Investigating the effect of MYCN overexpression and 17q gain on neural crest development using human pluripotent stem cells

Neuroblastoma is one of the most common paediatric tumours, it impacts 1 in 8000 live births and accounts for approximately 15% of mortalities in children. Tumours arise during embryonic development, in tissues derived from the trunk neural crest and sympathoadrenal lineage. Several genetic aberrations have been identified, two of the most common and severe are gain of the 17q chromosomal arm and amplification of the transcription factor MYCN. Current models either use neuroblastoma derived cell lines or animal models, these fail to fully recapitulate the initiating events of neuroblastoma in neural crest development in a human context. Here I have established an in vitro model of MYCN amplified neuroblastoma in humans, by combining the use of in vitro differentiation to trunk neural crest and sympathoadrenal lineage, with human pluripotent stem cells containing these gain of 17q and inducible MYCN overexpression. This model, has allowed for temporal dissection of tumour-initiating events that disrupt differentiation. Here I show that inducing MYCN overexpression in vitro alters differentiation to trunk neural crest and sympathetic neuron specification, promotes hallmarks of tumorigenesis and the formation of tumours. I have also shown that this phenotype is more severe when MYCN overexpression is combined with gain of 17q, suggesting that 17q gain combined with MYCN overexpression primes the cells for a more aggressive tumour phenotype. I have used RNA-sequencing to understand how the different genetic lesions alter the transcriptomics of the differentiation to trunk neural crest cells. I have also used ATAC-sequencing to study the chromatin accessibility along the differentiation and uncovered a 17q MYCN specific opening of chromatin regions associated with binding of the YAP protein. I then aimed to further understand and characterise this 17q MYCN specific YAP phenotype.