small molecule control of chromatin remodeling

Chemistry & Biology

Review

Small Molecule Control of Chromatin Remodeling

Aidan Finley1 and Robert A. Copeland1,*1Epizyme, Inc., 400 Technology Square, 4th Floor, Cambridge, MA 02139, USA*Correspondence: [email protected]://dx.doi.org/10.1016/j.chembiol.2014.07.024

Control of cellular transcriptional programs is based on reversible changes in chromatin conformation thataffect access of the transcriptional machinery to specific gene promoters. Chromatin conformation is inturn controlled by the concerted effects of reversible, covalent modification of the DNA and histone com-ponents of chromatin, along with topographical changes in DNA-histone interactions; all of these chro-matin-modifying reactions are catalyzed by specific enzymes and are communicated to the transcriptionalmachinery by proteins that recognize and bind to unique, covalent modifications at specific chromatin sites(so-called reader proteins). Over the past decade, considerable progress has been made in the discovery ofpotent and selective small molecule modulators of specific chromatin-modifying proteins. Here we reviewthe progress that has been made toward small molecule control of these mechanisms and the potential clin-ical applications of such small molecule modulators of chromatin remodeling.

All somatic cells within multicellular organisms contain the full

complement of DNA that constitutes the genome of that

organism. Nevertheless, beyond early embryonic development,

multicellular organisms are distinguished from single cell and

colony-forming organisms by the presence of a broad spectrum

of fit-for-purpose cells that have differentiated themselves from

their common progenitors. Cellular differentiation depends on

precise control of gene transcription programs, such that certain

genes are transcriptionally active whereas others are repressed,

depending on the cell type, cell cycle stage, and timing relative to

cellular division.

The regulatory system used to control transcriptional pro-

grams is commonly referred to as epigenetics (Allis et al.,

2007; Holliday, 1990), but is more correctly referred to as chro-

matin remodeling (Ptashne, 2013). At the molecular level, chro-

matin remodeling involves controlling the local structure of

chromatin at specific gene promoters by the concerted action

of a spectrum of chromatin-modifying proteins (CMPs) (Arrow-

smith et al., 2012; Copeland et al., 2009; Wilkins et al., 2014).

Chromatin is the amalgam of DNA and histone proteins that

make up chromosomes. Chromosomal DNA exists as stretches

of unassociated, double-stranded DNA, interspersed with nu-

cleosomes—loci of �140–150 base-pair stretches of DNA

coiled twice around a spool-like octamer core of histones, con-

sisting of two copies each of four core histone proteins (H2A,

H2B, H3, and H4). The coiling of DNA around the histone

core provides a mechanism for compaction of the ca. 2 m of

DNA that needs to be fit into the small volume of a cell nucleus.

Fully compacted chromatin is referred to a heterochromatin.

This conformational state, however, severely limits steric ac-

cess of gene promoter regions to transcription factors, poly-

merases and the rest of the transcriptional machinery. A more

relaxed (i.e., less compacted) state of chromatin, referred to

as euchromatin, also exists; in this latter state, the gene pro-

moter region is readily accessible to the transcriptional machin-

ery (Figure 1). Methylation of CpG islands of chromosomal DNA

directly affects gene transcription, whereas the conformational

switch between euchromatin and heterochromatin states in

1196 Chemistry & Biology 21, September 18, 2014 ª2014 Elsevier Lt

proximity to particular gene locations is effected by modifica-

tions to the histone proteins; these modifications involve a

collection of highly specific enzymes and associated recogni-

tion proteins, as detailed next.

CMPsThree major categories of chromatin modifications work in con-

cert to effect transcriptional regulation. These are: (1) methyl-

ation of chromosomal DNA, (2) posttranslational modifications

of specific amino acids on histones, and (3) ATP hydrolysis-

dependent alteration of DNA-histone interactions.

Methylation of chromosomal DNA is catalyzed by the DNA

methyltransferases (DNMTs) and methylation is reversed by

the ten-eleven translocation (TET) proteins (Figure 2). DNA

methylation results in transcriptional silencing of genes, in-

cluding a number of tumor suppressors (Baylin, 2005). The

DNMTs have been targeted for small molecule inhibitors as

cancer therapeutics; indeed, two DNMT-targeted drugs have

been approved as treatments for myelodysplastic syndrome,

decitibine (Dacogen) and azacitidine (Vidaza). DNA methylation

and the drugs targeting this modification have been extensively

reviewed in the literature (see, for example, Baylin, 2005 and

Dhe-Paganon et al., 2011). Among the covalent modifications

of histone proteins, the protein kinases that phosphorylate

histones and their inhibitors have also been extensively re-

viewed in the recent literature (see, for example, Rossetto

et al., 2012 and Suganuma and Workman, 2012). Hence, we

shall focus our attention for the remainder of the current

review on the various CMPs involved in histone acetylation/

deacetylation and methylation/demethylation reactions and on

the CMPs involved in ATP hydrolysis-dependent chromatin

remodeling.

Posttranslational Modification of Histone Proteins

Site-specific covalent modification of histones can affect the

conformation of chromatin by altering interactions between the

histones and the nucleosomal DNA (Arrowsmith et al., 2012;

Copeland et al., 2009). These modifications include covalent

attachment of small proteins, such as ubiquitin and SUMO, to

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Figure 1. Cartoon of the ConformationalTransition of Chromatin from the MoreOpen, Uncondensed Euchromatin State tothe More Condensed HeterochromatinStatePurple and red spheres represent individual his-tone proteins comprising the nucleosome corearound which the DNA (turquoise) is wrapped.Small circles are meant to represent covalentmodifications to the DNA and histones.

Chemistry & Biology

Review

specific lysines, as well as covalent attachment of chemical

groups to specific amino acids; these latter reactions include

lysine/arginine methylation, lysine acetylation, and serine/

threonine/tyrosine phosphorylation. All of these modifications

affect chromatin structure, hence gene transcription. The en-

zymes involved in placement of acetyl and methyl groups on

histones (referred to as writers) and the enzymes involved in

the specific removal of these chemical groups (referred to as

erasers) have been the most studied and are enzymes for which

small molecule modulators have been reported. As stated

above, we shall focus the remainder of our discussion on these

enzymes and the reactions catalyzed by them, together with

the proteins that recognize and bind to specific histonemodifica-

tions (referred to as readers) as initiating events in transcriptional

control.

