDH1 expression, and decreased ARID1A expression were observed. Detection of phosphorylated S6, 4EBP1 and ERK via immunohistochemistry confirmed PI3K pathway and ERK activation. Here, we report one of the first PDX models for RIG, which recapitulates the patient disease and is molecularly distinct from primary brain stem glioma. Genetic interrogation of this model has enabled the identification of potential therapeutic vulnerabilities in this currently incurable disease.

Background Medulloblastoma (MB) is the most common malignant paediatric brain tumour and a leading cause of cancer-related mortality and morbidity. Existing treatment protocols are aggressive in nature resulting in significant neurological, intellectual and physical disabilities for the children undergoing treatment. Clearly, there is an urgent need for improved, targeted therapies that minimize these harmful side effects. Methods We identified candidate drugs for MB using a network-based systems-pharmacogenomics approach: based on results from a functional genomics screen, we identified a network of interactions implicated in human MB growth regulation. We then integrated drugs and their known mechanisms of action, along with gene expression data from a large collection of medulloblastoma patients to identify drugs with potential to treat MB. Results Our analyses identified drugs targeting CDK4, CDK6, and AURKA as strong candidates for MB; all of these genes are well validated as drug targets in other tumour types. We also identified non-WNT MB as a novel indication for drugs targeting TUBB, CAD, SNRPA, SLC1A5, PTPRS, P4HB and CHEK2. Based upon these analyses we subsequently demonstrated that one of these drugs, the new microtubule stabilizing agent, ixabepilone, blocked tumour growth in vivo in mice bearing Sonic Hedgehog and Group 3 patient-derived xenograft tumours, providing the first demonstration of its efficacy in MB. Conclusions Our findings confirm that this data-driven systems pharmacogenomics strategy is a powerful approach for the discovery and validation of novel therapeutic candidates relevant to MB treatment, and along with data validating ixabepilone in PDX models of the two most aggressive subtypes of medulloblastoma, we present the network analysis framework as a resource for the field.

Children with medulloblastoma and ependymoma are treated with a multidisciplinary approach that incorporates surgery, radiotherapy, and chemotherapy, however, overall survival rates for patients with high-risk disease remain unsatisfactory. Data indicate that plant-derived cannabinoids are effective against adult glioblastoma, however, preclinical evidence supporting their use in pediatric brain cancers is lacking. Here we investigated the potential role for &Delta, 9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in medulloblastoma and ependymoma. Dose-dependent cytotoxicity of medulloblastoma and ependymoma cells was induced by THC and CBD in vitro, and a synergistic reduction in viability was observed when both drugs were combined. Mechanistically, cannabinoids induced cell cycle arrest, in part by the production of reactive oxygen species, autophagy, and apoptosis, however, this did not translate to increased survival in orthotopic transplant models despite being well tolerated. We also tested the combination of cannabinoids with the medulloblastoma drug cyclophosphamide, and despite some in vitro synergism, no survival advantage was observed in vivo. Consequently, clinical benefit from the use of cannabinoids in the treatment of high-grade medulloblastoma and ependymoma is expected to be limited. This study emphasizes the importance of preclinical models in validating therapeutic agent efficacy prior to clinical trials, ensuring that enrolled patients are afforded the most promising therapies available.

Medulloblastoma is a heterogeneous disease, comprising four molecular subgroups, termed WNT, SHH, Group 3 and Group 4. Each has distinct clinical, pathological, and molecular features. Surgical resection and craniospinal irradiation followed by chemotherapy are the mainstay of medulloblastoma treatment. Despite treatment intensification, survival has plateaued for the past two decades at around 70%. Certain sub-groups, namely those with SHH with p53 mutations and Group 3 with MYC amplification, have dismal prognoses. Since medulloblastoma patients that relapse are essentially incurable, ergo, the best opportunity to cure medulloblastoma is during frontline therapy. To increase cure rates, we employed an unbiased high-throughput drug screening approach, to identify novel drugs that enhance the effects of current treatments to aid front-line therapy. We identified prexasertib, an inhibitor of the cell cycle checkpoint kinases 1 and 2 (Chk1/2), as a promising candidate. Chk1/2 are critical regulators of the DNA-damage response. Given radiation is a potent inducer of DNA damage, we hypothesised that Chk1/2 inhibition with prexasertib may enhance radiation-induced cytotoxicity in medulloblastoma. In this study, we found that radiation induced a strong DNA damage response in multiple medulloblastoma models. In vitro, prexasertib reduced the colony-forming ability of medulloblastoma cells post-irradiation. In vivo, tumour-targeted radiation therapy and prexasertib each induced modest apoptosis in orthotopically-implanted medulloblastomas, which was significantly increased when co-administered. Importantly, this combination therapy also improved the survival of mice with medulloblastoma. Taken together, these data reveal significant anti-tumour effects when radiation is combined with Chk1/2 inhibition suggesting that this combination therapy may increase treatment efficacy.

MB-75. EXPLOITING THE CELL CYCLE TO IMPROVE TREATMENT EFFICACY IN MEDULLOBLASTOMA Raelene Endersby1, Jacqueline Whitehouse1, Hilary Hii1, Mani Kuchibhotla1, Brett Patterson1, Brooke Strowger1, Tobias Schoep1, and Nick Gottardo1,2; Telethon Kids Institute and University of Western Australia, Perth, WA, Australia; Princess Margaret Hospital, Subiaco, WA, Australia Medulloblastoma is themost common malignantbraincancerof childhood. It arises in the cerebellum and has a predilection to spread throughout the central nervous system. The most effective treatment is a combination of surgery, followed by radiotherapy and intensive cytotoxic chemotherapy; however, devastating long-term sequelae, due to the collateral damage of healthy tissue by these treatments, including developmental defects, psychosocial deficits and secondary tumours are frequently encountered by survivors, and for patients with metastatic disease prognosis are dismal. Using high-throughput, cell-based assays human medulloblastoma cells (n 1⁄4 6) were screened against a library of approximately 3200 compounds. Fifty effective compounds were identified and further in vitro assessment identified several drugs that enhanced the cytotoxic activity of clinically-used chemotherapeutics: cyclophosphamide, cisplatin, and gemcitabine. Cell cycle checkpoint kinase (CHK1/2) inhibitors (iCHKs), were further assessed in vivo using mice bearing intracranial implants of human medulloblastoma cells. When combined with DNA-damaging chemotherapeutics, iCHK treatment reduced tumour burden as measured by bioluminescence imaging and significantly increased survival of animals with medulloblastoma up to three-fold, in several different PDX models. Immunohistochemical assessment of tumours showed the combination treatment significantly decreased tumour proliferation and significantly induced apoptosis compared with controls. These data demonstrate our experimental approach has robustly identified effective new therapies for paediatric medulloblastoma, and our findings strongly suggest CHK1/2 inhibitors have promising potential to improve treatments for this disease. Neuro-Oncology 18:iii97–iii122, 2016. doi:10.1093/neuonc/now076.71 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.