Characterisation of Expression the Arginine Pathway Enzymes in Childhood Brain Tumours to Determine Susceptibility to Therapeutic Arginine Depletion.

Despite significant improvements in treatment and survival in paediatric cancers, outcomes for children with brain tumours remain poor. Novel therapeutic approaches are needed to improve survival and quality of survival. Extracellular arginine dependency (auxotrophy) has been recognised in several tumours as a potential therapeutic target. This dependency is due to the inability of cancer cells to recycle or synthesise intracellular arginine through the urea cycle pathway compared to normal cells. Whilst adult glioblastoma exhibits this dependency, the expression of the arginine pathway enzymes has not been delineated in paediatric brain tumours. We used immunohistochemical (IHC) methods to stain for arginine pathway enzymes in paediatric high-grade glioma (pHGG), low-grade glioma (pLGG), ependymoma (EPN), and medulloblastoma (MB) tumour tissue microarrays (TMAs). The antibodies detected protein expression of the metaboliser arginase (Arg1 and Arg2); recycling enzymes ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS1), and argininosuccinate lyase (ASL); and the transporter SLC7A1. Deficiency of OTC, ASS1, and ASL was seen in 87.5%, 94%, and 79% of pHGG samples, respectively, consistent with an auxotrophic signature. Similar result was obtained in pLGG with 96%, 93%, and 91% of tumours being deficient in ASL, ASS1, and OTC, respectively. 79%, 88%, and 85% of MB cases were ASL, ASS1, and OTC deficient whilst ASL and OTC were deficient in 57% and 91% of EPN samples. All tumour types highly expressed SLC7A1 and Arginase, with Arg2 being the main isoform, demonstrating that they could transport and utilise arginine. Our results show that pHGG, pLGG, EPN, and MB demonstrate arginine auxotrophy based on protein expression and are likely to be susceptible to arginine depletion. Pegylated arginase (BCT-100) is currently in phase I/II trials in relapsed pHGG. Our results suggest that therapeutic arginine depletion may also be useful in other tumour types and IHC analysis of patient tumour samples could help identify patients likely to benefit from this treatment.

Sequential Orbitrap Secondary Ion Mass Spectrometry and Liquid Extraction Surface Analysis-Tandem Mass Spectrometry-Based Metabolomics for Prediction of Brain Tumor Relapse from Sample-Limited Primary Tissue Archives.

We present here a novel surface mass spectrometry strategy to perform untargeted metabolite profiling of formalin-fixed paraffin-embedded pediatric ependymoma archives. Sequential Orbitrap secondary ion mass spectrometry (3D OrbiSIMS) and liquid extraction surface analysis-tandem mass spectrometry (LESA-MS/MS) permitted the detection of 887 metabolites (163 chemical classes) from pediatric ependymoma tumor tissue microarrays (diameter: <1 mm; thickness: 4 µm). From these 163 classes, 60 classes were detected with both techniques, whilst LESA-MS/MS and 3D OrbiSIMS individually allowed the detection of another 83 and 20 unique metabolite classes, respectively. Through data fusion and multivariate analysis, we were able to identify key metabolites and corresponding pathways predictive of tumor relapse, which were retrospectively confirmed by gene expression analysis with publicly available data. Altogether, this sequential mass spectrometry strategy has shown to be a versatile tool to perform high-throughput metabolite profiling on sample-limited tissue archives.

Assessment of Cannabidiol and Δ9-Tetrahydrocannabiol in Mouse Models of Medulloblastoma and Ependymoma.

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 Δ9-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.

A retrospective analysis of recurrent pediatric ependymoma reveals extremely poor survival and ineffectiveness of current treatments across central nervous system locations and molecular subgroups.

