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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer J. Author manuscript; available in PMC 2017 May 1.
Published in final edited form as:
PMCID: PMC4959441
NIHMSID: NIHMS775218

Therapeutic Approaches to RAS Mutation

Abstract

The study of oncogenic RAS mutations has led to crucial discoveries regarding cancer molecular biology and behavior, and has been integral in shaping the era of targeted cancer therapy. RAS mutations are one of the most common oncogenic drivers in human cancer, and intense efforts to find a clinically effective inhibitor is ongoing. Despite these efforts, targeting RAS mutations has remained elusive, so much so that some have termed oncogenic RAS mutations as “undruggable”. In this review, we will summarize current understanding of RAS biology, explore strategies to inhibit RAS oncoproteins and its downstream effectors, and discuss recently described complexities that have shed new light on this pursuit.

Keywords: RAS, synthetic lethality, MAPK, PI3K, oncogenic, colorectal cancer

Introduction

Rat sarcoma virus (RAS) proto-oncogenes encode 21,000-dalton membrane bound small GTPases that include Harvey-Ras (HRAS), Kirsten-Ras (KRAS), and neuroblastoma-Ras (NRAS), collectively called RAS [1]. Oncogenic mutant RAS isoforms were first described over three decades ago when they were implicated in the development of rat sarcomas [1-5]. RAS gene mutations are among the most frequently mutated genes in human cancers, found in approximately 30% of all tumor types and in approximately 50% of colorectal cancer (CRC) [6, 7]. Clinically, RAS mutations have proven to be an elusive target despite the substantial amount of research investigating strategies to inhibit oncogenic RAS mutations. In addition, oncogenic RAS mutations cause acquired resistance to anti-epidermal growth factor receptor (EGFR) therapies such as cetuximab and panitumumab [7-11]. Given its clinical relevance as an important predictive biomarker, the American Society of Clinical Oncology (ASCO) and National Comprehensive Cancer Network (NCCN) guidelines recommend RAS testing for KRAS and NRAS mutations in exon 2 (codons 12 and 13), 3 (codons 59 and 61), and 4 (codons 117 and 146) when managing patients with metastatic colorectal cancer. Due to the elusive nature of RAS biology, the National Cancer Institute organized a RAS initiative in 2013 aimed at developing effective therapies targeted at oncogenic RAS [12, 13].

The complexities of oncogenic RAS mutations

In a sentinel paper published in 1988, Vogelstein et al. first described the role of RAS activation in the development of CRC [14]. Upon DNA analysis of 80 adenomas and 92 CRC samples, the authors observed RAS gene mutations in 58% of adenomas larger than 1 cm and in 47% of carcinomas. RAS mutations appear to be an early event in CRC tumorigenesis [15]. Consistent with present day data, the majority were KRAS mutations, although 5 NRAS mutations were also found. The authors concluded that RAS is often a dominating oncogene and additional aberrations, such as loss of tumor suppressing genes, are responsible for development of CRC.

RAS mutations in human malignancies encompass a variety of tumor types. For example, KRAS mutations exist in greater than 90% of pancreatic cancers, 44-53% of colorectal cancers, and 17-33% of lung cancers, whereas NRAS and HRAS occur less frequently and are most often observed in melanoma, acute myeloid leukemia, and thyroid cancers (Table 1) [7, 16-19]. Once present, oncogenic RAS mutations act through many different pathways to elicit its tumor-promoting effects and render RAS insensitive to GTPase-activating proteins (GAPs) activity, promoting constitutive activation of RAS leading to a persistent “on” signal activating multiple downstream effector proteins [20]. Tumor cells subsequently develop an “oncogenic addiction” to RAS activity driving tumor cell survival, differentiation, and proliferation[21].

Table 1
Approximate frequency of RAS mutations in human cancers[1, 18, 54]

RAS biology: structure, function, and mutations

RAS functions as signal transducers important for regulating cell proliferation, differentiation, and survival in normal and malignant cells. RAS exists in an “on” or “off” state by cycling between GTP-bound active and GDP-bound inactive forms [22]. The RAS-mitogen activated protein kinase-ERK kinase-extracellular signal-related kinase (RAS-RAF-MEK-ERK) pathway is one of the best characterized signal transduction pathways and its aberrancies are commonly implicated in the development of multiple different cancer types including colorectal cancer (Figure 1a) [22].

Figure 1
RAS-mediated signal transduction pathways and post-translational modification of RAS. (A) The RAS-RAF-MEK-ERK is commonly implicated as the primary signaling pathway of RAS. Ligand activated RTKs in turn activate GEFs, which catalyze the formation of ...

In 1988, de Vos et al. first reported the amino acid sequence and tertiary structure of HRAS-GTP using crystallography [23]. Following this tertiary structural mapping, the description of the RAS-MAPK pathway largely unfolded throughout the 1990s. By the late 1990s, it was known that binding of the epidermal growth factor receptor (EGFR) tyrosine kinase to its ligand(s) recruits guanine nucleotide exchange factors (GEFs), such as Son-of-sevenless (SOS), to promote the formation of RAS-GTP. Crystal structure analysis of the interaction between RAS, GEFs, and GAPs show that GEFs catalyze formation of RAS-GTP, the active form, and GAPs catalyze conversion back to RAS-GDP, the inactive form [24, 25].

