NRC 3884artigo

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Critical roles of non-histone

protein lysine methylation in
human tumorigenesis
Ryuji Hamamoto, Vassiliki Saloura and Yusuke Nakamura
Abstract | Several protein lysine methyltransferases and demethylases have been identified
to have critical roles in histone modification. A large body of evidence has indicated that
their dysregulation is involved in the development and progression of various diseases,
including cancer, and these enzymes are now considered to be potential therapeutic targets.
Although most studies have focused on histone methylation, many reports have revealed
that these enzymes also regulate the methylation dynamics of non-histone proteins such as
p53, RB1 and STAT3 (signal transducer and activator of transcription 3), which have
important roles in human tumorigenesis. In this Review, we summarize the molecular
functions of protein lysine methylation and its involvement in human cancer, with a particular
focus on lysine methylation of non-histone proteins.
Sadenosyl-lmethionine
(AdoMet). A molecule
synthesized from methionine
and ATP by methionine adeno
syltransferase. The methylation
group attached to the
methionine sulphur atom in
AdoMet is chemically reactive.
This allows donation of this
group to an acceptor substrate
in transmethylation reactions.
Section of Hematology/
Oncology, Department of
Medicine, The University of
Chicago, 5841S.Maryland
Avenue, MC 2115 Chicago,
Illinois 60637, USA.
Correspondence to R.H.
e-mail: rhamamoto@
medicine.bsd.uchicago.edu
doi:10.1038/nrc3884
Nmethyl-lysine was first found in a bacterial flagel

lar protein in 1959 (REF.1) and, 5years later, this posttranslational modification (PTM) was also identified in
histone proteins2. Biochemically, Sadenosyl-lmethionine
(AdoMet) is the principal methyl donor in the methyl
transferase reaction and is the second most widely used
enzyme substrate following ATP3. This frequency indi
cates the importance of the methylation reaction in
various biochemical or metabolic pathways. Although
the physiological importance of protein lysine methyla
tion was unknown for many years, several protein lysine
methyltransferases (PKMTs) have now been identified,
and their physiological significance, particularly in
the field of epigenetics, has begun to be elucidated48.
The SET-domain proteins constitute a major group of
AdoMet-dependent PKMTs; nearly 50 human proteins
are categorized into this family, although not all of them
have known PKMT activity. In addition to the SETdomain proteins, several non-SET-domain proteins,
including DOT1like histone H3K79 methyltransferase
(DOT1L), are also reported to have PKMT activity 9.
Moreover, protein lysine methylation was thought to
be irreversible because the half-life of the histone meth
ylation was approximately equivalent to the half-life of
histones themselves10. However, the first protein lysine
demethylase lysine-specific demethylase1 (LSD1; also
known as KDM1A) was discovered in 2004 (REF.11), and
subsequently the Jumonji C (JmjC)-domain-containing
family was reported to have protein demethylase activ

ity 12. These findings indicate that protein lysine methyla
tion seems to be dynamically regulated.
Many reports have indicated that dysregulation of
PKMTs and protein lysine demethylases (PKDMs)
has substantial roles in tumorigenesis4,13,14, and these
enzymes are considered to be important targets for
the development of anticancer therapy 15,16. Functional
analyses of PKMTs and PKDMs have been carried out
primarily in the context of their role in epigenetic regu
lation. For instance, the PKMT EZH2, which methyl
ates histone H3 at lysine27 (H3K27), is a component
of Polycomb-repressive complexes and is overexpressed
in various types of cancer 17. Polycomb-repressive com
plexes play a fundamental part in controlling the expres
sion of downstream genes, including cyclin-dependent
kinase inhibitor 2A (CDKN2A), to promote cell cycle
progression in physiological and pathological condi
tions18. Similarly, several reports have demonstrated
that aberrations of other PKMTs and PKDMs promote
malignant transformation via histone methylationdependent transcriptional regulation4,13,1921.
Beyond histones, the biological and physiological
significance of non-histone lysine methylation in human
tumorigenesis has recently begun to be explored2227.
Accumulating evidence indicates that, similar to other
PTMs such as phosphorylation and acetylation, lysine
methylation may be important not only in epigenetic
110 | FEBRUARY 2015 | VOLUME 15
www.nature.com/reviews/cancer
2015 Macmillan Publishers Limited. All rights reserved
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regulation of gene expression but also in the regulation of
cellular signal transduction pathways. Furthermore, dys
regulation of non-histone lysine methylation seems to be
involved in the development and progression of cancer.
As anticancer drugs targeting PKMTs and PKDMs have
actively been developed in recent years, it is essential to
clarify the substrates of these enzymes to correctly under
stand the mechanism of action of thesedrugs.
Monoamine oxidase
A family of enzymes that
catalyse the oxidation of
monoamines. They belong to
the protein family of
flavin-containing amine
oxidoreductases.
a Lysine methylation
PKMT
H H
H N+
PKMT
H3C H
H N+
AdoMet AdoHcy
AdoMet AdoHcy
O
+H
3N
AdoMet
+H
Lysine
H3C CH3
H3C N+
AdoHcy
3N
PKMT
H3C CH3
H N+
+H
3N
+H
Dimethyl lysine
Monomethyl lysine
3N
Trimethyl lysine
b Lysine demethylation (LSD1: FAD-dependent amine oxidase)

H3C CH3
H N+
LSD1
FAD
H2O2
H2C
CH3
N+
FADH2
+H
3N
H2C=O
Formaldehyde
H3C H
H N+
O2
Dimethyl lysine
H2O
OH
H2C CH3
H N+
+H
3N
+H
3N
Imine intermediate
+H
3N
Monomethyl lysine
c Lysine demethylation (JmjC-containing proteins: hydroxylase)

H3C CH3
H N+
JKDM
Fe(II)
-ketoglutarate Succinate
+ O2
+ CO2
OH
H2C CH3
H N+
O
+H
3N
Dimethyl lysine
H2C=O
Formaldehyde
H CH3
H N+
O
+H
3N
O
+H
3N
Monomethyl lysine
Figure 1 | The chemical mechanism of protein lysine methylation. a | Protein lysine

methyltransferases (PKMTs) catalyse monomethylation, dimethylation
trimethylation
Natureand
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| Cancer
of the lysine amino group in an Sadenosyl-lmethionine (AdoMet)-dependent manner.
PKMTs consist of two main classes: the SET domain-containing family and the DOT1L
family. For methyl transfer to occur, the amine of the lysine substrate must be
deprotonated. To proceed, AdoMet and the lysine residue of the substrate must first be
bound to the catalytic pocket of the SET domain. Then, a nearby tyrosine residue
deprotonates the group of the lysine residue. The lysine chain then makes a
nucleophilic attack on the methyl group on the sulphur atom of the AdoMet molecule,
which transfers the methyl group to the lysine side chain. This process results in the
formation of an Nmethylated lysine and Sadenosyl-lhomocysteine (AdoHcy).
Despite lacking a SET domain, DOT1L family methyltransferases catalyse an overall
similar mechanism of methyl transfer. b | Lysine-specific demethylase 1 (LSD1)
concurrently catalyses the reduction of FAD to FADH2 and the oxidation of the
methylated lysine substrate, generating an imine intermediate. The formation of this
imine requires a free lone pair of electrons on lysine and, accordingly, LSD1 is only
capable of demethylating monomethyl and dimethyl, and not trimethyl, lysine residues.
c|The Jumonji C (JmjC)-driven demethylase reaction proceeds through the oxidative
decarboxylation of ketoglutarate coupled to hydroxylation of the methylation group.
This hydroxylated intermediate spontaneously decomposes to produce formaldehyde
and a lysine from which one methyl group is removed. JKDM, JmjC domain-containing
protein lysine demethylase.
In this Review, we highlight the current knowledge

of the functions of protein lysine methylation in human
cancer and describe, in particular, the significance of nonhistone lysine methylation. We also discuss the impor
tance of PKMTs and PKDMs as anticancer drug targets.
Biochemical characteristics of lysine methylation

Lysine methyltransferases. Monomethylation, dimeth
ylation and trimethylation of the lysine group are
catalysed by PKMTs in an AdoMet-dependent man
ner 28 (FIG.1a). Most PKMTs contain the SET domain,
but several non-SET-domain proteins such as DOT1L,
methyltransferase-like 10 (METTL10) and METTL21A
are also known to have lysine Nmethyltransferase activ
ity 9,29. The SET-domain PKMTs are thought to catalyse a
sequential bibi kinetic mechanism in which both sub
strate association and product release occur in a random
manner 28. Methylation increases the hydrophobic and
basic nature of the lysine residue, which allows other
proteins to recognize methylatedlysine.
Lysine demethylases. The PKDMs are a diverse group
of proteins that can be broadly categorized into two
functional enzymatic families. The first family includes
LSD1, which is a flavin-dependent monoamine oxidase
(FIG.1b). LSD2 (also known as KDM1B) is the only hom
ologue of LSD1 in the human genome30. These amine
oxidases can only demethylate monomethyl and dime
thyl lysine residues, and not trimethyl lysine residues,
because they require a lone pair of electrons that are only
present on monomethyl and dimethyl lysine residues.
The second family of PKDMs consists of JmjC domaincontaining proteins that use an oxygenase mechanism to
demethylate monomethyl, dimethyl and trimethyl lysine
residues (FIG.1c).
Functions of protein lysine methylation

The biological functions of protein lysine methylation in
human cancer are categorized into five different func
tions (FIG.2). We describe the detailed mechanism of
action of each groupbelow.
Effect on other protein modifications. Accumulating
evidence has revealed that protein lysine methylation
affects other PTMs, either directly or indirectly (FIG.2a).
For example, EZH2 methylates histone H2B at lysine120
(H2BK120), which is also known to be a site of ubiq
uitylation31. EZH2dependent H2BK120 methylation
competitively inhibits ubiquitylation and suppresses
the transcription of genes involved in cancer 31. In addi
tion, lysine methylation also affects phosphorylation and
acetylation of neighbouring or distant amino acids. In
this regard, methylated lysine residues seem to change
the affinity of enzymes such as kinases or phosphatases
for their substrates, which may alter PTMs at other sites
on the substrates32,33.
Proteinprotein interactions. Lysine methylation can reg
ulate proteinprotein interactions (FIG.2b). Biochemically,
lysine methylation leads to no change in charge and small
changes in size, but it substantially alters the hydration
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a Other protein modications
Competitive
inhibition
K Me
Indirect eect K Me
on other
modications
Ub
Ac
P S
b Proteinprotein interactions
Methylatedlysine-specic
binding proteins
localization
Me Me Nucleus
HSP70 AURKB
Activate
Chromodomain
Me
Me
K
Inhibition of
proteinprotein
interactions
d Subcellular
c Protein stability
HSP70
Me
Ub Ub
Ub Ub Ub
Inhibition of
Me
Me
K
polyubiquitylation
Protein degradation
by proteasomes
Me
Ub
Ub
Ub Ub
Ub
e Promoter binding
Me
HSP70
Transcription
factors
Methylation
activates
transcription
Promoter
Cytoplasm
Figure 2 | Molecular functions of lysine methylation in human tumorigenesis. The biological Nature
importance
of lysine
Reviews
| Cancer
methylation is primarily categorized into five groups. a | Lysine methylation affects other protein modifications directly
(through competitive inhibition) or indirectly. b | Lysine methylation regulates proteinprotein interactions. Methylatedlysine-specific binding proteins have been reported, and these proteins contain motifs that specifically recognize
methylated lysine residues such as chromodomains and Tudor domains. Methylated lysine can also negatively regulate
some proteinprotein interactions. c | Lysine methylation competitively inhibits the polyubiquitylation of lysine residues
and stabilizes the protein. Additionally, lysine methylation also promotes the polyubiquitylation of other lysine residues on
the same protein, leading to protein destabilization. d | Subcellular localization is regulated by lysine methylation. For
example, methylated heat shock protein 70 (HSP70) proteins are predominantly localized in the nucleus, whereas
unmodified versions are predominantly localized in the cytoplasm. e | Lysine methylation regulates promoter binding
affinity of transcription factors, thereby changing transcription levels of target genes. Ac, acetyl group; AURKB, Aurora
kinase B; P, phosphate group; Me, methyl group; Ub, ubiquitin.
energies and hydrogen bonding potential of the lysine

