Title: Drugable epigenetic control by modulation a transcriptional master regulator:switching neuroblastoma from a proliferative to a differentiation programme
Funder: Cancer Research UK

Abstract: Background: Neuroblastoma, the most common extracranial solid cancer in children, arises from noradrinergic neuroblasts of the sympathetic nervous system, and causes 15% of pediatric cancer deaths despite the use of aggressive multimodal chemotherapy. Neuroblastoma harbors few recurrent mutations; instead, its pathogenesis may be driven by epigenetic changes locking neuroblasts into a pro-proliferative developmental programme, which is further subverted by events such as N-MYC amplification that accompany high-risk disease. We aim to reactivate the stalled differentiation programme by using our recent findings about regulation of proliferation and differentiation in neuroblastoma. The transcriptional master regulator, ASCL1, plays a central role in controlling noradrinergic neuron differentiation during development. Importantly, we have recently showed that ASCL1 is phosphorylated by Cyclin-dependent Kinases (CDKs) in actively dividing cells, where it directs a pro-proliferative transcriptional programme; conversely ASCL1 dephosphorylation, resulting from CDK inhibition, directs a pro-differentiation programme. Here, we address the overarching hypothesis that phosphorylation of ASCL1 by CDKs controls the switch between proliferative and differentiation transcriptional programmes in neuroblastoma cells, and aim to identify reactivation of an ASCL1-driven differentiation programme as a novel neuroblastoma therapy. Aims: We will identify regulatory phosphorylation sites on ASCL1 and the kinases responsible. Then, we will test the hypothesis that phospho-ASCL1 associates with clustered transcriptional enhancers (CEs) promoting neuroblastoma proliferation but when dephosphorylated, ASCL1 binds to and activates differentiation-specific CEs. We will identify an ASCL1-dependent signature of differentiation and use it to test the ability of small-molecule CDK inhibitors to drive neuroblastoma differentiation in vitro and in vivo. We will also characterise functional interactions of ASCL1 with co-factors including MYCN and the epigenetic modulator BET proteins at pro-proliferative and differentiation-associated CEs genome-wide, and at selected key promoters. We will test small molecule BET inhibitors as a method of potentiating tumour differentiation in vitro and in vivo. Methods: We will take genome-wide approaches including ChIPseq, RNAseq and ATACseq, along with bioinformatic analysis, comparing ASCL1 targets with gene expression and outcome data from patient cohorts. We also use small molecule inhibitors of CDKs and BET proteins to assay differentiation targets genome-wide in multiple neuroblastoma lines and genetically modified mouse models of neuroblastoma. How research will be used: 1) To characterise the epigenetic basis of alternative ASCL1-dependent programmes of progenitor maintenance versus differentiation and its perturbation by NMYC, epigenetic modulation and kinase/BET inhibition. 2) To provide in vivo validation of the new therapeutic approach of epigenetic reactivation of normal developmental differentiation to treat neuroblastoma.


Research activity codes: 5.1 PHARMACEUTICALS;1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: CANCER AND NEOPLASMS