Through interaction with GEFs, RAS undergoes a conformational change in two flexible regions called Switch I (residues 32-38) and Switch II (residues 60-75) that favors GTP binding (Figure 2) [26]. RAS-GTP then binds to RAF serine/threonine kinases, such as B-RAF and C-RAF, leading to RAF phosphorylation and allosteric conformational changes. In turn, RAF phosphorylates and activates MEK 1/2 dual specificity protein kinase leading to phosphorylation and activation of ERK 1/2. Activated ERK 1/2 then translocates into the cell's nucleus and activates transcription factors such as E26 transformation-specific (Ets-family) proteins. Other targets of RAS include multiple downstream effector proteins such as phosphoinositide 3-kinases (PI3K), Ral guanine nucleotide dissociation stimulator (RalGDS) family proteins and phospholipase C [22, 27].

Figure 2
Structure of RAS. RAS exists as a molecular switch cycling between RAS-GDP and RAS-GTP, classically thought of as “off” and “on”, respectively. Recent data has shown that RAS-GTP may exist in equilibrium between two states ...

The observation that RAS-GTP exists in 2 states heightens the complexity of our understanding of RAS activity [27-30]. Regardless of the presence or absence of oncogenic mutations, RAS-GTP may exist in either State 1, or its inactive form, and State 2, or its active form. The state of RAS-GTP largely depends on the position of the Switch I and II regions. Switch I and II consist of flexible loops within the GTP-bound RAS protein and are the main interface for effector protein recognition. The flexibility of these loops presumably allows a broad range of sequence diversity in the RAS-binding domains of different effector proteins.

Post-translational lipid modification of RAS also remains an area of interest in understanding RAS biology and inhibition (Figure 1b). It has been observed that lipid modification of the C-terminus domain of RAS is crucial for intracellular membrane anchoring and downstream effector activation [31]. While the C-terminus of RAS isoforms are variable, they all terminate with the CAAX (C: cysteine, A: aliphatic amino acids, X: any amino acids) motif sequence [32]. Multiple enzymes covalently modify RAS during transportation from the nucleus to the intracellular membrane. First, farnesyltransferase(FTase) attaches a farnesyl moiety to the cytoplasmic RAS. This is followed by proteolytic cleavage of the AAX sequence by RAS-converting enzyme-1 (RCE1) and subsequent carboxymethylation on the newly formed C-terminal Cys186 by isoprenylcysteine carboxymethytransferase-1 (ICMT1). An additional step may occur where a palmitoyl moiety attaches to the Cys residue immediately upstream of the CAAX motif via palmitoyltransferase (PTase). Interestingly, distinct RAS isoforms may undergo different post-translational lipid modification altering RAS anchoring and its behavior. Despite much research in developing targeted therapies aimed at inhibiting these enzymes, nothing has proven to be efficacious to date (see below).

Oncogenic RAS mutations lead to a protein locked in the RAS-GTP state, allowing a constitutively active protein where GAPs are no longer able to accelerate its transition back to RAS-GDP, or “off”, state. Oncogenic mutations in the nucleotides 12, 13, and 61 of the G domain prevent GAPs interaction with RAS, allowing it to maintain a GTP-bound, active state.

Ras isoforms and post-translational behavior in cancer

A recent publication by Nussinov et al. demonstrated evidence that small GTPase Ras isoforms might exist in different signaling states according to splice variants and tissue type [33]. The KRAS gene encodes for two splice variants, K-Ras4A and K-Ras4B. While it has been observed that K-ras4B is the dominant isoform in human cancer, Tsai et al. recently showed both K-Ras isoforms may be equally expressed in human CRC [34]. While sequence analysis of the catalytic domains of H-Ras, N-Ras, K-Ras4A, and K-Ras4B show they are nearly identical, their hypervariable region (HVR) of their C-terminus domain may differ substantially. This variability in the HVR may lead to distinct interactions with the intracellular membrane, altering behavior and activity of the different Ras isoforms. There is also evidence that K-Ras4A may exist in two different states, and that depending on the tumor type the oncogenic K-Ras4A may be K-Ras4B-like, in which K-Ras4A is only farnesylated, or N-Ras-like, in which K-Ras4A is palmitoylated and farnesylated. Nussinov et al. suggest that adenocarcinomas such as colon, pancreatic, and lung cancer may express oncogenic K-Ras4A that mirrors a K-Ras4B-like state versus tumor types such mirroring an N-Ras-like state in melanoma and acute myeloid leukemia. This variation among differing K-Ras4A states may have important biological and clinical implications, as protein interactions and signaling transduction via K-Ras4A protein may differ depending on its state and tumor type. Clearly, the emerging complexities of RAS isoforms and their biology in different cancer types further complicate the ability to therapeutically target RAS.

Targeting RAS

Small molecule RAS inhibition has largely been centered around three main concepts, 1) inhibiting membrane targeting of RAS, 2) preventing the formation of RAS-GTP via blockade of RAS-GEF interaction, and 3) blocking RAS-effector protein interaction. Alternative inhibition of RAS mutations has included using combinational approaches, synthetic lethality, and immunotherapy. Below we discuss strategies for each approach.

Targeting RAS-membrane interaction

RAS activity is reliant upon correct intracellular localization to the inner leaflet of the plasma membrane. The transportation of cytoplasmic to membrane-bound RAS is regulated by post-translational lipid modifications. Targeting the enzymes responsible for these modifications was first described in the late 1990s. As previously mentioned, lipid modification occurs in the HVR of RAS. Only later the observation was made that only 15% homology existed in the HVR of different RAS isoforms, highlighting the presence of multiple different series in which RAS could undergo lipidations. To further complicate matters, certain RAS isoforms, such as K-Ras4B, undergo an additional palmitoylation step.

