Subscribe to PCMR and stay up-to-date with the only journal committed to publishing basic research in melanoma and pigment cell biology
As a member of the IFPCS or the SMR you automatically get online access to PCMR. Sign up as a member today at www.ifpcs.org or at www.societymelanomaresarch.org
If you wish to order reprints of this article, please see the guidelines here
EMAIL ALERTSReceive free email alerts and stay up-to-date on what is published in Pigment Cell & Melanoma Research – click here
The official journal of
INTERNATIONAL FEDERATION OF PIGMENT CELL SOCIETIES · SOCIETY FOR MELANOMA RESEARCH
PIGMENT CELL & MELANOMAResearch
To take out a personal subscription, please click hereMore information about Pigment Cell & Melanoma Research at www.pigment.org
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: [email protected]
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).
References
Abdel-Rahman, M.H., Pilarski, R., Cebulla, C.M., Massengill, J.B.,
Christopher, B.N., Boru, G., Hovland, P., and Davidorf, F.H. (2011).
Germline BAP1 mutation predisposes to uveal melanoma, lung
adenocarcinoma, meningioma, and other cancers. J. Med. Genet.
48, 856–859.Adzhubei, I.A., Schmidt, S., Peshkin, L., Ramensky, V.E., Gerasim-
ova, A., Bork, P., Kondrashov, A.S., and Sunyaev, S.R. (2010). A
method and server for predicting damaging missense mutations.
Nat. Methods 7, 248–249. doi:10.1038/nmeth0410-248.
Alexandrov, L.B., Nik-Zainal, S., Wedge, D.C. et al. (2013). Signatures
of mutational processes in human cancer. Nature 500, 415–421.Antonysamy, S., Condon, B., Druzina, Z. et al. (2013). Structural
context of disease-associated mutations and putative mechanism
of autoinhibition revealed by X-ray crystallographic analysis of the
EZH2-SET domain. PLoS ONE 8, 0084147.
Arrowsmith, C.H., Bountra, C., Fish, P.V., Lee, K., and Schapira, M.
(2012). Epigenetic protein families: a new frontier for drug
discovery. Nat. Rev. Drug Discov. 11, 384–400.Asangani, I.A., Harms, P.W., Dodson, L. et al. (2012). Genetic and
epigenetic loss of microRNA-31 leads to feed-forward expression
of EZH2 in melanoma. Oncotarget 3, 1011–1025.Bachmann, I.M., Halvorsen, O.J., Collett, K., Stefansson, I.M.,
Straume, O., Haukaas, S.A., Salvesen, H.B., Otte, A.P., and
Akslen, L.A. (2006). EZH2 expression is associated with high
proliferation rate and aggressive tumor subgroups in cutaneous
melanoma and cancers of the endometrium, prostate, and breast.
J. Clin. Oncol. 24, 268–273.Barski, A., Cuddapah, S., Cui, K., Roh, T.Y., Schones, D.E., Wang, Z.,
Wei, G., Chepelev, I., and Zhao, K. (2007). High-resolution profiling
of histone methylations in the human genome. Cell 129, 823–837.Beguelin, W., Popovic, R., Teater, M. et al. (2013). EZH2 is required
for germinal center formation and somatic EZH2 mutations
promote lymphoid transformation. Cancer Cell 23, 677–692.Ben-Porath, I., Thomson, M.W., Carey, V.J., Ge, R., Bell, G.W.,
Regev, A., and Weinberg, R.A. (2008). An embryonic stem cell-like
gene expression signature in poorly differentiated aggressive
human tumors. Nat. Genet. 40, 499–507.Bodor, C., Grossmann, V., Popov, N. et al. (2013). EZH2 mutations
are frequent and represent an early event in follicular lymphoma.
