Ras genes are frequently activated in cancer. Attempts to develop drugs that target mutant Ras proteins have, so far, been unsuccessful. Tumors
bearing these mutations, therefore, remain among the most difficult to treat. Most efforts to block activated Ras have focused on pathways
downstream. Drugs that inhibit Raf kinase have shown clinical benefit in the treatment of malignant melanoma. However, these drugs have failed to
show clinical benefit in Ras mutant tumors. It remains unclear to what extent Ras depends on Raf kinase for transforming activity, even though Raf
proteins bind directly to Ras and are certainly major effectors of Ras action in normal cells and in development. Furthermore, Raf kinase inhibitors can
lead to paradoxical activation of the MAPK pathway. MEK inhibitors block the Ras-MAPK pathway, but often activate the PI3’-kinase, and have shown
little clinical benefit as single agents. This activation is mediated by EGF-R and other receptor tyrosine kinases through relief of a negative feedback
loop from ERK. Drug combinations that target multiple points within the Ras signaling network are likely to be necessary to achieve substantial clinical
benefit. Other effectors may also contribute to Ras signaling and provide a source of targets. In addition, unbiased screens for genes necessary for
Ras transformation have revealed new potential targets and have added to our understanding of Ras cancer biology.
Keywords: Ras, targeted therapy, signal transduction, Raf, MAPK kinase
Ras genes are frequently mutated in
human cancers, and the proteins they
encode have been considered drug tar-
gets since they were first identified and
characterized 30 years ago. Yet, in 2011,
no drugs that target Ras proteins directly
or act on Ras-driven human cancers
have been developed successfully.
Indeed, tumors harboring Ras mutations
remain the most difficult to treat and are
excluded from treatment with specific
Ras proteins cycle between an inac-
tive GDP-bound “off” state and an active
GTP-bound “on” state and function as
molecular switches that mediate signal
transduction between cell surface growth
factor receptors and intracellular signal-
ing pathways.1 The activation of Ras pro-
teins, that is, the exchange of GDP with
GTP, is an intrinsically slow process
and is catalyzed by guanine nucleotide
exchange factors (GEFs). However, this
exchange is, in principle, reversible. The
inactivation, that is, the hydrolysis of the
γ-phosphate of GTP to GDP, is catalyzed
by GTPase-activating proteins (GAPs)
and is irreversible. Oncogenic mutations
occur most frequently in codons 12, 13,
and 61 and elsewhere. The resulting
oncogenic versions of Ras proteins are
resistant to GAP-mediated GTP hydroly-
sis, which renders them constitutively
active. Besides cycling between GDP-
and GTP-bound states, Ras proteins
undergo posttranslational processing.2-6
These modifications attach the proteins to
cellular membranes, which is essential
The catalytic domain, also referred to
as the G domain, is highly homologous
between the 3 Ras proteins, K-Ras,
H-Ras, and N-Ras, which are activated in
human cancer. The first 80 amino acids
are identical, and the next 85 amino acids
only differ by 5%. Analysis of the approx-
imately 50 crystal structures of H-Ras
and the recently solved x-ray structures
of K- and N-Ras confirms the remarkable
similarity of these proteins.7-9 For this
reason, it is obvious that the functional
differences between the 3 proteins are not
embedded in the G domain but in the
C-terminal hypervariable region (HVR),
which comprises the last 23 of 24 amino
acids. The Ras proteins share only 15%
homology in this region. The HVR can
be subdivided in 2 parts: the presumed
unstructured linker region (AA 166-179
in N- and H-Ras and AA 166-174 in
K-Ras) and the membrane-interacting
lipid anchor (AA 180-189 in N- and
H-Ras and AA 175-188 in K-Ras). Both
parts are involved in high affinity interac-
tions with lipid raft and nonraft plasma
membrane microdomains.10,11 Determina-
tion of x-ray structures of membrane-
bound Ras has been impossible. Therefore,
biophysical experiments and computer
simulations have been carried out in order
to study membrane-bound Ras. Computer
simulations with full-length H-Ras sug-
gested that the GTP-bound protein under-
goes a major conformational change at the
membrane, bringing the catalytic domain
in contact with the lipid bilayer. Basic resi-
dues in α4 have been implicated in these
The basic function of nucleotide bind-
ing and hydrolysis is carried out by the
approximately 20-kDa G domain. The G
domain is classified as a α/β protein, typi-
cal for nucleotide-binding proteins. The
critical regions involve a conserved
UCSF Helen Diller Family Comprehensive Cancer
Center, University of California, San Francisco, CA,
*Each of these authors contributed equally.
Frank McCormick, UCSF Helen Diller Family
Comprehensive Cancer Center, University of California,
San Francisco, 1450 3rd Street, San Francisco, CA
Therapeutic Strategies for Targeting Ras Proteins
Stephan Gysin*, Megan Salt*, Amy Young*, and Frank McCormick
Genes & Cancer
2(3) 359 –372
© The Author(s) 2011
Reprints and permission:
Genes & Cancer / vol 2 no 3 (2011)
phosphate-binding loop (P-loop, residues
10-17) and 2 switch regions (switch I, AA
25-40; switch II, AA 57-75) that bind the
nucleotide. It is these 3 regions that are
affected by oncogenic mutations. The
switch regions usually show a high
degree of flexibility when analyzed by
x-ray diffraction or by nuclear magnetic
resonance and electron paramagnetic res-
onance. The canonical switch mechanism
can be considered as a loaded-spring
mechanism, where the release of the
γ-phosphate after the GTP hydrolysis
allows the 2 switch regions to relax into
the GDP-specific conformation. Differ-
ent members of the Ras superfamily
show variations of this mechanism.4,13
The release of guanine nucleotides
from Ras proteins is a slow process and
is accelerated by GEFs, by several
orders of magnitude. The catalytic
mechanism involves a series of fast
reactions, which lead from a binary Ras-
nucleotide complex via a trimeric Ras-
nucleotide-GEF complex to a binary
nucleotide-free complex. These reac-
tions are reversible: GEFs act as cata-
lysts and increase the rate at which
equilibrium is reached. The position of
the equilibrium is determined by the
high affinity of Ras proteins for GDP or
GTP, the intracellular concentration of
nucleotides, and the affinities and con-
centrations of effector proteins that shift
the equilibrium towards the GTP-bound
state. GEFs interact with switch I and II
regions and insert residues into the
P-loop and the Mg2+-binding area. This
perturbation is considered to be the main
cause for the decreased affinity between
Ras proteins and nucleotides.14,15 Con-
versely, Ras-GAPs accelerate conver-
sion of active Ras-GTP back to Ras-GDP
dramatically. Ras-GAPs insert an argi-
nine side chain into the catalytic site and
thereby neutralize developing charges in
the transition state. Ras-GAPs stabilize
the switch II domain and allow the con-
served glutamine 61 to participate in
catalysis. Oncogenic mutations of gly-
cine 12 and glutamine 61 cause a pertur-
bation, which renders Ras catalytically
insensitive to GAP activity.16
Effector proteins of Ras show an
enhanced affinity for the GTP-bound
state. Some of the effector-binding
domains are preformed and undergo no
conformational change upon binding.