Multiple FTase inhibitors (FTIs) have shown promising preclinical data, some of which reached late phase clinical trials, but none have ultimately proven to be efficacious to date [35, 36]. James et al. showed that FTIs inhibited downstream effects of HRasV12-mutated Rat-1 cells while leaving unmutated cells unaffected [37]. Interestingly, FTI anti-tumor effects were not seen in KRasBV12-mutated Rat-1 cells, suggesting that response to FTI inhibition depends on the presence of distinct RAS isoforms [38]. In addition, preclinical data has shown that FTI effects may be circumvented via alternate prenylation by geranylgeranyl transferase I (GGTase I) [35]. Prenylation allows effective membrane localization of KRAS and NRAS in the presence of FTI. Targeting of GGTase I as a single-agent has failed to show any clinical efficacy to date [39]. Preclinical studies showed that dual inhibition of FTI and GGTase I blocks translocation of RAS, but also led to excessive toxicity[40]. Inhibitors targeting palmitoylation of HRAS and NRAS elicited phenotypic reversion in HRASG12V-transformed fibroblast cell line; however, their anti-tumor effects in human cancer cell lines or xenografts have yet to be determined [41]. Inhibition of other enzymes responsible for lipidation, including RCE1 and ICMT, have failed to show good preclinical data compared to other FTIs [42].

Given the lack of efficacy targeting inhibitors of enzymes for farnesylation and palmitoylation, attention has been placed on inhibiting transport proteins. Salirasib, a farnesylcystein mimetic interfering with the binding of farnesylated K-Ras4B to the Ras-escort protein Galectin, has at best shown modest clinical efficacy in early phase trials both as a single-agent and in combination with conventional chemotherapy [43, 44]. In addition, RAS dislodgement from the plasma membrane and subsequent cytoplasmic degradation was not clearly shown in human trials. Zimmermann et al. further optimized compounds to target transport and discovered deltarasin, a molecule capable of inhibiting the interaction of K-Ras4B with the escort protein phosphodiesterase 6 delta (PDEδ) [45]. Deltarasin demonstrated both in vitro and in vivo activity through inhibition of Ras-dependent signaling, although deltarasin's effects have been observed to be nonspecific and inhibit multiple other GTPases [45, 46].

Direct RAS inhibition

The paradigm for inhibiting mutated or overexpressed proteins in “oncogenic addicted” malignant cells are attractive for two major reasons, 1) deprivation of the oncogene with a specific targeted agent leads to cell arrest and death, and 2) normal cells without the “addiction” would not be harmed by the therapy. For these reasons, much attention has been made to the development of a direct RAS inhibitor. The development of direct RAS inhibitors have been fraught with road blocks, in which it has earned its reputation as “undruggable”, given the protein's extremely high (picomolar) affinity for guanine nucleotides, the 10-fold higher cellular concentration of GTP compared with GDP, and the lack of available drug binding sites on the activated RAS-GTP protein.

The usefulness of the inhibition of RAS-GDP to RAS-GTP remains unclear given the nature of constitutively activated oncogenic RAS mutations. However, some argue that certain cancers remain dependent upon wild-type (WT) RAS expression for cell maintenance and survival [47]. Through crystallography, multiple compounds have been isolated to target nucleotide exchange between GEFs and RAS-GDP. Optimization of structure-based derivatives by Ostrem et al. revealed a previously unrecognized pocket adjacent to the Switch 2 region, called the SII pocket [48]. Through allosteric binding, this group of agents increases the affinity of KRASG12C for GDP over GTP [49]. However, these agents have showed nonspecific binding in follow-up studies thus far.

To bypass this issue, investigators have attempted to develop agents that target one specific oncogenic RAS mutation through covalent inhibition [48, 50]. K-RasG12C, a mutation found in approximately half of all KRAS-mutant lung cancers, has recently become an exciting target due to the amenable nature for covalent modification of its thiol group Cys12. Lim et al. showed that SML-10-70-1 was capable of mimicking GDP and covalently binding with KRASG12C, trapping it in an inactive form. The ability for covalent bonding allows this compound to overcome the picomolar affinity of RAS for guanine nucleotides. SML-10-70-1, a pro-drug of SML-8-73-1, reportedly also demonstrated the ability to inhibit proliferation of KRasG12C bearing cells; however, further preclinical studies have shown that SML-10-70-1 may act nonspecifically given its anti-proliferative effects in other RAS mutation types [50].

Another agent that has shown promise is a potent acrylamide, named AA12. AA12 induced apoptosis in K-RasG12C cell lines in vitro, but not in cell lines with WT RAS [48]. Additional analysis has shown that AA12 may also act non-specifically. In an alternative approach, the inhibition of formation of the RAS-GTP complex has been studied using synthetic hydrogen bond surrogate (HBS) peptides. Designed by Bar-Sagi et al., HBS3 inhibited the Sos-Ras interaction and down-regulated the RAS-MAPK signaling in response to EGF stimulation [51]. Clearly, all of these agents listed above need to undergo optimization to allow suitable pharmacokinetic and pharmacodynamic properties prior to their use in the clinical setting. Despite these setbacks, there remains much excitement regarding the therapeutic potential for these classes of drugs.

Inhibition of downstream effector interactions

The most promising therapeutic strategy for targeting oncogenic RAS mutations thus far has been blockade of downstream effectors that interact with RAS due to the aforementioned difficulties of inhibiting oncogenic RAS mutations via transport or direct RAS targeting. Driven by the knowledge that RAS-GTP most prominently interacts with MAPK and phosphoinositide 3 kinase (PI3K) pathways, substantial preclinical and clinical research has been directed at the inhibition of the active components of these signal transduction pathways. Multiple agents already currently available that target different kinases in the MAPK and PI3K pathways.

