RELA-fused supratentorial (ST) ependymoma (EPN) is an aggressive subgroup with poor prognosis. Considering the putative role of Notch signaling in the maintenance of the cancer stem cells (CSC) phenotype in RELA-fused EPN, we investigated the expression of Notch pathway and its target genes in this subgroup. We also evaluated the effects of two Notch inhibitors (DAPT and RO4929097) on cell proliferation, apoptosis, colony formation, and CSCs markers gene expression on EPN cell line of the RELA-fused subgroup (BXD-1425). In addition, in silico signatures of the Notch genes and CSCs markers were analyzed on a large clinical dataset from GSE64415 study. We found that among the ST-EPN subgroups the Notch signaling (NOTCH1, JAG1, JAG2, and HES4) is specifically activated in the ST-EPN-RELA. Furthermore, treatment of the RELA-fused EPN cell line with the Notch inhibitors impaired the Notch signaling expression and revealed that Notch axis is not essential for cell proliferation and survival in this setting. NOTCH1 expression in ST-EPN was correlated with the CSCs markers VEGFA and L1CAM overexpression and JAG1 expression was correlated with the CCND1 and CDK6 overexpression. In addition, in vitro treatment with Notch inhibitors induced downregulation of CSCs markers. These findings indicate that Notch signaling can be involved in the ST-EPN-RELA CSCs maintenance by modulating the expression of genes responsible for cell phenotype and cell fate.

RELA融合幕上 (ST) 室管膜瘤 (EPN) 是一种预后不良的侵袭性亚组。考虑到 Notch 信号在维持 RELA 融合 EPN 中癌症干细胞 (CSC) 表型中的假定作用，我们研究了 Notch 通路及其靶基因在该亚组中的表达。我们还评估了两种 Notch 抑制剂（DAPT 和 RO4929097）对 RELA 融合亚组（BXD-1425）的 EPN 细胞系上细胞增殖、凋亡、集落形成和 CSCs 标志物基因表达的影响。此外，在来自 GSE64415 研究的大型临床数据集上分析了 Notch 基因和 CSC 标记的计算机特征。我们发现在 ST-EPN 子组中，Notch 信号（NOTCH1、JAG1、JAG2和HES4 ) 在 ST-EPN-RELA 中被特别激活。此外，用 Notch 抑制剂处理 RELA 融合的 EPN 细胞系会损害 Notch 信号表达，并表明 Notch 轴在这种情况下对细胞增殖和存活不是必需的。ST-EPN 中的NOTCH1表达与CSCs标志物VEGFA和L1CAM过表达相关，JAG1表达与CCND1和CDK6 相关过度表达。此外，用 Notch 抑制剂进行体外治疗会诱导 CSCs 标志物的下调。这些发现表明 Notch 信号可以通过调节负责细胞表型和细胞命运的基因的表达参与 ST-EPN-RELA CSCs 的维持。