This means that the recruitment of effec-
tor proteins by Ras-GTP at the membrane
is considered as the activation process. In
those cases where a large conformational
change takes place, it has been shown
that allosteric regulations of the effector
proteins are involved. One of the best-
characterized Ras effector proteins is the
Raf kinase. Its Ras-binding domain
(RBD) is a small domain that contains an
ubiquitin fold with an interprotein β-sheet
that is Ras-GTP sensitive.17 Other pro-
teins like Ral-GDS have a similar domain
and bind to Ras-GTP in a similar fashion.
Both Raf and Ral-GDS bind to the switch
I domain, as does the γ-subunit of PI3’-
kinase. In the latter case, Ras uses also its
switch II domain in order to bind the cata-
lytic subunit. These interactions cause a
structural change in PI3’-Kγ and affect
the binding to phospholipids and the cata-
lytic activity.18 Ras proteins undergo a
series of posttranslational processing
steps in order to become attached to cel-
lular membranes and become biologi-
cally functional,19 as shown in Figure 1.
The C-terminal membrane-interacting
lipid anchor region of HVR contains dis-
tinct motifs that are subject to these
modifications. In all 3 Ras proteins, the
cysteine of the CAAX box (Cys185 in
K-Ras 4A and 4B and Cys186 in N- and
H-Ras) becomes farnesylated under nor-
mal conditions (Figure 2). This reaction
is catalyzed by the enzyme farnesyl
transferase (FTase).20 The farnesyl 15
carbon chain becomes attached to the
cysteine via a stable thioether linkage.
Ras farnesylation is, therefore, an irre-
versible reaction. In the presence of
farnesyl transferase inhibitors (FTIs)
(see below), K-Ras and N-Ras become
alternatively prenylated by the attach-
ment of a 20-carbon geranylgeranyl
chain through geranylgeranyl transfer-
ase I (GGTase I). This has been a major
obstacle for the successful application
of FTIs in K-Ras– or N-Ras–driven
tumors,21 as discussed below. K-Ras 4A
and N-Ras have 1 additional cysteine
(Cys179 in K-Ras 4A and Cys181 in
N-Ras), whereas H-Ras has 2 additional
cysteines (Cys181 and Cys184) that
become palmitoylated. Ras palmi-
toylation is reversible and is carried out
by palmitoyl transferases (PTase).22 The
DHHC family of PTases has been char-
acterized, and it has also been shown
that Ras palmitoylation takes place at
the endoplasmic reticulum (ER)/Golgi
endomembrane system, whereas depal-
mitoylation, carried out by less-studied
acyl protein thioesterases, takes place at
the plasma membrane.23
N-Ras, H-Ras, and likely, K-Ras
4A undergo a palmitoylation/depalmi-
toylation cycle. This is an efficient way
of shuttling these proteins from the ER/
Golgi to the plasma membrane and
reverse.24 In contrast, K-Ras 4B contains
a polybasic lysine stretch in the adjacent
upstream region of the CAAX box. This
sequence mediates the interaction of
K-Ras 4B with acidic phospholipids in
the plasma membrane. Furthermore,
K-Ras 4B contains a serine residue
(Ser181) that is subject to phosphoryla-
tion. Phosphorylation by members of the
protein kinase C family had been pro-
posed, leading to dissociation of K-Ras
4B from the plasma membrane.25,26
The last 3 amino acids of the CAAX
box (–AAX) are subject to proteolytic pro-
cessing. In humans, the reaction is carried
out by a prenyl protein–specific endopro-
tease known as ras-converting enzyme 1
(RCE1). It is an integral membrane protein
of the ER. Rce1-null mice die between
embryonic day 15.5 and the first week of
life. The reasons for this lethality are not
clear.27,28 The final CAAX processing step
is carboxyl methylation. Isoprenylcyste-
ine-carboxyl-methyltransferase (ICMT) is
also an integral membrane protein of the
ER, which is unusual for methyltransfer-
ases. After modification by ICMT, the
fully processed Ras protein consists of a
methyl-esterified farnesyl or geranylgera-
nyl cysteine. ICMT is also essential for
mouse development, as mice lacking the
gene die by embryonic day 11.5.29,30
Therapeutic strategies for targeting Ras proteins / Gysin et al.
These 2 postprenylation events ren-
der the C-termini of Ras proteins even
more hydrophobic. Additionally, it has
been speculated that carboxyl methyla-
tion is also required for the binding with
interacting proteins. The carboxyl meth-
ylation reaction is reversible, and it is
conceivable that this could represent
another level of regulating Ras activity
or subcellular localization.31 However, a
Ras-specific methylesterase has not yet
Direct Therapeutic Attack
on the G Domain
Ras proteins bind GTP with picomolar
affinities. Furthermore, the concentra-
tion of GTP in cells approaches micro-
molar levels. Unlike protein kinases, in
which phosphoryl transfer from ATP to a
substrate is a rapid, catalytic process, the
role of GDP or GTP is to stabilize inac-
tive or active states of the Ras protein.
For these reasons, targeting mutant Ras
with nucleotide analogs does not appear
to be a promising approach. The search
for small molecules that bind to the sur-
face of Ras proteins has been almost as
challenging, as Ras does not have an
accessible active site or pocket to which
molecules are likely to bind. Neverthe-
less, Taveras and coworkers at Schering-
Plough (Kenilworth, NJ) were able to
identify a small molecule that binds to
pocket on Ras, without displacing bound
nucleotide. The compound, SCH 54292,
has not been developed clinically but
approaches may yet identify ways of tar-
geting Ras directly.32
Another approach to inactivate onco-
genic Ras could involve small molecules
that restore GTP hydrolysis in mutant
Ras. Analysis of the GTPase site suggests
this will also be technically extremely
challenging, as the GTPase site is occu-
pied by guanine nucleotide, and there
appears little room for a small molecule
to bind. Ideally, such a compound would
mimic the effects of GAP and insert an
arginine-like residue that would facilitate
GTP hydrolysis in the mutant protein.