side chains. Indeed, there are methyl lysine-binding pro
teins that have a special motif such as a chromodomain
for recognizing methylated lysine residues, called the
aromatic cage. This aromatic cage comprises a collec
tion of aromatic protein residues, often accompanied by
one or more neighbouring anionic residues34. The com
bination of favourable cation, electrostatic and van
der Waals interactions, as well as shape complementa
rity, provides methyl lysine-binding proteins with a high
degree of specificity for methylated lysine35. By contrast,
lysine methylation of MAPK kinase kinase 2 (MAP3K2)
inhibits its interaction with the serine/threonine protein
phosphatase 2A (PP2A) complex, which is a key negative
regulator of the MAPK pathway, implying that methyl
lysine can also block proteinprotein interactions33.
Oxygenase
An enzyme of the
oxidoreductase class that
catalyses the incorporation of
both atoms of molecular
oxygen into the substrate.
Protein stability. As lysine polyubiquitylation plays a

key part in protein degradation through the ubiquitin
proteasome pathway, lysine methylation may increase
the stability of proteins by preventing polyubiquityla
tion. Indeed, in Saccharomyces cerevisiae, lysine methyl
ated proteins show a significantly longer half-life than
non-methylated proteins. Moreover, 43% of methylated
lysine sites are predicted to be amenable to ubiquityla
tion, suggesting that methylated lysine residues might
block the action of ubiquitin ligases36 (FIG.2c). However,
the DCAF1DDB1CUL4 E3 ubiquitin ligase complex
recognizes monomethylated lysine and promotes poly
ubiquitylation of the other lysine residues on substrates
such as retinoic acid-related orphan nuclear receptor

(ROR)37, implying that lysine methylation may also
destabilize target proteins through regulation of distant
polyubiquitylation.
Subcellular localization. Typically, a nuclear localiza
tion signal consists of one or more short sequences of
positively charged lysine or arginine residues exposed
on the protein surface38. As both lysine and arginine
residues are critical for the nuclear localization of pro
teins, one could speculate that methylation of lysine or
arginine may affect subcellular localization. Indeed,
some lysine methylated proteins such as heat shock pro
tein70 (HSP70) and p53 are predominantly localized
in the nucleus, whereas unmodified versions of these
same proteins are localized in both the cytoplasm and
the nucleus23,39 (FIG.2d)
Promoter binding. Lysine methylation also regulates
the binding affinity of transcription factors for pro
moters, which changes the transcription levels of tar
get genes40 (FIG.2e). For example, lysine methylation of
p53 by the PKMT SETD7 and that of nuclear factor-B
(NFB) by the PKMT nuclear receptor-binding
SET domain-containing protein1 (NSD1) markedly
increased their promoter binding ability and activation
of downstream genes39,41. In a structural modelling
analysis of RELA (a subunit of NFB) in complex with
DNA, two methylated lysine residues on RELA were
shown to interact with DNA through hydrophobic
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contacts42. This result reveals that increased hydropho
bicity of lysine residues by methylation can enhance
the promoter binding ability of transcription factors.
Non-histone PKMT and PKDM substrates

So far, nearly 20 non-histone substrates have been dis
covered for PKMTs and PKDMs (TABLE1). Many PKMTs
and PKDMs are localized in the nucleus, and several
nuclear proteins, including transcription factors, can
serve as substrates of PKMTs and PKDMs. However,
several PKMTs or PKDMs are also localized in the cyto
plasm, and cytoplasmic proteins are also reported to
be substrates of these enzymes. Below, we highlight the
lysine methylation of non-histone proteins involved in
several tumour-associated signalling pathways.
Tudor domains
Protein domains originally
identified as a region of 50
amino acids found in the
Drosophila melanogaster
Tudor protein. The structurally
characterized Tudor domain in
human proteins recognizes
symmetrically dimethylated
arginine. This domain is also
reported as a methyl
lysine-binding protein module.
p53. p53 is one of the most important tumour sup

pressor genes involved in human tumorigenesis 43.
Chuikov etal. 39 demonstrated that SETD7 methyl
ates lysine 372 of p53 (p53K372) and that this meth
ylation enhances p53 stability and transcriptional
activity. K372methylated p53 is restricted to the
nucleus, although p53 is equally distributed between
the nuclear and cytosolic fractions 39. Subsequently,
Huang et al. 44 showed that the PKMT SET and
MYND-domain containing 2 (SMYD2) monometh
ylates lysine 370 of p53 (p53K370); this lysine residue
is located in the regulatory domain at the extreme
carboxyl terminus of p53 (FIG. 3a) . Knockdown
of SMYD2 by small interfering RNA enhances
p53mediated apoptosis in cancer cells44. In addition,
SMYD2dependent p53K370 methylation impairs
the expression of CDKN1A, an important down
stream target of p53, implying that SMYD2 represses
the function of p53 through K370 monomethylation.
Moreover, p53K372 methylation by SETD7 seems
to inhibit SMYD2depedent p53K370 methylation
through blocking the interaction between SMYD2
and p53 (REFS39,44) (FIG.3a).
The function of monomethylation and dimethylation
of p53 on lysine 370 seems to be different. For example,
LSD1 was reported to demethylate p53K370 and repress
p53 activity 45 (FIG.3a). In this case, LSD1 preferentially
reverses dimethylated p53K370 in cancer cells, although
it can remove both monomethylation and dimethylation
at lysine 370 in biochemical reactions invitro45. p53K370
dimethylation promotes the association of p53 with the
co-activator p53binding protein 1 (p53BP1) through
tandem Tudor domains in p53BP1 (REF.45), leading to
increased p53 function, including apoptosis induc
tion. These results indicate that monomethylation of
p53K370 is crucial for the repression of p53 activity,
whereas dimethylation of p53K370 seems to activate
p53. The complexity of methylation pathways in human
cancer is highlighted by the fact that both SMYD2 and
LSD1 affect the same lysine residue of p53, and that
both of these enzymes can promote oncogenesis and are
overexpressed in various types of cancer 22,46. It is plau
sible that their oncogenic actions on p53 may be syn
ergistic, although they could also have effects on other
cancer-related pathways.
Furthermore, Shi etal.47 reported that the protein

lysine methyltransferase SETD8 monomethylates p53
at lysine 382 and that this methylation suppresses
p53dependent transcription activation in cancer
cells. Taken together, these results indicate that lysine
methylation is an important regulator of p53 function.
RB1 and E2F. RB1 is a key cell cycle regulator and tumour
suppressor, and is dysfunctional in several cancer types48.
RB1 was identified as a binding partner for mitogenic
oncoproteins49 and was also discovered to be phosphoryl
ated in synchrony with the cell cycle50. Recently, we and
others reported that lysine methylation of RB1 seems to
be one of the substantial regulators of RB1 function and is
pivotal for cell cycle regulation22,51,52. Methylation at lysine
810 of RB1 by SMYD2 is likely to enhance phosphory
lation of RB1 at serines 807 and 811 (REF.22). Additionally,
although RB1 normally interacts with E2F (a family of
transcription factors that modulate important cellular
events, including cell cycle progression, apoptosis and
DNA damage response53) to suppress transcription of
E2F target genes, lysine 810 methylation of RB1 acceler
ates E2F transcriptional activity through enhancement of
RB1 phosphorylation and promotes cell cycle progression
(FIG.3b). Furthermore, we identified that lysine 442 of pro
tein phosphatase 1, regulatory subunit 12A (PPP1R12A;
also called MYPT1) in the myosin phosphatase holo
enzyme, which stimulates dephosphorylation of RB1, is
a target for methylation and demethylation catalysed by
SETD7 and LSD1, respectively 24 (FIG.3b). Demethylation
of PPP1R12A by LSD1 enhances PPP1R12A polyubiq
uitylation, which increases the proteasome-mediated
degradation of PPP1R12A and therefore the amount of
phosphorylated RB1. Subsequently, released E2F acti
vates transcription genes required for Sphase, and cell
cycle progression is enhanced24. Together, methylation
and demethylation dynamics seems to play an impor
tant part in cell cycle progression through the regulation
of RB1 activity.
Deregulated expression or activity of members
of the E2F family has also been detected in many
human cancers54. Kontaki etal.55 demonstrated that in
p53deficient cancer cells, methylation of lysine 185 of
E2F1 (E2F1K185) by SETD7 inhibits acetylation and
phosphorylation at distant positions and, in parallel,
induces polyubiquitylation and degradation of E2F1.
This process prevents E2F1 accumulation during
DNA damage and activation of its pro-apoptotic tar
get geneTP73 (FIG.3b). Moreover, LSD1 demethylates
E2F1K185, which is important to maintain a substan
tial pool of unmethylated E2F1 in cancer cells, which
can then be subjected to lysine acetyltransferase 2B
(KAT2B)-mediated hyperacetylation, as well as check
point kinase2 (CHEK2)-mediated phosphorylation,
following DNA damage55 (FIG.3b). These results imply
that in p53deficient cancer cells, LSD1 and SETD7
can influence DNA damage-induced cell death in a
manner that is in contrast to the aforementioned
p53dependent apoptosis induction regulated by
LSD1 and SETD7. These results suggest that antican
cer treatments that combine DNA-damaging agents

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Table 1 | Protein lysine methyltransferases and demethylases involved in human tumorigenesis and their substrates
Family
Enzyme
name (alias)
Domain
Subcellular Substrate
localization
Histone
Cancer type
Refs
Non-histone
Lysine methyltransferases
Polycomb
complex
EZH2 (KMT6)
SET, CXC and

SANT
Nucleus*
and
cytoplasm
H3K27 and
H2BK120
ROR and STAT3
AML, bladder cancer, breast

cancer, CCC, CML, CRC,
glioblastoma, lymphoma,
NSCLC, oesophageal cancer,
osteosarcoma, SCLC and RCC
17,31,37,74
SMYD
family
SMYD2
(KMT3C)
SET
Cytoplasm*
and nucleus
H3K4 and
H3K36
p53, RB1, PARP1,

HSP90AB1 and ER
Bladder cancer, breast

cancer, cervical cancer, CRC,
HCC, head and neck cancer,
lymphoma, oesophageal
cancer, ovarian cancer,
pancreatic cancer, prostate
cancer, seminoma and skin
cancer
22,25,27,44,
51,52,69,
111113
SMYD3
(KMT3E)
SET
Cytoplasm*
and nucleus
H3K4 and
H4K5
VEGFR1 and
MAP3K2
ACC, breast cancer, CCC,

cervical cancer, CRC,
HCC, lung cancer, MTC,
oesophageal cancer,
pancreatic and prostate
cancer
SET, PWWP,
AWS, PHD,
RING and
PostSET
Nucleus*
H3K36
and
chromosome
NFB
AML, glioblastoma, lung

cancer and multiple myeloma
122124
WHSC1
(MMSET and
NSD2)
SET, PWWP,
AWS, PHD,
RING and
PostSET
Nucleus*,
H3K36
cytoplasm
and
chromosome
Bladder cancer, breast

cancer, CCC, CML, HCC,
multiple myeloma, NSCLC,
oesophageal cancer
osteosarcoma, prostate
cancer, RCC and SCLC
125129
WHSC1L1
(NSD3)
SET, PWWP,
AWS, PHD,
RING and
PostSET
Nucleus*
H3K36
and
chromosome
Bladder cancer, breast cancer,

lymphoma and SCLC
130132
SETD1A
(KMT2F)
SET, NSET and

PRM
Nucleus*
H3K4
and
chromosome
HSP70
Bladder cancer, CRC, HCC,

lung cancer and RCC
23,133
SETD7
(KMT7)
SET and MORN
Nucleus*
H3K4
and
chromosome
PPP1R12A, p53,
NFB, E2F1, DNMT1
and STAT3
Breast cancer and multiple

myeloma
24,39,55,66,
73,134,135
SETD8
(KMT5A)
SET
Nucleus*
H4K20
and
chromosome
PCNA and p53
Bladder cancer, CML, HCC,

