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EZH2: an emerging role in melanoma biologyand strategies for targeted therapyJessamy Tiffen, Stuart J. Gallagher and Peter Hersey

Submit your next paper to PCMR online at http://mc.manuscriptcentral.com/pcmr

DOI: 10.1111/pcmr.12280

EZH2: an emerging role in melanoma biology andstrategies for targeted therapyJessamy Tiffen, Stuart J. Gallagher and Peter Hersey

Melanoma Research Group, Kolling Institute of Medical Research, University of Sydney, St Leonards, NSW,Australia

CORRESPONDENCE P. Hersey, e-mail: peter.hersey@sydney.edu.au

KEYWORDS EZH2/H3K27me3/histone methyltrans-ferase/melanoma/small molecule inhibitor/epigenetics

PUBLICATION DATA Received 4 March 2014,revised and accepted for publication 27 May 2014,published online 10 June 2014

doi: 10.1111/pcmr.12280

Summary

Histone modifications are increasingly being recognized as important epigenetic mechanisms that govern

chromatin structure and gene expression. EZH2 is the catalytic subunit of the polycomb repressive complex 2

(PRC2), responsible for tri-methylation of lysine 27 on histone 3 (H3K27me3) that leads to gene silencing. This

highly conserved histone methyltransferase is found to be overexpressed in many different types of cancers

including melanoma, where it is postulated to abnormally repress tumor suppressor genes. Somatic mutations

have been identified in approximately 3% of melanomas, and activating mutations described within the catalytic

SET domain of EZH2 confer its oncogenic activity. In the following review, we discuss the evidence that EZH2 is

an important driver of melanoma progression and we summarize the progress of EZH2 inhibitors against this

promising therapeutic target.

Introduction

It is increasingly recognized that the epigenetic state of

cancers may be dynamic as a result of epigenetic drift

within the tumor population as well as changes induced

by activation of oncogenic signaling pathways. It is

thought that these epigenetic changes may contribute

to the plasticity of cancers and development of resistance

to a wide range of therapies (van den Hurk et al., 2012).

The mechanisms involved in epigenetic gene regulation

include not only methylation of cytosines in DNA but also

by effects on histones, around which DNA is wrapped to

form nucleosomes. Several protein families have been

identified as mediating effects on histones by ‘marking’

them with acetyl or methyl groups. Indeed fifteen

chromatin states were described on the basis of different

marks with 9 acetyl and methyl groups (Arrowsmith

et al., 2012). These concepts have resulted in the

description of protein groups which are writers (e.g.,

methylases, including EZH2), erasers (e.g., histone

deacetylases; HDACs), or readers (e.g., bromodomain

proteins) of these marks.

As reviewed elsewhere, it is clear that much of the

dysfunctional protein transcription in cancers is due to

abnormalities in chromatin complexes (Dawson and

Kouzarides, 2012; Dawson et al., 2012). Loss-of-function

mutations in the chromatin remodeling SWI/SNF complex

proteins (ARID2A, ARID1A, SMARCA4) were identified in

approximately 13% of melanomas by whole-genome

sequencing studies (Hodis et al., 2012). Aberrant activity

of several histone methyltransferases has also been

implicated in melanoma (Table 1). These findings have

been made particularly relevant by the discovery of a

number of drugs that target components of the methyl-

transferase complexes and which promise to be effective

treatments for cancer (Popovic and Licht, 2012).

The EZH2 (enhancer of zeste homolog 2) protein is of

particular interest in epigenetic regulation of gene expres-

sion. It is part of the polycomb repressive complex 2

(PRC2) that is conserved across organisms from plants to

humans (O’Meara and Simon, 2012) and mediates its

effect by tri-methylation of lysine 27 on H3 histones

(H3K27me3) which is predominantly associated with

transcriptional repression (Cao et al., 2002). EZH2 is

essential in early development but downregulated in

normal adult tissues, and EZH2 knockout mice display

embryonic lethality post-implantation (O’Carroll et al.,

2001). Polycomb-mediated H3K27me3 has previously

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 1

Pigment Cell Melanoma Res. REVIEW

been recognized in the transcriptional silencing of differ-

entiation genes, for example Hox transcription factors,

and in the early stages of mammalian X-chromosome

inactivation (Jones and Gelbart, 1990). Several reviews

have drawn attention to the role of EZH2 in repressing

differentiation genes in embryonic stem (ES) cells and

thereby maintaining their pluripotency but allowing

expression of genes involved in cell division (Simon and

Lange, 2008). Parallels have been drawn to a potentially

similar role for EZH2 in cancer in promoting self-renewal

and impeding differentiation. For example, a direct link

was shown between poorly differentiated breast and

bladder cancer cells and the ES cell state as shown by

shared gene signatures defined by Oct4/Sox2/Nanog in

ES cells and suppression of PRC2 target genes in cancer

cells (Ben-Porath et al., 2008). In this sense, the genes

driving differentiation in ES cells may be regarded as

similar to tumor suppressor genes in cancer cells.

