Download Molecular Pathways: Protein Methyltransferases in Cancer

Published OnlineFirst August 19, 2013; DOI: 10.1158/1078-0432.CCR-13-0223
Clinical
Cancer
Research
Molecular Pathways
Molecular Pathways: Protein Methyltransferases in Cancer
Robert A. Copeland
Abstract
The protein methyltransferases (PMT) constitute a large and important class of enzymes that catalyze sitespecific methylation of lysine or arginine residues on histones and other proteins. Site-specific histone
methylation is a critical component of chromatin regulation of gene transcription—a pathway that is often
genetically altered in human cancers. Oncogenic alterations (e.g., mutations, chromosomal translocations,
and others) of PMTs, or of associated proteins, have been found to confer unique dependencies of cancer
cells on the activity of specific PMTs. Examples of potent, selective small-molecule inhibitors of specific PMTs
are reviewed that have been shown to kill cancers cells bearing such oncogenic alterations, while having
minimal effect on proliferation of nonaltered cells. Selective inhibitors of the PMTs, DOT1L and EZH2, have
entered phase I clinical studies and additional examples of selective PMT inhibitors are likely to enter the
clinic soon. The current state of efforts toward clinical testing of selective PMT inhibitors as personalized
cancer therapeutics is reviewed here. Clin Cancer Res; 19(23); 6344–50. 2013 AACR.
Background
Posttranslational modifications of histone proteins
play a critical role in defining the local structure of
chromatin (the complex of DNA and histone proteins
that make up chromosomes) and thereby control entire
programs of gene transcription within cells (1). Both
reversible small-molecule (e.g., acetylation, methylation,
and phosphorylation) and protein (e.g., ubiquitination
and sumoylation) modifications of histone are known to
affect nucleosome compacting within chromatin and
also to serve as recognition loci for binding of transcription factors, polymerases, and auxillary proteins that
either facilitate or antagonize gene transcription (2).
Among these various histone modifications (Fig. 1A),
methylation of lysine and arginine residues seems to
play a particularly important role in control of gene
transcription programs (3–5). These modifications are
catalyzed by a class of group-transfer enzymes known as
the protein methyltransferases (PMT; refs. 3, 6). The
PMT enzyme class is composed of two distinct families
of enzymes, based on their active site structures and on
the amino acid to which they transfer methyl groups: the
protein lysine methyltransferases (PKMT) and the protein arginine methyltransferases (PRMT; ref. 6). There is
one exception to this general structural bifurcation of the
PMT class. The enzyme DOT1L is biochemically a lysine
methyltransferase, but its active site structure is most
Author's Affiliation: Epizyme, Inc., Cambridge, Massachusetts
Corresponding Author: Robert A. Copeland, Epizyme, Inc., 400 Technology Square, Cambridge, MA 02139. Phone: 617-500-0707; Fax: 617-3490707; E-mail: [email protected]
doi: 10.1158/1078-0432.CCR-13-0223
2013 American Association for Cancer Research.
6344
closely aligned with that of the protein arginine methyltransferases (6). On the basis of active site structure, a
number of enzymes that had been previously annotated
as RNA methyltransferases can also be included within
the family of PRMTs, and some of these enzymes have
subsequently been shown biochemically to catalyze
both RNA and protein methylation (6, 7). Thus, the
PMT class is composed of 96 enzymes within humans.
Reaction fidelity is variable among the PMTs. In some
cases, a single enzyme is responsible for site-specific
methylation of a unique histone location (e.g., DOT1L
methylation of H3K79; see below). In other cases, several PMTs seem to methylate a common histone site but
may do so in a gene-specific manner (Fig. 1B). In yet
other cases, a single enzyme may methylate multiple
protein substrates.
The PMTs constitute an interesting target class for cancer
therapeutics both because of the critical role these enzymes
play in controlling cellular programs of gene transcription
and because, increasingly, bioinformatic surveillance of
cancer genome databases are showing that a number of
these enzymes are implicated in specific human cancers
(5, 8); in a recent review, for example, Copeland listed over
10 PMTs for which genetic alterations are found in specific
human cancers (8).