NSCLC and SCLC
47,75
SUV39
family
SUV39H2
(KMT1B)
SET, PreSET,
PostSET and
chromodomain
Nucleus*
H3K9 and
and
H2AXK134
chromosome
Bladder cancer, cervical

cancer, NSCLC, oesophageal
cancer, osteosarcoma,
prostate cancer and STT
32,136
EHMT
family
EHMT2
(KMT1C)
SET, PreSET,
PostSET and
ANK
Nucleus*
H3K9
and
chromosome
C/EBP

cancer, CCC, CML, NSCLC,
oesophageal cancer, prostate
cancer and SCLC
19,71,
137,138
MLL family
MLL2
(KMT2D)
SET, PHD, RING,

FYRN, FYRC and
HMG
Nucleus*
H3K4

CRC, lung cancer, melanoma
and MLL
139
MLL3
(KMT2C)
SET, PHD, RING,

FYRN, FYRC,
HMG, AT_hook,
C1 and PostSET
Nucleus*
H3K4
Breast cancer, glioblastoma,

melanoma, MLL, oesophageal
cancer, pancreatic cancer and
stomach cancer
140142
DOT1L
(KMT4)
AT_hook
Nucleus*
H3K79
MLL
143145
NSD family NSD1

(KMT3B)
SETD
family
DOT1L
family
4,13,26,33,
114121
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Table 1 (cont.) | Protein lysine methyltransferases and demethylases that are involved in human tumorigenesis and their substrates
Family
Enzyme
Domain
name (alias)
Subcellular Substrate
localization
Histone
Cancer type
Refs
Non-histone
Lysine demethylases
LSD1
family
LSD1
(KDM1A)
FAD-binding2
and SWIRM
Nucleus*
H3K4 and
H3K9
PPP1R12A, p53, ER
and STAT3
Bladder cancer, CRC and

SCLC
11,24,45,46
JMJD
family
JMJD1A
(KDM3A)
JmjC
Nucleus*
and
cytoplasm
H3K9
Bladder cancer, CCC, HCC,

NSCLC, osteosarcoma, SCLC
and RCC
20,146,147
JMJD2A
(KDM4A)
JmjC, JmjN, PHD

and Tudor
Nucleus*
H3K9 and
H3K36

CML, NSCLC, oesophageal
cancer, osteosarcoma, ovarian
cancer, prostate cancer, SCLC,
stomach cancer and uterine
cancer
JMJD2B
(KDM4B)
JmjC, JmjN, PHD

and Tudor
Nucleus*
H3K9
Bladder cancer, CRC, NSCLC,

oesophageal cancer, SCLC,
stomach cancer and RCC
JMJD3
(KDM6B)
JmjC
Nucleus*
H3K27
Glioblastoma
UTX
UTX
(KDM6A)
JmjC and TPR
Nucleus*
H3K27

CML, CRC, glioblastoma,
multiple myeloma, NSCLC,
oesophageal cancer,
pancreatic cancer, RCC and
SCLC and TALL
156159
JARID
family
JARID1B
(KDM5B)
JmjC, JmjN,
ARID, PHD,
ZFC5HC2 and
PLU1
Nucleus*
H3K4

cancer, cervix cancer, CML,
CRC, melanoma, NSCLC,
oesophageal cancer, SCLC
and RCC
160164
148151
21,152154
155
ACC, adenocarcinoma; AML, acute myeloid leukaemia; CCC, cholangiocarcinoma; C/EBP, CCAAT/enhancer binding protein-; CML, chronic myelogenous
leukaemia; CRC, colorectal cancer; DNMT1, DNA (cytosine5-)-methyltransferase 1; DOT1L, DOT1like histone H3K79 methyltransferase; EHMT, euchromatic
histone-lysine Nmethyltransferase; ER, oestrogen receptor-; H3K27, histone H3 lysine 27; HCC, hepatocellular carcinoma; HSP, heat shock protein; JARID,
Jumonji, AT-rich interactive domain; JMJD, Jumonji domain-containing; KMT, lysine N-methyltransferase; LSD1, lysine-specific demethylase 1; MAP3K2, MAPK
kinase kinase 2; MLL, mixed-lineage leukaemia; MTC, medullary thyroid cancer; NF-B, nuclear factor-B; NSCLC, non-small cell lung carcinoma; NSD, nuclear
receptor-binding SET domain-containing; PARP1, poly(ADP-ribose) polymerase 1; PCNA, proliferating cell nuclear antigen; PPP1R12A, protein phosphatase 1,
regulatory subunit 12A; RCC, renal cell carcinoma; ROR, retinoic acid-related orphan nuclear receptor; SCLC, small cell lung carcinoma; SETD, SET
domain-containing; SMYD, SET and MYND-domain-containing; STAT3, signal transducer and activator of transcription 3; STT, soft tissue tumour; SUV39H,
suppressor of variegation 39 homologue; TALL, Tcell acute lymphoblastic leukaemia; VEGFR1, vascular endothelial growth factor receptor 1.*The predominant
subcellular localization.
and drugs modulating LSD1 or SETD7 activity may

have inverse effects depending on the p53 status in
cancer cells.
Heat shock proteins. HSPs are overexpressed in a wide
range of human cancers and have substantial roles in
tumour cell proliferation, differentiation, invasion,
metastasis and recognition by the immune system56.
Increased expression of HSPs can also protect malignant
cells from activation of pro-apoptotic signalling, and this
may underlie the role of HSPs in tumour progression
and resistance to treatment 57. HSP70 and HSP90AB1
undergo several PTMs, and we previously identified that
they are also lysine methylated23,25.
HSP70 is a ubiquitous molecular chaperone that
functions in a myriad of biological processes, including
modulation of polypeptide folding, protein degrada
tion, subcellular translocation of proteins across mem
branes and proteinprotein interactions58. We reported
that dimethylation of HSP70 lysine 561 was markedly
increased in cancer cells, and methylated HSP70 was
predominantly localized in the nucleus. This is in contrast

to non-methylated HSP70, which is localized predomi
nantly in the cytoplasm23. The methylation of HSP70 is
catalysed by the PKMT SETD1A, which is overexpressed
in various types of cancer 23. The lysine 561 methylated
HSP70 preferentially binds to and activates Aurora
kinaseB in the nucleus, resulting in cell cycle progression
in cancer cells23 (FIG.2d). The importance of this modifica
tion in cancer was confirmed by immunohistochemical
analysis using an HSP70K571 dimethylation-specific
antibody, which revealed positive nuclear staining in 354
of 409 (86.6%) non-small cell lung carcinoma cases. In
addition to lung cancer, this positive staining of lysine
561 methylated HSP70 was commonly found in bladder
and kidney cancer tissues, whereas no such staining was
observed in non-neoplastic tissues.
HSP90 is an evolutionarily conserved molecular
chaperone that participates in the stabilization and acti
vation of more than 200 proteins, which are referred to as
HSP90 clients. Many of the client proteins of HSP90 are
essential for various cell signalling pathways, including

REVIEWS
a
53BP1
Me Me
p53
p53 inactivation
K370me2 to
K370me1
K370me1
SMYD2
LSD1
53BP1
LSD1
p53
Me
Inhibition of K370me1
p53 inactivation
K372me1
K382me1
SETD7
SETD8
Me Me
Me
370 KSKKGQSTSRHKK 382

p53
TA1 and TA2
PRR
DBDL
OD
CTD
ANK
PPP1R12A
CC
K442 Stabilizes PPP1R12A protein
Me
CDK4
SETD7
LSD1
High anity
PPP1R12A
dephosphorylates
RB1
SMYD2
Me
Enhances RB1
K810 hyperphosphorylation
RB1
Pocket A
Pocket B
SETD7
LSD1
CTD
SMYD2-dependent RB1
methylation activates E2F
Me
K185
E2F1
DBD
CC
MB
TD
Cell cycle progression
Regulation of E2F1-dependent
apoptosis in p53-decient cancer cells
c
VEGFR1
TK1
TM
IG-LD
K260me2
or K260me3
K831me2
SMYD3
Me
K831
Me
Inhibition of
PP2A interaction
TK2
Activation of
kinase activity
PP2A
K260
MAP3K2
PB1
Activation of oncogenic
RAS signalling
Kinase domain
Nature Reviews | Cancer
Figure 3 | Effects of lysine methylation on the pathways of p53, RB1 and protein kinases. a | Methylation of lysine
at residues 370, 372 and 382 in the carboxyterminal domain (CTD) of p53 has been identified. Lysine 370-dimethylated p53
(p53K370me2) can promote the association of p53 with the co-activator p53binding protein1 (53BP1) through tandem
Tudor domains in 53BP1. By contrast, lysine-specific demethylase 1 (LSD1) prevents the accumulation of p53K370me2 by
demethylating this site, thereby preventing the binding of 53BP1 to p53. Both LSD1 and the protein lysine
methyltransferase (PKMT) SET and MYND-domain containing 2 (SMYD2) produce p53K370me1 that results in the
inactivation of p53. Meanwhile, the PKMT SETD7 monomethylates K372 (K372me1), which inhibits K370me1 and activates
p53. SETD8 also inactivates p53 through monomethylation of K382 (K382me1). b | The RB1 pathway is regulated by lysine
methylation. Lysine methylation sites on RB1, E2F1 and protein phosphatase 1, regulatory subunit 12A (PPP1R12A) are
displayed. SETD7 monomethylates K442, which stabilizes PPP1R12A, and this methylation is reversibly demethylated by
LSD1. SMYD2 monomethylates K810 on RB1 and increases the affinity between cyclin-dependent kinase 4 (CDK4) and RB1,
which enhances hyperphosphorylation of RB1 and therefore activates E2F. SETD7 monomethylates K185 of E2F1
(E2F1K185me1), which prevents apoptotic functions of E2F1 in p53deficient cells during DNA damage, and E2F1K185me1
is demethylated by LSD1, promoting E2F1 stability and apoptosis. c | K831 of vascular endothelial growth factor receptor1
(VEGFR1) and K260 of MAPK kinase kinase 2 (MAP3K2) are methylated by SMYD3. VEGFR1K831 dimethylation by SMYD3
activates the kinase activity of VEGFR1. K260 of MAP3K2 is dimethylated and trimethylated by SMYD3, which activates
oncogenic RAS signalling through inhibition of the MAP3K2PP2A (protein phosphatase 2A) interaction. ANK, ankyrin
repeat; CC, coiled-coil domain; DBD, DNA-binding domain; IG-LD, immunoglobulin-like domain; MB, marked-box;
OD,oligomerization domain; PB1, Phox and Bem1p; PRR, proline-rich region; TA, transcription-activation domain;
TD,transactivation domain; TK, tyrosine kinase domain; TM, transmembrane domain.
REVIEWS
Nitrosylation
Nitrosylation, specifically
Snitrosylation, involves the
covalent incorporation of a
nitric oxide moiety into thiol
groups, to form Snitrosothiol.
Snitrosylation is a
physiologically important
post-translational modification
that affects a variety of
proteins involved in a number
of cellular processes.
adaptive responses to various stresses57,59. HSP90 and

