Map Krev

Oncogene (2007) 26, 32913310
& 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00
www.nature.com/onc
REVIEW
Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for

the treatment of cancer
PJ Roberts1 and CJ Der2
1
Division of Pharmacotherapy and Experimental Therapeutics, Lineberger Comprehensive Cancer Center, University of North
Carolina at Chapel Hill, Chapel Hill, NC, USA and 2Department of Pharmacology, Lineberger Comprehensive Cancer Center,
University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Mitogen-activated protein kinase (MAPK) cascades are

key signaling pathways involved in the regulation of
normal cell proliferation, survival and differentiation.
Aberrant regulation of MAPK cascades contribute to
cancer and other human diseases. In particular, the
extracellular signal-regulated kinase (ERK) MAPK pathway has been the subject of intense research scrutiny
leading to the development of pharmacologic inhibitors for
the treatment of cancer. ERK is a downstream component
of an evolutionarily conserved signaling module that is
activated by the Raf serine/threonine kinases. Raf
activates the MAPK/ERK kinase (MEK)1/2 dual-specicity protein kinases, which then activate ERK1/2. The
mutational activation of Raf in human cancers supports
the important role of this pathway in human oncogenesis.
Additionally, the Raf-MEK-ERK pathway is a key
downstream effector of the Ras small GTPase, the most
frequently mutated oncogene in human cancers. Finally,
Ras is a key downstream effector of the epidermal growth
factor receptor (EGFR), which is mutationally activated
and/or overexpressed in a wide variety of human cancers.
ERK activation also promotes upregulated expression of
EGFR ligands, promoting an autocrine growth loop
critical for tumor growth. Thus, the EGFR-Ras-RafMEK-ERK signaling network has been the subject of
intense research and pharmaceutical scrutiny to identify
novel target-based approaches for cancer treatment. In
this review, we summarize the current status of the
different approaches and targets that are under evaluation
and development for the therapeutic intervention of this
key signaling pathway in human disease.
Oncogene (2007) 26, 32913310. doi:10.1038/sj.onc.1210422
Keywords: mitogen-activated protein kinases; ERK;
Ras; epidermal growth factor receptor; small molecule
inhibitors; monoclonal antibodies
of death in the United States in people under the age

of 85 (Jemal et al., 2005). Although we have made great
strides in unraveling the mysteries of cancer genetics and
biology, the important task now facing researchers and
clinicians is to translate these discoveries into novel
therapeutics that will improve patient outcomes. As
many of the genetic alterations found in cancers involve
genes whose products are regulators of signal transduction (Hanahan and Weinberg, 2000), much of the focus
in the development of novel therapeutics has involved
inhibitors of signal transduction molecules, in particular
protein kinases (Arslan et al., 2006; Davies et al., 2006;
Sebolt-Leopold and English, 2006). The human kinome
is comprised of over 518 protein kinases (http://
kinase.com), with disease associations reported for over
150 kinases (http://www.cellsignal.com/reference/kinase_disease.asp). Presently, the Food and Drug Administration (FDA) has approved 10 protein kinase
inhibitors and over 100 kinase-targeted agents are
currently undergoing clinical evaluation.
Target-based therapies are widely considered to be the
future of cancer treatment and much attention has been
focused on developing inhibitors of the Rafmitogenactivated protein kinase (MAPK)/extracellular signalregulated kinase (ERK) kinase MEKERKMAPK
signaling pathway and its upstream activators (SeboltLeopold and Herrera, 2004). Evidence that ERK MAPK
signaling promotes cell proliferation, cell survival and
metastasis, along with the overwhelming frequency in
which this pathway is aberrantly activated in cancer, in
particular by upstream activation by the epidermal
growth factor receptor (EGFR) and the Ras small
guanosine triphosphatases (GTPases) (Figure 1), support
current efforts to identify approaches to inhibit this
pathway. In this review, we will summarize the current
status of the approaches and development of pharmacologic inhibitors to block EGFRRasRafMEKERK
MAPK signaling for the treatment of cancer.
Introduction
In 2005, the American Cancer Society reported that
cancer has surpassed heart disease as the leading cause
Correspondence: CJ Der, Department of Pharmacology, Lineberger
Comprehensive Cancer Center, University of North Carolina at
Chapel Hill, CB 7295, Chapel Hill, NC 27599-7295, USA.
E-mail: cjder@med.unc.edu
The RafMEKERK MAPK cascade

Mammalian cells possess four well characterized and
widely studied MAPKs (Figure 2). These cascades are
comprised of three protein kinases that act as a signaling
relay controlled, in part, by protein phosphorylation: a
MAPK kinase kinase (MAPKKK), a MAPK kinase
Targeting the Raf-MEK-ERK MAPK cascade

PJ Roberts and CJ Der
3292
EGFR overexpression:
Colorectal cancer (27-77%)
Pancreatic cancer (30-50%)
Lung cancer (40-80%)
Non-small cell lung cancer (14-91%)
TGF
Sos
EGFR*
Ras*
Grb2
Raf*
EGFR mutation:
NSCLC (10%)
Glioblastoma (20%)
MEK
Ras mutation:
Pancreatic cancer (90%)
Papillary thyroid cancer (60%)
Colon cancer (50%)
Non-small cell lung cancer (30%)
B-Raf mutation:
Melanoma (70%)
Papillary thyroid cancer (50%)
Colon cancer (10%)
ERK
*Mutated in human cancers

Figure 1 Oncogene activation of the ERK MAPK cascade. Mutationally activated B-Raf, Ras and mutationally activated (by
missense mutations in the cytoplasmic kinase domain in NSCLC or by extracellular domain truncations (e.g., VIII) in glioblastomas)
and/or overexpressed EGFR causes persistent activation of the ERK MAPK cascade in human cancers. Activated ERKs translocate
to the nucleus, where they phosphorylate and regulate various transcription factors leading to changes in gene expression. In
particular, ERK-mediated transcription can result in the upregulation of EGFR ligands, such as TGFa, thus creating an autocrine
feedback loop that is critical for Ras-mediated transformation and Raf-mediated gene expression changes.
(MAPKK) and a MAPK (Johnson and Lapadat, 2002).

The terminal serine/threonine kinases (MAPKs) are the
ERK1/2, the c-Jun amino-terminal kinases (JNK12/3;
also called SAPKs), p38 kinases (p38a/b/g/d) and
ERK5. Generally, the ERK pathway is activated by
growth factor-stimulated cell surface receptors, whereas
the JNK, p38 and ERK5 pathways are activated by
stress and growth factors.
MAPK cascades function downstream of cell surface
receptors and other cytoplasmic signaling proteins
whose functions are deregulated in cancer and other
human pathologic disorders. In light of the recent
success in the clinical development of small molecule
inhibitors of protein kinases, components of MAPK
cascades have been the subject of intense research and
drug discovery efforts. Of these, the p44 ERK1 and p42
ERK2 MAPKs have attracted intense research interest
because of their critical involvement in the regulation of
cell proliferation and survival. In particular, the mutational activation and/or overexpression of upstream
signaling components that activate the ERK MAPKs
(Figure 1), together with the substantial body of
experimental observations demonstrating the necessity
of this pathway in oncogene function, has validated
this pathway for drug discovery (Benson et al., 2006).
This has stimulated intensive efforts by the research
community and pharmaceutical industry to develop
inhibitors of ERK signaling for cancer treatment. The
validated involvement of the related JNK and p38
MAPK signaling cascades in cancer is less clearly
established, and consequently, the status of inhibitor
Oncogene
development for these MAPK cascades will only be

discussed briey.
Quite an extensive array of potent and specic
inhibitors of p38 are being evaluated in phase I and II
clinical trials (Dominguez et al., 2005; Hynes and
Leftheri, 2005; ONeill, 2006) (Table 1). They have been
developed primarily for the treatment of chronic
inammatory diseases (e.g., rheumatoid arthritis,
Crohns disease), although some trials are also evaluating possible applications in cancer. One consequence of
p38 inhibitors is blockage of p38-induced transcriptional
expression of genes that encode proinammatory
cytokines.
In contrast to p38 inhibitors, only a handful of JNK
inhibitors have been developed, and are being considered for the treatment of cancer, as well as inammatory, vascular, neurodegenerative and metabolic
disorders (Manning and Davis, 2003). Unlike the
considerable volume of preclinical studies that utilized
highly specic inhibitors of MEK1/2 (PD98059, U0126
and CI-1040/PD184352) or p38 (SB203580 and SB
202190) (Davies et al., 2000), the preclinical observations made with the SP600125 JNK inhibitor are likely
to be less informative regarding the biological roles of
JNK, owing to the considerable off-target activity of
this compound (Bain et al., 2003). One JNK inhibitor,
CC-401, is currently in phase II evaluation in acute
myelogenous leukemia, and has also been considered for
the treatment of respiratory diseases (Table 1).
The MAPKKK component of the ERK cascade is
comprised of the Raf serine/threonine kinases (c-Raf-1,

3293
Stimulus
Growth Factors
Stress, Cytokines
Growth Factors
Stress, Cytokines
Growth Factors
Stress,
Growth Factors
MAPKKK
A-Raf, B-Raf,
c-Raf-1, Mos,
Tpl-2
MLK3, TAK1,
MEKK4,
ASK1
MEKK1/4, DLK,
MLK1-4, LZK,
TAK1, ASK1, ZAK
MEKK2/3
MAPKK
MEK1/2
MKK3/6
MKK4/7
MEK5
MAPK
ERK1/2
p38///
JNK1,2,3
ERK5
Transcription
Factors, Etc.
Elk-1, Ets-2
RSK, MNK,
MSK, cPLA2
CHOP, ATF2,
MNK, MSK,
MEF2, Elk-1
Jun, ATF2,
RNPK, p53,
NFAT 4, Shc
MEF2
Figure 2 Mammalian MAPK cacades. There are four major mammalian MAPKKKMAPKKMAPK protein kinase cascades.
Whereas the ERK pathway is commonly activated by growth factors, the JNK, p38 and ERK5 pathways are activated by
environmental stress, including osmotic shock, ionizing radiation. Many of the substrates for MAPKs are nuclear transcription factors.
Interactions and substrates were compiled from information from STKE (http://stke.sciencemag.org/index.dtl).
Table 1 Germline mutation of the Ras-Raf-MEK-ERK cascade in human disorders

Syndrome
Protein
Mutations
References
Noonan syndrome
Costello syndrome
K-Rasa
H-Rasa
P34R, T58I, V14 I, V152G, D153V

G12A, G12C G12E, G12S, G12V,
G13C, G13D, K117R, A146T
Cardiofaciocutaneous
syndrome
K-Rasa
G60R, D153V
Carta et al. (2006); Schubbert et al. (2006)

Aoki et al. (2005); Estep et al. (2006); Gripp et al.
(2006); Kerr et al. (2006); van Steensel et al.
(2006); Carta et al. (2006)
Niihori et al. (2006); Schubbert et al. (2006)
B-Rafb
A246P, Q257R, S467A, F468S,

G469E, L485F, K499E, E501G,
E501K, N518D, F595L, G596V
F35S, Y130C
F57C
MEK1c
MEK2c
Niihori et al. (2006); Rodriguez-Viciana et al.

(2006)
Rodriguez-Viciana et al. (2006)
Rodriguez-Viciana et al. (2006)
Abbreviations: MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. aThe most common RAS mutations found in
human cancers are seen at residues G12, then Q61, and infrequently at G13; bOf the over 40 missense mutations, the V600E mutation in the kinase
domain accounts for B90% of human cancers; cNo MEK1/2 mutations have been identied in human cancers.
A-Raf and B-Raf) (Wellbrock et al., 2004; Schreck and

Rapp, 2006). Raf kinases phosphorylate and activate the
MEK1 and MEK2 dual-specicity protein kinases.
Although Raf has been reported to phosphorylate other
proteins, to date, the only validated physiologically
relevant substrates remain the two closely related MEK1
and MEK2 proteins. MEK1/2 (MAPKK) then phosphorylate and activate the ERK1 and ERK2 MAPKs.
Activated ERKs phosphorylate and regulate the activities of an ever growing roster of substrates that are
estimated to comprise over 160 proteins (Yoon and
Seger, 2006). The majority of ERK substrates are
nuclear proteins, but others are found in the cytoplasm
and other organelles. Activated ERKs can translocate to

the nucleus, where they phosphorylate and regulate
various transcription factors, such as Ets family
transcription factors (e.g., Elk-1), ultimately leading to
changes in gene expression (Zuber et al., 2000; Schulze
et al., 2004).
There is substantial evidence validating the importance of Raf and MEK in cancer progression and in
promoting cancer growth (Shields et al., 2000). The
importance of this pathway in oncogenesis was rst
suggested by the initial identication of Raf as potent
retrovirus oncogenes (Schreck and Rapp, 2006). Subsequently, laboratory generated constitutively activated
Oncogene

