Replication stress induces mitotic cell death through cohesion failure and telomere deprotection

VP Masamsetti1, KS Mak1, RRJ Low1, N Lamm-Shalem1, J Karlseder2, MT Hayashi3 and AJ Cesare1

  1. Genome Integrity Group, Children's Medical Research Institute, Westmead, NSW Australia
  2. Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA USA
  3. Hakubi Center for Advanced Research, Kyoto University, Kyoto, Japan

We previously demonstrated that mitotic arrest induces an Aurora B- and ATM-dependent telomere DNA damage response (DDR), resulting in mitotic cell death at telomere crisis and in cancer cells treated with mitotic poisons. Replication stress leads to mitotic chromosome segregation errors, and we therefore investigated if replication stress in genomic DNA could also induce mitotic arrest, and mitotic cell death, via the telomere DDR. Live-cell imaging in p53-compromised cells revealed that pharmacologically-induced, genomic DNA replication stress, induced Spindle Assembly Checkpoint (SAC)-dependent mitotic arrest and mitotic cell death. FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator) live-cell imaging confirmed that replication stress in early S-phase, resulted in mitotic arrest and mitotic cell death in the same cell cycle. Live cell imaging also demonstrated that over 75% of cell death events due to genomic replication stress occurred during mitosis. Cytogenetic analysis, and live-cell imaging, revealed two prominent mitotic chromosome phenotypes induced by replication stress: 1) cohesion failure initiated at the centromeres, resulting in separated sister chromatids preceding cell death; and 2) an SAC-, Aurora B-, and ATM-dependent telomere DDR occurring during mitotic arrest, which is regulated by TRF2. Suppression of the cohesion antagonist Wapl, efficiently suppressed mitotic cohesion failure and mitotic cell death due to replication stress, but did not affect the corresponding mitotic telomere DDR. Conversely, exacerbating or suppressing the mitotic telomere DDR, by depletion or over-expression of TRF2, sensitized or suppressed mitotic cell death, respectively, without impacting the corresponding cohesion failure. We have, therefore, discovered that genomic DNA replication stress primarily induces mitotic cell death, signalled by independent and parallel pathways of cohesion failure and telomere DDR activation. Cumulatively, these data imply that the telomere DDR has a much broader function in signalling cell death in response to genomic stress than previously appreciated, and suggests that telomeres impact tumour suppression in a length-independent manner.