Histone Acetylation. Histone lysine residues are acetylated by

the histone acetyltransferases (HATs). HATs acetylate lysine

residues using acetyl-CoA as a common acetyl group donor.

This results in charge neutralization of the lysine side chain,

altering the electrostatic interactions that can be made with

the negatively charged DNA (Figure 3A). Histone acetylation

generally results in exposure of gene promoter sites to tran-

scriptional machinery and activation of gene transcription

(Rice and Allis, 2001). Acetylation also creates a locus for

bromodomains to recognize ‘‘transcription ready’’ chromatin,

and thus recruitment of transcriptional machinery (Dey et al.,

2003).

There are 18 human HATs, divided into distinct families based

on sequence homology and structure. Type A (nuclear) HATs are

divided into three subfamilies, GNAT, p300/CB, and MYST

(Hodawadekar and Marmorstein, 2007; Andreoli et al., 2013).

Type B HATs are found in the cytoplasm and acetylate histones

prior to incorporation into chromatin (Andreoli et al., 2013).MYST

and p300/CPB enzymes also acetylate nonhistone proteins

(Friedmann and Marmorstein, 2013). HATs are large proteins

with multiple domains, and the complexity of structure is impor-

tant for substrate targeting and catalytic function (Lee and

Workman, 2007). Structures of several HAT catalytic domains

have been solved (Andreoli et al., 2013) and feature a conserved

core binding to acetyl-CoA despite low sequence conservation

and differing surrounding structures (Liu et al., 2008; Hodawade-

kar and Marmorstein, 2007; Marmorstein, 2001). This structural

understanding is bolstered by detailed kinetic and biochemical

studies of the HAT reaction mechanism. GCN5 family members

require a ternary complex (enzyme * acetyl-CoA * H3 histone)

before catalysis can occur, and this is conserved for the human

and yeast enzymes. The catalytic mechanism is, however, likely

specific to each HAT family/subfamily, as kinetic mechanisms

are different among the P300/CBP, MYST, and GNAT subfam-

ilies and tool inhibitory peptides specific for PCAF (another

Chemistry & Biology 2

type A HAT that associates with p300/CBP) were unable to

inhibit P300/CBP (Roth et al., 2001; Hodawadekar and Marmor-

stein, 2007).

Deacetylation of lysine is catalyzed by the histone deacety-

lases (HDACs). There are 18 human HDACs and these are sub-

divided into four subclasses (Gregoretti et al., 2004). Class I

contains the enzymes HDAC 1, 2, 3, and 8. Class IIa consists

of HDACs 4, 5, 7, and 9. Class IIb contains HDACs 6 and 10.

Class III is made up of the Sirtuins 1–7, whereas class IV contains

a single enzyme, HDAC 11. With the exception of the Sirtuins, all

of these enzymes use an active site zinc atom to active a coordi-

nated water molecule that, together with active site histidine and

aspartate side chains facilitate nucleophilic catalysis on the

acetyl-lysine substrate (Lombardi et al., 2011; Figure 3B). The

Sirtuins, on the other hand, use a distinct mechanism of catalysis

involving NAD+ attack of the bound acetyl-lysine substrate

(Walsh, 2006; Milne and Denu, 2008). For both the metal-utilizing

HDACs and the Sirtuins, substrate specificity is moderate and is

conferred by recognition elements outside the catalytic active

site of these enzymes.

Bromodomains (BRDs) are protein domains of approximately

110 amino acids in length with a predominantly a-helical struc-

ture (Haynes et al., 1992) configured as a left-handed up-and-

down helical bundle. Bromodomain-containing proteins are

found across several phyla from fruit flies to man, and in humans

there are 61 different BRD structures (Filippakopoulos et al.,

2012). Although the bromodomain family, including the bromo-

domain and extra-terminal domain (BET) proteins is large and

relatively diverse, with low overall sequence homology, all bro-

modomain proteins are defined by a well-ordered, deep, hydro-

phobic pocket into which the acetyl-lysine side chain binds

(Filippakopoulos and Knapp, 2014). Surface residues in the var-

iable loop regions contribute to substrate recognition, allowing

for considerable diversity in binding partners. In contrast to the

surface residues, the binding site for the acetylated lysine is

well conserved, with critical hydrophobic and aromatic residues

(Owen et al., 2000). These residues interact with the aliphatic

side chain of the lysine while a conserved asparagine residue

forms a key hydrogen bond with the acetyl carbonyl. Water mol-

ecules in the binding pocket also bridge interactions between the

acetylated lysine and the protein (Owen et al., 2000; Mujtaba

et al., 2007). These common features notwithstanding, BRDs

display considerable diversity of amino acid composition within

the binding pocket and this has provided a basis for the develop-

ment of domain-selective BRD inhibitors, as discussed below.

Histone Methylation. Lysine and arginine residues within his-

tones are methylated by the protein methyltransferases (PMTs;

Copeland et al., 2009). This enzyme class bifurcates into families

(Richon et al., 2011): the protein lysine methyltransferases

(PKMTs) and the protein arginine methyltransferases (PRMTs).

1, September 18, 2014 ª2014 Elsevier Ltd All rights reserved 1197

Figure 2. Reversible Methylation of Cytosine within CpG Islandsof DNAMethylation is catalyzed by the DNMT class of enzymes and reversed, in amultistep process, by the TET class of enzymes. See text for further details.SAM, S-adenosyl methionine; SAH, S-adenosyl homocysteine; a-KG, alpha-ketoglutarate.

Chemistry & Biology

Review

All PMTs use SAM as a universal methyl donor and perform

protein methylation via a SN2 reaction mechanism, involving

formation of a ternary enzyme-SAM-protein complex prior to

direct methyl transfer from SAM to the nitrogen atom of the

lysine or arginine side chain (Figures 4A and 4B). Despite

the common chemical reaction catalyzed by these enzymes,

the active site architecture varies considerably among them,

thus providing a structural basis for substrate specificity. As

discussed below, the structural diversity of PMT active sites

also provides a basis for potent and selective modulation of

these enzymes by small molecule ligands (Basavapathruni

et al., 2012).