3294
mutants of Raf and MEK were shown to potently

transform rodent broblasts and other cell types.
Furthermore, studies using genetic or pharmacologic
approaches have shown that MEK and ERK are
required for the transforming activities of Ras and
other oncogenes. More recently, mutationally activated
B-Raf has been identied in a variety of human cancers
(Davies et al., 2002) and the nding that mutationally
activated Ras and Raf occur in a non-overlapping
occurrence in melanomas, colorectal carcinomas, papillary thyroid carcinomas, serous ovarian carcinomas and
lung cancers suggests that Ras function is facilitated
primarily by activation of Raf (Rajagopalan et al., 2002;
Mercer and Pritchard, 2003; Singer et al., 2003; Vos
et al., 2003; Sieben et al., 2004). Interfering RNA
suppression of mutant B-Raf demonstrated the importance of continued B-Raf activity for the transformed
and tumorigenic growth of melanomas (Hingorani et al.,
2003; Sharma et al., 2005; Hoeich et al., 2006;
Sumimoto et al., 2006).
Recently, germline de novo mutational activation of
H-Ras and K-Ras, B-Raf, as well as MEK1 and MEK2,
has been found in patients that comprise a group of
Table 2
related developmental disorders (Costello, cardiofacio

cutaneous and Noonans syndromes) (Table 2), and
suggests that aberrant ERK activation will contribute to
other human disorders as well as cancer (Aoki et al.,
2005; Niihori et al., 2006; Rodriguez-Viciana et al.,
2006; Schubbert et al., 2006). These syndromes are
associated with similar consequences that include facial
dysmorphia, cardiomyopathy, as well as increased
incidence of cancer (Duesbery and Vande Woude,
2006). Interestingly, the mutational spectrum seen
in these syndromes differ from those seen in cancer,
and generally lead to weakly activated proteins.
This reects the likelihood that the more potent activating mutations found in cancer cannot be tolerated
during development. Taken together, these observations
provided strong support for the therapeutic value of
blocking ERK signaling in cancer and developmental
disorders.
Although the RafMEKERK cascade is typically
drawn as a simple linear, unidirectional cascade of
protein kinases, the more appropriate depiction of this
cascade is that it is a key core element of a complex
signaling network, with many other interactions
Small molecule MAPK inhibitorsa
Agent
Company
Target
Status
RO4402257
RO3201195
PH-797804
AZD-6703
GW681323
(SB-681323)
Hoffmann-LaRoche
Hoffmann-LaRoche
Pzer
AstraZeneca
GlaxoSmithKline
p38
p38
p38
p38
p38
Phase
Phase
Phase
Phase
Phase
VX-745
VX-702
Vertex Pharmaceuticals
Vertex Pharmaceuticals/
Kissei
Scios/Johnson & Johnson
p38
p38
Phase IIb
Phase II
p38a
Phase II
p38a
p38
p38
Phase I
Phase IIb
Phase I
RWJ-67657
BIRB-796
KC706
Scios/Johnson & Johnson

Takeda Pharmaceuticals
Pharmacopeia/
Bristol-Myers Squibb
Johnson & Johnson
Boehringer Ingelheim
Kemia
p38
p38a
p38
Phase Ib
Phase IIb/IIIb
Phase II
ARRY-797
AMG-548
CC-401
SP600125
CEP-1347
Sorafenib (Nexavar)
Array BioPharma
Amgen
Celgene
Celgene
Cephalon
Bayer/Onyx
p38
p38a
JNK
JNK1, JNK2, JNK3
MLK-1, MLK-2, MLK-3
B-Raf, Raf-1c
Phase I
Phase Ib
Phase II
Preclinical
Phase II/IIIb
Approved
Phase IIIII
Raf265 (CHIR-265)
PLX4032
PD0325901
AZD6244
(ARRY-142886)
ARRY-438162
Novartis
Roche/Plexxikon
Pzer
AstraZeneca/Array
BioPharma
Array BioPharma
A-Raf, B-Raf, c-Raf-1d

Mutant B-Raf(V600E)
MEK,MEK2
MEK,MEK2
Phase
Phase
Phase
Phase
MEK, MEK2
Phase I
Scio-469
Scio-323
TAK-715
PS540446
Indication/target population
II
I
II
I
II
I
I
II
II
Rheumatoid arthritis
Patients with coronary heart disease undergoing percutaneous coronary interventions;
neuropathic pain following nerve trauma
Rheumatoid arthritis, acute coronary
syndromes
Rheumatoid arthritis, multiple myeloma,
myelodysplastic syndromes
Inammatory disease
Inammatory and autoimmune diseases
Rheumatoid arthritis, metabolic disorders,
cardiovascular disease
Inammatory disease, cancer
Inammatory disease
AML, inammatory disease
Parkinsons disease, asthma
Parkinsons disease, Alzheimers disease
Advanced RCC
CRC, NSCLC, pancreatic cancer, melanoma,
ovarian cancer, AML, as well as other solid
and hematological malignancies
Locally advanced or metastatic melanoma
Cancer
Breast cancer, CRC, NSCLC, melanoma
NSCLC, melanoma, pancreatic cancer;
inammation
Abbreviations: AML, acute myelogenous leukemia; CRC, colorectal cancer; MEK, mitogen-activated protein kinase/extracellular signal-regulated
kinase kinase; NSCLC, non-small-cell lung cancer; RCC, renal cell cancer, aCompiled from studies cited in reviews that we have referenced in the
text, from company websites and from www.clinicaltrials.gov; AML bDiscontinued, cAlso inhibitory activity for VEGFR-2, PDGFR-b, Flt-3, c-Kit
and FGFR-1, dAlso inhibitory activity for VEGFR-2.
Oncogene

3295
(Figure 3) (Kolch, 2005). This complexity is best

represented at the level of Raf, where multiple signals
converge to regulate Raf activation. As described below,
a major activator of Raf kinases are the Ras small
GTPases. However, Raf activation is complex, with
Ras facilitating the plasma membrane association of
normally cytosolic Raf, where additional signaling
activities, including phosphorylation (e.g., p21-activated
protein kinases (PAK) serine/threonine and Src
family tyrosine kinases) and dephosphorylation (protein
phosphatase 2A) events are required to fully activate
the kinase function. Raf function is also regulated
by interaction with other proteins, including 14-3-3
proteins and heat shock protein 90 (Hsp90). Hence,
inhibitors of Raf and MEK are not likely to cause
similar consequences and show equivalent clinical
responses. These regulatory events also suggest
other approaches for antagonizing RafMEKERK
signaling.
Another layer of regulation of the RafMEKERK
cascade involves scaffolding proteins that help to
regulate the activity, specicity and spatial regulation
of MEK/ERK signaling in mammalian cells. These
include kinase suppressor of Ras 1 (KSR1), MEK
partner-1 (MP1), b-arrestins, IQ motif-containing
GTPase activating protein 1 (IQGAP1) and others
(Kolch, 2005). Of these, KSR has been one of the best
studied, and KSR function has been shown to be critical
for Ras activation of Raf. Mouse embryonic broblasts
(MEF) decient in KSR1 function showed impaired
Golgi
Late
Endosomes
MEK
Ras
Src
PAK
Sef
MEK1
ERK
IMP
Raf
RKIP
MP1
Hsp90
ERK1
KSR
MEK
Mos
Actin
ERK
MEK
MKP1
Plasma
Membrane
IQGAP1
ERK
Nuclear Elk-1, Fos, Myc, STAT3

Membrane Syk, CD120a, calnexin
Cytoskeletal paxillin, neurofilaments
Cytosolic - RSK
Figure 3 The RafMEKERK cascade is a core signaling

component of a complex signaling network. The ERK MAPK
cascade is regulated by a complex array of functionally diverse
proteins. In particular, Raf function is regulated by both positive
and negative signal inputs. In particular, scaffold proteins (e.g.,
KSR, Sef, MP1 and IQGAP1) bind to distinct subsets of
components of this cascade to create unique ERK MAPK signaling
complexes at specic and distinct subcellular locations that result in
preferential utilization of only some of the many possible ERK
substrates. This arrangement channels the unique input signals that
activate the pathway into the specic output signaling in distinct
spatio-temporal patterns. ERKs then phosphorylate multiple
targets, including nuclear, cytosolic, membrane-associated and
cytoskeletal substrates.
sensitivity to Ras transformation (Kortum and Lewis,

2004). Furthermore, antisense suppression of KSR1
expression was found to impair the growth of Ras
mutation-positive human tumor cells, supporting
KSR as a possible therapeutic target for blocking Ras
(Xing et al., 2003).
Ras family small GTPases are key upstream activators of

Raf
Ras proteins (H-, K- and N-Ras) function as a GDP/
GTP-regulated switch. GDP/GTP cycling is regulated
by guanine nucleotide exchange factors (RasGEFs; e.g.,
Sos) that promote formation of active Ras-GTP,
whereas GTPase-activating proteins (GAPs; e.g., NF1
neurobromin) stimulate GTP hydrolysis and formation
of inactive Ras-GDP (Mitin et al., 2005). In normal
quiescent cells, Ras is GDP-bound and inactive.
Extracellular stimuli (e.g., EGF) cause transient formation of the active, GTP-bound form of Ras. Activated
Ras-GTP binds to a spectrum of downstream effector
targets, of which the Raf kinases are the best characterized. Ras is mutationally activated in 30% of all cancers,
with pancreas (90%), colon (50%), thyroid (50%), lung
(30%) and melanoma (25%) having the highest prevalence (Malumbres and Barbacid, 2003). The mutant
Ras genes in human cancers encode mutated proteins
that harbor single amino-acid substitutions primarily at
residues G12 or Q61. Mutant Ras proteins are GAPinsensitive, rendering the proteins constitutively GTPbound and activated, leading to stimulus-independent,
persistent activation of downstream effectors, in particular, the RafMEKERK cascade (Figure 1).
Although there is considerable experimental evidence
that the RafMEKERK cascade is a critical mediator
of Ras-induced oncogenesis, recent studies have also
clearly demonstrated that Ras utilizes additional effectors to promote tumorigenesis (Repasky et al., 2004).
Currently, at least four other effector classes have
demonstrated roles in Ras transformation: the p110
catalytic subunits (p110a, b, g and d) of class I
phosphatidylinositol 3-kinases (PI3K), the Tiam1 Rac
small GTPase-specic GEF, the Ral small GTPasespecic GEFs (RalGDS, Rgl, Rgl2 and Rgl3) and
phospholipase C epsilon. The evidence for their
involvement and roles in Ras-mediated oncogenesis
has been summarized in recent reviews (Repasky et al.,
2004; Shaw and Cantley, 2006). The existence of nonRaf mechanisms of Ras-mediated oncogenesis prompts
the question of whether blocking the RafMEKERK
pathway alone be sufcient to effectively block oncogenic Ras function, or will concurrent inhibition of
multiple effector pathways be required? Alternatively, as
this protein kinase pathway is central to many signaling
networks beyond Ras, will blocking the RafMEKERK
pathway be too deleterious and result in signicant
normal cell toxicity? Instead, will blocking a subset of
downstream functions of the ERK pathway be a more
effective approach?
Oncogene

3296
The epidermal growth factor receptor: upstream

activation of Ras and the ERK cascade and a component
of a Ras-mediated autocrine growth loop
In addition to activating mutations of the Ras oncogene,
the ERK MAPK signaling pathway can also be
activated by perturbation of components upstream of
Ras. For example, aberrant overexpression or mutational activation of receptor tyrosine kinases (RTKs;
e.g., EGFR and HER2) can cause hyperactivation of
Ras leading to upregulated MAPK signaling (Lynch
et al., 2004; Stephens et al., 2004; Hynes and Lane,
2005). In particular, the EGFR is overexpressed or
mutationally activated in many human cancers (Grandis
and Sok, 2004). The regularity with which this signaling
cascade is activated suggests that it is critical in
oncogenesis and makes it an appealing pathway for
drug development. Another linkage between Ras and
the EGFR receptor is mediated by the upregulation of
expression of EGFR ligands by Ras signaling (Figure 1).
One important gene target of Ras activation involves
transcriptional activation of the gene for transforming
growth factor alpha (TGFa), a ligand for the EGFR.
Increased TGFa gene expression and secretion of TGFa
in turn causes persistent stimulation of the EGFR. The
upregulated expression and secretion of TGFa and
other EGFR ligands (e.g., heparin-binding EGF,
amphiregulin) has been observed in a wide variety of
Ras- or Raf-transformed cell types (McCarthy et al.,
1995; Gangarosa et al., 1997; Schulze et al., 2001). The
importance of this autocrine signaling loop for Ras
transformation has been demonstrated by the ability of
inhibitors of EGFR to block oncogenic Ras transformation. Additionally, the majority of Raf-induced changes
in gene expression were found to be dependent on
EGFR function (Schulze et al., 2004). Hence, the EGFR
can function both upstream, as well as downstream, of
Ras and the ERK MAPK cascade.
Inhibitors of the RafMEKERK cascade

Currently, inhibitors of the kinase function of Raf and
MEK represent the most studied and advanced
approaches for blocking ERK signaling, with several
inhibitors under evaluation in clinical trials and additional inhibitors in preclinical analyses (Table 1). To
date, no inhibitors of ERK1 or ERK2 have been
described.
Raf inhibitors
The three Raf kinases exhibit the same substrate
specicity, with MEK1 and MEK2 the only known
substrates. However, these highly related isoforms do
exhibit differences in regulation and biological function
(Wellbrock et al., 2004; Schreck and Rapp, 2006).
Furthermore, functions independent of MEK activation
have been described, although these activities remain
poorly characterized. Several structurally distinct classes
of compounds have been developed as potential Raf
Oncogene
kinase inhibitors (Smith et al., 2006)). In addition to

small molecule inhibitors of Raf kinase, other anti-Raf
efforts include the development of antisense inhibitors
of Raf expression, particularly ISIS-5132, a 20base
phosphorothioate DNA oligonucleotide that inhibits
c-Raf-1 protein expression. ISIS-5132 showed antitumor
activity in preclinical xenograph models and early
clinical trials (Monia et al., 1996; Cripps et al., 2002;
Tolcher et al., 2002; Oza et al., 2003). However, no
patient response or antitumor efcacy was seen in phase
II clinical trials (Cripps et al., 2002; Tolcher et al., 2002;
Oza et al., 2003) and it has been withdrawn from further
clinical evaluation. A related approach, using liposomeencapsulated antisense c-raf-1 oligonucleotide (Gokhale
et al., 2002), showed antitumor activity in xenograft
analyses (Mewani et al., 2004). Liposome entrapment
serves to protect the oligonucleotide from degradation
and to improve delivery. LErafAON has completed
initial phase I clinical trials where it was evaluated as
monotherapy and in combination with radiation or
chemotherapy (Rudin et al., 2004; Dritschilo et al.,
2006). Further clinical development of LErafAON is
ongoing.
Small molecule inhibitors of RasRaf interaction
(MCP1 and derivatives) have been described and shown
to have antitumor activity in cell culture studies (KatoStankiewicz et al., 2002). MCP1 was identied as an
inhibitor of Ras interaction with Raf in a yeast twohybrid-based screen. The exact mechanism by which
MCP1 functions is currently unclear and this information will be important for further clinical development
of this novel class of Raf inhibitors. Currently,
preclinical evaluation of MCP continues in human
tumor xenograft mouse models in combination with
other chemotherapeutic agents (Skobeleva et al., 2007).
Hsp90 functions as a chaperone that is required for
the stability and function of Raf and other oncogene
proteins, including HER2 and the Met RTK, as well as
steroid hormone receptors that include the androgen
and estrogen receptors (Neckers, 2006). The antitumor
activity of the antibiotic benzoquinone ansamycin
geldanamycin is due to binding to and promoting
Hsp90 degradation, and hence, indirectly inhibiting the
function of Raf and other client proteins by promoting
their proteosomal degradation. Interestingly, a recent
study found that Hsp90 is required for wild-type c-Raf-1
and A-Raf, but not B-Raf, stability (Grbovic et al.,
2006). However, mutant B-Raf showed dependency on
Hsp90 for function. Treatment of melanoma cells with
the geldanamycin analog 17-allylamino-17-demethoxygeldanamycin (17-AAG) caused degradation of mutant
B-Raf and inhibition of ERK, and showed antitumor
activity. Another study also found preferential sensitivity of mutant B-Raf to 17-AAG treatment (da Rocha
Dias et al., 2005). 17-AAG is currently in phase II
clinical trials (Sharp and Workman, 2006). Although
17-AAG has shown promising activities in preclinical
and clinical trials, the further clinical development may
be limited by problems with solubility, stability and
hepatotoxicity. Hsp90 inhibitors are relatively unique
antitumor agents in that they simultaneously inhibit