With the aid of nuclear magnetic resonance (NMR) spectroscopy and crystallography, much research has been dedicated to structure analysis and investigation of possible agents that bind to the activation site of RAS. While multiple compounds have been discovered, the therapeutic effects of these compounds largely remain a mystery. For example, sulindac, a nonsteroidal anti-inflammatory drug, was reported to inhibit HRAS-induced malignant transformation of MDCK-F3 cells and Ras-dependent Raf activation; however, there has been lack of experimental evidence to show anti-tumor effects [52, 53].

As previously described, RAS-GTP exists in dynamic equilibrium between state 1 (inactive form) and state 2 (active form), and it is now known that RAS-GTP may be vulnerable to inhibitor binding while in state 1 due to the presence of an exposed pocket, which is absent in state 2. Recently, the discovery of Kobe0065-family compounds, which inhibit Ras-Raf binding, has shown inhibited Ras-Raf association in cells and decreased phosphorylation on the RAS-RAF-MEK-ERK pathway [54]. Moreover, these compounds have also reportedly inhibited Ras-PI3K-AKT and RAS-RAL-GDS-RALA/B pathways, in addition to RAS-SOS interaction. It has also been observed that these compounds inhibited the proliferation of colorectal and pancreatic cancer cell lines carrying oncogenic Ras mutations and showed anti-tumor effects in a xenograft model of K-RasG12V mutated human colon cancer [55].

The RAL pathway has also been implicated in a variety of cancers and aberrant activation of this pathway through oncogenic RAS is felt to lead to more aggressive tumor phenotypes [56]. Through interaction of activated RAS with RAL-GDS (RAL-guanine nucleotide dissociation stimulator), the downstream effectors Ras-like GTPases, RALA and RALB, two homologous G-proteins, are activated that then promote cell survival and proliferation. Yan et al. recently discovered a unique pocket in RAL-GDP susceptible to allosteric inhibition and found a group of small molecules that blocked RAL-GDP nucleotide exchange that hindered activation of RALA or RALB and showed anti-tumor effects in RAL-depedent human cancer cell lines and xenograft models [57].

Inhibiting kinases in the MAPK pathway have also been in intense focus for therapeutic targets. RAF kinases interact with RAS through a RAS binding domain, leading to activation of MEK 1/2 followed by ERK that then acts on transcription factors to promote gene expression. Drugs targeting components of the MAPK pathway include BRAF and MEK inhibitors. While single-agent MEK inhibitors have not shown promise in BRAF-mutant mCRC, as demonstrated in a phase II study by Kopetz et al., preclinical and clinical activity with MEK inhibition has been observed in RAS mutant cancer [58]. In a phase I trial using single-agent trametinib, a MEK inhibitor, Infante et al. demonstrated an overall response rate of 10% in solid tumors that included KRAS-mutant pancreatic and NSCLC patients; however, none of the 13 patients with KRAS-mutant CRC showed a response [59]. In a phase II open label study, treatment with single agent MEK inhibition led to a 20% response rate in NRAS-mutant melanoma [60]. The combination of MEK inhibition and docetaxel in patients with KRAS mutant metastatic NSCLC has also shown benefit in a recently published phase II clinical trial [61]. Unfortunately, combination MEK plus conventional chemotherapy has not shown improved efficacy in KRAS-mutant pancreatic cancer [62].

Similarly, single-agent targeting of the PI3K pathway in RAS-driven malignancies has proven ineffective in the clinical setting. Negative feedback mechanisms, overexpression of alternate key regulator proteins, and reflexive activation of other downstream partners of RAS likely account for the bulk of this resistance [63-65]. Alternatively, preclinical data using dual inhibition of PI3K and MEK in KRAS-mutant cell lines and xenograft models have shown benefit, in addition to modest benefit observed in pancreatic cancer mouse models [64, 66]. This data has spurred ongoing preclinical and clinical research using a combination inhibitor strategy targeting kinases such as MEK, ERK, PI3K, AKT, and/or mTOR [67]. In another recently reported phase Ib trial, combination of a PI3K plus a MEK inhibitor demonstrated an overall response rate in 29% of patients with KRAS-mutated ovarian cancer [68]. This degree of response was not seen in lung or pancreatic cancer patients. The toxicity of this combination was also suspect as 65% of patients experienced a grade 3/4 adverse event, and the authors predict that the recommended phase 2 dose may require quick dose reduction in further trials. While this strategy has shown only modest clinical benefit with moderate toxicity thus far, this strategy has been adopted in many ongoing clinical trials with highly anticipated results [69, 70].

Synthetic lethality screens

Synthetic lethality occurs when combination of mutations in two or more genes lead to cell death, while a mutation in only one of these genes is viable. In oncogenic RAS mutant cancers, the targeting of a key-signaling molecule may elicit cell death, whereas normal cells would not be vulnerable. Large-scale knockdown of signaling molecules in oncogenic RAS mutant cancers have been studied [71-76]. As with other strategies, producing specific and reproducible anti-tumor effects has proven to be difficult.

Using large-scale screening of short hairpin RNA (shRNA) technology, Barbie et al. identified TBK1, a tyrosine kinase that acts downstream of the RAS/RALGDS/RALB axis, as a synthetically lethal partner of mutant KRAS [72, 77]. TBK1 appears to regulate growth KRAS mutant cells via the chemokine CCL5 and cytokine interleukin-6. Momelotinib, a dual JAK2 and TBK1 inhibitor, combined with a MEK inhibitor showed regression in growth of KRAS-driven lung cancers in xenograft models. This combination is currently being studied in early clinical trials (NCT02258607).

