Abstract SY28-02: A phosphorylation switch regulating proliferation and differentiation in Xenopus development and neuroblastoma

Abstract

Abstract Neuroblastoma is associated with stalling of normal differentiation, resulting in excessive proliferation of neuroblastic precursors. Here we show that Xenopus frog embryos can be used as a rapid and versatile model to investigate the molecular regulation of proliferation versus differentiation of neuroblasts and what may go wrong with this in neuroblastoma. We find that CDK-dependent phosphorylation of the transcriptional master regulator ASCL1 plays a critical role in controlling the balance between cell proliferation and differentiation in embryonic neuroblasts and in neuroblastoma, and can be modulated to reactivate differentiation of neuroblastoma cells. ASCL1 is a master regulator proneural transcription factor that controls the balance between proliferation and differentiation of neuroblasts and is expressed transiently during formation of noradrenergic neurons arising from the neural crest. Embryos of the frog Xenopus laevis provide a versatile, rapid, and inexpensive system to study the role of ASCL1 phosphorylation by cyclin-dependent kinases in restraining neuronal differentiation. We found that enhanced cyclin-dependent kinase activity or elevated MYCN levels are sufficient to lock neuroblasts into a noradrenergic precursor-like state by multisite phosphorylation of ASCL1 protein. Conversely, preventing phosphorylation of ASCL1 can potentiate neuronal differentiation even in the face of proproliferative cues. Thus, the in vivo use of Xenopus as a model organism led us to the hypothesis that neuroblastoma arises from neuroblasts that fail to undergo the normal developmental program of differentiation due to precocious phosphorylation of ASCL1. We have gone on to test this hypothesis directly in neuroblastoma cells. We show that ASCL1 is phosphorylated by cyclin-dependent kinases and that phospho-ASCL1 supports the proproliferative program in neuroblastoma cells. However, preventing CDK-dependent phosphorylation of ASCL1 results in changes in the genome-wide transcriptional program of neuroblastoma cells, leading to suppression of proproliferative targets and simultaneous activation of genes that drive cell cycle exit and differentiation. Mechanistically, ASCL1 ChIPSeq reveals enhanced binding of un(der)phosphorylated ASCL1 at sites associated with prodifferentiation targets. Moreover, PHOX protein binding at key downstream regulatory elements is also modulated by ASCL1 phosphorylation. Finally, we also show that chemical CDK inhibition is sufficient to drive differentiation of neuroblastoma cells in a manner dependent on endogenous ASCL1. Therefore, we conclude that CDK-dependent phosphorylation of ASCL1 acts as a critical fulcrum controlling the balance between proliferation and differentiation in Xenopus and in human cells, and thus offers a novel therapeutic opportunity for neuroblastoma. Citation Format: Anna Philpott, Fahad Ali, Luke Wylie, Daniel Marcos Corchado, Igor Chernukhin, Evangelia Papachristou, Tatiana Papkovskaia, Liam Lee, Christopher Weekes, Tom Hiscock, Clive D'Santos, Suzanne Turner, Jason Carroll. A phosphorylation switch regulating proliferation and differentiation in Xenopus development and neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY28-02.