1. Blocking RAS Location at the Plasma Membrane

The initial search for therapeutic approaches targeting oncogenic RAS in cancer was for the most part a sad history of trial and failure. Early crystallographic studies suggested that finding blockers of RAS action would be a tough task with improbable success because the analysis of RAS structure did not show surface pockets where small molecule inhibitors could bind and block oncogenic signals ^[1]. Due to the unyielding nature of the RAS proteins, the initial focus of anti-RAS cancer therapeutics was placed on targeting RAS plasma membrane localization, mediated through the binding of isoprenoid residues to its C terminal region. For this purpose, a wide range of farnesyl transferases inhibitors (FTIs) were developed in different laboratories with the goal of blocking RAS signaling from the plasma membrane ^[2]. In short, despite initial success blocking RAS farnesylation in tissue culture and preclinical models, FTIs failed completely in clinical trials (rev. in ^[3]), probably due to the fact that KRAS and NRAS can also be geranyl-geranylated (another form of prenylation used by cellular proteins to gain access to the membrane) ^[4]. The next logical step was, therefore, to design dual prenylation inhibitors, but dose-dependent toxicity has prevented these molecules from reaching clinical use so far ^[5]. Despite these uninspiring results, new attention has lately been put on the use of refined FTIs for cancer treatment, and as on April 2021, several clinical trials are being carried out; for example, testing Tipifarnib on tumors harboring HRAS mutations (see https://www.clinicaltrials.gov accessed on 5 April 2021, for more details).

Alternative strategies to delocalize RAS oncoproteins have also been developed recently. One of these involves the use of a novel inhibitor designed to disrupt the recently discovered interaction between KRAS and PDEδ, which is needed for KRAS placement in the inner side plasma membrane. This inhibitor, named Deltarasin was reported to prevent KRAS location to the plasma membrane and block pancreatic cancer cell proliferation ^[6]. A different approach takes advantage of RAS prenylation by binding covalent inhibitors to RAS through the action of the farnesyl transferase. This prevents the action of geranyl-geranyl transferases and mislocalizes KRAS in SW-620 colon cancer cells ^[7].

2. Inhibiting RAS Downstream Signaling

The lack of effectiveness of FTIs in clinical trials, together with the absence of discernible pockets on the RAS surface allowing direct binding of inhibitor, led to the assumption that RAS could be “undruggable” and for many years the search for drugs against RAS-driven tumors focused on the inhibition of downstream signaling pathways ^[8]. Most downstream RAS effectors are protein kinases, enzymes for which inhibitors are easily developed, as proven by the vast availability of inhibitors of the MAPK cascade. So far, among the inhibitors developed against RAF, MEK and ERK1/2, the RAF inhibitors hold the most promise for cancer treatment. In addition to its role as an effector of oncogenic RAS, RAF is also frequently mutated in cancer. Unfortunately, the first generation of RAF inhibitors showed high rates of drug resistance and subsequent analysis of the underlying resistance mechanisms uncovered a paradoxical hyperactivation of ERK, either through increased activation of WT RAF dimers in BRAF mutant cancers, or by transactivation of the drug-free RAF molecule after binding of the inhibitor to the other RAF monomer within a dimer ^[9][10]. New, second generation inhibitors have been developed in the last decade with the ability to brake the paradoxical ERK activation, and many of them are currently being analyzed in clinical trials ^[11]. In addition to RAF inhibitors, MEK and ERK inhibitors have also been developed, but these inhibitors have narrow therapeutic dose margin due to their effect on normal cells. Despite this, two MEK inhibitors (cobimetinib and trametinib) are currently being used for treatment of tumors harboring BRAF mutations ^[12].