Blood 122, 3165–3168.Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H.,
Tempst, P., Jones, R.S., and Zhang, Y. (2002). Role of histone H3
lysine 27 methylation in Polycomb-group silencing. Science 298,
1039–1043.Ceol, C.J., Houvras, Y., Jane-Valbuena, J. et al. (2011). The histone
methyltransferase SETDB1 is recurrently amplified in melanoma
and accelerates its onset. Nature 471, 513–517.Cerami, E., Gao, J., Dogrusoz, U. et al. (2012). The cBio cancer
genomics portal: an open platform for exploring multidimensional
cancer genomics data. Cancer Discov. 2, 401–404.Cha, T.-L., Zhou, B.P., Xia, W., Wu, Y., Yang, C.-C., Chen, C.-T., Ping,
B., Otte, A.P., and Hung, M.-C. (2005). Akt-mediated phosphory-
lation of EZH2 suppresses methylation of lysine 27 in histone H3.
Science 310, 306–310.Chen, X., He, D., Dong, X.D., Dong, F., Wang, J., Wang, L., Tang, J.,
Hu, D.N., Yan, D., and Tu, L. (2013). MicroRNA-124a is epigenet-
ically regulated and acts as a tumor suppressor by controlling
multiple targets in uveal melanoma. Invest. Ophthalmol. Vis. Sci.
54, 2248–2256.Davidovich, C., Zheng, L., Goodrich, K.J., and Cech, T.R. (2013).
Promiscuous RNA binding by Polycomb repressive complex 2. Nat.
Struct. Mol. Biol. 20, 1250–1257.Dawson, M.A., and Kouzarides, T. (2012). Cancer epigenetics: from
mechanism to therapy. Cell 150, 12–27.Dawson, M.A., Kouzarides, T., and Huntly, B.J.P. (2012). Targeting
epigenetic readers in cancer. N. Engl. J. Med. 367, 647–657.Ezhkova, E., Lien, W.H., Stokes, N., Pasolli, H.A., Silva, J.M., and
Fuchs, E. (2011). EZH1 and EZH2 cogovern histone H3K27
trimethylation and are essential for hair follicle homeostasis and
wound repair. Genes Dev. 25, 485–498.Fan, T., Jiang, S., Chung, N., Alikhan, A., Ni, C., Lee, C.C., and
Hornyak, T.J. (2011). EZH2-dependent suppression of a cellular
senescence phenotype in melanoma cells by inhibition of p21/
CDKN1A expression. Mol. Cancer Res. 9, 418–429.Goff, L.A., and Rinn, J.L. (2013). Poly-combing the genome for RNA
Nat. Struct. Mol. Biol. 20, 1344–1346 doi: 10.1038/nsmb.2728.
Grasso, C.S., Wu, Y.M., Robinson, D.R. et al. (2012). The mutational
landscape of lethal castration-resistant prostate cancer. Nature
487, 239–243.Harbour, J.W., Onken, M.D., Roberson, E.D., Duan, S., Cao, L.,
Worley, L.A., Council, M.L., Matatall, K.A., Helms, C., and
Bowcock, A.M. (2010). Frequent mutation of BAP1 in metastasiz-
ing uveal melanomas. Science 330, 1410–1413.Hodis, E., Watson, I.R., Kryukov, G.V. et al. (2012). A landscape of
driver mutations in melanoma. Cell 150, 251–263.Holling, T.M., Bergevoet, M.W., Wilson, L., van Eggermond, M.C.,
Schooten, E., Steenbergen, R.D., Snijders, P.J., Jager, M.J., and
van den Elsen, P.J. (2007). A role for EZH2 in silencing of
8 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Tiffen et al.
IFN-gamma inducible MHC2TA transcription in uveal melanoma. J.
Immunol. 179, 5317–5325.Hou, P., Liu, D., Dong, J., and Xing, M. (2012). The BRAFV600E
causes widespread alterations in gene methylation in the genome
of melanoma cells. Cell Cycle 11, 286–295.van den Hurk, K., Niessen, H.E., Veeck, J., van den Oord, J.J., van
Steensel, M.A., Zur Hausen, A., Van Engeland, M., and Win-
nepenninckx, V.J. (2012). Genetics and epigenetics of cutaneous
malignant melanoma: a concert out of tune. Biochim. Biophys.