3297
multiple, functionally diverse, signaling components

that promote oncogenesis. Hence, the precise biomarker
for dening and monitoring antitumor response may be
complicated.
To date, the most successful anti-Raf inhibitor has
been sorafenib (tosylate salt of BAY 43-9006; Nexavar),
an orally available compound that received FDA
approval in 2005 for the use in advanced renal cell
carcinoma (RCC). Sorafenib is a bi-aryl urea compound
(Smith et al., 2006) that was originally developed as an
inhibitor of Raf-1 (Lyons et al., 2001). Subsequent
analysis revealed that sorafenib was a potent inhibitor of
both wild-type and mutant (e.g., V600E, the most
frequent mutation found in human cancers) B-Raf
kinases in vitro. Crystallographic analyses of sorafenib
complexed with the kinase domain of B-Raf showed
that the inhibitor bound to the ATP-binding pocket and
prevented kinase activation, thus preventing substrate
binding and phosphorylation (Wan et al., 2004).
Shortly after clinical analyses begin, it was revealed
that sorafenib also showed very potent activity for other
protein kinases in vitro and in vivo, in particular, for the
proangiogenic RTKs such as VEGFR-2, VEGFR-3,
PDGFR-b, Flt-3, c-Kit and FGFR-1 (Wilhelm et al.,
2004). Although sorafenib demonstrated multikinase
inhibition, it was also found to maintain some specicity, as it did not inhibit other protein kinases such as
MEK1, ERK1, EGFR, HER2 and others.
In cell culture and mouse models representing a wide
range of tumor cell types, sorafenib exhibited broad
antitumor activity and was associated with reduced
MEK and ERK activation, supporting the possibility
that its antitumor activity involves, in part, inhibition of
Raf (Wilhelm et al., 2004). However, despite its
association with reduced ERK activation, sorafenib
antitumor activity must also be a consequence of its
ability to inhibit angiogenesis-related kinases as well as
other non-Raf kinases. This possibility is supported by
the potent antitumor activity that was independent of
Ras or B-Raf mutation status that was seen with
sorafenib in xenograft studies.
Phase I clinical trials established sorafenib as a safe
and well-tolerated oral agent with skin rash and
diarrhea as the most common adverse effects (Awada
et al., 2005) (Moore et al., 2005a). Results from phase I
clinical trials suggested clinical activity in several
patients with RCC, resulting in subsequent clinical trials
focused on RCC. Ultimately, this effort led to a large
phase III clinical trial that enrolled more than 900
patients with advanced RCC, who previously failed
prior systemic therapy. The primary endpoint of this
study was improved survival. This trial met its surrogate
endpoint of signicantly longer progression-free survival
in the sorafenib arm compared to the placebo arm of the
study. In addition, sorafenib treatment doubled the
disease progression-free survival from 12 to 24 months
when compared to the placebo-control arm (Escudier
et al., 2005; Nexavar website has updated survival
data). In addition to RCC, several single agent and
combination clinical studies are ongoing in hepatocelluar carcinoma, non-small-cell lung cancer (NSCLC),
prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma and hematological malignancies
(Hahn and Stadler, 2006; Rini, 2006).
The results of clinical trial analyses of sorafenib have
not provided sufcient information to conclude that
inhibition of Raf provides a clinical value. As B-Raf
mutations are not seen in RCC, and as sorafenib is not a
potent Raf inhibitor, the antitumor activity seen may be
attributed to the antiangiogenic, rather than anti-Raf,
activity of sorafenib. Although preclinical cell culture
and mouse model analyses showed that continued
expression of mutant B-Raf is critical for melanoma
growth and tumorigenicity, phase II clinical trial
analyses with sorafenib observed little or no antitumor
activity when evaluated as monotherapy for advanced
melanomas (Eisen et al., 2006). Some clinical efcacy
had been seen for melanomas when sorafenib was used
in combination, with clinical trials ongoing for advanced
melanomas with sorafenib combination with bevacizumab or carboplatin and paclitaxel. However, a recently
completed phase III trial administering sorafenib or
placebo tablets in combination with carboplatin and
paclitaxel in patients with advanced melanoma found no
difference in the primary end point of improving
progression-free survival.
RAF265 (formerly CHIR-265) is another orally
bioavailable Raf inhibitor currently being investigated
in phase I clinical trials in locally advanced or metastatic melanoma (http://www.clinicaltrials.gov/ct/show/
NCT00304525). RAF265 inhibits all three Raf isoforms
as well as mutated B-Raf. Like sorafenib, RAF265 may
also have antiangiogenic activity through inhibition of
VEGFR2. PLX4032 is a potent and selective inhibitor
of mutant B-Raf that is currently in phase I clinical
evaluation. Other Raf inhibitors are also in preclinical
evaluation and should be entering clinical evaluation in
the near future.
The clinical success of sorafenib and another
approved multikinase inhibitor (sunitinib) has prompted
a debate regarding the advantages and disadvantages
of highly specic versus broad specicity inhibitors
(Sebolt-Leopold and English, 2006). As cancer is a
multistep process, requiring multiple alterations, it is expected that effective cancer treatment requires concurrent
activities that target different defects (Hanahan and
Weinberg, 2000). The greater success of combination
chemotherapy is consistent with this premise. The ability
to optimize the pharmacokinetics and pharmacodynamic properties of a single agent with multiple
activities is also a great advantage for the successful
clinical development of a drug. In the development of
sorafenib, the intention was the identication of a Raf
inhibitor, with the activity against other protein kinases
fortuitous and unplanned. Hence, this has complicated a
full understanding of the mechanism of action of
sorafenib and the importance of its anti-Raf activities
for its clinical efcacy. Therefore, whether blocking Raf
will be a clinically effective approach will require future
clinical evaluation of more specic and potent Raf
kinase inhibitors. The clinical success of highly selective
protein kinase inhibitors, in particular monoclonal
Oncogene

3298
antibody (mAb)-based drugs (e.g., trastuzumab, bevacizumab), demonstrates that there is clinical value for
both highly selective and multitargeted inhibitors.
MEK inhibitors
MEK1 and MEK2 are closely related dual-specicity
kinases, capable of phosphorylating both serine/threonine and tyrosine residues of their substrates ERK1 and
ERK2. They are the only known catalytic substrates of
Raf kinases. The fact that ERK is the only known
substrate of MEK, when coupled with the observation
that ERK is commonly activated in both tumor cell lines
and patient tumors, has fueled strong interest in
developing pharmacological inhibitors of MEK as a
means to block ERK activation (Hoshino et al., 1999).
In contrast to sorafenib, small molecule inhibitors of
MEK1/2 are highly specic protein kinase inhibitors.
Although the rst two MEK inhibitors, PD98059 and
U0126, were highly specic (Davies et al., 2000) they
lacked the pharmaceutical properties needed to be
successful clinical candidates. Nonetheless, these compounds have been invaluable academic research tools
for dissecting the MEKERK pathway and have
provided enormous insight into the importance of
ERK MAPK signaling in cancer (Cox and Der, 2002b;
Sebolt-Leopold and Herrera, 2004).
The rst MEK inhibitor to enter clinical trials was
CI-1040 (PD184352), an orally active, highly potent and
selective inhibitor of MEK1 and MEK2 (SeboltLeopold et al., 1999). Preclinical evaluation found that
CI-1040 inhibited the growth of human colon cancer
cells and human melanoma cells in athymic nude mice
(Sebolt-Leopold et al., 1999; Collisson et al., 2003).
Subsequent phase I and II clinical trials reported the
most common toxicities were mild skin rash, diarrhea
and fatigue (Lorusso et al., 2005) (Rinehart et al., 2004).
During the phase I trial, a partial response was seen in
one patient with pancreatic cancer and 25% of patients
with a variety of tumors had stable disease for greater
than 3 months (Lorusso et al., 2005). Tumor tissues
from treated patients showed signicant reduction in
activated phosphorylated ERK, indicating that the
target was inhibited. These encouraging results
prompted a phase II study in patients with advanced
NSCLC, breast cancer, colorectal cancer (CRC) and
pancreatic cancer. Unfortunately, the results of this trial
were negative and CI-1040 was determined to have poor
pharmacokinetic properties (Rinehart et al., 2004).
However, when considered together with the signicant
body of positive preclinical data as well as early
indications from the phase I trial, it is still believed that
MEK is a valid therapeutic target for the treatment of
cancer. Thus, two second generation MEK1/2-specic
inhibitors (PD325901 and ADZ6244) believed to have
superior pharmacological and biopharmaceutical properties have been developed and are currently in clinical
trials (Table 1).
In contrast to the majority of protein kinase
inhibitors, MEK inhibitors are non-ATP competitive
inhibitors, which may account for their highly selective
Oncogene
properties. Structural studies with an analog of CI-1040

in complex with MEK1 or MEK2 showed inhibitor
binding did not perturb ATP binding, and instead,
bound to a unique inhibitor binding pocket adjacent to
the ATP-binding site (Ohren et al., 2004). Inhibitor
binding locked MEK in a catalytically inactive conformation. This recognition of MEK sequences that are
not shared with other protein kinases, and their
association with an inactivate conformation, account
for MEK inhibitor target selectively.
PD0325901 is a derivative of CI-1040 where several
slight modications to the chemical structure have
resulted in more than a 50-fold increase in potency
against MEK1/2, improved bioavailability, and longer
duration of target suppression compared to CI-1040
(Sebolt-Leopold and Herrera, 2004). Antitumor activity
for PD0325901 was demonstrated for a variety of tumor
xenografts and this inhibitor is now being evaluated in
phase I/II clinical trials with a focus on tumors expected
to have activated ERK MAPK signaling (Thompson
and Lyons, 2005; Solit et al., 2006).
AZD6244 (ARRY-142886) is an orally bioavailable
benzimidazole derivative known to potently inhibit
MEK1/2 in vitro and in cell-based assays (Lyssikatos
et al., 2004; Yeh et al., 2004). Like other MEK
inhibitors, AZD6244 is ATP non-competitive. Preclinical evaluation of AZD6244 showed antitumor activity in
several human xenograft models including colon,
pancreas, breast, NSCLC and melanoma (Lee et al.,
2004). Additionally, AZD6244 antitumor activity was
found to correlate with suppression of ERK activation,
which further validates that its mechanism of action is
MEK-dependent. Results from preclinical analysis have
been extremely promising and thus AZD6244 has
moved into clinical development. Recently, initial results
of a rst in human dose-ranging study to assess the
pharmacokinetics, pharmacodynamics and toxicities of
AZD6244 in patients with advanced solid tumors
concluded that AZD6244 is well tolerated, and the most
common treatment-related adverse events were rash,
diarrhea, nausea, fatigue, peripheral edema and vomiting. The best clinical response seen in the 57 patients was
stable disease (19 patients at the end of cycle two; nine of
whom achieved stable disease for 5 months or greater).
The pharmacokinetic and pharmacodynamic analyses
showed good systemic exposure, which correlated with
high levels of ERK inhibition in peripheral blood mononuclear cells (PBMCs) (http://www.arraybiopharma.
com). AZD6244 is now being evaluated in multiple
phase II trials in a variety of solid tumors.
Solit et al., (2006) recently reported that B-Raf
mutant tumors are exquisitely sensitive to MEK
inhibition (Solit et al., 2006). In this study, the authors
used genetic and pharmacological (CI-1040 and
PD0325901) approaches to evaluate MEK dependence
in a variety of tumor types and found that human tumor
cell lines possessing mutant B-Raf were much more
sensitive to MEK inhibition than cells with wild-type
B-Raf or mutant K-Ras. In their xenograft models,
they found B-Raf mutation-positive tumor xenografts
had completely abrogated tumor growth whereas

