Abstract 5447: Phosphorylation status of Ascl1 regulates neuroblastoma self-renewal and differentiation.

Abstract

Abstract Neuroblastoma (NB) is a tumor of infancy that accounts for 15% of all pediatric cancer mortality. NB bares striking similarity to undifferentiated neuroblasts of the sympathetic nervous system in gene expression profiles and histological appearance. In most neuroblastoma tumors, the cell cycle is hyperactive due to overexpression of cyclin D1 and amplification of MYCN. However, retinoic acid (RA) induces differentiation and cell cycle exit of some NB cell lines in vitro and provides significant benefit to patients in the clinic. As neuroblastoma appears to result from sustained proliferation and inhibition of differentiation, we sought to understand how cell cycle and differentiation are linked within NB. We focused our efforts on Ascl1, a proneural transcription factor that is both necessary and sufficient for neural differentiation of noradrenergic neurons and has been shown to be regulated by the cell cycle. Paradoxically, Ascl1 is both highly expressed and associated with poor prognosis in NB. However, we hypothesized that Ascl1 phosphostatus critically regulates its ability to induce differentiation. We show that Ascl1 is highly expressed and phosphorylated across multiple NB cell lines and is largely phosphorylated by CDKs. Phosphorylated Ascl1 actively promotes the G1-S transition by upregulating E2f, Skp2, and Cdk2. These targets all promote CDK activity, further maintaining Ascl1 phosphorylation. However, a phosphomutant form of Ascl1, where putative CDK sites are mutated, arrests cells in the G1 phase by preferentially upregulating Ebf3 and p27. These targets inhibit CDK activity promoting Ascl1 dephosphorylation and further upregulation of Ebf3 and p27. We propose a model whereby Ascl1 sits between two positive feedback loops, both forming a critical link between cell cycle and differentiation, but also as an agent to commit cells to either a self-renewing undifferentiated state or a postmitotic differentiated state. Given this model, we hypothesized that pharmacologic inhibition of CDK activity when combined with a pro-differentiation agent such as RA would synergistically cause differentiation in neuroblastoma. Indeed, CDK inhibitor and RA combination treatment causes increased differentiation in RA-susceptible cell lines and induces differentiation in RA-resistant cell lines. Given that most neuroblastoma tumors have an overactive cell cycle and also highly express Ascl1, combination therapy of CDK inhibitor and RA may provide generalized benefit to NB patients. Citation Format: Luke A. Wylie, Carol J. Thiele, Anna Philpott. Phosphorylation status of Ascl1 regulates neuroblastoma self-renewal and differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5447. doi:10.1158/1538-7445.AM2013-5447