The Role of the Proneural Transcription Factor ASCL1 in Neuroblastoma Cell Division and Differentiation

Neuroblastoma is the most common solid childhood cancer and typically has a very poor prognosis. Neuroblastoma is a ‘cancer of improper development’ and is thought to arise from sympathetic neuroblast precursors that fail to engage the neuronal differentiation programme; instead they are locked in a pro-proliferative developmental state. Neuroblastomas are epigenetically regulated, a core regulatory circuit (CRC) of transcription factors maintaining their highly proliferative, developmental state. In subtype MS neuroblastoma, tumours can spontaneously regress, in which case children are cured for life. The mechanisms behind spontaneous regression are poorly understood, but the main hypothesis is that the cells enter a terminal differentiation programme. Thus, harnessing the latent differentiation capacity of neuroblasts provides an exciting therapeutic avenue for drug induced cell differentiation and tumour clearance in neuroblastoma. ASCL1 is a master transcriptional regulator which modulates both proliferation and differentiation of sympathetic neuroblast precursor cells. During development, ASCL1 is transiently expressed and is downregulated as cells differentiate into mature sympathetic neurones. In high-risk neuroblastomas, the levels of ASCL1 remain high, supporting proliferation. The aim of this project was to understand the effect of ASCL1 deletion on neuroblastoma cell behaviour. CRISPR technology was used to remove ASCL1 from three different neuroblastoma cell lines to study the effect of losing ASCL1 in different cellular contexts. It was found that ASCL1 deletion results in slower cell growth, a phenotype consistent in all neuroblastoma cell lines tested. Studies show no difference in the transcription and expression of the CRC transcription factors, but instead their ability to bind to regulatory regions of chromatin is compromised. RIME analysis shows that ASCL1 binds components of the CRC on the chromatin suggesting ASCL1 could be directly recruiting targets. ASCL1 is considered a pioneer factor and ATAC-Seq analysis shows that chromatin accessibility is reduced in the ASCL1 knock-out lines, suggesting ASCL1 could be limiting both chromatin accessibility and directly recruiting transcription factors to the chromatin. In addition to these findings, when analysing RNA-Seq and ATAC-Seq data it appears ASCL1 maintains neuroblastoma cells in a state which is primed for differentiation. Taken together these results suggest ASCL1 has a dual role in neuroblastoma, supporting both the proliferative state and also poising cells for differentiation.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2021

Author dc:creator

• Parkinson, Lydia

Advisor dc:contributor.advisor

• Philpott, Anna

• Neuroblastoma
• ASCL1
• Differentiation
• Chromatin Accessibility

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.83261

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/335827