Corcoran et al. also used large scale screening of shRNA to discover genes that acted synergistically with MEK inhibition to induce apoptosis in RAS-driven cancer cells [71]. This screen identified BCL-XL, an anti-apoptotic gene, as a potential target. It has been observed that single-agent MEK inhibition induces expression of BIM, a pro-apoptotic gene, but this isn’t enough to induce apoptosis. When selumetinib was combined with anti-sense RNA to BLM-XL, KRAS mutant cell lines underwent apoptosis. This effect was reproduced when navitoclax, an inhibitor of antiapoptotic proteins BCL-2 and BCL-XL, was combined with selumetininb. A clinical trial combining navitoclax and trametinib, a MEK inhibitor approved for melanoma treatment, is currently underway (NCT02079740).

Eckhardt et al. has also shown that the Wnt pathway plays an important role in KRAS-mutant CRC [78]. Through shRNA screening, targeting the Wnt pathway increased sensitivity to selumitinib, and the combination of Wnt and MEK inhibition showed synergistic anti-tumor effects in in vitro and in vivo KRAS-mutant colorectal cancer models [78].

Another method investigating genetically engineered mice has been used to take advantage of synthetic lethality. Puyol et al. recently showed that interphase cyclin-dependent kinases (CDKs) were necessary for survival and proliferation in KRAS mutant NSCLC[76]. They also showed that loss of CDK4 halted cell proliferation in mutant cells and were able to recapitulate this effect by treating with a CDK4 inhibitor in a mouse model. Kwong et al. also demonstrated that combination of a cyclin-dependent kinase 4 (CDK4) inhibition with MEK inhibition led to significant effects [79]. This research has led to a clinical trial using palbociclib, a CDK4/6 inhibitor already FDA-approved for use in estrogen receptor-positive metastatic breast cancer, combined with PD-0325901, a MEK inhibitor (NCT02022982).

Lastly, there has been recent research showing that RAS-driven tumor dependence on autophagy, the digestion of intracellular components by fusion with lysosomes for nutrient support required for tumor homeostasis and growth [19, 80]. While studies using hydroxychloroquine, an autophagy inhibitor, have largely been unimpressive, more potent anti-autophagy agents are in development [81, 82].

Immunologic approaches

In the early 1990s, it was recognized that a T cell immune response could be induced through presentation of peptides from oncogenic RAS proteins [83, 84]. It was discovered that mice vaccinated with oncogenic RAS protein with codon 12 arginine were protected from developing tumors with the same mutation, but not from tumors harboring other RAS mutations and that CD8+ T cell depletion removed this effect [84]. Vaccination of patients with pancreatic cancer in vivo with RAS peptides of the same oncogenic RAS mutation also demonstrated a mutant RAS-specific T cell response, measured by interferon-ELISPOT, in 2 out of the 5 patients [85]. This did not lead to any antitumor responses, however. Similar outcomes were seen in pancreatic cancer patients who received antigen-pulsed antigen-presenting cells as the immunogen [86].

In another approach, patients were administered mutant RAS peptides together with granulocyte-macrophage colony-stimulating factor as a method for stimulating T cell immune response. While immune responses (measured by ELISPOT or proliferative assay) were observed in the majority of patients, no anti-tumor effects were appreciated. Vaccination therapy has also been tested in the adjuvant setting in RAS-mutant CRC and pancreatic cancer patients, but the authors concluded that they were unable to prove that the vaccine influenced disease recurrence rates [87].

While vaccine therapy has largely been disappointing, there is much optimism in harnessing checkpoint inhibitors to treat oncogenic RAS mutant cancers. Targets such as CTLA4, PD-1, and PD-L1 have led to dramatic responses in a variety of tumor types, and the basis for these responses appear to be polyclonal recognition of proteins and not from a single peptide [88]. There has been some discussion about whether adding vaccine therapy to this approach would compound therapeutic effects [18]. It remains to be seen whether immunotherapy with or without vaccination therapy will be efficacious in oncogenic RAS-mutant cancers.

Summary

The pursuit to understand RAS biology and discover RAS oncoprotein inhibitors has proven to be extremely difficult. With information gathered from each new failed attempt at RAS inhibition, the puzzle for finding clinically effective inhibitors becomes more complex. Initially, oncogenic RAS behavior was seemingly organized in a linear, unidirectional signal transduction pathway. We now know RAS biology is much more complex, and its effects are exerted in a highly dynamic, bi-directional network of kinases with a high degree of redundancy and feedback loops enabling RAS-driven cancers to develop therapeutic resistance regardless of therapeutic strategy.

Despite many obstacles in targeting oncogenic RAS mutations, researchers and clinicians remain optimistic in inhibiting RAS through a variety of strategies. While single inhibitor therapy has largely failed in RAS-driven cancers, dual inhibition with MEK and PI3K inhibitor therapy has shown some efficacy (and toxicity) in preclinical and clinical trials (see Table 2 for a list of selected current clinical trials). The discovery of a pocket in Switch II of the RAS-GTP protein is a promising target for potential agents to allosterically inhibit RAS. RAS trafficking protein targets such as PDEδ and Galectin have also been identified and may become a more relevant treatment option with ongoing research. Synthetic lethality has become a valuable method in both understanding RAS biology in addition to finding possible effective targets including the CKD, BCL-XL, and TBK1 families. One of the most promising approaches in drug discovery are the GDP analogue agents that covalently bind to specific RAS mutations locking RAS in an inactive form. Vaccine therapy has not demonstrated any clinical efficacy to date; however, checkpoint inhibitor therapy in combination with RAS mutant peptide vaccine therapy may offer a new approach in immunotherapy in RAS-driven cancers. Given the potential impact that RAS inhibition could have on millions of patients with cancer worldwide, it is no surprise that this is a rapidly evolving field with its own NCI-designated center to track progress (www.cancer.gov/research/key-initiatives/ras).

