Aberrant expression of tryptophan 2,3-dioxygenase (TDO) is a determinant of malignancy and immune response in gliomas in part through kynurenine (KYN)-mediated activation of the aryl hydrocarbon receptor (AhR). In the current study, we investigated the hypothesis that TDO activation in gliomas has a broad impact upon genome maintenance - promoting tolerance of replication stress (RS) and repair of DNA damage. We report that inhibition of TDO activity attenuated recovery from hydroxyurea (HU)-induced RS and increased the genotoxic effects of bis-chloroethylnitrosourea (BCNU), as fork progress was impeded when TDO-deficient glioma cells were treated with BCNU. Activation of the Chk1 arm of the replication stress response (RSR) was reduced when TDO activity was blocked prior to treatment with BCNU, whereas phosphorylation of serine 33 (pS33) on replication protein A (RPA) was enhanced - indicative of increased fork collapse. Restoration of KYN levels protected against some replication-associated effects of BCNU. Inhibition of TDO activity had a strong anti-proliferative effect on glioma-derived cells - enhancing the cytotoxic effects of BCNU. Analysis of results obtained using quantitative proteomics revealed TDO-dependent changes in several signaling pathways - including down-regulation of DNA repair factors and sirtuin signaling. Consistent with these observations, inhibition of TDO diminished SIRT7 recruitment to chromatin, which increased histone H3K18 acetylation - a key mark involved in 53BP1 recruitment to sites of DNA damage. Cells lacking TDO activity exhibited defective recruitment of 53BP1 to γH2AX foci, which corresponded with delayed repair of BCNU-induced DNA breaks. Addition of exogenous KYN increased the rate of break repair. The discovery that TDO activity modulates sensitivity to DNA damage by fueling SIRT7/53BP1 localization to chromatin and repair of BCNU-induced DNA damage highlights the potential for tumor-specific metabolic changes to influence genome stability and may have implications for glioma biology and treatment strategies.

中文翻译：

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色氨酸-2,3-双加氧酶的抑制作用损害神经胶质瘤细胞的DNA损伤耐受性和修复能力

色氨酸2，3-双加氧酶（TDO）的异常表达是神经胶质瘤中恶性肿瘤和免疫应答的决定因素，部分是由犬尿氨酸（KYN）介导的芳烃受体（AhR）激活引起的。在当前的研究中，我们调查了神经胶质瘤中TDO激活对基因组维护具有广泛影响的假设-促进复制压力（RS）的耐受性和DNA损伤的修复。我们报道抑制TDO活性减弱了从羟基脲（HU）诱导的RS的恢复，并增加了双氯乙基亚硝基脲（BCNU）的遗传毒性作用，因为当用BCNU治疗TDO缺陷型神经胶质瘤细胞时，叉子的进展受到了阻碍。当在用BCNU治疗之前阻断TDO活性时，复制应激反应（RSR）的Chk1臂的激活减少，而丝氨酸33（pS33）在复制蛋白A（RPA）上的磷酸化增强-指示叉塌陷增加。恢复KYN水平可防止BCNU的某些复制相关效应。抑制TDO活性对神经胶质瘤来源的细胞具有很强的抗增殖作用-增强了BCNU的细胞毒性作用。使用定量蛋白质组学获得的结果分析显示，TDO依赖于几种信号传导途径的变化-包括DNA修复因子和瑟土因信号传导的下调。与这些观察结果一致，抑制TDO减少了SIRT7对染色质的募集，从而增加了组蛋白H3K18乙酰化-这是53BP1对DNA损伤位点募集的关键标志。缺乏TDO活性的细胞显示出53BP1向γH2AX灶的募集缺陷，这与BCNU诱导的DNA断裂的修复延迟有关。添加外源性KYN可以提高断裂修复率。TDO活性通过促进SIRT7 / 53BP1定位于染色质并修复BCNU诱导的DNA损伤来调节对DNA损伤的敏感性，这一发现突出了肿瘤特异性代谢变化影响基因组稳定性的潜力，并且可能对神经胶质瘤生物学和治疗策略产生影响。