PMTs may play critical roles in cancer cells in general,
but their greatest value as therapeutic targets may be in
cases where the oncogenic alterations of PMTs (or of
other proteins that impact PMT activity; see below) associated with specific human cancers have been shown to
confer to cancer cells a unique dependency on the enzymatic activity of the altered PMT for proliferation and/or
survival (i.e., an addiction to PMT activity). Hence, a
reasonable working hypothesis is that selective inhibition
of the affected PMT would result in cell killing of cancer
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PMTs in Cancer
A
Ac
R
K
CH3
K
Ub K
Figure 1. Chromatin
modification affects gene
transcription. A, chromatin
consists of histone proteins
(red) around which
chromosomal DNA (blue) is
wrapped. The histone proteins
undergo a variety of
posttranslational modifications,
including methylation of
arginine (R) and lysine (K)
residues catalyzed respectively
by the PMT families of arginine
methyltrasferases (RMT) and
lysine methyltransferases
(KMT). These posttranslational
modifications affect the state of
compacting of nucleosomes
(histone-DNA units) along the
chromosomes. Regions of
chromosomes can transition
from the open, transcriptionally
permissive state referred to as
euchromatin to the more
compact, transcriptionally
respressive state known as
heterochromatin. B, PMTs
catalyze methylation of specific
lysine and arginine residues on
histones H3 and H4. The
enzymes that catalyze
methylation of a specific
histone residue are listed in
the boxes.
PMTs
• Arginine (RMTs)
• Lysine (KMTs)
S
PO4
CH3
CpG
Euchromatin Transcriptionally permissive
Heterochromatin Transcriptionally repressive
B
Site-specific methylation of histones H3 & H4
CARM1
PRMT6
H3
MLL1
MLL2
MLL3
MLL4
MLL5
SET1A
SET1B
ASH1
SET7/9
PRMT5
EZH1
EZH2
CARM1
NDS1
NSD2
NSD3
SET2
SMYD2
DOT1L
N-A R T K ... R K S T ... K ... R K ... K ... R K S ... K ... K ... -C
2 4
8 9
17
26 27
36 79
PRMT1
PRMT5
H4
SUV39H1
SUV39H2
G9a
GLP
SETDB1
CLL8
RIZ1
PRDM3
PRDM16
Pr-SET7/8
SUV4 20H1
SUV4-20H2
SET9
Pr-SET7
SUV4-20
NSD1
SMYD3
N-S G R G K G G K G L G K G G A K R H R K V ...... -C
1 3
5
20
© 2013 American Association for Cancer Research
cells bearing the genetic alteration with minimal affect on
nonaltered cells. This would be expected to provide a
significant therapeutic index in vivo, with potent elimination of affected tumor cells and limited attendant
safety concerns. Preclinical animal studies of potent,
selective inhibitors of two PMTs, DOT1L (9) and EZH2
(ref. 10; see below) have borne out these expectations and
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have paved the way to clinical testing of PMT inhibitors
in patients with cancer.
Active site structure and enzymatic mechanism of
methyl transfer
All PMTs use S-adenosyl methionine as a universal
methyl donor in much the same way that all kinases use
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6345
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Copeland
PKMTs
Histone
H3 C
NH3+
Histone
HMT
N
H
O
+
O
CH3
+
N CH3
H3C
+
HMT
N
H
O
Mono-methyl
lysine
Lysine
CH3
NH
HMT
N
H
O
H3C
+
NH2
O
N
H
O
O
O
Di-methyl
lysine
Tri-methyl
lysine
CH3 CH3
CH3
CH3
PRMTs
CH3
S
PMT
+
S
H 2N
–
–
CH3
NH2+
HN
HN
NH
HN
NH3+
NH3
SAM
SAH
N
H
O
O
Arginine
N
H3C
HN
HMT
+
HN
HMT
N
H
O
O
Mono-methyl
arginine
N
NH
HN
or
N
H
O
O
Symmetrical
di-methyl
arginine
N
H
O
O
Asymmetrical
di-methyl
arginine
© 2013 American Association for Cancer Research
Figure 2. Chemical mechanism of PMT catalysis (left) and the products of lysine (top right), and arginine (bottom right) methylation. SAM, S-adenosyl
methionine
ATP as a universal phosphate donor (Fig. 2). The crystal
structures of a number of PMTs have been solved to
atomic resolution and reveal a well-organized S-adenosyl
methionine–binding pocket, which is conformationally
distinct between the lysine and arginine methyltransferases (3, 6, 11). In both cases, however, the S-adenosyl
methionine–binding pocket is juxtaposed to a long channel into which the methyl-accepting amino acid side
chain binds. This arrangement allows for close proximity
and molecular orbital alignment between S-adenosyl
methionine and the nitrogen of lysine or arginine for
facile methyl transfer. All of these enzymes transfer the
methyl group directly from S-adenosyl methionine to the
acceptor amino acid, so that the enzymatic reaction
requires the simultaneous binding of both substrates for
transfer to occur (3). These features of the enzymatic
reaction of PMTs afford multiple opportunities for inhibitor interactions with the enzymes. Indeed, small-molecule PMT inhibitors of various binding modalities
have been reported: S-adenosyl methionine competitive,
methyl acceptor competitive, noncompetitive, etc. (5, 8).