Acta 1826, 89–102.Jones, R.S., and Gelbart, W.M. (1990). Genetic analysis of the
enhancer of zeste locus and its role in gene regulation in Drosophila
melanogaster. Genetics 126, 185–199.Kaneko, S., Son, J., Shen, S.S., Reinberg, D., and Bonasio, R. (2013).
PRC2 binds active promoters and contacts nascent RNAs in
embryonic stem cells. Nat. Struct. Mol. Biol. 20, 1258–1264.Kim, W., Bird, G.H., Neff, T., Guo, G., Kerenyi, M.A., Walensky, L.D.,
and Orkin, S.H. (2013). Targeted disruption of the EZH2-EED
complex inhibits EZH2-dependent cancer. Nat. Chem. Biol. 9, 643–650.
Knutson, S.K., Wigle, T.J., Warholic, N.M. et al. (2012). A selective
inhibitor of EZH2 blocks H3K27 methylation and kills mutant
lymphoma cells. Nat. Chem. Biol. 8, 890–896.Knutson, S.K., Warholic, N.M., Wigle, T.J. et al. (2013). Durable
tumor regression in genetically altered malignant rhabdoid tumors
by inhibition of methyltransferase EZH2. Proc. Natl Acad. Sci. 110,
7922–7927.Knutson, S.K., Kawano, S., Minoshima, Y. et al. (2014). Selective
inhibition of EZH2 by EPZ-6438 leads to potent antitumor Activity in
EZH2 mutant non-Hodgkin lymphoma. Mol. Cancer Ther. 21, 21.
Krauthammer, M., Kong, Y., Ha, B.H. et al. (2012). Exome sequenc-
ing identifies recurrent somatic RAC1 mutations in melanoma. Nat.
Genet. 44, 1006–1014.Limm, K., Ott, C., Wallner, S., Mueller, D.W., Oefner, P., Hellerbrand,
C., and Bosserhoff, A.K. (2013). Deregulation of protein methyla-
tion in melanoma. Eur. J. Cancer 49, 1305–1313.Luo, C., Tetteh, P.W., Merz, P.R. et al. (2013). miR-137 inhibits the
invasion of melanoma cells through downregulation of multiple
oncogenic target genes. J. Invest. Dermatol. 133, 768–775.MacGregor, S., Montgomery, G.W., Liu, J.Z. et al. (2011). Genome--
wide association study identifies a new melanoma susceptibility
locus at 1q21.3. Nat. Genet. 43, 1114–1118.Maerker, D.A., Zeschnigk, M., Nelles, J., Lohmann, D.R., Worm, K.,
Bosserhoff, A.K., Krupar, R., and Jagle, H. (2014). BAP1 germline
mutation in two-first grade family members with uveal melanoma.
Br. J. Ophthalmol. 98, 224–227.Margueron, R., Li, G., Sarma, K., Blais, A., Zavadil, J., Woodcock,
C.L., Dynlacht, B.D., and Reinberg, D. (2008). Ezh1 and Ezh2
maintain repressive chromatin through different mechanisms. Mol.
Cell 32, 503–518.McCabe, M.T., Graves, A.P., Ganji, G. et al. (2012a). Mutation of
A677 in histone methyltransferase EZH2 in human B-cell lym-
phoma promotes hypertrimethylation of histone H3 on lysine 27
(H3K27). Proc. Natl Acad. Sci. USA 109, 2989–2994.McCabe, M.T., Ott, H.M., Ganji, G. et al. (2012b). EZH2 inhibition as
a therapeutic strategy for lymphoma with EZH2-activating muta-
tions. Nature 492, 108–112.McHugh, J.B., Fullen, D.R., Ma, L., Kleer, C.G., and Su, L.D. (2007).
Expression of polycomb group protein EZH2 in nevi and melanoma.
J. Cutan. Pathol. 34, 597–600.Miranda, T.B., Cortez, C.C., Yoo, C.B., Liang, G., Abe, M., Kelly,
T.K., Marquez, V.E., and Jones, P.A. (2009). DZNep is a global
histone methylation inhibitor that reactivates developmental
genes not silenced by DNA methylation. Mol. Cancer Ther. 8,
1579–1588.
