胶质母细胞瘤（GBM）是一种高度恶性的脑肿瘤，治疗选择有限。最近的研究聚焦于表观遗传调控因子，如Enhancer of Zeste Homolog 2（EZH2），它通过表观遗传修饰对基因表达起调控作用。已经开发了EZH2抑制剂作为GBM的潜在治疗药物，但对这些抑制剂的耐药性仍然是一个相当大的挑战。本研究旨在研究核糖体S6蛋白激酶4（RSK4）在GBM中的作用及其与EZH2抑制剂耐药性的关联。我们首先通过使用EZH2抑制剂处理原代GBM细胞系诱导出耐药性，并观察到耐药细胞中与胶质母细胞瘤干细胞（GSCs）相关的干性标记的表达增加。我们还发现GBM患者样本中RSK4的高表达，并确定了高RSK4表达与预后差和GSC标记表达的相关性。进一步的实验显示，在耐药性GBM细胞中敲除RSK4可以恢复其对EZH2抑制剂的敏感性，并减少GSC标记的表达，从而减少其自我更新能力。从机制上来看，我们发现RSK4直接磷酸化EZH2，激活EZH2/STAT3途径，促进GBM对EZH2抑制剂的耐药性。我们还发现，在体外和体内实验中，将EZH2抑制剂与一种名为BI-D1870的RSK4抑制剂联合使用对GBM的发生和进展具有更好的抑制效果。总之，本研究证明RSK4增强癌症干性，并在GBM中介导对EZH2抑制剂的耐药性。将EZH2抑制剂与RSK4抑制剂的联合治疗是治疗GBM的有希望的潜在策略。综上所述，我们的结果强烈证明RSK4以PRC2非依赖的方式调节EZH2/STAT3通路，促进GSC的维持和EZH2抑制剂耐药性，表明RSK4是GBM的一个有希望的治疗靶点。© 2023. 作者（以独家许可）授予Springer Nature America, Inc.。

Glioblastoma (GBM) is a highly malignant type of brain tumor with limited treatment options. Recent research has focused on epigenetic regulatory factors, such as Enhancer of Zeste Homolog 2 (EZH2), which plays a role in gene expression through epigenetic modifications. EZH2 inhibitors have been developed as potential therapeutic agents for GBM, but resistance to these inhibitors remains a considerable challenge. This study aimed to investigate the role of ribosomal S6 protein kinase 4 (RSK4) in GBM and its association with resistance to EZH2 inhibitors. We first induced drug resistance in primary GBM cell lines by treatment with an EZH2 inhibitor and observed increases in the expression of stemness markers associated with glioblastoma stem cells (GSCs) in the drug-resistant cells. We also found high expression of RSK4 in GBM patient samples and identified the correlation of high RSK4 expression with poor prognosis and GSC marker expression. Further experiments showed that knocking down RSK4 in drug-resistant GBM cells restored their sensitivity to EZH2 inhibitors and decreased the expression of GSC markers, thus reducing their self-renewal capacity. From a mechanistic perspective, we discovered that RSK4 directly phosphorylates EZH2, activating the EZH2/STAT3 pathway and promoting resistance to EZH2 inhibitors in GBM. We also found that combining EZH2 inhibitors with an RSK4 inhibitor called BI-D1870 had better inhibitory effects on GBM occurrence and progression in both in vitro and in vivo experiments. In conclusion, this study demonstrates that RSK4 enhances cancer stemness and mediates resistance to EZH2 inhibitors in GBM. Combination treatment with EZH2 inhibitors and RSK4 inhibitors is a promising potential therapeutic strategy for GBM. Collectively, our results strongly demonstrate that RSK4 regulates the EZH2/STAT3 pathway to promote GSC maintenance and EZH2i resistance in a PRC2-independent manner, indicating that RSK4 is a promising therapeutic target for GBM.© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.