This diversity of inhibitor-binding modalities distinguishes the PMTs from other group-transfer enzymes
that have been targeted for cancer therapies (e.g., the
kinases) and may allow greater flexibility in choosing
inhibitors that offer the greatest chance for clinical translation (12).
Role of PMTs in regulation of gene transcription
How site-specific methylation of histone lysines and
arginines controls gene transcription is incompletely under-
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Clin Cancer Res; 19(23) December 1, 2013
stood at present, but it is clear that methylation at specific
histone sites affects downstream transcription in different
ways. For example, methylation of histone H3 on lysines 4
(H3K4) and 79 (H3K79) has been implicated in the transcriptional activation of genes. In contrast, methylation at
other locations, such as H3K9 and H3K27, are involved in
repression of gene transcription (13). These opposing
effects of methylation of different histone sites can play a
critical role in cancer tumorigenesis when alterations in the
location and amplitude of specific site methylation leads
to aberrant activation of oncogene transcription and/or
transcriptional repression of tumor suppressor genes. It
is also becoming clear that the effect of site-specific methylation of histone sites on transcription cannot be understood in isolation, but depends on the context of other
modifications of proximal histone sites (14). Finally,
emerging data suggest that methylation of one histone site
may enhance or antagonize methylation of other histone
sites. For example, data in multiple myeloma cells suggest
that elevated methylation of H3K36 antagonizes methylation of H3K27, and likewise diminution of H3K36 methylation may result in elevation of H3K27 methylation
levels (15).
Genetic alterations of PMT activity as drivers of cancer
A variety of genetic alterations are seen in members of
the PMT enzyme class associated with specific human
cancers. Ongoing surveillance of cancer genome databases is continuing to reveal additional examples of
amplifications and mutations within PMTs and within
PMT-associated proteins that seem to be specific to
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PMTs in Cancer
individual cancer types. Over the past decade, several
examples have emerged of alterations of PMT activity
that seem to be critical drivers of genetically defined
cancers (16). Some of these will be described here to
exemplify the diversity of diver alterations observed to
impinge on this enzyme class.
Indirect chromosomal translocation leading to ectopic
PMT activity. MML-rearranged leukemia is a devastating
form of acute leukemia that affects patients from infancy
through adulthood and is also prevalent among secondary
leukemia associated with the use of etoposide (and other
topoisomerase II inhibitors; ref. 17). This disease is universally associated with a chromosomal translocation (11q23)
affecting the MLL gene that encodes for the PMT MLL, which
normally catalyzes the methylation of the H3K4 residue
(18, 19). As a result of the translocation, the MLL protein
loses its enzymatic active site (hence, PMT activity) and is
fused to any of a number of proteins within the AF or ENL
protein families (20, 21). Gene localization is conferred by
recognition elements within the N-terminal region of the
MLL protein, and these are retained within the various
fusion proteins. Hence, the MLL fusion proteins localize to
the same gene locations as wild-type MLL, but lack the ability
to catalyze histone methylation at H3K4. It has been recognized for some time that this chromosomal translocation
plays a causal role in MML-rearranged leukemia, but the
molecular mechanism of pathogenesis was not clear. Early
attention focused on the loss of PMT activity associated with
the fusion proteins that results from the translocation, but
this was hard to reconcile with the residual H3K4 methylation activity of the wild-type MLL protein resulting from the
second, unaffected allele. In 2005, Okada and colleagues
(22) showed that another enzyme, DOT1L, binds to AF10,
one of the various fusion partner proteins associated with the
MLL translocation, and thereby recruits DOT1L to ectopic
gene locations at which the MLL fusion proteins reside.
Subsequent studies revealed that other AF and ENL fusion
partners were also able to directly, or indirectly recruit
DOT1L to MLL fusion–bound genes (23–25). The resulting
ectopic localization of DOT1L catalyzes H3K79 methylation
of genes otherwise under the control of MLL-catalyzed
H3K4 methylation, including proleukemogenic genes
such as HOXA9 and MEIS1 (22, 26, 27). Methylation of
H3K79 is a transcriptional activation mark. Thus, the ectopic
localization of DOT1L, due to the universal chromosomal
translocation in MML-rearranged leukemia, results in elevated transcription of a program of leukemogenic genes that in
turn drive this proliferative disease. It has been hypothesized
that MML-rearranged leukemia is uniquely dependent on
DOT1L enzymatic activity and that selective inhibition of
DOT1L would lead to specific cell killing of MML-rearranged
leukemia cells. This hypothesis was confirmed by gene
silencing approaches (26) and by the use of potent and
selective small-molecule inhibitors of DOT1L (see below;
refs. 28–30).
