Decitabine cytotoxicity is promoted by dCMP deaminase DCTD and mitigated by SUMO-dependent E3 ligase TOPORS

The nucleoside analog decitabine (or 5-aza-dC) is used to treat several hematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks, leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.

核苷类似物地西他滨（decitabine，又称5-aza-dC）可用于治疗多种血液系统恶性肿瘤。当其经三磷酸化修饰并掺入DNA后，5-aza-dC会诱导形成共价结合的DNA甲基转移酶1（DNMT1）-DNA蛋白质交联（DPC），进而引发DNA低甲基化。然而，5-aza-dC的临床疗效存在个体差异，且复发情况较为常见。本研究借助全基因组CRISPR/Cas9筛选技术，绘制了调控5-aza-dC药物敏感性的因子图谱。令人意外的是，研究发现dCMP脱氨酶（dCMP deaminase，DCTD）的缺失会使细胞对5-aza-dC产生耐药性，这提示5-aza-dUMP的生成具有细胞毒性。随后，本研究结合DCTD缺陷细胞中的后续遗传筛选结果，以及对DNMT1-DPC邻近蛋白质组的鉴定，发现泛素与SUMO1 E3连接酶TOPORS是一种新型DPC修复因子。TOPORS可被招募至经SUMO化修饰的DNMT1-DPC复合物中，并促进其降解。本研究表明，当DPC修复功能受损时，5-aza-dC诱导形成的DPC会引发细胞毒性；而在野生型细胞中，细胞毒性则源于核苷酸代谢紊乱。该研究为未来鉴定地西他滨治疗的预测性生物标志物奠定了基础。