Abstract A15: Epigenetic regulators maintain neuroblastoma cancer stem cells: Model to treatment

High risk neuroblastoma (NB) represents a major clinical challenge in pediatric oncology and accounts for 15% of pediatric cancer deaths. Our recent studies suggest that neuroblastoma tumors are heterogeneous and composed of multiple subpopulations including cancer stem cells (CSCs), marked by Granulocyte Colony Stimulating Factor Receptor (G-CSFR or CD114) positive cells. These CD114+ cells are shown to be highly tumorigenic, metastatic, self-renewing and can differentiate into non-tumorigenic progeny. As tumor subpopulations typically demonstrate distinct epigenetic modifications at histone H3K4 and H3K27, we evaluate these epigenetic marks at the CSF3R locus (gene encoding G-CSFR) by ChIP-qPCR. Results suggest that the CSF3R gene is switched ‘on’ in NB CSCs via reversible epigenetic histone modifications. The CSF3R promoter in CD114+ NB CSCs, shows increased activating H3K4me3 marks and reduced repressive H3K27me3 marks. In the CD114- bulk tumor population, we found the opposite pattern of increased H3K27me3 marks and reduced H3K4me3 marks. In addition, knockdown of EZH2 (the PRC2 methyltransferase responsible for H3K27me3 marks) increases CD114+ expression in both cell culture and murine xenograft models of neuroblastoma. Furthermore, gene expression studies revealed high levels of MLL1 (encoded by KMT2A), and Jumonji D3 (JMJD3, encoded by KDM6B) in CD114+ cells. MLL1 is a histone methyltransferase that catalyzes mono-, di- and tri-methylation on H3K4, while JMJD3 is an H3K27me3 demethylase. Blocking MLL1 and JMJD3 with specific small molecule inhibitors, MM-102 and GSK-J4 reduces overall survival of NB cells and also reduces the percentage of CD114+ cells in vitro. Importantly, targeting these epigenetic regulators rapidly down-regulate the G-CSF receptor expression and induces apoptosis specifically in CD114+ cells but not in CD114- cells, suggesting maintenance of NB CSCs by these epigenetic modifiers. In vivo studies of these epigenetic inhibitors in neuroblastoma murine xenograft models validate our in vitro studies and demonstrate highly significant reduction in overall tumor size and metastasis with correlated reduction of total CD114+ cells in resected tumors. Overall, we demonstrate that histone modifications play a key role in maintaining the NB cancer stem cells, and that specific epigenetic inhibitors are highly potent anti-tumor agents for high risk neuroblastoma. Our findings have direct translational impact and support the development of