Morin, R.D., Johnson, N.A., Severson, T.M. et al. (2010). Somatic
mutations altering EZH2 (Tyr641) in follicular and diffuse large
B-cell lymphomas of germinal-center origin. Nat. Genet. 42, 181–185.
Muto, T., Sashida, G., Oshima, M. et al. (2013). Concurrent loss of
Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic
disorders. J. Exp. Med. 210, 2627–2639.Ng, P.C., and Henikoff, S. (2001). Predicting deleterious amino acid
substitutions. Genome Res. 11, 863–874.Nicholas, C., Yang, J., Peters, S.B. et al. (2013). PRMT5 is upregu-
lated in malignant and metastatic melanoma and regulates
expression of MITF and p27(Kip1.). PLoS ONE 8, 0074710.
Ntziachristos, P., Tsirigos, A., van Vlierberghe, P. et al. (2012).
Genetic inactivation of the polycomb repressive complex 2 in T cell
acute lymphoblastic leukemia. Nat. Med. 18, 298–301.O’Carroll, D., Erhardt, S., Pagani, M., Barton, S.C., Surani, M.A., and
Jenuwein, T. (2001). The polycomb-group gene Ezh2 is required for
early mouse development. Mol. Cell. Biol. 21, 4330–4336.O’Meara, M.M., and Simon, J.A. (2012). Inner workings and
regulatory inputs that control Polycomb repressive complex 2.
Chromosoma 121, 221–234.Pardoll, D.M. (2012). The blockade of immune checkpoints in cancer
immunotherapy. Nat. Rev. Cancer 12, 252–264.Poetsch, M., Dittberner, T., and Woenckhaus, C. (2002). Frameshift
mutations of RIZ, but no point mutations in RIZ1 exons in
malignant melanomas with deletions in 1p36. Oncogene 21,
3038–3042.Popovic, R., and Licht, J.D. (2012). Emerging epigenetic targets and
therapies in cancer medicine. Cancer Discov. 2, 405–413.Qi, W., Chan, H., Teng, L. et al. (2012). Selective inhibition of Ezh2 by
a small molecule inhibitor blocks tumor cells proliferation. Proc.
Natl Acad. Sci. USA 109, 21360–21365.Robinson, G., Parker, M., Kranenburg, T.A. et al. (2012). Novel
mutations target distinct subgroups of medulloblastoma. Nature
488, 43–48.Scheuermann, J.C., De Ayala Alonso, A.G., Oktaba, K., Ly-Hartig, N.,
McGinty, R.K., Fraterman, S., Wilm, M., Muir, T.W., and Muller, J.
(2010). Histone H2A deubiquitinase activity of the Polycomb
repressive complex PR-DUB. Nature, 465, 243–247.Shen, H., and Laird, P.W. (2013). Interplay between the cancer
genome and epigenome. Cell 153, 38–55.Shen, X., Liu, Y., Hsu, Y.J., Fujiwara, Y., Kim, J., Mao, X., Yuan, G.C.,
and Orkin, S.H. (2008). EZH1 mediates methylation on histone H3
lysine 27 and complements EZH2 in maintaining stem cell identity
and executing pluripotency. Mol. Cell 32, 491–502.Sherr, C.J. (2001). The INK4a/ARF network in tumour suppression.
Nat. Rev. Mol. Cell Biol. 2, 731–737.Shi, B., Liang, J., Yang, X. et al. (2007). Integration of estrogen and
Wnt signaling circuits by the polycomb group protein EZH2 in
breast cancer cells. Mol. Cell. Biol. 27, 5105–5119.Simon, J.A., and Lange, C.A. (2008). Roles of the EZH2 histone
methyltransferase in cancer epigenetics. Mutat. Res. 647, 21–29.Sneeringer, C.J., Scott, M.P., Kuntz, K.W., Knutson, S.K., Pollock,
R.M., Richon, V.M., and Copeland, R.A. (2010). Coordinated
activities of wild-type plus mutant EZH2 drive tumor-associated
hypertrimethylation of lysine 27 on histone H3 (H3K27) in human
B-cell lymphomas. Proc. Natl Acad. Sci. USA 107, 20980–20985.de Souza, C.F., Xander, P., Monteiro, A.C., Silva, A.G., da Silva, D.C.,
Mai, S., Bernardo, V., Lopes, J.D., and Jasiulionis, M.G. (2012).