Direct chromosomal translocations involving PMTs.
WHSC1 (also referred to as MMSET or NSD2) is a PMT
that catalyzes the dimethylation of H3K36 (31, 32).
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Methylation of this histone site is associated with
regions that are transcriptionally active. The t(4;14)
chromosomal translocation occurs in a subset of about
15% of multiple myeloma (33, 34), and patients with
this translocation represent one of the worst prognostic
subgroups of multiple myeloma (35). The t(4;14) translocation results in massive overexpression of WHSC1
and of fibroblast growth factor receptor 3 (FGFR3) due
to the placement of the strong immunoglobulin H (IgH)
intronic Em enhancer and 30 enhancer in the promoter
regions of WHSC1 and FGFR3 genes, respectively. While
approximately 30% of t(4;14) patients have lost expression of FGFR3, 100% retain overexpression of WHSC1,
suggesting that WHSC1, rather than FGFR3, is the
primary driver of disease (35). The overexpression of
WHSC1 in t(4;14) translocated cells results in significantly elevated levels of dimethylated H3K36, as would
be expected from elevation of catalytic enzyme levels
(15). Genetic knockdown of WHSC1 or disruption of
the translocated allele in t(4;14) myeloma cells results in
inhibition of cellular proliferation and of tumorigenicity. As expected, genetic knockdown of WHSC1 shows an
accompanying reduction in global levels of H3K36me2
(15).
Point mutations affecting PMT activity. The most wellstudied example of tumorigenic point mutations affecting
PMT activity is that of mutations within the catalytic domain
of EZH2 in germinal center diffuse large B-cell lymphoma
and follicular lymphoma subsets of non-Hodgkin lymphoma (NHL; 36–38). EZH2 (or the closely related EZH1) is
the catalytic subunit of a multiprotein complex known
as PRC2 (polycomb repressive complex 2) that is responsible for mono-, di-, and trimethylation of the H3K27 site.
Trimethylated H3K27 is a translational repressive mark,
and hypertrimethylation of H3K27 has always been found
to lead to tumorigenesis in various human cancers, presumably due to the transcriptional repression of tumor
suppressor genes. Mutations at Tyr641 or Ala677 have been
found to occur heterozygously in NHL patient groups
(36, 37) and have been shown to be change-of-function
mutations with respect to the substrate specificity of the
enzyme (38). The wild-type enzyme is most active in
placing the first methyl group on to H3K27 and its catalytic
efficiency wanes with consecutive methylation cycles. In
direct contrast, the mutations associated with NHL show the
exact opposite substrate specificity; they are essentially
unable to put the first methyl group on H3K27, but once
that first methyl is on, the mutant enzymes are able to
put the second methyl on and are extremely efficient at
putting the third methyl group on. In the context of disease
heterozygosity, the wild-type and mutant enzymes work
in concert with one another to effect a tumorigenic
hypertrimethylated H3K27 phenotype (38). Two groups
have now shown that potent-, selective-, small-molecule
inhibitors of EZH2 selectively kill NHL cells bearing these
EZH2 mutations while having essentially no effect on
the proliferation of homozygous EZH2 wild-type cells
(39, 40).
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Copeland
Synthetic lethal associations with PMT activity. In addition to histone methylation, gene expression is also
regulated by remodeling of nucleosomes in an ATPdependent manner. Of the ATP-dependent chromatin
remodelers, SWI/SNF complexes are emerging as bona
fide tumor suppressors, as specific inactivating mutations
in several SWI/SNF subunits are found in human cancers
(41). For instance, the INI1 (also known as SMARCB1 or
hSNF5) subunit is inactivated in nearly all malignant
rhabdoid tumors and atypical teratoid rhabdoid tumors,
aggressive cancers of young children with no effective
therapy (42). Tumorigenesis in INI1-deficient malignant
rhabdoid tumors can be completely suppressed by tissue
specific codeletion of EZH2, suggesting an antagonistic
relationship between PRC2 and SWI/SNF activities (43).
