RAS Initiative study shines the spotlight on less-understood KRAS4a

A four-year effort undertaken by the NCI RAS Initiative has come to fruition, revealing a host of new information about a less-studied KRAS variant that plays a key role in cell biology and human cancers.  

The team, comprising specialists within the RAS Initiative’s hub—Frederick National Laboratory’s Cancer Research Technology Program (CRTP)—performed extensive biochemical and biophysical analyses of the protein KRAS4a. Their study appears in Science Advances. 

KRAS is a member of the RAS family of proteins. RAS proteins sit on the surface of cells throughout the body and perform crucial functions, but mutations in RAS proteins often cause abnormal functions that result in tumor development. Mutant RAS is implicated in more than 20% of all human cancers. 

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Mutant KRAS is a high-priority target for cancer treatment. However, most RAS research, including cancer drug development, has focused on KRAS4a’s sister protein KRAS4b, which appears to be more widespread in the body and—when mutated—more involved in cancer’s formation. Despite this, KRAS4a remains an important target: When mutated, it also causes cancer, often alongside mutant KRAS4b. 

“Whenever you develop a therapy against KRAS, you have to keep in mind that you have to hit both KRAS4a as well as KRAS4b and not just KRAS4b. If you just hit KRAS4b, it’s not the end of the solution,” said Dhirendra Simanshu, Ph.D., a senior investigator in CRTP who coordinated the study across multiple groups within the RAS Initiative. 

Understanding the differences 

The team combined several analytical methods spanning structural biology, cell biology, and data science to give one of the more complete pictures of KRAS4a to date. 

Of particular note were the findings of effects brought on by the presence of different amino acids in key positions within KRAS4a and KRAS4b, the team said. (Amino acids are the molecular building blocks that comprise proteins’ structures.) These building blocks serve as the basis for the shapes KRAS takes and the functions it performs. 

One difference occurs at the 151st amino acid. The team determined that the one that appears there in KRAS4a causes the protein to melt at temperatures five degrees Celsius lower than KRAS4b melts, which means KRAS4b is the more stable protein. However, under normal conditions, KRAS4a can still interact with other proteins at an efficiency similar to KRAS4b. 

The team found that another difference, occurring at the 153rd amino acid, influences how KRAS4a attaches to a protein called RAF1, a crucial interaction in cancers driven by mutant KRAS. This amino acid has a different size in each of the two forms of KRAS: KRAS4b has a smaller one, allowing RAF1 to approach closer than it can to KRAS4a. Position 153 serves to fine-tune the interaction of KRAS with RAF1. 

While small, these nuances have a large impact. Even the tiniest difference in chemical interactions and protein binding can influence whether a cancer drug is able to hit the protein. Most existing drugs targeting mutant KRAS take aim at KRAS4b, so KRAS4a may require different approaches. 

“It’s important to understand what the differences are between 4a and 4b, the two splice variants,” said Megan Rigby, an author on the study and a CRTP research associate. “Our study sheds light on that from a lot of different angles.” 

Robust methods, robust environment 

The use of multiple methods allowed the team to uncover much more than would be typically possible in a smaller study. X-ray crystallography revealed the structure of KRAS4a and allowed it to be compared to KRAS4b. Nuclear magnetic resonance (NMR), another method for determining molecular structure, made it possible to analyze the proteins’ structure in liquid solution. 

“In terms of complexity and structural insight, I think these methods are highly complementary,” said Gabriel Cornilescu, Ph.D., an author on the study and a CRTP scientist. “The differences [between the two KRAS proteins] are very subtle, so it took a lot of NMR and crystallography efforts to pinpoint [them].” 

The team also deployed a bioluminescence resonance energy transfer assay, which allowed them to look at KRAS4a and KRAS4b in cells as they interacted with other proteins in their natural state—a crucial angle that helped tie the findings to how they behave in humans. For further relevance, they analyzed large sets of data from The Cancer Genome Atlas database to illuminate the extent to which KRAS4a and KRAS4b occur in humans and, when mutated, contribute to cancer. 

While the findings have made KRAS4a less enigmatic, the team cautions that there’s still much left to determine. Understanding KRAS4b to the extent that it is today took time, so grappling with KRAS4a will likely be similar, although they expect progress could increase as more data become available. 

“Even though we answered some questions, other questions … we couldn’t answer because we don’t have data. So that’s what we want in the future,” said Ming Yi, Ph.D., a study author and senior bioinformatics scientist in CRTP. 

Still, the study represents a substantial step forward and serves as a testament to programs like the RAS Initiative, where expertise is colocated in one hub or connected by robust networks. For instance, Frederick National Laboratory’s Protein Expression Laboratory, which co-authored the paper and is also part of the initiative, painstakingly made more than 20 proteins and reagents that enabled the study. 

“A project of this scale involves lots of different experimental techniques and computational methodologies and informatics approaches,” said Matthew Whitley, Ph.D., first author on the study and a CRTP scientist, summing up the effort. 

“It’s really amazing to have that scale of expertise and that amount of equipment and resources basically available at the drop of a hat,” he added.