Where DNA Copying Fails Can Decide How Cancer Cells Respond to PARP Inhibitors

Not all DNA damage is equal. Every time a cell divides, it has to copy its DNA, and some stretches of the genome are harder to copy than others. Cancer cells depend on a protective response to get past those failures. A new study pinpoints where on chromosomes the problems arise and shows that blocking that response with PARP inhibitors lets damage build up at those sites, compromising the cancer cells’ survival.

The work, led by researchers at NYU Langone Health’s Perlmutter Cancer Center, was published in Nature Cell Biology under the title “Positional replication fork instability drives PARP inhibitor cytotoxicity.” Eli Rothenberg, PhD, a professor of biochemistry and molecular pharmacology at NYU Grossman School of Medicine, and Dipika Gupta, PhD, a postdoctoral scholar in his lab, are co-corresponding authors.

The team began with CRISPR screens in breast and small cell lung cancer models. The screens flagged ESCO2, a protein that helps hold newly copied chromosomes together, as a strong determinant of sensitivity to PARP inhibitors. Even partial loss of ESCO2 increased sensitivity in colorectal, ovarian, and lung cancer cells. The ESCO2-depleted cells kept the tested homologous recombination response. That DNA repair pathway is impaired in the BRCA-mutated cancers for which PARP inhibitors are typically used.

To find out why the cells were sensitive, the researchers turned to quantitative single-molecule imaging, an ultrasensitive microscopy approach developed in Rothenberg’s laboratory. It let them distinguish subtypes of replication-associated damage sites that other methods cannot readily separate, according to Rothenberg. Reducing ESCO2 increased G-quadruplexes, or G4s, which are folded DNA structures that can obstruct copying. The increase was concentrated at a small subset of replication sites, particularly in repetitive pericentromeric regions next to centromeres.

“The imaging enabled us to move beyond measuring damage across an entire cell,” Gupta said. “We could detect subtle replication problems, distinguish the sites involved, and identify the protective response that the cells relied on.”

That response depended on PARP1 activity and involved two other proteins. TIMELESS helps protect DNA replication, and DDX11 is an enzyme that can unwind G4 structures. PARP inhibition disrupted the response in ESCO2-depleted cells. G4s built up further, DNA synthesis declined, and damage increased near centromeres, which helps explain the cells’ sensitivity to the drugs.

“We often think of DNA damage in terms of quantity: More damage means a greater chance of killing a cancer cell,” Rothenberg said. The findings point instead to location as a factor in treatment response.

The authors said the results provide a foundation for developing biomarkers of cancer drug sensitivity. They also raise the possibility that other DNA-damaging treatments work partly because they are more likely to hit these sensitive genomic sites. Whether that holds in other genetic contexts and with other treatments remains to be tested.

Rothenberg is working with clinicians to apply the imaging approaches to patient-derived cancer cells. The goal is to identify patterns of replication stress and DNA damage that could help predict who responds to treatment. The study was conducted in cancer cell models, so further preclinical and clinical research is needed to establish whether these features can guide treatment decisions.

“Our work reveals a genomic Achilles’ heel—a small subset of sites where replication problems can have disproportionately severe consequences. Identifying those sites helps explain what cancer cells depend on to survive and why a particular treatment can be so effective,” added Rothenberg.

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