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Molecular characterisation of lower-grade glioma and investigation of its genomic aberrations influencing patient prognosis and therapeutic response(2022) Noor, HumairaThesisLower-grade gliomas (LGGs) are tumours of the Central Nervous System affecting young adults aged 17-44 years. The overall survival of LGG patients has not improved significantly over the last 30 years, and new therapeutic approaches are warranted to effectively treat this cancer. In this thesis, I have molecularly characterised a retrospective cohort of LGG specimen from 102 patients, including 29 patients with matched primary and recurrent tumours, in order to identify prognostic biomarkers, molecular subgroups with unfavourable prognosis and investigate their potential as therapeutic targets through pre-clinical studies. Specific hotspot tumour protein 53 (TP53) codon 273 mutations occurred in 33% of astrocytoma, which were associated with significantly improved survival in both univariate (p<0.05) and multivariate analysis (p<0.05). TP53 wildtype tumours were associated with the most unfavourable clinical outcome. Analysis of publicly available LGG datasets validated these findings and elucidated the mechanism of this prognostication involving enhanced chemosensitivity of TP53 codon 273 mutations. Integrated analysis uncovered interactions between Yes-associated protein 1 (YAP1) and TP53 mutation potentially plays a role in inducing this chemosensitivity. In silico analysis identified the over-expression of carbonic anhydrase 12 (CA12) mRNA in the TP53 wildtype astrocytoma sub-group with the most unfavourable prognosis. I attempted to establish LGG patient-derived neurosphere cell lines for in vitro drug efficacy studies with the CA12 inhibitor U-104 combined with temozolomide treatment. Drug efficacy studies were conducted on a panel of TP53 wildtype and TP53 mutant glioblastoma cell lines where U-104 monotherapy, but not combination therapy, was efficacious in both subgroups. U-104 monotherapy also showed efficacy in LGG neurospheres. Molecular characterisation revealed that 72.9% of astrocytomas and 63.2% of oligodendrogliomas followed alternative lengthening of telomere (ALT) mechanism to maintain their telomere lengths. Neither ALT nor telomerase mechanisms were prognostic factors in astrocytoma, while, ALT was a significantly associated with a longer progression-free survival in oligodendroglioma. Cyclin a1 (CCNA1) was identified as a potential ALT-associated gene in the analysis of in-house RNA sequencing data. While CCNA1 methylation was not found to be directly associated with ALT upon investigation, CCNA1 methylation showed a strong trend for association with ALT-associated aberrations in ATRX, and progression-free survival in both the in-house astrocytoma cohort and TCGA dataset. Moreover, CCNA1 methylation levels were significantly associated with increasing astrocytoma grade, and it may be involved in tumour progression and aggressiveness. In conclusion, this thesis identified therapeutically actionable biomarkers, determinant of chemosensitivity, and prevalence of TMMs in LGG.
Investigating novel therapeutic strategies in DIPG – copper chelation and its effect on epigenetics, metabolomics, and kinase signalling cascades(2023) Michniewicz, FilipThesisDiffuse Intrinsic Pontine Glioma (DIPG) is an incurable paediatric brain cancer, characterised by aggressive diffuse growth in the pontine region of the brain, preventing surgical resection and requiring targeted therapeutic intervention. Over 80% of DIPG patients possess epigenetic alteration H3 K27M, which substitutes a lysine for a methionine in the n-terminus tail of Histone H3, preventing the binding of methyl groups and therefore induces H3 K27 hypomethylation. This alteration partners with other mutations, notably TP53, PIK3CA, PDGFRα and other receptor tyrosine kinase associated alterations and ACVR1, complicating the application of targeted therapies. Over 200 clinical trials investigating various combinations of therapies have thus far failed to improve survival, with the majority of patients passing within a year of diagnosis. Copper is an essential metal ion which is widely incorporated into proteins and enzymes, which take advantage of its redox activity to catalyse reactions. Interestingly, copper is highly accumulated within the brain, is upregulated in several cancers, including brain cancers and is increasingly targeted by therapeutics. In particular, copper is known to influence RTK signalling in cancer, and in Wilson’s Disease has been linked to epigenetic alterations. A recent study also highlighted the redox ability of the H3-H4 tetramer. Upon this basis, it was hypothesised that reducing copper may represent a viable therapeutic strategy for DIPG. Interrogation of patient transcriptomic data revealed correlation between copper chaperones and epigenetic genes, and increased expression of copper chaperone MT1X. Copper reduction through copper chelator tetraethylenepentamine (TEPA) induced apoptosis in 3 DIPG cell lines and dose-dependently reduced phosphorylation of RTK associated proteins. Transcriptomic, proteomic and metabolomic analyses further revealed copper chelation activated inflammasome, impacted cell cycle proteins, impacted citric acid, cysteine and methionine, fatty acid, lipid, purine and pyrimidine metabolism, and dysregulated epigenetic mechanisms. Copper chelation also improved survival in an orthotopically injected PDX model of DIPG, and completely cleared the tumour in 25% of treated mice. This thesis outlines that copper chelation is a potential therapeutic strategy for DIPG, however further work is required to find combinations and more clearly define its cellular effects.
New therapeutic strategies for DIPG – understanding the role of the super BBB in the brainstem and ways to overcome it.(2023) Ung, CaitlinThesisDiffuse Intrinsic Pontine Glioma (DIPG) is an aggressive paediatric high-grade glioma with no effective treatments. The blood-brain barrier (BBB) is a vascular network in the brain that separates peripheral circulation and critical brain tissue, tightly regulating the passage of molecules, ions, and cells. However, this presents a significant challenge to drug delivery, as 98% of all small molecules are excluded from the BBB. Some studies have determined that there are regional differences in BBB permeability, as well as some brain tumours locally altering the vasculature. Few studies have been conducted to understand how DIPG affects the vasculature and whether the brainstem itself could be the reason behind treatment failure. Investigations into whether DIPG location could affect therapeutic efficacy were conducted using in vivo mouse models. It was found that temsirolimus, an mTOR inhibitor, had significantly higher efficacy in the cortex compared to the brainstem with the same DIPG model, shown through increased survival, higher drug levels and improved targeting. Single-cell RNA sequencing was performed on isolated vasculature from DIPG injected mice, in the cortex or brainstem to identify transcriptomic alterations in response to DIPG. Significantly altered pathways were identified in endothelial cells related to apoptosis, ribosomal translation, and inflammatory pathways when DIPG was present in the brainstem. Leakage studies showed a significant reduction in permeability in both the brainstem and cortex injected with DIPG compared to control, suggesting that DIPG induced changes in the vasculature that resulted in a tighter BBB. The pathways identified were then targeted with inhibitors to modulate the BBB. S63845, an MCL-1 inhibitor, omacetaxine, a ribosomal translation inhibitor, S-NGR-TNFα, a vascular targeted TNFα, and IFNγ, were assessed in vitro through BBB transwell assays and western blots to identify their effect on BBB permeability. S63845 significantly altered protein expression of claudin-5 and increased permeability and barrier integrity in an in vitro BBB model. In vivo studies with DIPG models showed that S63845 and S-NGR-TNFα increased the BBB permeability of tracer dye Texas-red dextran. Overall, these studies indicate that the BBB in the brainstem with DIPG is exhibiting a tighter phenotype through effects on endothelial cell pathways associated with BBB permeability. This results in reduced therapeutic efficacy and may contribute to treatment failure. S63845 and S-NGR-TNFα represent promising BBB modulators that may improve the delivery of therapeutics through the restricted BBB, giving potential to enhance the activity of anti-DIPG therapeutics that were previously considered impenetrable.