DNA “replication stress” describes any phenomenon that alters the rate of DNA replication. Endogenous replication stress in pre-cancerous tissues drives oncogenic progression through the propagation of genome instability. Cancer cells, however, must maintain an active replication stress response to prevent the onset of lethal genome instability. Many, if not most, genotoxic chemotherapeutics induce tumour cell death specifically through direct or indirect induction of replication stress.
Our lab has an interest in the cellular mechanisms that counteract replication stress to maintain genome stability, and which execute cell death when replication stress becomes overwhelming. We have recently produced manuscripts describing two independent discoveries on this topic: 1) That replication stress induces mitotic cell death through independent pathways of cohesion fatigue and telomere deprotection1, and 2) that the ATR and mTOR kinase regulate nuclear filamentous actin to alter nuclear architecture and repair replication stress2. Whist these works identified novel mechanisms regulating genome stability when replication stress is encountered, the genetic regulators of both pathways remain to be identified.
To identify genetic regulators of the replication stress response we carried out multiple CRISPR/Cas9 screens. Our positive-selection screen was designed specifically to identify genes that regulate replication stress-induced mitotic death. Additionally, we carried out drop-out screens under conditions of acute or chronic dosing of Aphidicolin, a replication stress inducing drug, to identify genes involved in replication stress repair. Together, these screens have produced a list of candidate genes which are known to be involved in processes including cohesion regulation, chromatin remodelling, actin regulation and translation initiation, as key regulators of the DNA replication stress response. Several hits have been validated and their mechanism(s) of action is under investigation.