Perhaps abnormally high levels of EZH2 found in cancer

cells may shift expression profiles to promote a return to

a stem cell-like state.

Whether EZH2 has this role in melanoma is yet to be

fully explored, but there is increasing evidence of the

importance of EZH2 in melanoma. High EZH2 expression

was shown to be associated with more malignant forms

of melanoma (Bachmann et al., 2006; McHugh et al.,

2007), leading to the repression of several important

tumor suppressors in melanoma such as RAP1GAP

(Asangani et al., 2012) and p21/CDKN1A (Fan et al.,

2011). Somatic mutations including gain-of-function alter-

ations in EZH2 were reported in ~3% of samples in

several large-scale exome sequencing studies, identifying

drivers of melanoma progression (Alexandrov et al.,

2013; Hodis et al., 2012; Krauthammer et al., 2012).

The recent success of small molecule inhibitors of mutant

EZH2 in lymphoma (McCabe et al., 2012b) has made it

even more important to understand the role of this

molecule in melanoma.

EZH2 as part of a major repressive complex

Polycomb complexes have recently emerged as essential

chromatin regulators in the control of tissue development

and tumorigenesis. Polycomb proteins form complexes

known as polycomb repressive complexes (PRC) 1 and 2

that inhibit transcription by compacting chromatin and

blocking the recruitment of transcriptional initiation

machinery (Cao et al., 2002). EZH1/2 is the crucial

enzymatic subunit of the PRC2 (Figure 1) that facilitates

H3K27 methylation, but cannot function independently

Table 1. Histone methyltransferases implicated in melanoma

Histone

methyltransferase

Histone

modification

Transcriptional

activation or

repression Consequence

CARM1/PRMT6 H3R2 Repression Downregulated in melanoma leading to activation of ERK signaling (Limm et al., 2013)

PRMT5 H3R8 Repression Overexpressed in melanoma, regulates MITF and p27 (Nicholas et al., 2013)

SETDB1 H3K9 Repression Identified within the melanoma susceptibility locus 1q21.3 as recurrently amplified

(MacGregor et al., 2011)

Cooperates with BRAF (V600E) to accelerate melanoma formation in zebrafish (Ceol

et al., 2011)

RIZ1 H3K9 Repression Frameshift mutations identified in melanoma (Poetsch et al., 2002).

EZH2 H3K27 Repression Activating mutations identified within the catalytic SET domain (Hodis et al., 2012),

(Krauthammer et al., 2012), (Alexandrov et al., 2013)

Upregulation in melanoma associated with more aggressive tumor subtypes and

decreased survival (Bachmann et al., 2006), (McHugh et al., 2007)

Overexpression represses the p21/CDKN1A cell cycle inhibitor in melanoma

(Fan et al., 2011).

Overexpression decreases T cell-mediated immune responses in uveal melanoma

(Holling et al., 2007)

Knockdown of BRAFV600E reduces expression of EZH2 and DNMT1 in melanoma

cells (Hou et al., 2012)

Ectopic overexpression of miR-124a, a negative regulator of EZH2, decreased cell

growth, migration, invasion and in vivo tumor growth, similar to EZH2 knockdown in

uveal melanoma (Chen et al., 2013)

Decreased miR-137 and miR101, negative regulators of EZH2, associated with poor

survival in stage IV melanoma patients. Ectopic overexpression of miRs decreased

invasion, migration, proliferation and increased apoptosis, phenocopying EZH2

knockdown (Luo et al., 2013)

Decreased miR-31, a negative regulator of EZH2, reported in melanoma. Ectopic

overexpression of miR-31 decreased cell migration, invasion, EZH2 expression and

derepressed the tumor suppressor RAP1GAP (Asangani et al., 2012)

NSD1 H4K20 Repression Overexpressed in metastatic melanoma (de Souza et al., 2012)

2 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Tiffen et al.

