Pre-clinical investigation of the effect of combining the cytotoxic agent Temozolomide with a dual mTORC2 inhibitor for the treatment of glioblastoma and establishment of an imageable surgical resection model of glioblastoma

Glioblastoma is the most common primary brain tumour. It is an incurable disease and is commonly referred to as the worst of the “incurable cancers” by neurosurgeons and neuro-oncologists. It is heterogeneous on a cellular and genetic level. The genetic mutations that occur in glioblastoma lead to dysregulation of several principal pathways, the most commonly dysregulated pathways involve mTOR. mTOR is involved in the control of several cellular processes including autophagy and apoptosis. Despite maximal treatment, median survival remains abysmal ranging from 15 to 16 months. Current standard of care consists of two phases. The first phase involves maximal safe surgical resection. This intervention reduces the tumour burden and improves the efficacy of the second phase. This second phase involves chemotherapy and radiotherapy commonly referred to as the “Stupp protocol”. This phase employs a common strategy of inducing apoptosis in an attempt to control the residual disease. Within this thesis we explore both phases of treatment for glioblastoma. Firstly, we established the in vitro efficacy of the mTOR inhibitor, AZD 8055 using the glioblastoma cell line U87MG, and glioblastoma tissue derived from a patient in Norway referred to as patient 3. We then assessed the in vivo efficacy of AZD 8055 in combination with temozolomide, using the patient derived glioblastoma tumour material from patient 3, in an orthotopic bioluminescence murine model. We demonstrated that although AZD 8055 is a strong inhibitor of the mTOR pathway when used as a monotherapy or combined with temozolomide, this did not translate into either increased survival or a reduction in tumour growth in vivo. By interrogating the points of crosstalk between autophagy and apoptotic pathways, we hypothesized that mTOR inhibition induced a protective autophagy which antagonised the apoptosis induced by temozolomide. Secondly, we validated a novel animal model of surgical resection. We established that orthotopic implantation of glioblastoma cells can be safely resected, and that the residual tumour, which has invaded the surrounding brain parenchyma, can be reliability imaged in vivo. We advocated the use of this model for the investigation of local delivery of novel therapies into the surgical resection cavity or in the assessment of systemic therapeutic agents targeting residual tumour that has migrated into the normal brain parenchyma

Targeting the RhoGEF βPIX/COOL-1 in Glioblastoma: Proof of Concept Studies

Glioblastoma (GBM), a highly invasive and vascular malignancy is shown to rapidly develop resistance and evolve to a more invasive phenotype following bevacizumab (Bev) therapy. Rho Guanine Nucleotide Exchange Factor proteins (RhoGEFs) are mediators of key components in Bev resistance pathways, GBM and Bev-induced invasion. To identify GEFs with enhanced mRNA expression in the leading edge of GBM tumours, a cohort of GEFs was assessed using a clinical dataset. The GEF βPix/COOL-1 was identified, and the functional effect of gene depletion assessed using 3D-boyden chamber, proliferation, and colony formation assays in GBM cells. Anti-angiogenic effects were assessed in endothelial cells using tube formation and wound healing assays. In vivo effects of βPix/COOL-1-siRNA delivered via RGD-Nanoparticle in combination with Bev was studied in an invasive model of GBM. We found that siRNA-mediated knockdown of βPix/COOL-1 in vitro decreased cell invasion, proliferation and increased apoptosis in GBM cell lines. Moreover βPix/COOL-1 mediated endothelial cell migration in vitro. Mice treated with βPix/COOL-1 siRNA-loaded RGD-Nanoparticle and Bev demonstrated a trend towards improved median survival compared with Bev monotherapy. Our hypothesis generating study suggests that the RhoGEF βPix/COOL-1 may represent a target of vulnerability in GBM, in particular to improve Bev efficacy

Somatic variants as a cause of drug-resistant epilepsy including mesial temporal lobe epilepsy with hippocampal sclerosis

