Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells.

The trapping of Poly-ADP-ribose polymerase (PARP) on DNA caused by PARP inhibitors (PARPi) triggers acute DNA replication stress and synthetic lethality (SL) in BRCA2-deficient cells. Hence, DNA damage is accepted as a prerequisite for SL in BRCA2-deficient cells. In contrast, here we show that inhibiting ROCK in BRCA2-deficient cells triggers SL independently from acute replication stress. Such SL is preceded by polyploidy and binucleation resulting from cytokinesis failure. Such initial mitosis abnormalities are followed by other M phase defects, including anaphase bridges and abnormal mitotic figures associated with multipolar spindles, supernumerary centrosomes and multinucleation. SL was also triggered by inhibiting Citron Rho-interacting kinase, another enzyme that, similarly to ROCK, regulates cytokinesis. Together, these observations demonstrate that cytokinesis failure triggers mitotic abnormalities and SL in BRCA2-deficient cells. Furthermore, the prevention of mitotic entry by depletion of Early mitotic inhibitor 1 (EMI1) augmented the survival of BRCA2-deficient cells treated with ROCK inhibitors, thus reinforcing the association between M phase and cell death in BRCA2-deficient cells. This novel SL differs from the one triggered by PARPi and uncovers mitosis as an Achilles heel of BRCA2-deficient cells.

PARP Activity Fine-tunes the DNA Replication Choreography of Chk1-depleted Cells.

Checkpoint Kinase 1 (Chk1) prevents DNA damage by adjusting the replication choreography in the face of replication stress. Chk1 depletion provokes slow and asymmetrical fork movement, yet the signals governing such changes remain unclear. We sought to investigate whether poly(ADP-ribose) polymerases (PARPs), key players of the DNA damage response, intervene in the DNA replication of Chk1-depleted cells. We demonstrate that PARP inhibition selectively alleviates the reduced fork elongation rates, without relieving fork asymmetry in Chk1-depleted cells. While the contribution of PARPs to fork elongation is not unprecedented, we found that their role in Chk1-depleted cells extends beyond fork movement. PARP-dependent fork deceleration induced mild dormant origin firing upon Chk1 depletion, augmenting the global rates of DNA synthesis. Thus, we have identified PARPs as novel regulators of replication fork dynamics in Chk1-depleted cells.

Mus81-Eme1-dependent aberrant processing of DNA replication intermediates in mitosis impairs genome integrity.

Chromosome instability (CIN) underpins cancer evolution and is associated with drug resistance and poor prognosis. Understanding the mechanistic basis of CIN is thus a priority. The structure-specific endonuclease Mus81-Eme1 is known to prevent CIN. Intriguingly, however, here we show that the aberrant processing of late replication intermediates by Mus81-Eme1 is a source of CIN. Upon depletion of checkpoint kinase 1 (Chk1), Mus81-Eme1 cleaves under-replicated DNA engaged in mitotic DNA synthesis, leading to chromosome segregation defects. Supplementing cells with nucleosides allows the completion of mitotic DNA synthesis, restraining Mus81-Eme1-dependent DNA damage in mitosis and the ensuing CIN. We found no correlation between CIN arising from nucleotide shortage in mitosis and cell death, which were selectively linked to DNA damage load in mitosis and S phase, respectively. Our findings imply the possibility of optimizing Chk1-directed therapies by inducing cell death while curtailing CIN, a common side effect of chemotherapy.

CDK-Independent and PCNA-Dependent Functions of p21 in DNA Replication.

p21Waf/CIP1 is a small unstructured protein that binds and inactivates cyclin-dependent kinases (CDKs). To this end, p21 levels increase following the activation of the p53 tumor suppressor. CDK inhibition by p21 triggers cell-cycle arrest in the G1 and G2 phases of the cell cycle. In the absence of exogenous insults causing replication stress, only residual p21 levels are prevalent that are insufficient to inhibit CDKs. However, research from different laboratories has demonstrated that these residual p21 levels in the S phase control DNA replication speed and origin firing to preserve genomic stability. Such an S-phase function of p21 depends fully on its ability to displace partners from chromatin-bound proliferating cell nuclear antigen (PCNA). Vice versa, PCNA also regulates p21 by preventing its upregulation in the S phase, even in the context of robust p21 induction by irradiation. Such a tight regulation of p21 in the S phase unveils the potential that CDK-independent functions of p21 may have for the improvement of cancer treatments.

Unscheduled MRE11 activity triggers cell death but not chromosome instability in polymerase eta-depleted cells subjected to UV irradiation.

The elimination of DNA polymerase eta (pol η) causes discontinuous DNA elongation and fork stalling in UV-irradiated cells. Such alterations in DNA replication are followed by S-phase arrest, DNA double-strand break (DSB) accumulation, and cell death. However, their molecular triggers and the relative timing of these events have not been fully elucidated. Here, we report that DSBs accumulate relatively early after UV irradiation in pol η-depleted cells. Despite the availability of repair pathways, DSBs persist and chromosome instability (CIN) is not detectable. Later on cells with pan-nuclear γH2AX and massive exposure of template single-stranded DNA (ssDNA), which indicate severe replication stress, accumulate and such events are followed by cell death. Reinforcing the causal link between the accumulation of pan-nuclear ssDNA/γH2AX signals and cell death, downregulation of RPA increased both replication stress and the cell death of pol η-deficient cells. Remarkably, DSBs, pan-nuclear ssDNA/γH2AX, S-phase arrest, and cell death are all attenuated by MRE11 nuclease knockdown. Such results suggest that unscheduled MRE11-dependent activities at replicating DNA selectively trigger cell death, but not CIN. Together these results show that pol η-depletion promotes a type of cell death that may be attractive as a therapeutic tool because of the lack of CIN.

Persistent double strand break accumulation does not precede cell death in an Olaparib-sensitive BRCA-deficient colorectal cancer cell model.

The poly (adenosine diphosphate (ADP)-ribosyl) polymerase inhibitors (PARPi) selectively kill cancer cells with BRCA1 or BRCA2 (BRCA)-mutations. It has been proposed that cell death induction after PARPi depends on unrepaired double strand breaks (DSBs) that accumulate due to the homologous recombination deficiency of BRCA-mutated cells. Such accumulation of DSBs is inferred mainly from the high levels of DNA damage markers like phosphorylated histone H2AX. Herein, we developed a model of isogenic cell lines to show that depletion of BRCA causes PARPi-triggered cell death, replication stress (phosphorylated-H2AX and 53BP1 foci), and genomic instability. However, persistent DSBs accumulation was not detected under the same experimental conditions. Hence, at least in this cellular model, the trigger for cell death in PARPi-treated BRCA-depleted samples is not the accumulation of unrepaired DSBs. Instead, cell death better correlates with a rapid and aberrant resolution of DSBs by error-prone pathways that leads to severe chromosomic aberrations. Therefore, our results suggest that in PARPi-treated BRCA-deficient cells, chromosome aberrations may dually trigger both genomic instability and cell death.