(Tan et al., 2014). Together with other PRC2 members

SUZ12, EED (both essential), JARID2, and RBBP4/7, the

complex facilitates mono-, di- or tri-methylation of lysine

27 on histone 3 (H3K27me1, H3K27me2, H3K27me3).

Whereas H3K27me1 is reportedly associated with gene

activation, H3K27me2/3 leads to gene silencing (Barski

et al., 2007).

The H3K27me3 modification deposited by EZH1/2

recruits and provides docking sites for PRC1 (Figure 1).

PRC1 mono-ubiquitinates histone H2A at lysine 119

(H2AK119ub) via its enzymatic core unit RNF2, thus

blocking the recruitment of transcription initiation factors

and RNA polymerase II elongation (Shen and Laird, 2013).

PRC1 and 2 thereby maintain the chromatin organization

for gene repression.

An additional polycomb complex known as PR-Dub

(polycomb repressive deubiquitinase, Figure 1) has been

shown to counteract the PRC1 by removing ubiquitin

from H2AK119, thus reactivating gene expression (Sche-

uermann et al., 2010). Importantly, the catalytic subunit

of PR-Dub is BAP1, in which germline inactivating

mutations occur in uveal melanoma (Abdel-Rahman et al.,

2011; Maerker et al., 2014). Somatic inactivating muta-

tions in BAP1 have also been identified in as many as

84% (26/31) of metastasizing uveal melanoma (Harbour

et al., 2010). This presumably contributes to unrestrained

division of uveal melanoma cells, via abnormal silencing

of tumor suppressor genes by imbalanced PRC1 activity.

A paralogue of EZH2 is known to exist in noncanonical

PRC2 called EZH1. Mouse ES cells that underwent

conditional ablation of EZH2 displayed a global loss of

H3K27me2/3 as expected. However, the cells were still

able to maintain mono- and some tri-methylation of

H3K27 at PRC2 targeted sites, suggesting that EZH1

offered compensatory methylation activity (Shen et al.,

2008). Margueron et al. (2008) went on to demonstrate

that although EZH1 displays histone methyltransferase

activity in a subset of PRC2 target genes, its activity is

lower compared to EZH2. Its pattern of expression also

differs with EZH1 being ubiquitously expressed, whereas

EZH2 is found in proliferating tissues. It is thought that

while EZH2 is involved in establishment of H3K27

methylation, EZH1 is necessary for maintenance of this

mark on developmental genes in stem cells (Shen et al.,

2008). Although little is known about the implications of

EZH1 mutations in cancer, 2% of melanoma cases in the

TCGA database carry somatic mutations including one

nonsense mutant that truncates prior to the SET domain

(Cerami et al., 2012). Unlike EZH2, no EZH1 mutants

have been identified as gain-of-function thus far, but it will

be important to understand their role in the interplay

between EZH1 and EZH2 in PRC2 and when considering

EZH inhibitors as therapy.

Insights have been gained into the role of EZH1/2 in

skin development using conditional knockout mice. Con-

sistent with earlier studies, complete abolishment of

H3K27me3 and changes in skin phenotype were only

seen in double knockouts, suggesting functional redun-

dancy between EZH1 and EZH2 (Ezhkova et al., 2011).

Although the hair follicles of EZH1/2-null mice showed

progressive degeneration due to defective proliferation

and increased apoptosis, the same effect was not

observed in epithelial progenitors (Ezhkova et al., 2011).

Both lineages, however, displayed dramatic upregulation

of PRC2 targeted Ink4a/Arf and Ink4b genes in the EZH1/

2 double knockouts (Ezhkova et al., 2011). In mice, Ink4a/

b encodes the p16/15 cell cycle inhibitors and Arf

encodes the p19 inhibitor of the p53 suppressor MDM2

(Sherr, 2001). Whether neural progenitor or melanoma

cells display the same EZH2-mediated repression of the

human equivalent CDKN2A locus is yet to be deter-

mined.

Interactions with other epigeneticregulators

Independent of its histone methyltransferase role, EZH2

has been shown to cooperate with other enzymes

involved in transcriptional silencing. In ‘active’ chromatin,

H3K27 may initially be occupied by an acetyl group

which must be removed by an HDAC for methylation

and silencing to occur. Indeed, co-immunoprecipitation

Me3

Ub

Me3

Ub

Me3

UbH3H2A

H2BH4H3

H2A

H2BH4

PR-DUBASXL1/2

(3.2/4)BAP1(1.9)

OFF

RNF2(0.5)

BMI1(0)

CBX7(1.6)

PRC1

RING1(0.5)

H3K27Me3

EZH1/2(2/3)

SUZ12(0.8)

EED(0.5)

RBBP4/7(0.8/0.2)

PRC2

KDM6A/B(0.5/2)

Me3

H3K27Me3

H2AK119Ub

Ub

Figure 1. The polycomb repressive

complexes and somatic mutation rates in

melanoma.

