Melanoma Mutagenesis and Aberrant Cell Signaling · additional targeted therapies possible....

October 2013, Vol. 20, No. 4 Cancer Control 261

IntroductionMalignant melanoma is the most fatal type of skin

cancer. In 2013, approximately 76,690 new cases

of invasive melanoma will be diagnosed and 9,480

deaths will occur.1 The incidence of melanoma among

men and women is increasing. Because of the uni-

formly poor prognosis of this disease, efforts focusing

on combining surgical management with systemic

treatments are ongoing.

Significant progress has been made in the field of

immunotherapy. Ipilimumab, a human monoclonal

antibody that targets cytotoxic T-lymphocyte antigen 4

(CTLA-4), is the first agent to improve overall survival

(OS) in patients with metastatic melanoma.2 Prior to

this discovery, no therapy in a phase III randomized

From the Departments of Surgery (DMB, CEA) and Medical Oncol-ogy (RDC) at the Memorial Sloan-Kettering Cancer Center, New York, New York.

Submitted September 29, 2012; accepted March 5, 2013.

Address correspondence to Richard D. Carvajal, MD, Memorial Sloan-Kettering Cancer Center, 300 East 66th Street, No. 1071, New York, NY 10065. E-mail: [email protected]

Dr Carvajal receives grant/research support from Novartis Phar-maceutical Inc and serves as a consultant for Morphotec Inc. No significant relationship exists between the remaining authors and the companies/organizations whose products or services may be referenced in this article.

Expanding knowledge of the molecular

alterations in melanoma is making

additional targeted therapies possible.

Melanoma Mutagenesis and Aberrant Cell Signaling

Danielle M. Bello, MD, Charlotte E. Ariyan, MD, PhD, and Richard D. Carvajal, MD

Background: Malignant melanoma is the most fatal type of skin cancer. Traditional melanoma classification

has been based on histological subtype or anatomical location. However, recent evidence suggests that melanoma

comprises a group of diseases characterized by distinct molecular mutations. These mutations affect disease

behavior but provide unique opportunities for targeted therapy.

Methods: In this review, several signaling pathways in melanoma are described as well as how mutations of

BRAF, NRAS, KIT, GNAQ, and GNA11 genes cause aberrant signaling and malignant transformation.

Results: Multiple genes affecting both the mitogen-activated protein kinase (MAPK) and phosphatidylinositol

3-kinase (PI3K) pathway are mutated in melanoma. Melanomas harboring the BRAF V600E mutation have

demonstrated sensitivity to both RAF and MAPK/extracellular signal regulated kinase (ERK) inhibitors in

vitro and in vivo. In addition, KIT-mutant melanomas, often arising from mucosal, acral, and chronically

sun-damaged skin surfaces, have shown clinical response to several inhibitors of the type III transmembrane

receptor tyrosine kinase KIT. Uveal melanoma, which often harbors GNAQ or GNA11 mutations, may also be

sensitive to MAPK/ERK or protein kinase C/PI3K pathway inhibition.

Conclusions: Emerging knowledge of these molecular alterations has led to clinical advances in patients with

melanoma. The study of known mutations and identification of new potential targets must continue in an

effort to develop more effective therapies for this disease.

Tenzin Norbu Lama. Yaks (detail).

262 Cancer Control October 2013, Vol. 20, No. 4

controlled trial improved OS in patients with advanced

melanoma. Ipilimumab potentiates the immunologi-

cal response to melanoma, both as monotherapy and

in combination with other treatments.3 CTLA-4 is an

immune checkpoint molecule that downregulates T-

cell signaling to inhibit the costimulatory CD28-B7

pathway, limiting T-cell responses and contributing to

tolerance to self-antigens.2,4 By blocking this molecule,

ipilimumab prevents the downregulation of T cells

and increases the immune response against tumor

antigens. In addition, programmed death 1 (PD-1)

protein, an inhibitory receptor on activated T cells, and

PD-1 ligand (PD-L1), which is selectively expressed

on tumor cells, are thought to play critical roles in the

ability of a tumor to overcome the immune system.

PD-1 is another immune-checkpoint molecule that

mediates immunosuppression to cancer cells. Block-

ade of PD-1 binding to PD-L1 enhances the immune

response.5 A phase I trial of nivolumab, a fully hu-

man immunoglobulin G4 (IgG4) anti-PD-1 antibody,

demonstrated objective responses (either partial or

complete) in 18% of patients with non–small cell lung

cancer, 28% of patients with melanoma, and 27% of

patients with renal cell cancer.6 PD-L1, the primary

ligand of PD-1, is upregulated on solid tumors. PD-L1

impedes cytokine production and inhibits the cytolytic

activity of PD-1–expressing, tumor-infiltrating CD4+

and CD8+ T cells.7-11 A phase I trial of MDX-1105, a

human PD-L1 monoclonal antibody, demonstrated du-

rable and objective responses in 6% to 17% of patients

with advanced cancers, including non–small cell lung

cancer, melanoma, and renal cell cancer.11

Emerging knowledge of several mutations in mel-

anoma has also led to dramatic clinical advances in the

use of molecularly targeted therapy in patients with

melanoma harboring these mutations. Vemurafenib, a

small molecule inhibitor of mutant BRAF, was recently

approved by the US Food and Drug Administration

(FDA) for the treatment of melanoma harboring the

BRAF V600E mutation. A significant impact was dem-

onstrated on progression-free survival (PFS) and OS

in a phase III trial of patients with melanoma and the

BRAF V600E mutation.12

In this review, we describe several signaling

pathways in melanoma and discuss how mutations

of BRAF, NRAS, KIT, GNAQ, and GNA11 cause aberrant

signaling and malignant transformation.

BRAF MutationsOncogenic Mechanism of Action

V-raf murine sarcoma viral oncogene homolog B1

(BRAF) is a member of the rapidly accelerated fibro-

sarcoma (RAF) family of serine/threonine kinases,

which also contains enzymes ARAF and CRAF (RAF1).

It is part of the rat sarcoma (RAS)/RAF/mitogen-

activated protein kinase (MAPK)/extracellular-signal–

regulated kinase (ERK [MEK]) signal transduction

pathway. The MAPK cascade receives activation sig-

nals from upstream tyrosine kinases, cytokines, and

G-protein coupled receptors, leading to cell growth

and differentiation (Fig 1). RAS, a G protein attached

to the inner layer of the plasma membrane, is respon-

sible for direct activation of BRAF. The other pathway

kinases, including RAF, MEK, and ERK, are all located

in the cytosol. In melanocytes, growth factors such as

fibroblast growth factor and stem cell factor lead to

temporary activation of the MAPK cascade, causing a

modest mitogenic effect. However, cell proliferation

can occur if robust and prolonged activation of ERK

is present, which requires a combination of multiple

growth factors.13,14

A high frequency of activating mutations of the

RAS/RAF/MEK/ERK pathway exists in melanoma.

BRAF is the most commonly mutated gene in this

malignancy, with a frequency ranging from 50% to

70%,15-19 depending on the distinct subset of mela-

noma examined (Table 120,21). More than 80% of BRAF

mutations result from a substitution of valine to glu-

tamic acid (Val600Glu, designated as BRAF V600E).

The BRAF V600E mutation destabilizes the inactive

conformation of the BRAF kinase, shifting the equi-

librium to the constitutively active state.22 The BRAF

V600E mutation then leads to continuous downstream

signaling of the MAPK pathway and resultant ERK

activation, causing a cell proliferation and melanoma

survival advantage. Other BRAF mutations include

V600K (1798 1799 GT > AA; 5% to 6%), followed by

V600R (1798 1799 GT > AG; 1%), V600E2 (1799 1800

AG > AA; 0.7%), and V600D (1799 1800 AG > AT).20,23,24

Other rare mutations affecting different codons of the

BRAF gene have also been described. The frequency

of the BRAF V600K mutation is as high at 20% in

some populations.16 In addition to melanoma, BRAF

mutations have been found in many other cancers,

including ovarian carcinoma, colorectal carcinoma,

and papillary thyroid cancer.25

Oncogenic BRAF V600E leads to the malignant

transformation of human melanocytes in vivo and in

vitro.26 The melanocyte expression of BRAF V600E

leads to the spontaneous melanoma formation in

transgenic mice. However, this finding is observed

in conjunction with inactivation of the tumor suppres-

sor phosphatase and tensin homolog (PTEN), sug-

gesting that mutant BRAF may be an early initiator

of melanoma formation and additional concomitant

events are required for full malignant transforma-

tion.27 Vredeveld et al28 recently demonstrated that

PTEN depletion or increased protein kinase B (Akt)

activation abrogated mutant BRAF V600E-induced se-

nescence in fibroblasts and melanocytes, suggesting

that activation of the phosphatidylinositol 3-kinase

(PI3K) pathway contributes to the progression of nevi


to melanomas. Clinically, oncogenic BRAF V600E is

found in approximately 50% of melanomas that occur

on skin lacking chronic sun-induced damage (CSD;