Substrate permissiveness varies among the PMT enzymes,

as does the degree of enzyme redundancy for methylation of

a particular histone site (Copeland, 2013; Copeland et al.,

2009; Richon et al., 2011). For example, the enzyme DOT1L

is the only human enzyme known to methylate histone H3

at lysine 79 (H3K79) and this position is the only site

methylated by DOT1L. Likewise, the multi-protein complex

polycomb repressive complex 2 (PRC2), containing the cata-

lytic subunits EZH1 or EZH2, represent the only H3K27 methyl-

transferases in humans; PRC2 is also known to methylate only

H3K27. On the other hand, some histone sites are methylated

by several PMTs (e.g., H3K36). While the PKMTs tend to

demonstrate a high degree of substrate specificity, the PRMTs

tend to display greater permissiveness of substrate utilization,

sometimes methylating multiple arginine residues on multiple

proteins.

The transcriptional consequences of histone methylation

depend on the specific site being methylated and also on the

number of methyl groups placed on the amino acid. Lysine can

accept 1, 2 or 3 methyl groups and arginine can be monomethy-

lated on one nitrogen, symmetrically dimethylated, or asymmet-

rically dimethylated (Figure 4B); each of these states of

methylation can confer different biological consequences.

Methylation of histone sites can be transcriptionally activating

or repressive, depending on the site of the methylation. For


example, trimethylation at H3K27 suppresses gene transcrip-

tion, whereas trimethylation of H3K79 is transcriptionally acti-

vating (Copeland, 2013; Copeland et al., 2009).

Methyl groups are oxidatively released from lysine by the

lysine demethylases (KDMs; Arrowsmith et al., 2012; Forneris

et al., 2005; Tsukada et al., 2006). The KDM class clusters into

two families: the lysine-specific demethylase (LSD) family and

the Jumonji domain-containing family (JmjC; Arrowsmith et al.,

2012). Two flavin-dependent enzymes comprise the LSD family,

LSD1 and LSD2 (Figure 4C). The JmjCs constitute a larger family

of 27 enzymes that all use iron and 2-oxoglutarate to oxygenate

the methyl group (Figure 4D). KDMs act primarily on histones,

but can also demethylate nonhistone proteins. At least one

JmJC family KDM, JMJD6, has been reported to directly de-

methylate methyl-arginine residues (Chang et al., 2007) and the

protein arginine deiminases (PADs) can also lead to demethyla-

tion of arginine via conversion of arginine to citrulline (Cuthbert

et al., 2004; Wang et al., 2004).

Methyl-lysine is an abundant histone modification, and a

diversity of methyl-lysine reader domains exist (Wigle and Cope-

land, 2013). Over 200 methyl-lysine reader domains have been

described to date (James and Frye, 2013). The methyl-lysine

readers (KMe readers) are divided into two families: PHD zinc

finger domains, and the ‘‘Royal Family,’’ comprising Tudor,

Agenet, MBT, CHROMO domain, WD40 repeats (WDR5 and

EED) and PWWP domains (Wigle and Copeland, 2013; James

and Frye, 2013). Recently, another protein domain, the bromo

adjacent homology (BAH) domain of the ORC1 protein has

been shown to also be a KMe reader specific to H4K20 dimethy-

lation (Kuo et al., 2012). The KMe reader domains are typically

comprised of less than 100 residues and fold to form pockets

that contain an electron-rich cage of two to four aromatic

residues (the aromatic cage) that interact with the lysine

through p-cation, hydrogen bonding, and van der Waals inter-

actions. The methylation state of the lysine is recognized by

the presence of from zero to two acidic amino acids, at the

base of the pocket, that interact with the nitrogen of the lysine

side-chain.

ATP Hydrolysis-Dependent Changes to DNA-Histone

Interactions

ATP hydrolysis-dependent chromatin remodeling is exemplified

by the SWI/SNF complexes (Shain and Pollack, 2013; Wang

et al., 2014; Wilson and Roberts, 2011). These complexes are

composed of 9–12 protein subunits in humans; the exact

composition of subunits defines the specific complex and its

gene targets. Subunits of the SWI/SNF complex consist of: (1)

one of two mutually exclusive ATP-hydrolyzing subunits known

as BRM (also known as SMARCA2) or BRG1 (also known as

SMARCA4), (2) a set of highly conserved core subunits, such

as SNF5 (also known as SMARCB5), INI1, and BAF47, and (3)

a variable set of subunits involved in targeting, assembly and

cell lineage-specific functions.

SWI/SNF complexes remodel chromatin by catalyzing sliding

and ejection or insertion of histone octamer cores (Wilson and

Roberts, 2011). Sliding refers to the following sequence of steps.

First, the SWI/SNF complex binds at a specific position on nucle-

osomal DNA. Disruption of key contacts between the DNA and

histone proteins then ensues. This is followed by translocation

of the DNA, using the energy derived from ATP hydrolysis. A


Figure 3. The Chemistry of Histone Acetylation(A) Reaction mechanism for the histone acetyltransferases (HATs).(B) Reaction mechanism for the metal-dependent histone deacetylases (HDACs).(C) Reaction mechanism for the Sirtuin histone deacetylases.

Chemistry & Biology 21, September 18, 2014 ª2014 Elsevier Ltd All rights reserved 1199

Chemistry & Biology

Review

A

B

C

D

Figure 4. The Chemistry of Histone Methylation(A) The common methyl group donor SAM is converted to SAH by all proteinmethyltransferases (PMTs).(B) Stepwise methylation of the ε-nitrogen of lysine side chains by the proteinlysine methyltransferases (PKMTs) and of the side chain guanidino group ofarginine by the protein arginine methyltransferases (PRMTs).(C) Reaction mechanism for the flavin-dependent lysine demethylases (LSDs).(D) Reaction mechanism for the iron-dependent lysine demethylases (JmjC).

Chemistry & Biology

Review

DNA loop is thus formed and this can be propagated around

the nucleosome to create sites of increased access to DNA

binding factors. Thus, transcription factors and the like can


bind to specific gene promoter locations because of SWI/SNF

catalysis. The SWI/SNF complex is also known to catalyze the

ejection and insertion of histone octamers into nucleosomes,

although the mechanistic details of these reactions are not well

understood.