3299
xenographs of Ras mutation-positive tumor cells were

only partially inhibited, perhaps reecting the fact that
Ras also utilizes non-Raf effector pathways to promote
oncogenesis. Whether Ras or B-Raf mutation status will
accurately identify the patients who will respond to
MEK inhibitor treatment, and whether suppression of
ERK activity is an accurate measure of drug inhibition
of MEK, are issues that remain to be determined.
In addition to small molecule inhibitors of MEK
kinase activation, bacterial toxins have been identied
that inhibit MEK function by unique biochemical
mechanisms. Anthrax lethal factor (LF) is a protease,
a component of Bacillus anthracis exotoxin, the Grampositive bacterium responsible for the disease anthrax.
LF, together with a second exotoxin component
(protective antigen), comprise lethal toxin (LeTx). LeTx
inactivates multiple MAPKKs, including MEK1 and
MEK2 by proteolytic cleavage and inactivation of
kinase function (Bodart et al., 2002). LeTx was shown
to block the transformed and tumorigenic growth of
Ras-transformed rodent broblasts (Duesbery et al.,
2001). LeTx also showed preferential inhibition of
growth of melanoma cell lines that harbored mutated
B-Raf and elevated ERK activity (Abi-Habib et al.,
2005). Yersinia outer protein J (YopJ) is another
bacterial toxin that inhibits MEK function. The
bacterial pathogen Yersinia pestis, the causative agent
for the plague (Black Death), uses a type III secretion
system to inject YopJ and other virulence factors into
target cells. YopJ was shown to bind directly to MEK1,
MEK2 and other MAPKKs and block their phosphorylation and activation (Orth et al., 1999). YopJ
functions as an acetyltransferase, using acetyl-coenzyme
A (CoA) to modify the critical serine and threonine
residues in the activation loop of MEKs and thereby
blocking MAPKKK phosphorylation and activation
(Mukherjee et al., 2006). YopJ also causes acetylation of
a threonine residue in the activation loop of inhibitor of
nuclear Factor kappa B (NF-kB) (IkB) kinase b,
preventing its phosphorylation and inactivation of
IkB, thus preventing nuclear translocation and activation of the NF-kB transcription factor. As inhibition of
NF-kB also has an antitumor consequences (Karin,
2006), YopJ can inhibit concurrently at least two
important pathways that promote oncogenesis. The
unique biochemical activities of these bacterial proteins
may identify novel approaches for blocking MEKERK
signaling.
Inhibitors of Ras
As the most commonly mutated oncogene in cancer,
Ras has been thought of as the Holy Grail of cancer
drug development (Cox and Der, 2002b). As the key
biochemical defect of mutated Ras proteins is the GAPinsensitivity and persistent GTP binding, early efforts
attempted to develop GAP-based approaches to reactivate the intrinsic GTP hydrolysis activity or to
antagonize GTP binding, but with no success reported
in these efforts. Instead, over the years two main

strategies have been vigorously pursued to identify
anti-Ras inhibitors. As described above, the rst
approach has been the development of inhibitors of
Ras downstream effector signaling, with efforts focused
on the ERK MAPK pathway. The second approach has
focused in blocking the post-translational modications
that promote Ras membrane association.
For proper signaling, Ras proteins must be localized
to the inner surface of the plasma membrane where they
interact with upstream activators (receptor tyrosine
kinases) and downstream effectors. Ras proteins are
synthesized as inactive, cytosolic proteins that quickly
undergo a series of post-translational modications,
which targets Ras to the plasma membrane (Figure 4)
(Basso et al., 2006). This series of modications is
initiated by enzymes that recognize the Ras carboxylterminal CAAX tetrapeptide (C cysteine, A aliphatic
amino acid, and X terminal amino acid) motif. The
rst step is catalysed by the cytosolic farnesyltransferase (FTase) enzyme and results in the covalent
addition of a farnesyl isoprenoid lipid to the cysteine
residue of the CAAX sequence. Next, the AAX peptide
is cleaved by the Ras converting enzyme 1 (Rce1)
endoprotease. Finally, isoprenylcysteine-O-carboxyl
methyltransferase (ICMT) covalently attaches a methyl
group to the farnesylated cysteine residue (Sebti and
Der, 2003). Both Rce1 and ICMT are associated with
the endoplasmic reticulum.
Each modication is important for increasing Ras
afnity for membranes and inhibition of the initial
CAAX-signaled processing step (farnesylation) has been
shown to inhibit all subsequent steps. With this knowledge, drug discovery efforts have focused on the
development of FTase inhibitors (FTIs) in hopes that
they would render Ras completely cytosolic and
inactive. Although FTIs have shown some success in
treating hematologic cancers, their use as Ras inhibitors
has been complicated by the fact that K-Ras and N-Ras
(the Ras isoforms commonly mutated in cancer) undergo alternative prenylation by a related enzyme, geranylgeranyltransferase I (GGTaseI), when FTase
activity is inhibited by FTIs (Figure 4) (Sebti and Der,
2003; Rowinsky, 2006). Hence, in contrast to popular
perception, FTIs are not effective inhibitors of Ras
function. Nevertheless, although FTIs are no longer
considered Ras inhibitors (except for H-Ras), they have
continued to progress through preclinical and clinical
development, and preclinical studies are focused on
identifying the true targets that contribute to the
antitumor activities of FTIs. To date, many potent
and specic FTIs have been developed, with SCH-66336
(lonafarnib; Sarasar) and R115777 (tipifarnib; Zarnestra) the most advanced and are currently in phase II/III
clinical trials for hematologic and other cancers (Lancet
et al., 2007). Finally, inhibitors of GGTaseI have also
been considered, to block the activities of K-Ras and
N-Ras when treated with FTIs.
Initial evaluation of the importance of each of the three
post-translational modications of Ras concluded that
inhibition of AAX proteolysis or carboxylmethylation
Oncogene

3300
Normal Ras Processing
PM
Alternative K/N-Ras Processing

PM
Ras
-C-OMe
ICMT
ER
-C-OMe
Ras
-C
Ras
-CAAX
ICMT
Ras
-C
Ras
-CAAX
ER
Rce1
Rce1
FTI
FTase
Cytosol
Ras
Ras
FTase
-CAAX
Farnesyl isoprenoid
Cytosol
GGTaseI
Ras
-CAAX
Geranylgeranyl isoprenoid
Figure 4 Post-translational processing of Ras. Normal Ras

processing Under normal physiological conditions Ras undergoes
three CAAX-motif signaled post-translational modications catalysed by the cytosolic farnesyltransferase (FTase), which catalyses
covalent addition of the C15 farnesyl isoprenoid. This modication
is followed by reactions catalysed by the endoplasmic reticulum
(ER)-associated Ras converting enzyme 1 (Rce1) endoprotease and
isoprenylcysteine-O-carboxyl methyltransferase (ICMT). The
CAAX modications, together with a second membrane targeting
signal upstream of the CAAX motif (palmitoylated cyteines, for HRas, N-Ras and K-Ras4A; lysine-rich sequence, for K-Ras4B).
Alternative K/N-Ras processing When FTase activity is blocked
by farnesyltransferase inhibitors (FTIs), N-Ras and K-Ras (the
Ras isoforms most commonly mutated in cancer) will undergo
alternative prenylation by the related geranylgeranyltransferase I
(GGTase) enzyme, which adds the more hydrophobic C20
geranylgeranyl isoprenoid to the cysteine residue of the CAAX
motif. As alternatively prenylated Ras retains normal membrane
association and function, FTIs fail to block the function of these
Ras isoforms. H-Ras does not undergo alternative prenylation, and
consequently, its function is effectively blocked by FTI treatment.
was not sufcient to fully block Ras transformation

and thus attention was focused on developing inhibitors
of FTase (Cox and Der, 2002a). However, with the
realization that FTIs are not effective anti-Ras drugs,
attention has shifted back to evaluating Rce1 and ICMT
as targets for inhibiting Ras membrane association and
localization (Clarke and Tamanoi, 2004; Winter-Vann
and Casey, 2005). Studies by Young, Casey and coworkers found that conditional deletion of either Rce1
or ICMT in broblasts derived from Rce1- or ICMTdecient MEFs impaired their sensitivity to K-Rasmediated transformation. Although knockout of Rce1
or ICMT function was critical for mouse development,
lack of Rce1 or ICMT expression was not essential for
the growth of adult tissue or for MEFs in cell culture
(Kim et al., 1999; Bergo et al., 2000). When mutated
K-Ras(12V) was expressed ectopically in broblasts
isolated from Rce1- or ICMT-decient mouse embryos,
K-Ras membrane association was partially impaired,
Ras activation of ERK was reduced, and Ras-mediated
growth transformation was impaired (Bergo et al., 2001,
2002, 2004).
Further evidence for the importance of ICMT in
Ras transformation came from studies by Casey and
co-workers, who found that the anti-neoplastic drug
Oncogene
methotrexate, an inhibitor of DNA synthesis, may also

act, in part, by inhibition of ICMT to block Ras transformation (Winter-Vann et al., 2003). Furthermore,
this group recently reported the identication of a selective small-molecule inhibitor of ICMT, 2-(5-(3-methylphenyl)-1-octyl-1H-indol-3-yl)acetamide (cysmethynil)
(Winter-Vann and Casey, 2005). Early preclinical
evaluation indicated that cysmethynil treatment inhibited cell growth in an ICMT-dependent fashion,
resulted in cytosolic mislocalization of Ras proteins
in MDCK cells and blocked EGFR-mediated signaling. In addition, cysmethynil treatment was shown to
inhibit anchorage-independent growth of DKOB8 cells
(Winter-Vann and Casey, 2005).
Although an obvious concern with Rce1 and Icmt
inhibitors is the fact that the number of substrates for
these enzymes is extensive, with several hundred CAAXterminating proteins that are known or putative
substrates for FTase or GGTaseI (Reid et al., 2004),
the fact that a number of these CAAX-terminating
proteins also display functions in oncogenesis (e.g.,
Rheb, Ral, RhoC and Rac1b) also suggest that their
multi-targeted actions may also be advantageous for
cancer treatment. However, it is possible that ICMTmediated methylation may be critical for the function of
only farnesylated proteins, thus greatly reducing the
number of targets for ICMT inhibitors (Michaelson
et al., 2005). Finally, ICMT-mediated methylation is
also needed for the function of a subset of Rab family
small GTPases, regulators of vesicular trafcking
(Leung et al., 2006), thus expanding the candidate
targets for ICMT inhibitors beyond CAAX-terminating
proteins. Some Rab proteins have also been implicated
in oncogenesis (Cheng et al., 2005).
Inhibitors of EGFR signaling

The EGFR family consists of four transmembrane
receptors: EGFR (HER1/erbB-1), HER2 (erbB-2/neu),
HER3 (erbB-3) and HER4 (erbB-4) (Mendelsohn and
Baselga, 2006; Scaltriti and Baselga, 2006). Multiple
ligands bind to and activate EGFR, HER3 and HER4.
Ligands for EGFR include EGF, TGFa, HB-EGF,
betacellulin and epiregulin. Ligand binding results in the
formation of receptor homodomers and heterdimers
leading to subsequent receptor activation and ultimately
the activation of downstream signal transduction pathways (Yarden, 2001; Wiley, 2003).
The best characterized cytoplasmic signaling pathway
activated by the EGFR is the ERK MAPK pathway,
and ERK activation has been utilized as a biomarker for
EGFR inhibitor action. Additional EGFR-activated
pathways include the PI3K and AKT serine/threonine
kinase, signal transducer and activator of transcription
(STAT), as well as protein kinase C and phospholipase
D pathways (Citri et al., 2003). EGFR activation of
these pathways results in enhanced proliferation, angiogenesis, invasion and metastasis, as well as inhibition of apoptosis (Jimeno and Hidalgo 2006). Aberrant

3301
activation of the EGFR signaling commonly occurs in

cancer (NSCLC, CRC, breast cancer, gastric cancer and
others), and multiple mechanisms describing its activation have been reported including EGFR overexpression,
EGFR gene amplication, acquisition of activating
mutations and overexpression of EGFR ligands (Baselga
and Arteaga, 2005).
Two strategies to inhibit EGFR signaling have been
successfully developed and include mAbs directed
against the extracellular domain of EGFR (Table 3)
and small molecule tyrosine kinase inhibitors (TKIs) of
the intracellular tyrosine kinase domain (Table 4). By
blocking ligandreceptor interactions, mAbs inhibit
receptor homo- and heterodimerization resulting in
receptor internalization and inhibition of EGFR signaling pathways. Additionally, the clinical activity of some
EGFR mAbs may also be attributed to their ability to
stimulate an immune response. Alternatively, EGFR
receptor TKIs bind to the intracellular tyrosine kinase
domain preventing ATP binding and receptor activation, thus blocking EGFR activation of ERK and other
signaling pathways. Mab and small molecule inhibitors
of EGFR possess shared and distinct features that
distinguish their mechanistic and clinical activities
(Table 5). Hence, it is believed that their use in
combination may act synergistically and improve antiEGFR therapy.
As with other signal-transduction inhibitors, EGFR
inhibitors lack the severe myelosuppressive toxicities
seen with conventional cytotoxic drugs. Toxicities are
Table 3
most evident in tissues that are dependent on EGFR

function, in particular the skin (Lacouture, 2006). This
includes a papulopustular rash that affects the face and
upper trunk, abnormalities in hair growth, and dry and
itchy skin. There is incomplete evidence that the strength
of skin rash may be a good indication of drug activity
and possibly favorable patient response and survival
(Perez-Soler and Saltz, 2005).
Several EGFR mAbs have been developed that
recognize the extracellular domain of the EGFR
(Figure 5). However, as they recognize distinct sequences and vary in binding afnities and composition
(Figure 5), they also vary in their biological activities.
The rst FDA-approved EGFR mAb was cetuximab, a
chimeric monoclonal IgG1 antibody initially approved for
use in combination with irinotecan for the treatment of
EGFR-detectable metastatic CRC refractory to irinotecan, as well as monotherapy for the treatment of EGFRdetectable metastatic CRC in patients intolerant to
irinotecan (Cunningham et al., 2004). More recently,
cetuximab has received additional approval for use in
combination with radiation to treat inoperable squamous
cell cancer of the head and neck (Bonner et al., 2006).
Finally, although EGFR expression was an initial basis for
patient selection, patient response has not correlated with
the degree of overexpression. Thus, a reliable biomarker
for dening patient response remains elusive.
Although cetuximab has proven to be an effective
treatment in the aforementioned indications and continues to be investigated for use in other tumor types,
Monoclonal antibody EGFR-directed therapeutic agentsa
Compound
Generic (Trade)
name
Characteristics
Company
Status
Tumors
IMC-C225
Cetuximab
(Erbitux)
Chimeric IgG1
Imclone/Bristol-Myers
Squibb/Merck KGaA
Approved
CRC, SCCHN
ABX-EGF
Panitumumab
(Vectibix)
Fully human IgG2
Abgenix/Amgen
Phase IIIII Pancreatic cancer, NSCLC, breast

cancer, and other solid tumors
Approved
CRC
Phase IIII
EMD-72000
h-R3
Matuzumab
Nimotuzumab
(TheraCIM)
mAb 2F8
Zalutumumab
(HuMax-EGFr)
rhuMAb 2C4 Pertuzumab
(Omnitarg)
ch806
MDX-214
Phase III
NSCLC, rectal cancer, bladder

cancer, breast cancer and ovarian
cancer
Various solid tumors including
NSCLC, cervical cancer and
gastric cancer
Various solid tumors with a focus
in NSCLC
SCCHN, NSCLC
Phase II
Breast cancer, ovarian cancer
Phase I
Advanced solid tumors
Phase II
Humanized IgG1
EMD Pharmaceuticals/ Phase II