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 3

EZH2 biology and targeted inhibition in melanoma

experiments have demonstrated an interaction between

HDAC and the EED subunit of the PRC2 and that

transcriptional repression could be alleviated by an HDAC

inhibitor (van der Vlag and Otte, 1999).

Additionally, experimental evidence suggests a physi-

cal interaction between EZH2 and DNA methyltransfe-

rases (Vire et al., 2006) (DNMTs, Figure 2). This

interaction was shown to be critical for DNMTs to

methylate and silence CpG sites at EZH2 target gene

promoters. An example of this in melanoma may lie in the

RAP1GAP gene, known to be targeted by EZH2 and

whose expression is downregulated due to promoter

methylation (Zheng et al., 2009). This effectively doubles

the silencing mechanism at EZH2 target genes: first,

indirectly through PRC-mediated histone methylation and

chromatin condensation and second, by direct CpG

hypermethylation of gene promoters to maintain more

permanent silencing. Both these interactions raise the

possibility for combination therapy using EZH2 inhibitors

plus HDAC or DNMT inhibitors.

Genetic abnormalities of EZH2 in cancer

In normal development, EZH2 expression typically

declines after birth and remains low in many adult

mammalian tissues (Zhang et al., 2012). Its overexpres-

sion in several types of cancer means that EZH2 has long

been considered a potential oncogene (Simon and Lange,

2008). It is proposed that amplification of EZH2 leads to

an abnormal accumulation of the H3K27me3 mark, which

in turn represses the expression of critical tumor sup-

pressors, such as cell cycle inhibitors, pro-apoptotic,

senescence, and differentiation genes. Supporting this

view, EZH2 is amplified in 5% of cutaneous melanoma

samples that show putative copy number gains, and

EZH2 mRNA is upregulated in 9% of cases in the TCGA

dataset (Cerami et al., 2012). Previously, hotspot muta-

tions (Y641 and A677) within the catalytic SET domain of

EZH2 that result in its constitutive activation have been

identified in 22% of lymphomas (McCabe et al., 2012a;

Morin et al., 2010; Sneeringer et al., 2010; Yap et al.,

2011). This event has been reported to occur early in the

clonal evolution of lymphoma (Bodor et al., 2013), sup-

porting its role as a driver of tumorigenesis and not

merely a passenger mutation. The equivalent tyrosine

residue in an alternate EZH2 transcript (Y646, transcript

ID: ENST00000320356) was found to be mutated (Fig-

ure 2, Table 2) in several studies of driver mutations in

melanoma and is described in both the TCGA dataset and

cosmic database (Alexandrov et al., 2013; Hodis et al.,

2012; Krauthammer et al., 2012).

Recently, the crystal structure of EZH2’s catalytic SET

domain has been resolved, helping to explain the signif-

icance of these mutations (Antonysamy et al., 2013; Wu

et al., 2013). Wild-type EZH2 has a preference for

unmethylated or monomethylated H3K27 as there is little

room in the binding pocket to accept additional methyl

groups (Wu et al., 2013). However, the substitution of

Y641 for another amino acid alleviates this conformational

constraint, allowing the acceptance of a second or third

methyl group leading to transcriptional repression (Wu

et al., 2013).

However, it is important to note that loss-of-function

mutations have also been described in EZH2 and other

members of the PRC2 in hematological malignancies

(Muto et al., 2013; Ntziachristos et al., 2012). This sug-

gests that these complexes can also function as tumor

suppressors. It will therefore be critical to distinguish

between these types of mutations in patients to deter-

mine whether EZH2 inhibitors are an appropriate course

of treatment in personalized medicine.

Deregulation of EZH2 in cancer

H3K27me3 is a reversible marker that is regulated

dynamically by the balance between EZH2 methyltrans-

DNMT interaction CXC SET

EED interaction

1 751300 450 600150

P751S

G709S

Y646SY646FY646FY646NY646NY646NY646N

C535WG464E

P431S

S412S

A226VD142V

P132S

R34PR347Q

S229LT4L S538L

R216Q

R360G

TCGA data setHodis et al , Cell 2012Krauthammer et al, Nature Genetics 2012

I645splice

Alexandrov et al, Nature 2013

Figure 2. Schematic representation of

EZH2 protein structural domains and

mutations identified in melanoma.