proteins called cochaperones form the dynamic com
plex known as the HSP90 chaperone machinery 60.
Cancer cells use the HSP90 chaperone machinery to pro
tect an array of mutated and overexpressed oncoproteins
from misfolding and degradation. Therefore, HSP90 is
recognized as a critical facilitator of oncogene addiction
and cancer cell survival61. HSP90 function is regulated by
various PTMs such as acetylation, phosphorylation and
nitrosylation. We also reported methylation of lysines 531
and 574 of HSP90AB1 by SMYD2. The lysine methyla
tion of HSP90AB1 is important for its homodimerization
and its interaction with stress-induced phosphoprotein1
(STIP1) and cell division cycle 37 (CDC37), which are
cochaperones of HSP90AB1 in human cancer cells,
resulting in enhancement of cancer cell growth25.
Protein kinases (VEGFR1 and MAP3K2). Vascular
endothelial growth factor receptor 1 (VEGFR1), a recep
tor tyrosine kinase, mediates signalling that is involved
in cell proliferation and angiogenesis62. We found that
SMYD3 methylates lysine 831 of VEGFR1, which is
located in the tyrosine kinase domain, and this meth
ylation enhances the kinase activity of VEGFR1 both
invitro and invivo26 (FIG.3c). VEGFR1 in cancer cells
is implicated in liver metastasis of colorectal cancer 63,
and exogenous expression of VEGFR1 enhances the
migration and invasion of pancreatic cancer cells64.
Hence, therapeutic approaches targeting VEGFR1 meth
ylation by SMYD3 can directly attenuate kinase activ
ity of VEGFR1 in cancer cells and benefit patients by
inhibiting invasion and metastasis of cancercells.
SMYD3 can methylate MAP3K2, a member of the ser
ine/threonine protein kinase family 33. SMYD3mediated
methylation of MAP3K2 at lysine 260 activates the RAS
RAFMEKERK signalling module, and SMYD3 deple
tion synergizes with a MEK inhibitor to block RAS-driven
tumorigenesis33 (FIG.3c). A clinical implication of this find
ing is the identification of SMYD3 as a candidate thera
peutic target to treat pancreatic and lung cancers driven
by RAS, as well as potentially other RAS-driven tumours.
The complete loss of SMYD3 function has no apparent
phenotype in mice, implying that SMYD3 inhibitors
would have minimal adverse effects as chemotherapeutic
agents33. Consequently, we could envisage a therapeutic
strategy comprising inhibitors of RAF or MEK (which
are already currently used for anticancer therapy) with
an SMYD3 inhibitor, which could mitigate potential side
effects by lowering the overall dosage needed for each
agent and also limit the development of resistance.
Transcription factors (NFB, ER, C/EBP and
STAT3). The NFB transcription factor regulates multi
ple biological functions, including inflammation, immu
nity, cell proliferation and apoptosis65. SETD7 methylates
lysine 37 of RELA in the nucleus, which enhances the
promoter binding affinity of RELA. This methylation
seems to have a critical role in the induction of a sub
set of NFBdependent genes in response to tumour
necrosis factor (TNF) stimulation66. In addition, Lu
etal.41 showed that NSD1 methylates lysines 218 and 221
of RELA and activates NFB activity. Given that the

K218A, K221A or combined K218A and K221A muta
tion of RELA substantially diminished its DNA-binding
ability, it seems that methylation of several lysine residues
on NFB plays an important part in NFBdependent
transcriptional regulation through increasing its bind
ing to promoters of downstream genes. Interestingly,
these two methylation sites of RELA are demethylated
by the PKDM Fbox and leucine-rich repeat protein11
(FBXL11), and overexpression of FBXL11 inhibits
NFB activation and retards the growth of HT29 colon
cancer cells41 (FIG.4a). Furthermore, SETD6 methylates
RELA on lysine 310 (RELAK310), and this methyla
tion renders RELA inert and attenuates RELA-driven
transcriptional programmes, including inflammatory
responses in primary immune cells67. Monomethylated
RELAK310 is recognized by the ankyrin repeat of
euchromatic histone-lysine Nmethyltransferase 1
(EHMT1), and this interaction stabilizes EHMT1 activ
ity. As EHMT1 works as a transcriptional repressor
through generating monomethylated and dimethylated
H3K9 at euchromatin, the interaction of EHMT1 with
RELA attenuates transcription of NFB target genes
(FIG.4a). Taken together, NFB activity is intricately
regulated by lysine methylation of RELA in a positive
or negative manner depending on the methylationsite.
Oestrogen receptor- (ER), a ligand-activated
transcription factor involved in human breast cancer 68,
is also a substrate of PKMTs. Upon oestrogen stimula
tion, ER recruits several co-regulators to the oestrogen
response elements that modulate activation or repres
sion of target genes. SMYD2 methylates lysine 266 of
ER (ERK266) both in cell-free biochemical assays and
in breast cancer cells, and this SMYD2mediated ER
methylation negatively regulates acetylation of lysines
266 and 268. Given that acetylation of these lysine resi
dues promotes ER transcriptional activity in response
to the ER ligand 17oestradiol through enhancing the
DNA-binding activity of ER69, SMYD2dependent
lysine 266 methylation attenuates the chromatin recruit
ment of ER to prevent ER target gene activation under
an oestrogen-depleted condition69. On oestrogen stim
ulation, ERK266 methylation is diminished, thereby
enabling p300/CREB-binding protein (CBP) to acetylate
ER, which can activate ER target genes. Importantly,
ERK266 is also demethylated by LSD1. These results
suggest a model in which SMYD2 and LSD1 control the
dynamics of ERK266 methylation and its crosstalk with
acetylation of lysines 266 and 268, thereby modulating
ER functions in breast cancer cells69 (FIG.4b).
CCAAT/enhancer-binding protein- (C/EBP) is a
member of the basic leucine zipper family of transcription
regulators that regulates tissue-specific gene expression,
proliferation and differentiation in numerous cell types.
Increased C/EBP expression has been detected in breast
cancer, ovarian tumours and colorectal tumours, and
C/EBP is required for tumour progression in the epider
mis70. Pless etal.71 demonstrated that lysine 39 of C/EBP
(C/EBPK39), which is located in the amino-terminal
transactivation domain, is methylated by EHMT2, leading
to repression of C/EBP transcriptional activity (FIG.4c).

REVIEWS
a
K37me1 K218me1; K221me2

SETD7
NSD1
FBXL11
Me
Me
K37
Gene activation
in response to
TNF stimulation
PP2A
Me
Me
K221
Me
PHF20
TA2
ER
TA1
K310
Me Suppression of NF-Bregulated genes through
High
EHMT1-dependent
anity
H3K9 methylation
DBD Hinge
STAT3
DBD LZ
II
NTD
Me
SETD7
EHMT2
Agonistantagonist
distinction
Promotes STAT3
activity in GSCs
CC
K140
Represses
transcriptional
activity
Me
Ligand-binding
K180me3
EZH2
EHMT1
Me
K180
K39
Ac
Transcriptional
activity
TAs
I
Prevents ER target
gene activation under
an oestrogendepleted condition
p300
K266 K268
IPT_NF-B
Constitutive
activation of
NF-B activity
C/EBP
LSD1
SMYD2
K218 K221
RHD-n
RELA
b K266me1; K266me2
K310me1
SETD6
DBD
LD
SH2
CTD
Negatively regulates
transcription of
some specic genes
LSD1
K140me2
e
PCNA
f
PCNA_N
PARP1
PCNA_C
K248
Me
Interaction
Automodication domain
DBD
Stabilizes
PCNA protein
NLS
Catalytic domain
K528
Me
High anity
SETD8
K248me1
FEN1
Enhances poly(ADP-ribose)
formation after oxidative stress
SMYD2
K528me1
Figure 4 | Effects of lysine methylation on transcription factors and nuclear proteins. a | Lysine 37 of RELA, a subunit
Nature Reviews | Cancer
of nuclear factor-B (NFB) , is monomethylated by SETD7, which activates NFB-dependent genes in response to
tumour necrosis factor (TNF) stimulation. Nuclear receptor-binding SET domain-containing protein 1 (NSD1)
monomethylates K218 (K218me1) and dimethylates K221 (K221me2), resulting in constitutive NFB activation through
interaction with PHF20, which disrupts the recruitment of PP2A to RELA, and these sites are reversibly demethylated by
Fbox and leucine-rich repeat protein 11 (FBXL11). SETD6 monomethylates K310 of RELA and suppresses
NFB-regulated genes through euchromatic histone-lysine Nmethyltransferase1 (EHMT1)dependent H3K9
methylation. b | SMYD2mediated oestrogen receptor (ER) methylation prevents ER target gene activation under an
oestrogen-depleted condition, and lysine-specific demethylase 1 (LSD1) demethylates this site. c | K39 of CCAAT/
enhancer-binding protein- (C/EBP) is methylated by EHMT2, which represses C/EBP transcriptional activity. d | K140 of
signal transducer and activator of transcription 3 (STAT3) is dimethylated by SETD7, which negatively regulates the
transcription of various STAT3 target genes. STAT3K140me2 is reversibly demethylated by LSD1. EZH2 trimethylates K180
of STAT3, which promotes STAT3 activity in glioblastoma stem-like cells (GSCs). e | SETD8 monomethylates K248 of
proliferating cell nuclear antigen (PCNA), and this methylation stabilizes the PCNA protein and enhances the interaction
between PCNA and flap endonuclease 1 (FEN1). f | SMYD2 monomethylates K528 of poly(ADP-ribose) polymerase1
(PARP1) and enhances poly(ADP-ribose) formation after oxidative stress. CC, coiled-coil domain; CTD, carboxy-terminal
domain; DBD, DNA-binding domain; IPT, immunoglobulin-plexin-transcription; LD, linker domain; LZ, leucine zipper
domain; NLS, nuclear localization signal; NTD, amino terminal domain; PCNA_C, PCNA C-terminal domain; PCNA_N,
PCNA N-terminal domain; PHF20, PHD finger protein 20; RHD, REL homology domain; SH2, Src homology 2 domain;
TA,transcription-activation domain; Y, conserved tyrosine domain.
Mechanistically, methylation of C/EBP by EHMT2 may

create a new binding site for a repressive protein com
plex or enhance the interaction between EHMT2 and
C/EBP, which promotes H3K9 methylation by EHMT2
in the vicinity of C/EBP targetgenes.
Signal transducer and activator of transcription3
(STAT3) is a latent transcription factor that acts as an
oncoprotein in several cancers72. Intriguingly, STAT3 is
dimethylated on lysine 140 by SETD7 when it is bound

to the subset of promoters that it activates following its
tyrosine phosphorylation, and this methylation is revers
ibly demethylated by LSD1 (REF.73) (FIG.4d). In this case,
STAT3 is methylated in the nucleus and not in the cyto
sol, in particular only when it is part of a promoter-bound
complex. Moreover, dimethylation only negatively affects
subsets of STAT3activated genes, implying that lysine
REVIEWS
Okazaki fragments
Short, newly synthesized DNA
fragments that are formed on
the lagging template strand
during DNA replication. The
fragments are produced
because of the need for DNA
polymerase to always
synthesize in a 5-to-3
direction and are subsequently
ligated together to form a
continuous strand.
methylation may contribute to specific transcriptional

regulation. Furthermore, lysine 180 on STAT3 is also tri
methylated by EZH2. EZH2dependent STAT3 methyla
tion activates STAT3 functions by increasing its tyrosine
phosphorylation in glioblastoma stem-like cells, and this
EZH2STAT3 pathway seems to be one of the key signal
ling nodes in glioblastoma stem-like cells74. Given that
inhibition of EZH2 inhibits the expression of Polycomb
target genes and diminishes STAT3 activity, it might be a
potential therapeutic strategy in glioblastoma.
Other nuclear proteins (PCNA and PARP1). We reported
previously that the PKMT SETD8 methylates lysine 248
of proliferating cell nuclear antigen (PCNA), one of the
key regulators in DNA replication and cell cycle pro
gression75. Methylation of lysine 248 by SETD8 stabilizes
PCNA proteins through inhibition of polyubiquityla
tion and substantially enhances the interaction between
PCNA and the flap endonuclease FEN1 (REF.75). Loss
of PCNA methylation retards the maturation of Okazaki
fragments and slows DNA replication. Moreover, cells
expressing a PCNA mutant are unable to be methylated
and are more susceptible to hydrogen peroxide-induced
DNA damage75. There are increased levels of methyl
ated PCNA in cancer cells, and there is a correlation
between the expression levels of SETD8 and PCNA in
human cancer tissues75 (FIG.4e). PCNA has been widely
recognized as a tumour marker for cancer progression
and poor patient prognosis because of its function in
cell proliferation76. Given this, SETD8dependent PCNA
methylation is likely to promote tumorigenesis. Hence,
inhibition of PCNA methylation by SETD8 might be a
rational approach to treatcancer.
Poly(ADP-ribose) polymerase 1 (PARP1) catalyses the
transfer of an ADP ribose unit from its substrate, NAD+,
to protein acceptors such as histones and PARP1 itself.
The pivotal roles of PARP1 in the DNA repair pathway
prompted researchers to investigate the effect of PARP1
inhibition on DNA-damaging anticancer therapies.
Indeed, inhibition of PARP1 was shown to enhance the
cytotoxicity of DNA-damaging agents to cancer cells77. We
identified that SMYD2 methylates lysine 528 of PARP1,
which is located in the catalytic domain. Lysine 528meth
ylated PARP1 shows increased poly(ADP-ribosyl)ation
activity after oxidative stress27 (FIG.4f). As SMYD2 deple
tion results in the reduction of PARP1 enzymatic activity,
SMYD2 inhibition might increase the susceptibility of
cancer cells to DNA-damaging chemotherapy.
Somatic mutations of PKMTs and PKDMs