also called non–CSD melanoma). BRAF mutations

are less common in melanomas on skin with marked

CSD (CSD melanoma), suggesting that the deleteri-

ous effects of ultraviolet light may be unrelated to the

development of BRAF mutations.21,23,29

Constitutive activation of the MAPK cascade via

mutant BRAF V600E leads to growth of melanoma

cells through the upregulation of cyclin D1 expres-

sion and by the downregulation of p27 KIPI, a cell

cycle inhibitor. BRAF V600E also serves to promote

melanoma survival by regulating expression and

function of many proapoptotic and antiapoptotic

proteins such as those from the Bcl-2 family (BMF,

BIM, BAD) and Mcl-1.30-34 Melanoma migration and

capability for invasion are also enhanced by mutant

BRAF. Continuous activation of the MAPK cascade

promotes the invasion and migration through altera-

tions in the cytoskeletal organization, upregulation

of matrix metalloprotease expression, and activation

of migratory machinery.35,36 This occurs through the

downregulation of the cyclic guanosine monophos-

phate (cGMP) phosphodiesterase PDE5A, which fa-

cilitates cell invasion and contractility by increasing

intracellular cGMP levels leading to calcium release

into the cytosol and myosin light chain phosphoryla-

tion.37 Melanoma migration and motility are enhanced

by the effect of mutant BRAF on the expression of

RND3/RhoE/Rho8, which serves to mediate crosstalk

between the MAPK and Rho/Rock/LIM domain kinase

(LIMK)/Cofilin pathways.38,39

Oncogenic BRAF may also play a role in the im-

munological response to melanoma. The inhibition of

BRAF or MEK signaling in melanoma cells increases

the expression of highly immunological differentiation

antigens such as melanoma antigen (Melan-A) and

MART-1, leading to enhanced T-cell recognition.40,41

This increased recognition of melanoma by the im-

mune system suggests that a combination of BRAF

inhibitors with immunotherapeutic agents such as

Fig 1. — Major signaling pathways in melanoma and oncogenic mutation frequency. MEK1 and MEK2 are also known as MAP2K1 and MAP2K2, respec-tively. ERK1 and ERK2 are also known as MAPK3 and MAPK1, respectively. Akt1 = protein kinase B, ERK = extracellular-signal-regulated kinase, GEFT = RAC/CDC42 exchange factor, GNAQ = guanine nucleotide-binding protein G(q), GPCR = G-protein coupled receptor, IκB = inhibitor of kappa B, LIMK = LIM domain kinase, MAPK = mitogen-activated protein kinase, MEK = MAPK/ERK kinase, MLC = myosin light chain, MPK1 = MAPK phosphatase 1, mTOR = mammalian target of rapamycin, NFκB = nuclear factor kappa light-chain enhancer of activated B cells, PAK = p21 protein (CDC42/RAC)-activated kinase, PI3K = phosphoinositide 3 kinase, PTEN = phosphatase and tensin homolog, RAC = RAS-related C3 botulinum toxin substrate, RAF = rapidly accelerated fibrosarcoma, RAS = rat sarcoma, RHO = RAS homolog, ROCK = Rho-associated, coiled-coil containing protein kinase, RTK = receptor tyrosine kinase (eg, type III transmembrane receptor tyrosine kinase [KIT], fibroblast growth factor receptor 1 [also known as FLT2], PDGF, VEGF), UM = uveal melanoma. Modified from Romano E, Schwartz GK, Chapman PB, et al. Treatment implications of the emerging molecular classification system for melanoma. Lancet Oncol. 2011;12(9):913-922. Reprinted from The Lancet with permission from Elsevier © 2011.

RAS

RAF

ERK1/2

PTEN

MEK1/2 MPK1

PI3K

Akt1

mTOR

I B

NF B

GEFT

RHO

ROCK

MLC

RAC

PAK

LIMK

GPCR

30%-50% Loss

30% Amplified

Growth Factors

NRAS 15% Mutated

GNAQ Mutated 50% of UMRTK

Translation

BRAF 50%-70% Mutated

Cell Membrane

Cytoplasm

Transcription

(CDK4, CCND1, BCL2)

Survival, Proliferation, Metastases, Neoangiogenesis


ipilimumab may be an effective therapeutic strategy

against this type of cancer.42

Therapeutic Considerations

Given the relevance of the MAPK pathway in melano-

ma, efforts have focused on the development of path-

way inhibitors in the treatment of this disease. Mutant

BRAF demonstrates greater sensitivity to both RAF

and MEK inhibitors compared with wild-type BRAF

or oncogenic NRAS melanoma cell lines.43 Therefore,

inhibitors of RAF and MEK are in various stages of

development for the treatment of melanomas with

BRAF mutations.

Sorafenib, a small multitargeted kinase inhibitor

of BRAF and CRAF (RAF1), was one of the first small

molecule inhibitors to be tested in melanoma. It is

an oral biaryl urea with additional activity against

vascular endothelial growth factor receptor 2 and 3

(VEGFR2/3), the receptor-type tyrosine-protein kinase

FLT3, the type III transmembrane receptor tyrosine

kinase (KIT), and platelet-derived growth factor recep-

tor (PDGFR).44 Although encouraging effects were

observed in early-phase trials, particularly in combi-

nation with chemotherapeutic agents such as carbo-

platin and paclitaxel, randomized trials failed to show

clinical benefit in front-line and second-line settings.45

The next generation of RAF inhibitors offers the

potential for increased efficacy against BRAF-mutant

melanoma. Vemurafenib is an adenosine triphosphate

(ATP)-competitive selective RAF kinase inhibitor of

RAF1, BRAF, and oncogenic BRAF V600E, with half

maximal inhibitory concentration (IC50) values of 44

nmol/L, 110 nmol/L, and 44 nmol/L, respectively.46

In cell lines harboring mutant BRAF, vemurafenib

causes both gap 1 (G1) cell-cycle arrest and apopto-

sis as well as regression in BRAF V600E melanoma

xenograft models. However, unexpected results were

documented when small-molecule BRAF inhibitors

were used to treat non–BRAF V600E melanoma. In

vitro, inhibition of BRAF wild-type melanoma in the

presence of an oncogenic RAS mutation leads to the

paradoxical activation of the MAPK pathway through

homodimeric or heterodimeric complex formation of

CRAF (RAF1) with wild-type BRAF or kinase-dead

BRAF, resulting in potent ERK phosphorylation

(Fig 2).47-50 This paradoxical activation of RAS after

the inhibition of BRAF resulted in the increased

capacity of melanoma cells to proliferate and invade

via FAK activity in addition to the increased survival

of mutant NRAS cell lines.51 This suggests that the

use of BRAF inhibitors to treat patients with wild-type

melanoma or melanoma harboring NRAS mutations

may lead to enhanced tumorigenicity.

Clinically, this paradoxical pathway activation in

wild-type BRAF cells may explain how RAF inhibitors,

such as sorafenib, vemurafenib, and dabrafenib, trig-

ger the formation of hyperkeratotic lesions, which are

frequently seen with their use. These lesions include

squamous cell carcinoma, keratoacanthomas, verrucal

keratosis, and plantar hyperkeratosis. Several stud-

ies have reported mutations in genes encoding RAS

proteins in 30% to 70% of cutaneous squamous cell

carcinoma excised from patients treated with BRAF

inhibitors, further emphasizing how the paradoxical

activation of the MAPK pathway in wild-type BRAF

cells (eg, keratinocytes) via RAF inhibitors may lead

to the development of cutaneous neoplasms.52-56

The emergence of other types of tumors has also

been attributed to the use of RAF inhibitors. One

study examined 22 cutaneous melanocytic lesions

that had either emerged or considerably changed in

morphology in 19 patients undergoing treatment with

type I RAF inhibitors for BRAF-mutant melanoma.57

Of these lesions, 12 newly detected melanomas were

found in 11 patients within 27 weeks of initiating a

BRAF blockade. Ten new nevi were also seen in these

patients, of which 9 were dysplastic. All melanocytic

lesions were wild-type BRAF. Molecular examination

Table 1. — Frequencies of Selected Somatic Gene Mutations in Melanoma According to the COSMIC Database

Mutated Gene Frequency in Cutaneous Melanoma (%)

BRAF 46

CDKN2A 26

NRAS 18

P53 16

PTEN 15

MEK (MAP2K1) 9

KIT 8

CTNNB1 4

APC 4

CDK4 1

GNA11 1 (36% of UM)

GNAQ 0.4 (36% of UM)

Distribution of mutations in melanoma varies by site of origin and presence or absence of chronic sun damage. These numbers represent an approximate percentage based on COSMIC cutaneous melanoma data. Data from Forbes SA, Bindal N, Bamford S, et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 2011;39 (Database): D945-950. http://www.sanger.ac.uk/cosmic

COSMIC = Catalogue of Somatic Mutations in Cancer. UM = uveal melanoma.


revealed an increased expression of cyclin D1 in these

new primary melanomas compared with nevi (P =

.01 and P = .03, respectively). In addition, a report

of a patient with BRAF-mutant metastatic melanoma

undergoing treatment vemurafenib revealed acceler-

ated growth of a previously unsuspected RAS-mutant

leukemia.58 Exposure to RAF inhibition induced hy-

peractivation of ERK signaling and the proliferation

of the leukemic cell population, an effect that was

reversed on drug withdrawal. These findings sup-

port the concept that BRAF inhibitors induce growth

and tumorigenesis in RAF wild-type cells. Increased

surveillance for the emergence of new cutaneous and

noncutaneous neoplasms in patients receiving RAF

inhibitors may be appropriate moving forward.

The BRIM-3 study, a phase III clinical trial of

vemurafenib, consisted of 675 patients with previ-

ously untreated BRAF V600E-mutant melanoma who

were randomized to receive either vemurafenib (960

mg orally twice a day) or dacarbazine (1,000 mg/m2

of body-surface area intravenously every 3 weeks).12

The trial was halted early due to the clear efficacy of

vemurafenib over dacarbazine. At a 6-month analysis,

the OS rate was 84% for patients treated with vemu-

rafenib compared with 64% for those treated with da-

carbazine. Those treated with vemurafenib achieved

an overall response rate of 48% and a median PFS of

5.3 months compared with a 5% response rate and

a 1.6-month median PFS for dacarbazine. In 2011,

the FDA approved vemurafenib for the treatment of

metastatic BRAF V600E-mutant melanoma. Updated

results of the BRIM-3 study were recently present-

ed.59 Median follow-up times on vemurafenib and

dacarbazine were 10.5 and 8.4 months, respectively.

The median OS was 13.2 months with vemurafenib

compared with 9.6 months with dacarbazine. One-

year OS rates were 55% for vemurafenib and 43% for

dacarbazine, demonstrating that patients with BRAF

V600E-mutant melanoma treated with vemurafenib

continued to have improved OS rates.

Another BRAF inhibitor, dabrafenib, also demon-

strated dramatic responses in BRAF V600E/K-mutant

melanoma. Dabrafenib has a greater than 100-fold

selectivity for BRAF V600E-mutant cell lines and dem-

onstrates dose-dependent inhibition of MEK and ERK

phosphorylation associated with tumor regression in

xenograft models. The phase I study of this agent

consisted of 184 patients, of which 156 had BRAF-

mutant melanoma.60 At the recommended phase

II dose (150 mg twice daily), 18 of the 36 patients

D) Mutant RAS

Cell Membrane

Cytoplasm

RTK

ERK1/2

MEK1/2

RAF

RAS

Cell Growth

C) Mutant RAF,

RAF Inhibitor

B) Mutant RAF.

