Scientists at Dana-Farber Cancer Institute have created a powerful technology platform for rapidly eliminating almost any desired protein from cells – an advance with applications to better understanding and potentially treating cancer and other diseases.
The system can remove a protein in as little as one hour, compared to previous methods requiring many hours or a day or two, so that the effects of its absence can be quickly assessed, the scientists report in Nature Chemical Biology. The dTAG system, as it’s called, can be used to deplete proteins in living mice, which hasn’t generally been possible with other systems.
“It’s a great way to rapidly degrade a protein of interest for target validation, and also for basic biology, to study the acute loss of function of a protein,” said Nathanael Gray, PhD. He is corresponding author along with James Bradner, MD, formerly of Dana-Farber and now president of the Novartis Institutes for BioMedical Research.
“We can use this platform to answer cancer-related questions in a time-scale that has not been possible with other genetic strategies, fundamentally changing the types of biologic questions that we now pursue,” said Behnam Nabet, PhD, co-first author of the report.
The advance comes at a time of massive interest and development in the fast-moving field of targeted protein degradation. The tens of thousands of proteins in each cell – referred to in aggregate as the proteome -- control its workings, and many diseases, including cancer, are due to abnormal or malfunctioning proteins. Drugs that block abnormal proteins, such as those that regulate cell growth and division, are standard treatments for cancer, but the great majority of proteins – including some major cancer-causing proteins such as KRAS -- can’t be “drugged” with current methods.
Targeted protein degradation works differently: rather than block or inhibit a protein, it eliminates it from the cell. It exploits the cell’s mechanism for recycling and eliminating, or degrading, proteins that are misshapen, worn-out, or no longer needed. Such proteins are designated for removal by being tagged with a small protein called ubiquitin, like a Post-It note saying “this is garbage.” Ubiquitin is attached to the target protein with the guidance of an adaptor protein called an E3 ligase. The protein is then steered to a disposal-like structure called a proteasome, where it is chewed up into small fragments.
Scientists have learned to hijack this system. They use two-ended molecules, called degraders, to recruit a desired protein to an E3 ligase, designating it for disposal by the proteasome.
The dTAG system devised by the Dana-Farber chemical biologists uses molecules that recruit the CRBN E3 ligase complex to target proteins that are fused to an engineered variant of a binding protein called FKBP12. “With targets that work well, you can get complete degradation in an hour – much faster than with other methods where you might need a day or two,” said Gray.
To demonstrate the system’s applicability to live animals, the investigators attached the engineered variant of FKBP12 to luciferase, a luminescent protein, and transferred them into the bone marrow of mice. They then administered a dTAG degrader molecule, and observed that within four hours the glowing signal of luciferase was dimmed, indicating that the protein had been degraded. Subsequently, 28 hours later, the glowing signal returned, showing that the cells containing luciferase were making the protein again.
The Dana-Farber scientists also used the system to degrade a mutant form of the KRAS cancer protein in mouse cells in the laboratory. They demonstrated that rapidly depleting mutant KRAS from the cells altered the downstream cancer-related activity of pathways controlled by KRAS, showing that chemical degradation of the protein is potentially a new strategy for treating KRAS-driven cancers.
The authors said they envision that the dTAG system “will broadly enable biological exploration and target validation in cells and in mice.”
The research was supported by an American Cancer Society Postdoctoral Fellowship PF-17-010-01-CDD, the Claudia Adams Barr Program in Innovative Basic Cancer Research, Damon Runyon Cancer Research Foundation, the Hale Center for Pancreatic Cancer Research, and the Katherine L. and Steven C. Pinard Research Fund.