For differentiation-defective malignancies, compounds that modulate transcription, such as retinoic acid and histone deacetylase (HDAC) inhibitors, are of particular interest. HDAC inhibitors are currently under investigation for the treatment of a broad spectrum of cancer diseases. However, one clinical drawback is class-specific toxicity of unselective inhibitors, limiting their full anticancer potential. Selective targeting of individual HDAC isozymes in defined tumor entities may therefore be an attractive alternative treatment approach. We have previously identified HDAC family member 8 (HDAC8) as a novel target in childhood neuroblastoma. Using small-molecule inhibitors, we now demonstrate that selective inhibition of HDAC8 exhibits antineuroblastoma activity without toxicity in two xenograft mouse models of MYCN oncogene-amplified neuroblastoma. In contrast, the unselective HDAC inhibitor vorinostat was more toxic in the same models. HDAC8-selective inhibition induced cell cycle arrest and differentiation in vitro and in vivo. Upon combination with retinoic acid, differentiation was significantly enhanced, as demonstrated by elongated neurofilament-positive neurites and upregulation of NTRK1. Additionally, MYCN oncogene expression was downregulated in vitro and tumor cell growth was markedly reduced in vivo. Mechanistic studies suggest that cAMP-response element-binding protein (CREB) links HDAC8- and retinoic acid-mediated gene transcription. In conclusion, HDAC-selective targeting can be effective in tumors exhibiting HDAC isozyme-dependent tumor growth in vivo and can be combined with differentiation-inducing agents.

Neuroblastoma is an embryonic solid tumor of neural crest origin and accounts for 11% of all cancer-related deaths in children. Novel therapeutic strategies are therefore urgently required. MYCN oncogene amplification, which occurs in 20% of neuroblastomas, is a hallmark of high risk. Here, we aimed to exploit molecular mechanisms that can be pharmacologically addressed with epigenetically modifying drugs, such as histone deacetylase (HDAC) inhibitors. Grainyhead-like 1 (GRHL1), a gene critical for Drosophila neural development, belonged to the genes most strongly responding to HDAC inhibitor treatment of neuroblastoma cells in a genomewide screen. An increase in the histone H4 pan-acetylation associated with its promoter preceded transcriptional activation. Physically adjacent, HDAC3 and MYCN colocalized to the GRHL1 promoter and repressed its transcription. High-level GRHL1 expression in primary neuroblastomas correlated on transcriptional and translational levels with favorable patient survival and established clinical and molecular markers for favorable tumor biology, including lack of MYCN amplification. Enforced GRHL1 expression in MYCN-amplified neuroblastoma cells with low endogenous GRHL1 levels abrogated anchorage-independent colony formation, inhibited proliferation, and retarded xenograft growth in mice. GRHL1 knockdown in MYCN single-copy cells with high endogenous GRHL1 levels promoted colony formation. GRHL1 regulated 170 genes genome-wide, and most were involved in pathways regulated during neuroblastomagenesis, including nervous system development, proliferation, cell-cell adhesion, cell spreading, and cellular differentiation. In summary, the data presented here indicate a significant role of HDAC3 in the MYCN-mediated repression of GRHL1 and suggest drugs that block HDAC3 activity and suppress MYCN expression as promising candidates for novel treatment strategies of highrisk neuroblastoma. Cancer Res; 74(9); 2604-16. Ó2014 AACR.