No Inhibitors

A) Wild-Type Cells,

No Inhibitors

RTK

Tumor Growth

RAS

BRAFv600E BRAFv600E

ERK1/2

MEK1/2

RTK

Cell Death,

Tumor Shrinkage

RAS

BRAFv600E BRAFv600E

ERK1/2

MEK1/2

DRUG DRUG

CRAF

RTK

ERK1/2

WTBRAF CRAF

CRAF

CRAF

KinaseDead BRAF

MutantRAS

oror

or

Tumor Growth

MEK1/2

DRUG

DRUG

DRUG

Fig 2. — BRAF-mutant and WT cell responses to RAF inhibition. (A) Normal activation of the wild-type cell RAS/RAF/MEK/ERK pathway promotes cell growth. Normally, RAS enzymes activate signaling when bound to guanosine triphosphate. (B) Mutations of the MAPK pathway, especially those of onco-genic BRAF V600E in melanoma, produce excessive activation and eventual tumor formation and growth. In melanoma cells that harbor BRAF V600E, RAS/GTP activation is low, and ERK signaling is predominantly activated by dimers of the mutant kinase. (C) In the presence of a BRAF inhibitor that blocks RAF activation, MEK/ERK signaling in BRAF V600E-mutant cells is suppressed, causing cell death and tumor shrinkage. (D) In RAS-mutant cells that contain wild-type BRAF, RAF inhibitors block phosphorylation of RAF monomers (BRAF or CRAF [RAF1]), forming homodimeric and heterodimeric complexes (BRAF/CRAF or CRAF/CRAF [RAF1]). In addition, kinase-dead BRAF, which cannot be phosphorylated, forms a dimeric complex with CRAF (RAF1). The formation of these dimers causes excessive downstream activation stimulated by oncogenic RAS, resulting in a paradoxical hyperactivation of MEK and ERK by the RAF inhibitor and subsequent tumor growth. BRAF = v-raf murine sarcoma viral oncogene homolog B1, ERK = extracellular-signal-regulated kinase, GTP = guanosine trisphosphate, MAPK = mitogen-activated protein kinase, MEK = MAPK/ERK kinase, MLC = myosin light chain, RAF = rapidly accelerated fibrosarcoma, RAS = rat sarcoma, RTK = receptor tyrosine kinase, WT = wild type.


(69%) with BRAF-mutant melanoma demonstrated

confirmed clinical responses. Of the 27 patients with

BRAF V600E-mutant melanoma, 15 (56%) had a con-

firmed response. In BRAF V600E-mutant melanoma,

responses were durable, with 17 patients (47%) on

treatment for more than 6 months.

A single-arm phase II study of dabrafenib also

showed promising clinical results. The BREAK-2

study enrolled 92 patients, in which 76 harbored BRAF

V600E and 16 harbored BRAF V600K mutations.61

The objective response rate was 59% in the BRAF

V600E group, with two responses seen in the BRAF

V600K cohort. The median PFS rates were 27 weeks

and 20 weeks, respectively. Results from BREAK-3,

a phase III randomized, multicenter trial comparing

dabrafenib with dacarbazine in 250 patients with

previously untreated BRAF V600E-mutant melanoma

showed improved PFS and objective response rates

with dabrafenib.62 The median PFS rates were 5.1 and

2.7 months, respectively. Confirmed response rates

were 53% and 19% for dabrafenib and dacarbazine,

respectively. This study further emphasized the ef-

ficacy of RAF inhibitors for the treatment of BRAF-

mutant melanoma.

Of note, the original phase I study of dabrafenib

by Falchook et al60 was also significant because its

results demonstrated the potential for therapeutic ef-

ficacy of targeted RAF inhibition against melanoma

brain metastases. Of the 10 patients with BRAF V600E-

mutant melanoma and untreated brain metastases who

were given dabrafenib, brain metastases in most pa-

tients reduced in size, with complete resolution of the

metastases in 4 patients. Intracranial disease reduc-

tion was accompanied by extracranial disease reduc-

tion. Although patients with melanoma metastatic to

the brain typically survive less than 5 months, all 10

patients in this study were alive at 5 months and 2

patients had durable antitumor activity with survival

beyond 1 year. Although dabrafenib was designed

to prevent blood-brain barrier penetration due to the

potential neurotoxic effects on abundant wild-type

BRAF in the brain, disruption of the blood-brain bar-

rier by melanoma metastases could be the mechanism

by which dabrafenib accesses these lesions.

The successes of vemurafenib59 and dabrafenib

against cerebral metastatic disease in melanoma

support the inclusion of patients with BRAF-mutant

melanoma and brain metastases in future trials.60,63

Results from the BREAK-MB trial, a multicenter, phase

II study examining dabrafenib in patients with BRAF

V600E/K-mutant melanoma and brain metastases,

also highlighted the efficacy of RAF inhibition on

these lesions (NCT01266967).64 Of the 139 patients

with a BRAF V600E mutation, objective responses

were observed in 29 of 74 patients (39%) who had

no previous treatment for their brain metastases and

20 of 65 patients (31%) who had received prior local

treatment. In 33 patients with a BRAF V600E muta-

tion, 15% had an objective response to dabrafenib.

Therapeutic regimens combining targeted agents with

different locoregional modalities are also being ex-

plored. A phase II prospective trial of dabrafenib

with stereotactic radiosurgery for the treatment of

BRAF V600E-mutant melanoma brain metastases com-

menced in April 2013 (NCT01721603).

BRAF ResistancePrimary and secondary resistance mechanisms ex-

ist in which tumors can evade treatment. Primary

resistance implies initial refractoriness of a tumor to

treatment, which may be due to an alteration in part

of the gene locus not targeted by the drug. Second-

ary, or acquired, resistance denotes tumor progression

following an initial response to the drug.

Primary Resistance: Although the reasons be-

hind primary resistance remain unclear, it has been

demonstrated in preclinical studies of BRAF V600E

melanoma cell lines showing a wide range of IC50

values to various BRAF inhibitors.65-68 One molecule

thought to play a role in intrinsic BRAF inhibitor re-

sistance in melanoma is PTEN.31,65 Loss of PTEN, a

negative regulator of Akt, was demonstrated in a sub-

set of BRAF-mutant melanoma resulting in Akt activa-

tion.31 When BRAF is inhibited, increased Akt signal-

ing is present, limiting the accumulation of FOXO3a

in the nucleus and leading to decreased apoptosis.

When BRAF melanoma cell lines are treated with

selumetinib, a small molecule inhibitor of MEK1/2,

insulin-like growth factor-1 (IGF-1)–mediated Akt

signaling increases, thus decreasing apoptosis. This

increased Akt signaling and decreased cell death can

be overcome by combination therapy using MEK

and Akt or mammalian target of rapamycin (mTOR)

1/2 inhibitors.69 In addition, concomitant PTEN and

retinoblastoma protein (RB) loss results in cell cycle

checkpoint dysregulation and intrinsic resistance to

BRAF and MEK inhibitors.65

Cyclin D and protein kinase D3 (PRKD3) may

also play a role in the primary resistance to BRAF

inhibitors. Inhibition of BRAF in cell lines leads to

the weakening of MEK/ERK signaling, a reduction in

cyclin D1 expression, and cell cycle arrest.66 However,

a small subset of BRAF-mutant melanoma cell lines

harbor cyclin D1 amplifications. Cell lines that possess

both amplifications in cyclin D1 and BRAF mutations

demonstrate intrinsic resistance to BRAF inhibition

and continue to freely enter the cell cycle even when

a BRAF inhibitor is administered.68 PRKD3 may also

play a role in intrinsic resistance to RAF inhibitors.

PRKD3 knockdown by small interfering RNA (siRNA)

reduced the IC50 of the RAF inhibitors in melanoma

cells lines. MAPK pathway signaling was also blocked


after the RAF inhibitors were administered. Melanoma

cell lines harboring the BRAF V600E mutation, which

also had PTEN loss, demonstrated that PRKD3 caused

a reactivation of the PI3K/Akt cascade following RAF

inhibition, further adding to resistance.70

The tumor microenvironment may also play a

role in innate tumor resistance to therapy. Strauss-

man et al71 reported that stroma-mediated resistance

to targeted agents was common. In BRAF-mutant

melanoma, stromal cell secretion of hepatocyte growth

factor (HGF) resulted in the activation of MET and

reactivation of the MAPK and PI3K/Akt signaling path-

ways, conferring immediate resistance to RAF inhibi-

tion. Analysis of the immunohistochemistry of BRAF-

mutant melanoma tumors demonstrated the stromal

cell expression of HGF and revealed a relationship

between HGF expression and the innate resistance

to RAF inhibitor treatment. Thus, dual inhibition by

RAF and HGF or MET inhibitors represents a potential

therapeutic combination in BRAF-mutant melanoma.

Secondary Resistance: Acquired or secondary

resistance is common in patients treated with small-

molecule BRAF inhibitors. Despite impressive re-

sponse rates with these drugs, complete responses

have been rare and usually of a short duration (ap-

proximately 7 months), and secondary resistance ul-

timately developed in most cases.12,72 Unlike other

targeted therapies such as imatinib in chronic myeloid

leukemia and gastrointestinal stromal tumors (GISTs)

in which acquisition of secondary mutations in the

kinase domain leads to loss of drug efficacy, no similar

gatekeeper mutations were found in BRAF inhibitor-

resistant cell lines or biopsies taken from patients

who failed vemurafenib.73,74 Multiple mechanisms

play a role in acquired resistance to BRAF inhibitors

such as the constitutive signaling of receptor tyrosine

kinases (IGF-1R and PDGFR-α), increased expression

of COT (MAP3K8,TPL-2) in the MAPK cascade, NRAS

and MEK1 mutation acquisition, and resultant BRAF

alterations.68,75-79 The significance and frequency of

these mechanisms of resistance in melanoma remain

unknown and are under clinical investigation.77,80-84

Poulikakos et al82 identified a new mechanism of

RAF-acquired resistance. They generated five inde-

pendent vemurafenib-resistant melanoma cell lines

by exposing the lines to high doses of the drug for 2

months. Three of the five clones were found to have

an in-frame deletion of exons 4 to 8 (61kDa), includ-

ing a domain critical for RAF activation, the RAS-

binding domain. This BRAF V600E variant, p61 BRAF

V600E, showed enhanced dimerization in cells with

low levels of RAS activation compared with full-length

BRAF V600E. In cells that express p61 BRAF V600E,

ERK signaling was resistant to RAF inhibition. Muta-

tions that eliminated the dimerization of p61 BRAF

V600E restored RAF inhibitor sensitivity. In addition,

BRAF splice variants lacking the RAS-binding domain

were identified in the tumors of 6 of the 19 patients

with acquired RAF inhibitor resistance.82 These data

suggest that effective RAF inhibition is dependent on

levels of RAS–guanosine trisphosphate (GTP) too low

to support RAF dimerization, and alterations in BRAF

structure that alter its function in the MAPK pathway

affect sensitivity to targeted agents.

MEK and ERK as Therapeutic Targets: High

constitutive activation of the MAPK pathway exists

in the vast majority of melanomas. Because of this,

as well as the eventual resistance of BRAF V600E-

mutant melanoma to BRAF inhibition, recent efforts

have focused on other targets in the MAPK cascade.

