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

Yannick P Kok,Marcel A T M Van Vugt,Marieke Everts,Sergi Guerrero Llobet,Rudolf S N Fehrmann,Nathalie Van Den Tempel,Pepijn M Schoonen,Arkajyoti Bhattacharya

doi:10.1038/s41389-020-00270-2

Yannick P Kok, Marcel A T M Van Vugt + Show 6 more

Open Access

https://doi.org/10.1038/s41389-020-00270-2

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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.

Highlights

A common hallmark of cancer is the acquisition of genomic gains and losses as well as complex genomic rearrangements, collectively termed genomic instability[1]
Overexpression of cyclin E1 or Cdc25A leads to slower replication kinetics and mitotic defects Cyclin E1 is often found to be overexpressed in cancers, in triple negative breast cancer (TNBC) and high-grade ovarian cancers[7,8], which is accompanied by higher CCNE1 mRNA expression levels in these cancers (Supplementary Fig. 1A)
We evaluated the effects of Cdc25A overexpression, as this protein leads to Cyclin-dependent kinase-2 (CDK2) hyperactivation, albeit through an alternative mechanism (Fig. 1a)

Summary

Introduction

A common hallmark of cancer is the acquisition of genomic gains and losses as well as complex genomic rearrangements, collectively termed genomic instability[1]. There are various factors that induce replication stress, a common cause in the context of cancer is the increased activity or elevated expression of oncogenes[4,5,6]. CCNE1 amplification has been linked to induction of replication stress, by causing collisions between the replication and transcription machineries[17], and by triggering aberrant firing of replication origins, which subsequently leads to depletion of the nucleotide pool[3,17]. Combined, these effects can lead to stalling or collapse of replication forks[4]. In good agreement with these observations, Cyclin E1 overexpression was demonstrated to exclusively induce genome instability in tumors lacking functional p5318–20

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