Synthetic lethality of EZH2 and DNMT Inhibition suppresses neuroblastoma proliferation via MYCN destabilization

Enhancer of Zeste Homolog 2 (EZH2) is highly expressed in various cancers and is thought to contribute to cancer malignancy through the methylation of lysine 27 on histone H3 (H3K27). Therefore, EZH2 is an H3K27 methylase and a target of cancer epigenetic treatments. Here, we investigated the effects and functions of a small-molecule EZH2 inhibitor (EZH2i) in aggressive neuroblastoma (NB). To identify genes associated with the sensitivity to EZH2i, we classified NB cell lines into sensitive and resistant groups. EZH2i suppressed proliferation and induced cell cycle arrest in EZH2-sensitive NB cells. Transcriptome analysis revealed the de-repression of gene sets related to differentiation and cell cycle arrest. To investigate whether the gene repression in resistant cells is mediated by mechanisms other than H3K27 methylation, methylome analysis was conducted to evaluate the significance of DNA methylation. The effect of combined treatment with an EZH2 inhibitor and a DNA methylation inhibitor (DNMTi) on the growth of resistant NB cells was subsequently assessed both in vitro and in vivo, even in cells resistant to EZH2 inhibition. Genes such as TRIM63, VSTM2L, GPNMB, and TIMP3, which are de-repressed by EZH2is in sensitive cells. Furthermore, the promoters of some of these genes are hypermethylated in EZH2i-resistant cells and NB tissues. Additionally, the combined treatment with EZH2i and 5-aza-dC significantly inhibited cell proliferation with MYCN destabilization at protein level, c-MYC suppression at RNA and protein level and induced a robust differentiation phenotype. Our study identified gene expression profiles that can distinguish the responsiveness to EZH2i in neuroblastoma, and demonstrated that DNMTi not only de-repression in gene expression but also suppress neuroblastoma growth through inhibition of MYC. These results confirm the importance of epigenetic regulation in NB biology and provide information for developing epigenetic therapies for patients with advanced NB.