The RAS Chase: Gaining Ground Against the Toughest Oncogene

Philip A. Philip, MD, PhD

Nearly 35 years after its discovery as the first human oncogene, RAS remains a frustrating target in anticancer therapy even as knowledge about the molecular drivers of malignancies continues to expand at a rapid pace. Thus far, no therapies aimed directly at RAS mutations have been developed.

Nevertheless, a concerted effort by the National Cancer Institute (NCI) is starting to unravel the RAS mystery by building the scientific foundation for researchers to move forward. Researchers are producing a more detailed roadmap of the cellsignaling networks set into motion by mutations in the gene along with a more accurate databank for mutations in the 180 genes in the pathway. Notably, a new method for producing a key RAS protein from insect cells has yielded higher-quality material for experiments.

The focus on RAS is understandable, considering its important role in tumorigenesis. More than 30% of all tumors have some sort of mutation in the RAS family of genes, according to Sara Hook, PhD, RAS program officer at the NCI.

From Rats to Humans

“If we had effective inhibitors to RAS, or the RAS pathway, it could radically change cancer treatment just because of the sheer volume of patients who would be affected,” Hook said in an interview with OncologyLive.The observation that RAS genes could cause cancer was initially discovered in rat sarcomas; hence the name, RA for rat and S for sarcoma. These genes were later identified in humans in 1982. RAS is a family of oncogenes that are present in all cells in the body and were the first oncogenes identified in human cancer cells.¹ Oncogenes work as an on/off switch for cell division, and RAS mutations affect this switch, not allowing the cells to switch off and causing them to grow uncontrollably, leading to the development of cancer.

“In normal cells, the RAS gene is under very tight control in terms of how it behaves, but in the cancer cell, when it’s mutated, it becomes out of control. It’s independently sending signals to the nucleus to not only make cells divide and proliferate without any control, but also to spread and evade the effects of anticancer drug therapy,” Philip A. Philip, MD, PhD, explained in an interview. Philip is leader of GI and Neuroendocrine Oncology, and vice president of Medical Affairs at the Barbara Ann Karmanos Cancer Center in Detroit.

There are three RAS genes in humans: KRAS, HRAS, and NRAS. Mutations within KRAS are found more often in human cancers than are mutations within HRAS or NRAS.1 Stephen et al wrote in 2014 that KRAS mutations may be more effective in oncogenesis than HRAS and NRAS because of its structure. Only KRAS has “stemlike properties” on certain cell types. KRAS also seems to be critical in development.1 Mice that were lacking KRAS died during formation of the embryos, while those that did not have HRAS or NRAS did not die at that stage. KRAS is found to be more frequently altered in solid tumors: 95% of pancreatic cancers, 45% of colorectal cancers, and 35% of lung cancers, according to the NCI (Figure). By contrast, NRAS is found to be more frequently altered in hematological cancers: 30% of acute myeloid leukemias and 15% of melanomas. HRAS is found to be altered in 15% of bladder cancer.

The Research Bandwagon

“RAS oncogenes are the worst oncogenes,” Frank McCormick, PhD, FRS, DSc (Hon), national program advisor for the NCI’s RAS Initiative, has said. McCormick is a professor emeritus at UCSF Hellen Diller Family Comprehensive Cancer Center in San Francisco.When RAS mutations were initially discovered, there was a flurry of research into new drug possibilities, but these did not lead to effective treatments. Initially, there was hope that farnesyltrans- ferase inhibitors (FTIs) were the answer. However, testing found that, although FTIs did block HRAS, the strategy did not prove effective in phase II trials for lung and pancreatic cancers, or in phase III trials for colorectal and pancreatic cancers.²

“There were some clinical trials in the early 2000s that did not succeed,” Hook said. Researchers found that the RAS protein already had an escape mechanism, she explained, and that the cells had found a way to get around the drugs.

Almost 20 years have passed since the initiation of those failed trials and researchers have continued to learn more about RAS mutations. This, along with the advancements in technology since the original research, has led to a new interest in RAS. In response, the NCI established the RAS Initiative in 2013, with the goal of discovering more about RAS genes and how they can be targeted for anticancer treatment. The program, which the NCI funds at $10 million annually, uses a large-scale open-model collaborative approach organized through a research hub at the Frederick National Laboratory for Cancer Research in Maryland.