Several preclinical studies have demonstrated that

the inhibition of MEK in melanoma cells led to G1-

phase cell cycle arrest, which was associated with

cyclin D inhibition, increased p27KIPI expression,

and decreased phosphorylation of RB1 protein.43,85,86

Early clinical testing of CI1040 and PD-0325901,

two small molecule MEK inhibitors, did not show suf-

ficient antitumor activity and proved too toxic to move

beyond phase I/II trials. CI1040, an ATP noncom-

petitive inhibitor against MEK1/2, did not show any

partial or complete responses in a multicenter phase II

trial.87 Similarly, the MEK1 inhibitor PD-0325901 did

not demonstrate any objective responses in a phase II

study of patients with lung cancer.88 A phase I trial of

PD-0325901, which included patients with melanoma,

was halted early due to toxicity (NCT00147550).89

However, promising results have targeted MEK in

patients with advanced melanoma. Selumetinib is a

selective non–ATP competitive inhibitor of MAP2K1

(MEK1) and MAP2K2 (MEK2).90 In both preclinical

and clinical studies, selumetinib showed favorable

pharmacological characteristics with limited toxico-

logical effects. In the phase I trial of selumetinib, 1

patient with BRAF-mutant melanoma treated with se-

lumetinib demonstrated a complete durable response

that persisted for more than 15 months.91 In a phase

II study in which selumetinib was compared with

temozolomide in unselected patients with melanoma,

6 of 104 patients treated with selumetinib achieved

a partial response.92 Although no difference in PFS

occurred, patients with BRAF-mutant lesions had an

11% response rate, and 5 of the 6 responding patients

had tumors harboring oncogenic BRAF V600E.

Trametinib, a potent, selective inhibitor of

MAP2K1, has also demonstrated inhibition of phos-

phorylated ERK, resulting in cell death.93 In a phase I

study of 20 patients with BRAF-mutation melanoma, 5

achieved a partial response and a reduction in tumor

burden by 50%.94 Eight patients had stable disease.

Eleven of these patients had BRAF-mutant melanoma,

with 3 achieving partial responses, 5 achieving stable

disease, and 3 demonstrating progressive disease. The


phase II trial also produced promising results.95 Of

the 57 patients with BRAF-mutant melanoma previ-

ously treated with immunotherapy or chemotherapy,

complete responses were seen in 2 patients, partial

responses in 13, and stable disease in 29. The dis-

ease control rate was 75%, with a response rate of

25%. This was similar between BRAF mutation types

V600E and V600K. In patients previously untreated

with a BRAF inhibitor, the median PFS was 4 months,

with a median duration of response of 5.7 months.

However, in patients previously treated with a BRAF

inhibitor, response rates were reduced when patients

were treated with trametinib. The phase III open-label

trial results of 322 patients with metastatic melanoma

with BRAF V600E or BRAF V600K mutations random-

ized to receive trametinib or chemotherapy (dacarba-

zine or paclitaxel) were recently published.96 Patients

who progressed on chemotherapy were allowed to

cross over to receive the MEK inhibitor. The median

PFS was 4.8 months in the trametinib group and 1.5

months in the chemotherapy group. Six-month OS

rates were 81% for the trametinib group and 67% for

the chemotherapy group despite patient crossover.

Therefore, although BRAF inhibitors still yield the best

clinical responses to date, trametinib demonstrated

the greatest clinical efficacy of the MEK inhibitors

thus far evaluated in cases of BRAF-mutant melanoma.

ERK inhibitors such as AEZS-131 and SCH772984

are currently being developed and tested. Data on the

clinical efficacy of these compounds are not yet pub-

lished; however, the results of such studies may be of

future use in treating resistant BRAF-mutant melanoma.

Advances in the field of mutational screening

identifying novel, actionable mutations in RAF and

other pathway molecules are likely to present thera-

peutic targets for MEK and ERK inhibition, highlighted

by the work of Dahlman et al97 in which a somatic

BRAF L597R mutation was identified in exon 15 in

wild-type BRAF V600E melanoma during whole ge-

nome sequencing. In vitro, this BRAF L597R-mutant

melanoma showed sensitivity to MEK inhibition. In

addition, a patient with BRAF L597R-mutant melano-

ma had radiographically proven tumor regression to

treatment with TAK-733, a MEK inhibitor, suggesting

that routine screening and therapy of BRAF L597R mu-

tations should be initiated. Although single-mutation

interrogation studies lack depth and whole genome

sequencing is costly, the authors advocate for multi-

plex tests to allow for broader screening capabilities

by testing for common mutations concurrently such

as BRAF (V600E/K/R/D), NRAS (G12/13, Q61), KRAS

(G12, Q61), and several known alterations in Akt,

MEK, EGFR, ERBB2 (HER2/neu), and PIK3CA. One

such assay simultaneously screens for several known

melanoma mutations such as BRAF (V600E/K/M/R/D),

NRAS (G12/13, Q61), KIT (W557, V559, L576, K642,

and D816), GNAQ (Q209), and GNA11 (Q209) and

provides similar advantages, thus allowing for both

cost effectiveness and comprehensiveness.97 These

tests are advantageous because they concurrently ex-

amine several genes and require a smaller quantity of

DNA per test. Thus, moving forward, these assays will

allow for a more efficient identification of commonly

mutated genes and reserve the more costly, whole

genome sequencing for screening less frequent mu-

tations, which may represent future pharmacological

targets in wild-type melanoma.

Combination Therapy: Due to the limited du-

ration of clinical efficacy and the ultimate develop-

ment of resistance to BRAF inhibitors, dual pathway

inhibition and therapeutic combinations of different

pharmacological classes of are currently being in-

vestigated in the treatment of BRAF V600E-mutant

melanoma. A phase I/II trial is underway in patients

with BRAF V600E-mutant melanoma examining the

combination treatment of the BRAF inhibitor, vemu-

rafenib, and the anti-CTLA-4 monoclonal antibody

ipilimumab (NCT01400451).

Because MEK inhibition alone has not been found

to induce high levels of cell death, dual pathway in-

hibition or combination therapies may be needed to

increase efficacy. Reactivation of MAPK signaling is

commonly found in BRAF inhibitor-resistant mela-

noma; however, the combination of MEK and BRAF

inhibitors has been effective in overcoming resistance

mediated by specific molecular aberrations such as

COT overexpression, MEK1 mutations, RAS muta-

tions, and BRAF truncations.66,77,82,83 This suggests

that treatment with combination therapy, in which

multiple alterations in more than one pathway are

overcome, may be required to effectively treat BRAF-

mutant melanoma.

A phase I/II trial combining the inhibitors of

BRAF and MEK demonstrated promising results.98 In

BRAF-mutant tumors, including melanoma, combina-

tion therapy using the MEK inhibitor trametinib and

the BRAF inhibitor dabrafenib in 16 evaluable patients

resulted in 13 patients with a partial response, 3 with

stable disease, and an objective response rate of 81%.

Another trial combining the MEK inhibitor GDC-0973

with vemurafenib is underway (NCT01271803).

BRAF V600E may be a negative regulator of the

Akt pathway. The presence of the BRAF V600E muta-

tion suppresses MEK inhibitor (RG7167)- or mTOR in-

hibitor-induced Akt phosphorylation and downstream

activation. Aberrant activation of the Akt pathway

by PTEN loss overrides the negative impact of BRAF

V600E on phosphorylated Akt, suggesting that induc-

tion of the PI3K/Akt cascade through the loss of PTEN

controls the sensitivity to targeted inhibition.99,100 Due

to the dual activation of RAS/RAF/MEK/ERK and the

PI3K/Akt cascades in melanoma progression and the


role of enhanced Akt signaling in BRAF resistance,

clinical trials have commenced to test combinations

of agents targeting both the MAPK and PI3K path-

ways (NCT01347866, NCT01363232, NCT01392521,

NCT01337765, NCT01390818).

Combinations of MEK and PI3K inhibition or MEK

inhibition combined with chemotherapy agents such

as paclitaxel have shown some enhanced antitumor

effects.85,101 IGF-1R mediated resistance has been ef-

fectively treated with dual MEK and PI3K inhibition,

while PDGFR-α mediated resistance has been over-

come by inhibiting the mTOR/PI3K/Akt pathway.84,102

Lastly, the PI3K/mTOR inhibitor BEZ235 in combina-

tion with the MEK inhibitor MEK162 is undergoing

clinical evaluation for the treatment of melanoma

(NCT01337765).27,84,101,103

NRAS MutationsOncogenic Mechanism of Action

The RAS family of GTPases consists of the homolo-

gous proteins KRAS, HRAS, and NRAS. The N-termi-

nus is common to all proteins; the C-terminal end,

or the hypervariable region, is where RAS proteins

structurally diverge.104 These proteins are small GT-

Pases that alternate between the inactive form bound

to guanosine diphosphate (GDP) and the active form

bound to GTP.105,106 RAS is a regulator of cellular

responses to extracellular stimuli, including growth

factors. When growth factors bind to cell-surface

receptors, intracellular binding sites are created for

molecules to then recruit and activate guanine nucleo-

tide-exchange factors (GNEFs). These activate RAS by

displacing guanine nucleotides, allowing the passive

binding of GTP in the cytosol to RAS. GTP-RAS can

then stimulate multiple effector pathways, including

PI3K, RAF, and Ral guanine nucleotide-dissociation

stimulator to regulate cell proliferation, survival, and

differentiation.107,108 RAS proteins are negatively regu-

lated by GTPase activating proteins (GAPs). These

proteins profoundly enhance intrinsic GTPase activity

by providing stability to the high-energy transition

state that occurs during the hydrolysis reaction of

RAS-GTP. Glutamine at position 61 is required for

GTP hydrolysis, and the substitution of any other

amino acid at this position prevents hydrolysis. Simi-

larly, replacing glycine at position 12 of RAS with

any other amino acid except proline also results in

constitutively activated RAS.109

RAS-GTP binds to and activates several classes

of effector molecules, including class 1 PI3K, RAF

kinases, Ral guanine exchange factors (RalGEFs), the

RAC exchange factor TIAMI, and phospholipase C.110

The RAF/MEK/ERK (MAPK) cascade is the most thor-

oughly described RAS effector pathway.108 Strong

evidence exists to support that the RAF/MEK/ERK

pathway is critical for RAS-induced cell proliferation,

migration, and survival.111 When triggered by RAS, the

three RAF serine/threonine kinases ARAF, BRAF, and

RAF1 serve to activate the MEK/ERK kinase cascade.

ERK kinases can phosphorylate substrates located in

both the nucleus and the cytosol, including transcrip-

tion factors such as JUN and ETS domain-containing

protein (ELK1). Activation of these regulators of

transcription can lead to the expression of proteins

that influence cell cycle progression such as cyclin

D1, which is required for RAS-dependent malignant

transformation.112-114

RAS also plays a role in the PI3K/Akt cascade.