Many somatic mutations have been discovered in
PKMTs and PKDMs through whole-genome sequenc
ing or exome sequencing of cancers (see Supplementary
informationS1 (table)). For example, the missense muta
tions Y641 and A677 in EZH2 are frequently observed
in diffuse large B cell lymphoma (DLBCL), and these
mutations significantly increase the methyltransferase
activity of EZH2, resulting in enhanced proliferation of
cancer cells15. In addition, the E1099K missense muta
tion in the PKMT WolfHirschhorn syndrome candi
date 1like1 (WHSC1), which is found in 14% of t(12;21)
ETV6RUNX1containing acute lymphoblastic leukae

mia cases, enhances the methyltransferase activity of
WHSC1 (REF.78). It is crucial to consider these tumourspecific mutations for the development of anticancer
treatment. Furthermore, somatic mutations in mixedlineage leukaemia (MLL) lysine methyltransferase fam
ily members and in the PKDM UTX (also known as
KDM6A) have frequently been reported in various types
of cancer7990, indicating the importance of these enzymes
in human tumorigenesis. So far, the effect of dysregulation
of PKMTs and PKDMs caused by somatic mutations has
only been discussed in the context of their roles in epige
netic regulations through histone methylation or demeth
ylation. Thus, additional studies to clarify non-histone
substrates and their effects in human cancer areneeded.
Protein lysine methylation as a therapeutic target

Accumulating evidence clearly indicates that the inhibition
of PKMTs or PKDMs shows promise for the development
of anticancer therapy. In 2005, Greiner etal.91 identified
that chaetocin, the first described inhibitor of a PKMT,
specifically inhibits suppressor of variegation 39 homo
logue1 (SUV39H1; also known as SU(VAR)39) both
invitro and invivo.
Subsequently, BIX01294, which is an inhibitor of the
PKMT EHMT2 (REF.92), was shown to effectively suppress
the growth of cancer cell lines19 (TABLE2). Analysis of its
crystal structure shows that BIX01294 lies in a loca
tion occupied by the histone H3 peptide of lysine 4 to
arginine 8 (H3K4H3R8) that is next to the methylation
site of lysine 9 (REF.93), implying that BIX01294 occu
pies the substrate-binding site. This mode of action is
markedly different from that of kinase inhibitors because
most of the anticancer drugs targeting kinases discov
ered so far block phosphotransferase activity by com
peting with ATP94. In 2011, the novel EHMT2 inhibitor
UNC0638 with a half-maximal inhibitory concentration
(IC50) value of <15nM was developed95 (TABLE2). This
compound had high cellular potency and low toxicity 95.
The SMYD2specific inhibitor AZ505 with an IC50
value of 120nM was also reported in 2011; notably, p53
peptides were used for high-throughput screening and
hit validation96. The subsequent crystal structure analysis
of SMYD2 with p53 substrate peptides in complex with
AZ505 clarified that a single AZ505 molecule is bound in
the peptide-binding groove of SMYD2 (REF.96). This result
reveals that AZ505 is a substrate competitive inhibitor 96.
The critical point of this study is that a non-histone pro
tein was used as a substrate of the PKMT for drug devel
opment. When we carefully consider the nature of PKMTs
or PKDMs relevant to tumorigenesis, drug development
strategies that account for their non-histone substrates are
essential. In particular, when targeting PKMTs or PKDMs
that are predominantly localized in the cytoplasm, some
non-histone substrates should be used for drug discovery.
In late 2012, two anticancer drugs targeting EZH2,
both of which inhibit EZH2 carrying a cancer-specific
mutation, were reported. One is GSK126 that targets
Y641- and A677mutated EZH2 for the treatment of
DLBCL15 (TABLE2). GSK126 is a potent, highly selec
tive, AdoMet-competitive chemical compound, which

REVIEWS
Table 2 | Selected inhibitors targeting protein lysine methyltransferases and demethylases
Inhibitor
name
Chemical structure
BIX01294
HN
H3C
Target
enzyme
IC50 value
(enzyme)
Cancer type
Phase
EHMT2
0.18M
None
Preclinical
EHMT2
<15nM
None
Preclinical
SMYD2
0.12M
None
Preclinical
EZH2
0.5nM (Ki)
DLBCL
Preclinical
EZH2
24nM (Ki)
Non-Hodgkin
lymphoma
Preclinical
EZH2
11nM
(Ki=2.5nM)
Non-Hodgkin
lymphoma
PhaseI/II
DOT1L
80pM (Ki)
Relapsed/refractory
AML, ALL or MLL
with rearrangement
of the MLL gene,
including 11q23
or partial tandem
duplication
PhaseI
N
N
O
CH3
O CH3
UNC0638
HN
N
CH3
N
O
N
AZ505
HO
Cl
H
N
N
H
Cl
O
HN
O
GSK126
O
NH
NH
O
HN
EPZ005687
N N
H
N
HN
H
N
EPZ6438
N
O
O
H
N
H2N
HO
OH
N
O
H
N
OR
EPZ5676
H2N
N
N
N
N
NH
HO
OH
GSK2879552
Not available
LSD1
Not available
Relapsed/refractory
SCLC
PhaseI
ORY1001
Not available
LSD1
Not available
AML
PhaseI/IIA
ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; DLBCL, diffuse large B cell lymphoma; DOT1L, DOT1like histone H3K79 methyltransferase;
IC50, half-maximal inhibitory concentration; Ki, inhibition constant; LSD1, lysine-specific demethylase 1; MLL, mixed-lineage leukaemia; SCLC, small cell lung
cancer; SMYD2, SET and MYND domain-containing 2.
REVIEWS
effectively inhibits the proliferation of EZH2mutant
DLBCL cell lines and xenografts in mice15. The other is
EPZ005687, which also targets Y641- and A677mutated
EZH2 found in non-Hodgkin lymphoma97 (TABLE2).
Recently, a new inhibitor (EPZ6438) specific for mutated
EZH2 has been reported that has superior potency and
properties to EPZ005687, including better oral bioavail
ability 98 (TABLE2). A PhaseI/II clinical trial of EPZ6438
has begun for patients with non-Hodgkin lymphoma.
By contrast, 10% of patients with myelodysplastic syn
drome (MDS) harbour lossoffunction mutations in
EZH2 (REF.99), and EZH2 mutations in these patients are
associated with significantly worse prognosis, although
this is not due to transformation to acute myeloid leu
kaemia 100. Patients with MDS without monosomy
7/del(7q) chromosome anomalies also have reduced
expression of EZH2 in CD34+ cells101, which implies the
possibility that EZH2 functions as a tumour suppressor
in MDS, and similar results are also reported in myelo
proliferative neoplasm102. Along this line, patients with
colorectal cancer who have EZH2 overexpression have a
good prognosis17. These results highlight that although
there is no doubt that it is an ideal therapeutic target,
caution is necessary in developing anticancer therapies
targetingEZH2.
EPZ5676, a potent and selective aminonucleoside
inhibitor of the PKMT DOT1L 103,104, has also been
studied in a PhaseI clinical trial for the treatment of
patients with acute leukaemia in which the MLL gene
has undergone rearrangement or tandem duplication
(TABLE2). The data derived from these clinical studies
are expected to provide important information regard
ing further possibilities and potential issues in the
development of anticancer drugs targetingPKMTs.
With regard to PKDMs as candidates for anticancer
therapy development, inhibitors targeting LSD1 have
been actively studied. LSD1 is significantly overexpressed
in human cancer, including small cell lung carcinoma,
and has a crucial role in the growth of cancer cells
through the regulation of chromatin functions46. Because
of the similarity in the catalytic and structural proper
ties, drugs targeting monoamine oxidase (MAO) were
first investigated as LSD1 inhibitors. For example, tranyl
cypromine, an MAO inhibitor used as an antidepressant
drug, can inhibit LSD1 (REF.105), and tranylcypromine
or its analogues show antitumour activity when admin
istered alone or in combination with all-trans-retinoic
acid in leukaemia models106. Given that anti-MAO drugs
are not specific to LSD1 and induce substantial toxicity
invivo, further refined LSD1specific inhibitors have
recently been developed107. In particular, a PhaseI clini
cal trial of GSK2879552, an LSD1specific inhibitor, has
been initiated for patients with relapsed/refractory small
cell lung cancer (TABLE2). Additionally, a PhaseI clinical
trial of the novel LSD1 inhibitor ORY1001 has begun for
acute myeloid leukaemia (TABLE2).
As mentioned above, in addition to the LSD1 fam
ily proteins, JmjC-containing proteins also have PKDM
activity. In 2012, Kruidenier etal.108 reported a selective
JmjC H3K27 demethylase inhibitor. The authors indicated
the relevance and tractability of demethylase inhibition
and that targeting the JmjC-containing proteins may have

broad therapeutic application108. Indeed, several JmjCcontaining demethylases have been reported as candidates
for anticancer therapy (TABLE1), and inhibitors targeting
the Jumonji-type demethylase activity showed antitu
mour effects even though no drugs have yet been evalu
ated in a clinical trial109. Further investigation may reveal
a great potential of Jumonji-type demethylase inhibitors
as antitumouragents.
Conclusions and outlook

Although the first protein lysine methylation was dis
covered in 1959, its physiological significance remained
unknown for a long time. In the twenty-first century, the
discovery of several PKMTs and PKDMs markedly accel
erated a deeper understanding of the role of protein lysine
methylation in many biological processes, particularly
its vital role in epigenetics and also its involvement in
various human diseases, including cancer. In this regard,
although it was difficult to identify methylation sites
of novel substrates, the progress of proteomic analysis
using mass spectrometry has enabled the identification
of methylation sites to become feasible both invitro and
invivo. Consequently, lysine methylation is now widely
recognized as a fundamental PTM. Pang etal.36 reported
45 high-confidence lysine methylation sites in 40 of 2,607
(1.53%) S.cerevisiae proteins, even though their func
tions are mostly unknown. This result demonstrates the
possibility that many substrates of human PKMTs or
PKDMs remain uncharacterized. Importantly, several
PKMTs or PKDMs are localized in both the cytoplasm
and the nucleus; in particular, SMYD2 and SMYD3 pre
dominantly localize in the cytoplasm. In fact, a couple of
cytoplasmic proteins have been identified as substrates
of SMYD2 or SMYD3 (REFS25,26,33). These facts provide
hints that important cytoplasmic substrates of PKMTs or
PKDMs remain to be identified. Historically, the research
on protein lysine methylation was started to analyse its
epigenetic functions, focusing on histone methylation,
and there is no doubt as to its importance for epigenetic
regulation. This is also confirmed by the fact that many
researchers still use the terms histone methyltransferase
and histone demethylase for enzymes relevant to the
protein lysine methylation. Moreover, in addition to its
epigenetic function, further functional analyses may
unveil a wide range of functions of lysine methylation
on non-histone proteins and its relevance to human
cancer. In this regard, a large number of non-histone
proteins have been identified as substrates of histone
acetyltransferases and histone deacetylases (HDACs),
and many of those are the products of oncogenes or
tumour suppressor genes and are directly involved in
human tumorigenesis110. As HDAC inhibitors are now
considered an emerging class of anticancer therapeutics,
detailed examinations of the functions of non-histone
substrates is essential to correctly understand the mecha
nism of actions of HDAC inhibitors110. For the successful
development of PKMT and PKDM inhibitors for cancer
therapy, comprehensive functional analyses to identify
critical substrates of these enzymes in tumorigenesis are
also crucial.