4 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Tiffen et al.

ferase activity and opposing demethylases. To reverse

the writer activity of EZH2, the demethylase erasers

KDM6A (lysine demethylase, Figure 1) and KDM6B

demethylate the repressive H3K27me3 mark to allow

transcriptional activation of genes required in a particular

cell type (Shen and Laird, 2013). They therefore have a

tumor-suppressive role by counteracting EZH2 activity.

Interestingly, many solid tumors exhibit loss-of-function

mutations in KDM6A (Grasso et al., 2012; Robinson

et al., 2012), supporting the suggestion that an accumu-

lation of H3K27me3 plays an important role in malignant

transformation. Analysis of the cBioportal TCGA mela-

noma dataset reveals a significant tendency toward co-

occurrence of EZH2 and KDM6B alterations with an odds

ratio of 2.95 and P value of 0.031 (Cerami et al., 2012).

This suggests a possible mechanism of EZH2 deregula-

tion in which aberrant methylation remains unopposed by

inactivating the demethylation activity of KDM6B.

EZH2 activity is regulated in a number of ways including

by post-translational modifications, microRNAs, and tran-

scription factors. A number of pathways central to

melanoma biology are involved in EZH2 activity, including

MAP kinase, AKT, and E2F1. The most common mela-

noma mutation, BRAFV600E, that activates the MAP

kinase pathway has also been associated with increased

expression of EZH2 (Hou et al., 2012). Knockdown of

BRAFV600E was shown to profoundly reduce the

expression levels of EZH2 (Hou et al., 2012), suggesting

that deregulated BRAF activation may contribute to the

abnormal overexpression of EZH2 seen in melanoma.

Interestingly, cBioportal predicts a tendency toward

co-occurrence for BRAF and EZH2 alterations with an

odds ratio of 3.03 and P value of 0.00008 (Cerami et al.,

2012). This raises the possibility that combination therapy

using both an EZH2 and BRAF inhibitor may produce

synergistic killing. Alternatively, EZH2 inhibition may

provide a suitable course of action in the event of BRAF

inhibitor resistance.

In addition, EZH2 can be inhibited by phosphorylation of

EZH2 protein on serine 21 by activation of AKT signaling

pathway and hence release gene silencing (Cha et al.,

2005).

EZH2 is known to be regulated by E2F1, a transcription

factor which is downstream of p16INK4a and p14ARF

tumor suppressors commonly inactivated in melanoma

(Wu et al., 2010). Upregulation of E2F1 leads to increased

levels of EZH2 that in turn represses the pro-apoptotic

factor Bim. RNA interference (RNAi) against EZH2 was

then used to show that Bim expression and apoptosis

could be restored in cancer cell lines, in an E2F1-

dependent manner (Wu et al., 2010). Although these

studies were not performed in melanoma cells, the pRb/

E2F pathway may be implicated in EZH2 regulation given

its frequent deregulation in melanoma.

A recent study showed that loss of microRNA-31 was

associated with upregulation of EZH2 in melanoma that

could be reversed by ectopic overexpression of microR-

NA-31 (Asangani et al., 2012). This led to inhibition of

migration and invasion of melanoma cells lines and the

de-repression of the EZH2 target tumor suppressor gene,

RAP1GAP (Asangani et al., 2012). Similar experiments

have revealed other microRNAs that have been attributed

to EZH2 regulation in melanoma (Table 1), including miR-

137 (Luo et al., 2013) and miR-124a in uveal melanoma

(Chen et al., 2013).

EZH2 expression is associated withprogression of melanoma

There is a growing body of evidence to support a

significant role for EZH2 in melanoma pathogenesis

(Figure 3). In addition to the activating mutations

identified in melanoma, immunohistochemistry studies

revealed an incremental increase in EZH2 protein levels

from benign nevi to metastatic melanoma (Asangani

et al., 2012; McHugh et al., 2007). A similar study

showed high levels of EZH2 were associated with

increased proliferation (Ki-67 staining), thicker primary

melanomas, and increased invasion (Bachmann et al.,

2006). Furthermore, the 5-year survival rate of EZH2-

high patients was 48% compared with 71% in the

EZH2-low group (Bachmann et al., 2006). Collectively,

these results implicate a role for EZH2 in melanoma

progression.

