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(2021)ThesisPeripheral neuroblastic tumors (PNTs) represent a spectrum of tumors derived from the neural crest and account for 10% of paediatric malignancies. PNTs comprise four histological subtypes, including neuroblastoma (NB). Half of high-risk NB patients relapse after chemotherapy, and relapsed patients seldom respond to therapy afterward. This study aimed to identify chemoresistance mechanisms and effective combination therapies against chemoresistant NB. In the first results chapter, intratumoural heterogeneity in PNT subtypes were first investigated using single cell RNA sequencing (scRNA-seq) on 7 primary PNTs spanning all histological subtypes. This revealed extensive transcriptome heterogeneity among PNTs and identified malignant neuroblasts differentiating through a novel “transitional” state. This state resembled known chemoresistant cell phenotypes and predicted poor NB patient prognoses. In the second results chapter, drug resistance mechanisms in NB were explored in vitro using longitudinal IMR-32 cisplatin-resistant cell line models. Following scRNA-seq, resistant cells undergoing cytoskeletal remodelling were identified and then characterised in vitro. Additionally, tumour samples from 5 high-risk NB patients at diagnosis and post-chemotherapy were analysed with scRNA-seq, where an enrichment of oncogenic cell states was observed. In the third results chapter, potential combination therapies were identified using a combinatorial drug screen against the chemoresistant SK-N-BE(2)-C cell line. The highest synergy manifested between two histone methyltransferase inhibitors, GSK343 and SGC0946. Mechanistic studies revealed the induction of ER stress programs by the combination treatment. The administration of this combination therapy in vivo reduced NB tumour growth. In the fourth results chapter, an integrative approach was used to identify mitochondrial drug targets in NB, whereby the mitochondrial translocase SLC25A5 was prioritized. Targeting SLC25A5 with the selective inhibitor, PENAO, reduced cell viability in NB cell lines, however TP53 mutations conferred chemoresistance. The histone deacetylase inhibitor SAHA combined with PENAO overcame mutant TP53 mediated chemoresistance to synergistically reduce NB cell and tumour growth. Collectively, this thesis has identified transcriptional cell states that drive chemoresistance and combination therapies that overcome chemoresistant NB, thereby contributing novel therapeutic targets and treatment avenues for NB patients.
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(2022)ThesisNeuroblastoma (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.
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(2021)ThesisAdvanced neuroblastoma is associated with MYCN amplification and gene amplification of MYCN has been shown to correlate with poor prognosis in patients. The MYC family of proteins have been widely researched due to their important roles in the development of various cancers including neuroblastoma. Pharmaceutical companies and researchers have attempted to develop novel small molecule inhibitors which are able to target c-MYC and MYCN directly. However there has been limited success in the development of these inhibitors, and to date no MYCN direct inhibitors are in clinical use or clinal trials. It is known that MYCN regulates the transcription of several genes that are involve in cell proliferation and cell death including both tumour suppressor and oncogenes. Therefore, is has been suggest that rather targeting MYCN directly, it may be a better approach toidentify and target proteins which regulate or directly bind to MYCN as potential therapeutic targets. The hypotheses for this thesis is that USP5 acts in an oncogenic manner by directly interacting with MYCN and regulate MYCN stability to promote neuroblastoma tumorigenesis and that novel small molecule compounds are potent and selective in targeting MYCN and USP5 in MYCN-driven neuroblastoma tumorigenesis. Three aims have been established to assess these hypotheses. The first aim is to determine the functional role of USP5 in neuroblastoma tumorigenesis using various phenotypical assays in vitro and in vivo models. The rationale of this aim is to investigate whether USP5 is a potential therapeutic target for the treatment of neuroblastoma. Several studies have reported that USP5 acts in an oncogenic manner in various adult cancers by regulating different cellular pathways. Therefore, aim two of the study is to determine whether USP5 function is in MYCN dependent manner and to study the mechanism of USP5 as an oncogenic protein in neuroblastoma. By addressing this aim this thesis will explore USP5’s role in neuroblastoma tumorigenesis and provide insight into the molecular mechanism which used by USP5 to stabilize MYCN. The third aim of this thesis is to evaluate the efficacy and study the drug action of novel USP5 and MYCN inhibitors for the treatment of neuroblastoma. This aim will demonstratethe importance of developing novel compounds with clinical potential for better targeted treatment options. In summary, the purpose of this thesis is to investigate the role of USP5 in MYCN-drive neuroblastoma and highlight a novel therapeutic option for the treatment of MYCN amplified neuroblastoma by targeting the interaction between USP5 and MYCN.