REVIEWS
Ambler,R.P. & Rees,M.W. -Nmethyl-lysine in
bacterial flagellar protein. Nature 184, 5657 (1959).
2. Murray,K. The occurrence of -Nmethyl lysine in
histones. Biochemistry 3, 1015 (1964).
3. Clarke,S.G. & Tamanoi,F. (eds) The Enzymes: Protein
Methyltransferases Vol. 24 (Academic Press, 2006).
4. Hamamoto,R. etal. SMYD3 encodes a histone
methyltransferase involved in the proliferation of
cancer cells. Nature Cell Biol. 6, 731740 (2004).
This is the pioneering report to describe that
dysregulation of a PKMT can cause tumorigenesis.
5. Rea,S. etal. Regulation of chromatin structure by
site-specific histone H3 methyltransferases. Nature
406, 593599 (2000).
This is the first report to show the existence of a
PKMT.
6. Tachibana,M., Sugimoto,K., Fukushima,T. &
Shinkai,Y. SET domain-containing protein, G9a, is a
novel lysine-preferring mammalian histone
methyltransferase with hyperactivity and specific
selectivity to lysines 9 and 27 of histone H3. J.Biol.
Chem. 276, 2530925317 (2001).
7. Schlichter,A. & Cairns,B.R. Histone trimethylation by
Set1 is coordinated by the RRM, autoinhibitory, and
catalytic domains. EMBO J. 24, 12221231 (2005).
8. Ringrose,L., Ehret,H. & Paro,R. Distinct
contributions of histone H3 lysine 9 and 27
methylation to locus-specific stability of polycomb
complexes. Mol. Cell 16, 641653 (2004).
9. Feng,Q. etal. Methylation of H3lysine 79 is
mediated by a new family of HMTases without
a SET domain. Curr. Biol. 12, 10521058 (2002).
10. Bannister,A.J., Schneider,R. & Kouzarides,T. Histone
methylation: dynamic or static? Cell 109, 801806
(2002).
11. Shi,Y. etal. Histone demethylation mediated by the
nuclear amine oxidase homolog LSD1. Cell 119,
941953 (2004).
This is the first report to show the existence of a
PKDM.
12. Tsukada,Y. etal. Histone demethylation by a family of
JmjC domain-containing proteins. Nature 439,
811816 (2006).
13. Hamamoto,R. etal. Enhanced SMYD3 expression is
essential for the growth of breast cancer cells. Cancer
Sci. 97, 113118 (2006).
14. Kotake,Y. etal. pRB family proteins are required for
H3K27 trimethylation and Polycomb repression
complexes binding to and silencing p16INK4 tumor
suppressor gene. Genes Dev. 21, 4954 (2007).
15. McCabe,M.T. etal. EZH2 inhibition as a therapeutic
strategy for lymphoma with EZH2activating
mutations. Nature 492, 108112 (2012).
This study describes the importance of somatic
mutations in PKMTs in developing anticancer drugs.
16. Fiskus,W. etal. Highly effective combination of LSD1
(KDM1A) antagonist and pan-histone deacetylase
inhibitor against human AML cells. Leukemia 28,
21552164 (2014).
17. Takawa,M. Validation of the histone methyltransferase
EZH2 as a therapeutic target for various types of
human cancer and as a prognostic marker. Cancer Sci.
102, 12981305 (2011).
18. Sasaki,M., Yamaguchi,J., Itatsu,K., Ikeda,H. &
Nakanuma,Y. Over-expression of polycomb group
protein EZH2 relates to decreased expression of
p16INK4a in cholangiocarcinogenesis in hepatolithiasis.
J.Pathol. 215, 175183 (2008).
19. Cho,H.S. etal. Enhanced expression of EHMT2 is
involved in the proliferation of cancer cells through
negative regulation of SIAH1. Neoplasia 13,
676684 (2011).
20. Cho,H.S. etal. The JmjC domain-containing histone
demethylase KDM3A is a positive regulator of the
G1/S transition in cancer cells via transcriptional
regulation of the HOXA1 gene. Int. J.Cancer 131,
E179189 (2012).
21. Toyokawa,G. etal. The histone demethylase JMJD2B
plays an essential role in human carcinogenesis
through positive regulation of cyclin-dependent kinase
6. Cancer Prev. Res. (Phila) 4, 20512061 (2011).
22. Cho,H.S. etal. RB1 methylation by SMYD2 enhances
cell cycle progression through an Increase of RB1
phosphorylation. Neoplasia 14, 476486 (2012).
23. Cho,H.S. etal. Enhanced HSP70 lysine methylation
promotes proliferation of cancer cells through
activation of Aurora kinase B. Nature Commun. 3,
1072 (2012).
This study provides clear evidence that protein
lysine methylation plays a critical part in the
regulation of subcellular localization.
1.
24. Cho,H.S. etal. Demethylation of RB regulator

MYPT1 by histone demethylase LSD1 promotes cell
cycle progression in cancer cells. Cancer Res. 71, 16
(2011).
25. Hamamoto,R., Toyokawa,G., Nakakido,M., Ueda,K.
& Nakamura,Y. SMYD2dependent HSP90
methylation promotes cancer cell proliferation by
regulating the chaperone complex formation. Cancer
Lett. 351, 126133 (2014).
26. Kunizaki,M. etal. The lysine 831 of vascular
endothelial growth factor receptor 1 is a novel target
of methylation by SMYD3. Cancer Res. 67,
1075910765 (2007).
27. Piao,L. etal. The histone methyltransferase SMYD2
methylates PARP1 and promotes poly(ADP-ribosyl)
ation activity in cancer cells. Neoplasia 16, 257264.
e2 (2014).
28. Smith,B.C. & Denu,J.M. Chemical mechanisms of
histone lysine and arginine modifications. Biochim.
Biophys. Acta 1789, 4557 (2008).
29. Shimazu,T., Barjau,J., Sohtome,Y., Sodeoka,M. &
Shinkai,Y. Selenium-based Sadenosylmethionine
analog reveals the mammalian seven--strand
methyltransferase METTL10 to be an EF1A1 lysine
methyltransferase. PLoS ONE 9, e105394 (2014).
30. Fang,R. etal. Human LSD2/KDM1b/AOF1 regulates
gene transcription by modulating intragenic H3K4me2
methylation. Mol. Cell 39, 222233 (2010).
31. Kogure,M. etal. The oncogenic polycomb histone
methyltransferase EZH2 methylates lysine 120 on
histone H2B and competes ubiquitination. Neoplasia
15, 12511261 (2013).
32. Sone,K. etal. Critical role of lysine 134 methylation
on histone H2AX for H2AX production & DNA repair.
Nature Commun 5, 5691 (2014).
33. Mazur,P.K. etal. SMYD3 links lysine methylation of
MAP3K2 to Ras-driven cancer. Nature 510, 283287
(2014).
This study provides strong evidence that a PKMT
regulates the function of a cytoplasmic protein.
34. Khorasanizadeh,S. Recognition of methylated
histones: new twists and variations. Curr. Opin. Struct.
Biol. 21, 744749 (2011).
35. Daze,K.D. & Hof,F. The cationpi interaction at
proteinprotein interaction interfaces: developing and
learning from synthetic mimics of proteins that bind
methylated lysines. Acc. Chem. Res. 46, 937945
(2013).
36. Pang,C.N., Gasteiger,E. & Wilkins,M.R.
Identification of arginine- and lysine-methylation in the
proteome of Saccharomyces cerevisiae and its
functional implications. BMC Genomics 11, 92 (2010).
This study comprehensively analyses lysine
methylation of yeast proteins and identifies
numerous substrates.
37. Lee,J.M. etal. EZH2 generates a methyl degron
that is recognized by the DCAF1/DDB1/CUL4 E3
ubiquitin ligase complex. Mol. Cell 48, 572586
(2012).
38. Hodel,M.R., Corbett,A.H. & Hodel,A.E. Dissection
of a nuclear localization signal. J.Biol. Chem. 276,
13171325 (2001).
39. Chuikov,S. etal. Regulation of p53 activity through
lysine methylation. Nature 432, 353360 (2004).
This paper provides the first evidence that p53 is a
substrate of a PKMT.
40. Stark,G.R., Wang,Y. & Lu,T. Lysine methylation of
promoter-bound transcription factors and relevance to
cancer. Cell Res. 21, 375380 (2011).
41. Lu,T. etal. Regulation of NFB by NSD1/
FBXL11dependent reversible lysine methylation of
p65. Proc. Natl Acad. Sci. USA 107, 4651 (2010).
42. Lu,T. etal. Role of lysine methylation of NFB in
differential gene regulation. Proc. Natl Acad. Sci. USA
110, 1351013515 (2013).
43. Nakamura,Y. Isolation of p53target genes and their
functional analysis. Cancer Sci. 95, 711 (2004).
44. Huang,J. etal. Repression of p53 activity by
Smyd2mediated methylation. Nature 444, 629632
(2006).
45. Huang,J. etal. p53 is regulated by the lysine
demethylase LSD1. Nature 449, 105108 (2007).
This study provides the first evidence that a PKDM
can also demethylate a non-histone protein.
46. Hayami,S. etal. Overexpression of LSD1 contributes
to human carcinogenesis through chromatin
regulation in various cancers. Int. J.Cancer 128,
574586 (2011).
47. Shi,X. etal. Modulation of p53 function by
SET8mediated methylation at lysine 382. Mol. Cell
27, 636646 (2007).
48. Goodrich,D.W. The retinoblastoma tumor-suppressor

