Article

NRAS mutant melanoma - undrugable?

University of California San Francisco, Department for Dermatology, Mt. Zion Cancer Research Center, California, USA.
Oncotarget (Impact Factor: 6.36). 04/2013; 4(4):494-5. DOI: 10.18632/oncotarget.970
Source: PubMed
ABSTRACT
Mutations in the three rat sarcoma (RAS) family members NRAS (neuroblastoma-RAS), HRAS (Harvey-RAS) and KRAS (Kirsten-RAS) are found in one third of human cancers. Among the first oncogenes discovered in cutaneous melanoma was NRAS, which is mutant in up to 20% of tumors causing aberrant signaling in several downstream cascades. Despite, being a highly relevant therapeutic target, design of small molecules selectively inhibiting mutant NRAS in melanoma, to date, remains an unsolved challenge. The end?

Full-text

Available from: Christian Posch, Mar 14, 2014
Oncotarget 2013; 4: 494-495494
www.impactjournals.com/oncotarget
www.impactjournals.com/oncotarget/
Oncotarget, April, Vol.4, No 4
NRAS mutant melanoma – undrugable?
Christian Posch and Susana Ortiz-Urda
Mutations in the three rat sarcoma (RAS) family
members NRAS (neuroblastoma-RAS), HRAS (Harvey-
RAS) and KRAS (Kirsten-RAS) are found in one third
of human cancers. Among the rst oncogenes discovered
in cutaneous melanoma was NRAS, which is mutant in
up to 20% of tumors causing aberrant signaling in several
downstream cascades. Despite, being a highly relevant
therapeutic target, design of small molecules selectively
inhibiting mutant NRAS in melanoma, to date, remains an
unsolved challenge. The end?
RAS proteins are molecular switches mediating
signals from ligand activated receptor tyrosine kinases
(RTK) to the nucleus through a complex network of
downstream signaling cascades. Although NRAS, KRAS
and HRAS share structural and functional similarities,
recent ndings suggest distinctive subcellular localization
and compartmentalized signalling of these isoforms. This
is thought to contribute to differences in protein function in
the RAS family, but may also explain signaling variations
and predominance of certain RAS mutations across
different cancer types. Whereas KRAS mutations are
frequent in colorectal cancer, lung cancer and pancreatic
cancer, NRAS mutations are by far the predominant
alteration among RAS isoforms in melanoma. The
majority of NRAS mutations are found in codon 61
impairing the enzymatic activity of RAS to cleave GTP
to GDP. Other, less frequent mutations are found in codon
12 and 13 preventing the association of GAPase activating
proteins (GAP), which accelerate the weak hydrolytic
potential of RAS. As a result, NRAS remains in its active,
GTP-bound state driving cell proliferation, survival and
motility making NRAS an important therapeutic target in
melanoma. What are the challenges in designing effective
inhibitors of mutant RAS?
So far, several different strategies of directly
targeting RAS have not resulted in effective therapeutics.
One approach is based on the concept of inhibiting the
association of RAS with GTP. Unlike kinases, where ATP
binds and activates at low micromolar concentrations, the
afnity for GTP to RAS is in the low picomolar range
making the development of specic GTP antagonists
to date impossible. A second approach is based on
restoring the enzymatic activity of mutant RAS with
GAP-like molecules that enhance RAS-GAP association
and promote cleavage of GTP. So far, endeavours to
directly inhibit RAS have not translated into a clinical
success, thus, the central focus of research efforts
has become indirect inhibition of RAS. This entails a
growing understanding of essential post translational
modications (farnesylation), the membrane association
and the complex downstream signaling network of
RAS. Farnesylation is a lipid modication necessary
for RAS function. Several farnesylate inhibitors (FTI)
entered clinical studies, but failed to conrm the high
pre-clinical, anti tumor activity. Although FTIs potently
block farnesylation in HRAS models, an unexpected
biochemical difference among the RAS isoforms revealed
alternative posttranslational modications that can
substitute farnesylation, largely limiting the use of FTIs
as anti-RAS therapeutics. The localization of RAS to
the plasma membrane is also critical for the interaction
of RAS with various downstream effectors. It is hoped
that interference with docking proteins such as prenyl
binding sites on the plasma membrane may help to prevent
stimulus independent signaling by mutant RAS.
