Researchers have discovered a novel pathway that explains how cancer cells become resistant to chemotherapy, which in turn offers a potential solution to prevent chemoresistance.
Experimental DNA fibers with fluorescence (pictured) were used to reveal the speed of DNA replication forks.
The research describes for the first time how a type of enzyme – previously known for its role in DNA repair – prevents DNA damage in cancer cells and makes them tolerant to chemotherapy drugs.
“It provides us with tools to manipulate and then break chemoresistance in cancer cells,” said Marcus Smolka, interim director of the Weill Institute of Cell and Molecular Biology and professor of molecular biology and genetics at the College of Agriculture and Life Sciences.
Diego Dibitetto, a former postdoctoral fellow in Smolka’s lab and currently at the University of Bern in Switzerland, is the first author of the publication.
Many anti-cancer drugs work by creating blocks on the DNA of cancer cells as they replicate. During replication, strands of DNA intertwined in a double helix separate into two individual strands, allowing each strand to be copied, eventually resulting in two new double helixes. The junction where this separation and copying occurs is called the replication fork, which unwraps the double helix.
If these replication forks were cars on a road, you could think of chemotherapy drugs as obstacles that disrupt the flow of cars, stopping replication and breaking DNA. But cancer cells have a way of slowing down these forks, which allows them to avoid such collisions and protect their DNA, leading to drug tolerance.
This study reports for the first time how a kinase (enzyme) called DNA-PKcs acts as a sensor when a fork is stressed due to blockages, promoting fork slowing and chemoresistance.
DNA-PKcs is known for its role in DNA repair related to immune system antibody formation and radiation resistance. But this is the first time the kinase has been linked to slowing down a replication fork, a process called fork inversion.
It’s a whole new way of thinking about how this kinase works. In this case, it doesn’t repair DNA; it slows down forks to prevent it from stopping in the first place.”
Marcus Smolka, Interim Director of the Weill Institute for Cell and Molecular Biology and Professor of Molecular Biology and Genetics at the College of Agriculture and Life Sciences
The results open the door to new cancer treatments as DNA-PKcs inhibitors already exist and are used in clinical trials along with radiotherapy. In these treatments, radiation damages the DNA of cancer cells, and the thought was that inhibiting DNA-PKcs would limit cell repair. But DNA-PKcs inhibitors don’t work well in this context because cancer cells have other ways to repair themselves.
This study provides early evidence that a DNA-PKcs inhibitor could be effective in combination with chemotherapy regimens, where chemotherapy drugs would block DNA replication and the inhibitor would prevent the slowing of replication forks that leads to chemoresistance.
In the study, researchers used an assay to detect DNA-PKcs kinase at replication forks. Then they used a DNA fiber assay using fluorescent colors so that the faster the replication forks moved, the longer the fibers became. In the presence of chemotherapeutic agents, the fibers were short, indicating slowed replication forks. But when inhibitors were added, the fibers stayed longer, indicating the forks were moving at higher speeds.
Co-author Massimo Lopes, an expert in replication stress at the University of Zurich, took pictures that confirmed that the replication forks stopped reversing and slowed down in the presence of the kinase inhibitors. The team also showed that cancer cells became diseased or were broken down when chemotherapy and inhibitors were used together.
Finally, BRCA2-deficient breast cancers can become resistant to chemotherapy drugs used to treat them, and fork reversal was known to be involved in resistance. In this study, when the researchers applied DNA-PKcs inhibitors to BRCA2-deficient breast cancer cells that were resistant to treatment, the cells regained their sensitivity to the treatment.
“This is another way to confirm that the ability to prevent slowing and fork reversal by DNA-PKcs inhibitors appears to be a really good way to manipulate chemoresistance,” Smolka said.
In future work, the research team will study how cells perceive replication fork stress and which proteins DNA-PKcs interact with to slow down these forks.
Co-author is Sven Rottenberg, cancer therapy resistance researcher at the University of Bern.
The study was funded by the Fleming Research Foundation, the National Institutes of Health, the Swiss National Science Foundation, the European Union and the Wilhelm Sander Foundation.
Dibitetto, D. et al. (2022) DNA-PKcs promote fork reversal and chemoresistance. molecular cell. doi.org/10.1016/j.molcel.2022.08.028.