Combined inhibition of histone methyltransferases EZH2 and DOT1L is an effective therapy for neuroblastoma

The child cancer, neuroblastoma (NB), is characterised by a low incidence of mutations and strong oncogenic embryonal driver signals. Many new targeted epigenetic modifier drugs have failed in human trials as monotherapy. We performed a high‐throughput, combination chromatin‐modifier drug screen against NB cells. We screened 13 drug candidates in 78 unique combinations. We found that the combination of two histone methyltransferase (HMT) inhibitors: GSK343, targeting EZH2, and SGC0946, targeting DOT1L, demonstrated the strongest synergy across 8 NB cell lines, with low normal fibroblast toxicity. High mRNA expression of both EZH2 and DOT1L in NB tumour samples correlated with the poorest patient survival. Combination HMT inhibitor treatment caused activation of ATF4‐mediated endoplasmic reticulum (ER) stress responses. In addition, glutathione and several amino acids were depleted by HMT inhibitor combination on mass spectrometry analysis. The combination of SGC0946 and GSK343 reduced tumour growth in comparison to single agents. Our results support further investigation of HMT inhibitor combinations as a therapeutic approach in NB. SK‐N‐BE(2)‐C and KELLY neuroblastoma cell lines were seeded in T25 flasks. Following 24 h of incubation cell culture medium was replaced with medium containing either DMSO, 12.8 μM SGC0946, 12.8 μM GSK343 or a combination of both drugs. Treated cells were incubated for 6 h, followed by total RNA extraction and quantitation. RNA from treated samples were then subject to microarray profiling via the PrimeView Human Genome U219 Array (#901605 Applied Biosystems) according to manufacturer instructions, by the Ramaciotti Centre for Genomics (UNSW, Australia).

儿童癌症神经母细胞瘤（neuroblastoma, NB）以突变发生率低、存在强效致癌胚胎驱动信号为典型特征。多款新型靶向表观遗传修饰药物作为单药疗法开展人类临床试验时均以失败告终。我们针对神经母细胞瘤细胞完成了一项高通量染色质修饰药物联合筛选实验：对13种候选药物的78种独特组合进行了筛选。结果发现，两种组蛋白甲基转移酶（histone methyltransferase, HMT）抑制剂的联合使用——靶向EZH2的GSK343与靶向DOT1L的SGC0946——在8株神经母细胞瘤细胞系中展现出最强的协同效应，且对正常成纤维细胞的毒性较低。神经母细胞瘤肿瘤样本中EZH2与DOT1L的mRNA高表达均与患者最差的生存率呈显著相关。联合使用这两种组蛋白甲基转移酶抑制剂可激活ATF4介导的内质网（endoplasmic reticulum, ER）应激应答。此外，质谱分析结果显示，该联合用药方案会导致谷胱甘肽与多种氨基酸发生耗竭。与单药治疗相比，SGC0946与GSK343的联合给药可显著抑制肿瘤生长。本研究结果支持进一步探索组蛋白甲基转移酶抑制剂联合疗法用于神经母细胞瘤治疗的可行性。
将SK-N-BE(2)-C与KELLY神经母细胞瘤细胞系接种于T25培养瓶中。孵育24小时后，将细胞培养基更换为分别含有二甲基亚砜（DMSO）、12.8μM SGC0946、12.8μM GSK343或两种药物联合的培养基。处理后的细胞继续孵育6小时，随后进行总RNA提取与定量。将处理后样本的RNA通过PrimeView人类基因组U219芯片（#901605，应用生物系统公司（Applied Biosystems））进行基因芯片表达谱分析，实验严格遵循制造商提供的操作说明书，由澳大利亚新南威尔士大学拉马乔蒂基因组学中心（Ramaciotti Centre for Genomics, UNSW Australia）完成。