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AIMS Glioblastoma (GBM) is the most common primary malignant brain tumour in adults. Despite aggressive treatment, a resistant tumour recurs in practically all patients. We therefore aimed to better understand the mechanisms driving this treatment resistance through investigating changes in gene expression across pairs of primary and recurrent GBM tumours. METHOD We generated or acquired bulk tumour RNA sequencing data for primary and first recurrent tumours from 107 patients who received standard treatment. Differential expression analysis between primary and recurrent samples found that the most dysregulated genes were involved in neurodevelopment and neurodifferentiation. We therefore used a publicly available ChIP-seq database to identify DNA binding factors for which binding sites are enriched in the promotors of genes with the largest expression changes from primary to recurrent. RESULTS Jumonji and AT-Rich Interacting Domain 2 (JARID2) was the most strongly enriched for binding to promotors of dysregulated genes. 65 patients showed an up-regulation and 42 showed a down-regulation of genes bound by this protein. The same set of JARID2 bound genes were found to be dysregulated in each direction, and correlated with the largest source of variation between samples in their response to treatment. Further enrichment analyses indicated that ‘Up’ responders may resist treatment through reduced proliferation and increased interaction with the tumour microenvironment, whereas ‘Down’ responders instead rely on a shift to mesenchymal cell states. CONCLUSION These results indicate that GBM tumours can be split into two subtypes that transcriptionally reprogramme in different directions through treatment and may benefit from different treatment approaches.

Summary Glioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To understand this problem more clearly we used a combination of natural killer (NK) cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem‐like cells, a source of post‐treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM‐infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of transforming growth factor (TGF)‐β‐mediated inhibition. This NK cell inhibition is accompanied by expression of multiple immune checkpoint molecules on T cells. Single‐cell transcriptomics demonstrated that both tumour and haematopoietic‐derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co‐expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognized by the immune system but that anti‐tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more probably benefit from combination immunotherapies directed against multiple immunosuppressive pathways., Our manuscript challenges the current paradigm that Glioblastoma (GBM) is a poor candidate for immunotherapy, therefore impacting on clinical approaches. We identify NK cells as a target for immunosuppression in GBM, with multiple suppressive pathways within GBM human tumours. We also identify a group of GBM patients with higher immunological activity, who will benefit from immunotherapies that target these immunosuppressive pathways.

Background Brain metastases (BrM) develop in 20–40% of cancer patients and represent an unmet clinical need. Limited access of drugs into the brain because of the blood-brain barrier is at least partially responsible for therapeutic failure, necessitating improved drug delivery systems. Methods Green fluorescent protein (GFP)-transduced murine and nontransduced human hematopoietic stem cells (HSCs) were administered into mice (n = 10 and 3). The HSC progeny in mouse BrM and in patient-derived BrM tissue (n = 6) was characterized by flow cytometry and immunofluorescence. Promoters driving gene expression, specifically within the BrM-infiltrating HSC progeny, were identified through differential gene-expression analysis and subsequent validation of a series of promoter-green fluorescent protein-reporter constructs in mice (n = 5). One of the promoters was used to deliver tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) to BrM in mice (n = 17/21 for TRAIL vs control group). Results HSC progeny (consisting mostly of macrophages) efficiently homed to macrometastases (mean [SD] = 37.6% [7.2%] of all infiltrating cells for murine HSC progeny; 27.9% mean [SD] = 27.9% [4.9%] of infiltrating CD45+ hematopoietic cells for human HSC progeny) and micrometastases in mice (19.3–53.3% of all macrophages for murine HSCs). Macrophages were also abundant in patient-derived BrM tissue (mean [SD] = 8.8% [7.8%]). Collectively, this provided a rationale to optimize the delivery of gene therapy to BrM within myeloid cells. MMP14 promoter emerged as the strongest promoter construct capable of limiting gene expression to BrM-infiltrating myeloid cells in mice. TRAIL delivered under MMP14 promoter statistically significantly prolonged survival in mice (mean [SD] = 19.0 [3.4] vs mean [SD] = 15.0 [2.0] days for TRAIL vs control group; two-sided P = .006), demonstrating therapeutic and translational potential of our approach. Conclusions Our study establishes HSC gene therapy using a myeloid cell–specific promoter as a new strategy to target BrM. This approach, with strong translational value, has potential to overcome the blood-brain barrier, target micrometastases, and control multifocal lesions.

BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

INTRODUCTION Glioblastoma multiforme remains one of the most difficult to treat cancers due to the highly infiltrative nature of cancer cells. Targeting the invasive properties of glioblastoma presents a novel treatment avenue. Recently, specific GSK-3 beta (GSK3b) inhibitors such as Bio-Indirubin (BIO), have been described that inhibit the migratory activity of glioma cells. In this study, the GSK-3b downstream targets were investigated.

Summary: Many Next Generation Sequencing analyses involve the basic manipulation of input sequence data before downstream processing (e.g. searching for specific sequences, format conversion or basic file statistics). The rapidly increasing data volumes involved in NGS make any dataset manipulation a time-consuming and error-prone process. I have developed fqtools; a fast and reliable FASTQ file manipulation suite that can process the full set of valid FASTQ files, including those with multi-line sequences, whilst identifying invalid files. Fqtools is faster than similar tools, and is designed for use in automatic processing pipelines. Availability and implementation: fqtools is open source and is available at: https://github.com/alastair-droop/fqtools. Supplementary information: Supplementary data are available at Bioinformatics online. Contact: a.p.droop@leeds.ac.uk

INTRODUCTION: There is a pressing need for new therapeutic approaches for the treatment of glioblastoma (GBM). MicroRNAs are single-stranded 22-24nt non-coding RNAs which function by reducing translation or causing degradation of target mRNAs. MiR-1300 was identified in a high-throughput miRNA mimic library screen as a promising therapeutic candidate, inducing cell death. METHOD: To validate the cytotoxic effect of miR-1300, a panel of four established cell lines (U251,LN229,U373,KNS42)and two patient-derived stem-like cell lines were utilised for RTqPCR, imaging and flow-cytometry to assess endogenous expression levels, cellular morphology, apoptosis, and cell cycle delay respectively. Bioformatic approaches identified potential miR-1300 targets as mediators of this effect and validation of this involvement in the cytotoxic phenotype using siRNA knock-down is being investigated. Validation of the direct targeting by miR-1300 is being performed using 3' UTR Luciferase assays. RESULTS: Expression, of miR-1300, by transfection of oligonucleotide mimics, in our panel of GBM cells consistently led to G2M arrest followed by apoptosis within 72h. The associated phenotype is binucleation, indicative of cytokinesis failure.Preliminary data suggest this may be mediated through ECT2, a predicted target of miR-1300 known to be involved in formation of the cleavage furrow. CONCLUSION: We have identified a highly potent pro-apoptotic microRNA that may be exploitable for GBM therapy. Importantly, there is no detectable endogenous miR-1300 expression in any GBM samples or cell lines. On-going work will confirm the targets of miR-1300 that drive this effect as well as exploring potential approaches to deliver this as a biotherapeutic agent.

MicroRNAs are short non-coding RNAs that act as micro-managers of cellular function. Their dysregulation is well documented in many cancers. TCGA microRNA expression data is available to identify the prognostic potential of microRNAs in malignant glioma. Using the LASSO statistical approach for 475 glioblastomas in the TCGA dataset we identified a nine-microRNA signature that predicts overall (p = 2.26e-09) and progression-free survival (p = 9.91e-08) with superior power than conventional molecular markers, including MGMT methylation status. This signature has significance in all molecular subtypes of glioblastoma with the exception of the non-G-CIMP proneural group and validated in a separate validation cohort (p = 4.50E-02). The signature also predicts survival in grade II and III disease (p = 5.20e-03). The great majority of microRNAs in the signature have previously been shown to play a role in glioma biology. In addition, when we compared patient groups at the extremes of survival in grade III astrocytoma (poor prognosis n = 10, survival 48 months) and glioblastoma (poor prognosis n = 14, survival 48 months), we found that microRNA profiles aligned with specific neuro-developmental pathways were associated with outcomes. Specifically, poorer prognosis disease was associated with profiles aligned towards glial-restricted and oligodendrocyte precursor (OP) cells. Furthermore, correlation of microRNA expression patterns of OPs with expression of 39 grade III astrocytomas and 558 glioblastomas predicts survival with high significance (p= 5.50e-06), indicating that the more tightly a grade III or IV tumor correlates with an OP profile, the poorer the outcome. These observations indicate that microRNAs have predictive power in malignant glioma. MicroRNA expression patterns may also reveal important aspects of underlying tumor biology. This data, coupled with the superior stability of microRNAs over mRNAs in clinical specimens, suggests they may represent attractive candidates as novel biomarkers for the disease.