Merck KGaA
Humanized IgG1
YM Biosciences/
CIMAB S.A.
Medarex/Genmab
Fully human IgG1

Humanized mAb inhibits
HER2 dimerization with other
HER family members
Chimeric anti-EGFR
vIIIb IgG2
Genentech/HoffmannLa Roche
Ludwig Institute/
Life Science
Pharmaceuticals
EGF fusion with Fab fragment of Medarex
a fully human anti-CD89
(IgA FcR) mAbc
Phase III
Abbreviations: CRC, colorectal cancer; SCCHN, squamous cell carcinoma of the head and neck; NSCLC, non-small-cell lung cancer. aCompiled
from studies cited in reviews that we have referenced in the text, from company websites, and from www.clinicaltrials.gov, bGenerated against the
deletion mutant of EGFR lacking exons 27 of the extracellular domain, cActivation of cytotoxic neutrophils via CD89 stimulation.
Oncogene

3302
Table 4 Small molecule tyrosine kinase inhibitor EGFR-directed therapeutic agentsa
Compound
Generic
(Trade) name
Characteristics
Company
Status
Tumors
ZD1839
Getinib
(Iressa)
EGFR-specic; reversible quinazoline
AstraZeneca
Approvedb
NSCLC
OSI-774
Erlotinib
(Tarceva)
GW572016/GW2016 Lapatinib
(Tykerb)
EKB-569
PKI-166/PKI116
ARRY-334543
BMS-599626
CI-1033
HKI-272
AEE788
Canertinib
ZD6474
Vandetanib
(Zactima)
XL647
Phase IIIII Breast cancer, hepatocellular

carcinoma, esophageal cancer,
SCCHN, brain and CNS
tumors, and other solid tumors
EGFR-specic, reversible quinazoline OSI Pharmaceu- Approved
NSCLC, pancreatic cancer
ticals/Genentech/
Roche
Phase IIIII CRC, brain and CNS tumors,
bladder cancer, RCC, SSCHN,
prostate cancer and other solid
tumors.
EGFR, HER2; reversible quinazoline GlaxoSmithKPhase III
Breast cancer, RCC, head and
line
neck cancer
EGFR, HER2; irreversible quinazoline Wyeth
Phase III
Breast cancer, NSCLC
EGFR, HER2 pyrrolotriazine
Novartis
Phase I
Prostate, RCC
EGFR, HER2 reversible
Array
Phase I
Advanced tumors
pyridopyrimidine
Biopharma
Pan-HER,reversible pyrrolotriazine
Bristol-Myers
Phase I
Squibb
Pan-HER, irreversible quinazoline
Pzer
Phase I/II
NSCLC, breast cancer
Pan-HER, irreversible quinazoline
Wyeth
Phase II
breast cancer, NSCLC
EGFR, HER2 and VEGFR2;
Novartis
Phase I
reversible pyrrolotriazine
EGFR, VEGFR2, RET; reversible
AstraZeneca
Phase III
NSCLC, medullary thyroid
quinazoline
cancer
EGFR, HER2, VEGFR2, EphB4
Exelixis
Phase II
NSCLC
Abbreviations: CRC, colorectal cancer; CNS, central nervous system; NSCLC, non-small-cell lung cancer; SCCHN, squamous cell carcinoma of
the head and neck; RCC, renal cell cancer. aCompiled studies cited in reviews that we have referenced in the text, from company websites and from
www.clinicaltrials.gov; bCurrently approved for patients with past treatment; not available to new patients.
Table 5
Properties of monoclonal antibody and small molecule tyrosine kinase inhibitors of EGFR
Characteristics
Monoclonal antibodies
Tyrosine kinase inhibitors
mAb versus TKI
Mechanism of action
Blockade of EGFR ligand binding,

receptor internalization, Inhibition of
receptor dimerization, stimulation of
antibody-dependent immune response
(IgG1 isotypes only)
Multiple mechanisms of mAb may lead

to enhanced antitumor activity and more
robust EGFR inhibition, whereas
TKIs can inhibit ligand-independent
constitutively activated EGFR.
Route of
administration
Frequency of
administration
Size
Specicity
Intravenous
Binding to or adjacent to ATPbinding pocket to inhibit kinase

activity and phosphorylation of
downstream substrates, preventing
activation of the ERK MAPK and
other signaling pathways.
Oral
Weekly to every 3 weeks
Daily
Large and bulky

Highly specic, no off-target activities
Small molecule
Likely to have off-target activity,
ranging from limited to very
signicant.
Yes
Effectiveness against
constitutively active
EGFR
No
Common toxicities
Rash, fatigue, infusion reactions

(for some)
Rash, fatigue and diarrhea
Oral administration of TKIs decreases

time in the clinic
mAbs have longer elimination halife,
allowing for less frequent dosing
TKIs have better tumor penetration.
mAb specicity may reduce toxicity, while
TKI off-target activity may enhance
anti-tumor activity
TKIs are active on mutated, constitutively
active EGFRs that no longer rely on
ligand activation; mAbs will not be
effective in these tumors.
Toxicity proles are similar with the
EGFR-specic mAb and TKIs; however,
the pan-HER and TKIs with additional
antiangeogenesis activities may exhibit
greater toxicities.
Abbreviations: EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase.
one of its major drawbacks is the associated risk of

anaphylactic reactions during cetuximab infusion
(Cunningham et al., 2004; Bonner et al., 2006). This
Oncogene
has led to the development of two humanized EGFR

(matuzumab and nimotuzumab) and two fully human
(panitumumab and zalutumumab) mAbs.

3303
Mouse (-o mab)
Chimeric (-xi mab)
Humanize (-zu mab)
Human (-u mab)
100% Mouse
225
~33% Mouse
Cetuximab
~10% Mouse
Matuzumab
Nimotuzumab
100% Human
Panitumumab
Zalutumumab
Figure 5 EGFR monoclonal antibodies. Nomenclature of mAbs has been devised for assigning generic or nonproprietary names. All
monoclonal antibodies end with the sufx -mab, whereas different inxes are used to differentiate the agents depending on the
structure and function of the therapeutic agent. The inx immediately preceding the -mab sufx denotes the animal origin of the
antibody (o, mouse; xi, chimera; zu, humanized; u, human). The rst mAbs were produced in mice and are recognized as foreign by the
human immune system (e.g., 225), which can lead to increased clearance and allergic/anaphylactic reactions. Therefore, sequences that
comprise the constant regions of the antibody, which are not involved in epitope recognition, may be replaced with human sequences,
to create mAbs that are chimeric mousehuman proteins (e.g., IMC-C225 was derived from 225) or humanized mAbs. Recently, the
use of transgenic mice with human immunoglobulin heavy and light chain coding sequences (e.g., XenoMouse) has facilitated the
generation of fully human mAbs. The inx preceding the source of the antibodies refers to the disease condition the agent is used to
treat. Most of these consist of a consonant, vowel and another consonant. To simplify the naming scheme, the nal consonant is
commonly dropped if the following inx begins with a consonant (such as -zu- or -xi-). For mAbs used for the treatment of cancer
-tu(m)- is used as the inx to designate tumor (* designates EGFR mAbs that are FDA approved).
Panitumumab (Vectibix, Amgen, Inc.) was approved

in 2006 for the treatment of patients with EGFR
expressing, metastatic CRC with disease progression
on or following uoropyrimidine-, oxaliplatin- and
irinotecan-containing chemotherapy regimens. Panitumumab is a fully humanized mAb that is similar to
cetuximab except that panitumumab is an IgG2 antibody
that does not elicit antibody-dependent cell-mediated
cytotoxicity (ADCC) (Yang et al., 1999). In the pivotal
phase III trial that led to its approval, panitumumab treatment signicantly improved progression-free
survival and resulted in an 8% overall response rate
(Gibson et al., 2006). The most common toxicities seen
include rash, fatigue, hypomagnesemia, abdominal pain
and diarrhea. As expected with a fully humanized mAb,
no grade 3/4 infusion-related reactions were seen in
this trial.
Matuzumab is a humanized IgG1 anti-EGFR mAb.
As a humanized mAb matuzumab, like panitumumab, is
expected to have reduced infusion-related anaphylactic
reactions. Furthermore, as an IgG1 antibody matuzumab-induced potent ADCC against tumor cells in vivo,
which distinguishes matuzumab from the recently
approved panitumumab (Bier et al., 1998). Although
results from several phase II trials are expected within
the next year, initial phase I trials have shown
matuzumab is well tolerated, with rash and diarrhea
the most common toxicities. In these trials, evaluation of
both tumor tissue and skin biopsies determined that
matuzumab inhibited phosphorylation of EGFR, ERK
and AKT (Tabernero et al., 2003; Salazar et al., 2004;
Vanhoefer et al., 2004; Doi et al., 2005). In phase I trials,
activity has been evaluated in colorectal, cervical and
esophageal cancers and in squamous cell cancer of the
head and neck. Current therapeutic targets in phase II

trials include cervical and gastric cancers, and NSCLC.
Pertuzumab is a novel mAb with a unique mechanism
of action designed to inhibit the heterodimerization of
HER2 with EGFR and other HERs. Similar to other
EGFR mAb, the most common toxicities include rash
and diarrhea (Agus et al., 2005). To date several phase II
trials evaluating pertuzumab have been completed.
Initial results evaluating pertuzumab as monotherapy
in metastatic breast cancer and hormone-refractory
prostate cancer were not promising as there was limited
evidence of activity in breast cancer patients and no
prostate-specic antigen responses in the prostate cancer
(Cortes et al., 2005; de Bono et al., 2005). However,
more promising results in refractory or recurrent
ovarian cancer have been seen in which clinical activity
was seen in 15% of patients (Gordon et al., 2005).
Although single-agent use of pertuzumab has shown
limited clinical activity, our experience with targetedtherapies along with preclinical xenograft data suggest
that pertuzumab may have greater clinical benet when
used in combination with other therapies (Friess et al.,
2005). Thus, current phase II trials of pertuzumab are
focused on combination therapy, including a study of
pertuzumab in combination with erlotinib in patients
with locally advanced or metastatic NSCLC.
Two EGFR small molecule kinase inhibitors have
received approval for use in NSCLC (Table 4). The rst
was getinib, which received accelerated approval as
third-line monotherapy in NSCLC based on its 12%
response rate and 43% rate of tumor control. However,
getinib was subsequently evaluated in a large phase III
trial comparing getinib to placebo in NSCLC patients
who had failed previous treatment regimens, and results
Oncogene

3304
from this trial showed no improvement in survival

compared to placebo resulting in a FDA-mandated
labeling change. Currently, getinib is only approved for
the treatment of cancer patients who have previously
received and beneted from treatment. Ongoing clinical
trials are evaluating the use of getinib for the treatment
of other cancers.
As apposed to getinib, erlotinib has demonstrated
improved survival in a randomized phase III placebocontrolled trial. Although the overall response rate was
only 9% in the erlotinib arm, erlotinib prolonged overall
survival (6.7 vs 4.7 months) and increased 1-year
survival (31 vs 22%). These results led to FDA approval
in 2004. More recently, erlotinib has also gained FDA
approval for use in combination with gemcitabine for
the rst-line treatment of patients with locally advanced,
unresectable or metastatic pancreatic cancer (Moore
et al., 2005b).
As is seen with some recent mAb-based EGFR
strategies (e.g., zatumumab), newer generation TKIs
are now being developed to inhibit multiple RTKs
(Table 4). In particular, laptinib (Tykerb) is a dual
EGFR and HER2 tyrosine kinase inhibitor that has
been extensively evaluated in multiple solid tumors
including breast, NSCLC, CRC, head and neck cancer,
hepatocellular carcinoma and billary carcinoma (Nelson
and Dolder, 2006). Initial evaluation of this compound
revealed diarrhea and skin rash as the most common
toxicities (Nelson and Dolder, 2006). Although the
clinical activity of this agent has been evaluated in
multiple tumor types, the main focus has been in the
treatment of breast cancer a disease known to rely on
both HER-2 and EGFR signaling. Numerous phase I
and II clinical trials have been reported, including
several looking at the use of lapatinib in refractory
metastatic breast cancer and in rst line treatment of
advanced breast cancer (Moy and Goss, 2006). An
international, multicenter, randomized, open label phase
III trial in patients with documented HER2 overexpressing refractory advanced or metastatic breast
cancer treated with lapatinib in combination with
capecitabine versus capecitabine alone was stopped
after the interim analysis. Of the 321 evaluable patients,
161 were treated in the combination arm and 160 in the
monotherapy arm. Median time to progression in the
combination arm was 8.5 months, compared with 4.5
months in the capecitabine alone arm (Tykerb at ASCO
2006, http://www.gsk.com/investors/presentations_webcasts.html). Although these data need to mature so that
an overall survival advantage can be determined, this
compound is likely to become part of the standard of
care in breast cancer and possibly other tumor types as
well. The results of this phase III trial are so promising
that it has been made available through an Expanded
Access Protocol of lapatinib combined with capecitabine in metastatic breast cancer in a non-randomized,
open label, uncontrolled trial.
In addition to lapatinib, EKB-569 and PKI-166 are
two additional dual EGFR/HER2 inhibitors in clinical
trials (Table 4). Furthermore, canertinib (CI-1033),
BMS-599626 and HKI-272 are pan-HER inhibitors,
Oncogene
whereas several EGFR inhibitors also inhibit RTKs

