Overexpression of Cyclin E1 or Cdc25A leads to replication stress, mitotic aberrancies, and increased sensitivity to replication checkpoint inhibitors

Abstract

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.

Shaping the BRCAness mutational landscape by alternative double-strand break repair, replication stress and mitotic aberrancies

“…Moreover, the genomic scars resulting from the increased dependence of BRCAness tumours on alternative repair pathways indicate that targeting these pathways, for example through RAD52 or Pol θ inhibition, may further sensitize BRCAness tumours to DNA damaging agents ( 216 , 217 ). Moreover, forcing cells with DNA damage into mitosis before DNA lesions are resolved, for example by inhibition of ATR, may provide an alternative therapeutic vulnerability ( 165 , 218 , 219 ). Our understanding of mutational signatures therefore not only has the potential to expand patient selection possibilities, but may also aid in the development of new therapeutic strategies.…”

TP53loss initiates chromosomal instability in high-grade serous ovarian cancer

“…Moreover, it should be noted that such aneuploidy tolerance studies utilised experimental induction of chromosome mis-segregation in cells lacking p53. However, the emergence of aneuploid clones with TP53 loss has been observed in untreated mammary epithelial and RPE-1 cells (Kok et al, 2020;Salehi et al, 2020;Soto et al, 2017). In addition, multiple cellular processes were deregulated in response to p53 inactivation in transformed murine embryonic fibroblasts, including ploidy control (Valente et al, 2020).…”

“…Initial studies using the near-diploid colorectal cancer cell line HCT116, suggested that p53-loss is not sufficient to cause CIN (Bunz et al, 2002). More recently, however, suppressing p53 in hTERT-immortalized RPE-1 cells did generate abnormal karyotypes (Kok et al, 2020;Soto et al, 2017). Furthermore, p53 inactivation in transformed murine embryonic fibroblasts deregulated multiple cellular processes affecting DNA damage response, mitosis and ploidy control (Valente et al, 2020).…”

An mRNA expression-based signature for oncogene-induced replication-stress

“…Interestingly, patients with the largest decrease in tumor size upon WEE1 inhibitor treatment showed enrichment for CCNE1 amplification 18 . In line with this notion, overexpression of CCNE1 sensitized TNBC cell lines to WEE1 inhibition 14,19 . Furthermore, an unbiased genomic screen identified regulators of CDK2 as determinants of WEE1 inhibitor sensitivity 20 .…”