BACKGROUND: Relapse occurs in 50% of pediatric ependymoma cases and has poor prognosis. Few studies have investigated the clinical progress of relapsed disease, and treatment lacks a standardized approach. METHODS AND MATERIALS: We analyzed 302 pediatric ependymoma cases. Tumor, demographic, and treatment variables were investigated for association with relapse risk, time to recurrence, and survival after relapse. DNA methylation profiling was performed for 135/302 cases, and predominant subgroups were EPN_PFA (n = 95) and EPN_RELA (n = 24). Chromosome 1q status was ascertained for 185/302 cases by fluorescent in-situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), and DNA methylation profiles. RESULTS: Sixty-two percent of cases relapsed, with a median of two recurrences with no difference between posterior fossa and supratentorial locations (66% vs 55% relapse rate). One hundred seventeen (38%) cases relapsed within two years and five (2%) beyond 10 years. The late relapses were clinically heterogeneous. Tumor grade and treatment affected risk and time to relapse variably across subgroups. After relapse, surgery and irradiation delayed disease progression with a minimal impact on survival across the entire cohort. In the EPN_PFA and EPN_RELA groups, 1q gain was independently associated with relapse risk (subhazard ratio [SHR] 4.307, P = 0.027 and SHR 1.982, P = 0.010, respectively) while EPN_PFA had increased relapse risk compared with EPN_RELA (SHR = 0.394, P = 0.018). CONCLUSIONS: Recurrent pediatric ependymoma is an aggressive disease with poor outcomes, for which current treatments are inadequate. We report that chromosome 1q gain increases relapse risk in common molecular subgroups in children but a deeper understanding of the underlying biology at relapse and novel therapeutic approaches are urgently needed.

A role for ABCB1 in prognosis, invasion and drug resistance in ependymoma.

Three of the hallmarks of poor prognosis in paediatric ependymoma are drug resistance, local invasion and recurrence. We hypothesised that these hallmarks were due to the presence of a sub-population of cancer stem cells expressing the multi-drug efflux transporter ABCB1. ABCB1 gene expression was observed in 4 out of 5 paediatric ependymoma cell lines and increased in stem cell enriched neurospheres. Functional inhibition of ABCB1 using vardenafil or verapamil significantly (p ≤ 0.05-0.001) potentiated the response to three chemotherapeutic drugs (vincristine, etoposide and methotrexate). Both inhibitors were also able to significantly reduce migration (p ≤ 0.001) and invasion (p ≤ 0.001). We demonstrate that ABCB1 positive patients from an infant chemotherapy-led trial (CNS9204) had a shorter mean event free survival (EFS) (2.7 versus 8.6 years; p = 0.007 log-rank analysis) and overall survival (OS) (5.4 versus 12 years; p = 0.009 log-rank analysis). ABCB1 positivity also correlated with reduced event free survival in patients with incompletely resected tumours who received chemotherapy across CNS9204 and CNS9904 (a radiotherapy-led SIOP 1999-04 trial cohort; p = 0.03). ABCB1 is a predictive marker of chemotherapy response in ependymoma patients and vardenafil, currently used to treat paediatric pulmonary hypertension in children, could be repurposed to reduce chemoresistance, migration and invasion in paediatric ependymoma patients at non-toxic concentrations.

Modulating autophagy as a therapeutic strategy for the treatment of paediatric high-grade glioma.

Paediatric high-grade gliomas (pHGG) represent a therapeutically challenging group of tumors. Despite decades of research, there has been minimal improvement in treatment and the clinical prognosis remains poor. Autophagy, a highly conserved process for recycling metabolic substrates is upregulated in pHGG, promoting tumor progression and evading cell death. There is significant crosstalk between autophagy and a plethora of critical cellular pathways, many of which are dysregulated in pHGG. The following article will discuss our current understanding of autophagy signaling in pHGG and the potential modulation of this network as a therapeutic target.

In search of effective therapies to overcome resistance to Temozolomide in brain tumours.

Glioblastoma multiforme is the most common and lethal brain tumour-type. The current standard of care includes Temozolomide (TMZ) chemotherapy. However, inherent and acquired resistance to TMZ thwart successful treatment. The direct repair protein methylguanine DNA methyltransferase (MGMT) removes the cytotoxic O6-methylguanine (O6-MeG) lesion delivered by TMZ and so its expression by tumours confers TMZ-resistance. DNA mismatch repair (MMR) is essential to process O6-MeG adducts and MMR-deficiency leads to tolerance of lesions, resistance to TMZ and further DNA mutations. In this article, two strategies to overcome TMZ resistance are discussed: (1) synthesis of imidazotetrazine analogues - designed to retain activity in the presence of MGMT or loss of MMR; (2) preparation of imidazotetrazine-nanoparticles to deliver TMZ preferably to the brain and tumour site. Our promising results encourage belief in a future where better prognoses exist for patients diagnosed with this devastating disease.