Table 2
Selected current clinical trials using targeted agents in RAS-mutant cancers

References

1. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. [PubMed]
2. Capon DJ, Seeburg PH, McGrath JP, et al. Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations. Nature. 1983;304:507–513. [PubMed]
3. Bos JL. ras oncogenes in human cancer: a review. Cancer Res. 1989;49:4682–4689. [PubMed]
4. Bos JL, Verlaan-de Vries M, Jansen AM, et al. Three different mutations in codon 61 of the human N-ras gene detected by synthetic oligonucleotide hybridization. Nucleic Acids Res. 1984;12:9155–9163. [PMC free article] [PubMed]
5. Perucho M, Goldfarb M, Shimizu K, et al. Human-tumor-derived cell lines contain common and different transforming genes. Cell. 1981;27:467–476. [PubMed]
6. Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer. 2003;3:11–22. [PubMed]
7. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487:330–337. [PMC free article] [PubMed]
8. Sorich MJ, Wiese MD, Rowland A, et al. Extended RAS mutations and anti-EGFR monoclonal antibody survival benefit in metastatic colorectal cancer: a meta-analysis of randomized, controlled trials. Ann Oncol. 2015;26:13–21. [PubMed]
9. Van Cutsem E, Kohne CH, Hitre E, et al. Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med. 2009;360:1408–1417. [PubMed]
10. Amado RG, Wolf M, Peeters M, et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol. 2008;26:1626–1634. [PubMed]
11. Douillard JY, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. N Engl J Med. 2013;369:1023–1034. [PubMed]
12. Thompson H. US National Cancer Institute's new Ras project targets an old foe. Nat Med. 2013;19:949–950. [PubMed]
13. National Cancer Institute The RAS Initiative: Development of the RAS Initiative at the Frederick National Laboratory for Cancer Research (FNLCR)
14. Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med. 1988;319:525–532. [PubMed]
15. Bettington M, Walker N, Clouston A, et al. The serrated pathway to colorectal carcinoma: current concepts and challenges. Histopathology. 2013;62:367–386. [PubMed]
16. Ryan DP, Hong TS, Bardeesy N. Pancreatic adenocarcinoma. N Engl J Med. 2014;371:2140–2141. [PubMed]
17. Forbes SA, Beare D, Gunasekaran P, et al. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Res. 2015;43:D805–811. [PMC free article] [PubMed]
18. Singh H, Longo DL, Chabner BA. Improving Prospects for Targeting RAS. J Clin Oncol. 2015;33:3650–3659. [PubMed]
19. Cox AD, Fesik SW, Kimmelman AC, et al. Drugging the undruggable RAS: Mission possible? Nat Rev Drug Discov. 2014;13:828–851. [PMC free article] [PubMed]
20. Karnoub AE, Weinberg RA. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol. 2008;9:517–531. [PMC free article] [PubMed]
21. Weinstein IB. Cancer. Addiction to oncogenes--the Achilles heal of cancer. Science. 2002;297:63–64. [PubMed]
22. Wennerberg K, Rossman KL, Der CJ. The Ras superfamily at a glance. J Cell Sci. 2005;118:843–846. [PubMed]
23. de Vos AM, Tong L, Milburn MV, et al. Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. Science. 1988;239:888–893. [PubMed]
24. Boriack-Sjodin PA, Margarit SM, Bar-Sagi D, Kuriyan J. The structural basis of the activation of Ras by Sos. Nature. 1998;394:337–343. [PubMed]
25. Scheffzek K, Ahmadian MR, Kabsch W, et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science. 1997;277:333–338. [PubMed]
26. Milburn MV, Tong L, deVos AM, et al. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science. 1990;247:939–945. [PubMed]
27. Pacold ME, Suire S, Perisic O, et al. Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma. Cell. 2000;103:931–943. [PubMed]
28. Pai EF, Krengel U, Petsko GA, et al. Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. Embo j. 1990;9:2351–2359. [PubMed]
29. Nassar N, Horn G, Herrmann C, et al. The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue. Nature. 1995;375:554–560. [PubMed]
30. Huang L, Hofer F, Martin GS, Kim SH. Structural basis for the interaction of Ras with RalGDS. Nat Struct Biol. 1998;5:422–426. [PubMed]
31. Shima F, Ijiri Y, Muraoka S, et al. Structural basis for conformational dynamics of GTP-bound Ras protein. J Biol Chem. 2010;285:22696–22705. [PMC free article] [PubMed]
32. Cox AD, Der CJ. Ras family signaling: therapeutic targeting. Cancer Biol Ther. 2002;1:599–606. [PubMed]
33. Nussinov R, Tsai CJ, Chakrabarti M, Jang H. A New View of Ras Isoforms in Cancers. Cancer Res. 2016;76:18–23. [PMC free article] [PubMed]
34. Tsai FD, Lopes MS, Zhou M, et al. K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif. Proc Natl Acad Sci U S A. 2015;112:779–784. [PubMed]
35. Whyte DB, Kirschmeier P, Hockenberry TN, et al. K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem. 1997;272:14459–14464. [PubMed]
36. Wang Y, Kaiser CE, Frett B, Li HY. Targeting mutant KRAS for anticancer therapeutics: a review of novel small molecule modulators. J Med Chem. 2013;56:5219–5230. [PMC free article] [PubMed]
