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

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.