For the pedagogic purposes of this review, we have described

the various mechanisms of chromatin modification as discrete

reactions, each with unique biochemical and biological conse-

quences. It is important, however, to recognize that there can

be considerable interplay and crosstalk between these varied

mechanisms. For example, it is clear that DNA methylation

affects the degree and location of histone post-translational

modifications and that the two work in concert to control tran-

scription (Collings et al., 2013; Rivera et al., 2014). Also, methyl-

ation of histone H3 at lysine 79 (H3K79) by DOT1L appears to

depend critically on ubiquitylation of the nucleosome on histone

H2B (Wang et al., 2013). Histone methylation at sites H3K36 and

H3K27 appear to be antagonistic to one another, so that methyl-

ation at one site diminishes the ability of the alternative site to be

methylated (Zheng et al., 2012) The activity of the SWI/SNF com-

plex may be modulated by acetylation/deacetylation by HATs/

HDACs and chromatin modification by the SWI/SNF complex

is antagonistically related to methylation of the H3K27 site by

PRC2 (Knutson et al., 2013; Wilson et al., 2010). Finally, there

are several examples of proteins that contain domains leading

to multiple forms of chromatin interaction and remodeling. For

example, the SWI/SNF complex not only contains an ATP-

hydrolysis subunit, but also contains bromodomains that are

critical for recognition of acetyl-lysines and at least one

CHROMO domain; these histone mark-recognition elements of

SWI/SNF likely play an important role in homing of the complex

to specific gene locations that have been appropriately marked

by acetylation and (perhaps) methylation (Tang et al., 2010;

Wilson et al., 2014). Thus, the various forms of chromatin

modification work not as isolated biochemical reactions, but

rather in concert with one another to provide a combinatorial,

chemical ‘‘code’’ that regulates chromatin structure, hence

gene transcription.

Chromatin Modification and Human DiseaseCMPs are genetically altered in a number of human diseases,

most notably in various human cancers. Hence, the develop-

ment of small molecule modulators of specific CMPs as thera-

peutics has become a focal point for drug discovery research.

The genetic alterations of CMPs in human disease and their

pathogenic significance have been reviewed a number of times

in the recent literature (Arrowsmith et al., 2012; Baylin and Jones,

2011; Campbell and Tummino, 2014; Copeland, 2013; Copeland

et al., 2009, 2010, 2013; Helin and Dhanak, 2013; Wigle and

Copeland, 2013). Table 1 summarizes some examples of patho-

genic genetic alterations of CMPs. The reader is referred to the

review articles cited above for additional information on this

topic.

Small Molecule Modulators of Chromatin-ModifyingProteinsThe importance of chromatin modification in human diseases

has spurred significant interest in the discovery of small molecule

modulators of CMPs. A pivotal start for the field was provided by


Table 1. Genetic Alterations of Chromatin-Modifying Proteins in Human Diseases

Target Genetic Alteration or Disease Association Indication Key Referencea

PMT

PRC2 (EZH2) heterozygous activating mutations occurring at Y641,

A677 and A687 that result in hypertrimethylation of

H3K27

lymphoma (1–3)

deletion of miR-101 leads to EZH2 overexpression prostate cancer

deletion of SNF5 leads to EZH2 dependency malignant rhabdoid tumors

heterozygous mutations at Y153, H694Y, and

P132S

Weaver syndrome

PRC2 (EZH2, SUZ12,

EED, RBBP4)

SUZ12 fusion protein (JJAZ1/JAZF1) endometrial stromal sarcoma (4–6)

SUZ12 Inactivating mutations T cell precursor acute lymphoblastic

leukemia (ALL)

EED Inactivating mutations acute myeloid leukemia (AML)

DOT1L 11q23 chromosomal translocations fusing MLL1

(without its catalytic SET domain) to DOT1L binding

partners such as AF4, AF9, AF-10, and ENL leading

to aberrant H3K79 methylation

acute leukemia (both ALL and AML) (7)

CALM-AF10 and SET-NUP214 fusions are known to

mistarget DOT1L

NSD1 t(5;11)(q35;p15.5) translocation create NSD1-NUP98

fusions

AML (9, 10)

exon 22 mutations (p.Asp2119Valfs*31) Sotos syndrome

NSD2 (WHSC1/MMSET) t(4:14)(p16;q32) chromosomal translocations that

places WHSC1 gene under the control of the IGH

promoter and results in the overexpression of

WHSC1

multiple myeloma (8, 97)

Glu1099-to-Lys (E1099K) variant is hyperactivating ALL

NSD3 (WHSC1L1) t(8;11)(p11.2;p15) chromosomal translocations fuses

WHSC1L1 to NUP98

AML (11,12)

8p11–12 focal amplifications breast cancer, squamous cell lung

cancer

SETDB1 1q21 amplifications melanoma (13)

SMYD2 1q32 amplifications esophageal squamous cell

carcinoma

(14)

EHMT2 (G9A) H3K9 gene silencing required for apoptotic hair

cell death

sensorineural hearing loss (15, 16)

regulates HoxA9 transcription AML

SETD2 (HYPB, KMT3A) mutations Sotos-like overgrowth syndrome (98, 99)

clear cell renal cell carcinoma

(ccRCC)

SETD7 polymorphisms Type 1 Diabetes (T1D) and diabetic

complications

(17)

SMYD3 upregulation prostate cancer (18–20,100)

tandem repeat polymorphisms in promoter region

(VNTR)

esophageal squamous cell

carcinoma

tandem repeat polymorphisms in E2F-1 binding

element (CCGCC)

colorectal cancer and hepatocellular

carcinoma (HCC)

Type 1 PRMT (1,2,3, 6,8) PRMT1 methylates SRSF1, upregulated in ALL pediatric ALL (18, 21, 22)

PRMT1 upregulated in asthma asthma

PRMT1 methylation of FUS is linked to

heritable ALS

ALS

PRMT8 upregulated prostate cancer

(Continued on next page)


Chemistry & Biology

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Table 1. Continued


CARM1 (PRMT4) methylates SWI/SNF core subunit BAF155 colon, breast, and prostate cancer (23–25)

necessary for AR function glycogen storage diseases

necessary for glycogen gene expression program in

skeletal muscle

acute coronary syndrome

involved in chemokine activation (IP-10, MCP-1, IL-8)