Recently, Knutson and colleagues (44) have shown that
potent, selective inhibitors of EZH2 are effective in eradicating INI1-deficient malignant rhabdoid tumors in cell
culture and in a mouse xenograft model. Hence, although
the tumorigenetic driver of malignant rhabdoid tumors
occurs in a pathway distinct from any PMT, it nevertheless
creates a synthetic lethal relationship that confers a
unique sensitivity to selective EZH2 inhibition in this
disease.
Clinical–Translational Advances
Preclinical in vivo activity of selective PMT inhibitors
Selective inhibitors of several PMTs have been
described, and compounds targeting two particular PMTs,
DOT1L and EZH2, have been reported to show dramatic
antitumor activity in rodent models of disease (9,
10, 40, 44). The DOT1L tool compound EPZ004777 was
the first PMT inhibitor reported to show antitumor effects
in a disseminated mouse model of MLL-rearranged leukemia (28). Subsequent pharmacologic optimization
within this chemotype series resulted in the clinical candidate DOT1L inhibitor EPZ-5676, which was also shown
to eradicate MLL-rearranged tumors (100% tumor
growth inhibition) in a rat xenograft model (9, 45). Two
distinct EZH2 inhibitors have been shown to display
significant, dose-dependent antitumor activity in mouse
xenograft models of EZH2 mutant–bearing NHL tumors
(60%–100% tumor growth inhibition in various tumor
models) and of INI1-deficient malignant rhabdoid
tumors (100% tumor growth inhibition; refs. 10, 40, 44).
In all of these studies, the compounds were well tolerated
by the rodents at doses that resulted in significant tumor
growth inhibition, with no acute toxicity or weight loss
observed. These results provide a strong foundation for
the clinical testing of DOT1L and EZH2 inhibitors in
genetically defined patients with cancer and also portend
the general use of PMT inhibitors as personalized cancer
therapeutics. Also, in all of these studies, the investigators
were able to show a correlative relationship between
tumor growth inhibition and diminution of the relevant
histone methyl mark in tumor tissue as well as in surrogate tissues such as skin and peripheral blood mononu-
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Clin Cancer Res; 19(23) December 1, 2013
clear cells. The ability to measure drug-induced loss of
site-specific histone methylation in surrogate tissue by
noninvasive means provides a cogent basis for pharmacodynamics assessment of target engagement in clinical
trials.
Clinical trials
At the time of this writing, two PMT inhibitors have
entered phase I human clinical trials: the DOT1L inhibitor EPZ-5676 and the EZH2 inhibitor EPZ-6438. The
primary goal of both of these trials is to assess the safety of
the drugs and to establish the maximum tolerated dose
for each.
The EPZ-5676 phase I trial is divided into two parts: a
dose-escalation portion to establish maximum tolerated
dose and an expansion cohort of patients treated at the
maximum tolerated dose. To accelerate dose escalation,
eligibility is not restricted to genetically defined patients;
patients with hematologic malignancies (mainly acute
myelogenous leukemia and acute lymphocytic leukemia)
are eligible for this portion of the trial. Once the maximum tolerated dose has been established, the expansion
cohort study will begin exclusively in patients with
leukemia involving the MLL rearrangement at 11q23. In
this manner, the drug may be tested as early as possible
in the genetically defined patients for whom it was
designed.
The EZH2 inhibitor EPZ-6438 is being studied in a
phase I trial of relapsed or refractory malignancies that
have failed all standard therapy. Again, the primary goal
here is to establish the safety and define the maximum
tolerated dose of the drug. As with the EPZ-5676 trial, a
secondary objective is to test EPZ-6438 as early as
possible in the genetically defined patients for whom
the drug was designed. Hence, once the maximum
tolerated dose has been established, a phase IIa trial is
planned exclusively in NHL patients bearing mutations
in EZH2.
No data have yet been reported for either of these
trials. Nevertheless, these trials represent a true watershed in the development of PMT inhibitors as personalized cancer therapeutics. The cancer community will be
looking forward with great interest toward the outcomes
of these trials. It is clear that these trials represent the
vanguard of a much larger array of PMT inhibitors that
will be entering clinical trials in the near future for
testing against genetically defined groups of patients with
cancer.
Disclosure of Potential Conflicts of Interest
R.A. Copeland has an ownership interest (including patents) in Epizyme Inc. and is a consultant/advisory board member of Mersana.
Acknowledgments
The author thanks Dr. Roy M. Pollock for helpful suggestions.
Received June 24, 2013; revised July 21, 2013; accepted August 7, 2013;
published OnlineFirst August 19, 2013.
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PMTs in Cancer
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