OBJECTIVE: The contribution of somatic variants to epilepsy has recently been demonstrated, particularly in the etiology of malformations of cortical development. The aim of this study was to determine the diagnostic yield of somatic variants in genes that have been previously associated with a somatic or germline epilepsy model, ascertained from resected brain tissue from patients with multidrug-resistant focal epilepsy. METHODS: Forty-two patients were recruited across three categories: (1) malformations of cortical development, (2) mesial temporal lobe epilepsy with hippocampal sclerosis, and (3) nonlesional focal epilepsy. Participants were subdivided based on histopathology of the resected brain. Paired blood- and brain-derived DNA samples were sequenced using high-coverage targeted next generation sequencing to high depth (585— and 1360—, respectively). Variants were identified using Genome Analysis ToolKit (GATK4) MuTect-2 and confirmed using high-coverage Amplicon-EZ sequencing. RESULTS: Sequence data on 41 patients passed quality control. Four somatic variants were validated following amplicon sequencing: within CBL, ALG13, MTOR, and FLNA. The diagnostic yield across 41 patients was 10%, 9% in mesial temporal lobe epilepsy with hippocampal sclerosis and 20% in malformations of cortical development. SIGNIFICANCE: This study provides novel insights into the etiology of mesial temporal lobe epilepsy with hippocampal sclerosis, highlighting a potential pathogenic role of somatic variants in CBL and ALG13. We also report candidate diagnostic somatic variants in FLNA in focal cortical dysplasia, while providing further insight into the importance of MTOR and related genes in focal cortical dysplasia. This work demonstrates the potential molecular diagnostic value of variants in both germline and somatic epilepsy genes

Nanocomposite formulation for a sustained release of free drug and drug-loaded responsive nanoparticles: an approach for a local therapy of glioblastoma multiforme

Abstract Malignant gliomas are a type of primary brain tumour that originates in glial cells. Among them, glioblastoma multiforme (GBM) is the most common and the most aggressive brain tumour in adults, classified as grade IV by the World Health Organization. The standard care for GBM, known as the Stupp protocol includes surgical resection followed by oral chemotherapy with temozolomide (TMZ). This treatment option provides a median survival prognosis of only 16–18 months to patients mainly due to tumour recurrence. Therefore, enhanced treatment options are urgently needed for this disease. Here we show the development, characterization, and in vitro and in vivo evaluation of a new composite material for local therapy of GBM post-surgery. We developed responsive nanoparticles that were loaded with paclitaxel (PTX), and that showed penetration in 3D spheroids and cell internalization. These nanoparticles were found to be cytotoxic in 2D (U-87 cells) and 3D (U-87 spheroids) models of GBM. The incorporation of these nanoparticles into a hydrogel facilitates their sustained release in time. Moreover, the formulation of this hydrogel containing PTX-loaded responsive nanoparticles and free TMZ was able to delay tumour recurrence in vivo after resection surgery. Therefore, our formulation represents a promising approach to develop combined local therapies against GBM using injectable hydrogels containing nanoparticles

Pumilio-1 mediated translational control of claudin-5 at the blood-brain barrier

Abstract Claudin-5 is one of the most essential tight junction proteins at the blood-brain barrier. A single nucleotide polymorphism rs10314 is located in the 3’-untranslated region of claudin-5 and has been shown to be a risk factor for schizophrenia. Here, we show that the pumilio RNA-binding protein, pumilio-1, is responsible for rs10314-mediated claudin-5 regulation. The RNA sequence surrounding rs10314 is highly homologous to the canonical pumilio-binding sequence and claudin-5 mRNA with rs10314 produces 25% less protein due to its inability to bind to pumilio-1. Pumilio-1 formed cytosolic granules under stress conditions and claudin-5 mRNA appeared to preferentially accumulate in these granules. Added to this, we observed granular pumilio-1 in endothelial cells in human brain tissues from patients with psychiatric disorders or epilepsy with increased/accumulated claudin-5 mRNA levels, suggesting translational claudin-5 suppression may occur in a brain-region specific manner. These findings identify a key regulator of claudin-5 translational processing and how its dysregulation may be associated with neurological and neuropsychiatric disorders. Graphical Abstrac

Validation of an imageable surgical resection animal model of Glioblastoma (GBM).

