KDM2B 编码包含 JmjC 结构域的组蛋白赖氨酸脱甲基酶，该酶在包括 TNBC 在内的多种类型的肿瘤中充当癌基因。这项研究的目的是解决我们早期工作结果与癌症的相关性，该研究表明 KDM2B 的过度表达通过调节抗氧化机制使小鼠胚胎成纤维细胞 (MEF) 抵抗氧化应激。我们主要采用了多组学策略，包括RNA-Seq、定量TMT蛋白质组学、基于质谱的全局代谢组学、ATAC-Seq和ChIP-seq，探讨KDM2B在抗氧化应激和中间代谢中的作用。使用生物信息学工具对这些数据和来自现有患者数据集的数据进行了分析，包括外显子-内含子分裂分析 (EISA)、FLUFF 和聚类分析。我们采用的主要遗传策略是使用 shRNA 进行基因沉默。使用 CellROX 染色后，通过流式细胞术测量 ROS，并使用市售试剂盒通过生化测定测量各种代谢物。如图所示，通过 qRT-PCR 和免疫印迹监测基因表达。在基底样乳腺癌细胞系中敲低 KDM2B 会降低 GSH 水平，并使细胞对 ROS 诱导剂、GSH 靶向分子和 DUB 抑制剂敏感。为了解决 GSH 调节机制，我们敲低了 MDA-MB-231 细胞中的 KDM2B，并使用多组学策略检查了敲低的影响。结果表明，KDM2B 在 ncPRC1.1 的背景下发挥作用，调节表观遗传和转录因子网络，这些因子控制许多代谢酶，包括参与 SGOC、谷氨酸和 GSH 代谢的酶。他们还表明，KDM2B 增强了 MYC 和 ATF4 的染色质可及性和表达，并且它与 MYC 和 ATF4 一致结合，MYC 和 ATF4 是大量转录活性基因（包括许多编码代谢酶）的启动子。此外，MYC 和 ATF4 结合位点在其可及性取决于 KDM2B 的基因中富集，并且对表达高水平或低水平 KDM2B 的 TNBC 队列进行了分析，但相似水平的 MYC 和 ATF4 确定了 MYC 靶标的子集，其表达与KDM2B 的表达。对同一队列中的基底细胞样 TNBC 的进一步分析表明，高水平表达所有三种调节因子的肿瘤表现出明显的代谢特征，导致预后不良。本研究将 KDM2B、ATF4 和 MYC 连接在一个转录网络中，该转录网络调节多种代谢酶的表达，包括那些控制相互关联的 SGOC、谷氨酸和 GSH 代谢途径的酶。通过所有三个因素，许多转录活性基因的启动子共同占据，染色质可及性取决于 KDM2B 的基因中 MYC 结合位点的富集，以及 KDM2B 水平与 MYC 靶标子集表达的相关性肿瘤中表达相似水平 MYC 的基因表明 KDM2B 调节 MYC 的表达和转录活性。重要的是，所有三个因素的协同表达也定义了预后不良的 TNBC 的独特代谢子集。总体而言，这项研究确定了 SGOC 调节的新机制，提出了 TNBC 中新的 KDM2B 依赖性代谢脆弱性，并为 KDM2B 在转录表观遗传调节中的作用提供了新的见解。版权所有 © 2023。由 Elsevier Inc. 出版。

KDM2B encodes a JmjC domain-containing histone lysine demethylase, which functions as an oncogene in several types of tumors, including TNBC. This study was initiated to address the cancer relevance of the results of our earlier work, which had shown that overexpression of KDM2B renders mouse embryonic fibroblasts (MEFs) resistant to oxidative stress by regulating antioxidant mechanisms.We mainly employed a multi-omics strategy consisting of RNA-Seq, quantitative TMT proteomics, Mass-spectrometry-based global metabolomics, ATAC-Seq and ChIP-seq, to explore the role of KDM2B in the resistance to oxidative stress and intermediary metabolism. These data and data from existing patient datasets were analyzed using bioinformatic tools, including exon-intron-split analysis (EISA), FLUFF and clustering analyses. The main genetic strategy we employed was gene silencing with shRNAs. ROS were measured by flow cytometry, following staining with CellROX and various metabolites were measured with biochemical assays, using commercially available kits. Gene expression was monitored with qRT-PCR and immunoblotting, as indicated.The knockdown of KDM2B in basal-like breast cancer cell lines lowers the levels of GSH and sensitizes the cells to ROS inducers, GSH targeting molecules, and DUB inhibitors. To address the mechanism of GSH regulation, we knocked down KDM2B in MDA-MB-231 cells and we examined the effects of the knockdown, using a multi-omics strategy. The results showed that KDM2B, functioning in the context of ncPRC1.1, regulates a network of epigenetic and transcription factors, which control a host of metabolic enzymes, including those involved in the SGOC, glutamate, and GSH metabolism. They also showed that KDM2B enhances the chromatin accessibility and expression of MYC and ATF4, and that it binds in concert with MYC and ATF4, the promoters of a large number of transcriptionally active genes, including many, encoding metabolic enzymes. Additionally, MYC and ATF4 binding sites were enriched in genes whose accessibility depends on KDM2B, and analysis of a cohort of TNBCs expressing high or low levels of KDM2B, but similar levels of MYC and ATF4 identified a subset of MYC targets, whose expression correlates with the expression of KDM2B. Further analyses of basal-like TNBCs in the same cohort, revealed that tumors expressing high levels of all three regulators exhibit a distinct metabolic signature that carries a poor prognosis.The present study links KDM2B, ATF4, and MYC in a transcriptional network that regulates the expression of multiple metabolic enzymes, including those that control the interconnected SGOC, glutamate, and GSH metabolic pathways. The co-occupancy of the promoters of many transcriptionally active genes, by all three factors, the enrichment of MYC binding sites in genes whose chromatin accessibility depends on KDM2B, and the correlation of the levels of KDM2B with the expression of a subset of MYC target genes in tumors that express similar levels of MYC, suggest that KDM2B regulates both the expression and the transcriptional activity of MYC. Importantly, the concerted expression of all three factors also defines a distinct metabolic subset of TNBCs with poor prognosis. Overall, this study identifies novel mechanisms of SGOC regulation, suggests novel KDM2B-dependent metabolic vulnerabilities in TNBC, and provides new insights into the role of KDM2B in the epigenetic regulation of transcription.Copyright © 2023. Published by Elsevier Inc.