Genome methylation defines EZH2 inhibitor resistance in neuroblastoma [I]

EZH2 is a H3K27 methylase and a target of cancer epigenetic treatments. We recently reported the oncogenic roles and NTRK1 (TRKA) epigenetic regulation by EZH2 in the MYCN-amplified aggressive neuroblastomas (NB) (Li et al., ONCOGENE, 2018). Here, we investigated the effects and function of small molecule EZH2 inhibitor (EZH2i) on aggressive NB model cell lines. We examined the antitumor effects of EZH2i using WST assay and colony formation assay. By EZH2i treatments, suppression of proliferation was observed in SK-N-SH and SK-N-BE cells (sensitive cells) dose-dependently, whereas it was not observed in IMR32 and NGP cells (resistant cells). FACS analysis showed apoptosis and G0/G1 cell cycle arrest in sensitive cells. Transcriptome analysis and GSEA indicated significant changes were observed in the gene set related to cell cycle arrest and differentiation in the sensitive cells. We selected genes induced at mRNA level by EZH2i only in the sensitive cells and confirmed tumor suppressor function in NB cells. Almost of the EZH2i-induced gene promoters were marked by H3K27me3 in MYCN-amplified NB cell lines. Interestingly, a part of the EZH2i-induced gene promoters have CpG islands and methylated in NB tumor samples registrated in databases. Further, combination of EPZ6438 and 5-aza-deoxycitide, resulted in effective suppression of proliferation in the EPZ6438-resistant 3 NB cell lines. Transcriptome/methylome analysis of the EPZ6438 and 5-aza-deoxycitide-treated NB cells revealed the combinational epigenetic regulation of the tumor suppressors and oncogene expression. Finally, methylome analysis of the promoter regions, e.g. VSTM2L, GPNMB, and TIMP3 CpG islands can be biomarkers of EPZ6438-registancy in unfavorable NB patients. These responsible CpG island methylation as biomarkers for the application of EZH2i/DNMTi combination therapy. RNA was extracted 3 (SK-N-BE, SK-N-SH, NGP) or 4 (IMR32) days after EPZ-6438 1 µM treatment, and a microarray analysis was performed using the Agilent platform of 8x60 K design ID G4851B (Agilent Single Color. 39494, Agilent Technologies). Two hundred nanograms of total RNA was labeled with Cyanine3 using a Low Input Quick-Amp Labeling Kit (one color, Agilent Technologies) according to the manufacturer’s instructions. Purified labeled total RNA was hybridized to the expression microarray. Hybridization, the scanning of microarrays, and data extraction from scanned images were conducted according to Agilent protocol version 6.9.