Mining gene expression signature for the detection of pre-malig-
nant melanocytes and early melanomas with risk for metastasis.
PLoS ONE 7, 11.
Tan, J., Yang, X., Zhuang, L., Jiang, X., Chen, W., Lee, P.L., Karuturi,
R.K., Tan, P.B., Liu, E.T., and Yu, Q. (2007). Pharmacologic
disruption of Polycomb-repressive complex 2-mediated gene
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 9
EZH2 biology and targeted inhibition in melanoma
repression selectively induces apoptosis in cancer cells. Genes
Dev. 21, 1050–1063.Tan, J.Z., Yan, Y., Wang, X.X., Jiang, Y., and Xu, H.E. (2014). EZH2:
biology, disease, and structure-based drug discovery. Acta Phar-
macol. Sin. 35, 161–174.Vire, E., Brenner, C., Deplus, R. et al. (2006). The Polycomb group
protein EZH2 directly controls DNA methylation. Nature 439, 871–874.
van der Vlag, J., and Otte, A.P. (1999). Transcriptional repression
mediated by the human polycomb-group protein EED involves
histone deacetylation. Nat. Genet. 23, 474–478.Wu, Z.L., Zheng, S.S., Li, Z.M., Qiao, Y.Y., Aau, M.Y., and Yu, Q.
(2010). Polycomb protein EZH2 regulates E2F1-dependent apop-
tosis through epigenetically modulating Bim expression. Cell Death
Differ. 17, 801–810.Wu, H., Zeng, H., Dong, A. et al. (2013). Structure of the catalytic
domain of EZH2 reveals conformational plasticity in Cofactor and
substrate binding sites and explains oncogenic mutations. PLoS
ONE 8, 0083737.
Xu, K., Wu, Z.J., Groner, A.C. et al. (2012). EZH2 oncogenic activity in
castration-resistant prostate cancer cells is Polycomb-independent.
Science 338, 1465–1469.
Yao, S., Zhu, Y., and Chen, L. (2013). Advances in targeting cell
surface signalling molecules for immune modulation. Nat. Rev.
Drug Discov. 12, 130–146.Yap, D.B., Chu, J., Berg, T. et al. (2011). Somatic mutations at EZH2
Y641 act dominantly through a mechanism of selectively altered
PRC2 catalytic activity, to increase H3K27 trimethylation. Blood
117, 2451–2459.Young, M.D., Willson, T.A., Wakefield, M.J., Trounson, E., Hilton,
D.J., Blewitt, M.E., Oshlack, A., and Majewski, I.J. (2011).
ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate
with transcriptional activity. Nucleic Acids Res. 39, 7415–7427.Zhang, J., Bardot, E., and Ezhkova, E. (2012). Epigenetic regulation of
skin: focus on the Polycomb complex. Cell. Mol. Life Sci. 69, 2161–2172.
Zheng, H., Gao, L., Feng, Y., Yuan, L., Zhao, H., and Cornelius, L.A.
(2009). Down-regulation of Rap1GAP via promoter hypermethyla-
tion promotes melanoma cell proliferation, survival, and migration.
Cancer Res. 69, 449–457.Zhou, J., Bi, C., Cheong, L.L., Mahara, S., Liu, S.C., Tay, K.G., Koh,
T.L., Yu, Q., and Chng, W.J. (2011). The histone methyltransferase
inhibitor, DZNep, up-regulates TXNIP, increases ROS production,
and targets leukemia cells in AML. Blood 118, 2830–2839.
10 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Tiffen et al.
Top Related