Type II PRMTs (5,7) PRMT5 overexpression glioblastoma (15, 26)

ovarian cancer

Putative PMT

NSUN2 5p amplification breast cancer (27, 28, 102)

homozygous truncating mutation (Q227X) mental retardation

METTL1 SNPs multiple sclerosis 101

KDM

KDM4C (JMJD2C, GASC1) 9p23–24 amplifications esophageal squamous cell

carcinoma, squamous cell lung

cancer

(12, 29, 30, 39–41)

t(9;14)(9p24.1q32) translocations creating fusions

to IGH overexpression

medulloblastoma, basal breast

cancer

SNPs

KDM1B (LSD2, AOF1) 6p22 amplifications urothelial carcinomas (31)

KDM6 (UTX) inactivating mutations of UTX lead to pro-oncogenic

hypertrimethylation of H3K27 and dependence

on EZH2

multiple blood and solid cancers (32)

KDM1A (LSD1) overexpression AML (103, 33–35)

hypomorphic allele (point mutations in tower domain) heart development defects

mRNA overexpression ovarian cancer

SNPs (rs671357, rs587168) HCC, salt-sensitive hypertension

KDM4A (JMJD2A) site-specific copy gain/re-replication ovarian cancer (36–38)

overexpression breast cancer

lung cancer

KDM6B (JMJD3) overexpression driven by EBV oncogene Hodgkin’s lymphoma (42)

KDM5A (JARID1A) amplifications breast cancer (104, 48, 49)

t(11;21;12)(p15;p13;p13) translocations create fusions

of JARID1A PHD domain and NUP98

AML

KDM2B (FBXL10, JHDM1B,

PCCX2)

overexpression pancreatic cancer 105

PAD

PADI2 SNPs rheumatoid arthritis (43–45)

overexpression breast cancer

PADI4 SNPs cutaneous basal cell carcinoma (46, 47)

rheumatoid arthritis

Methyl Reader

PHF23 t(11;17)(p15;13) translocations create fusions of

PHF23’s PHD domain to NUP98

AML (48, 50)

PHF1 t(1;6)(p34;p21) translocation create MEAF6-PHF1

fusions, which may misdirect HAT activity toward

PHF1 targets

endometrial stromal sarcoma (51)

SFMBT1 copy number loss ventriculomegaly (52)

HAT

(A) GCN5 (KAT2A) increase in occupancy at regulated promoters spinocerebellar ataxia type 7

(SCA7)

(53, 54)

loss of function retinopathy


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Table 1. Continued


(A) PCAF (KAT2B) Asn386Ser polymorphism HCC (55, 56)

promoter methylation esophageal squamous cell

carcinoma

(A) P300 (KAT3B) C-terminal truncations without catalytic HAT activity diffuse large B-cell lymphoma

(DLBCL)

(57–59)

inactivating mutations cervical cancer

fusion with MLL (t(11;22)(q23;q13) serous endometrial tumors

frameshift mutation AML, Rubinstein-Taybi syndrome,

Cornelia de Lange syndrome

(A) CBP (CREBBP) translocations (t(11;16)(q23;p13.3) with MLL AML (60–63)

translocations t(8;16) with MOZ Rubinstein-Taybi syndrome

point mutations including truncating protein product

duplication (16p13.3)

mental retardation, DLBCL

C-terminal truncations without catalytic HAT activity relapsed ALL

sequence or deletion mutations in HAT domain adenoid cystic carcinoma

(A) TIP60 (KAT5) monoallelic loss

Point mutation (Y327F)

lymphoma, H&N, and mammary

carcinomas

(64, 65)

(A) MOZ (MYST3) fusion gene (inv(8)(p11q13) with TIF2 AML (66, 67)

homozygous (C-terminal deletion) and heterozygous

(deletion of exons 3-7) mutations of Moz

DiGeorge syndrome

(A) MORF (MYST4, KAT6B) MORF/CBP fusion gene—t(10;16)(q22;p13) AML (68–70)

heterozygous mutations (nonsense, truncating,

frameshift)

Ohdo syndrome SBBYS variant

genitopatellar syndrome

HDAC

HDAC 1,2,3,8 (class I) HDAC8 loss of function Cornelia de Lange syndrome (71, 72)

X-linked intellectual disability

HDAC 4,5,7,9 (Class IIa) HDAC4 point mutations brachydactyly-mental retardation (73, 74)

HDAC9 amplification CNS lymphoma

HDAC 6, 10 (Class IIb) HDAC6 point mutation adult-onset Alexander disease (75)

HDAC6 SNP (c.281A > T) X-linked dominant chondrodysplasia

Sirtuins 1-7 (Class III) SIRT1 polymorphisms cardiovascular disease (76–78)

Type 1 diabetes (T1D)

Parkinson’s disease

BRD

BRD4 balanced translocation (t15:19) NUT midline carcinoma (79, 80, 106)

localization to N-terminal domain of AR castration-resistant prostate cancer

transcriptional pause release heart failure

SWI/SNF

SWI/SNF (ARID1A, ARID1B,

SMARCA4, SMARCC1,

SMARCC2, SMARCD1,

SMARCD2, SMARCD3,

ACTL6A, ACTL6B)

ADNP mutation autism spectrum disorder (81–85)

AIRD1B (truncating mutations) intellectual disability (Coffin Siris

syndrome)

SMARCB1 mutations (biallelic) rhabdoid tumors

PBRM1, ARID1A (truncating), SMARCA4 mutations ccRCC

AIRD1A, AIRD1B, PRBM1, SMARCA2, SMARCA4

(alterations, deletions, mutations, and rearrangements)

Burkitt’s lymphoma, pancreatic

cancer

DNMT

DNMT1 heterozygous mutations affecting DNMT1 folding autosomal dominant hereditary

sensory neuropathy type IE

(86, 87)

autosomal dominant cerebellar

ataxia, deafness, and narcolepsy



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Table 1. Continued


DNMT3A somatic mutations (Arg883) AML (88–90)

somatic mutations (reduced enzymatic activity or

aberrant affinity to histone H3)

MDS

heterozygous mutations (Arg882) chronic myelomonocytic

DNMT3L polymorphisms (R271Q) leading to hypomethylation impaired spermatogenesis (91, 92)

TET

TET2 loss of function mutation overexpression systemic mastocytosis, MDS,

AML, CLL

(93–95)

TET1 loss of function mutation AML (96)

ALS, amyotrophic lateral sclerosis; TET, ten-eleven translocation; MDS, myelodysplastic syndromes; CLL, chronic lymphocytic leukemia.aPlease see the Supplemental Information for key references associated with this table.