gene, the exception that proves the rule. Oncogene
25, 52335243 (2006).
49. Munger,K. etal. Complex formation of human
papillomavirus E7 proteins with the retinoblastoma
tumor suppressor gene product. EMBO J. 8,
40994105 (1989).
50. Buchkovich,K., Duffy,L.A. & Harlow,E. The
retinoblastoma protein is phosphorylated during
specific phases of the cell cycle. Cell 58, 10971105
(1989).
51. Saddic,L.A. etal. Methylation of the retinoblastoma
tumor suppressor by SMYD2. J.Biol. Chem. 285,
3773337740 (2010).
52. Carr,S.M., Munro,S., Kessler,B., Oppermann,U. &
La Thangue,N.B. Interplay between lysine
methylation and Cdk phosphorylation in growth
control by the retinoblastoma protein. EMBO J. 30,
317327 (2011).
53. Tsantoulis,P.K. & Gorgoulis,V.G. Involvement of E2F
transcription factor family in cancer. Eur. J.Cancer 41,
24032414 (2005).
54. Chen,H.Z., Tsai,S.Y. & Leone,G. Emerging roles of
E2Fs in cancer: an exit from cell cycle control. Nature
Rev. Cancer 9, 785797 (2009).
55. Kontaki,H. & Talianidis,I. Lysine methylation
regulates E2F1induced cell death. Mol. Cell 39,
152160 (2010).
56. Ciocca,D.R. & Calderwood,S.K. Heat shock proteins
in cancer: diagnostic, prognostic, predictive, and
treatment implications. Cell Stress Chaperones 10,
86103 (2005).
57. Trepel,J., Mollapour,M., Giaccone,G. & Neckers,L.
Targeting the dynamic HSP90 complex in cancer.
Nature Rev. Cancer 10, 537549 (2010).
58. Kampinga,H.H. & Craig,E.A. The HSP70 chaperone
machinery: J proteins as drivers of functional
specificity. Nature Rev. Mol. Cell Biol. 11, 579592
(2010).
59. Wandinger,S.K., Richter,K. & Buchner,J. The Hsp90
chaperone machinery. J.Biol. Chem. 283,
1847318477 (2008).
60. Pratt,W.B. & Toft,D.O. Regulation of signaling
protein function and trafficking by the hsp90/
hsp70based chaperone machinery. Exp. Biol. Med.
(Maywood) 228, 111133 (2003).
61. Whitesell,L. & Lindquist,S.L. HSP90 and the
chaperoning of cancer. Nature Rev. Cancer 5,
761772 (2005).
62. Olsson,A.K., Dimberg,A., Kreuger,J. &
Claesson-Welsh,L. VEGF receptor signalling in
control of vascular function. Nature Rev. Mol. Cell Biol.
7, 359371 (2006).
63. Fan,F. etal. Expression and function of vascular
endothelial growth factor receptor1 on human
colorectal cancer cells. Oncogene 24, 26472653
(2005).
64. Abdelrahim,M. etal. Regulation of vascular
endothelial growth factor receptor1 expression by
specificity proteins 1, 3, and 4 in pancreatic cancer
cells. Cancer Res. 67, 32863294 (2007).
65. Silverman,N. & Maniatis,T. NFB signaling pathways
in mammalian and insect innate immunity. Genes Dev.
15, 23212342 (2001).
66. Ea,C.K. & Baltimore,D. Regulation of NFB activity
through lysine monomethylation of p65. Proc. Natl
Acad. Sci. USA 106, 1897218977 (2009).
67. Levy,D. etal. Lysine methylation of the NFB subunit
RelA by SETD6 couples activity of the histone
methyltransferase GLP at chromatin to tonic
repression of NFB signaling. Nature Immunol. 12,
2936 (2011).
68. Ali,S. & Coombes,R.C. Estrogen receptor in human
breast cancer: occurrence and significance.
J.Mammary Gland Biol. Neoplasia 5, 271281
(2000).
69. Zhang,X. etal. Regulation of estrogen receptor by
histone methyltransferase SMYD2mediated protein
methylation. Proc. Natl Acad. Sci. USA 110,
1728417289 (2013).
70. Grimm,S.L. & Rosen,J.M. The role of C/EBP in
mammary gland development and breast cancer.
J.Mammary Gland Biol. Neoplasia 8, 191204
(2003).
71. Pless,O. etal. G9amediated lysine methylation
alters the function of CCAAT/enhancer-binding
protein-. J.Biol. Chem. 283, 2635726363
(2008).
72. Chen,Y. etal. STAT3, a poor survival predicator, is
associated with lymph node metastasis from breast
cancer. J.Breast Cancer 16, 4049 (2013).
REVIEWS
73. Yang,J. etal. Reversible methylation of promoterbound STAT3 by histone-modifying enzymes. Proc.
Natl Acad. Sci. USA 107, 2149921504 (2010).
74. Kim,E. etal. Phosphorylation of EZH2 activates
STAT3 signaling via STAT3 methylation and promotes
tumorigenicity of glioblastoma stem-like cells. Cancer
Cell 23, 839852 (2013).
This study provides clear evidence that non-histone
lysine methylation is an important target of cancer
therapy invivo.
75. Takawa,M. etal. Histone lysine methyltransferase
SETD8 promotes carcinogenesis by deregulating
PCNA expression. Cancer Res. 72, 32173227
(2012).
76. Zhao,H. etal. Targeting tyrosine phosphorylation of
PCNA inhibits prostate cancer growth. Mol. Cancer
Ther. 10, 2936 (2011).
77. Chen,A. PARP inhibitors: its role in treatment of
cancer. Chin. J.Cancer 30, 463471 (2011).
78. Jaffe,J.D. etal. Global chromatin profiling reveals
NSD2 mutations in pediatric acute lymphoblastic
leukemia. Nature Genet. 45, 13861391 (2013).
This study describes the importance of somatic
mutations in PKMTs for developing anticancer
drugs.
79. Yin,S. etal. Exome sequencing identifies frequent
mutation of MLL2 in non-small cell lung carcinoma
from Chinese patients. Sci. Rep. 4, 6036 (2014).
80. Gossage,L. etal. Clinical and pathological impact of
VHL, PBRM1, BAP1, SETD2, KDM6A, and JARID1c in
clear cell renal cell carcinoma. Genes Chromosomes
Cancer 53, 3851 (2014).
81. Cleary,S.P. etal. Identification of driver genes in
hepatocellular carcinoma by exome sequencing.
Hepatology 58, 16931702 (2013).
82. Dubuc,A.M. etal. Aberrant patterns of H3K4 and
H3K27 histone lysine methylation occur across
subgroups in medulloblastoma. Acta Neuropathol.
125, 373384 (2013).
83. Jankowska,A.M. etal. Mutational spectrum analysis
of chronic myelomonocytic leukemia includes genes
associated with epigenetic regulation: UTX, EZH2, and
DNMT3A. Blood 118, 39323941 (2011).
84. Morin,R.D. Frequent mutation of histone-modifying
genes in non-Hodgkin lymphoma. Nature 476,
298303 (2011).
85. Wang,X.X. etal. Somatic mutations of the mixedlineage leukemia 3 (MLL3) gene in primary breast
cancers. Pathol. Oncol. Res. 17, 429433 (2011).
86. Gui,Y. Frequent mutations of chromatin remodeling
genes in transitional cell carcinoma of the bladder.
Nature Genet. 43, 875878 (2011).
87. Grasso,C.S. etal. The mutational landscape of lethal
castration-resistant prostate cancer. Nature 487,
239243 (2012).
88. Lindberg,J. etal. The mitochondrial and autosomal
mutation landscapes of prostate cancer. Eur. Urol. 63,
702708 (2013).
89. Ho,A.S. etal. The mutational landscape of adenoid
cystic carcinoma. Nature Genet. 45, 791798 (2013).
90. Kandoth,C. etal. Mutational landscape and
significance across 12 major cancer types. Nature
502, 333339 (2013).
91. Greiner,D., Bonaldi,T., Eskeland,R., Roemer,E. &
Imhof,A. Identification of a specific inhibitor of the
histone methyltransferase SU(VAR)39. Nature Chem.
Biol. 1, 143145 (2005).
92. Kubicek,S. etal. Reversal of H3K9me2 by a smallmolecule inhibitor for the G9a histone
methyltransferase. Mol. Cell 25, 473481 (2007).
93. Chang,Y. etal. Structural basis for G9alike protein
lysine methyltransferase inhibition by BIX01294.
Nature Struct. Mol. Biol. 16, 312317 (2009).
This study determined the crystal structure of the
catalytic SET domain of G9alike protein in complex
with the specific inhibitor BIX01294.
94. Liu,Y. & Gray,N.S. Rational design of inhibitors that
bind to inactive kinase conformations. Nature Chem.
Biol. 2, 358364 (2006).
95. Vedadi,M. etal. A chemical probe selectively inhibits
G9a and GLP methyltransferase activity in cells.
Nature Chem. Biol. 7, 566574 (2011).
96. Ferguson,A.D. etal. Structural basis of substrate
methylation and inhibition of SMYD2. Structure 19,
12621273 (2011).
This study describes a drug development strategy
for inhibitors of a PKMT using a non-histone
protein as a substrate.
97. Knutson,S.K. etal. A selective inhibitor of EZH2
blocks H3K27 methylation and kills mutant lymphoma
cells. Nature Chem. Biol. 8, 890896 (2012).
98. Knutson,S.K. etal. Selective inhibition of EZH2 by

EPZ6438 leads to potent antitumor activity in
EZH2mutant non-Hodgkin lymphoma. Mol. Cancer
Ther. 13, 842854 (2014).
99. Nikoloski,G. etal. Somatic mutations of the histone
methyltransferase gene EZH2 in myelodysplastic
syndromes. Nature Genet. 42, 665667 (2010).
100. Bejar,R. etal. Validation of a prognostic model and
the impact of mutations in patients with lower-risk
myelodysplastic syndromes. J.Clin. Oncol. 30,
33763382 (2012).
101. Jerez,A. etal. Loss of heterozygosity in 7q myeloid
disorders: clinical associations and genomic
pathogenesis. Blood 119, 61096117 (2012).
102. Muto,T. etal. Concurrent loss of Ezh2 and Tet2
cooperates in the pathogenesis of myelodysplastic
disorders. J.Exp. Med. 210, 26272639 (2013).
103. Basavapathruni,A. etal. Nonclinical pharmacokinetics
and metabolism of EPZ5676, a novel DOT1L histone
methyltransferase inhibitor. Biopharm. Drug Dispos.
35, 237252 (2014).
104. Klaus,C.R. etal. DOT1L inhibitor EPZ5676 displays
synergistic antiproliferative activity in combination
with standard of care drugs and hypomethylating
agents in MLL-rearranged leukemia cells.
J.Pharmacol. Exp. Ther. 350, 646656 (2014).
References 103 and 104 describe the DOT1L
inhibitor EPZ5676, which is currently in clinical
trials.
105. Schmidt,D.M. & McCafferty,D.G.
Trans2phenylcyclopropylamine is a mechanismbased inactivator of the histone demethylase LSD1.
Biochemistry 46, 44084416 (2007).
106. Schenk,T. etal. Inhibition of the LSD1 (KDM1A)
demethylase reactivates the all-trans-retinoic acid
differentiation pathway in acute myeloid leukemia.
Nature Med. 18, 605611 (2012).
107. Wang,J. etal. Novel histone demethylase LSD1
inhibitors selectively target cancer cells with
pluripotent stem cell properties. Cancer Res. 71,
72387249 (2011).
108. Kruidenier,L. etal. A selective jumonji H3K27
demethylase inhibitor modulates the proinflammatory
macrophage response. Nature 488, 404408
(2012).
This report provides evidence that a Jumonji-type
demethylase is a good target for drug discovery.
109. Wang,L. etal. A small molecule modulates Jumonji
histone demethylase activity and selectively inhibits
cancer growth. Nature Commun. 4, 2035 (2013).
110. Singh,B.N. etal. Nonhistone protein acetylation as
cancer therapy targets. Expert Rev. Anticancer Ther.
10, 935954 (2010).
111. Komatsu,S. etal. Overexpression of SMYD2 relates
to tumor cell proliferation and malignant outcome of
esophageal squamous cell carcinoma. Carcinogenesis
30, 11391146 (2009).
112. Abu-Farha,M. etal. The tale of two domains:
proteomics and genomics analysis of SMYD2, a new
histone methyltransferase. Mol. Cell Proteom. 7,
560572 (2008).
113. Brown,M.A., Sims,R.J., 3rd, Gottlieb,P.D. &
Tucker,P.W. Identification and characterization of
Smyd2: a split SET/MYND domain-containing histone
H3 lysine 36specific methyltransferase that interacts
with the Sin3 histone deacetylase complex. Mol.
Cancer 5, 26 (2006).
114. Silva,F.P. etal. Enhanced methyltransferase activity
of SMYD3 by the cleavage of its Nterminal region in
human cancer cells. Oncogene 27, 26862692
(2008).
115. Foreman,K.W. etal. Structural and functional
profiling of the human histone methyltransferase
SMYD3. PLoS ONE 6, e22290 (2011).
116. Dong,S.W. etal. Effect of the downregulation of
SMYD3 expression by RNAi on RIZ1 expression and
proliferation of esophageal squamous cell carcinoma.
Oncol. Rep. 32, 10641070 (2014).
117. Sponziello,M. etal. Epigenetic-related gene
expression profile in medullary thyroid cancer revealed
the overexpression of the histone methyltransferases
EZH2 and SMYD3 in aggressive tumours. Mol. Cell
Endocrinol. 392, 813 (2014).
118. Vieira,F.Q. etal. Deregulated expression of
selected histone methylases and demethylases in
prostate carcinoma. Endocr. Relat. Cancer 21,
5161 (2014).
119. Wang,S.Z. etal. Knockdown of SMYD3 by RNA
interference inhibits cervical carcinoma cell growth
and invasion invitro. BMB Rep. 41, 294299
(2008).
120. Zeng,B. etal. Epigenetic regulation of miR124 by