The capacity of mutant Ras to hydrolyze
GTP was demonstrated by Ahmadian and
coworkers, who constructed GTP analogs
covalently attached to positively charged
groups that facilitated GTP hydrolysis.
While this approach appears encourag-
ing, identifying a small molecule that can
bind and interact with mutant Ras in cells
remains a daunting challenge.33
Strategies to Interfere with
Ras prenylation, proteolytic processing,
and carboxyl methylation are mechanisms
that appear to represent reasonable targets
for therapeutic intervention. Drugs that
interfere with Ras prenylation have been
developed over the last 2 decades. FTIs
can be subdivided in 3 different classes:
A) CAAX peptidomimetics that compete
Figure 1. Ras processing.
Figure 2. C-termini of Ras proteins.
Genes & Cancer / vol 2 no 3 (2011)
with Ras-CAAX for FTase; B) nonpepti-
(FPP) analogs that compete with FPP for
binding to FTase; or C) bisubstrate inhibi-
tors, which are combinations of A and B.34
Attempts to covalently modify CAAX
cysteines of Ras proteins have also been
described with limited results so far.35 The
first inhibitor approaches focused on the
development of competitors for the pro-
tein substrate. However, some of these
peptides were not very cell permeable or
became rapidly degraded in the cell.
CAAX peptidomimetics in which the AA
portion was replaced with benzodiazepine
(C-BZA-M) or aminomethylbenzoic acid
(C-AMBA-M) were good inhibitors of
FTase and considerably more stable.36
Later, high-throughput screening led to
the identification of small molecule inhib-
itors. Two of those compounds made it
into clinical evaluation: lonafarnib and
tipifarnib. Lonafarnib (SCH66336) is a
nonpeptidic CAAX-competitive inhibitor
that is selective for FTase (IC50 = 1.9 nM).
Tipifarnib (R115777) is also selective for
FTase with an IC50 of 7.9 nM.37,38
FTIs inhibit cell growth of a large
variety of cancer cell lines in vitro and
also in vivo as tumor xenografts.39 In
particular, FTIs were shown to prevent
H-Ras farnesylation and reverse H-Ras–
driven cell transformation.40 FTIs induce
tumor growth inhibition rather than
regression when used as monotherapies.
However, K-Ras, the major Ras onco-
gene in human cancers, and N-Ras are
subject to alternative prenylation by
GGTase I in FTI-treated cells.21 This
resulted in a persistent membrane local-
ization of K-Ras and N-Ras and con-
comitant upregulation of downstream
signaling. The fact that K-Ras and
N-Ras became cross-prenylated had
been often cited as the main reason why
FTI monotherapy showed very poor
efficacy in clinical trials. FTIs might
be more effective in combination
with cytotoxic, STI-571, or hormonal
Another problem hampering develop-
ment of FTIs is the lack of reliable genetic
markers of response. FTI activities do not
correlate with K- or N-Ras mutational
status. Furthermore, FTIs clearly have
targets other than Ras proteins that might
be responsible for the effects seen in pre-
clinical models. For example, the inhibi-
tion of RhoB farnesylation seems to be
important for FTI antitumor activity.
FTIs also inhibit farnesylation of Rheb1
and Rheb2 GTPases that play a role in
tuberous sclerosis. Furthermore, some
FTIs potently inhibit GGTase II (Rab
GTPase) as well as farnesylation of other
farnesylated proteins like lamins, the cen-
tromeric proteins CENPE and CENPF,
PxF, and HDJ2.42,43
The alternative prenylation of K-Ras
and N-Ras by GGTase I led to the devel-
opment of GGTase I inhibitors (GGTIs).
Because geranylgeranylated proteins are
more numerous than farnesylated pro-
teins, this strategy is likely to result in
widespread toxicity. Indeed, GGTIs at
doses sufficient to prevent K-Ras pre-
nylation in the presence of FTIs were
found to be lethal in a mouse model.44
The development of dual prenylation
inhibitors led to similar conclusions as
with FTIs. Again, the observed antitu-
mor activity did not correlate with the
inhibition of K-Ras prenylation, which
suggests that these inhibitors have a
variety of targets in cells.45
Another strategy to interfere with
Ras prenylation is to inhibit the formation
of isoprenoids FPP and GGPP in the
mevalonate pathway. Statins inhibit
the rate-limiting enzyme 3-hydroxy-
3-methylglutaryl-CoA (HMG-CoA). Biphos-
phonates inhibit 2 other critical enzymes
in the pathway: isopentyl diphosphate
(IPP) isomerase and FPP synthase that
are required for FPP or GPP synthesis.
Interference with the mevalonate path-
way shows antitumor activity in some
cancers, but here also, this effect cannot
be solely attributed to the inhibition of
The function of the postprenylation
processing enzymes RCE1 and ICMT in
tumorigenesis has been investigated in
cells and mice in which the genes for
these enzymes have been disrupted. The
lack of RCE1 caused mislocalization of
Ras proteins. A conditional deletion of
RCE1 in fibroblasts was shown to reduce
Ras-induced transformation in cells.48
However, interference with RCE1 func-
tion in tumor cells or cancer models has
only shown modest effects. In hemato-
poietic cells of mice, simultaneous inacti-
vation of RCE1 and activation of K-Ras
led to acceleration of myeloproliferative
The development of RCE1 inhibitors
first concentrated on substrate analogs
like modified CAAX peptides. A nitro-
phenyl modification of the second ali-
phatic amino acid in the CAAX peptide
behaves as a competitive inhibitor. The
activity of such compounds in cell-based
assays has not been reported so far.50
The yeast RCE1 enzyme Rce1p and the
nonrelated CAAX-protease Ste24p can
independently promote the proteolytic
cleavage of –AAX of prenylated CAAX
box proteins in yeast. Recently, a class
of inhibitors was found that are dual-
specificity inhibitors. Such peptidyl
could be engineered so that they exhibit
selectivity for either of these enzymes.
The same group screened a small mole-
cule library that yielded 9 compounds
being able to inhibit Rce1p in the low
micromolar range. Some of these inhibi-
tors were effective in disrupting yeast
The effects of interfering with RCE1
function were modest, but more striking
effects were seen through blocking the
activity of ICMT. Inactivation of ICMT
inhibited cell growth and K-Ras–
induced oncogenic transformation both
in soft agar assays as well as in a nude
mouse model. Cells had a strongly
reduced level of RhoA and increased
levels of p21. Interference with ICMT
function inhibited transformation by
B-Raf V600E, an event that is thought to
be largely Ras independent.53 Further-
more, disrupting ICMT ameliorated
K-Ras–induced myeloproliferative dis-
ease.54 The anticancer drug methotrex-
ate induces higher levels of homocysteine
that causes hypomethylation in cells:
treatment with methotrexate reduced
Therapeutic strategies for targeting Ras proteins / Gysin et al.
