Data_Sheet_11_Lysine Methyltransferase Inhibitors Impair H4K20me2 and 53BP1 Foci in Response to DNA Damage in Sarcomas, a Synthetic Lethality Strategy.PDF

BackgroundChromatin is dynamically remodeled to adapt to all DNA-related processes, including DNA damage responses (DDR). This adaptation requires DNA and histone epigenetic modifications, which are mediated by several types of enzymes; among them are lysine methyltransferases (KMTs).
MethodsKMT inhibitors, chaetocin and tazemetostat (TZM), were used to study their role in the DDR induced by ionizing radiation or doxorubicin in two human sarcoma cells lines. The effect of these KMT inhibitors was tested by the analysis of chromatin epigenetic modifications, H4K16ac and H4K20me2. DDR was monitored by the formation of γH2AX, MDC1, NBS1 and 53BP1 foci, and the induction of apoptosis.
ResultsChaetocin and tazemetostat treatments caused a significant increase of H4K16 acetylation, associated with chromatin relaxation, and increased DNA damage, detected by the labeling of free DNA-ends. These inhibitors significantly reduced H4K20 dimethylation levels in response to DNA damage and impaired the recruitment of 53BP1, but not of MDC1 and NBS1, at DNA damaged sites. This modification of epigenetic marks prevents DNA repair by the NHEJ pathway and leads to cell death.
ConclusionKMT inhibitors could function as sensitizers to DNA damage-based therapies and be used in novel synthetic lethality strategies for sarcoma treatment.

背景：染色质会发生动态重塑，以适配所有与DNA相关的生物学过程，包括DNA损伤应答（DNA damage response, DDR）。该适配过程依赖于DNA与组蛋白的表观遗传修饰，这类修饰由多种酶类介导，其中赖氨酸甲基转移酶（lysine methyltransferases, KMTs）便是此类酶之一。
方法：本研究采用赖氨酸甲基转移酶抑制剂——查托霉素（chaetocin）与他泽司他（tazemetostat, TZM），探究其在电离辐射或多柔比星诱导的DNA损伤应答中的作用，实验使用两株人肉瘤细胞系。通过分析染色质表观遗传修饰H4K16ac与H4K20me2的水平，评估此类抑制剂的作用效果；通过检测γH2AX、MDC1、NBS1及53BP1灶点的形成情况，以及细胞凋亡的诱导情况，对DNA损伤应答进行监测。
结果：经查托霉素与他泽司他处理后，H4K16乙酰化水平显著升高，该修饰与染色质松弛状态相关，且通过游离DNA末端标记检测到的DNA损伤程度也显著增加。此类抑制剂可显著降低DNA损伤应答过程中的H4K20二甲基化水平，并削弱53BP1在DNA损伤位点的招募能力，但对MDC1与NBS1的招募无显著影响。这类表观遗传标记的改变会阻碍非同源末端连接（non-homologous end joining, NHEJ）通路介导的DNA修复，最终引发细胞死亡。
结论：赖氨酸甲基转移酶抑制剂可作为DNA损伤类治疗的增敏剂，有望应用于肉瘤治疗的新型合成致死策略中。