37. James GL, Brown MS, Cobb MH, Goldstein JL. Benzodiazepine peptidomimetic BZA-5B interrupts the MAP kinase activation pathway in H-Ras-transformed Rat-1 cells, but not in untransformed cells. J Biol Chem. 1994;269:27705–27714. [PubMed]
38. James G, Goldstein JL, Brown MS. Resistance of K-RasBV12 proteins to farnesyltransferase inhibitors in Rat1 cells. Proc Natl Acad Sci U S A. 1996;93:4454–4458. [PubMed]
39. Berndt N, Hamilton AD, Sebti SM. Targeting protein prenylation for cancer therapy. Nat Rev Cancer. 2011;11:775–791. [PMC free article] [PubMed]
40. Lobell RB, Omer CA, Abrams MT, et al. Evaluation of farnesyl:protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models. Cancer Res. 2001;61:8758–8768. [PubMed]
41. Dekker FJ, Rocks O, Vartak N, et al. Small-molecule inhibition of APT1 affects Ras localization and signaling. Nat Chem Biol. 2010;6:449–456. [PubMed]
42. Baines AT, Xu D, Der CJ. Inhibition of Ras for cancer treatment: the search continues. Future Med Chem. 2011;3:1787–1808. [PMC free article] [PubMed]
43. Riely GJ, Johnson ML, Medina C, et al. A phase II trial of Salirasib in patients with lung adenocarcinomas with KRAS mutations. J Thorac Oncol. 2011;6:1435–1437. [PubMed]
44. Laheru D, Shah P, Rajeshkumar NV, et al. Integrated preclinical and clinical development of S-trans, trans-Farnesylthiosalicylic Acid (FTS, Salirasib) in pancreatic cancer. Invest New Drugs. 2012;30:2391–2399. [PMC free article] [PubMed]
45. Zimmermann G, Papke B, Ismail S, et al. Small molecule inhibition of the KRAS PDEdelta interaction impairs oncogenic KRAS signalling. Nature. 2013;497:638–642. [PubMed]
46. Chandra A, Grecco HE, Pisupati V, et al. The GDI-like solubilizing factor PDEdelta sustains the spatial organization and signalling of Ras family proteins. Nat Cell Biol. 2012;14:148–158. [PubMed]
47. Lim KH, Ancrile BB, Kashatus DF, Counter CM. Tumour maintenance is mediated by eNOS. Nature. 2008;452:646–649. [PMC free article] [PubMed]
48. Ostrem JM, Peters U, Sos ML, et al. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature. 2013;503:548–551. [PMC free article] [PubMed]
49. Maurer T, Garrenton LS, Oh A, et al. Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity. Proc Natl Acad Sci U S A. 2012;109:5299–5304. [PubMed]
50. Lim SM, Westover KD, Ficarro SB, et al. Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor. Angew Chem Int Ed Engl. 2014;53:199–204. [PMC free article] [PubMed]
51. Patgiri A, Yadav KK, Arora PS, Bar-Sagi D. An orthosteric inhibitor of the Ras-Sos interaction. Nat Chem Biol. 2011;7:585–587. [PMC free article] [PubMed]
52. Muller O, Gourzoulidou E, Carpintero M, et al. Identification of potent Ras signaling inhibitors by pathway-selective phenotype-based screening. Angew Chem Int Ed Engl. 2004;43:450–454. [PubMed]
53. Waldmann H, Karaguni IM, Carpintero M, et al. Sulindac-derived Ras pathway inhibitors target the Ras-Raf interaction and downstream effectors in the Ras pathway. Angew Chem Int Ed Engl. 2004;43:454–458. [PubMed]
54. Shima F, Matsumoto S, Yoshikawa Y, et al. Current status of the development of Ras inhibitors. J Biochem. 2015;158:91–99. [PubMed]
55. Shima F, Yoshikawa Y, Ye M, et al. In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction. Proc Natl Acad Sci U S A. 2013;110:8182–8187. [PubMed]
56. Lim KH, O'Hayer K, Adam SJ, et al. Divergent roles for RalA and RalB in malignant growth of human pancreatic carcinoma cells. Curr Biol. 2006;16:2385–2394. [PubMed]
57. Yan C, Liu D, Li L, et al. Discovery and characterization of small molecules that target the GTPase Ral. Nature. 2014;515:443–447. [PMC free article] [PubMed]
58. Kopetz S, Desai J, Chan E, et al. Phase II Pilot Study of Vemurafenib in Patients With Metastatic BRAF-Mutated Colorectal Cancer. J Clin Oncol. 2015;33:4032–4038. [PMC free article] [PubMed]
59. Infante JR, Fecher LA, Falchook GS, et al. Safety, pharmacokinetic, pharmacodynamic, and efficacy data for the oral MEK inhibitor trametinib: a phase 1 dose-escalation trial. Lancet Oncol. 2012;13:773–781. [PubMed]
60. Ascierto PA, Schadendorf D, Berking C, et al. MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study. Lancet Oncol. 2013;14:249–256. [PubMed]
61. Janne PA, Shaw AT, Pereira JR, et al. Selumetinib plus docetaxel for KRAS-mutant advanced non-small-cell lung cancer: a randomised, multicentre, placebo-controlled, phase 2 study. Lancet Oncol. 2013;14:38–47. [PubMed]
62. Infante JR, Somer BG, Park JO, et al. A randomised, double-blind, placebo-controlled trial of trametinib, an oral MEK inhibitor, in combination with gemcitabine for patients with untreated metastatic adenocarcinoma of the pancreas. Eur J Cancer. 2014;50:2072–2081. [PubMed]
63. Ihle NT, Lemos R, Jr., Wipf P, et al. Mutations in the phosphatidylinositol-3-kinase pathway predict for antitumor activity of the inhibitor PX-866 whereas oncogenic Ras is a dominant predictor for resistance. Cancer Res. 2009;69:143–150. [PMC free article] [PubMed]
