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Figure 1. Scheme of the critical role of RAS in biological processes that regulate cell proliferation, survival and autophagy, and potential
therapeutic targets (created with BioRender.com)

mutations are virtually ever-present, precursor lesions - pancreatic intraepithelial neoplasia (PanIN) -
contain RAS mutations which increase in frequency as they progress stepwise to malignancy. Though
generally cooperative with other oncogenes during malignant transformation, these mutations are capable
of neoplastic growth in the absence of further genetic abnormalities [13,14] . K-RAS is the isoform most
frequently mutated and in addition to being almost inevitably mutated in pancreatic cancers, K-RAS
[3]
mutations are present in approximately half of colorectal cancers and a third of lung cancers . H-RAS
mutations are found in salivary gland cancers (15% of salivary gland cancers), cervical cancers (9%) and
urinary tract cancers (9%). N-RAS mutations are found in melanoma (17%), haematologic malignancies
(10%) and thyroid cancers (7%) . Not only are RAS mutations implicated in carcinogenesis, but also they
[15]
are strongly associated with treatment resistance [16,17] , and have been shown to be adverse prognostic
markers in cancer . RAS-mutated colorectal cancer will not benefit from EGFR-directed treatment with
[18]
cetuximab or panitumumab. Similarly, in lung cancer RAS status has been shown to be an independent
[19]
predictor for EGFR tyrosine kinase inhibition .

APPROACHES TO RAS INHIBITION
Given the central role of RAS both in carcinogenesis and tumour progression, the RAS oncoprotein is an
important therapeutic target. Table 1 provides a summary of current trials of Ras inhibitors. Despite decades
of efforts, however, it has proven extremely difficult to synthesise clinically effective direct inhibitors of RAS
oncoproteins. This has been attributed to the high affinity of RAS towards GDP and GTP (in contrast to the
low affinity of ATP for protein kinases, for example), and lack of deep hydrophobic pockets that would
allow the binding of small molecules . In addition, given variation in the frequency of isoform mutations
[7]
within different cancer types, and in the specific mutations involved, there may not be a single effective RAS
inhibitor for all RAS-mutated cancers . Historical efforts to target RAS which have made it to the clinic
[4]
could be broadly summarized as those focusing on RAS plasma membrane localisation and those
attempting to indirectly block RAS by inhibiting the downstream effector signalling.

RAS membrane localisation
In order to carry out their role, RAS proteins must become membrane-bound. This involves a complex
series of post-translational modifications. Three enzymatic steps are necessary for RAS to associate with
membranes - (1) prenylation of the CAAX box by farnesyltransferase (FTase); (2) cleavage of the terminal
AAX residues by RAS converting enzymes RCE1; and (3) methylation of the cysteine residues of the CAAX
box by isoprenylcysteine carboxyl methyltransferase ICMT. Farnesylation, the addition of farnesyl groups to
RAS, is a critical step in creating a hydrophobic domain in RAS that allows the protein to associate with the
plasma membrane, and therefore to be biologically active. Farnesyltransferases (FTases) are the enzymes
responsible for this step, and they were an early target in efforts to inhibit RAS function . Two
[20]
farnesyltransferase inhibitors (FTIs), lonafarnib and tipifarnib, were investigated in Phase III trials either as
monotherapy or in combination with chemotherapy in a number of different RAS-mutated tumours.
Despite Phase I and II clinical trials showing some antitumour activity and low toxicity, no improvement in
overall survival was reported in Phase III trials [21-23] . One reason for lack of “pan-RAS” efficacy for the FTI
class is that K-RAS and N-RAS membrane localisation can be achieved in the absence of
farnesyltransferases, via geranylgeranyl transferases. Attempts were made to target these enzymes with
geranylgeranyltransferase inhibitors, but these were ineffective and associated with toxicity . In contrast,
[24]
H-RAS is not a substrate for geranylgeranyl transferase and therefore its membrane localization could be
suppressed solely by FTIs . Tipifarnib has demonstrated preclinical activity against a wide panel of H-
[25]