Chemistry & Biology

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the crystal structure of a HDAC with the early inhibitors trichos-

tatin A and suberoylanilide hydroxamic acid (SAHA) bound in

the active site (Finnin et al., 1999). Since then, the field has grown

to see potent, selective inhibitors reported for several of the CMP

classes discussed above. Moreover, inhibitors of HDACs and of

DNMTs are now approved drugs for specific cancer indications

and additional CMP inhibitors have recently entered human clin-

ical trials for the treatment of specific cancers.

Below we describe the current state of efforts toward identifi-

cation of potent, selective small molecule modulators of CMPs,

exemplified by the most advanced compounds for several of the

CMP classes. A number of contemporary reviews have been

published on protein kinase inhibitors [see, for example, (Kollar-

eddy et al., 2012)] and DNMT hypomethylating agents (vide

supra). Since these topics have been well covered within recent

reviews, we shall not reiterate this information here. Instead, we

focus our attention on small molecule modulators of histone

acetylation and methylation.

HAT Inhibitors

Relatively few potent HAT inhibitors have been reported. Early

examples of inhibitors were substrate mimetics and natural

product derivatives of modest potency, selectivity and cell

permeation. High throughput library screening has yielded iso-

thiazolones and benzythiazine sulfonamide scaffolds as inter-

esting starting points for inhibitor discovery. Recently, C646

(Figure 5A) a potent (Ki = 400 nM), selective pyrazolone-based in-

hibitor of the HAT p300/CBP was identified. Intracellular inhibi-

tion of p300/CBP by C646 led to cell growth inhibition, providing

a clear rationale for further exploration of p300/CBP and other

HATs as potential therapeutic targets (Bowers et al., 2010;

Dekker et al., 2014). C646 provides a useful tool compound

with some demonstration of intracellular activity. This com-

pound, however, is not a pharmacologically tractable therapeu-

tic agent. Hence, additional inhibitor discovery efforts are clearly

needed for the HAT target class.

HDAC Modulators

Nonselective HDAC inhibitors have been reported that all act

through chelation of the active site zinc atom of these enzymes.

Two such compounds, vorinostat (SAHA, Zolinza) and romidep-

sin (Istodax) have been approved for the treatment of refractory

cutaneous T cell lymphoma and peripheral T cell lymphoma

(Prince et al., 2009). Vorinostat contains a hydroxamic acid as


the zinc-chelating moiety (Finnin et al., 1999), wherease romi-

depsin is administered as a disulfide-containing, cyclic peptide

prodrug (Furumai et al., 2002). Glutathione-mediated reduction

of the disulfide bond of Istodax liberates a free thiol moiety that

acts to chelate the active site zinc of HDACs (Figure 5B). Both

drugs inhibit a broad spectrum of HDAC enzymes; vorinostat is

reported to inhibit essentially all of the zinc-utilizing HDACs

with similar potency, whereas romidepsin is somewhat more

selective, inhibiting all class I HDACs.

The utility of these drugs for cancers beyond T cell lym-

phomas has been limited in part due to a number of dose-

limiting adverse effects, such as thrombocytopenia and anemia

(Mann et al., 2007). This has led to the suggestion that more

enzyme-selective inhibitors may provide efficacy while mini-

mizing adverse side effects, a hypothesis that has yet to be

tested clinically. Additionally, selective HDAC inhibitors may

also be effective therapeutics for indications beyond cancer,

such as autoimmune diseases and schizophrenia (Bridle et al.,

2013; Kurita et al., 2012).

The most advanced, isozyme-selective compound to be re-

ported to date is the HDAC6 selective inhibitor ACY-1215

(Figure 5B). This compound inhibits HDAC6 with a half-maximal

inhibitory concentration (IC50) of 5 nM; it displays >10-fold selec-

tivity with respect to HDAC1, 2, and 3 and shows no inhibitory

activity against other HDACs (Santo et al., 2012). ACY-1215 is

currently being tested in a phase I/II open-label, multicenter

study as monotherapy and in combination with bortezomib and

dexamethasone for the treatment of relapsed or relapsed/refrac-

tory multiple myeloma (http://clinicaltrials.gov, NCT01323751).

Other examples of selective HDAC inhibitors include BRD8430,

a HDAC1/2 selective inhibitor (IC50 = 69 and 560 nM for HDAC1

and 2, respectively) that induces differentiation in neuroblastoma

(Frumm et al., 2013) and PCI-34051 (Balasubramanian et al.,

2008), a selectiveHDAC8 inhibitor (Ki = 10 nM) being investigated

for potential use in T cell lymphomas (Figure 5B).

Bromodomain Inhibitors

TheBRD class includes a family of protein domains referred to as

the bromodomain and BET proteins. The BET family contains the

proteins BRD2, BRD3, BRD4, and BRDT (bromodomain testis-

specific protein). The BRDs are defined by a well-ordered,

deep, hydrophobic pocket into which the acetyl-lysine side chain

binds. This pocket provides a highly favorable locus for small


http://clinicaltrials.gov

A

B

C

Figure 5. Small Molecule Modulators ofHistone Acetylation(A) Histone acetyltransferase (HAT) inhibitors.(B) Metal-dependent histone deacetylase (HDAC)inhibitors.(C) BET domain protein antagonists.

Chemistry & Biology

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molecule modulator binding; indeed all of the BRD modulators

that have been reported bind within the pocket in a manner

competitive with acetyl-lysine.

A number of BRD binding molecules have been reported and

several of these have been advanced into human clinical trials.

The first BRD antagonist to enter clinical study was RVX-208

(Figure 5C), a preferential BRD2 inhibitor that was tested for

use in atherosclerosis. RVX-208 was well tolerated in patients

at the doses tested (Picaud et al., 2013). Optimization of the thie-

notriazolodiazepene core, first identified by Mitsubishi Tanabe

scientists, led to JQ1 (Figure 5C). This compound selectively

binds to BET family BRDs, such as BRD4, with minimal binding

to non-BET family BRD proteins (Filippakopoulos et al., 2010).

