Bente studied the consequences of DNA replication stress, an increasingly recognized hallmark of many cancer cells. To model DNA replication stress she used mouse embryonic fibroblasts (MEFs) that lack the G1/S phase checkpoint by ablation of the three Rb-proteins. These so-called triple knockout (TKO) cells, which also overexpressed Bcl2 (TKO-Bcl2 MEFs), can enter S-phase in the absence of mitogenic signaling, but do so at the expense of severe DNA replication stress evidenced by slow replication speed, reduced origin firing, frequent DNA breakage and G2-like cell cycle arrest.
Surprisingly, concomitant loss of p53 restored origin firing, reduced DNA breakage and allowed proliferation in the absence of mitogenic signaling.
This provided a novel rationale for the frequent co-occurrence of Rb and p53 pathway inactivation in the process of tumorigenesis; loss of p53 does not only attenuates cell cycle arrest or apoptosis, but also reduces replication-stress induced DNA damage.
However, mitogen-independently proliferating TKO-Bcl2-p53KO MEFs still experienced replication stress, depicted by slow replication and sensitivity to inhibitors of the DNA Damage Response. To identify mechanisms that are essential for mitogen-independent proliferation, Bente performed an shRNA drop-out screen and identified a critical role of the helicase RECQL in preserving the integrity of stalled replication forks by preventing MRE11-dependent DNA breakage.
Another protein she found to be essential for mitogen-independent proliferation is the cohesin-antagonist WAPL. WAPL promotes RAD51-dependent repair and restart of broken replication forks at the expense of sister chromatid cohesion. In collaboration with the Oncogenomics group of Rob Wolthuis at VUMC-CCA, she also observed that cohesion loss is frequent in cancer cells and that induction of replication stress is sufficient to trigger cohesion loss in untransformed cells. Also the cohesion-loaders NIPBL and MAU2 were required for cell growth in replication stress conditions. However, unlike WAPL, they were not essential for the restart of broken replication forks.
Understanding the molecular mechanisms that cancer cells rely on for mitigating the deleterious consequences of DNA replication stress might eventually provide new therapeutic opportunities to restrain tumor growth.