Ascl1 phospho-status regulates neuronal differentiation in a Xenopus developmental model of neuroblastoma

Neuroblastoma (NB), although rare, accounts for 15% of all pediatric cancer mortality. Unusual among cancers, NBs lack a consistent set of gene mutations and excluding large-scale chromosomal rearrangements, and the genome appears to be largely intact. Indeed, many interesting features of NB suggest it has less in common with adult solid tumours but instead has characteristics of a developmental disorder. NB arises overwhelmingly in infants under 2 years during a specific window of development, and histologically NB bares striking similarity to undifferentiated neuroblasts of the sympathetic nervous system, its likely cells of origin. Hence, NB could be considered a disease of development arising when neuroblasts of the sympathetic nervous system fail to undergo proper differentiation, but instead are maintained precociously as progenitors with the potential for acquiring further mutations eventually resulting in tumour formation. To explore this possibility, we require a robust and flexible developmental model to investigate the differentiation of NB's presumptive cell of origin. Here we use Xenopus frog embryos to characterise differentiation of anteroventral noradrenergic (AVNA) cells, cells derived from neural crest. We find these cells share many characteristics with their mammalian developmental counterparts, and also with NB cells. We find that the transcriptional regulator Ascl1 is expressed transiently in normal AVNA cell differentiation but its expression is aberrantly maintained in NB cells, where it is largely phosphorylated on multiple sites. We show that Ascl1's ability to induce differentiation of AVNA cells is inhibited by its multi-site phosphorylation at serine-proline motifs, while overexpression of Cyclin/CDKs and MYCN inhibit wild-type Ascl1-driven AVNA differentiation, but not differentiation driven by a phosphomutant form of Ascl1. This suggests that maintainance of Ascl1 in its multiply phosphorylated state may prevent terminal differentiation in NB, which may offer new approaches for differentiation therapy in NB.