such efforts fruitless (1). The search for small-molecule mimetics of GAP active on mutant RAS
proteins was likewise unsuccessful. These early
experiences, coupled with the lack of well-defined
hydrophobic pockets on the surface of RAS proteins, contributed to a perception that RAS may be
not tractable (8). This perception led to the pursuit
of indirect strategies to target proteins that promote RAS membrane interaction or effector signaling. Later, the concept of synthetic lethality was
applied to identify genetic interactors with mutant
RAS. With a rebirth in the interest in cancer cell
metabolism, a search for RAS-dependent metabolism began (9, 10). Only recently has the field
returned to the issue of direct RAS inhibitors,
with unexpected findings that suggest that RAS
may be a tractable drug target after all. Below, we
highlight key recent findings in these areas.
Targeting RAS plasma
With the recognition that RAS oncogenic activity is dependent on the protein’s association
with the inner face of the plasma membrane,
and the subsequent identification of the posttranslational modifications that modulated this
association, came the next important direction
for anti-RAS drug discovery (11). The RAS isoforms are synthesized initially as cytosolic, inactive proteins. The RAS C-terminal CAAX (C,
cysteine; A, aliphatic amino acid; X, terminal
amino acid) tetrapeptide motif gives signals
for a series of posttranslational modifications (12).
The first is farnesyltransferase-catalyzed cova-
lent addition of a farnesyl moiety to the cysteine
residue of the CAAX motif. The second, which
occurs at the cytosolic surface of the endoplas-
mic reticulum, is the proteolytic removal of the
last three amino acids by RAS converting en-
zyme 1 (RCE1). Last, isoprenylcysteine carboxyl
methyltransferase (ICMT) facilitates methyl trans-
fer to the C-terminal amino acid to negate the
negative charge and prevent plasma membrane
repulsion. The CAAX-signaled modifications, to-
gether with the addition of a palmitate fatty acid
(HRAS, KRAS4A, and NRAS) and/or polylysine se-
quences (KRAS4A and KRAS4B), promote RAS
association with the plasma membrane (Fig. 2).
Given the essential role of the farnesyl lipid
modification for all subsequent posttranslational
modifications and for RAS oncogenic activity,
an intense search for FTIs was initiated in 1990.
Many were developed and demonstrated to potently block HRAS-driven growth of cancer cells
in vitro and in various mouse models of cancer.
Two inhibitors (lonafarnib and tipifarnib) advanced to phase III clinical trials and were tested
in cancers with KRAS mutations (pancreatic,
colorectal, and lung cancer) but disappointingly
showed no efficacy in cancers with high-frequency
KRAS mutations. In retrospect, this result was foreshadowed by earlier cell culture studies. In contrast to HRAS, KRAS4B and NRAS had been found
to retain membrane association in the presence
of FTIs because these isoforms were modified by
the related geranylgeranyl isoprenoid (13).
The experience with FTIs diminished the pharmaceutical industry’s interest in anti-RAS drug
discovery, particularly with respect to targeting
RAS membrane association. Cautiously, in recent years, the field is again revisiting this issue,
with a focus on new targets. Several studies have
applied unbiased functional screens for previously
unidentified components that facilitate KRAS4B
plasma membrane association. One chemical library screen identified fendiline, an L-type calcium channel blocker, as a selective inhibitor of
KRAS4B but not HRAS or NRAS membrane association (14). Fendiline works relatively nonspecifically by inhibiting acid sphingomyelinase,
causing a reduction in plasma membrane phos-phatidylserine levels (15).
One of the more intriguing recently identified
targets is the prenyl-binding protein phospho-
diesterase d (PDEd). PDEd acts as a solubilizing
factor that facilitates the transit of RAS proteins
to either the Golgi or the recycling endosomes,
and from there, RAS is shuttled via directed ves-
icular transport to the plasma membrane (16)
(Fig. 2). Deltarasin and deltazinone are two small
molecules with different chemical scaffolds that
can occupy the farnesyl-binding pocket of PDEd.
When these molecules were used to inhibit PDEd,
RAS was found to shift from the plasma mem-
brane to the endomembranes; this in turn re-
duced oncogenic signaling and impaired the
tumorigenic growth of RAS-mutant cancer cells
in a xenograft model (17, 18). In principle, tar-
geting PDEd would overcome the concerns en-
countered with FTIs; however, because PDEd
regulates the function of other farnesylated
proteins, inhibitors of PDEd may be compro-
mised by off-target RAS-independent activities.
Because virtually all proteins that influence RAS
membrane association are likely to have other
substrates, the relative nonspecificity of targeting
RAS membrane association remains a concern.
Targeting RAS downstream
Blocking effector signaling is one of the most attractive and intensely pursued anti-RAS strategies.
However, with at least 11 catalytically diverse downstream effector families, the key questions are
which effectors should be targeted and whether
concurrent inhibition of multiple effectors is required. The two effector pathways that have attracted the greatest attention are the RAF-MEK-ERK mitogen-activated protein kinase (MAPK)
cascade and the PI3K-AKT-m TOR pathway (19, 20).
Mutations in genes encoding components of each
pathway (BRAF and PIK3CA) are known to drive
human cancer development, and their gene products are druggable protein kinases.
Numerous inhibitors against each component
of both the RAF-MEK-ERK and PI3K-AKT-m TOR
effector pathways have been developed and are
under clinical evaluation. The evidence to date
suggests that the RAF pathway is the more critical effector in RAS-dependent cancer growth
(19). For example, in PDAC, a substantial fraction
of the rare cancers harboring wild-type KRAS
also harbor either BRAF missense mutations or
deletion mutations (21–23). In contrast, PI3K mutations can co-occur with RAS mutations, arguing
that RAS may not potently activate PI3K signaling. When evaluated in mouse models, mutant
BRAF, but not PI3K, phenocopied mutant KRAS
and drove the initiation and progression of PDAC
(24). In the developmental disorders characterized by germline RAS mutations (the so-called
“RASopathies”), mutations in the RAF-MEK-ERK
cascade are also found. Last, only components
of the RAF-MEK-ERK pathway, and not other
effectors, could counteract the loss of RAS function
and restore the growth of mouse embryo fibroblasts deficient in all Ras alleles (24). Because
this pathway is the key effector for RAS in these
divergent settings, we focus on recent developments in the therapeutic targeting of the RAF-MEK-ERK MAPK cascade.
The first kinases in the MAPK pathway that
is activated by RAS-GTP are the RAF serine-threonine kinases (ARAF, BRAF, and CRAF/RAF1).
The only well-validated RAF substrates are the
Fig. 1. Five general strategies for anti-RAS drug development. (i) Molecules that directly bind RAS
disrupt its interaction with guanine nucleotide exchange factors or with effectors such as the RAF serine-
threonine kinases. Also shown are four indirect approaches that target (ii) proteins modulating RAS
spatial organization and association with the plasma membrane (e.g., farnesyltransferase and PDEd),
(iii) RAS effector signaling (e.g., RAF and PI3K), (iv) synthetic lethal interactors of mutant RAS, and (v) RAS-
regulated metabolic processes in cancer cells. G R A