RAS-GTP binds to class I PI3K at the catalytic subunit,

which results in the transfer of PI3K to the plasma

membrane where it becomes activated.115,116 PI3K

phosphorylates phosphatidylinositol-4,5-biphosphate

(PIP2) to generate phosphatidylinositol-3,4,5-triphos-

phate (PIP3), which further triggers downstream ki-

nase activation of ligands such as 3-phosphoinosit-

ide-dependent protein kinase 1 (PDK1) and Akt.117

Akt is a kinase that phosphorylates, and therefore

inactivates, several proapoptotic proteins, serving to

enhance survival of many cell types.118,119

Approximately 30% of all human cancers contain

oncogenic RAS mutations. KRAS mutations are most

prevalent in human neoplasms, including pancreatic,

colorectal, endometrial, biliary tract, lung, and cervi-

cal malignancies.120 Mutations in NRAS, which codes

for a small GTP-binding protein, are usually found in

primary melanoma and melanoma metastases, with

approximately 15% to 18% of cases containing muta-

tions.20,121-124 The most frequent sites of somatic mis-

sense mutations are exons 1 (codon 12) and 2 (codon

61), or, less commonly, exon 13.125,126 More than 80%

of HRAS and NRAS mutations involve position Q61;

however, approximately 70% of NRAS mutations in

melanoma occur at position G12.127 These mutations

lead to impaired GTPase activity, the accumulation

of RAS-GTP, and a lack of sensitivity to the normal

regulation of RAS signaling by guanine nucleotide

exchange factors (GEFs; Sos1/2, RASGRP1-4, and RAS-

GRF1/2 proteins) and GAPs (nuclear factor 1 [NF1]

and p120GAP).110,128

Both NRAS and BRAF mutations can be found in

benign neoplasms such as cutaneous nevi, suggesting

that other genetic alterations are required for malig-

nant transformation of these lesions.17 The vast major-

ity of activating mutations BRAF and NRAS are mutu-

ally exclusive, which suggests that they may function

on the same pathway.18,124,129,130 PTEN loss, a com-

mon genetic alteration in melanoma, is uncommon in

NRAS-mutant melanomas. This finding in melanoma

cell lines suggests that PTEN and the neuroblastoma

RAS viral oncogene homolog (NRAS) may function in

a common molecular cascade.131 Together, these find-

ings indicate that NRAS-mutant melanomas activate


both the RAS/RAF/MEK/ERK pathway and the PI3K/

Akt pathway to induce malignant transformation.

NRAS and BRAF mutations are not equally dis-

tributed among all types of melanoma. Curtin et al21

analyzed genome alterations in DNA copy number

and BRAF and NRAS mutational status in four distinct

groups of melanoma that differed by levels of ultra-

violet light exposure, anatomical location, or both.

These types of melanoma — melanoma arising from

CSD skin, melanoma arising from skin without CSD,

acral melanoma, and mucosal melanoma — demon-

strated varying rates of NRAS and BRAF mutations.

In skin lacking damage due to prolonged ultravio-

let light, mutations in NRAS and BRAF occurred in

81% of melanomas. Alternatively, acral or mucosal

melanomas often were wild-type for BRAF and NRAS

mutations, but they demonstrated an increase in DNA

copy number for cyclin-dependent kinase 4 (CDK4)

and cyclin D1, downstream substrates of NRAS.21,29

The majority of melanomas found on CSD skin were

also wild-type for NRAS and BRAF mutations (76%)

and were significantly more likely to have gains in-

volving the cyclin D1 gene (P = .001), losses involv-

ing chromosome 4q (P = .004), and gains involving

regions of chromosome 22 (P = .004) than melanomas

from non–CSD skin, suggesting that the development

of BRAF and NRAS mutations may be unrelated to

ultraviolet damage.21

Several reports suggest that NRAS-mutant tumors

are distinct from BRAF-mutant melanoma in both

pathological and clinical behavior, and several studies

have shown that NRAS-mutant tumors are correlated

with greater thickness when compared with BRAF

V600E-mutant or wild-type tumors.123,129,130 Conflict-

ing evidence exists on whether or not NRAS-mutant

status correlates with survival outcomes. Multiple

studies have found on multivariate analyses that the

presence of an NRAS mutation, when compared with

wild-type tumors, is an adverse prognostic factor for

melanoma-specific survival (hazard ratio [HR], 2.96;

P = .04)129 and OS rates (HR, 2.05: P = .005).132 NRAS

was also found to correlate with higher rates of mi-

tosis.129 Other studies examining hundreds of pri-

mary melanoma specimens found that NRAS-mutant

melanomas were more often located on the extremi-

ties.130,132 In addition, in comparison to wild-type

tumors, one study found that melanomas containing

NRAS and BRAF mutations were more likely to have

advanced stage at diagnosis, according to the Ameri-

can Joint Committee on Cancer classification.130 This

study found no survival difference between wild-type,

NRAS-mutant, or BRAF-mutant melanoma. One pos-

sible explanation for the correlation of mutant NRAS

with increased tumor thickness is that activating RAS

mutations may be associated with the vertical growth

phase of melanoma. Evidence to support this theory

includes the phenotypical changes seen with RAS on-

cogene transfection into melanoma cell lines, which

include an epithelioid cell morphology, increased

protease production, and increased cell motility, all

of which are features of the vertical growth phase of

melanoma.29,133

Oncogenic NRAS may play a pivotal role in mela-

nomas containing less common BRAF (non–V600E)

mutations. Davies et al19 showed that when melanoma

cell lines were microinjected with Y13-259, a RAS-

targeting monoclonal antibody, proliferation was not

inhibited in cell lines containing BRAF V600D/E muta-

tions. However, in cell lines containing less common

BRAF mutations (G464V, G463V, and L596V), of which

two also harbored RAS mutations, proliferation was

inhibited by Y13-259. This finding suggests that BRAF

V600E-mutant melanoma does not require NRAS for

BRAF signaling, whereas other BRAF mutations still

require RAS interaction for activation.


The role of oncogenic NRAS in the growth and pro-

gression of melanoma suggests the importance of

developing therapies to target mutant NRAS in mela-

noma. Therapeutic approaches to the treatment of

NRAS-mutant melanoma include (1) targeting the

downstream signaling pathways activated by NRAS,

(2) inhibiting NRAS expression through siRNA, and

(3) inhibiting the membrane translocation of NRAS.

Direct inhibition of NRAS has been challenging. RAS

possesses a high affinity for GTP. That fact, combined

with the high concentrations of GTP in the cytosol,

has hindered the development of small molecules

to prevent the buildup of RAS-GTP because of the

complexity in repairing the defective GTPase activity

in mutant NRAS and in disallowing its return to an

inactive GDP-RAS form.134

RAS activation is heavily dependent on translo-

cation to the inner layer of the cell membrane. This

localization requires farnesylation, a post-translational

process mediated by enzymes called farnesyltrans-

ferases.135 Therefore, farnesyltransferase inhibitors

(FTIs) have been an area of interest in targeting RAS.

Niessner et al136 examined the role of lonafarnib, one

such FTI, alone and in combination with MAPK or Akt

pathway inhibitors on cell proliferation, survival, and

tumor growth of melanoma cell lines, two of which

harbored NRAS mutations. Lonafarnib alone or in

combination with Akt pathway inhibitors did not alter

melanoma growth or survival. Combinations of lona-

farnib and MAPK pathway inhibitors demonstrated

some decrease in cell growth. In particular, the com-

bination of lonafarnib and sorafenib demonstrated

synergistic effects on the inhibition of melanoma cell

growth, with enhanced apoptosis and suppressed tu-

mor growth. Lonafarnib affected mTOR signaling,


although it had no effect on MAPK or Akt signaling.

These findings suggest that lonafarnib inhibits mTOR

signaling and enhances sorafenib-induced cell death

in melanoma cells.

MEK as a Therapeutic Target: MEK and ERK

both rely on RAS for activation. Thus, they present

attractive downstream therapeutic targets in NRAS-

mutant melanoma. Solit et al43 demonstrated that

some melanoma cell lines harboring NRAS mutations

are sensitive to MEK inhibition. Dedicated trials in

patients with NRAS-mutant melanoma have not been

conducted; however, clinical studies of MEK inhibi-

tors included patients with NRAS-mutant melanoma.

Although few studies revealed no clinical efficacy or

survival benefit, more recent data suggest that MEK

inhibition may be of clinical use.92,137 In a phase

II study, MEK162, a selective inhibitor of MEK1/2,

demonstrated antitumor effects in patients with both

BRAF- and NRAS-mutant melanoma.138 Among the

29 patients evaluable for efficacy, 13 had tumors har-

boring an NRAS mutation. In addition to patients

with BRAF-mutant melanoma who demonstrated a

response to MEK162 by Response Evaluation Crite-

ria in Solid Tumors (RECIST) criteria, 2 patients with

confirmed partial responses, 1 with an unconfirmed

partial response, and 4 with stable disease were seen

in the mutant NRAS group. This was the first trial in

which a targeted molecular agent showed activity

against NRAS-mutant melanoma.

In melanoma, multiple mechanisms by which tu-

mors are able to acquire resistance to therapy have

been described.139 NRAS oncogenic mutations can be

acquired or NRAS can be upregulated as part of the

BRAF V600E resistance to RAF inhibitors. Subsequent

knockdown of acquired mutant NRAS in these cells

reduced the reactivated signaling through MEK and

ERK and reduced growth of BRAF inhibitor-resistant

tumors.81 Often ERK signaling is reactivated as part

of these resistance mechanisms, which has led to

trials assessing the combination of MEK and BRAF

inhibitors. This combination may be effective for

the treatment of BRAF-mutant tumors secondarily

harboring acquired NRAS mutations. However, it is

unlikely that BRAF inhibitors such as vermurafenib or

dabrafenib in combination with MEK inhibitors have

therapeutic activity in primary NRAS-mutant tumors.

This is due to the fact that in NRAS-mutant tumors in

which BRAF mutations are lacking, BRAF inhibitors

are unable to block RAF signaling.50,51

Recent data suggest that other combinations of

targeted agents, such as CDK4 and MEK, may be effec-

tive for the treatment of NRAS-mutant melanomas.140

Using an inducible mouse model of NRAS-mutant

melanoma, inhibition of MEK with either selumetinib

or trametinib induced apoptosis and caused tumor

stasis but did not trigger cell cycle arrest. By contrast,

genetic extinction of NRAS Q61K resulted in both

cell cycle arrest and apoptosis, causing tumor regres-

sion. Further investigation using network modeling

concluded that CDK4, a regulator of the G1/S cell-

cycle checkpoint, was a key driver of the phenotypi-

cal difference between MEK inhibition and mutant

NRAS extinction. In vivo, the combination of MEK

inhibition using selumetinib or trametinib and CDK4

inhibition using palbociclib, a dual selective inhibitor

of CDK4 and CDK6, led to both cell cycle arrest and

apoptosis and resulted in clear tumor regression.140

This therapeutic synergy was apparent both in a xe-

nograft model and in NRAS-mutant human melanoma

cell lines, suggesting novel pathway targets for the

future treatment of NRAS-mutant melanoma.

