Ewing的肉瘤是一种高度恶性的儿童肿瘤，尽管经过了许多化疗方案的强化，但在过去的二十年中其疗效几乎没有改变。因此，寻找新的治疗选择是至关重要的。本研究旨在探讨联合抑制ATR和核糖核苷酸还原酶（RNR）这两个有前途的靶点在Ewing肉瘤细胞中的作用效果。通过细胞死亡、线粒体去极化和细胞周期分布的流式细胞术分析，以及通过caspase 3/7活性测定、免疫印迹和实时RT-PCR等方法，评估了ATR抑制剂VE821与RNR抑制剂triapine和didox的联合作用对三种具有不同TP53状态（WE-68、SK-ES-1、A673）的Ewing肉瘤细胞系的影响。通过组合指数分析评估了抑制剂之间的相互作用。单独应用ATR或RNR抑制剂产生轻度至中度的效果，而它们的联合使用则产生了强烈的协同作用。ATR和RNR抑制剂诱导协同细胞死亡，并协同诱导线粒体去极化、caspase 3/7活性和DNA断裂，表明细胞死亡的凋亡形式。所有影响都与功能性p53无关。此外，VE821与triapine的联合使用提高了p53水平，并在p53野生型Ewing肉瘤细胞中诱导p53靶基因表达（CDKN1A，BBC3）。我们的研究揭示了联合靶向ATR和RNR对Ewing肉瘤具有体外作用，并因此将ATR和RNR抑制剂的联合作为治疗这种难治性疾病的新策略进行体内探索。©2023作者。

Ewing's sarcoma is a highly malignant childhood tumour whose outcome has hardly changed over the past two decades despite numerous attempts at chemotherapy intensification. It is therefore essential to identify new treatment options. The present study was conducted to explore the effectiveness of combined inhibition of two promising targets, ATR and ribonucleotide reductase (RNR), in Ewing's sarcoma cells.Effects of the ATR inhibitor VE821 in combination with the RNR inhibitors triapine and didox were assessed in three Ewing's sarcoma cell lines with different TP53 status (WE-68, SK-ES-1, A673) by flow cytometric analysis of cell death, mitochondrial depolarisation and cell cycle distribution as well as by caspase 3/7 activity determination, by immunoblotting and by real-time RT-PCR. Interactions between inhibitors were evaluated by combination index analysis.Single ATR or RNR inhibitor treatment produced small to moderate effects, while their combined treatment produced strong synergistic ones. ATR and RNR inhibitors elicited synergistic cell death and cooperated in inducing mitochondrial depolarisation, caspase 3/7 activity and DNA fragmentation, evidencing an apoptotic form of cell death. All effects were independent of functional p53. In addition, VE821 in combination with triapine increased p53 level and induced p53 target gene expression (CDKN1A, BBC3) in p53 wild-type Ewing's sarcoma cells.Our study reveals that combined targeting of ATR and RNR was effective against Ewing's sarcoma in vitro and thus rationalises an in vivo exploration into the potential of combining ATR and RNR inhibitors as a new strategy for the treatment of this challenging disease.© 2023. The Author(s).