3. Direct RAS Inhibitors

The initial notion that RAS was undruggable began to change with the discovery of pockets on the RAS surface that could be used to design new specific inhibitors capable of binding directly to the RAS oncoproteins. Based on structural and crystallographic studies showing that HRAS proteins cycle between two structural states (called state 1 and state 2) and that RAS proteins are unable to bind to effectors while in state 1, the existence of a groove in the surface of HRAS in state 1 was predicted that would be large enough to bind small molecule inhibitors potentially capable of blocking RAS-effector interaction ^[13]. This seminal report started a race to find and develop new small molecules that could block RAS interaction with either its effectors, guanine nucleotides, or its GEF activators. In this regard, many reports have been published describing the isolation of small molecule RAS inhibitors, either blocking RAS/effector interactions ^[14] or inhibiting the interaction with SOS proteins (rev. in ^[15]). Happily, some of these inhibitors have already overcome most barriers in drug development and are being tested in the clinic. For example, Sulindac and Rigosertib (drugs that inhibit RAS/RAF interaction) have reached Phase 3 clinical trials for several human malignancies, and BI-1701963 is the first inhibitor of RAS/SOS interaction to reach clinical trials (for more information see https://www.clinicaltrials.gov, accessed on 5 April 2021).

One of the most relevant developments in the race towards blocking RAS in cancer came from the discovery of a small-molecule covalent inhibitor of KRAS^G12C that strongly and irreversibly bound to the mutated cysteine and prevented guanine nucleotide exchange, RAS activation and cancer cell growth ^[16]. This initial drug had limited inhibitory capacity but opened the gate for the development of new, more potent inhibitors in the following years that are able to bind to both RAS.GDP and RAS.GTP complexes, even independently of the specific oncogenic mutation carried, in different biological and tumoral contexts (rev. in ^[17]). At the beginning of 2021 there were 15 clinical trials listed in Clinical Trials.gov dedicated to analyzing the effectiveness and toxicity of KRAS^G12C inhibitors in solid tumors; especially lung cancers, where this mutation is frequent. Two of these inhibitors (AMG 510 ^[18] and MRTX849 ^[19]) have reached phase 3 clinical trials but, unfortunately, resistance mechanisms have already been described and further work will be needed to overcome them in the future ^[19].

4. Future Perspectives on RAS Therapy

RAS research is entering its fifth decade, behind more than forty years of discoveries that have revealed a vast amount of information on these small GTPases. From the discovery of the viral oncogenes, and the isolation of their cellular counterparts, to the latest work on RAS inhibition and signaling, the knowledge on these proteins has allowed to design new strategies to target their role in cancer. Thus, new avenues to inhibit RAS translocation to the plasma membrane, to block downstream signaling pathways, or to directly inhibit their activity through small inhibitor binding, have come out in the last ten years, and covalent KRAS^G12C inhibitors might be the first drugs targeting RAS directly that may reach FDA approval for clinical use.

Despite these recent advances, there is still a long way to go to effectively block RAS signaling in cancer. KRASG12C mutations comprise only a small percentage of RAS alterations in cancer and new drugs specifically targeting other RAS mutations are needed. Furthermore, resistance to drugs targeting RAS will likely limit their efficacy in the clinic ^[20], and effective treatments may probably require combinatorial therapies. Another concern regarding the development of effective RAS inhibitors is toxicity. Recent research had shown that complete abrogation of most members of RAS signaling pathways in mice leads to death; predicting a high toxicity for inhibitors that effectively block them ^[21], a problem that can be extended to most chemotherapeutic tools.

Another relevant consideration for therapy of RAS-driven cancer is the long-known ability of RAS oncogenes to cause accumulation of genomic/chromosomal instability that critically contributes to progression through the evolutionary phases of tumorigenesis ^[22][23]. This suggests that future therapeutic approaches against RAS-driven cancers should not only focus on searching for new biochemical inhibitors directly binding to RAS surface pockets, but also for wider approaches capable of targeting various other mechanistic aspects and hallmarks of RAS-dependent cancer evolution ^[24], as well as reducing systemic toxicity by specifically releasing the chemotherapeutic drugs to the tumors ^[25].