影响致癌转录和 DNA 损伤反应的表观遗传失调是癌症的一个标志。组蛋白去甲基化酶 KDM4B 是调节这些过程的因子，在乳腺癌中雌激素受体介导的转录和 DNA 修复中发挥着重要作用。然而，致癌磷酸信号转导如何影响表观遗传调控尚不完全清楚。在这里，我们发现核糖体 S6 激酶 (RSK)（Ras/MAPK 途径的下游效应子）对 KDM4B 的磷酸化对于 KDM4B 响应 DNA 损伤的功能至关重要。KDM4B 敲除乳腺癌细胞系是通过 CRISPR/Cas9 生成的介导的基因编辑。通过慢病毒介导的基因转移，在敲除细胞中重新表达野生型或磷酸位点突变的 KDM4B。基因敲低是通过RNA干扰实现的。 DNA 双链断裂 (DSB) 是通过电离辐射或激光微辐射诱导的。通过免疫荧光分析 DSB 位点的蛋白质积累。通过体外和体内激酶测定评估 RSK 对 KDM4B 的磷酸化。通过 RT-PCR 和蛋白质印迹分析基因和蛋白质表达水平。通过克隆存活测定检查细胞对电离辐射的敏感性。RSK 在 Ser666 位点磷酸化 KDM4B，并通过 RSK 耗竭或 RSK 抑制剂抑制磷酸化，消除 KDM4B 在 DNA 双链断裂 (DSB) 位点的积累。 KDM4B 敲除细胞或用 RSK 抑制剂处理的细胞中 DSB 修复显着延迟。用拟磷突变体 S666D 替换内源 KDM4B 可恢复被 RSK 抑制剂抑制的 KDM4B 积累和 DSB 修复，表明 KDM4B 特定丝氨酸残基上的 RSK 在 RSK 抑制剂对 DSB 修复的影响中发挥着关键作用。由于这些异常反应，抑制 KDM4B 磷酸化增加了细胞对电离辐射的敏感性。总体而言，本研究发现了 RSK 对 DNA 损伤反应的新功能，这为其抑制剂在癌症治疗中提供了额外的作用.© 2024。作者。

Epigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage.KDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay.RSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation.Overall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy.© 2024. The Author(s).