involved in tumor angiogenesis. Although targeting
multiple protein kinases may enhance antitumor activity,
it may also result in greater normal cell toxicity.
Biomarkers: verifying target inhibition and predicting

patient response
The important issues that MAPK drug discovery faces
are those that have accompanied the new era of targetbased anticancer drug discovery. Historically, oncology
drug development has focused on determining the
maximum tolerated dose (MTD), safety prole and
efcacy of a compound whereas mechanistic studies and
efforts to preselect responders were almost never
undertaken. In fact, the mechanisms of action that
accounts for the antitumor activity of many of our
commonly used chemotherapeutic agents used today are
still poorly understood. However, with an ever-growing
understanding of cancer biology, new targeted-based or
mechanism-based therapies are making there way into
the clinic. These compounds differ greatly from the
majority of the conventional cytotoxic antineoplastic
drugs in that they are rationally designed to mechanistically inhibit a particular protein target that is
hypothesized to be critical for cancer growth and
progression. Although these novel agents offer great
promise for signicant improvements in cancer chemotherapy, they have also brought researchers and
clinicians several new challenges and issues for drug
discovery. First, the identication of useful and predictive biomarkers has become an important endeavor.
The development of methodology to accurately verify
that a targeted therapy is actually blocking the function
of its target is crucial for effective clinical evaluation. If
the compound efciently inhibits the target but has no
clinical response, then it allows researchers to conclude
that the target is not important for that particular
cancer. For example, will a reduction in ERK activation
be a reliable marker for monitoring the effectiveness of
inhibitors of Raf or MEK to block target function? Will
ERK inhibition correlate with patient response?
A related issue involves the tissue source for
monitoring drug action. The most direct and relevant
measure of target inhibition is to use pre- and posttreatment tumor samples to measure the biomarker of
interest. The obvious limitation of this approach is that
tumor tissue is not readily accessible in many types of
cancer and putting patients through multiple biopsies is
not reasonable. Therefore, other methods to measure
target inhibition have been devised and include the use
of surrogate tissues such as PBMCs, skin and buccal
mucosa (Parulekar and Eisenhauer, 2004). However,
while feasible, whether drug inhibition in the surrogate
tissue accurately reects drug activity in the tumor
remains an important unresolved issue.
A second crucial issue is the identication of genetic
or biochemical markers that dene the patient population that will respond to inhibitor treatment. Despite

3305
their target-based development, patient response to

signal transduction inhibitors remains disappointingly
poor and modest. For example, EGFR is overexpressed
in 5080% of NSCLCs yet initial clinical trials evaluating EGFR TKIs found only a 10% response rate.
Furthermore, early correlative studies were unable to
establish a positive correlation between phospho-EGFR
immunohistochemistry and response, thus complicating
our ability to predict which patients will respond.
Eventually, retrospective analysis revealed the presence
of EGFR mutations in the majority of responders thus
identifying a molecular marker with a high-predictive
value of response (Lynch et al., 2004). However, more
recent analyses have found that EGFR mutation status
may not be as strongly associated with patient response
as initially believed 17045403 (Jimeno and Hidalgo,
2006). Additionally, repeated studies have shown that
patients with K-Ras mutations are resistant to EGFR
inhibition in both single-agent and combined therapy
regimens (Eberhard et al., 2005; Pao et al., 2005). To
further complicate patient selection, recent correlative
analysis suggests that in addition to EGFR/K-Ras
mutational analysis, EGFR gene amplication and
protein expression may also serve as important molecular markers (Cappuzzo et al., 2005). Specically,
EGFR mutations may be predictive of response,
whereas EGFR gene amplication and protein expression may be better markers of survival. An important
direction in this area of research is the determination of
gene array proles that may be more effective at
predicting patient response to a particular drug.
Finally, as the successful application of Raf or MEK
inhibitors will almost certainly require their use in
combination with other drugs, what other signaling
inhibitors or conventional cytotoxic drugs should be
used? As the potential combinations are daunting,
effective preclinical analyses are needed to better focus
clinical trial design. Will our current preclinical cell
culture or mouse models allow reliable determination of
the best combination approaches? Although still the
standard of the pharmaceutical industry, the value of
human tumor xenograft mouse models in predicting
drug activity in the patient remains a hotly debated issue
(Sausville and Burger, 2006). Although the new generation of genetically engineered mouse models are
optimistically believed to provide more accurate systems
for anticancer agent evaluation, this remains to be
validated (Sharpless and Depinho, 2006). With limited
patient populations for phase I/II clinical trials, and
limited research and development budgets, this remains
an important limitation in anticancer drug discovery.
Conclusions and future directions

The considerable genetic and experimental observations
provide strong validation that inhibitors of the ERK
MAPK cascade will provide effective antineoplastic
agents for the treatment of a wild range of human
cancers, including those where our current regimen of
drugs are disappointingly ineffective. Many inhibitors of
EGFR, Ras, Raf and MEK have been developed that
target different components of ERK signaling, with a
handful of agents already approved and added to our
expanding repertoire of anticancer agents. However,
many questions and issues remain, and whether
inhibitors of ERK signaling will provide drugs that
signicantly advance cancer treatment is far from
certain. As these efforts continue, in parallel, research
efforts have also revealed a considerably greater
complexity to the once simple linear RafMEKERK
signaling cascade. These complexities suggest that
targeting this pathway will not be as straightforward
as once imagined. It has become evident that cancer cell
resistance will limit the effectiveness of target-based
signal-transduction inhibitors as it has with conventional cytotoxic anticancer drugs. Therefore, developing
mechanistically distinct inhibitors of the same targets
will be essential. Additionally, as the clinical analyses of
these inhibitors progress, in parallel, we have also
developed a greater appreciation of the complex biology
and genetics of the cancer cell, and a better understanding
of how clinical trial design can be improved. Hence, our
learning curve in the development of target-based anticancer drugs is steep and we are in the very early part of
that curve. Despite the continued twists and turns and
bumps in the path of drug discovery, optimism remains
cautiously high that we are still heading in the right
direction. Finally, as reected in current efforts, cell
surface available molecules and protein kinases continue
to be the most favored targets for anticancer drug
discovery. Perhaps an important emphasis for future
MAPK drug discovery should be to consider targets that
are not classically considered attractive and druggable
targets (Overington et al., 2006), yet are clearly important
modulators of MAPK function.
Acknowledgements
We thank Misha Rand for assistance in gure and manuscript
preparation, and Robert Campbell for helpful discussions. We
apologize to all colleagues whose work could not be cited due
to space limitations. Research in the authors laboratory
was supported by grants from the National Institutes of
Health (to CJD).
References
Abi-Habib RJ, Urieto JO, Liu S, Leppla SH, Duesbery NS,
Frankel AE. (2005). BRAF status and mitogen-activated
protein/extracellular signal-regulated kinase kinase 1/2
activity indicate sensitivity of melanoma cells to anthrax
lethal toxin. Mol Cancer Ther 4: 13031310.
Agus DB, Gordon MS, Taylor C, Natale RB, Karlan B,
Mendelson DS et al. (2005). Phase I clinical study of
pertuzumab, a novel HER dimerization inhibitor, in

patients with advanced cancer. J Clin Oncol 23: 25342543.
Aoki Y, Niihori T, Kawame H, Kurosawa K, Ohashi H, Tanaka
Y et al. (2005). Germline mutations in HRAS proto-oncogene
cause Costello syndrome. Nat Genet 37: 10381040.
Arslan MA, Kutuk O, Basaga H. (2006). Protein kinases as drug
targets in cancer. Curr Cancer Drug Targets 6: 623634.
Oncogene

3306
Awada A, Hendlisz A, Gil T, Bartholomeus S, Mano M, de
Valeriola D et al. (2005). Phase I safety and pharmacokinetics of BAY 43-9006 administered for 21 days on/7 days
off in patients with advanced, refractory solid tumours. Br J
Cancer 92: 18551861.
Bain J, McLauchlan H, Elliott M, Cohen P. (2003). The
specicities of protein kinase inhibitors: an update. Biochem J
371: 199204.
Baselga J, Arteaga CL. (2005). Critical update and emerging
trends in epidermal growth factor receptor targeting in
cancer. J Clin Oncol 23: 24452459.
Basso AD, Kirschmeier P, Bishop WR. (2006). Lipid
posttranslational modications. Farnesyl transferase inhibitors. J Lipid Res 47: 1531.
Benson JD, Chen YN, Cornell-Kennon SA, Dorsch M, Kim S,
Leszczyniecka M et al. (2006). Validating cancer drug
targets. Nature 441: 451456.
Bergo MO, Ambroziak P, Gregory C, George A, Otto JC,
Kim E et al. (2002). Absence of the CAAX endoprotease
Rce1: effects on cell growth and transformation. Mol Cell
Biol 22: 171181.
Bergo MO, Gavino BJ, Hong C, Beigneux AP, McMahon M,
Casey PJ et al. (2004). Inactivation of Icmt inhibits
transformation by oncogenic K-Ras and B-Raf. J Clin
Invest 113: 539550.
Bergo MO, Leung GK, Ambroziak P, Otto JC, Casey PJ,
Gomes et al. (2001). Isoprenylcysteine carboxyl methyltransferase deciency in mice. J Biol Chem 276: 58415845.
Bergo MO, Leung GK, Ambroziak P, Otto JC, Casey PJ,
Young SG. (2000). Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells. J Biol Chem 275:
1760517610.
Bier H, Hoffmann T, Haas I, van Lierop A. (1998). Anti(epidermal growth factor) receptor monoclonal antibodies
for the induction of antibody-dependent cell-mediated
cytotoxicity against squamous cell carcinoma lines of the
head and neck. Cancer Immunol Immunother 46: 167173.
Bodart JF, Chopra A, Liang X, Duesbery N. (2002). Anthrax,
MEK and cancer. Cell Cycle 1: 1015.
Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen
RB et al. (2006). Radiotherapy plus cetuximab for
squamous-cell carcinoma of the head and neck. N Engl J
Med 354: 567578.
Cappuzzo F, Hirsch FR, Rossi E, Bartolini S, Ceresoli GL,
Bemis L et al. (2005). Epidermal growth factor receptor gene
and protein and getinib sensitivity in non-small-cell lung
cancer. J Natl Cancer Inst 97: 643655.
Carta C, Pantaleoni F, Bocchinfuso G, Stella L, Vasta I,
Sarkozy A et al. (2006). Germline missense mutations affecting
KRAS Isoform B are associated with a severe Noonan
syndrome phenotype. Am J Hum Genet 79: 129135.
Cheng KW, Lahad JP, Gray JW, Mills GB. (2005). Emerging
role of RAB GTPases in cancer and human disease. Cancer
Res 65: 25162519.
Citri A, Skaria KB, Yarden Y. (2003). The deaf and the dumb:
the biology of ErbB-2 and ErbB-3. Exp Cell Res 284: 5465.
Clarke S, Tamanoi F. (2004). Fighting cancer by disrupting
C-terminal methylation of signaling proteins. J Clin Invest
113: 513515.
Collisson EA, De A, Suzuki H, Gambhir SS, Kolodney MS.
(2003). Treatment of metastatic melanoma with an orally
available inhibitor of the Ras-Raf-MAPK cascade. Cancer
Res 63: 56695673.
Cortes J, Baselga J, Kellokumpu-Lehtinen P, Bianchi G,
Cameron D, Miles D et al. (2005). Open label, randomized,
Oncogene
phase II study of pertuzumab (P) in patients (pts) with

metastatic breast cancer (MBC) with low expression of
HER2. ASCO Meeting Abstracts 22(16S) (abstract 3068).
Cox AD, Der CJ. (2002a). Farnesyltransferase inhibitors:
promises and realities. Curr Opin Pharmacol 2: 388393.
Cox AD, Der CJ. (2002b). Ras family signaling: therapeutic
targeting. Cancer Biol Ther 1: 599606.
Cripps MC, Figueredo AT, Oza AM, Taylor MJ, Fields AL,
Holmlund JT et al. (2002). Phase II randomized study of
ISIS 3521 and ISIS 5132 in patients with locally advanced or
metastatic colorectal cancer: a National Cancer Institute
of Canada clinical trials group study. Clin Cancer Res 8:
21882192.
Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H,
Santoro A et al. (2004). Cetuximab monotherapy and
cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 351: 337345.
da Rocha Dias S, Friedlos F, Light Y, Springer C, Workman
P, Marais R. (2005). Activated B-RAF is an Hsp90 client
protein that is targeted by the anticancer drug 17allylamino-17-demethoxygeldanamycin. Cancer Res 65:
1068610691.
Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S
et al. (2002). Mutations of the BRAF gene in human cancer.
Nature 417: 949954.
Davies M, Hennessy B, Mills GB. (2006). Point mutations of
protein kinases and individualised cancer therapy. Expert
Opin Pharmacother 7: 22432261.
Davies SP, Reddy H, Caivano M, Cohen P. (2000). Specicity
and mechanism of action of some commonly used protein
kinase inhibitors. Biochem J 351: 95105.
de Bono JS, Bellmunt J, Droz JP, Miller K, Zugmaier G,
Sternberg C et al. (2005). An open label, phase II,
multicenter, study to evaluate the efcacy and safety of
pertuzumab (P) in chemotherapy naive patients (pts) with
hormone refractory prostate cancer (HRPC). ASCO Meeting Abstracts 22(16S) (abstract 4609).
Doi T, Ohtsu A, Saijo N, Takiuchi H, Ohhashi Y, Weber D
et al. (2005). A phase I study of a humanized monoclonal
anti-epidermal growth factor receptor (EGFR) antibody
EMD72000 (Matuzumab) administered weekly in Japanese
patients with advanced solid tumors; safety, PK and PD
results of skin biopsies. ASCO Meeting Abstracts 22(16S)
(abstract 3077).
Dominguez C, Powers DA, Tamayo N. (2005). p38 MAP
kinase inhibitors: many are made, but few are chosen. Curr
Opin Drug Discov Devel 8: 421430.
Dritschilo A, Huang CH, Rudin CM, Marshall J, Collins B,
Dul JL et al. (2006). Phase I study of liposome-encapsulated
c-raf antisense oligodeoxyribonucleotide infusion in combination with radiation therapy in patients with advanced
malignancies. Clin Cancer Res 12: 12511259.
Duesbery N, Vande Woude G. (2006). BRAF and MEK
mutations make a late entrance. Sci STKE 2006: pe15.
Duesbery NS, Resau J, Webb CP, Koochekpour S, Koo HM,
Leppla SH et al. (2001). Suppression of ras-mediated
transformation and inhibition of tumor growth and
angiogenesis by anthrax lethal factor, a proteolytic inhibitor
of multiple MEK pathways. Proc Natl Acad Sci USA 98:
40894094.
Eberhard DA, Johnson BE, Amler LC, Goddard AD, Heldens
SL, Herbst RS et al. (2005). Mutations in the epidermal
growth factor receptor and in KRAS are predictive and
prognostic indicators in patients with non-small-cell lung
cancer treated with chemotherapy alone and in combination
with erlotinib. J Clin Oncol 23: 59005909.