64. Engelman JA, Chen L, Tan X, et al. Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers. Nat Med. 2008;14:1351–1356. [PMC free article] [PubMed]
65. Dan S, Okamura M, Seki M, et al. Correlating phosphatidylinositol 3-kinase inhibitor efficacy with signaling pathway status: in silico and biological evaluations. Cancer Res. 2010;70:4982–4994. [PubMed]
66. Alagesan B, Contino G, Guimaraes AR, et al. Combined MEK and PI3K inhibition in a mouse model of pancreatic cancer. Clin Cancer Res. 2015;21:396–404. [PMC free article] [PubMed]
67. Ebi H, Faber AC, Engelman JA, Yano S. Not just gRASping at flaws: finding vulnerabilities to develop novel therapies for treating KRAS mutant cancers. Cancer Sci. 2014;105:499–505. [PMC free article] [PubMed]
68. Bedard PL, Tabernero J, Janku F, et al. A phase Ib dose-escalation study of the oral pan-PI3K inhibitor buparlisib (BKM120) in combination with the oral MEK1/2 inhibitor trametinib (GSK1120212) in patients with selected advanced solid tumors. Clin Cancer Res. 2015;21:730–738. [PubMed]
69. Shapiro GI, Rodon J, Bedell C, et al. Phase I safety, pharmacokinetic, and pharmacodynamic study of SAR245408 (XL147), an oral pan-class I PI3K inhibitor, in patients with advanced solid tumors. Clin Cancer Res. 2014;20:233–245. [PubMed]
70. Tolcher AW, Bendell JC, Papadopoulos KP, et al. A phase IB trial of the oral MEK inhibitor trametinib (GSK1120212) in combination with everolimus in patients with advanced solid tumors. Ann Oncol. 2015;26:58–64. [PubMed]
71. Corcoran RB, Cheng KA, Hata AN, et al. Synthetic lethal interaction of combined BCL XL and MEK inhibition promotes tumor regressions in KRAS mutant cancer models. Cancer Cell. 2013;23:121–128. [PMC free article] [PubMed]
72. Barbie DA, Tamayo P, Boehm JS, et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature. 2009;462:108–112. [PMC free article] [PubMed]
73. Scholl C, Frohling S, Dunn IF, et al. Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells. Cell. 2009;137:821–834. [PubMed]
74. Kumar MS, Hancock DC, Molina-Arcas M, et al. The GATA2 transcriptional network is requisite for RAS oncogene-driven non-small cell lung cancer. Cell. 2012;149:642–655. [PubMed]
75. Luo J, Emanuele MJ, Li D, et al. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell. 2009;137:835–848. [PMC free article] [PubMed]
76. Puyol M, Martin A, Dubus P, et al. A synthetic lethal interaction between K-Ras oncogenes and Cdk4 unveils a therapeutic strategy for non-small cell lung carcinoma. Cancer Cell. 2010;18:63–73. [PubMed]
77. Zhu Z, Aref AR, Cohoon TJ, et al. Inhibition of KRAS-driven tumorigenicity by interruption of an autocrine cytokine circuit. Cancer Discov. 2014;4:452–465. [PMC free article] [PubMed]
78. Spreafico A, Tentler JJ, Pitts TM, et al. Rational combination of a MEK inhibitor, selumetinib, and the Wnt/calcium pathway modulator, cyclosporin A, in preclinical models of colorectal cancer. Clin Cancer Res. 2013;19:4149–4162. [PMC free article] [PubMed]
79. Kwong LN, Costello JC, Liu H, et al. Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma. Nat Med. 2012;18:1503–1510. [PMC free article] [PubMed]
80. Kimmelman AC. Metabolic Dependencies in RAS-Driven Cancers. Clin Cancer Res. 2015;21:1828–1834. [PMC free article] [PubMed]
81. Wolpin BM, Rubinson DA, Wang X, et al. Phase II and pharmacodynamic study of autophagy inhibition using hydroxychloroquine in patients with metastatic pancreatic adenocarcinoma. Oncologist. 2014;19:637–638. [PMC free article] [PubMed]
82. Mahalingam D, Mita M, Sarantopoulos J, et al. Combined autophagy and HDAC inhibition: a phase I safety, tolerability, pharmacokinetic, and pharmacodynamic analysis of hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with advanced solid tumors. Autophagy. 2014;10:1403–1414. [PMC free article] [PubMed]
83. Jung S, Schluesener HJ. Human T lymphocytes recognize a peptide of single point-mutated, oncogenic ras proteins. J Exp Med. 1991;173:273–276. [PMC free article] [PubMed]
84. Fenton RG, Taub DD, Kwak LW, et al. Cytotoxic T-cell response and in vivo protection against tumor cells harboring activated ras proto-oncogenes. J Natl Cancer Inst. 1993;85:1294–1302. [PubMed]
85. Gjertsen MK, Bakka A, Breivik J, et al. Vaccination with mutant ras peptides and induction of T-cell responsiveness in pancreatic carcinoma patients carrying the corresponding RAS mutation. Lancet. 1995;346:1399–1400. [PubMed]
86. Gjertsen MK, Bakka A, Breivik J, et al. Ex vivo ras peptide vaccination in patients with advanced pancreatic cancer: results of a phase I/II study. Int J Cancer. 1996;65:450–453. [PubMed]
87. Toubaji A, Achtar M, Provenzano M, et al. Pilot study of mutant ras peptide-based vaccine as an adjuvant treatment in pancreatic and colorectal cancers. Cancer Immunol Immunother. 2008;57:1413–1420. [PubMed]
88. Snyder A, Wolchok JD, Chan TA. Genetic basis for clinical response to CTLA-4 blockade. N Engl J Med. 2015;372:783. [PubMed]