single-cell sequencing of RPE1-TP53 mutant cells overexpressing Cdc25a or Cyclin E1

Genomically instable tumors, including triple-negative breast cancers frequently show elevated expression of oncogenes, including Cyclin E1, which interfere with normal DNA replication. Oncogene-induced replication stress causes genomic instability and has been linked to tumorigenesis. To survive high levels of replication stress, tumors increasingly depend on pathways, which allow them to deal with replication-induced DNA lesions and may provide therapeutically actionable vulnerabilities. We aimed to uncover the consequences Cyclin E1 or Cdc25A overexpression on mitotic progression, and to assess the consequences thereof on the sensitivity to inhibitors of the WEE1 and ATR replication checkpoint kinases.We modeled oncogene-induced replication stress using inducible Cyclin E1 or Cdc25A in non-transformed retinal pigment epithelium cells (RPE-1), either in a TP53 wild-type or TP53 mutant background. Single-fiber DNA analysis showed that Cyclin E1 or Cdc25A overexpression induced delayed replication. Notably, the replication-derived DNA lesions induced by Cyclin E1 or Cdc25A overexpression were transmitted into mitosis and caused chromosome segregation defects and mitotic catastrophe. In line with these findings, single cell sequencing revealed that overexpression of Cyclin E1 or Cdc25A results in genetic instability. Inhibition of ATR and WEE1 exacerbated the mitotic aberrancies induced by Cyclin E1 or Cdc25A overexpression, and caused cytotoxicity in these cells. Notably, loss of p53 further increased ATR or WEE1 inhibitor sensitivity and increased the mitotic aberrancies in Cyclin E1-overexpressing cells. Conversely, downregulation of Cyclin E1 rescued replication kinetics and resulted in decreased sensitivity to inhibitors of ATR and WEE1.Combined, this study shows that Cyclin E1 or Cdc25A-induced replication stress leads to mitotic segregation defects and genomic instability. The mitotic defects are exacerbated by inhibition of ATR or WEE1, and point at mitotic catastrophe as an underlying mechanism for the cytotoxic effects of targeting replication checkpoint kinases, and suggest Cyclin E1 overexpression as a criterion in selecting patients for treatment with such agents.

基因组不稳定的肿瘤（包括三阴性乳腺癌）常表现出癌基因表达上调，其中包括细胞周期蛋白E1（Cyclin E1），该蛋白会干扰正常DNA复制过程。癌基因诱导的复制应激会引发基因组不稳定，且已被证实与肿瘤发生密切相关。为在高水平复制应激下存活，肿瘤愈发依赖特定通路以应对复制诱导的DNA损伤，这类通路也可能成为可靶向治疗的薄弱环节。本研究旨在探究细胞周期蛋白E1（Cyclin E1）或细胞分裂周期蛋白25A（Cdc25A）过表达对有丝分裂进程的影响，并评估其对WEE1与ATR复制检验点激酶抑制剂敏感性的影响。本研究采用可诱导表达Cyclin E1或Cdc25A的非转化视网膜色素上皮细胞（RPE-1）构建癌基因诱导的复制应激模型，分别设置TP53野生型与TP53突变型两种背景。单纤维DNA分析结果显示，Cyclin E1或Cdc25A过表达会引发复制延迟。值得注意的是，Cyclin E1或Cdc25A过表达所诱导的复制源性DNA损伤会传递至有丝分裂阶段，进而引发染色体分离缺陷与有丝分裂灾难。与上述发现一致，单细胞测序结果显示，Cyclin E1或Cdc25A过表达会导致基因组不稳定。抑制ATR与WEE1会加重Cyclin E1或Cdc25A过表达所诱导的有丝分裂异常，并对上述细胞产生细胞毒性。值得注意的是，p53缺失会进一步提升Cyclin E1过表达细胞对ATR或WEE1抑制剂的敏感性，并加重其有丝分裂异常。反之，下调Cyclin E1的表达可恢复复制动力学，并降低细胞对ATR与WEE1抑制剂的敏感性。综上，本研究证实，Cyclin E1或Cdc25A诱导的复制应激会引发有丝分裂分离缺陷与基因组不稳定。ATR或WEE1抑制会加重上述有丝分裂缺陷，提示有丝分裂灾难是靶向复制检验点激酶的细胞毒性效应的潜在机制，并表明Cyclin E1过表达可作为筛选此类药物治疗患者的生物标志物。