NUT midline carcinoma is a rare, genetically defined form of

squamous carcinoma for which there is currently no effective

treatment. Patients with NUT midline carcinoma are identified

by the occurrence of a chromosomal translocation affecting

the nuclear protein in testis (NUT) gene at the 15q14 location

(French, 2010). In approximately two-thirds of patients, the

translocation results in a fusion protein between NUT and

BRD4 (Stelow, 2011). Preclinical studies with JQ1 have shown

that the compound induces tumor regression and a significant

survival advantage in a mouse model of NUT midline carcinoma

involving the NUT-BRD4 fusion. Optimization efforts have led to

the JQ1 analog TEN-010 (also known as JQ2; structure not avail-

able), which is currently in phase I clinical testing (http://

clinicaltrials.gov, NCT01987362). Contemporaneous work by

the group at GlaxoSmithKline led to the identification of GSK

I-BET762 (GSK525762; Figure 5C), a benzodiazepine that also

selectively inhibits members of the BET-family of BRDs. GSK

I-BET762 displays Ki values of 50–60 nM for BRD2, BRD3, and

BRD4 with minimal effect on non-BET family BRDs (Mirguet

et al., 2013). This compound is currently in phase I clinical

testing for NUT midline carcinoma and other cancers (http://

clinicaltrials.gov, NCT01587703). A similar compound, OTX015

(Figure 5C) is also in phase I clinical testing in acute leukemia

and other hematological malignancies (http://clinicaltrials.gov,

Chemistry & Biology 21, September 18, 2014

NCT01713582). The sponsor of this trial

(Oncoethix) recently reported preliminary

results from their dose escalation studies.

Thrombocytopenia was observed as a

dose-limiting toxicity in patients with other

hematological malignancies at a dose of

80 mg every day, but not when dosed at

40 mg twice daily, suggesting that the

toxicity may be schedule dependent.

Two complete responses and one partial

response were observed in patients with

leukemia or lymphoma and some evi-

dence of response was observed in

another four patients out of a total of

42 patients treated (see http://oncoethix.com). A fourth BET-

family selective BRD inhibitor, CPI-0610, has entered phase I

clinical testing in patients with progressive lymphoma (http://

clinicaltrials.gov, NCT01949883); little additional information on

this compound is currently available. All of the BET-family selec-

tive BRD inhibitors have also shown antiproliferative activity in a

spectrum of solid tumor cell lines. These compounds also share

the ability to silence MYCN expression; amplification of MYCN

has been suggested to be a marker of BET-family selective

BRD inhibitor sensitivity. Indeed, MYCN amplification is being

used as a patient stratification biomarker within the GSK

I-BET762 clinical trial.

PMT Inhibitors

Selective inhibitors of specific PMTs have been reported that

bind within the SAM/SAH pocket, within the protein substrate

pocket or within allosteric sites on these proteins (Copeland

et al., 2013; Wigle and Copeland, 2013).

Potent, selective, SAM-competitive inhibitors have been re-

ported for several of the PKMT enzymes. Most notably, SAM-

competitive inhibitors of the enzymes DOT1L and EZH2 have

advanced to human clinical trials in cancer indications (Cope-

land, 2013). DOT1L catalyzes the methylation of the H3K79 res-

idue, leading to transcriptional activation of affected genes. In

MLL-rearranged leukemia (MLL-r), DOT1L is aberrantly recruited

to ectopic gene locations as a result of binding of the enzyme to

the various MLL-fusion proteins that result from the 11q23 chro-

mosomal translocation that is a universal hallmark of the disease.

EPZ-4777 (Figure 6A) was the first potent, selective DOT1L inhib-

itor demonstrated to selectively kill leukemia cells bearing the

11q23 chromosomal translocation in cell culture and in a murine

model of MLL-r (Daigle et al., 2011). Further optimization of

the amino-nucleoside core of EPZ-4777 led to EPZ-5676

(Figure 6A), the first PMT inhibitor to enter human clinical trials.

EPZ-5676 is a ca. 80 pM inhibitor of DOT1L that is >37,000-

fold selective against other human PMTs. In rat subcutaneous

xenografts of human MLL-r cells, EPZ-5676 treatment led to

essentially complete and durable tumor regression (Daigle

ª2014 Elsevier Ltd All rights reserved 1205






http://oncoethix.com



A

B

C

Figure 6. Small Molecule Modulators of Histone Methylation(A) Protein methyltransferase (PMT) inhibitors.(B) Lysine demethylase (KDM) inhibitors.(C) KMe reader domain protein antagonists.

1206 Chemistry & Biology 21, September 18, 2014 ª2014 Elsevier Ltd All rights reserved

Chemistry & Biology

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Chemistry & Biology

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et al., 2013). EPZ-5676 is currently in a phase I study in

advanced hematologic malignancies, including acute leukemia

with rearrangement of the MLL gene (http://clinicaltrials.gov,

NCT01684150). Earlier in 2014, the study sponsor (Epizyme) re-

ported objective responses in MLL-r patients within the context

of this ongoing trial.

Several groups have reported potent, selective, SAM-compet-

itive inhibitors of EZH2 (Figure 6A). Among these, EPZ-6438 (also

known as E7438) is the first EZH2 inhibitor to enter human clinical

trials as a single agent in subjects with advances solid tumors or

with B cell lymphomas (http://clinicaltrials.gov, NCT01897571

(Knutson et al., 2013, 2014). More recently, a second EZH2 inhib-

itor, GSK2816126, has likewise entered clinical testing to inves-

tigate the safety, pharmacokinetics, pharmacodynamics, and

clinical activity of the drug in subjects with relapsed/refractory

diffuse large B cell and transformed follicular lymphoma (http://

clinicaltrials.gov, NCT02082977). Both of these compounds are

nanomolar inhibitors of EZH2 with high selectivity against other

human PMTs. In preclinical studies, both EPZ-6438 and

GSK2816126 demonstrated robust tumor growth inhibition in

xenograft models of lymphomas bearing mutations in EZH2

(Knutson et al., 2014; McCabe et al., 2012; Van Aller et al.,

2014). EPZ-6438 is an orally bioavailable compound that is being

administered orally twice per day, while GSK2816126 is being

administered by intravenous infusion over 2 hours, twice weekly

for 3 weeks of a 28-day dosing cycle. In addition to the non-

Hodgkin lymphoma indication, EZH2 inhibitors have been shown

in preclinical models to effect tumor growth inhibition in INI-1-

deficient tumors, such as malignant rhabdoid tumors and have

also been implicated in additional solid tumor malignancies

(Knutson et al., 2013)

Protein substrate-competitive inhibitors of the PKMTs

EHMT1/2 (e.g., UNC0642 and A-366; Liu et al., 2013; Sweis

et al., 2014), SMYD2 (e.g., AZ505; Ferguson et al., 2011) and

SETD7 (e.g., PFI-2; Structural Genomics Consortium, Protein

Data Bank code 4JLG), and of the PRMT CARM1 (e.g., Methyl-

gene compound 7a; Allan et al., 2009) and BMS compound 7f

(Huynh et al., 2009) have been reported (Figure 6A). None of

these compounds, however, have yet been reported to demon-

strated efficacy in animal models of disease.