Knockdown studies of EZH2 in melanoma reduced

proliferation, restored a senescent-like phenotype, and

inhibited the growth of xenografts in mice (Fan et al.,

2011). It was demonstrated that EZH2 depletion led to

Table 2. EZH2 mutations in melanoma and predicted functional

consequence

EZH2

mutation

SIFT predictiona

(score)

PolyPhen-2 predictionb

(score)

T4L Tolerated (0.06) Possibly damaging (0.843)

R34P Damaging (0.01) Probably damaging (1)

P132S Damaging (0) Possibly damaging (0.889)

D142V Damaging (0) Possibly damaging (0.553)

R216Q Tolerated (0.23) Possibly damaging (0.510)

A226V Damaging (0) Probably damaging (0.998)

S229L Tolerated (0.11) Possibly damaging (0.619)

R347Q Tolerated (0.51) Benign (0.034)

R360G Tolerated (0.11) Possibly damaging (0.688)

S412S Tolerated (1) Benign (0)

P431S Tolerated (0.71) Possibly damaging (0.623)

G464E Tolerated (0.49) Possibly damaging (0.9)

C535W Damaging (0) Probably damaging (1)

S538L Damaging (0.05) Probably damaging (0.999)

Y646N/F/S Damaging (0) Probably damaging (0.98)

G709S Damaging (0) Probably damaging (1)

P751S Damaging (0) Probably damaging (0.998)

aSIFT, prediction of deleterious amino acid substitutions (Ng and

Henikoff, 2001).bPolyPhen-2, prediction of functional effects of human nsSNPs

(Adzhubei et al., 2010).

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 5

EZH2 biology and targeted inhibition in melanoma

the reactivation of p21/CDKN1A in a p53-independent

manner, leading to cell cycle arrest (Fan et al., 2011).

In addition, EZH2 overexpression has been implicated

in dampening efficient T cell-mediated immune respo-

nses in uveal melanoma (Holling et al., 2007). These

studies showed that CIITA, which is encoded by the

MHC2TA gene and is essential for downstream transcrip-

tional activation of MHC-II genes, was repressed by

H3K27me3 but that expression could be restored by

EZH2 knockdown. Recently, much attention has focused

on the success of targeting immune checkpoint regula-

tors in melanoma with monoclonal antibodies against

antigens such as PD1/PDL1 and CTLA4 (Pardoll, 2012;

Yao et al., 2013). Perhaps epigenetic EZH2 inhibition in

combination with one of these immunology approaches

may represent a strategy to enhance T cell-mediated

killing in uveal melanoma.

EZH2 inhibitors for targeted treatment ofcancer

Given the role of aberrant methyltransferase activity in

cancer, EZH2 represents a promising drug target via small

molecule inhibition. Several EZH2 inhibitors are currently

showing varying degrees of success in both preclinical

and clinical studies.

The first described inhibitor of EZH2, a molecule known

as DZNep, worked indirectly by inhibition of S-adenosyl-

homocysteine hydrolase (SAH). This caused degradation

of EZH2 protein along with other essential members of

the PRC2 complex, SUZ12, EED, and caused down-

stream H3K27 demethylation (Tan et al., 2007). Although

the drug showed promising results in vitro including cell

cycle inhibition and apoptosis in cultured leukemia cells

(Zhou et al., 2011), the specificity of the drug was called

into question when it was demonstrated to deplete

multiple histone methylation marks (Miranda et al., 2009).

In a more targeted approach, Novartis developed an

inhibitor of EZH2 that affected the activity of the gene via

S-adenosylmethionine (SAM) competition. The drug

known as EI1 was shown to reduce H3K27 methylation

and reactivate target genes, without depleting EZH2

levels (Qi et al., 2012). Although the drug effectively

reduced methylation in both wild-type and mutant EZH2

B-cell lymphoma cell lines, cell growth inhibition by EI1

was restricted to the mutants (Qi et al., 2012).

Epizyme has produced a similar inhibitor of EZH2 that

also works via SAM competition. The compound known

as EPZ005678 is 500-fold more selective to EZH2

compared with 15 different methyltransferases and 50-

fold more selective to EZH2 versus its EZH1 paralogue

(Knutson et al., 2012). The drug was once again shown to

selectively kill lymphoma cells harboring EZH2 activating

mutations (Knutson et al., 2012). EPZ-6438 is another

small molecule inhibitor of EZH2 that has shown promise

in the treatment of malignant rhabdoid tumors (MRTs)

(Knutson et al., 2013). Specifically, MRT cells harboring

inactivating mutations of SMARCB1 are thought to be

dependent on EZH2, thus treatment with EPZ-6438

induced apoptosis and differentiation in these cells and

caused xenograft regression in mice (Knutson et al.,

2013). EPZ-6438 is currently the only EZH2 inhibitor that

has entered clinical trials. In June 2013, Epizyme

announced the enrollment of the first patient in a phase

1/2 clinical trial to assess safety, tolerability, and pharma-

cokinetics of EPZ-6438. Patients entered into the trial are

non-Hodgkin’s lymphoma patients harboring the activat-

ing EZH2 mutation.