Ras methylation by almost 90% and
caused mislocalization and a decreased
activity of ERK 1/2 and Akt.55 More
recently, synthetic as well as natural
small molecule inhibitors were found
that block ICMT, induce apoptosis, and
reduce tumor growth in a variety of
Taken together, strategies that com-
promise Ras-CAAX box processing
have, for the most part, not been reward-
ing. However, the actual proof of con-
cept that these drugs specifically
interfere with Ras membrane interaction
and could be clinically useful is lacking.
Prenylation and postprenylation inhibi-
tors generally lack the specificity to
inhibit signaling of specific Ras pro-
teins, especially K-Ras. However, new
approaches to block Ras through pro-
voking mislocalization are being inves-
tigated and may still hold promise for
Inhibition of Ras Expression
The idea of preventing Ras expression by
antisense or RNA interference is promis-
ing, but the successful application of this
technology is currently limited by lack
of efficient delivery, uptake, and gene
silencing. In cell lines, oncogenic
mutation–specific, small interfering RNAs
were able to silence K-Ras expression
and inhibit tumor cell growth.57 A more
recent study using anti–K-Ras RNA
interference demonstrated that tumor cell
lines can be classified as K-Ras depen-
dent or K-Ras independent. This depen-
dency correlated with their state of
Epithelial tumor cells were dependent on
K-Ras, whereas cell lines expressing
mesenchymal markers were relatively
independent on the oncogene. This find-
ing showed that it is important to know
the biological phenotype of a tumor in
order to apply the correct strategy for Ras
Downstream of Ras
Developing therapeutic agents to directly
block oncogenic Ras activity has thus
far been a challenging and unsuccessful
endeavor, for reasons discussed above.
Therefore, a great deal of effort has been
applied to developing therapies that tar-
get effector pathways downstream of
Ras (Fig. 3). Constitutive activation of
downstream effector pathways by onco-
genic Ras results in the uncontrolled
growth, proliferation, and survival of
cancer cells. Understanding which effec-
tor pathways are required for Ras-driven
oncogenesis is critical for determining
which pathways should be targeted for
therapeutic purposes. Many Ras effector
pathways are comprised of kinase cas-
cades, providing multiple nodes for
potential therapeutic intervention. While
several Ras effectors have been identi-
fied and comprehensively described,59-61
below, we discuss 2 of the best-charac-
terized Ras effector pathways: the Raf-
pathways. Importantly, both pathways
are integral to Ras-driven transforma-
tion, and small-molecule compounds
targeting these pathways are currently
under clinical investigation.62,63
The Raf-MEK-ERK Pathway
The Raf-MEK-ERK signal transduction
pathway, also known as the MAPK cas-
cade, was the first Ras effector signaling
pathway identified. Raf serine/threonine
kinases (A-Raf, B-Raf, and C-Raf/Raf-1)
specifically interact with GTP-bound
Ras, resulting in the activation of Raf
protein kinase activity.64-67 Upon activa-
tion by Ras, Raf phosphorylates and acti-
vates the serine/threonine kinase MEK,
which in turn phosphorylates and acti-
vates the serine/threonine kinase ERK.
This series of signaling events results
in the activation of transcriptional regula-
tors that promote a wide variety of
cellular events, including cell cycle pro-
gression and cell proliferation.59,60,68
The requirement for Raf-MEK-ERK
signaling in Ras-mediated transforma-
tion and tumorigenesis has been well
mutants of Raf-1, MEK, and ERK
inhibit Ras-driven transformation, high-
lighting the importance of this signaling
cascade downstream of Ras.73-76 In sup-
port of these findings, mutations in the
effector loop of H-Ras V12 that abro-
gate its ability to bind Raf-1 eliminate its
transforming potential in mammalian
cells, demonstrating the requirement for
Raf-1 activity downstream of activated
Ras.69 In addition, the growth inhibition
induced by overexpression of dominant-
negative Ras N17 can be overcome by
expression of constitutively active Raf-
1.77 Finally, cells that lack Ras proteins
altogether can be rescued from growth
arrest by expression of activated alleles
of Raf, MEK, or ERK proteins, again
Figure 3. Pathways downstream of Ras.
Genes & Cancer / vol 2 no 3 (2011)
showing that the Raf-MEK-ERK path-
way is downstream from Ras in mam-
malian cells, as it is in Caenorhabditis
elegans and Drosophila melanogaster
and other model organisms.78
Baccarini and colleagues recently
demonstrated that Raf-1 is required
for the initiation and maintenance of
squamous cell carcinoma in 2 separate
models of Ras-driven tumorigenesis.79
In the first model, Ras activation is
achieved through a classic chemical
carcinogenesis protocol in which tumors
are initiated through the topical applica-
tion of 7,12-dimethylbenz[a]anthracene
(DMBA), which causes an activating
mutation in codon 61 of H-Ras. Tumor
development is then promoted through
the topical application of 12-O-tetradec-
anoylphorbol 13-acetate (TPA). In the
second model, activation of the Ras
pathway is achieved by expression of a
dominant-active form of Son of Seven-
less (SOS), specifically in the epidermis.
In both models, ablation of Raf-1 leads
to the regression of established Ras-
driven tumors, suggesting that Raf-1
might serve as an appropriate target of
therapeutic intervention downstream of
activated Ras. Interestingly, in these
models, the ability of Raf-1 to promote
and maintain skin tumors is dependent
on the inhibition of the RhoGTPase tar-
get Rok-α rather than the activation
of the canonical MEK/ERK signaling
More recently, activating mutations in
various components of the MAPK signal-
ing cascade have been identified in
patients with related genetic develop-
mental disorders.68,80 For example, germ-
line gain-of-function mutations in KRAS,
BRAF, MEK1, and MEK2 have been
observed in patients with cardiofaciocu-
taneous (CFC) syndrome.81,82 Activating
mutations in Ras-Raf-MEK-ERK path-
way components are present in patients
with similar neurocardiofaciocutaneous
syndromes, including Noonan, LEOP-
ARD, and Costello syndromes.80,83,84
These findings provide genetic evidence
that the Raf-MEK-ERK pathway func-
tions downstream of Ras.