Tissue metabolite profiles for the characterisation of paediatric cerebellar tumours.

Paediatric brain tumors are becoming well characterized due to large genomic and epigenomic studies. Metabolomics is a powerful analytical approach aiding in the characterization of tumors. This study shows that common cerebellar tumors have metabolite profiles sufficiently different to build accurate, robust diagnostic classifiers, and that the metabolite profiles can be used to assess differences in metabolism between the tumors. Tissue metabolite profiles were obtained from cerebellar ependymoma (n = 18), medulloblastoma (n = 36), pilocytic astrocytoma (n = 24) and atypical teratoid/rhabdoid tumors (n = 5) samples using HR-MAS. Quantified metabolites accurately discriminated the tumors; classification accuracies were 94% for ependymoma and medulloblastoma and 92% for pilocytic astrocytoma. Using current intraoperative examination the diagnostic accuracy was 72% for ependymoma, 90% for medulloblastoma and 89% for pilocytic astrocytoma. Elevated myo-inositol was characteristic of ependymoma whilst high taurine, phosphocholine and glycine distinguished medulloblastoma. Glutamine, hypotaurine and N-acetylaspartate (NAA) were increased in pilocytic astrocytoma. High lipids, phosphocholine and glutathione were important for separating ATRTs from medulloblastomas. This study demonstrates the ability of metabolic profiling by HR-MAS on small biopsy tissue samples to characterize these tumors. Analysis of tissue metabolite profiles has advantages in terms of minimal tissue pre-processing, short data acquisition time giving the potential to be used as part of a rapid diagnostic work-up.

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target.

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. ß-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

Overcoming multiple drug resistance mechanisms in medulloblastoma.

INTRODUCTION: Medulloblastoma (MB) is the most common malignant paediatric brain tumour. Recurrence and progression of disease occurs in 15-20% of standard risk and 30-40% of high risk patients. We analysed whether circumvention of chemoresistance pathways (drug export, DNA repair and apoptotic inhibition) can restore chemotherapeutic efficacy in a panel of MB cell lines. RESULTS: We demonstrate, by immunohistochemistry in patient tissue microarrays, that ABCB1 is expressed in 43% of tumours and is significantly associated with high-risk. We show that ABCB1, O6-methylguanine-DNA-methyltransferase (MGMT) and BCL2 family members are differentially expressed (by quantitative reverse transcription polymerase chain reaction, Western blotting and flow cytometry) in MB cell lines. Based on these findings, each pathway was then inhibited or circumvented and cell survival assessed using clonogenic assays. Inhibition of ABCB1 using vardenafil or verapamil resulted in a significant increase in sensitivity to etoposide in ABCB1-expressing MB cell lines. Sensitivity to temozolomide (TMZ) was MGMT-dependent, but two novel imidazotetrazine derivatives (N-3 sulfoxide and N-3 propargyl TMZ analogues) demonstrated ≥7 fold and ≥3 fold more potent cytotoxicity respectively compared to TMZ in MGMT-expressing MB cell lines. Activity of the BAD mimetic ABT-737 was BCL2A1 and ABCB1 dependent, whereas the pan-BCL2 inhibitor obatoclax was effective as a single cytotoxic agent irrespective of MCL1, BCL2, BCL2A1, or ABCB1 expression. CONCLUSIONS: ABCB1 is associated with high-risk MB; hence, inhibition of ABCB1 by vardenafil may represent a valid approach in these patients. Imidazotetrazine analogues of TMZ and the BH3 mimetic obatoclax are promising clinical candidates in drug resistant MB tumours expressing MGMT and BCL2 anti-apoptotic members respectively.

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion.

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3-4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.