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Page 2 Conroy et al. Cancer Drug Resist 2021;4:543-58 https://dx.doi.org/10.20517/cdr.2021.07

commonly mutated oncogenes in human cancer with approximately one-third of all cancers driven by these
[3,4]
oncoproteins, including 40%-50% of colorectal cancer and over 90% of pancreatic cancers . The discovery
of oncogenes and the elucidation of intracellular signalling pathways heralded an era of targeted therapy
[5,6]
that has greatly improved the outlook for many cancers . New agents targeting receptor kinases and their
downstream mediators demonstrated the ability to stabilise and shrink tumours, with side effects that were
frequently milder than those associated with standard cytotoxic chemotherapy. Throughout this enormous
paradigm shift in cancer therapeutics, however, RAS stood apart, dominant and seemingly undruggable .
[4]
The development of direct RAS inhibitors proved very challenging. RAS has a high affinity towards GDP
and GTP and a lack of deep hydrophobic pockets which would allow binding of small molecules . Subtle
[7]
differences in structure and variable activation of RAS proteins added greatly to the complexity and
[8]
attention largely focused on downstream inhibition of the transduced signalling pathways . In recent years,
however, there have been some very promising developments in direct RAS targeting which would suggest
that this has real potential as a therapeutic avenue. Here we aim to review current efforts at RAS inhibition
in the context of both RAS family biology and the historical efforts which attempted, largely without
success, to perturb its role as a major oncogenic driver.

RAS STRUCTURE AND FUNCTION
[9]
The RAS superfamily of genes has about 36 members, which encode for 39 proteins . Three RAS genes, H-
RAS, K-RAS and N-RAS, encode four protein isoforms: H-RAS, K-RAS4A, K-RAS4B and N-RAS. K-
RAS4B is the predominant isoform and is referred to simply as K-RAS in this article. RAS proteins are small
GTPases, of about 21kD molecular weight, and are monomeric proteins that have a central role in cell
differentiation, adhesion, migration, proliferation and survival . RAS proteins convey signals from growth
[10]
factors and extracellular components, and are upstream of signalling pathways including the ERK pathway
and the PI3K/mTOR survival pathway. As illustrated in Figure 1, they function as a membrane-bound
molecular switch, alternating between an inactive GDP-bound state and an active GTP-bound state. This
alternation is mediated by guanine nucleotide exchange factors (GEFs) and GTPase activating/accelerating
proteins (GAPs). GEFs are activated by an upstream mitogenic signal, and they in turn cause an inactive
RAS to shed its GDP and bind a GTP, which has a 10-fold higher cellular concentration than GDP, thereby
becoming activated. This period of activity terminates when the intrinsic GTPase activity of RAS-GTP is
enhanced by GAPs, leading to hydrolysis of the bound GTP. In normal cells, a tight equilibrium is
maintained between the active and inactive states.

Pathogenesis
The normal function of RAS, as described above, can be deranged by mutations which unbalance this
equilibrium. Single point missense mutations in codons G12 (most commonly), G13 or Q61 are responsible
for converting proto-oncogenes to oncogenes. These mutations favour GTP binding and lead to constitutive
activation of RAS, with reduction or loss of GTPase activity. These codons are implicated as their amino
acid residues are found in the cavity where GTPase catalytic activity operates . The consequence of this
[10]
aberrantly activated RAS is prolonged oncogenic signalling rather than short, controlled bursts of
activation . There is subsequent activation of downstream signalling molecules such as PI3K, RAF and
[11]
Rin1. By contrast, mutations in other RAS codons, or any nonsense mutations, are likely to inhibit rather
than enhance the activity of RAS, and do not provide a survival advantage. As well as uncontrolled
proliferation, the mutagenic RAS oncogene has been implicated in tumour immune resistance by causing
intrinsic - as opposed to adaptive - upregulation of programmed death ligand 1 .
[12]
RAS in cancer
Approximately 25%-30% of cancers contain mutations in one of the RAS isoforms, and they are considered
an early genetic event in tumour progression . For example, in pancreatic adenocarcinoma, where RAS
[4]