Oncogene-induced replication stress, for instance as a result of Cyclin E1 overexpression, causes genomic instability and has been linked to tumorigenesis. To survive high levels of replication stress, tumors depend on pathways to deal with these DNA lesions, which represent a therapeutically actionable vulnerability. We aimed to uncover the consequences of Cyclin E1 or Cdc25A overexpression on replication kinetics, mitotic progression, and the sensitivity to inhibitors of the WEE1 and ATR replication checkpoint kinases. We modeled oncogene-induced replication stress using inducible expression of Cyclin E1 or Cdc25A in non-transformed RPE-1 cells, either in a TP53 wild-type or TP53-mutant background. DNA fiber analysis showed Cyclin E1 or Cdc25A overexpression to slow replication speed. The resulting replication-derived DNA lesions were transmitted into mitosis causing chromosome segregation defects. Single cell sequencing revealed that replication stress and mitotic defects upon Cyclin E1 or Cdc25A overexpression resulted in genomic instability. ATR or WEE1 inhibition exacerbated the mitotic aberrancies induced by Cyclin E1 or Cdc25A overexpression, and caused cytotoxicity. Both these phenotypes were exacerbated upon p53 inactivation. Conversely, downregulation of Cyclin E1 rescued both replication kinetics, as well as sensitivity to ATR and WEE1 inhibitors. Taken together, Cyclin E1 or Cdc25A-induced replication stress leads to mitotic segregation defects and genomic instability. These mitotic defects are exacerbated by inhibition of ATR or WEE1 and therefore point to mitotic catastrophe as an underlying mechanism. Importantly, our data suggest that Cyclin E1 overexpression can be used to select patients for treatment with replication checkpoint inhibitors.

癌基因诱导的复制应激，例如由于细胞周期蛋白 E1 过表达，导致基因组不稳定，并与肿瘤发生有关。为了在高水平的复制压力下存活下来，肿瘤依赖于处理这些 DNA 损伤的途径，这代表了治疗上可操作的弱点。我们旨在揭示细胞周期蛋白 E1 或 Cdc25A 过表达对复制动力学、有丝分裂进展以及对 WEE1 和 ATR 复制检查点激酶抑制剂的敏感性的后果。我们使用细胞周期蛋白 E1 或 Cdc25A 在TP53野生型或TP53非转化 RPE-1 细胞中的诱导表达来模拟致癌基因诱导的复制应激-突变背景。DNA 纤维分析显示细胞周期蛋白 E1 或 Cdc25A 过表达减慢复制速度。由此产生的复制衍生的 DNA 损伤被传递到有丝分裂，导致染色体分离缺陷。单细胞测序显示，细胞周期蛋白 E1 或 Cdc25A 过表达后的复制应激和有丝分裂缺陷导致基因组不稳定。ATR 或 WEE1 抑制加剧了细胞周期蛋白 E1 或 Cdc25A 过表达诱导的有丝分裂异常，并引起细胞毒性。p53 失活后，这两种表型都会加剧。相反，细胞周期蛋白 E1 的下调拯救了复制动力学以及对 ATR 和 WEE1 抑制剂的敏感性。总之，细胞周期蛋白 E1 或 Cdc25A 诱导的复制应激导致有丝分裂分离缺陷和基因组不稳定。这些有丝分裂缺陷因 ATR 或 WEE1 的抑制而加剧，因此将有丝分裂灾难作为潜在机制。重要的是，我们的数据表明 Cyclin E1 过表达可用于选择接受复制检查点抑制剂治疗的患者。