3307
Eisen T, Ahmad T, Flaherty KT, Gore M, Kaye S, Marais R
et al. (2006). Sorafenib in advanced melanoma: a Phase II
randomised discontinuation trial analysis. Br J Cancer 95:
581586.
Escudier B, Szczylik C, Eisen T, Stadler WM, Schwartz B,
Shan M et al. (2005). Randomized phase III trial of the Raf
kinase and VEGFR inhibitor sorafenib (BAY 43-9006) in
patients with advanced renal cell carcinoma (RCC). J Clin
Oncol (Meeting Abstracts), (16S) (abstract LBA4510).
Estep AL, Tidyman WE, Teitell MA, Cotter PD, Rauen KA.
(2006). HRAS mutations in Costello syndrome: detection of
constitutional activating mutations in codon 12 and 13 and
loss of wild-type allele in malignancy. Am J Med Genet A
140: 816.
Friess T, Scheuer W, Hasmann M. (2005). Combination
treatment with erlotinib and pertuzumab against human
tumor xenografts is superior to monotherapy. Clin Cancer
Res 11: 53005309.
Gangarosa LM, Sizemore N, Graves-Deal R, Oldham SM,
Der CJ, Coffey RJ. (1997). A raf-independent epidermal
growth factor receptor autocrine loop is necessary for Ras
transformation of rat intestinal epithelial cells. J Biol Chem
272: 1892618931.
Gibson TB, Ranganathan A, Grothey A. (2006). Randomized
phase III trial results of panitumumab, a fully human antiepidermal growth factor receptor monoclonal antibody, in
metastatic colorectal cancer. Clin Colorectal Cancer 6:
2931.
Gokhale PC, Zhang C, Newsome JT, Pei J, Ahmad I, Rahman
A et al. (2002). Pharmacokinetics, toxicity, and efcacy of
ends-modied raf antisense oligodeoxyribonucleotide encapsulated in a novel cationic liposome. Clin Cancer Res 8:
36113621.
Gordon MS, Matei D, Aghajanian C, Matulonis UA, Brewer
MA, Fleming GF et al. (2005). Clinical activity of
pertuzumab (rhuMab 2C4) in advanced, refractory or
recurrent ovarian cancer (OC), and the role of HER2
activation status. ASCO Meeting Abstracts 22(16S): (abstract
5051).
Grandis JR, Sok JC. (2004). Signaling through the epidermal
growth factor receptor during the development of malignancy. Pharmacol Ther 102: 3746.
Grbovic OM, Basso AD, Sawai A, Ye Q, Friedlander P, Solit
D et al. (2006). V600E B-Raf requires the Hsp90 chaperone
for stability and is degraded in response to Hsp90 inhibitors.
Proc Natl Acad Sci USA 103: 5762.
Gripp KW, Lin AE, Stabley DL, Nicholson L, Scott Jr CI,
Doyle D et al. (2006). HRAS mutation analysis in Costello
syndrome: genotype and phenotype correlation. Am J Med
Genet A 140: 17.
Hahn O, Stadler W. (2006). Sorafenib. Curr Opin Oncol 18:
615621.
Hanahan D, Weinberg RA. (2000). The hallmarks of cancer.
Cell 100: 5770.
Hingorani SR, Jacobetz MA, Robertson GP, Herlyn M,
Tuveson DA. (2003). Suppression of BRAF(V599E) in
human melanoma abrogates transformation. Cancer Res 63:
51985202.
Hoeich KP, Gray DC, Eby MT, Tien JY, Wong L, Bower J
et al. (2006). Oncogenic BRAF is required for tumor
growth and maintenance in melanoma models. Cancer Res
66: 9991006.
Hoshino R, Chatani Y, Yamori T, Tsuruo T, Oka H, Yoshida
O et al. (1999). Constitutive activation of the 41-/43-kDa
mitogen-activated protein kinase signaling pathway in
human tumors. Oncogene 18: 813822.
Hynes J, Leftheri K. (2005). Small molecule p38 inhibitors:

novel structural features and advances from 2002-2005. Curr
Top Med Chem 5: 967985.
Hynes NE, Lane HA. (2005). ERBB receptors and cancer:
the complexity of targeted inhibitors. Nat Rev Cancer 5:
341354.
Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor
A et al. (2005). Cancer statistics, 2005. CA Cancer J Clin 55:
1030.
Jimeno A, Hidalgo M. (2006). Pharmacogenomics of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors.
Biochim Biophys Acta 1766: 217229.
Johnson GL, Lapadat R. (2002). Mitogen-activated protein
kinase pathways mediated by ERK, JNK, and p38 protein
kinases. Science 298: 19111912.
Karin M. (2006). Nuclear factor-kappaB in cancer development and progression. Nature 441: 431436.
Kato-Stankiewicz J, Hakimi I, Zhi G, Zhang J, Serebriiskii I,
Guo L et al. (2002). Inhibitors of Ras/Raf-1 interaction
identied by two-hybrid screening revert Ras-dependent
transformation phenotypes in human cancer cells. Proc Natl
Acad Sci USA 99: 1439814403.
Kerr B, Delrue MA, Sigaudy S, Perveen R, Marche M,
Burgelin I et al. (2006). Genotypephenotype correlation
in Costello syndrome: HRAS mutation analysis in 43 cases.
J Med Genet 43: 401405.
Kim E, Ambroziak P, Otto JC, Taylor B, Ashby M, Shannon
K et al. (1999). Disruption of the mouse Rce1 gene results in
defective Ras processing and mislocalization of Ras within
cells. J Biol Chem 274: 83838390.
Kolch W. (2005). Coordinating ERK/MAPK signalling
through scaffolds and inhibitors. Nat Rev Mol Cell Biol 6:
827837.
Kortum RL, Lewis RE. (2004). The molecular scaffold KSR1
regulates the proliferative and oncogenic potential of cells.
Mol Cell Biol 24: 44074416.
Lacouture ME. (2006). Mechanisms of cutaneous toxicities to
EGFR inhibitors. Nat Rev Cancer 6: 803812.
Lancet JE, Gojo I, Gotlib J, Feldman EJ, Greer J, Liesveld JL
et al. (2007). A phase II study of the farnesyltransferase
inhibitor tipifarnib in poor-risk and elderly patients with
previously untreated acute myelogenous leukemia. Blood
109: 13871394.
Lee P, Wallace E, Yeh T, Poch G, Litwiler K, Pheneger T et al.
(2004). ARRY-142886, a potent and selective MEK
inhibitor: III) Efcacy in murine xenograft models correlates
with decreased ERK phosphorylation. AACR Meeting
Abstracts (abstract 897).
Leung KF, Baron R, Seabra MC. (2006). Thematic review
series: lipid posttranslational modications. geranylgeranylation of Rab GTPases. J Lipid Res 47: 467475.
Lorusso PM, Adjei AA, Varterasian M, Gadgeel S, Reid J,
Mitchell DY et al. (2005). Phase I and pharmacodynamic
study of the oral MEK inhibitor CI-1040 in patients with
advanced malignancies. J Clin Oncol 23: 52815293.
Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto
RA, Brannigan BW et al. (2004). Activating mutations in
the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to getinib. N Engl J Med
350: 21292139.
Lyons JF, Wilhelm S, Hibner B, Bollag G. (2001). Discovery
of a novel Raf kinase inhibitor. Endocr Relat Cancer 8:
219225.
Lyssikatos J, Yeh T, Wallace E, Marsh V, Bernat B, Gross S
et al. (2004). ARRY-142886, a potent and selective MEK
inhibitor: I) ATP-independent inhibition results in high
Oncogene

3308
enzymatic and cellular selectivity. AACR Meeting Abstracts
45: (abstract 896b).
Malumbres M, Barbacid M. (2003). RAS oncogenes: the rst
30 years. Nat Rev Cancer 3: 459465.
Manning AM, Davis RJ. (2003). Targeting JNK for therapeutic benet: from junk to gold? Nat Rev Drug Discov 2:
554565.
McCarthy SA, Samuels ML, Pritchard CA, Abraham JA,
McMahon M. (1995). Rapid induction of heparin-binding
epidermal growth factor/diphtheria toxin receptor expression by Raf and Ras oncogenes. Genes Dev 9: 19531964.
Mendelsohn J, Baselga J. (2006). Epidermal growth factor
receptor targeting in cancer. Semin Oncol 33: 369385.
Mercer KE, Pritchard CA. (2003). Raf proteins and cancer:
B-Raf is identied as a mutational target. Biochim Biophys
Acta 1653: 2540.
Mewani RR, Tang W, Rahman A, Dritschilo A, Ahmad I,
Kasid UN et al. (2004). Enhanced therapeutic effects of
doxorubicin and paclitaxel in combination with liposomeentrapped ends-modied raf antisense oligonucleotide
against human prostate, lung and breast tumor models. Int
J Oncol 24: 11811188.
Michaelson D, Ali W, Chiu VK, Bergo M, Silletti J, Wright L
et al. (2005). Postprenylation CAAX processing is required
for proper localization of Ras but not Rho GTPases. Mol
Biol Cell 16: 16061616.
Mitin N, Rossman KL, Der CJ. (2005). Signaling interplay in
Ras superfamily function. Curr Biol 15: R563574.
Monia BP, Sasmor H, Johnston JF, Freier SM, Lesnik EA,
Muller M et al. (1996). Sequence-specic antitumor activity
of a phosphorothioate oligodeoxyribonucleotide targeted to
human C-raf kinase supports an antisense mechanism of
action in vivo. Proc Natl Acad Sci USA 93: 1548115484.
Moore M, Hirte HW, Siu L, Oza A, Hotte SJ, Petrenciuc O
et al. (2005a). Phase I study to determine the safety and
pharmacokinetics of the novel Raf kinase and VEGFR
inhibitor BAY 43-9006, administered for 28 days on/7 days
off in patients with advanced, refractory solid tumors. Ann
Oncol 16: 16881694.
Moore MJ, Goldstein D, Hamm J, Figer A, Hecht J, Gallinger
S et al. (2005b). Erlotinib plus gemcitabine compared to
gemcitabine alone in patients with advanced pancreatic
cancer. A phase III trial of the National Cancer Institute of
Canada Clinical Trials Group [NCIC-CTG]. ASCO Meeting
Abstracts 22(16S): (abstract 1).
Moy B, Goss PE. (2006). Lapatinib: Current status and future
directions in breast cancer. Oncologist 11: 10471057.
Mukherjee S, Keitany G, Li Y, Wang Y, Ball HL, Goldsmith
EJ et al. (2006). Yersinia YopJ acetylates and inhibits kinase
activation by blocking phosphorylation. Science 312: 1211
1214.
Neckers L. (2006). Using natural product inhibitors to validate
Hsp90 as a molecular target in cancer. Curr Top Med Chem
6: 11631171.
Nelson MH, Dolder CR. (2006). Lapatinib: a novel dual
tyrosine kinase inhibitor with activity in solid tumors. Ann
Pharmacother 40: 261269.
Niihori T, Aoki Y, Narumi Y, Neri G, Cave H, Verloes A
et al. (2006). Germline KRAS and BRAF mutations in
cardio-facio-cutaneous syndrome. Nat Genet 38: 294296.
ONeill LA. (2006). Targeting signal transduction as a
strategy to treat inammatory diseases. Nat Rev Drug
Discov 5: 549563.
Ohren JF, Chen H, Pavlovsky A, Whitehead C, Zhang E,
Kuffa P et al. (2004). Structures of human MAP kinase
Oncogene
kinase 1 (MEK1) and MEK2 describe novel noncompetitive

