Genome maintenance is required for cellular viability, and failure to preserve genomic integrity is associated with an increased risk of diseases, such as cancer. To ensure genomic stability, cells have checkpoints that control cell cycle progression in the event of DNA damage or incomplete DNA replication. The DNA replication checkpoint is regulated by the ATR-CHK1 pathway that stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Two distinct models have been proposed to explain how ATR stabilizes stalled forks: 1) through local modulation of fork remodelers, such as SMARCAL1 inhibition, and 2) through inhibition of CDK-dependent pathways, such as inhibition of the AURKA-PLK1 pathway, which prevent cell cycle progression. However, it remains unclear which stabilization function is essential for fork stability and whether specific sites in the genome depend on one function over the other.

In an effort to test if an essential part of fork stabilization is mediated through inhibiting CDK-dependent pathways, such as inhibiting premature activation of the AURKA-PLK1 pathway, we established a system to hyper-activate the AURKA-PLK1 pathway to determine if it is sufficient to cause fork collapse. We found that fork collapse was not achievable solely through Aurora A overexpression nor with overexpression of its co-activators, TPX2 and BORA, but rather that CDK1 activation was also required. To test if CDK1-activation is sufficient to promote fork collapse, we inhibited WEE1, which short-circuits the cell cycle checkpoint function of ATR without inhibiting its fork-proximal activity. Using flow cytometry based fork collapse assays and genome-wide detection of RPA accumulation using RPA ChIP-Seq, we show that WEE1 and ATR inhibition cause similar levels of fork collapse at overlapping genomic locations in a CDK1-depdendent manner under conditions of partial replication inhibition (low dose aphidicolin). Notably, treatment with WEE1 inhibitor (WEE1i) alone was also sufficient to cause replication fork collapse, and did so more rapidly and to a higher degree than treatment with ATR inhibitor (ATRi) alone. Interestingly, clear differences in site specificity were observed when WEE1i was combined with ATRi, suggesting that particular sites in the genome may be slightly more dependent on the local functions of ATR than others. Thus, cell cycle checkpoint abrogation by WEE1i is sufficient to cause replication fork collapse in a manner similar to ATRi; however, site-specific roles for ATR remain. Together our findings indicate that the cell cycle checkpoint of ATR is key in stabilizing replication forks at a majority of sites in the genome. These findings could be leveraged to develop cancer treatments that exploit combinations of oncogenic genomic breakage signatures with that of WEE1 or ATR inhibitors.