The identification of activating BRAF
mutations in cancer supports a role for
Raf-MEK-ERK signaling in oncogene-
sis.85,86 Interestingly, in melanoma and
colorectal cancer, a pattern of mutual
exclusivity between RAS and BRAF
mutation has emerged, suggesting that
mutation of either gene may be function-
ally equivalent in the pathogenesis of
these malignancies.87 However, in the
case of BRAF mutation, activation of
additional oncogenic signaling path-
ways such as the PI3’K pathway may
also be required.88
Attempts to target the Raf-MEK-ERK
signaling pathway for therapeutic pur-
poses have focused largely on the devel-
opment of Raf and MEK kinase
inhibitors. Sorafenib (Nexavar, Bayer,
Leverkusen, Germany) was the first Raf
kinase inhibitor to be tested in clinical tri-
als and is now US Food and Drug Admin-
istration (FDA) approved for the
treatment of renal cell carcinoma and
hepatocellular carcinoma.89 Although
sorafenib was designed to inhibit Raf-1
kinase activity, it also has activity against
additional cancer targets including
VEGF-R2, PDGFR, Flt-3, c-kit, and
FGFR-1.90 In fact, the success of
sorafenib as a cancer therapy has largely
been attributed to its inhibitory effects on
tumor angiogenesis,89 particularly for
renal cell carcinoma, which is largely
driven by hyperactive VEGF-R signal-
ing. In support of this, the VEGF-R2
inhibitor Sutent (Pfizer, New York City,
NY) is equally effective in treating this
disease. In contrast, Sutent (Pfizer) failed
to show efficacy in hepatocellular carci-
noma, suggesting that sorafenib’s effects
in this disease may indeed be mediated
through inhibition of Raf kinase. Further-
more, clinical responses to sorafenib cor-
relate well with levels of MAPK signaling
in this disease.91 Activation of this path-
way in hepatocellular carcinoma is
caused by loss of the negative regulatory
proteins Spred and Sprouty92,93 rather
than by oncogenic Ras.
When B-Raf was identified as a
major oncogene in human cancers,85
sorafenib was tested for clinical efficacy
in this disease. However, no clinical
benefit was observed. This may be
because sorafenib interacts with the
inactive form of Raf kinase and is less
effective against B-Raf V600E than
wild-type B-Raf. This prompted the
development of second-generation Raf
inhibitors, which demonstrate elevated
specificity for B-Raf V600E.62,94 While
these inhibitors potently suppress Raf-
MEK-ERK signaling and cell growth in
cancer cells expressing B-Raf V600E,
they paradoxically have the opposite
effect in cancer cells with wild-type
B-Raf, including those with oncogenic
Ras mutations.95-98 The promotion of
Raf-MEK-ERK signaling in Ras mutant
cancer cells by Raf inhibitors has been
reviewed extensively62,98,99 and precludes
the use of these inhibitors for the treat-
ment of Ras mutant cancers. Further-
more, although the use of the pan-RAF
inhibitor PLX-4032 in melanoma patients
harboring BRAF V600E mutations pro-
duced promising clinical results,100-102
recent studies have identified multiple
mechanisms of Raf inhibitor resistance,
including enhanced receptor tyrosine
kinase signaling as well as mutational
activation of NRAS.103-105 The finding
that mutational activation of NRAS can
bypass the effects of Raf inhibition sug-
gests that targeting Raf in the context of
an activating Ras mutation may not be
beneficial, despite the evidence in cell
culture and animal models that suggest
Several small-molecule compounds
have been developed to potently and
selectively inhibit the activity of MEK.
Studies in cancer cell lines and animal
models demonstrate that B-Raf mutation
predicts sensitivity to these agents,
although a subset of Ras mutant cell
lines displays sensitivity as well.106,107
Despite these promising preclinical
results, the outcome of early clinical tri-
als was underwhelming in part because
of the limited bioavailability and dose-
limiting toxicity of the compounds.62,108
Several compounds with improved
pharmaceutical properties are currently
under clinical investigation and hold
promise for the treatment of Ras mutant
tumors.108 Defining the factors that
underlie MEK inhibitor sensitivity and
resistance in Ras mutant cancers is of
great interest and will aid in determining
which patients will benefit most from
Therapeutic strategies for targeting Ras proteins / Gysin et al.
therapy. Likewise, it will be critical to
determine whether toxicities that limited
dosing are on target or off target. In this
respect, it is of interest that the MEK
inhibitor PD0325901, which is consid-
ered a relatively specific compound (it
acts through an allosteric mechanism
rather than as an ATP competitor), was
able to block growth of cells in culture
that had been engineered to grow in the
absence of Ras-Raf-MEK-ERK activity,
strongly suggesting that it has off-target
effects that could, in principle, have
accounted for clinical toxicity.109
The PI3’K Pathway
The phosphoinositide 3-kinase (PI3’K)
pathway is another well-studied signaling
cascade downstream of Ras. Class IA
PI3’Ks are heterodimeric lipid kinases
comprised of a p85 regulatory subunit
and a p110 catalytic subunit. The p110
catalytic subunit of PI3’K was identified
as a Ras effector when it was found to
preferentially associate with GTP-bound
Ras through its RBD.110,111 Although
PI3’K can be activated by upstream
receptor tyrosine kinases in a Ras-inde-
pendent manner, association with and
activation by Ras-GTP have proven to be
a principal mechanism of PI3’K regula-
tion. PI3’K catalyzes the conversion of
(PIP2) to the second-messenger phospha-
tidylinositol (3,4,5)-trisphosphate (PIP3).
A primary downstream effector of PIP3 is
the serine/threonine kinase Akt, which
activates a host of signaling programs to
promote cell growth, survival, and
PI3’K signaling is often upregulated
in tumor cells, indicating its importance
in the pathology of cancer. Hyperactiva-
tion of the pathway can be achieved
through a variety of mechanisms, includ-
ing gain-of-function mutation in PIK3CA,
which encodes the p110α catalytic sub-
unit of PI3’K.114-120 PTEN is a lipid phos-
phatase that negatively regulates PI3’K
signaling, and its expression is often lost
in cancers, providing yet another method
by which PI3’K signaling can be deregu-
lated.121-123 Additionally, increased activ-
ity of upstream regulators can also
activate the PI3’K signaling pathway,
and this can be achieved through ampli-
fication or activation of upstream recep-
tor tyrosine kinases or via oncogenic
Ras mutation.113,115 Interestingly, although
Ras mutation drives PI3’K activity, onco-
genic mutations in RAS and PIK3CA
often coexist in
cers.113,124-126 It is unclear whether these
amplify common downstream pathways
or function independently to activate
Several lines of experimental evi-
dence suggest that Ras mutant tumors
depend on the activation of the PI3’K
pathway. For example, PI3’K activity is
necessary for the transformation of
mouse embryonic fibroblasts by Ras.127
In addition, the interaction between Ras
and PI3’K is essential in a mouse model
of Ras-driven tumor formation.128 Col-
lectively, these studies demonstrate a
requirement for PI3’K activity down-
stream of oncogenic Ras and suggest that
targeting PI3’K in Ras mutant cancers
may have important antitumor effects.