PI3K/Akt as a Therapeutic Target: RAS has

multiple downstream effectors and is involved in both

the MEK/ERK and PI3K pathways. Recent evidence

suggests that the dual inhibition of the MEK/ERK and

PI3K pathways may be effective for the treatment of

RAS-mutant tumors due to interdependence of the two

pathways.141,142 Evidence of pathway codependence

includes the finding that BRAF or NRAS mutations

promote the formation of benign nevi, but the activa-

tion of ERK inhibits further tumor development. Akt3

activation is then required to bypass the effect of ERK

and allow for the progression to melanoma.27,103 In

addition, Akt3 has been shown to promote melanoma

proliferation through the phosphorylation of Ser364

and Ser428 on BRAF V600E, reducing the levels of

BRAF V600E to levels that promote tumor spread.103

Jaiswal et al143 provided further evidence to support

the use of this dual pathway inhibition. Using an in

vivo mouse model of NRAS-mutant melanoma, they

showed that inhibiting NRAS alone or the downstream

effectors of mutant NRAS leads to cell cycle arrest and

cell death. This further emphasizes the concept that

to treat melanoma tumors containing mutant NRAS,

the sustained downstream inhibition of Akt and ERK

is required.

mTOR as a Therapeutic Target: mTOR, a serine/

threonine kinase, is activated downstream in the PI3K

pathway and stimulates cell proliferation through con-

trol of cell-cycle progression regulators and may pres-

ent another target in NRAS-mutant melanoma. A phase

II clinical trial in patients with advanced melanoma

examined sorafenib in combination with tipifarnib, an

FTI, or temsirolimus, an mTOR inhibitor, and failed to

show significant clinical efficacy.144 However, patient

mutational status was not assessed in this study, thus

limiting the ability to determine whether the drugs

would be effective for the treatment of NRAS-mutant

melanoma. Lastly, although no therapies exist that

specifically target mutant NRAS, clinical trials are cur-

rently underway exploring the use of dual therapy

against downstream substrates of NRAS.


KIT MutationsOncogenic Mechanism of Action

The type III transmembrane receptor tyrosine kinase

KIT gene was first described in 1987 by Yarden et

al145 and Woodman et al.146 Structurally, KIT is char-

acterized by five unique domains: a glycosylated

extracellular ligand-binding domain containing five

immunoglobulin-like repeats (encoded by exons 1–9),

a hydrophobic transmembrane domain (encoded by

exon 10), an intracellular segment consisting of a jux-

tamembrane domain (encoded by exon 11), and two

intracellular tyrosine kinase domains separated by a

kinase insert region (encoded by exons 12–21).146,147

Distinct domains serve unique functional roles. The

juxtamembrane domain serves an autoinhibitory role;

when a ligand is absent, it prevents KIT activation.

Activation occurs with binding of stem cell factor, a

glycosylated transmembrane protein and the ligand

of KIT, to the extracellular domain, causing the KIT

tyrosine kinase receptor to dimerize. This dimeriza-

tion results in autophosphorylation of the intracellular

tyrosine kinase domains. Activated KIT then initiates

signaling through a variety of downstream pathways,

including in the MAP/MEK/ERK, PI3K/Akt, and JAK/

STAT pathways.146-148 Loss-of-function studies have

shown that KIT activation and downstream pathway

signaling may be important in multiple types of cells,

including germ cells, mast cells, hematopoietic stem

cells, and the interstitial cells of Cajal.149-151

Several studies have demonstrated the significant

role of KIT in the development of normal melanocytes.

Mutations resulting in loss of functional KIT in mice

and humans lead to loss of pigmentation of fur and

skin/hair, respectively.152-155 Although now recognized

as an oncogene, KIT was initially believed to serve as

a melanoma tumor suppressor. Transfection of the

highly metastatic melanoma cell line A375SM with

KIT led to reduced tumor growth and metastases in

mice injected with A375SM-KIT cells. In addition, KIT-

positive melanoma tumor cells demonstated a higher

level of apoptosis when stem cell factor was given.156

Other studies demonstrated that loss of KIT expres-

sion correlated with malignant melanocytic transfor-

mation, invasion, and metastases.157-159

Much of the current knowledge of KIT and its

function as an oncogene has come through experience

with other tumor models, most significantly GISTs.

Activating mutations in KIT have been identified both

in and outside of the juxtamembrane domain in GIST

tumors.160,161 Imatinib mesylate, a tyrosine kinase in-

hibitor known to target KIT,162 was administered in a

phase II trial in which patients with unresectable or

metastatic GISTs received 400 or 600 mg of the drug

daily.163 In this trial, 79 out of 147 patients (53.7%)

had a partial response and 41 patients (27.9%) had

stable disease. This trial revolutionized the concept

of targeted therapy and advocated for the use and

efficacy of targeted therapy in the treatment of ma-

lignancies harboring specific mutations.

More recently, KIT mutations have been identified

in melanoma. Similar to BRAF and NRAS mutations,

KIT mutations in melanoma have different frequencies

depending on the distinct type of melanoma. Although

BRAF and NRAS mutations most often arise from non–

CSD skin,21 KIT-activating mutations and/or increased

copy number are more common in acral and mucosal

melanomas and in those arising from CSD skin.164-166

In addition, distinct anatomical locations may have

unique molecular characteristics.167,168 For example,

vulvar melanomas have been found to contain a higher

rate of KIT mutations than melanomas arising from

other sites, including vaginal melanomas.168

The prevalence of KIT mutations in melanoma

varies by domain. Exon 9, which encodes for the

extracellular domain, is mutated in 6% of cases. Exon

11, which encodes for the juxtamembrane domain, is

mutated in 46% of cases. Exon 13, which encodes for

the proximal tyrosine kinase domain, is mutated in

19% of cases. Lastly, exons 17 and 18, which encode

for the distal tyrosine kinase domain, are mutated in

10% and 19%, respectively.169


Of the 102 melanomas analyzed by Curtin et al,165 the

majority of KIT mutations identified affected exon 11,

encoding the juxtamembrane domain. Mutations in

this domain, which is responsible for the autoinhi-

bition of KIT when unbound to its ligand, result in

enhanced ligand-independent receptor dimerization

and activation.170 GIST tumors that possess exon 11

mutations of KIT are sensitive to imatinib treatment,171

suggesting that KIT-mutant melanoma may similarly

respond to this targeted therapy.

Studies have examined KIT sensitivity to tyro-

sine kinase inhibitors in vitro. One such study by An-

tonescu et al172 examined anal tumors with KIT exon

11 L576P substitutions. The authors demonstrated

sensitivity of exon 11 KIT L576P-mutant tumors to

dasatinib as well as to imatinib. Another study simi-

larly showed KIT L576P-mutant cell line sensitivity to

dasatinib but not to imatinib, nilotinib, or sorafenib.173

Other studies demonstrated in vitro sensitivity of KIT-

mutated acral and mucosal melanomas to KIT inhibi-

tors sunitinib174 and imatinib.175

Early Clinical Trials and Case Reports of KIT

Treatment: Early phase II trials of imatinib in the

treatment of metastatic melanoma demonstrated lim-

ited clinical responses to therapy. This was largely at-

tributed to the fact that the presence of KIT mutations

in melanoma was not known at the time these studies

were initiated; thus, patient selection by molecular

criteria was not possible.176 Several studies assessed for


KIT expression by immunohistochemistry in a subset

of enrolled patients, with many demonstrating weakly

positive KIT staining.177,178 Similar findings were docu-

mented in a phase II study of dasatinib for the treat-

ment of patients with melanoma.179 Patients were not

selected for enrollment based on documented muta-

tional status; however, 1 patient who harbored an exon

13 KIT mutation did exhibit a response to treatment.

Although these early phase II clinical trials were

a disappointment in the efforts to develop targeted

treatments for melanoma, smaller case reports sup-

port the ongoing investigation of targeted molecular

therapy against KIT-mutant tumors. Two unique case

reports involving 2 women 79 years of age with recur-

rent metastatic rectal melanoma treated with imatinib

were documented to have significant benefit.180,181 The

first woman was found to have a 7-codon duplication

in exon 11 of KIT and strong immunohistochemistry

staining for KIT.180 With a dose of 400 mg daily of ima-

tinib, she achieved measurable reduction in metastatic

burden, as well as symptomatic relief of her rectal and

vaginal bleeding. The second patient had an exon

11 KIT L576P missense mutation.181 She also had a

documented reduction in metastatic burden and im-

provement in her performance status with treatment.

In several other case reports, patients harboring mela-

noma characterized by KIT mutations demonstrated

major durable responses to treatment with other KIT

tyrosine kinase inhibitors such as nilotinib, dasatinib,

sunitinib, and imatinib.173,182-184

Selected Clinical Trials of Mutant KIT: Based

on the findings in these case reports that suggest the

efficacy of imatinib in KIT-mutant melanoma, three

subsequent phase II trials were conducted. Unlike

prior studies, documentation of somatic alterations of

KIT (either mutation or amplification) was required

for enrollment.

In one study led by the Memorial Sloan-Kettering

Cancer Center, 25 patients with melanoma arising

from acral, mucosal, or CSD skin were treated with

imatinib 400 mg twice daily.169 Of the evaluable 25

patients, 6 achieved a radiographic response, and

4 experienced durable responses, each lasting for

more than 1 year. Two patients achieved complete

responses lasting 94 and 95 weeks, 2 durable partial

responses lasting 53 and 89 weeks, and 2 transient

partial responses lasting 12 and 18 weeks. Specific

KIT mutations corresponded to clinical responses.

Exon 11 KIT L576P and exon 13 KIT K642E mutations

were stronger predictors of clinical response. All 6

patients who responded to imatinib carried an exon

11 KIT L576P or an exon 13 KIT K642E mutation.

Although 2 patients who progressed harbored exon

11 KIT L576P mutations, both patients with complete

responses to imatinib had this alteration as well as

concomitant amplification.

