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. Overall design: 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.