Although the aforementioned studies
emphasize a role for PI3’K signaling in
Ras-mediated tumorigenesis, prelimi-
nary data suggest that Ras mutant tumors
are insensitive to single-agent PI3’K
inhibitors. In fact, in vitro experiments
have uncovered Ras mutation as a domi-
nant predictor of resistance to PI3’K
inhibitors.129-131 In addition, murine lung
cancers driven by oncogenic K-Ras do
not respond to treatment with a single-
agent dual PI3’K-mTOR inhibitor.132
Therefore, while PI3’K activity is an
important driver of Ras-mediated trans-
formation and tumorigenesis in cell cul-
ture and animal models, inhibition of
PI3’K pathway activity alone is likely
insufficient for the treatment of established
tumors harboring RAS mutations.63,133
Dual Inhibition of the
Raf-MEK-ERK and PI3’K
The limited response of Ras mutant can-
cer cells to single-agent pathway inhibi-
tors suggests that dual inhibition of
Raf-MEK-ERK and PI3’K signaling
may be necessary to block the growth of
Ras-driven tumors. The efficacy of a
single-agent pathway inhibitor is often
hindered by the release of negative feed-
back loops on the reciprocal pathway.
For example, treatment of Ras mutant
cancer cells with potent and specific
MEK inhibitors results in increased
phosphorylation of the PI3’K pathway
effector Akt.107,134,135 Upregulation of
PI3’K signaling in response to MEK
inhibition is due to the release of nega-
tive feedback from ERK to the EGF
receptor.136 In light of the crosstalk
between the Raf-MEK-ERK and PI3’K
signaling pathways, it has been proposed
that dual inhibition of both pathways
may be required to evade these feedback
loops. In support of this hypothesis,
combined inhibition of MEK and PI3’K
signaling in Ras mutant cancer cells is
superior to single-agent inhibition in
vitro and in vivo and results in a syner-
gistic decrease in cell viability and
increase in apoptosis.107,134,135 Further-
more, while dual pathway inhibition was
also more effective than single-agent
pathway inhibition in cancer cells driven
by activated receptor tyrosine kinase
signaling, the most pronounced syner-
gistic effect was observed in cancer cells
harboring oncogenic RAS mutations.107
Studies utilizing transgenic mouse
models provide additional support for this
therapeutic approach. In a mouse model of
lung cancer driven by oncogenic K-Ras,
single-agent treatment with a dual PI3’K-
mTOR inhibitor had no effect on tumor
growth. Additionally, single-agent treat-
ment with a MEK inhibitor caused only
modest tumor regression. However,
combined treatment with both pathway
inhibitors resulted in synergistic tumor
The necessity for dual pathway inhibi-
tion is most evident in cancers that harbor
coexisting oncogenic mutations in RAS
and PIK3CA. Reports indicate that muta-
tional activation of PIK3CA in KRAS
mutant cancer cells confers resistance to
MEK inhibition.137,138 Indeed, treatment
with single-agent MEK or Akt inhibitors
had no significant effect on tumor growth
in a xenograft model with coexisting
KRAS and PIK3CA mutations. However,
Genes & Cancer / vol 2 no 3 (2011)
combined treatment with both inhibitors
was effective at suppressing tumor
Collectively, these data indicate that
dual inhibition of Raf-MEK-ERK and
PI3’K signaling might be clinically ben-
eficial in Ras mutant tumors and provide
a rationale for the design of future clini-
cal trials to test combinations of path-
way inhibitors. Further, these studies
emphasize that the efficacy of targeted
therapeutics is genotype dependent and
underscore the importance of stratifying
patients by tumor genotype prior to
Additional Ras Effectors
as Potential Targets in
While Raf-MEK-ERK and PI3’K repre-
sent the best-characterized effector path-
ways utilized during tumor development
following activation of Ras oncogenes,
several additional effectors have been
implicated in Ras-driven tumorigenesis
including RalGDS, Tiam1, and PLCε.
Each of these proteins has been shown
to interact directly with Ras proteins,
thus prompting the question of their role
in Ras mutant tumors.60
The discovery that Ral guanine nucle-
otide dissociation stimulator (RalGDS)
was able to interact with activated Ras
proteins motivated the initial investiga-
tion of this effector arm of Ras signaling
during tumorigenesis.139-143 RalGDS is a
GEF, stimulating the dissociation of GDP
from its target Ral proteins and allowing
for binding of GTP and subsequent acti-
vation.142 In rodent cells in culture, Ral-
GDS or the downstream Ral proteins
cooperated with Ras oncogenes to induce
transformation but were unable to do so
on their own.61,144,145 Subsequent studies
in human cells utilizing Ras effector–
binding mutants demonstrated that acti-
vation of the RalGDS pathway was
sufficient to transform human epithelial
kidney cells.146 This pathway is thought
to play a role in mediating proliferation
and cell survival downstream of an acti-
vated Ras oncogene, as depletion of RalA
impairs anchorage-independent prolifer-
ation of Ras mutant pancreatic tumor
cells, while RalB was found to be required
for survival in a number of tumor cell
lines.142,147,148 Recently, Ral proteins have
been implicated in the development of
melanoma and myeloid malignancies, 2
cancers known to harbor frequent muta-
tions in Ras oncogenes. RalA is activated
in several melanoma cell lines with onco-
genic N-Ras mutations, and RalA knock-
down inhibited the tumorigenicity of
these cell lines. Furthermore, studies in
immortalized melanocytes showed that
RalGEF was able to recapitulate several
tumorigenic traits seen after transforma-
tion with N-Ras, including anchorage-
independent growth, consistent with
previous studies of this pathway.149,150
Expression of a Ras effector–binding
mutant only able to activate RalGDS in
hematopoietic cells demonstrated that
activation of this pathway alone was
able to inhibit neutrophil differentiation
and prolong proliferative potential.151
Finally, in vivo evidence for RalGDS as
a relevant Ras effector comes from data
generated using a multistage model of
skin carcinogenesis, leading to the
development of squamous cell tumors
harboring H-Ras mutations. Using this
protocol in mice with homozygous dele-
tion of RalGDS resulted in reduced
tumor incidence, size, and progression
to malignancy compared to wild-type
mice.61,152 Together, these data support a
role for RalGDS both in vitro and in vivo
as an important effector pathway uti-
lized by oncogenic Ras to drive tumori-
genesis that could potentially be
exploited for therapeutic intervention,
although the absence of somatic muta-
tions in this effector pathway makes its
precise role less clear than the Raf-
MEK-ERK and PI3’-kinase pathways.