Similar results were shown in a phase II trial

of Chinese patients with metastatic melanoma har-

boring KIT alterations treated with imatinib 400 mg

daily (NCT00881049).185 Of 43 evaluable patients, 10

(23.3%) had a partial response, 13 (30.2%) had stable

disease, and 20 (46.5%) demonstrated progressive dis-

ease (46.5%). Similar to the findings by Carvajal et al,169

9 of the 10 patients demonstrating partial responses

harbored mutations in exon 11 or 13. These findings

suggest that patients with specific KIT mutations, such

as exon 11 and 13, may benefit from targeted therapy

more than those without such mutations.

Second-generation dual Src/Bcr/Abl receptor tyro-

sine kinase inhibitors such as nilotinib and dasatinib

are also being studied. Dasatinib has demonstrated

preclinical activity in KIT-mutant melanoma, espe-

cially in patients harboring the exon 11 L576P muta-

tion. Presentation of preliminary results of a phase II

trial of dasatinib in patients with unresectable locally

advanced or stage IV mucosal, acral, or solar mela-

nomas demonstrated poor response in patients with

wild-type KIT.186 Of the 54 patients in the trial (26

mucosal, 13 acral, 15 solar melanomas), 1 had a com-

plete response (unknown KIT status), 2 achieved a

partial response, 13 had stable disease, 29 had disease

progression, and 6 were unevaluable. Forty of the 54

patients died. KIT status was assessed in 42 patients,

with 39 wild-type KIT (93%) and 3 KIT-mutant tumors

(7%). All patients with wild-type KIT and 2 of the

3 patients with mutant KIT died. This prompted a

modification to include KIT-mutant patients, given the

lack of efficacy in patients with wild-type KIT mela-

noma. Accrual to this amended protocol is ongoing.

KIT Resistance: Primary and secondary resis-

tance to imatinib can arise. Potential mechanisms of

secondary resistance include the development of sec-

ondary KIT or NRAS mutations, genomic KIT amplifi-

cations, activation of downstream pathway signaling,

and decreased imatinib plasma concentrations due

to inhibitor-binding proteins.187 Overcoming these

mechanisms may require the selection of alternative

KIT inhibitors specific for resistant mutant tumors,

combination therapy of several KIT inhibitors, or a

dual blockade with KIT and other pathway inhibitors.

Several lessons on secondary resistance mecha-

nisms of KIT can be learned from the GIST model to

improve efficacy in the future treatment of melanoma.

In GIST, the development of secondary mutations,

especially those in the tyrosine kinase domains en-

coded by exons 13 and 17, leads to the development

of secondary resistance.188 Study results in which

patients with acral, mucosal, or CSD melanoma were

tested for KIT and other molecular mutations revealed

that patients with KIT mutations had clinically effec-

tive (both partial and complete) responses to treat-

ment with sunitinib.184 A patient with mutant KIT


who initially had no other detectable mutations in

BRAF or NRAS responded to treatment with sunitinib;

however, the patient progressed after 7 months and

the resistant tumor was found to harbor a new NRAS

Q61K mutation in addition to the previously present

KIT mutation. Subsequent treatment with imatinib was

not effective. Thus, the development of additional

mutations may contribute to tyrosine kinase inhibitor

resistance in melanoma.

Si et al189 reported a patient with nasal cavity mu-

cosal melanoma harboring an exon 11 KIT mutation.

Although the patient initially achieved a partial re-

sponse to imatinib, she progressed after 1 year. Sub-

sequent evaluation of the tumor recurrence showed no

new mutations; however, the increased phosphoryla-

tion of Akt and ERK1/2 was seen in samples obtained

following imatinib treatment compared with tumor

samples taken before imatinib treatment. No increased

expression of cyclin D or phosphorylated MEK1/2

was present, suggesting that the mTOR pathway was

selectively activated. When treated with the mTOR

inhibitor everolimus, the patient achieved a partial

response, with an overall tumor shrinkage of 35%.

In GIST, treatment with alternative KIT inhibitors

following progression on imatinib has been effective.190

Such a strategy in melanoma is being tested in a phase

II study for KIT-mutant melanoma in which nilotinib

is being administered after resistance or intolerance to

prior KIT inhibitor therapy (NCT01028222). Nilotinib

has similar potency to imatinib but differs in that it is

rapidly transported into the cell via passive diffusion

as opposed to the time-dependent, active transport of

imatinib. Combining KIT tyrosine kinase inhibitors

with chemotherapy, immunotherapy, or other targeted

therapies may also present other opportunities to treat

patients who develop resistance to initial therapy.

GNAQ and GNA11 MutationsOncogenic Mechanism of Action

Uveal melanoma (UM), the most common primary

malignancy of the eye, accounts for approximately 5%

of all melanomas.191,192 It originates from melanocytes

of the choroid plexus, ciliary body, or iris of the eye

and may involve more than one of these anatomical

structures.193 Unlike cutaneous melanoma, UM is not

characterized by mutations in BRAF, NRAS, or KIT194;

it has unique cytogenetic alterations195 and has a pro-

pensity to hematogenously metastasize to the liver.196

Despite the lack of activating mutations in BRAF,

NRAS, and KIT, activation of the MAPK pathway in

UM is critical to the development and progression

of this malignancy.197 Guanine nucleotide-binding

protein Q polypeptide (GNAQ) encodes for the alpha

subunit of a heterotrimeric G protein (Gaα, which

couples transmembrane domain receptors to intra-

cellular signaling pathways such as MAPK in 45%

to 50% of patients with UM.198,199 The alpha subunit

serves as the molecular control for the G protein,

which is active when bound to GTP and inactivated

when GTP is hydrolyzed to GDP.200,201 If alterations

of specific glutamine or arginine residues exist in the

alpha subunit that contact the GTP molecule, then its

fundamental GTPase activity is obstructed. Thus, the

alpha subunit locks the G protein in a constitutively

active, GTP-bound state (Fig 3).201,202 An activating

somatic mutation in this critical glutamine in exon

5, the RAS-like domain of GNAQ, located at position

209 (Q209) in Ga, is mutated to either a leucine or

proline in melanocytic lesions (designated as GNAQ

Q209L/P).199 Van Raamsdonk et al199 sequenced the

entire coding region of GNAQ in both benign and

malignant melanocytic tumors. They found 46% of

primary UMs contained mutations in the glutamine

residue at position 209 of GNAQ. In mice, this muta-

tion leads to the melanocytic proliferation and cooper-

ates with other oncogenes to cause malignant trans-

formation.199,203 Onken et al204 also independently

identified activating mutations of GNAQ at position

209 in 49% of primary UM specimens, including 22%

of iris melanomas and 54% of posterior UMs.

Human melanocytes, when transfected with mu-

tant GNAQ Q209L, exhibited increased levels of phos-

phorylated ERK compared with control cells trans-

fected with wild-type GNAQ or an empty vector.199

These transfected human melanocytes containing

GNAQ Q209L demonstrated anchorage-independent

growth. When nude mice were injected with mela-

nocytes transfected with GNAQ Q209L, heavily pig-

mented tumors developed, which was not observed

with cells transfected with wild-type GNAQ. In ad-

dition, knockdown of GNAQ in GNAQ-mutated UM

cell lines resulted in a reduction of phosphorylated

ERK levels, a reduction in cell number, and a loss

of anchorage-independent growth. These findings

are also demonstrated when OMM1.3, a UM cell line

containing the GNAQ Q209L mutation, is treated with

the MEK inhibitor UO126.199

In patients with UM that is wild-type for GNAQ,

more than 50% possess a gain-of-function mutation

in GNA11, a paralog of GNAQ.205 GNA11, similar

to GNAQ, encodes for a member of the q class of

G-protein alpha subunits and signals through phos-

pholipase C (PLC) and protein kinase C (PKC). The

GNA11 exon 5 mutation most commonly seen results

in a Q209L substitution, which is identical to that seen

in GNAQ. GNAQ and GNA11 hypermorphic mutations

result in amplified numbers of intradermal melano-

cytes in mouse models.203 Similar to findings seen

with GNAQ transfection, Melan-A cells transfected

with mutant GNA11 lead to the activation of phos-

phorylated ERK.205 A study by Van Raamsdonk et al205

found that the majority of benign uveal nevi and 83%


of UMs contained either the GNAQ Q209 mutation or

the GNA11 R183 mutation in a mutually exclusive pat-

tern. These mutations cause constitutive activation of

the Gαq or Gα11 subunits by blocking their intrinsic

GTPase activity required to return them to the inac-

tive state. However, these mutations alone are not

adequate for complete malignant transformation to

melanoma, suggesting that GNAQ/GNA11 mutations

may be initiating events in UM progression.199

Conversely, other mutations such as BAP1 tend to

occur later in UM progression and have been linked to

the development of metastatic disease. BRCA1-asso-

ciated protein 1 (BAP1) is a deubiquitinating enzyme

that forms part of a tumor-suppressor heterodimeric

complex.206 In sequencing for metastasis-related mu-

tations in UM, Harbour et al207 discovered that inac-

tivating somatic mutations of BAP1 on chromosome

3q21.1 were present in 26 of 31 (84%) metastasizing

UM tumors. Of these 26 mutations, 15 caused pre-

mature protein termination and 6 altered the ubiq-

uitin carboxy-terminal hydrolase (UCH) domains.

These mutations usually occur in metastatic tumors

that have also lost the other copy of chromosome 3,

implicating BAP1 as a recessive cancer gene. BAP1

germline mutations, associated with a familial cancer

syndrome involving UM and cutaneous melanoma as

well as mesothelioma, have also been described.208,209

Thus, BAP1 appears to be involved in the metasta-

sis development of UM and may represent a future

therapeutic target.


Current efforts in the development of targeted therapy

for melanoma harboring GNAQ/GNA11 mutations fo-

cus on the inhibition of downstream signaling arising

from these mutations.210,211 Similar to the difficulties

in targeting mutations in the RAS family of oncogenes,

directly targeting mutant GNAQ or GNA11 may prove

problematic.196 Mutations in GNAQ and GNA11 alter

the intrinsic GTPase activity that would normally al-

low the proteins to return to their inactivated forms in

which they are bound to GDP. Potential downstream

targets include MEK, which is involved in the MAPK

pathway activated by GNAQ/GNA11 mutations, PLC,

which is activated by Gq, and PKC, which is activated

downstream of PLC.210,211

MEK as a Therapeutic Target: UM cell lines

containing GNAQ or GNA11 mutations are character-

ized by elevated levels of phosphorylated ERK. When

treated with increasing concentrations of selumetinib,

wild-type GNAQ cells demonstrated resistance; how-

ever, GNAQ- and GNA11-mutant cell lines exhibited a

Fig 3. — Mechanism of GNAQ signaling. GNAQ polypeptide encodes the alpha subunit of heterotrimeric G proteins, which couples the cell surface, 7-trans-membrane domain receptors to intracellular signaling pathways. Ligand binding triggers receptor activation that catalyzes the exchange of GTP for GDP bound to the inactive G protein alpha subunit, resulting in a conformational change and dissociation of the G protein beta-gamma subunits. A somatic mutation of glutamine in exon 5 located at position 209 (Q209) in G-alpha subunit inactivates the catalytic domain, preventing hydrolysis of GTP to GDP and thus fixing GNAQ in its constitutively active, GTP-bound, oncogenic state. GDP = guanine diphosphate, GNAQ = guanine nucleotide-binding protein Q, GTP = guanosine triphosphate.