McCabe et al. (2012b) described the most potent

inhibitor of EZH2 thus far. The drug known as GSK126

was reported to be 1000-fold more selective against

EZH2 than 20 other SET or non-SET domain containing

methyltransferases and over 150-fold more specific to

EZH2 than EZH1. Additionally, the drug had an excep-

tionally low nanomolar efficacy with a Ki of 0.5–3 nM

(McCabe et al., 2012b). GSK126 in diffuse large B-cell

lymphoma cell lines was shown to cause antiproliferative

effects, induce cell cycle inhibition, and activate apoptosis

via derepression of PCR2 silenced genes including TXNIP

and TNFRSF21(McCabe et al., 2012b). Additionally,

• Activating mutations in catalytic SET domain.

•↓ regulatory MiRs

• Aberrant BRAF signaling?

• Defective H3K27 demethylation?

• ↑ E2F1 activity?

•↓ AKT mediated phosphorylation?

EZH2 deregulation

•↑ Expression in cells

• ↑ H3K27me3 mediated tumor suppressor gene silencing

• PRC2 independent activation of oncogenes?

EZH2 •Aggressive tumor types, ↓ survival

• ↑ DNA methylation and silencing?

• ↓ RAP1GAP, CDKN1A/2A, BIM? tumor suppressor genes

• ↓ differentiation, maintenance of stem cell like state?

• ↓ T cell response in uvealmelanoma

Consequence in melanoma Figure 3. Summary of the predicted

methods of EZH2 deregulation in

melanoma.

6 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Tiffen et al.

GSK126 proved to be highly effective in cell lines

harboring EZH2 activating mutations and significantly

reduced the growth of tumor xenograft models in nude

mice without any toxic side effects (McCabe et al.,

2012b). Other EZH2 inhibitors identified by GlaxoSmithK-

line in a high-throughput screen include GSK343 and

GSK503 (Beguelin et al., 2013). These drug compounds

are structurally similar to GSK126. They were shown to

block germinal center formation and function in mice and

inhibit the transformation of B cells bearing EZH2 mutant

alleles, mirroring the effect of EZH2 conditional knockout

mice (Beguelin et al., 2013).

Importantly, in vivo studies on EZH2 inhibitors were

able to demonstrate a direct correlation between tumor

growth inhibition and decreased levels of H3K27me3 in

surrogate tissues like blood mononuclear cells as well as

tumors (Knutson et al., 2014). Measuring methylation

levels in PBMC’s may therefore represent a noninvasive

biomarker of response to EZH2 inhibitors.

A different approach has been the development of a

peptide that mimics the binding domain of the PRC2

subunit EED, thus preventing the interaction between

EZH2 and EED (Kim et al., 2013). This is particularly

relevant as the peptide can also inhibit the action of EZH1,

which may provide compensatory activity in the absence

of EZH2. The peptide was shown to impair proliferation of

EZH2 mutant lymphoma and EZH2 overexpressing breast

and prostate cancer cell lines (Kim et al., 2013). Although

not as effective as small molecule inhibitors in reduction

of H3K27me3, the peptide was specific, reduced EZH2

stability, and increased cell death. The two molecules

used in combination showed a strong synergistic inhibi-

tion of EZH2-dependent cancer growth (Kim et al., 2013).

Relatively few studies have been carried out on solid

cancers, but it is reasonable to suggest that small

molecule inhibitors of EZH2 may be effective in treating

subsets of melanoma. In particular, melanoma cell lines

harboring the Y646 EZH2 mutation that is not common in

other types of cancers except lymphomas. Despite the

relatively low frequency of this mutation in melanoma

patients, it is possible that wild-type melanoma cells that

overexpress EZH2 may also be responsive to EZH2

inhibitors.