BACKGROUND: Glioblastoma (GBM) is the most common and malignant primary brain tumour having a median survival of just 12-18 months following standard therapy protocols. Local recurrence, post-resection and adjuvant therapy occurs in most cases. NEW METHOD: U87MG-luc2-bearing GBM xenografts underwent 4.5mm craniectomy and tumour resection using microsurgical techniques. The cranial defect was repaired using a novel modified cranial window technique consisting of a circular microscope coverslip held in place with glue. RESULTS: Immediate post-operative bioluminescence imaging (BLI) revealed a gross total resection rate of 75%. At censor point 4 weeks post-resection, Kaplan-Meier survival analysis revealed 100% survival in the surgical group compared to 0% in the non-surgical cohort (p=0.01). No neurological defects or infections in the surgical group were observed. GBM recurrence was reliably imaged using facile non-invasive optical bioluminescence (BLI) imaging with recurrence observed at week 4. COMPARISON WITH EXISTING METHOD(S): For the first time, we have used a novel cranial defect repair method to extend and improve intracranial surgical resection methods for application in translational GBM rodent disease models. Combining BLI and the cranial window technique described herein facilitates non-invasive serial imaging follow-up. CONCLUSION: Within the current context we have developed a robust methodology for establishing a clinically relevant imageable GBM surgical resection model that appropriately mimics GBM recurrence post resection in patients.</p

A clinically relevant computed tomography (CT) radiomics strategy for intracranial rodent brain tumour monitoring

Abstract Here, we establish a CT-radiomics based method for application in invasive, orthotopic rodent brain tumour models. Twenty four NOD/SCID mice were implanted with U87R-Luc2 GBM cells and longitudinally imaged via contrast enhanced (CE-CT) imaging. Pyradiomics was employed to extract CT-radiomic features from the tumour-implanted hemisphere and non-tumour-implanted hemisphere of acquired CT-scans. Inter-correlated features were removed (Spearman correlation > 0.85) and remaining features underwent predictive analysis (recursive feature elimination or Boruta algorithm). An area under the curve of the receiver operating characteristic curve was implemented to evaluate radiomic features for their capacity to predict defined outcomes. Firstly, we identified a subset of radiomic features which distinguish the tumour-implanted hemisphere and non- tumour-implanted hemisphere (i.e, tumour presence from normal tissue). Secondly, we successfully translate preclinical CT-radiomic pipelines to GBM patient CT scans (n = 10), identifying similar trends in tumour-specific feature intensities (E.g. ‘glszm Zone Entropy’), thereby suggesting a mouse-to-human species conservation (a conservation of radiomic features across species). Thirdly, comparison of features across timepoints identify features which support preclinical tumour detection earlier than is possible by visual assessment of CT scans. This work establishes robust, preclinical CT-radiomic pipelines and describes the application of CE-CT for in-depth orthotopic brain tumour monitoring. Overall we provide evidence for the role of pre-clinical ‘discovery’ radiomics in the neuro-oncology space

Targeting the RhoGEF βPIX/COOL-1 in glioblastoma: proof of concept studies.

Glioblastoma (GBM), a highly invasive and vascular malignancy is shown to rapidly develop resistance and evolve to a more invasive phenotype following bevacizumab (Bev) therapy. Rho Guanine Nucleotide Exchange Factor proteins (RhoGEFs) are mediators of key components in Bev resistance pathways, GBM and Bev-induced invasion. To identify GEFs with enhanced mRNA expression in the leading edge of GBM tumours, a cohort of GEFs was assessed using a clinical dataset. The GEF βPix/COOL-1 was identified, and the functional effect of gene depletion assessed using 3D-boyden chamber, proliferation, and colony formation assays in GBM cells. Anti-angiogenic effects were assessed in endothelial cells using tube formation and wound healing assays. In vivo effects of βPix/COOL-1-siRNA delivered via RGD-Nanoparticle in combination with Bev was studied in an invasive model of GBM. We found that siRNA-mediated knockdown of βPix/COOL-1 in vitro decreased cell invasion, proliferation and increased apoptosis in GBM cell lines. Moreover βPix/COOL-1 mediated endothelial cell migration in vitro. Mice treated with βPix/COOL-1 siRNA-loaded RGD-Nanoparticle and Bev demonstrated a trend towards improved median survival compared with Bev monotherapy. Our hypothesis generating study suggests that the RhoGEF βPix/COOL-1 may represent a target of vulnerability in GBM, in particular to improve Bev efficacy