Medicine & Health

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  • (2022) Salib, Alice
    Neuroblastoma (NB) is a solid tumor of the developing sympathetic nervous system that occurs during childhood and has a less than 50% survival rate for high-risk patients. Amplification of the MYCN oncogene remains the single most important genetic predictor of poor prognosis. Many of the protumorigenic functions of the oncogene MYCN are attributed to its regulation of global gene expression programs. Alternative splicing is another important regulator of gene expression and has been implicated in NB development, however, the molecular mechanisms behind this remain unknown. In this thesis, the core spliceosomal protein, SNRPD3, was found to be significantly up-regulated in cell and animal models of NB initiation and progression. Moreover, high SNRPD3 expression correlated with poor patient prognosis and is an independent prognostic factor. Further investigation revealed that SNRPD3 was not only a MYCN transcriptional target but together with MYCN formed a protein complex with PRMT5, the protein responsible for methylating SNRPD3. RNA-sequencing revealed an overall increase in the number of genes being differentially spliced when MYCN was overexpressed, while depletion of SNRPD3 in the presence of overexpressed MYCN was sufficient to induce additional increases in differential splicing, particularly genes involved in the cell cycle. SNRPD3 knockdown in the presence of MYCN was accompanied with marked loss of cell viability, suggesting that SNRPD3 maintains a splicing balance for MYCN, and prevents detrimental differential splicing of cell cycle genes in NB. Further analysis revealed BIRC5 and CDK10 to be among the topmost differentially spliced cell cycle genes, both of which play a role in the G2/M phase of the cell cycle. Flow cytometry analysis revealed that depletion of SNRPD3 resulted in G2/M cell cycle arrest. Furthermore, SNRPD3 was an oncogenic co-factor for MYCN in vitro and in vivo. As such, SNRPD3 represents a therapeutic vulnerability for NB cells in the presence of high MYCN expression. Indeed, the PRMT5 inhibitor, JNJ64619178 and GSK3326595, that prevents SNRPD3 methylation was cytotoxic in NB cell lines with high SNRPD3 and MYCN expression. Overall, this thesis provides mechanistic insight into the function of SNRPD3 in MYCN-driven NB and highlights the importance of SNRPD3-dependent balanced splicing. Furthermore, this thesis presents preliminary data for a novel therapeutic approach for neuroblastoma patients using PRMT5 inhibitors, currently in clinical trials.

  • (2023) Michniewicz, Filip
    Diffuse 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.