Activation of Ras signaling has also
been linked to breakdown of phos-
phoinositides through its ability to bind
and activate phospholipase Cε.153,154 Acti-
vated PLCε catalyzes cleavage of PI(4,5)
P2 into inositol-1,4,5-trisphosphate (IP3)
and diacylglycerol (DAG), which subse-
quently promote the release of Ca2+ and
the activation of protein kinase C (PKC),
respectively. Although studies have
shown conflicting results in vitro,155 the
importance of this Ras effector pathway
during tumorigenesis is supported in
vivo by data generated again using the
multistage mouse model of skin carcino-
genesis. In these conditions, PLCε-null
mice showed delayed onset of the char-
acteristic squamous tumors resulting
from this protocol as well as markedly
reduced tumor incidence.156 Further-
more, tumors that did form in mice lack-
ing PLCε also failed to undergo
malignant progression to carcinomas.
While the understanding of the relation-
ship between Ras oncogenes and the
requirement for PLCε is incomplete,
these data suggest it may be an avenue to
pursue to expand the list of potential
drug targets in Ras mutant cancers.
Finally, Tiam1, a GEF that stimulates
the activation of Rac, has also been
implicated in tumorigenesis as an impor-
tant Ras effector using the DMBA/TPA
skin carcinogenesis model.157 Previ-
ously, Tiam1 had been shown to bind
directly to active Ras, leading to its acti-
vation and subsequent stimulation of
Rac activity.158-160 Tiam1-deficient mice
are resistant to the development of
DMBA/TPA-induced, Ras-driven skin
tumors. Furthermore, the small number
of tumors produced using this protocol
grew much slower than the tumors
formed in wild-type mice.157 Interest-
ingly, however, the few tumors that
grow in Tiam1-null mice show increased
invasiveness, suggesting there may be
different roles for Tiam1 at different
stages of tumor progression. Taken
together, each of these effectors repre-
sents a potential drug target for Ras-
driven tumors, given that deletion of
each leads to impaired tumorigenesis in
models of Ras-driven cancers. The true
promise of each of these effectors, how-
ever, will have to be further explored
using additional models of Ras-driven
tumorigenesis to define the extent to
which generalizations can be made
about the usage and requirements of
these noncanonical effector pathways.
Therapeutic strategies for targeting Ras proteins / Gysin et al.
Lethal Targets in Cells
with Mutant Ras
While rationally targeting Ras itself or
specific Ras effector pathways provides
one therapeutic strategy, an alternative is
to exploit vulnerabilities created specifi-
cally by the presence of a mutant Ras
oncogene. Several potential targets have
been identified in both cell culture sys-
tems and mouse models that are required
for the initiation or maintenance of Ras
mutant tumor cells. Considering that
Tiam1 has been implicated in Ras-driven
tumorigenesis, it is perhaps not unex-
pected that a role for the GTPase for
which Tiam1 serves as a GEF, Rac1, has
also been identified in Ras mutant tumors.
The relevance of Rac1 in Ras transforma-
tion was initially described in cell culture
systems in which dominant-negative
Rac1 was able to inhibit focus formation
by Ras oncogenes, while activated Rac1
was able to enhance Ras transformation
in addition to growth in soft agar and
motility.71,161,162 More recently, mouse
models have provided additional support
for the role of Rac1 in Ras-driven tumors
in vivo. Deletion of Rac1 in keratinocytes
and subsequent treatment with the
DMBA/TPA skin tumorigenesis protocol
demonstrated a role for this GTPase in
the development of these H-Ras–driven
cancers based on an observed decrease in
hyperproliferation of keratinocytes in
cells lacking Rac1.163 Furthermore, con-
ditional deletion of Rac1 in combination
with activation of K-Ras in a mouse
model of lung cancer led to a dramatic
reduction in cell proliferation with a
reduction in the number of tumors.164
Additionally, loss of Rac1 alone is dis-
pensable for proliferation but is required
in the context of activated Ras defining a
synthetic lethal interaction in these cells.
While Rac1 may be important for the
growth of Ras-driven tumors, the exact
role and mechanism of activation in
these cells remain to be fully elucidated,
as several pathways can lead to Rac
activity, which in turn can drive a num-
ber of downstream cellular behaviors.
These data support a role for Rac1 in
Ras-driven tumorigenesis, providing
another member of a growing list of
potential sites for therapeutic intervention
and warranting further investigation of
this pathway in Ras mutant cells.
Recently, a synthetic lethal interaction
has been defined between the presence of
a K-Ras oncogene and genetic deletion of
Cdk4. Cdk4 has been implicated previ-
ously in models of breast tumorigenesis
driven by alternate oncogenes165,166; how-
ever, Puyol et al. recently demonstrated
that genetic or conditional deletion of
Cdk4 led to a senescent response specifi-
cally in lung cells expressing an activated
K-Ras oncogene.167 Additionally, treat-
ment with a pharmacological inhibitor of
Cdk4 showed a reduction in the growth
of K-Ras–driven tumors, further support-
ing a role for Cdk4 as a therapeutic target
in this disease. Furthermore, a similar
requirement for Cdk4 was identified in a
mouse model of H-Ras–driven breast
tumorigenesis.168 Conversely, coexpres-
sion of Cdk4 and oncogenic Ras in
normal epidermal cells leads to the devel-
opment of squamous cell carcinoma–like
invasive neoplasia, and Cdk4 expression
was found to circumvent Ras-induced
growth arrest in primary human keratino-
cytes, suggesting a requirement for Cdk4
during Ras-driven tumorigenesis in this
system as well.169 Finally, in mouse mod-
els, expressing activated H-Ras in mela-
nocytes showed increased incidence of
spontaneous cutaneous melanoma when
crossed onto a Cdk4(R24C) background,
where Cdk4 is insensitive to inhibition by
p15INK4B and p16INK4A.170 Further-
more, treatment of these mice with
DMBA/TPA led to an increase in the
number of nevi and melanomas com-
pared to Cdk4 wild-type mice, suggesting
a cooperative interaction between onco-
genic Ras and Cdk4 during tumor devel-
opment. Data from these different
systems implicate Cdk4 as a promising
therapeutic target in Ras-driven cancers.
