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Abstract
Neuroblastoma is the most common extracranial solid tumour in the paediatric population, and is a highly aggressive disease that originates from the precursor cells of the postganglionic sympathetic nervous system. A number of genetic factors are associated with the biology of this tumour, of which the most relevant is the MYCN oncogene. This oncogene encodes a transcription factor whose deregulated expression and amplification occurs in approximately 25% of primary neuroblastomas, and represents a powerful independent prognostic marker in this disease. Despite advances in the therapy of childhood malignancies, neuroblastoma remains a clinical challenge, with survival rates of high-risk patients at less than 40%. This thesis, therefore, describes the investigation of novel small molecule inhibitors of the MYCN transcriptional pathway, as potentially therapeutic tools for the treatment of neuroblastoma.
A primary screen of a 34,000 chemical compound library was conducted to identify novel small molecule inhibitors of MYCN, and utilised a novel cell-based method to identify inhibitors without discriminating against their mechanisms of action. This cell-based approach allowed for the identification of molecules that potentially inhibit MYCN directly, at either the DNA or protein interface, or indirectly through upstream signalling events. Examination of the small molecules for their MYCN-inhibiting activity was first addressed by observing the effects of hit compounds on the toxicity and cell physiology of neuroblastoma cells. These studies, which included the determination of MYCN and downstream target gene expression, cell cycle analysis, cell viability studies and analysis of the cell morphology of BE(2)-C neuroblastoma cells, demonstrated growth inhibitive effects that were suggestive of cell cycle arrest and modest cellular differentiation. Subsequent examination of two compounds (N147A7 and N77A7), which exhibited relative stability in rat liver microsomal assays, in a MYCN transgenic mouse model of neuroblastoma demonstrated no inhibition of tumour development or progression. The absence of efficacy in the mouse model and the observation that one compound formed a precipitate upon intraperitoneal injection, suggested that these compounds were limited by their solubility and pharmacokinetic properties, which are common problems in the development of new drugs.
In order to overcome this hurdle, focused chemical libraries of compounds that were structurally similar to the original hit compounds were constructed. These libraries were screened to identify compounds with potentially improved efficacy over the parent compound. One promising compound, M606, was examined for its effects on neuroblastoma cells, including clonogenicity, MYCN and downstream target gene expression and toxicity, and characterised for its mechanism of action. M606 demonstrated selective toxicity to neuroblastoma cell lines in a MYCN-dependent manner, confirming its selectivity for the target gene of interest. Characterisation studies revealed a close relationship between the MYCN and HIF1α (hypoxia-inducible factor 1 alpha) transcriptional pathways, with M606 treatment resulting in MYC downregulation and conversely, upregulation of HIF1α. The results suggested that M606 acts by stabilising HIF1α through the inhibition of prolyl hydroxylase, thus preventing HIF1α from degradation.
M606 was examined for its potential as a therapy, by undertaking a number of in vitro, and in vivo assays, in combination with current chemotherapeutic drugs used in the treatment of neuroblastoma. These studies demonstrated the capacity of this small molecule compound to act in combination with either cisplatin or vincristine, and treatment of MYCN transgenic mice with these drug combinations resulted in an increase in the delay to tumour development and progression. Thus, these studies demonstrated the ability to isolate and characterise a novel small molecule inhibitor of MYCN with potential therapeutic value in the treatment of neuroblastoma.
Finally, studies were undertaken to isolate small molecule inhibitors of a downstream target of MYCN, MRP4 (multidrug resistance-associated protein 4). The relevance of MRP4 in the malignant phenotype of neuroblastoma is evidenced by the correlation of its overexpression with a poor prognosis and outcome. Chemical library screening led to the identification of highly specific and potent inhibitors of this drug transporter. In particular, two compounds, hits 46 and 52, were found to be able to mimic the effects of MRP4 siRNA on neuroblastoma cells, by inducing neurite formation and inhibiting the rate of growth of these cells in the absence of any chemotherapy. The identification of compounds with varied mechanisms of MRP4 inhibition has the potential to be further explored as therapies across a range of diseases overexpressing this multidrug transporter.
In conclusion, this study provides evidence for the potential to develop novel molecular targeted therapies for the treatment of neuroblastoma, using high-throughput screening methods. This approach enabled the identification of small molecule inhibitors of the MYCN transcriptional pathway. One molecule in particular, M606, demonstrated selective toxicity and growth inhibition against MYCN-overexpressing neuroblastoma cells, as well as clinical potential when used in combination with chemotherapeutics currently used in the treatment of neuroblastoma. In addition, investigation of the mechanism of action of this molecule highlighted the advantage of utilising a cell-based screening method over a more rational drug design approach. Furthermore, this study emphasises the importance of the MYCN oncogenic pathway, as well as its transcriptional target, MRP4 in the malignant phenotype of neuroblastoma. Novel inhibitors of MRP4 that were identified also demonstrated potential clinical utility for the treatment of neuroblastoma