GTP GDP

GDP

G Protein Coupled Receptor

Cell Membrane

Ligand

Exon 5 Q209L or

Q209P Mutation


high degree of sensitivity to the drug at IC50s in the

range of 100 to 250 nmol/L.196,212 Recently, Ambrosini

et al212 identified the MEK-dependent transcriptional

output unique to GNAQ-mutant cells, with MEK in-

hibition resulting in the downregulation of a unique

set of genes involved in cell proliferation, tumor cell

invasion, and drug resistance.

Given the known MEK-ERK pathway activation

in UM as a result of GNAQ and GNA11 mutations, in-

hibition of this pathway is of interest. A multicenter

phase I study of trametinib, a reversible, selective

allosteric inhibitor of MEK1/2 activation and kinase

activity, was performed in patients with advanced

melanoma.213 Sixteen of the 97 patients enrolled had

UM, 6 of whom had a documented GNAQ or GNA11

mutation. Of the 16 patients with UM, most of whom

were heavily pretreated, none had a documented par-

tial or complete response, although stable disease was

noted in 4 patients (25%).

A phase II randomized clinical trial of temozolo-

mide vs hydrogen sulfate selumetinib has been initi-

ated in patients with either wild-type or mutant GNAQ

or GNA11 metastatic UM (NCT01143402).197 Accrual

to the GNAQ/GNA11-mutant cohort of the trial was

completed, with study results anticipated to be avail-

able in the near future.

PKC as a Therapeutic Target: Through its

multiple cellular functions, such as the regulation

of proliferation, apoptosis, differentiation, angiogen-

esis, and tumor development, PKC provides another

therapeutic target through which GNAQ- and GNA11-

mutant melanoma can be treated. PKC molecules

are serine/threonine kinases that exist in multiple

functionally distinct isoforms.214,215 The conventional

PKCs (PKCα, PKCβ, and PKCγ) are activated by dia-

cylglycerol (DAG) and phospholipids and are calci-

um-dependent, while the novel PKCs (PKCδ, PKCε, PKCθ, and PKCη) are activated by DAG and phos-

pholipids and, by contrast, are calcium-independent.

Lastly, the atypical PKCs (PKCλ and PKCζ) do not

require DAG or calcium for activation.

Recent work by Wu et al216,217 demonstrated the

significance of PKC as a therapeutic target. Through

the treatment of both wild-type and GNAQ-mutant UM

cell lines with the PKC inhibitors sotrastaurin and en-

zastaurin, they showed that PKC inhibitors selectively

block UM viability in a mutation-dependent manner.

Sotrastaurin and enzastaurin, through the induction

of G1 cell cycle arrest and apoptosis, significantly in-

hibited the growth of UM cells harboring GNAQ muta-

tions, while wild-type UM growth was not appreciably

altered. Sotrastaurin suppressed the expression and

phosphorylation of PKC α, PKCβ, PKCδ, PKCε, and

PKCθ in GNAQ-mutated UM cells. Additional findings

from small hairpin RNA (shRNA)-mediated knock-

down studies revealed that these PKC isoforms are

functionally important for UM cells harboring GNAQ

mutations.216 Furthermore, the inhibition of PKC in

GNAQ-mutant UM resulted in the inhibition of the

MAPK pathway by targeting the PKC/ERK1/2 and

PKC/NF-κB pathways. Thus, PKC inhibition offers

novel therapeutic potential for UM with GNAQ muta-

tions. A phase I clinical trial examining sotrastaurin

for the treatment of UM is underway (NCT01430416),

and trials to explore the PKC blockade in combina-

tion with other pathway inhibitors, such as the PI3Ka

inhibitor BYL719, are in development.

PI3K/Akt as a Therapeutic Target: PI3K ac-

tivation has been shown in UM cell lines. PI3K is

activated by both G-protein receptors and receptor

tyrosine kinases. Activated PI3K is responsible for

the conversion of PIP2 to PIP3, which then mediates

the translocation Akt to the membrane where it is

activated. Akt is involved in several signaling path-

ways of cell proliferation and survival. PTEN, which

converts PIP3 to PIP2, antagonizes PI3K signaling and

decreases Akt activation.211 A decrease in PTEN levels

or loss of PTEN expression has been demonstrated in

aggressive primary UMs and portends a worse survival

when compared with normal levels of PTEN.219

Inhibition of PI3K with LY294002 in UM cell lines

resulted in decreased cell proliferation. Recent work

by Khalili et al218 showed that knockdown of GNAQ or

GNA11 resulted in decreased MAPK phosphorylation

in UM cell lines containing GNAQ or GNA11 muta-

tions, respectively, but not in wild-type cells, confirm-

ing that activated GNAQ or GNA11 signals through

the MEK/MAPK pathway in GNAQ- or GNA11-mutant

UM, respectively. They also demonstrated that loss of

GNAQ or GNA11 mutations had no significant effect

on Akt phosphorylation in either GNAQ- or GNA11-

mutant or wild-type cells. Inhibition of MEK, and

therefore MAPK signaling, resulted in the reciprocal

activation of Akt regardless of GNAQ- or GNA11-mu-

tant status. They further demonstrated that MEK or

PI3K inhibition alone achieved cell-cycle arrest and

reduced growth in most UM cells but resulted in small

amounts of apoptotic death. However, combination

treatment with MEK and PI3K inhibition resulted in

a significant amount of apoptosis, which was most

appreciable in GNAQ-mutant cells, but it was also

evident in the majority of GNA11-mutant cells. These

preclinical data support the use of combination target-

ed therapy in the treatment of UM containing GNAQ/

GNA11 mutations.219 Clinical trials assessing the safety

and efficacy of dual-pathway inhibition for advanced

UM are currently in development.

mTOR as a Therapeutic Target: mTOR, a serine/

threonine kinase activated downstream in the PI3K

pathway, stimulates cell proliferation by controlling

cell-cycle progression regulators. There are two dis-

tinct mTOR complexes: mTORC1 (mTOR complex 1,


rapamycin sensitive) and mTORC2 (mTOR complex

2, rapamycin insensitive). mTORC is activated via the

PI3K pathway through the tuberous sclerosis com-

plex (TSC) and Akt.220 Activated Akt phosphorylates

TSC2, causing a dissociation of the TSC1/TSC2 com-

plex, thereby inhibiting TSC2 from acting as a GTPase-

activating protein. This allows the G-protein Rheb to

remain in a GTP-bound state and activate mTORC1. In

addition, mTORC1 can be activated by Akt through the

phosphorylation of PRAS40, thus relieving the PRAS40

inhibition of mTORC1.221,222 When UM cell lines are

treated with rapamycin, an mTOR inhibitor, at levels

that completely inhibit downstream mTOR signaling,

they exhibit a reduction in cell proliferation levels

by 9% to 21%.223 This is likely due to the fact that

mTOR inhibition induces Akt activation through the

loss of the mTOR pathway-dependent inhibition (nega-

tive feedback) of IGF-1R signaling. IGF-1R inhibition

terminates mTOR inhibition-mediated Akt activation

and confers sensitivity to mTOR inhibition in cancer

cells.224 Therefore, IGF-1R–mediated feedback activa-

tion of PI3K signaling appears to confer resistance

to mTOR inhibitors, and mTOR inhibition alone is

unlikely to be effective alone in the treatment of UM.

A recent study examining combination treatment

of UM cells with MEK and mTOR inhibitors demon-

strated that efficacy was linked to tumor genotype.225

The ATP-competitive mTOR kinase inhibitor AZD8055

and the MEK inhibitor selumetinib did not show co-

operative antitumor effects in wild-type cells. In both

BRAF- and GNAQ-mutant cells, combination therapy

suppressed cell viability; however, apoptotic death

was seen in BRAF-mutant cells. This distinction may

reflect differences in how BRAF and GNAQ mutations

activate MAPK signaling. BRAF directly activates MEK

and ERK activity, while GNAQ activates MAPK through

PKC activation, which can facilitate cell survival signals

through several pathways in parallel to the MAPK

pathway. When GNAQ was suppressed in GNAQ-mu-

tant cells, higher resultant apoptosis was present when

treated with combination AZD8055/selumetinib, sug-

gesting that GNAQ activity in these cells activates MEK

and mTOR-independent prosurvival signals. Further

study is needed for the development of agents directly

targeting GNAQ and GNA11, as well as other down-

stream targets, to achieve better therapeutic efficacy.

ConclusionsAlthough it has long been considered a clinically di-

verse disease, advanced melanoma has been, until

recently, uniformly treated. This has been an unsuc-

cessful therapeutic strategy both in controlling disease

progression and in improving OS rates. Increasing evi-

dence suggests that future successful melanoma treat-

ment requires a better understanding of the inherent

biological and molecular alterations associated with

melanoma. As demonstrated by numerous preclinical

and clinical studies, one treatment will not work for

all tumors or all molecular types. The development of

targeted therapy aimed at individual molecular altera-

tions, specific to each individual melanoma, is likely

to result in more successful clinical outcomes.

The MAPK and PI3K pathways have emerged as

key aberrant pathways in melanoma for which spe-

cific inhibitors now exist. However, the complexity

of these cascades is an ongoing challenge in terms of

developing novel therapies. In addition to overcom-

ing mechanisms of resistance, future directions for the

development of effective melanoma therapies include

the identification of novel oncogenic mutations in the

subset of wild-type melanoma for currently known

molecular drivers, which may predict sensitivity or

resistance to specific pharmacological agents. Mul-

tiagent therapy using molecular inhibitors, molecular

inhibitors combined with chemotherapy, or pathway

inhibition combined with immunotherapy may be all

that is needed for the effective treatment of this het-

erogeneous disease.

Current progress and increased knowledge into

the biological basis of melanoma will aid in the cre-

ation and advancement of new therapeutic agents,

allowing therapy to be tailored to specific patients

based on the molecular characteristics of their tumors

that is likely to yield insightful results for the future

treatment of melanoma.

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Melanoma Mutagenesis and Aberrant Cell Signaling · additional targeted therapies possible....

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