Discovery of new pathway offers potential solution to prevent cancer chemoresistance

Researchers have discovered a new pathway that explains how cancer cells become resistant to chemotherapies, which in turn offers a potential solution to preventing drug resistance.

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, making them tolerant to chemotherapy drugs.

“It provides us with tools to manipulate and then break down chemoresistance in cancer cells,” said Marcus Smolka, acting director of the Weill Institute of Cellular and Molecular Biology and professor of molecular biology and genetics at the College of agriculture and life sciences.

Diego Dibitetto, a former postdoctoral researcher in Smolka’s lab who is currently at the University of Bern in Switzerland, is the paper’s first author.

Many cancer drugs work by creating blocks in the DNA of cancer cells as they replicate. During replication, the DNA strands entwined in a double helix separate into two individual strands so that each strand can be copied, ultimately leading to two new double helices. The junction where this splitting and copying occurs is called a replication fork, which unzips the double helix.

If these replication forks were cars on a road, chemotherapy drugs can be imagined as roadblocks that interfere with the traffic of cars, thereby 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, and promotes fork slowing and chemoresistance.

DNA-PKcs is known for its role in DNA repair related to immune system antibody generation 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 the action of this kinase. It’s not about repairing the DNA in this case; it slows down the forks to prevent breaks from happening in the first place.”


Marcus Smolka, acting 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 being used for clinical trials alongside radiotherapy. In these treatments, radiation damages the DNA of cancer cells, and it was thought that inhibiting DNA-PKcs would limit cell repair. But DNA-PKcs inhibitors don’t work well in this setting 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 chemotherapies, where the chemotherapy drugs would create blocks to DNA replication, and the inhibitor would prevent forks from slowing down replication that leads to drug resistance.

In the study, the researchers used an assay to detect DNA-PKcs kinase at replication forks. Then they used a DNA fiber test with fluorescent colors, so the faster the replication forks moved, the longer the fibers became. In the presence of chemotherapy drugs, the fibers were short, indicating slowed replication forks. But when inhibitors were added, the fibers remained longer, indicating that the forks were moving at faster speeds.

Co-author Massimo Lopes, an expert in replication stress at the University of Zurich, took images that confirmed that replication forks no longer reversed and slowed down in the presence of the kinase inhibitors. The team also proved that cancer cells became diseased or degraded when chemotherapy and inhibitors were applied together.

Finally, BRCA2-deficient breast cancers can become resistant to the chemotherapy drugs used to treat them, and fork inversion was known to be implicated in resistance. In this study, when researchers applied DNA-PKcs inhibitors to BRCA2-deficient breast cancer cells that were resistant to treatment, the cells regained sensitivity to treatment.

“This is another way to confirm that the ability to prevent fork-slowing and reversal with DNA-PKcs inhibitors appears to be a very good way to manipulate chemoresistance,” Smolka said. .

In future work, the research team will study how cells sense stress from replication forks and which proteins DNA-PKc interacts with to slow down these forks.

Sven Rottenberg, a cancer therapy resistance researcher at the University of Bern, is co-author.

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.

Source:

Journal reference:

Dibitetto, D. et al. (2022) DNA-PKcs promotes fork inversion and chemoresistance. Molecular cell. doi.org/10.1016/j.molcel.2022.08.028.

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