CYP3A5 promotes glioblastoma stemness and chemoresistance through fine-tuning NAD+/NADH ratio

Background. Glioblastoma multiforme (GBM) exhibits a cellular hierarchy with a subpopulation of stem-like cells known as glioblastoma stem cells (GSCs), that drive tumor growth and contribute to treatment resistance. NAD(H) emerged as a crucial factor influencing GSCs. Methods. A multistep process of machine learning algorithms was implemented to construct the glioma stemness-related score (GScore). Further in silico and patient tissue analyses validated the predictive ability of the GScore and identified a potential target, CYP3A5. Loss-of-function or gain-of-function genetic experiments were performed to assess the impact of CYP3A5 on the self-renewal and chemoresistance of GSCs both in vitro and in vivo. Mechanistic studies were conducted using nontargeted metabolomics, RNA-seq, seahorse, transmission electron microscopy, immunofluorescence, flow cytometry, ChIP?qPCR, RT?qPCR, western blotting, etc. The efficacy of pharmacological inhibitors of CYP3A5 was assessed in vivo. Results. Based on the proposed GScore, we identify a GSC target CYP3A5, which is highly expressed in GSCs and temozolomide (TMZ)-resistant GBM patients. This elevated expression of CYP3A5 is attributed to transcription factor STAT3 activated by EGFR signaling or TMZ treatment. Depletion of CYP3A5 impairs self-renewal and TMZ resistance in GSCs. Mechanistically, CYP3A5 maintains mitochondrial fitness to promote GSC metabolic adaption through the NAD?/NADH-SIRT1-PGC1a axis. Additionally, CYP3A5 enhances the activity of NAD-dependent enzyme PARP to augment DNA damage repair. Treatment with CYP3A5 inhibitor alone or together with TMZ effectively suppresses tumor growth in vivo. Conclusion. Together, this study suggests that GSCs activate STAT3 to upregulate CYP3A5 to fine-tune NAD?/NADH for the enhancement of mitochondrial functions and DNA damage repair, thereby fueling tumor proliferation and conferring TMZ resistance, respectively. Thus, CYP3A5 represents a promising target for GBM treatment. Overall design: To invertigate the role of CYP3A5 in stemness maintenance and chemoresistance of GSCs, we established U87 spheres in which target gene has been knocked out by sgRNA.

背景：多形性胶质母细胞瘤（Glioblastoma multiforme, GBM）具有细胞层级结构，其中存在一类被称为胶质母细胞瘤干细胞（glioblastoma stem cells, GSCs）的干细胞样亚群，该亚群可驱动肿瘤生长并介导治疗耐药。烟酰胺腺嘌呤二核苷酸（NAD(H)）作为影响GSCs的关键因子逐渐受到关注。
方法：本研究采用多步机器学习算法构建胶质瘤干性相关评分（glioma stemness-related score, GScore）。进一步通过计算机模拟分析与患者组织样本分析验证了GScore的预测能力，并筛选出潜在靶点CYP3A5。通过功能丧失与功能获得性遗传学实验，在体外及体内水平评估了CYP3A5对GSCs自我更新能力及化疗耐药性的影响。机制研究采用非靶向代谢组学、RNA测序（RNA-seq）、Seahorse分析仪、透射电子显微镜（transmission electron microscopy）、免疫荧光（immunofluorescence）、流式细胞术（flow cytometry）、染色质免疫沉淀定量PCR（ChIP-qPCR）、逆转录定量PCR（RT-qPCR）、蛋白质免疫印迹（western blotting）等技术手段。此外，在体内评估了CYP3A5药理学抑制剂的抗肿瘤疗效。
结果：基于构建的GScore，我们鉴定出GSCs的靶点CYP3A5，其在GSCs及对替莫唑胺（temozolomide, TMZ）耐药的GBM患者组织中呈高表达。CYP3A5的高表达归因于EGFR信号通路或TMZ处理激活的转录因子STAT3。敲除CYP3A5会削弱GSCs的自我更新能力及TMZ耐药性。机制层面，CYP3A5通过NAD+/NADH-SIRT1-PGC1α信号轴维持线粒体稳态，进而促进GSCs的代谢适应。此外，CYP3A5可增强NAD依赖型酶PARP的活性，以提升DNA损伤修复能力。单独使用CYP3A5抑制剂或联合TMZ处理，可在体内有效抑制肿瘤生长。
结论：综上，本研究表明GSCs可通过激活STAT3上调CYP3A5的表达，进而精准调控NAD+/NADH平衡以增强线粒体功能及DNA损伤修复能力，分别促进肿瘤增殖并赋予TMZ耐药性。因此，CYP3A5可作为GBM治疗的潜在靶点。
研究设计：为探究CYP3A5在GSCs干性维持及化疗耐药中的作用，我们构建了通过sgRNA敲除靶基因的U87肿瘤球模型。