A final mechanism of selective PMT inhibitor binding is exem-

plified by the recently reported series of urea-containing allo-

steric inhibitor of PRMT3 (Figure 6A). These compounds bind

at a PRMT3 dimer interface and cause structural rearrangements

of the protein that are allosterically communicated to the enzyme

active site. Themost potent inhibitor in this series displays a Ki of

230 nM and is highly selective for PRMT3, owing to the unique

nature of the compound binding pocket (Siarheyeva et al.,

2012). Whether inhibitors of this type can be pharmacologically

optimized and whether this approach can be more generally

applied to the PMT enzyme class remains to be determined.

KDM Inhibitors

The active sites of the flavin-dependent LSDs bear considerable

similarity to the FAD-dependent monoamino oxidases. Hence,

covalent modifiers of the flavin cofactor of monoamine oxidases,

such as tranylcypromine, also inactivate the LSDs by the same

mechanism. This has been capitalized on to develop LSD-

selective inactivators. Two such compounds, ORY-1001 (Maes

et al., 2013) and GSK2879552 (Kruger et al., 2013; Figure 6B),


have entered human clinical trials for relapsed or refractory

acute leukemia (ORY-1001) and for relapsed/refractory small

cell lung carcinoma (GSK2879552; FAD-http://clinicaltrials.gov,

NCT02034123).

The JmjCs are likewise amenable to small molecule inhibition.

In the case of these enzymes, all known inhibitors derive the

majority of their binding energy from iron chelation (e.g., GSK-

J1; Kruidenier et al., 2012; Figure 6B). Enzyme selectivity has

been modest for JmJC inhibitors and no reported compounds

have progressed beyond biochemical and cell culture assays.

Nevertheless, the pathobiological relevance of the JmjCs

makes them important targets for further inhibitor discovery

and optimization.

Methyl Reader Modulators

The KMe reader domains (vide supra) have only recently become

the focus of small molecule modulator discovery efforts. Only a

few examples of KMe reader domain ligands have been reported

(Wigle and Copeland, 2013), the most potent of which is

UNC1215 (James et al., 2013), a substrate-competitive inhibitor

of L3MBTL3 (Kd = 120 nM; Figure 6C).

Future DirectionsInitiation of clinical trials for isozyme-selective HDAC inhibitors,

BRD antagonists, LSD1 inhibitors, and PMT inhibitors ushers in

an exciting period of testing the safety, pharmacokinetics, and

ultimately the effectiveness of these novel therapeutic modal-

ities. These will hopefully augment the existing CMP-targeted

drugs approved for used in clinical oncology that today consists

exclusively of DNMT hypomethylating agents and broad-spec-

trum HDAC inhibitors. It will be interesting to see how these

CMP drugs, which display such promising effectiveness in pre-

clinical animal models, affect disease as single agents in the

context of human clinical settings.

Beyond the use of these various CMP-targeted drugs as single

agents, there is a growing body of data to suggest that these

agents may be even more effective in treating human cancers

when they are appropriately combined with other CMP modal-

ities, existing standard of care drugs and even drugs that act

to modulate allied intracellular signaling cascades (Johnston

et al., 2013; Klaus et al., 2013). The power of such combinations

of CMP modulators with other therapeutic agents is only begin-

ning to be realized, and deserves considerably more preclinical

and clinical exploration.

Beyond the direct treatment of diseases, such as genetically

defined cancers, small molecule CMPmodulators show promise

as constituents of mixed small molecule/macromolecule cock-

tails that are capable of inducing pluripotent stem cells (iPSCs)

by reprogramming of differentiated, somatic cells. Reprogram-

ming to iPSCs was first shown with a cocktail of four transcrip-

tion factors: OCT4, SOX2, KLF4, and c-Myc (the Yamanaka

factors). Several groups have demonstrated that small molecule

modulators of the kinases ALK5 and MEK and of specific CMPs

can replace individual Yamanaka factors to effect iPSC reprog-

ramming. For example, the PMT inhibitor BIX-01294 can substi-

tute for SOX2 and valproic acid, a pan-HDAC inhibitor, can

substitute for c-Myc (Moschidou et al., 2012). More recently

(Onder et al., 2012) the DOT1L inhibitor EPZ-4777 was shown

to substitute for two Yamanaka Factors, KIF4 and c-Myc. This

same group suggested that selective inhibitors of another PMT







Chemistry & Biology

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enzyme, SUV39H1, would also be effective in promoting iPSC

formation, based on results from shRNA studies. The ability to

induce cellular reprogramming to form iPSCs using small,

organic compounds that can be readily synthesized, in large

scale and at reasonable costs, suggests a practical approach

to the development of regenerative medicine applications.

The continued discovery and development of potent, selec-

tive, small molecule modulators of CMPs may provide new

avenues to novel therapeutic agents for a spectrum of human

diseases. In addition to these clinical applications, the availability

of such CMP modulators will continue to create new opportu-

nities to interrogate the biological consequences of chromatin

modification as well as revealing unanticipated interfaces be-

tween chromatin modification and other cellular signaling

pathways.

SUPPLEMENTAL INFORMATION

Supplemental Information includes references for Table 1 and can be foundwith this article online at http://dx.doi.org/10.1016/j.chembiol.2014.07.024.

ACKNOWLEDGMENTS

The authors thank Drs. Edward Olhava, Tim Wigle, P. Ann Boriack-Sjodin,Robert Gould, and James E. Bradner for helpful discussions and commentson this work. Both authors are employees and stockholders of Epizyme, Inc.

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