Mechanism of cytarabine-induced neurotoxicity [HTGTS]

Postmitotic neurons are known to harbor high levels of methylated cytosine and its oxidized intermediates such as 5hmC, but the functional relevance of these epigenetic modifications of DNA are poorly understood. We show that some but not all cytidine analogs, such as cytarabine, cause DNA double strand breaks (DSB) during TET-mediated active 5mC demethylation by interrupting TDG-dependent base excision repair. These DSBs are frequently converted into indels and translocations by DNA ligase 4. In vivo, Purkinje and Golgi cells in the cerebellum are the only neuronal populations that exhibit high levels of DNA damage to cytarabine. In Purkinje cells, TET targets gene bodies with the highest expression marked by enhancer-associated histone modifications. Many of these genes control movement coordination which explains the long recognized cerebellar neurotoxicity of cytarabine. We show that other cytidine analogs, such as gemcitabine cause only single strand breaks in neurons, which are repaired by DNA ligase 3 with minimal toxicity. Our findings uncover a mechanistic link between TET-mediated DNA demethylation, base excision repair and gene expression in neurons and provide a rational explanation for the different neurotoxicity profiles of an important class of antineoplastic agents. The high-throughput experiments used in this study included a CRISPR interference screen in iPSC-derived neurons; SAR-seq in iPSC-derived neurons; S1/END-seq in iPSC-derived neurons, cortical neurons, and Purkinje neurons; LAM-HTGTS in iPSC-derived neurons; targeted deep sequencing (amplicon-seq) in iPSC-derived neurons; ATAC-seq in Purkinje neurons; RNA-seq in iPSC-derived neurons, cortical neurons, Purkinje and Golgi neurons; and CUT&RUN in Purkinje and Golgi neurons; Pyridine borane sequencing (PS-seq) in iPSC-derived neurons and Purkinje neurons.

已知有丝分裂后神经元（Postmitotic neurons）中富集高水平的甲基化胞嘧啶（methylated cytosine）及其氧化中间体（如5hmC），但人们对这些DNA表观遗传修饰（epigenetic modifications）的功能相关性仍知之甚少。本研究证实，部分而非全部胞苷类似物（如阿糖胞苷（cytarabine））可在TET介导的5-甲基胞嘧啶（5mC）活性去甲基化过程中，通过干扰胸腺嘧啶DNA糖苷酶（TDG）依赖的碱基切除修复（TDG-dependent base excision repair），引发DNA双链断裂（double strand breaks, DSB）。此类DNA双链断裂常被DNA连接酶4（DNA ligase 4）介导转化为插入缺失（indels）与染色体易位（translocations）。在体内环境中，小脑（cerebellum）内的浦肯野细胞（Purkinje cells）与高尔基细胞（Golgi cells）是唯一对阿糖胞苷表现出高水平DNA损伤的神经元群体。在浦肯野细胞中，TET靶向那些以增强子关联组蛋白修饰（enhancer-associated histone modifications）为标记的高表达基因体，其中诸多基因参与运动协调（movement coordination）调控，这也解释了长期以来被证实的阿糖胞苷小脑神经毒性（neurotoxicity）。本研究同时发现，其他胞苷类似物如吉西他滨（gemcitabine）仅会在神经元中引发单链断裂（single strand breaks），此类断裂可通过DNA连接酶3（DNA ligase 3）完成修复，且神经毒性极低。本研究结果揭示了神经元中TET介导的DNA去甲基化、碱基切除修复与基因表达之间的机制性关联（mechanistic link），并为一类重要抗肿瘤药物（antineoplastic agents）的差异化神经毒性谱提供了合理解释。本研究采用的高通量实验技术包括：诱导多能干细胞（induced pluripotent stem cell, iPSC）衍生神经元的CRISPR干扰筛选（CRISPR interference screen）；iPSC衍生神经元的SAR-seq；iPSC衍生神经元、皮层神经元（cortical neurons）及浦肯野神经元的S1/END-seq；iPSC衍生神经元的LAM-HTGTS；iPSC衍生神经元的靶向深度测序（扩增子测序，amplicon-seq）；浦肯野神经元的转座酶可及性染色质测序（ATAC-seq）；iPSC衍生神经元、皮层神经元、浦肯野细胞及高尔基细胞的RNA测序（RNA-seq）；浦肯野细胞与高尔基细胞的CUT&RUN技术；以及iPSC衍生神经元与浦肯野神经元的吡啶硼烷测序（Pyridine borane sequencing, PS-seq）。