kinase inhibition. Nat Struct Mol Biol 11: 11921197.
Orth K, Palmer LE, Bao ZQ, Stewart S, Rudolph AE, Bliska
JB et al. (1999). Inhibition of the mitogen-activated protein
kinase kinase superfamily by a Yersinia effector. Science
285: 19201923.
Overington JP, Al-Lazikani B, Hopkins AL. (2006). How
many drug targets are there? Nat Rev Drug Discov 5:
993996.
Oza AM, Elit L, Swenerton K, Faught W, Ghatage P, Carey
M et al. (2003). Phase II study of CGP 69846A (ISIS 5132)
in recurrent epithelial ovarian cancer: an NCIC clinical
trials group study (NCIC IND.116). Gynecol Oncol 89:
129133.
Pao W, Wang TY, Riely GJ, Miller VA, Pan Q, Ladanyi M
et al. (2005). KRAS mutations and primary resistance
of lung adenocarcinomas to getinib or erlotinib. PLoS Med
2: e17.
Parulekar WR, Eisenhauer EA. (2004). Phase I trial design for
solid tumor studies of targeted, non-cytotoxic agents: theory
and practice. J Natl Cancer Inst 96: 990997.
Perez-Soler R, Saltz L. (2005). Cutaneous adverse effects with
HER1/EGFR-targeted agents: is there a silver lining? J Clin
Oncol 23: 52355246.
Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW,
Vogelstein B, Velculescu VE. (2002). Tumorigenesis:
RAF/RAS oncogenes and mismatch-repair status. Nature
418: 934.
Reid TS, Terry KL, Casey PJ, Beese LS. (2004). Crystallographic analysis of CaaX prenyltransferases complexed
with substrates denes rules of protein substrate selectivity.
J Mol Biol 343: 417433.
Repasky GA, Chenette EJ, Der CJ. (2004). Renewing the
conspiracy theory debate: does Raf function alone to
mediate Ras oncogenesis? Trends Cell Biol 14: 639647.
Rinehart J, Adjei AA, Lorusso PM, Waterhouse D, Hecht JR,
Natale RB et al. (2004). Multicenter phase II study of the
oral MEK inhibitor, CI-1040, in patients with advanced
non-small-cell lung, breast, colon, and pancreatic cancer.
J Clin Oncol 22: 44564462.
Rini BI. (2006). Sorafenib. Expert Opin Pharmacother 7:
453461.
Rodriguez-Viciana P, Tetsu O, Tidyman WE, Estep AL,
Conger BA, Cruz MS et al. (2006). Germline mutations in
genes within the MAPK pathway cause cardio-faciocutaneous syndrome. Science 311: 12871290.
Rowinsky EK. (2006). Lately, it occurs to me what a long,
strange trip its been for the farnesyltransferase inhibitors.
J Clin Oncol 24: 29812984.
Rudin CM, Marshall JL, Huang CH, Kindler HL, Zhang C,
Kumar D et al. (2004). Delivery of a liposomal c-raf-1
antisense oligonucleotide by weekly bolus dosing in patients
with advanced solid tumors: a phase I study. Clin Cancer
Res 10: 72447251.
Salazar R, Tabernero J, Rojo F, Jimenez E, Montaner I,
Casado E et al. (2004). Dose-dependent inhibition of the
EGFR and signalling pathways with the anti-EGFR
monoclonal antibody (MAb) EMD 72000 administered
every three weeks (q3w). A phase I pharmacokinetic/
pharmacodynamic (PK/PD) study to dene the optimal
biological dose (OBD). ASCO Meeting Abstracts 22(14S):
(abstract 2002).
Sausville EA, Burger AM. (2006). Contributions of human
tumor xenografts to anticancer drug development. Cancer
Res 66: 33513354 discussion 3354.

3309
Scaltriti M, Baselga J. (2006). The epidermal growth factor
receptor pathway: a model for targeted therapy. Clin Cancer
Res 12: 52685272.
Schreck R, Rapp UR. (2006). Raf kinases: oncogenesis and
drug discovery. Int J Cancer 119: 22612271.
Schubbert S, Zenker M, Rowe SL, Boll S, Klein C, Bollag G
et al. (2006). Germline KRAS mutations cause Noonan
syndrome. Nat Genet 38: 331336.
Schulze A, Lehmann K, Jefferies HB, McMahon M, Downward J. (2001). Analysis of the transcriptional program
induced by Raf in epithelial cells. Genes Dev 15: 981994.
Schulze A, Nicke B, Warne PH, Tomlinson S, Downward J.
(2004). The transcriptional response to Raf activation is
almost completely dependent on mitogen-activated protein
kinase kinase activity and shows a major autocrine
component. Mol Biol Cell 15: 34503463.
Sebolt-Leopold JS, Dudley DT, Herrera R, Van Becelaere K,
Wiland A, Gowan RC et al. (1999). Blockade of the MAP
kinase pathway suppresses growth of colon tumors in vivo.
Nat Med 5: 810816.
Sebolt-Leopold JS, English JM. (2006). Mechanisms of drug
inhibition of signalling molecules. Nature 441: 457462.
Sebolt-Leopold JS, Herrera R. (2004). Targeting the mitogenactivated protein kinase cascade to treat cancer. Nat Rev
Cancer 4: 937947.
Sebti SM, Der CJ. (2003). Opinion: searching for the elusive
targets of farnesyltransferase inhibitors. Nat Rev Cancer 3:
945951.
Sharma A, Trivedi NR, Zimmerman MA, Tuveson DA, Smith
CD, Robertson GP. (2005). Mutant V599EB-Raf regulates
growth and vascular development of malignant melanoma
tumors. Cancer Res 65: 24122421.
Sharp S, Workman P. (2006). Inhibitors of the HSP90
molecular chaperone: current status. Adv Cancer Res 95:
323348.
Sharpless NE, Depinho RA. (2006). The mighty mouse:
genetically engineered mouse models in cancer drug development. Nat Rev Drug Discov 5: 741754.
Shaw RJ, Cantley LC. (2006). Ras, PI(3)K and mTOR
signalling controls tumour cell growth. Nature 441: 424430.
Shields JM, Pruitt K, McFall A, Shaub A, Der CJ. (2000).
Understanding Ras: it aint over til its over. Trends Cell
Biol 10: 147154.
Sieben NL, Macropoulos P, Roemen GM, Kolkman-Uljee
SM, Jan Fleuren G, Houmadi R et al. (2004). In ovarian
neoplasms, BRAF, but not KRAS, mutations are restricted
to low-grade serous tumours. J Pathol 202: 336340.
Singer G, Oldt III R, Cohen Y, Wang BG, Sidransky D,
Kurman RJ et al. (2003). Mutations in BRAF and KRAS
characterize the development of low-grade ovarian serous
carcinoma. J Natl Cancer Inst 95: 484486.
Skobeleva N, Menon S, Weber L, Golemis GA, Khazak V.
(2007). In vitro and in vivo synergy of MCP compounds with
MAPK pathway- and microtuble-targeting inhibitors. Mol
Cancer Ther In Press.
Smith RA, Dumas J, Adnane L, Wilhelm SM. (2006). Recent
advances in the research and development of RAF kinase
inhibitors. Curr Top Med Chem 6: 10711089.
Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, Basso
A et al. (2006). BRAF mutation predicts sensitivity to MEK
inhibition. Nature 439: 358362.
Stephens P, Hunter C, Bignell G, Edkins S, Davies H, Teague
J et al. (2004). Lung cancer: intragenic ERBB2 kinase
mutations in tumours. Nature 431: 525526.
Sumimoto H, Hirata K, Yamagata S, Miyoshi H, Miyagishi
M, Taira K et al. (2006). Effective inhibition of cell growth
and invasion of melanoma by combined suppression of

BRAF (V599E) and Skp2 with lentiviral RNAi. Int J Cancer
118: 472476.
Tabernero J, Rojo F, Jimenez E, Montaner I, Santome L,
Guix M et al. (2003). A phase I PK and serial tumor and
skin pharmacodynamic (PD) study of weekly (q1w), every 2week (q2w) or every 3-week (q3w) 1-hour (h) infusion
EMD72000, a humanized monoclonal anti-epidermal
growth factor receptor (EGFR) antibody, in patients (pt)
with advanced tumors. ASCO Meeting Abstracts 22:
(abstract 770).
Thompson N, Lyons J. (2005). Recent progress in targeting the
Raf/MEK/ERK pathway with inhibitors in cancer drug
discovery. Curr Opin Pharmacol 5: 350356.
Tolcher AW, Reyno L, Venner PM, Ernst SD, Moore M,
Geary RS et al. (2002). A randomized phase II and
pharmacokinetic study of the antisense oligonucleotides
ISIS 3521 and ISIS 5132 in patients with hormonerefractory prostate cancer. Clin Cancer Res 8: 25302535.
van Steensel MA, Vreeburg M, Peels C, van Ravenswaaij-Arts
CM, Bijlsma E, Schrander-Stumpel CT et al. (2006).
Recurring HRAS mutation G12S in Dutch patients with
Costello syndrome. Exp Dermatol 15: 731734.
Vanhoefer U, Tewes M, Rojo F, Dirsch O, Schleucher N,
Rosen O et al. (2004). Phase I study of the humanized
antiepidermal growth factor receptor monoclonal antibody
EMD72000 in patients with advanced solid tumors that
express the epidermal growth factor receptor. J Clin Oncol
22: 175184.
Vos MD, Martinez A, Ellis CA, Vallecorsa T, Clark GJ.
(2003). The pro-apoptotic Ras effector Nore1 may serve as a
Ras-regulated tumor suppressor in the lung. J Biol Chem
278: 2193821943.
Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D,
Good VM et al. (2004). Mechanism of activation of the
RAF-ERK signaling pathway by oncogenic mutations of
B-RAF. Cell 116: 855867.
Wellbrock C, Karasarides M, Marais R. (2004). The RAF
proteins take centre stage. Nat Rev Mol Cell Biol 5: 875885.
Wiley HS. (2003). Trafcking of the ErbB receptors and its
inuence on signaling. Exp Cell Res 284: 7888.
Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong
H et al. (2004). BAY 43-9006 exhibits broad spectrum oral
antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor
progression and angiogenesis. Cancer Res 64: 70997109.
Winter-Vann AM, Casey PJ. (2005). Post-prenylation-processing enzymes as new targets in oncogenesis. Nat Rev Cancer
5: 405412.
Winter-Vann AM, Kamen BA, Bergo MO, Young SG,
Melnyk S, James SJ et al. (2003). Targeting Ras signaling
through inhibition of carboxyl methylation: an unexpected
property of methotrexate. Proc Natl Acad Sci USA 100:
65296534.
Xing HR, Cordon-Cardo C, Deng X, Tong W, Campodonico
L, Fuks Z et al. (2003). Pharmacologic inactivation of kinase
suppressor of ras-1 abrogates Ras-mediated pancreatic
cancer. Nat Med 9: 12661268.
Yang XD, Jia XC, Corvalan JR, Wang P, Davis CG,
Jakobovits A. (1999). Eradication of established tumors by
a fully human monoclonal antibody to the epidermal growth
factor receptor without concomitant chemotherapy. Cancer
Res 59: 12361243.
Yarden Y. (2001). The EGFR family and its ligands in human
cancer. Signalling mechanisms and therapeutic opportunities.
Eur J Cancer 37(Suppl 4): S3S8.
Oncogene

3310
Yeh T, Wallace E, Lyssikatos J, Winkler J. (2004). ARRY142886, a potent and selective MEK inhibitor: II) Potency
against cellular MEK leads to inhibition of cellular
proliferation and induction of apoptosis in cell lines with
mutant Ras or B-Raf. AACR Meeting Abstracts 45:
(abstract 3889).
Oncogene
Yoon S, Seger R. (2006). The extracellular signal-regulated

kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24: 2144.
Zuber J, Tchernitsa OI, Hinzmann B, Schmitz AC, Grips M,
Hellriegel M et al. (2000). A genome-wide survey of RAS
transformation targets. Nat Genet 24: 144152.

Map Krev

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Map Krev

Uploaded by

Copyright:

Available Formats

Oncogene (2007) 26, 32913310

Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for

Mitogen-activated protein kinase (MAPK) cascades are

of death in the United States in people under the age

The RafMEKERK MAPK cascade

Targeting the Raf-MEK-ERK MAPK cascade

*Mutated in human cancers

(MAPKK) and a MAPK (Johnson and Lapadat, 2002).

development for these MAPK cascades will only be

Targeting the Raf-MEK-ERK MAPK cascade

Table 1 Germline mutation of the Ras-Raf-MEK-ERK cascade in human disorders

P34R, T58I, V14 I, V152G, D153V

Carta et al. (2006); Schubbert et al. (2006)

A246P, Q257R, S467A, F468S,

Niihori et al. (2006); Rodriguez-Viciana et al.

A-Raf and B-Raf) (Wellbrock et al., 2004; Schreck and

and other organelles. Activated ERKs can translocate to

Targeting the Raf-MEK-ERK MAPK cascade

mutants of Raf and MEK were shown to potently

related developmental disorders (Costello, cardiofacio

Small molecule MAPK inhibitorsa

Scios/Johnson & Johnson

A-Raf, B-Raf, c-Raf-1d

Targeting the Raf-MEK-ERK MAPK cascade

(Figure 3) (Kolch, 2005). This complexity is best

Nuclear Elk-1, Fos, Myc, STAT3

Figure 3 The RafMEKERK cascade is a core signaling

sensitivity to Ras transformation (Kortum and Lewis,

Ras family small GTPases are key upstream activators of

Targeting the Raf-MEK-ERK MAPK cascade

The epidermal growth factor receptor: upstream

Inhibitors of the RafMEKERK cascade

kinase inhibitors (Smith et al., 2006)). In addition to

Targeting the Raf-MEK-ERK MAPK cascade

multiple, functionally diverse, signaling components

Targeting the Raf-MEK-ERK MAPK cascade

properties. Structural studies with an analog of CI-1040

Targeting the Raf-MEK-ERK MAPK cascade

xenographs of Ras mutation-positive tumor cells were

in these efforts. Instead, over the years two main

Targeting the Raf-MEK-ERK MAPK cascade

Alternative K/N-Ras Processing

Figure 4 Post-translational processing of Ras. Normal Ras

was not sufcient to fully block Ras transformation

methotrexate, an inhibitor of DNA synthesis, may also

Inhibitors of EGFR signaling

Targeting the Raf-MEK-ERK MAPK cascade

activation of the EGFR signaling commonly occurs in

most evident in tissues that are dependent on EGFR

Monoclonal antibody EGFR-directed therapeutic agentsa

Fully human IgG2

Phase IIIII Pancreatic cancer, NSCLC, breast

NSCLC, rectal cancer, bladder

Breast cancer, ovarian cancer

Advanced solid tumors

Advanced solid tumors

EMD Pharmaceuticals/ Phase II

Fully human IgG1

Targeting the Raf-MEK-ERK MAPK cascade

EGFR-specic; reversible quinazoline

Phase IIIII Breast cancer, hepatocellular

Tyrosine kinase inhibitors

mAb versus TKI

Blockade of EGFR ligand binding,