Impediments that slow the rate of DNA replication are collectively referred to as “replication stress”. Replication stress is the main driver of genome instability in oncogenesis and is recognized as a hallmark of cancer. Much of the oncogenic effect of DNA replication stress comes through inappropriate chromosome segregation errors during mitosis. Within the clinic, pharmacological induction of replication stress is an established frontline approach for chemotherapeutic cancer treatment. Lethal replication stress has previously been associated with “mitotic catastrophe”, a broad descriptor encompassing the complex and poorly understood mechanisms that connect genomic insult to mitotic disruption and cell death. Despite the clinical relevance of mitotic catastrophe and lethal DNA replication stress, no clear mechanisms of replication stress-induced cell death are established. In this study we examined the connection between lethal replication stress, cell death, and genomic instability.
Using cell and molecular biology, assayed primarily by live-cell and fixed imaging, we identified that pharmacological induction of replication stress in p53-competent IMR90 human fibroblasts resulted in mitotic bypass and growth arrest in tetraploid G1-phase cells. Whereas treatment of multiple p53-compromised human cell lines with the same dosage of replication stress inducing drugs, led to cell death, with ≥ 80% of cell death events occurring during mitosis (i.e. mitotic catastrophe). We found that in response to lethal replication stress, p53-compromised cells exhibited a prolonged S/G2 phase followed by Wings apart-like protein homolog (WAPL)- and spindle assembly checkpoint (SAC)-dependent mitotic arrest in the same cell cycle. The mitotic arrest drove Bax and Bak-dependent mitotic cell death signalled via WAPL-induced cohesion fatigue. Bax and Bak null cells escaped mitotic cell death at the consequence of mitotic slippage, whereas depleting WAPL and suppressing cohesion fatigue rescued cell death at the consequence of inappropriate chromosome segregation and aneuploidy. Simultaneously, and independent of Bax/Bak and WAPL-mediated cell death, a parallel mitotic cell death pathway was engaged through non-canonical mitotic telomere deprotection. This pathway of mitotic telomere deprotection was regulated by the SAC, Aurora B kinase, the telomere protein TRF2, and the DNA damage response kinase ATM. These data identify that replication stress-induced mitotic catastrophe is signalled through a dominant pathway of cohesion fatigue, and a minor pathway of telomere deprotection, and reveals how genome instability arises when these mechanisms are evaded.