hepatitis C virus core protein promotes migration and
invasion of intrahepatic cholangiocarcinoma cells by
targeting SMYD3. FEBS Lett. 586, 32713278
(2012).
121. Guo,N. etal. Hepatitis C virus core upregulates the
methylation status of the RASSF1A promoter through
regulation of SMYD3 in hilar cholangiocarcinoma cells.
Acta Biochim. Biophys. Sin. (Shanghai) 43, 354361
(2011).
122. Wang,G.G., Cai,L., Pasillas,M.P. & Kamps,M.P.
NUP98NSD1 links H3K36 methylation to HoxA
gene activation and leukaemogenesis. Nature Cell
Biol. 9, 804812 (2007).
123. Panarello,C., Rosanda,C. & Morerio,C. Cryptic
translocation t(5;11)(q35;p15.5) with involvement of
the NSD1 and NUP98 genes without 5q deletion in
childhood acute myeloid leukemia. Genes
Chromosomes Cancer 35, 277281 (2002).
124. Hollink,I.H. etal. NUP98/NSD1 characterizes a novel
poor prognostic group in acute myeloid leukemia with
a distinct HOX gene expression pattern. Blood 118,
36453656 (2011).
125. Toyokawa,G. etal. Histone lysine methyltransferase
Wolf-Hirschhorn syndrome candidate 1 is involved
in human carcinogenesis through regulation of
the Wnt pathway. Neoplasia 13, 887898
(2011).
126. Martinez-Garcia,E. etal. The MMSET histone methyl
transferase switches global histone methylation and
alters gene expression in t(4;14) multiple myeloma
cells. Blood 117, 211220 (2011).
127. Ezponda,T. etal. The histone methyltransferase
MMSET/WHSC1 activates TWIST1 to promote an
epithelial-mesenchymal transition and invasive
properties of prostate cancer. Oncogene 32,
28822890 (2013).
128. Kim,J.Y. etal. Multiple-myeloma-related WHSC1/
MMSET isoform REIIBP is a histone
methyltransferase with transcriptional repression
activity. Mol. Cell. Biol. 28, 20232034 (2008).
129. Pei,H. etal. MMSET regulates histone H4K20
methylation and 53BP1 accumulation at DNA
damage sites. Nature 470, 124128 (2011).
130. Kang,D. etal. The histone methyltransferase
WolfHirschhorn syndrome candidate 1like 1
(WHSC1L1) is involved in human carcinogenesis.
Genes Chromosomes Cancer 52, 126139
(2013).
131. Zhou,Z., Thomsen,R., Kahns,S. & Nielsen,A.L.
The NSD3L histone methyltransferase regulates cell
cycle and cell invasion in breast cancer cells.
Biochem. Biophys. Res. Commun. 398, 565570
(2010).
132. Kim,S.M. etal. Characterization of a novel
WHSC1associated SET domain protein with H3K4
and H3K27 methyltransferase activity. Biochem.
Biophys. Res. Commun. 345, 318323 (2006).
133. Salz,T. etal. hSETD1A regulates Wnt target genes
and controls tumor growth of colorectal cancer cells.
Cancer Res. 74, 775786 (2014).
134. Hu,H.Y. etal. Set9, NFB, and microRNA21
mediate berberine-induced apoptosis of human
multiple myeloma cells. Acta Pharmacol. Sin. 34,
157166 (2013).
135. Subramanian,K. etal. Regulation of estrogen receptor
by the SET7 lysine methyltransferase. Mol. Cell 30,
336347 (2008).
136. OCarroll,D. etal. Isolation and characterization of
Suv39h2, a second histone H3 methyltransferase
gene that displays testis-specific expression. Mol. Cell.
Biol. 20, 94239433 (2000).
137. Lehnertz,B. etal. The methyltransferase G9a
regulates HoxA9dependent transcription in AML.
Genes Dev. 28, 317327 (2014).
138. Zhong,X. etal. Overexpression of G9a and MCM7 in
esophageal squamous cell carcinoma associated with
poor prognosis. Histopathology http://dx.doi.
org/10.1111/his.12456 (2014).
139. Natarajan,T.G. etal. Epigenetic regulator MLL2
shows altered expression in cancer cell lines and
tumors from human breast and colon. Cancer Cell. Int.
10, 13 (2010).
140. Xia,M. etal. Downregulation of MLL3 in
esophageal squamous cell carcinoma is required for
the growth and metastasis of cancer cells. Tumour
Biol. http://dx.doi.org/10.1007/s13277-014-2616-3
(2014).
141. Li,B. etal. Association of MLL3 expression with
prognosis in gastric cancer. Genet. Mol. Res. 13,
75137518 (2014).

REVIEWS
142. Ruault,M., Brun,M.E., Ventura,M., Roizes,G. & De
Sario,A. MLL3, a new human member of the TRX/
MLL gene family, maps to 7q36, a chromosome region
frequently deleted in myeloid leukaemia. Gene 284,
7381 (2002).
143. Okada,Y. etal. hDOT1L links histone methylation to
leukemogenesis. Cell 121, 167178 (2005).
144. Nguyen,A.T., Taranova,O., He,J. & Zhang,Y. DOT1L,
the H3K79 methyltransferase, is required for
MLLAF9mediated leukemogenesis. Blood 117,
69126922 (2011).
145. Bernt,K.M. & Armstrong,S.A. A role for DOT1L in
MLL-rearranged leukemias. Epigenomics 3, 667670
(2011).
146. Yamada,D. etal. Role of the hypoxia-related gene,
JMJD1A, in hepatocellular carcinoma: clinical impact
on recurrence after hepatic resection. Ann. Surg.
Oncol. 19, S355S364 (2012).
147. Guo,X. etal. The expression of histone demethylase
JMJD1A in renal cell carcinoma. Neoplasma 58,
153157 (2011).
148. Kogure,M. etal. Deregulation of the histone
demethylase JMJD2A is involved in human
carcinogenesis through regulation of the G1/S
transition. Cancer Lett. 336, 7684 (2013).
149. Berry,W.L., Shin,S., Lightfoot,S.A. & Janknecht,R.
Oncogenic features of the JMJD2A histone
demethylase in breast cancer. Int. J.Oncol. 41,
17011706 (2012).
150. Wang,H.L. etal. Expression and effects of JMJD2A
histone demethylase in endometrial carcinoma.
Asian Pac. J.Cancer Prev. 15, 30513056 (2014).
151. Hu,C.E., Liu,Y.C., Zhang,H.D. & Huang,G.J.
JMJD2A predicts prognosis and regulates cell growth
in human gastric cancer. Biochem. Biophys. Res.

Commun. 449, 17 (2014).
152. Sun,B.B. etal. Silencing of JMJD2B induces cell
apoptosis via mitochondria-mediated and death
receptor-mediated pathway activation in colorectal
cancer. J.Dig. Dis. 15, 491500 (2014).
153. Chen,L. etal. Jumonji domain-containing protein 2B
silencing induces DNA damage response via STAT3
pathway in colorectal cancer. Br. J.Cancer 110,
10141026 (2014).
154. Zhao,L. etal. JMJD2B promotes epithelialmesenchymal transition by cooperating with -catenin
and enhances gastric cancer metastasis. Clin. Cancer
Res. 19, 64196429 (2013).
155. Ene,C.I. etal. Histone demethylase Jumonji D3
(JMJD3) as a tumor suppressor by regulating p53
protein nuclear stabilization. PLoS ONE 7, e51407
(2012).
156. Nickerson,M.L. etal. Concurrent alterations in
TERT, KDM6A, and the BRCA pathway in
bladder cancer. Clin. Cancer Res. 20,
49354948 (2014).
157. Ntziachristos,P. etal. Contrasting roles of histone 3
lysine 27 demethylases in acute lymphoblastic
leukaemia. Nature 514, 513517 (2014).
158. Shen,Y. etal. Expression and significance of histone
H3K27 demethylases in renal cell carcinoma. BMC
Cancer 12, 470 (2012).
159. van Haaften,G. etal. Somatic mutations of the histone
H3K27 demethylase gene UTX in human cancer.
Nature Genet. 41, 521523 (2009).
160. Hayami,S. etal. Overexpression of the JmjC histone
demethylase KDM5B in human carcinogenesis:
involvement in the proliferation of cancer cells
through the E2F/RB pathway. Mol. Cancer 9, 59

(2010).
161. Yamamoto,S. etal. JARID1B is a luminal lineagedriving oncogene in breast cancer. Cancer Cell 25,
762777 (2014).
162. Kano,Y. etal. Jumonji/Arid1b (Jarid1b) protein
modulates human esophageal cancer cell growth. Mol.
Clin. Oncol. 1, 753757 (2013).
163. Ohta,K. etal. Depletion of JARID1B induces cellular
senescence in human colorectal cancer. Int. J.Oncol.
42, 12121218 (2013).
164. Radberger,P., Radberger,A., Bykov,V.J.,
Seregard,S. & Economou,M.A. JARID1B protein
expression and prognostic implications in uveal
melanoma. Invest. Ophthalmol. Vis. Sci. 53,
44424449 (2012).
Acknowledgements
The authors express great gratitude to the past and present

members of Y.N.s laboratory.
Competing interests statement
The authors declare competing interests: see Web version for

details.
DATABASES
National Cancer Institute Drug Dictionary:
http://www.cancer.gov/drugdictionary
Pathway Interaction Database: http://pid.nci.nih.gov
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REVIEWS

Critical roles of non-histone

Nmethyl-lysine was first found in a bacterial flagel

family was reported to have protein demethylase activ

110 | FEBRUARY 2015 | VOLUME 15

b Lysine demethylation (LSD1: FAD-dependent amine oxidase)

c Lysine demethylation (JmjC-containing proteins: hydroxylase)

Figure 1 | The chemical mechanism of protein lysine methylation. a | Protein lysine

In this Review, we highlight the current knowledge

Biochemical characteristics of lysine methylation

Functions of protein lysine methylation

NATURE REVIEWS | CANCER

VOLUME 15 | FEBRUARY 2015 | 111

energies and hydrogen bonding potential of the lysine

Protein stability. As lysine polyubiquitylation plays a

such as retinoic acid-related orphan nuclear receptor

112 | FEBRUARY 2015 | VOLUME 15

Non-histone PKMT and PKDM substrates

p53. p53 is one of the most important tumour sup

Furthermore, Shi etal.47 reported that the protein

NATURE REVIEWS | CANCER

VOLUME 15 | FEBRUARY 2015 | 113

SET, CXC and

ROR and STAT3

AML, bladder cancer, breast

p53, RB1, PARP1,

Bladder cancer, breast

ACC, breast cancer, CCC,

AML, glioblastoma, lung

Bladder cancer, breast

Bladder cancer, breast cancer,

SET, NSET and

Bladder cancer, CRC, HCC,

SET and MORN

Breast cancer and multiple

PCNA and p53

Bladder cancer, CML, HCC,

Bladder cancer, cervical

AML, bladder cancer, breast

SET, PHD, RING,

Bladder cancer, breast cancer,

SET, PHD, RING,

Breast cancer, glioblastoma,

NSD family NSD1

114 | FEBRUARY 2015 | VOLUME 15

Bladder cancer, CRC and

Bladder cancer, CCC, HCC,

JmjC, JmjN, PHD

Bladder cancer, breast cancer,

JmjC, JmjN, PHD

Bladder cancer, CRC, NSCLC,

JmjC and TPR

Bladder cancer, breast cancer,

AML, bladder cancer, breast

and drugs modulating LSD1 or SETD7 activity may

predominantly localized in the nucleus. This is in contrast

NATURE REVIEWS | CANCER

VOLUME 15 | FEBRUARY 2015 | 115

370 KSKKGQSTSRHKK 382

TA1 and TA2

Cell cycle progression

adaptive responses to various stresses57,59. HSP90 and

of RELA and activates NFB activity. Given that the