Downstream targets of EZH2

Chromatin immunoprecipitation studies using an

H3K27me3 antibody, followed by sequencing (ChIP-seq)

have identified 2613 genes enriched for thismark inmouse

embryonic stem cells (Young et al., 2011). Although this

figure includes tumor suppressors, an important question

remains as to what other genes will be inadvertently

upregulated by EZH2 inhibition that may have detrimental

effects? Further studies are required to assess the long-

term effects of EZH2 inhibition on gene expression and

whether they are reversible. Interestingly, the number of

genes enriched for H3K27me3 varied considerably

between different cell types (Young et al., 2011), suggest-

ing EZH2 regulation is likely to be time and context

dependent. Nevertheless, available evidence suggests

that EZH2 inhibitors act preferentially on cancer cells that

overexpress EZH2 or harbor activatingmutations (Knutson

et al., 2012; McCabe et al., 2012b; Qi et al., 2012), thus

sparing normal cells in which EZH2 expression is low.

Opposing roles of EZH2

Expression array studies in lymphoma cells treated with

EZH2 inhibitors have shown mostly increases in gene

expression (McCabe et al., 2012b; Qi et al., 2012), as

would be expected given its silencing role. However,

there was a small subset of genes that decreased

expression following EZH2 inhibition (McCabe et al.,

2012b). Indeed, ChIP-seq studies used to map

H3K27me3 binding sites within the genome revealed 3

distinct patterns of enrichment (Young et al., 2011). The

first occurring within the body of a gene, associated with

transcriptional repression, and the second at transcription

start sites associated with H3K4me3 (active transcription)

or a bivalent state poised for gene transcription. Thirdly,

an enrichment peak was identified within the promoters

of genes, associated with active transcription (Young

et al., 2011). In light of this, it has been suggested that

EZH2 may be able to function independently of the PRC2

as a transcriptional activator, in breast (Shi et al., 2007)

and prostate cancer (Xu et al., 2012).

The H3K27me3 independent function of EZH2 was also

recently highlighted in two studies that showed a direct

interaction between EZH2 and genome-wide RNA (Goff

and Rinn, 2013). This high-affinity yet low-specificity RNA

binding surprisingly occurred from actively transcribed

genes marked by H3K4me3 and H3K36me3 and not the

repressive H3K27me3 modification (Davidovich et al.,

2013; Kaneko et al., 2013). Furthermore, the catalytic

activity of PRC2 was shown to be unnecessary for this

EZH2-RNA tethering, as depletion of the SUZ12 subunit

was unable to abolish the effect (Davidovich et al., 2013).

If, however, any existing H3K27me3 was detected on the

promoter, EZH2 would become activated to deposit

additional H3K27me3, thus maintaining the repressed

state (Davidovich et al., 2013). The studies proposed a

model in which PRC2 is able to ‘scan’ gene promoters to

detect local epigenetic states and determine whether

repression is appropriate (Goff and Rinn, 2013). Deci-

phering the complexity of EZH2 and the multiple roles it

may play in melanoma cell biology remains a challenging

yet promising prospect.

Future directions

In recent years, significant progress has been made to

understand the epigenetic actions of histone meth-

yltransferases in cancer. In particular, the critical catalytic

subunit of the PRC2 EZH2 has revealed itself as a

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 7

EZH2 biology and targeted inhibition in melanoma

promising therapeutic target in cancer, given its dysregu-

lation in cancers including melanoma. Despite this

progress, many questions still remain around the use of

small molecule inhibitors of EZH2 in clinical practice.

Although activating mutations appear to be detected in

only a small subset of melanoma, overexpression of

EZH2 may be more frequent. The underlying cause of the

latter needs further exploration especially in relation to

aberrant signaling pathways in melanoma. Many ques-

tions about the pathobiology of EZH2 in melanoma

remain unanswered. For example, do activating muta-

tions or overexpression of EZH2 define particular clinical

or pathological forms of melanoma? What is the relation-

ship between EZH2-mediated histone methylation and

DNA methylation and will targeting one be sufficient to

overcome tumor suppressor gene silencing? Further

understanding of this process may help guide combina-

tion treatments with EZH2 inhibitors. Relatively little

information is available about the role of the PRC2

complex in suppression of immune responses, but given

the important role of immunotherapy in treatment of

melanoma, this also requires examination. Clearly, there

is much work to do before the true value of EZH2

inhibitors in treatment of melanoma can be assessed, but

currently available information suggests they may be an

important extension to currently available treatments of

this disease.

Acknowledgements

We would like to thank Dr Kavitha Gowrishankar for her assistance in

editing this manuscript. This work was supported by program grant

633004 from the Australian National Health and Medical Research

Council (NHMRC).

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