A model of the interaction of processed KRAS-FME protein (cyan) with a lipid nanodisc (green and yellow).

A scientific innovation at the Frederick National Lab has opened the way for a new line of experiments in the decades-old quest for a drug to fight cancers triggered by mutant RAS proteins – which underlie one-third of all malignancies, including those of the pancreas, lung, and colon.

Using modified insect cells, a research group at the national lab recently invented a more accurate laboratory replica of a RAS protein, KRAS4b, that can be produced at high grade and in quantities adequate for research studies that were impossible previously.

The research advance, published in Nature Scientific Reports, describes a method for manufacturing KRAS4b sufficient for advancing the research and for screening compounds that might be used correct the mutant protein’s malfunction. These include studies involving X-ray crystallography, nuclear magnetic resonance spectroscopy, and high-throughput screening for drug candidates.

"Our approach is an improvement not only in the quality and accuracy of the protein that can be made, but also in the purity and quantity of KRAS4b," said Bill Gillette, Ph.D., principal scientist and group leader with the NCI RAS Initiative at the Frederick National Lab.

Gillette said the new approach places KRAS4b in its native environment, the cell membrane, where it carries out its vital functions and where, if mutated, it pushes the cell toward uncontrolled growth, which is characteristic of cancer. They call this a fully processed KRAS protein.

"We and others have already begun experiments with this new version of KRAS4b that are aimed at studying KRAS4b and the proteins to which it binds in the presence of the membrane," Gillette said. "Since this is where KRAS4b is active, this will allow a more biologically relevant platform to screen for compounds which can disrupt interactions that might lead to cell proliferation."

From left to right: Bill Gillette (Principal Scientist, Group Leader, Protein Production, PEL), Peter Frank (Research Associate III, Protein Production, PEL), Dom Esposito (Director, PEL).

Team leader Andy Stephen, Ph.D., RAS Biochemistry/Biophysics, said KRAS must be bound to the cell membrane to activate the next protein in the signaling network, RAF kinase. Having this laboratory model now available, Stephen said it will be possible to investigate the detailed molecular mechanism of that process and for developing novel therapeutics. His group has taken this protein, combined it with lipid nanoparticles, and used NMR spectroscopy and other biophysical methods to determine the shape and relative position of KRAS on the membrane, an initial step in understanding its function.

"The next step will be to take the RAF kinase and include it in these experiments to understand how these proteins work together at the membrane to initiate signaling," Stephen said. "None of our experiments would be possible without the fully processed KRAS protein."

In addition, the research will aid numerous other RAS investigators around the world. "We have already sent materials for making processed KRAS to nine different laboratories in three countries," said Dom Esposito, head of the Reference Reagents Group, NCI RAS Initiative.

The research involved a close collaboration of among a team of scientists.

"I am proud that our efforts will provide the community with a quality reagent to improve research and hopefully facilitate the discovery of potential therapeutics," said Carissa Grose, group leader, Cloning and Nucleic Acids.

"I feel fortunate to have joined the group at a time where the work has been very collaborative. There is still a great deal of excitement within the RAS Initiative and I hope that passing future milestones will maintain this energy as we learn the complexities of these oncogenes," she said.

Gillette said being a part of something like this could only happen with cooperation from scientists from several different areas.

"It is incredibly thrilling to have been a part of this team," Gillette said. "There were so many parts to the work that all needed to come into place for it to work. On a personal note, with many of my immediate family having already succumbed to the cancers for which KRAS4b mutations are involved, there is an entirely different level of satisfaction that I have felt having made a small contribution to advancing a field toward the hopeful development of new therapies, as there are currently none."

Esposito said the goal of creating high yields of KRAS4b protein similar to human cells was a longshot.

"It was a great feeling—going into the project we knew that this was pretty much the Holy Grail for our program," Esposito said. "If we could make this reagent in large amounts and with high quality, we could really make an impact both on basic RAS biology and drug discovery. It took a lot of hard work by a large team to finally nail it down, so seeing this manuscript in print is a real tribute to the team effort."

Research associates Peter Frank and Shelley Perkins worked out critical aspects of the purification. Cammi Bittner (recently retired) was critical in figuring out the optimal parameters for expressing the protein in insect cells. Scientist II Zhaojing Meng and senior scientist Oleg Chertov used mass spectrometry to help confirm that the proper modifications were made to the final proteins. Research associates II Jen Mehalko and Vanessa Wall worked to generate RAS plasmid constructs as part of the RAS Reference Reagents group.

"Dom (Esposito) had the initial idea that we could do this, and Carissa (Grose) made it possible by engineering the baculovirus to express the enzymes that were necessary for the critical modification," Gillette said.

The laboratory is continuing to modify the system to further improve the expression of KRAS4b as well as other proteins relevant to the research. The Frederick National Lab is continuing the work under the NCI RAS Initiative to improve the protein expression system for KRAS4b and for other proteins relevant to the research.

The Frederick National Lab is an integral part of the NCI RAS Initiative, a coordinated research and development initiative created by the National Cancer Institute to further scientific understanding of cancers driven by mutations in the RAS family of genes.

Top image: A model of the interaction of processed KRAS-FME protein (cyan) with a lipid nanodisc (green and yellow). The KRAS-FME structure was solved at the FNLCR by Dhirendra Simanshu as a complex of KRAS-FME and a binding partner, PDE-delta. Here the PDE-delta has been removed and replaced with the NMR structure of a nanodisc as solved by Mitsu Ikura at the University of Toronto to demonstrate a possible interacting surface for the KRAS protein and the plasma membrane.​

Last updated: September 17, 2015