A New KRAS Protein

“A team is researching RAS and they are developing assays for the pharmaceutical community and academic researchers,” Hook said. “They have developed many different reagents that they send to other researchers to help their studies of RAS. The research team is focused on the worst culprit of the RAS family of genes, KRAS. We actually didn’t know the structure of full length KRAS protein until very recently.”In an update at the 2016 American Association for Cancer Research Annual Meeting, McCormick said one of the RAS Initiative’s most significant achievements of the past year has been to produce a fully processed KRAS4b protein for research.^3,4

The new protein has been engineered to “mimic essential in vitro activities of KRAS,” in contrast to existing methods that do not accurately replicate the proteins functioning in human cancers, Gillette et al reported.⁴

“In order to fully understand the structure and function of KRAS4b in its native environment, a high yield and high quality method for production of processed protein is essential,” they wrote.⁴ This is particularly important because of the manner in which RAS genes function, McCormick indicated in his AACR presentation. “KRAS and RAS biology in general all happens in the plasma membrane. That’s where RAS gets activated or inactivated and that’s where RAS proteins interact with their effectors,” he said.

Pancreatic Cancer Mission

“There’s a lot we don’t understand about the interaction between RAS and the membrane but this is really critical in RAS signaling, and if we understood the molecular details of how RAS proteins snuck up to the membrane and are reflected by the membrane, it might provide new opportunities for therapeutic intervention,” he said.The need to investigate the mechanisms of KRAS activity are most pressing in pancreatic cancer. “Pancreas cancer kills 40,000 or more people every year, and KRAS is present in almost all of them,” Philip said. “It’s possible that if we find a treatment that could influence the effects of mutated KRAS or target KRAS itself, it could prolong the life of patients with pancreatic cancer.”

Eileen M. O’Reilly, MD, agrees. “I think, potentially, RAS in pancreatic cancer is sort of perceived as the holy grail because it’s such a fundamental driver mutation that occurs in these cancers.” O’Reilly is associate director for Clinical Research, David M. Rubenstein Center for Pancreatic Cancer Research, at Memorial Sloan Kettering Cancer Center, in New York.

What is particularly interesting when looking at the RAS mutations in different cancers, is that the mutation is an initiating event in pancreatic cancer, but accumulative in others. Alteration of KRAS is found very early on in pancreatic cancer and is the important oncogenic driver and source in this type of cancer.

Why No Therapy Yet?

“So if the RAS Initiative is going to come up with a drug, it better work in pancreatic cancer,” Susan E. Bates, MD, said in a National Institutes of Health (NIH) webcast. ⁵ “This is one of the most drug-resistant cancers we have.” Bates was head of the Molecular Therapeutics Section at NCI until August 2015, and currently is at Columbia University Medical Center in New York.Despite the large investment put toward RAS research and the renewed interest in it, there are no drugs ready yet for clinical trials. Aside from the RAS mutations’ ability to escape drug mechanisms, staying one step ahead so to speak, the actual protein structure makes it a difficult target for drugs. “Part of what makes RAS so difficult for drugs to bind is that it lacks good ‘pockets’—nooks and crannies in the protein structure where a small molecule can tightly attach,” Thompson et al wrote in a Nature Medicine article.⁶

“At this point in time, there are no FDA-approved drugs that target KRAS mutation,” Philip said. “The studies that have been done over the years were also to target the molecules that are what we call downstream, or the molecules (eg, MEK) that were influenced by ‘overactivity’ of RAS.

Newest RAS Research Findings

“The idea was to try to target these molecules because of inability to hit RAS itself,” said Philip. “At this time, we do not yet have any success in any of that.” Philip points out that there are several novel drugs that are in development, but they are still very experimental.E. Premkumar Reddy, PhD, a professor at the Department of Oncological Sciences at the Icahn School of Medicine at Mount Sinai, has described the difficulty in getting a drug to bind to the RAS structures by comparing the structures to Lego blocks that fit each other perfectly.⁷ The proteins all fit in nicely to the RAS binding domains, he explained in a video, and there is no room for a drug to get inside this perfect fit.

However, Reddy and colleagues recently reported a promising discovery: the anticancer agent rigosertib is small enough to slip inside the structure, bind to the domain, and block the proteins from latching on and activating the on-signal.⁸

Does RAS Mutation Affect Patient Outcome?

“Rigosertib’s mechanism of action represents a new paradigm for attacking the intractable RAS oncogenes,” Reddy said in a release. “Our current focus is to use the information from our studies with rigosertib to design the next generation of small molecule RAS-targeting therapies, and we are excited to have recently identified several compounds which we think improve on the qualities of rigosertib.”Another complexity in targeting RAS mutations is understanding the impact these aberrations have on the course of a cancer.

