Novel Protein May Provide Tool to Target Elusive RAS Oncogene

John P. O’Bryan, PhD

Associate Professor, Pharmacology

The University of Illinois at Chicago

For nearly 30 years, researchers have focused on identifying ways to pharmacologically inhibit RAS, one of the most frequently activated oncogenes in human cancers.

RAS genes include 3 members, HRAS, KRAS, and NRAS, each encoding proteins that function as regulated molecular switches, cycling between an “on” and “off” state. In cancer, mutations to RAS lock the protein in an active, or “on,” state, leading to chronic activation of downstream signaling pathways that stimulate proliferation.

Unfortunately, direct RAS inhibitors have been elusive because, unlike many other drug targets, RAS proteins lack deep pockets where putative inhibitors can insert to block activity. While a number of RAS inhibitors have been isolated, none have reached FDA approval status due to issues with specificity or lack of activity toward mutant RAS in tumors. Given the “addiction” of many tumors to mutant RAS for their survival, there has been renewed interest in developing new ways to inhibit RAS in cancer. Indeed, the National Cancer Institute launched the RAS Initiative in 2013 to attack the problem of RAS inhibition specifically.

Our team of researchers at the University of Illinois at Chicago have discovered a previously unrecognized surface on RAS that is important for the signaling and transforming activity of this oncogene. Our findings, published in Nature Chemical Biology, suggest a novel means of blocking the action of genetic mutations in cancer (2017;13[1]:62-68). (Figure)

Figure. NS1 Monobody as Anticancer Agent

RAS proteins bind to the nucleotide guanosine diphosphate (GDP) in the “off” state and become “active” through aberrant signaling to bind with guanosine triphosphate (GTP). The monobody NS1 can disrupt RAS functions by binding to HRAS or KRAS proteins and inhibiting the hetreodimerization and activation of CRAF and BRAF proteins.

This method could potentially lead to new therapeutic approaches to treat cancers caused from a RAS mutation, including pancreatic, lung, and colon cancers. Such approaches may also be of benefit in cancers that lack specific oncogenic mutations in RAS but depend on RAS activity for their survival. Targeting wild-type RAS may also help unlock new therapeutic options.

Our strategy to studying RAS was unique. We used monobody technology to identify regions of RAS that are critical for its function. Monobodies are engineered, synthetic binding proteins designed to recognize a specific protein of interest.

Developed by Shohei Koide, PhD, of NYU Langone's Laura and Isaac Perlmutter Cancer Center, a collaborator on this study, this technology has been used to inhibit a diverse array of signaling proteins. Unlike conventional antibodies, monobodies are much smaller, are resistant to the reducing environment inside a cell, and can be readily used as genetically encoded inhibitors.

We isolated a monobody, termed NS1, which bound selectively and with high affinity to HRAS and KRAS, but not NRAS. Using the NS1 monobody, we inhibited oncogenic HRAS and KRAS signaling and cellular transformation. This specificity was due to a minor sequence difference in NRAS versus HRAS and KRAS that prevented NS1 binding to the NRAS protein.

We found that NS1 blocks RAS function by binding to a surface of the protein previously unrecognized as important for RAS function. Further work revealed that this surface of RAS was critical for 2 RAS proteins interacting with each other in the process of activating their downstream targets.

Ways to Translate Findings

Structural analysis of the NS1:RAS complex revealed potential regions of RAS that might be targeted with small molecules to mimic the effect of NS1. These findings provide the most compelling support for the idea that RAS proteins function as molecular dimers or clusters, which has been a long-standing point of debate. This work reveals a new approach to targeting RAS inhibition, namely inhibition of RAS dimerization and clustering.So what do these findings mean for oncologists and their patients? For many patients with cancer, current treatment relies on the use of nonspecific and highly toxic drugs that differentially kill tumor cells as opposed to healthy cells because of the increased proliferative capacity of tumor cells. Nevertheless, these chemotherapy strategies result in significant nonspecific killing of normal cells and toxicity.

These new findings provide a potential approach to block the oncogenic mutation in the cancer by specifically targeting the oncogenic RAS mutant that drives the tumor, resulting in potentially higher specificity and lower toxicity to such therapy.

Indeed, researchers have observed a great deal of success with targeted therapies directed at specific oncogenic kinases present in tumors. However, such RAS-specific treatments are still in the future. While NS1 is a powerful research tool for understanding the role of RAS signaling in cancer, its use as a therapeutic modality is limited. As a relatively large protein, monobodies such as NS1 are not able to passively enter the cell like a typical drug or small-molecule therapeutic. In addition, monobodies, like any protein, are subject to degradation when injected into the body. Thus, the goal of this work is to identify a small molecule that can mimic the effect of NS1 and then be formulated into a viable drug candidate.

Although this process is likely to take a number of years, these findings, coupled with recent results from other research groups, suggest that researchers are zeroing in on effective ways to pharmacologically inhibit RAS. Kevan Shokat, PhD, of UCSF’s Helen Diller Family Comprehensive Cancer Center, and colleagues have isolated small molecular inhibitors that target a specific oncogenic RAS mutant present in many types of solid tumors. Although these inhibitors block only 1 particular oncogenic RAS mutant, the so-called G12C mutant, their work suggests that pharmacological targeting of RAS activity is possible.

In addition to providing insights into new approaches to inhibit RAS, the NS1 monobody represents a powerful tool to further probe RAS function. Our group has found that use of NS1 has highlighted specific differences in the way each RAS isoform functions. These results may provide additional insights into ways to selectively antagonize individual RAS isoforms.