Recently, the focus of indirect RAS inhibition has
shifted to interfere with the complex network of activated
downstream cascades such as the mitogen activated
protein kinase (MAPK), phosphoinsitol 3-kinase (PI3K),
phospholipid C (PLC), RAL and the cell cycle pathway
among others (Figure 1). Although a recent clinical
trial with MEK162, a potent MEK inhibitor, has shown
some activity in patients with NRAS mutant melanoma
(Ascierto PA et al.), innate or acquired tumor resistance
to single-targeted agents is inevitable. However, there is
reasonable hope that the concept of combined selective
pathway inhibition may be effective. Our group has
recently demonstrated the importance of MAPK and
PI3K/mTOR signaling in a large collection of primary
and metastatic, patient derived melanoma samples as
well as in 10 human NRAS mutant melanoma cell lines
(Posch C et al.). Blocking with specic inhibitors in these
two pathways synergistically decreased cell viability in
vitro and regressed NRAS mutant xenografts in vivo. It is
important to notice that only a certain ratio of the MEK to
the PI3K/mTOR inhibitor showed synergism across all 10
cell lines. Although a direct line between preclinical results
and applications in vivo cannot be drawn, this nding
suggests that the most effective balance of two drugs
might not be at the maximum tolerated concentration of
those drugs in vivo.
Another group discovered CDK4 as a coextinction
target with MEK in NRAS mutant melanoma (Kwong
LN et al). Based on an elegant, mainly computational
analysis of large data sets they found that the combination
of a selective MEK and CDK
4,6
inhibitor regressed tumors
Page 1
Oncotarget 2013; 4: 494-495495
www.impactjournals.com/oncotarget
of two independent NRAS mutant cell lines in a mouse
xenograft model. However, the constitutive CDKN2A
knockout mouse model used in this study and the fact,
that alterations in the cell cycle pathway are common
genetic events in human melanoma, make it unlikely
that NRAS status alone is a marker for effective therapy
with MEK+CDK
4,6
inhibition. For successful translation
into clinics it will be essential to test more NRAS mutant
cell lines for the activity of inhibitor combinations and
to rene and fully characterize the genetic prole of
melanoma cells that are most likely to respond.
Both discussed combinations for pathway
interference (MEK+PI3K/mTOR and MEK+CDK
4,6
) are
not tumor specic therapies and bear the risk of severe
side effects, as most cell types and tissues signal through
these pathways and will also be affected. Still, the concept
of oncogene addiction and over-activation of certain
cascades allows for some selectivity to inhibit mainly
tumor cells. It is to date the most promising strategy to
interfere with currently undrugable targets such as mutant
NRAS in melanoma.
Christian Posch: University of California San Francisco,
Department for Dermatology, Mt. Zion Cancer Research
Center, California, USA
Susana Ortiz-Urda: University of California San Francisco,
Department for Dermatology, Mt. Zion Cancer Research
Center, California, USA
Correspondence: Christian Posch, email poschc@derm.
ucsf.edu
Received: April 10, 2013;
Published: April 13, 2013
Figure 1: Model of NRAS signaling in melanoma. Wild type NRAS (top left) cycles between an inactive GDP-bound and active
GTP-bound state, whereas mutations in G12, G13 and Q61 prevent hydrolysis of GTP, locking mutant NRAS in its GTP-bound, active
state (top right) which results in permanent, stimulus independent downstream signaling. NRAS-GTP activates downstream effectors of the
MAPK and PI3K/mTOR pathway (dark blue boxes). Schematic of the cell cycle pathway (light blue) and interaction sites of the specic
inhibitors in the different cascades (red lines). (RTK: receptor tyrosine kinases, GAP: GAPase activating protein, GEF: guanosine exchange
factor, MEKi: MEK inhibitor, PI3K/mTORi: PI3K/mTOR inhibitor, CDKi
4,6
: CDK
4,6
inhibitor)
NRAS
wt
NRAS
wt
RAF
AKT
Proliferation
E2F
Rb
GTP
PI3K/mTORi
MEK
ERK
NRAS
mut
GDP
NRAS
mut
GTP
Survival
Metabolism
Motility
Invasion Cell cycle progression
GDP
PIP3
PI3K
mTOR1
mTOR2
S6
CDKN2A
CDK4
CyclinD1
Rb
E2F
P
PKCα
RTK
RTK
GAP
GAP
GEF
GEF
MEKi
CDKi
4,6
wild type mutant
Growth
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    • "NRAS Q61R appears to be the more frequent NRAS mutation in melanoma with about 40– 67 % to of NRAS mutations [20, 24]. NRAS targeting is a new field in melanoma treatment and there is no consensus on the NRAS inhibitors to date25262728 . Nevertheless , the determination of NRAS mutational status is already of interest in melanoma treatment strategies. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: The determination of NRAS and BRAF mutation status is a major requirement in the treatment of patients with metastatic melanoma. Mutation specific antibodies against NRAS(Q61R) and BRAF(V600E) proteins could offer additional data on tumor heterogeneity. The specificity and sensitivity of NRAS(Q61R) immunohistochemistry have recently been reported excellent. We aimed to determine the utility of immunohistochemistry using SP174 anti-NRAS(Q61R) and VE1 anti-BRAF(V600E) antibodies in the theranostic mutation screening of melanomas. Methods: 142 formalin-fixed paraffin-embedded melanoma samples from 79 patients were analyzed using pyrosequencing and immunohistochemistry. Results: 23 and 26 patients were concluded to have a NRAS-mutated or a BRAF-mutated melanoma respectively. The 23 NRAS (Q61R) and 23 BRAF (V600E) -mutant samples with pyrosequencing were all positive in immunohistochemistry with SP174 antibody and VE1 antibody respectively, without any false negative. Proportions and intensities of staining were varied. Other NRAS (Q61L) , NRAS (Q61K) , BRAF (V600K) and BRAF (V600R) mutants were negative in immunohistochemistry. 6 single cases were immunostained but identified as wild-type using pyrosequencing (1 with SP174 and 5 with VE1). 4/38 patients with multiple samples presented molecular discordant data. Technical limitations are discussed to explain those discrepancies. Anyway we could not rule out real tumor heterogeneity. Conclusions: In our study, we showed that combining immunohistochemistry analysis targeting NRAS(Q61R) and BRAF(V600E) proteins with molecular analysis was a reliable theranostic tool to face challenging samples of melanoma.