In colorectal cancers, studies have shown that patients with RAS mutations on certain exons will not respond well to anti-epidermal growth factor receptor (EGFR) therapy such as cetuximab. The American Society of Clinical Oncology has recommended that all patients with metastatic colorectal cancer who are candidates for anti- EGFR antibody therapy should have their tumor analyzed for mutations in both KRAS and NRAS exons 2, 3, and 4.⁹

In pancreatic cancer, there is some evidence that patients with specific KRAS mutations may have poorer outcomes.¹⁰

However, O’Reilly said that there is optimism for meaningful breakthroughs. For example, perhaps mutated RAS could one day be useful as a screening tool. “Researchers are looking at cell-free DNA—DNA that has broken off from cells—which is circulating in the blood,” she said.

“The principle behind looking for this is that it may be a detectable version of the tumor before it’s evident radiologically,” O’Reilly said. “This may be a way of screening.” If successful, this could also become a way to identify patients with early recurrence after standard treatment.

A KRAS mutation is found in about one-third of lung cancers, more so in lung adenocarcinoma than in squamous cell non—small cell lung cancer (NSCLC).

One study of 106 patients with adenocarcinoma of the lung found that only smokers in the group had a KRAS mutation, and a larger study of 482 patients with adenocarcinoma of the lung showed KRAS aberrations in more smokers than never-smokers.¹¹

At one point, researchers believed that patients with NSCLC with KRAS may have a poorer outcome, but studies to this effect have been conflicting.

Several studies did show that KRAS was a significant prognostic marker in some instances, but a retrospective analysis did not back this up, saying that there was no prognostic or predictive value.¹² More study also needs to be done on whether the presence of KRAS may have an adverse effect on patients with NSCLC who receive adjuvant chemotherapy. While there may be some indications that this may be so, this has yet to be validated by a prospective, randomized clinical trial.¹²

“KRAS mutations in NSCLC, despite being the most common, remain the most intriguing and elusive of therapeutic targets,” the authors wrote. “At present, targeted treatment is not available for KRAS-mutated NSCLC outside clinical trials. However, novel agents targeting downstream effector signaling pathways are under clinical development.”¹²

The RAS Initiative has pushed RAS research back into the forefront of cancer research. As the quest to solve the RAS puzzle continues, only time will tell the role that attacking these mutations might play in the future.

References

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3. McCormick F. The NCI RAS Initiative at the Frederick National Laboratory for Cancer Research. Presented at: 2016 AACR Annual Meeting; April 16-20, 2016; New Orleans, LA.
4. Gillette WK, Esposito D, Blanco MA, et al. Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions. Sci Rep. 2015;5:15916. doi:10.1038/srep15916.
5. McCormick F. Demystifying medicine 2015—Ras: a cancer mechanism and target [videocast]. National Institutes of Health. http://goo. gl/HWc6wd. Broadcast February 10, 2015. Accessed April 28, 2016.
6. Thompson H. US National Cancer Institute’s new Ras project targets an old foe. Nat Med. 2013;19(8):949-950.
7. Reddy EP. Mount Sinai Hospital video discussing RAS mutations and binding. https://goo.gl/Ke0t60. Posted April 22, 2016. Accessed April 28, 2016.
8. Athuluri-Divakar SK, Vasquez-Del Carpio R, Dutta K, et al. A small molecule RAS-mimetic disrupts RAS association with effector proteins to block signaling. Cell. 2016;165(3)643-655.
9. Allegra CJ, Rumble RB, Hamilton SR, et al. Extended RAS gene mutation testing in metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy: American Society of Clinical Oncology Provisional Clinical Opinion Update 2015. J Clin Oncol. 2016;34(2):179-185.
10. Holderfield M. New RAS assays and inhibitor screens. Presented at 2015 AACR Annual Meeting; April 18-22, 2015; Philadelphia, PA.
11. Sequist LV, Neal JW. Personalized, genotype-directed therapy for advanced non-small cell lung cancer. UpToDate. http://goo.gl/ mKvu26. Updated: February 11, 2016. Accessed April 28, 2016.
12. Korpanty GJ, Graham DM, Vincent MD, Leighl NB. Biomarkers that currently affect clinical practice in lung cancer: EGFR, ALK, MET, ROS-1, and KRAS. Front Oncol. 2014;4 204. doi:10.3389/ fonc.2014.00204.