Several other candidates have emerged
as important mediators of the transform-
ing effects of oncogenic Ras, including
NF-κB, cyclin D1, and myc. NF-κB has
been recently reported to be required for
the development of tumors in mouse
models of lung tumorigenesis.171,172 Mey-
lan et al. demonstrated that inhibition of
NF-κB signaling led to an apoptotic
response in p53-null lung cancer cell
lines, while inhibition of the pathway in
vivo in the context of K-RasG12D–driven
lung tumorigenesis showed reduced
tumor development both at the time of
tumor initiation or after tumor progres-
sion.172 Additionally, Basseres et al.
showed that deletion of NF-κB subunit
p65/RelA reduced the number of K-Ras–
induced lung tumors both in the presence
and absence of p53, and tumors that
emerged in the absence of p65/RelA
showed a higher number of apoptotic
cells, reduced spread, and showed lower
grade.171 The requirement for cyclin D1
has also been suggested in Ras mutant
tumors. Initially, observations that acti-
vated Ras led to overexpression of cyclin
D1 motivated investigation of the depen-
dence of Ras on cyclin D1.173,174 Subse-
quently, deficiency in cyclin D1 was
shown to decrease tumor development in
several different systems of Ras-driven
tumorigenesis, including grafting of ret-
roviral Ras-transduced keratinocytes and
phorbol ester treatment of Ras transgenic
mice, and in a 2-stage model of skin car-
cinogenesis.174 Finally, a dependence on
myc in Ras mutant tumors has been sug-
gested based on data that in a mouse
model of Ras-induced lung adenocarci-
noma, expression of a dominant-negative
myc mutant led to rapid regression of
both incipient and established tumors,
suggesting a requirement for myc in the
cells.175 Each of the examples discussed
above provides the basis for exploring
new avenues of intervention in cells har-
boring activated Ras alleles that may
demonstrate therapeutic efficacy specifi-
cally in these tumor cells.
High-Density RNAi Screens
for Synthetic Lethality in Ras
Numerous studies have sought to define
the molecular requirements that underlie
Ras-driven tumorigenesis in order to
inform potential sites of intervention.
Genes & Cancer / vol 2 no 3 (2011)
Recent high-throughput approaches
have provided an expanded list of poten-
tial therapeutic targets for Ras-driven
tumors. Using loss-of-function RNAi
screens, several groups have identified
proteins that, when lost, elicit a synthetic
lethal response with mutant Ras onco-
genes while leaving cells with wild-type
Ras proteins unaffected. Scholl et al. uti-
lized a subset of the RNAi Consortium
Lentiviral shRNA Library targeting
1,011 human genes to identify STK33, a
member of the calcium/calmodulin-
dependent protein kinase subfamily of
serine/threonine protein kinases, as a
target that is selectively required for via-
bility and proliferation in the context of
mutant KRAS.176 They found that sup-
pression of STK33 across a panel of
K-Ras mutant versus wild-type cell lines
demonstrated synthetic lethality inde-
pendently of tissue origin. Furthermore,
introduction of exogenous mutant KRAS
resulted in newly acquired dependence
on STK33. Having never been impli-
cated in cancer previously and being
insufficient for tumor initiation and
maintenance alone, STK33 represents
an example of a component of a signal-
ing pathway that becomes aberrantly
required in the presence of KRAS muta-
tions. STK33 may promote viability in
KRAS-dependent cells through regula-
tion of S6K1-induced inactivation of the
proapoptotic protein BAD.
Using a similar lentiviral shRNA
screening strategy in a panel of cancer
cell lines, Barbie et al. identified sup-
pression of TBK1, a noncanonical IκB
kinase as a second synthetic lethal inter-
action with K-Ras oncogenes. Viability
in cell lines with endogenous mutations
in KRAS was selectively reduced fol-
lowing TBK1 knockdown. Furthermore,
growth of tumor xenografts was inhib-
ited in KRAS mutant cells expressing
TBK1 shRNAs, while TBK1 shRNA
showed little to no effect on the tumor-
forming ability of cells with wild-type
KRAS.177 Introduction of oncogenic
KRAS in immortalized human lung epi-
thelial cells led to acquired sensitivity to
the knockdown of TBK1. Activation of
TBK1 may be linked to NF-κB–driven
survival signals downstream of onco-
genic K-Ras, consistent with the work
discussed previously implicating NF-κB
as playing an essential role in K-Ras
Finally, using a retroviral shRNA
library, a comparable K-Ras synthetic
lethal screen defined several mitotic genes
that were selectively required in cells har-
boring mutations in K-Ras. These included
several components of the anaphase-
promoting complex, proteasome and polo-
like kinase 1 (PLK1), expanding the list of
strategies for therapeutic intervention in
these cells.178 The authors suggest that
based on these findings, Ras oncogenes
may lead to increased dependence on key
mitotic proteins for survival when com-
pared to non-Ras transformed cells,
although the precise mechanism underly-
ing this phenomenon remains to be com-
pletely understood. Recently, similar
approaches have also identified WT1 and
Snail2 as candidate proteins required
in Ras mutant cells.179,180 These high-
throughput genome-wide screening strate-
gies hold great value in providing rapid
exploration of specific requirements of
cells for growth and survival and may help
to uncover previously unappreciated drug
targets in tumors with various genetic
Efforts to attack Ras proteins directly
are ongoing, based on new ways of
developing compounds based on struc-
tural considerations and on a better
understanding of Ras processing and
membrane localization. These efforts
are still in early exploratory phases of
drug discovery. Targeting downstream
pathways, in contrast, is now a major
focus of clinical research, as a rich pipe-
line of drug candidates that target pro-
teins within with MAPK and PI3’-kinase
pathways undergoes clinical evaluation.
In parallel, new ways of identifying pro-
teins that Ras depends on for malignant
transformation are being evaluated,
based on recent advances in RNA inter-
ference technology. Indeed, siRNA is
being developed for systemic therapy
and may eventually enter the main-
stream of clinical research opportuni-
ties. Successful targeting of other major
oncogenic drivers, such as EGF-R and
BCR-ABL, only serves to underscore
the importance of targeting Ras and
encourages these exploratory and clini-
cal efforts. Thirty years after Ras genes
were identified, the urgency of devising
strategies for treating Ras-driven can-
cers has only increased.
Declaration of Conflicting Interests
The author(s) declared the following potential con-
flicts of interest with respect to the research, author-
ship, and/or publication of this article: Dr. McCormick
is a consultant to ONYX, and this review discusses
the use of sorafenib. Drs. Gysin, Salt, and Young
declare no conflicts of interest.
This work was supported by Daiichi Sankyo Co. Ltd.
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