Achaete-scute homolog 1 (Ascl1) is a proneural transcription factor that plays a central role in regulating differentiation of noraderinergic neuron precursors during embryonic development, the cell type from which neuroblastoma is thought to arise. In a developmental model, we have shown that Ascl1 transcriptional activity is inhibited by multi-site phosphorylation on serine/proline sites, potentially by cdk kinases that drive cell cycle progression. Ascl1 is highly expressed in multiple neuroblastoma cell lines but the endogenous protein is usually phosphorylated. We are characterizing how phospho-regulation of Ascl1 by cyclin-dependent kinases regulates its ability to drive differentiation by genome-wide characterization of its targets. We are also exploring Ascl1 phospho-regulation of chromatin and co-factor binding.
Using chemical inhibitors and western blotting to characterise the phospho-regulation of Ascl1 in SHSY5Y neuroblastoma cells, we see that phosphorylation of Ascl1 is cell cycle regulated, and that Ascl1 is a target of the mitotic kinase Cdk1. Expression of a phospho-mutant Ascl1 or addition of Cdk1 inhibitor both cause cell cycle exit and morphological clustering, reminiscent of ganglion formation during development. We are also using inducible expression of wild-type and phosphomutant Ascl1 in these cells to characterize phosphorylation status-related morphological changes, DNA binding alterations (by ChIP-seq) and genome-wide transcriptome changes (by RNA-seq). We have identified over 6000 transcripts that are upregulated when Ascl1 is overexpressed. 1780 of there are specifically upregulated by phospho-mutant Ascl1 and these are highly enriched for differentiation-specific genes. Many of these genes are bound directly by Ascl1.
In summary, we find that Ascl1 is phosphorylated by Cdk1, while its dephosphorylation potentiates neuroblastoma cell differentiation. These findings indicate that cdk inhibitors may have therapeutic benefit by both arresting cell cycle and potentiating differentiation. Moreover, we additionally identify potential new biomarkers of neuroblastoma differentiation.