    Full-text · Article · Jul 2015 · Diagnostic Pathology
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    • "The most common NRAS mutation is at codon 61; this prevents RAS GTP hydrolysis, causing the NRAS protein to be constitutively active [19,20]. Less common mutations at codon 12 and 13 prevent the association of GAP proteins with the NRAS complex [21]. Association with the inner face of the plasma membrane is necessary for RAS function. "
    [Show abstract] [Hide abstract] ABSTRACT: Melanoma is the least common form of skin cancer, but it is responsible for the majority of skin cancer deaths. Traditional therapeutics and immunomodulatory agents have not shown much efficacy against metastatic melanoma. Agents that target the RAS/RAF/MEK/ERK (MAPK) signaling pathway-the BRAF inhibitors vemurafenib and dabrafenib, and the MEK1/2 inhibitor trametinib-have increased survival in patients with metastatic melanoma. Further, the combination of dabrafenib and trametinib has been shown to be superior to single agent therapy for the treatment of metastatic melanoma. However, resistance to these agents develops rapidly. Studies of additional agents and combinations targeting the MAPK, PI3K/AKT/mTOR (PI3K), c-kit, and other signaling pathways are currently underway. Furthermore, studies of phytochemicals have yielded promising results against proliferation, survival, invasion, and metastasis by targeting signaling pathways with established roles in melanomagenesis. The relatively low toxicities of phytochemicals make their adjuvant use an attractive treatment option. The need for improved efficacy of current melanoma treatments calls for further investigation of each of these strategies. In this review, we will discuss synthetic small molecule inhibitors, combined therapies and current progress in the development of phytochemical therapies. Copyright © 2015. Published by Elsevier Ireland Ltd.
    Full-text · Article · Jan 2015 · Cancer Letters
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    • "Their lack of efficacy in these diseases has focused attention on our current lack of ability to selectively inhibit oncogenic K-Ras [12, 91]. Similarly, mutant N-Ras that drives a significant fraction of malignant melanomas and some leukemias remains problematic despite the recent encouraging results with MEK inhibition as a downstream intervention [92], which is discussed further below. On the other hand, and in view of the immense investment that has been put into the development and testing of FTIs, it is perhaps surprising that they do not seem to have been systematically tested in cancers that are driven by mutated H-Ras. "
    [Show abstract] [Hide abstract] ABSTRACT: The ability to selectively and directly target activated Ras would provide immense utility for treatment of the numerous cancers that are driven by oncogenic Ras mutations. Patients with disorders driven by overactivated wild-type Ras proteins, such as type 1 neurofibromatosis, might also benefit from progress made in that context. Activated Ras is an extremely challenging direct drug target due to the inherent difficulties in disrupting the protein:protein interactions that underlie its activation and function. Major investments have been made to target Ras through indirect routes. Inhibition of farnesyl transferase to block Ras maturation has failed in large clinical trials. Likely reasons for this disappointing outcome include the significant and underappreciated differences in the isoforms of Ras. It is still plausible that inhibition of farnesyl transferase will prove effective for disease that is driven by activated H-Ras. The principal current focus of drugs entering clinic trial is inhibition of pathways downstream of activated Ras, for example, trametinib, a first-in-class MEK inhibitor. The complexity of signaling that is driven by activated Ras indicates that effective inhibition of oncogenic transduction through this approach will be difficult, with resistance being likely to emerge through switch to parallel pathways. Durable disease responses will probably require combinatorial block of several downstream targets.
    Full-text · Article · Oct 2013
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