From www.bloodjournal.org by guest on July 24, 2016. For personal use only.

Review Series

ADVANCES IN ACUTE MYELOID LEUKEMIA

Epigenetics and approaches to targeted epigenetic therapy in acute

myeloid leukemia
Bas J. Wouters and Ruud Delwel
Department of Hematology, Erasmus Medical Center, Rotterdam, The Netherlands

Acute myeloid leukemia (AML) is the most
common type of acute leukemia in adults.
AML is a heterogeneous malignancy char-
acterized by distinct genetic abnormalities.
Recent discoveries have highlighted an
additional important role of dysregulated
epigenetic mechanisms in the pathogene-
sis of the disease. In contrast to genetic
changes, epigenetic modifications are fre-
quently reversible, which provides opportu-
nities for targeted treatment using specific
inhibitors. In this review, we will provide an
overview of the current state of epigenetics
and epigenetic therapy in AML and will
describe perspectives on how to identify
promising new approaches for epigenetic
targeted treatment. (Blood. 2016;127(1):
42-52)

Introduction
Acute myeloid leukemia (AML) is the most common type of acute
leukemia in adults.1 Despite current treatment protocols involving
intensive chemotherapy and the judicious use of stem cell trans-
plantation, AML is still fatal in approximately half of younger patients
and in about 80% of patients over the age of 60 as a result of primary
refractoriness, relapse, or treatment-related mortality.2 Consequently,
there is a need for more specific and less toxic drugs that are rationally
designed to target leukemia-specific abnormalities.
The term epigenetics refers to changes in gene expression that are
inheritable by cell division but not caused by changes in the DNA
sequence itself.3 Examples include DNA modifications, such as cyto-
sine methylation, modifications of histone proteins, or RNA-associated
gene silencing. Recent discoveries have shed light on the important role
of dysregulated epigenetic mechanisms in the pathogenesis of AML.4-6
Several genes playing key roles in epigenetic regulation show recurrent
somatic alterations in AML. It is however apparent that epigenetic
dysregulation in AML is more widespread than can be explained by
recurrent somatic mutations alone.7 For instance, disturbed genome-
wide patterns of DNA methylation also exist in AML subtypes not
linked to mutations in known epigenetic modifiers.8 In addition to
somatic changes in coding regions of genes, alterations in enhancer
elements have been reported that perturb normal regulation of gene
expression.9
Epigenetic modifications are frequently reversible. Because of this
inherent plasticity, epigenetic dysregulation offers potential avenues for
targeted treatment using specific inhibitors of histone-modifying pro-
teins or proteins driving or maintaining DNA methylation. In support of
this, both experimental evidence as well as clinical observations under-
line the concept that epigenetic mutations and alterations contribute to a
preleukemic state but are usually not sufficient to cause full-blown acute
leukemia.10 This would indicate that these defects are already present
in early clones and targeting those abnormalities may help to eradicate
the disease.
In this review, we give an overview of the current state of epigenetics
and epigenetic therapy in AML and will describe perspectives on how
to identify promising new approaches for epigenetic-targeted treatment.
Modes of epigenetic (dys)regulation in AML
We first provide an outline of various modes of epigenetic regulation in
hematopoietic cells and how these may be disturbed in AML. This has
been summarized in Table 1. Several recent reviews have provided
excellent descriptions of the various abnormalities in epigenetic-
regulating enzymes in hematopoietic malignancies in general and AML
in particular, to which the reader is referred for more detail.4-6
DNA methylation
The process of DNA methylation involves the addition of a methyl
group to the 5-carbon position of cytosines in CpG dinucleotides,
yielding 5-methylcytosine. Aberrant patterns of DNA methylation
in malignancies were initially particularly studied in the context of
so-called “CpG islands” in gene promoters. Hypermethylation of
cytosines in CpG islands is associated with silencing of tumor-
suppressor genes.47 It is now increasingly clear that aberrant DNA
methylation patterns outside CpG islands may be equally important
in leukemogenesis, and that hypomethylation may be as relevant as
hypermethylation. Newly identified regions of interest include gene
bodies and so-called “CpG shores” (regions at 2 kb at either side of CpG
islands). In addition, even (long distant) enhancer regions are frequently
regulated by methylation. DNA methylation is established by DNA
methyltransferases (DNMTs). Recurrent mutations in DNMT3A, one of
the de novo DNA methyltransferases, are found in 6% to 36% of AML
patients and are most commonly heterozygous (Table 1). DNMT3A
mutations appear to be early events in leukemogenesis. This is evident
by studies demonstrating that DNMT3A mutations in AML patients
may also be present in T-lymphocytes derived from the same patient.48
These findings indicate that the mutation can arise in a primitive cell that
is still able to give rise to both myeloid as well as lymphoid cells. In
addition, it was recently demonstrated that DNMT3A mutations can
be found in (elderly) individuals without an apparent hematologic
malignancy, indicating that these mutations may be involved in clonal
hematopoiesis, which in some individuals precedes the development of
overt leukemia.10

Submitted July 22, 2015; accepted September 25, 2015. Prepublished online © 2016 by The American Society of Hematology
as Blood First Edition paper, December 10, 2015; DOI 10.1182/blood-2015-
07-604512.

42 BLOOD, 7 JANUARY 2016 x VOLUME 127, NUMBER 1

 From www.bloodjournal.org by guest on July 24, 2016. For personal use only.

BLOOD, 7 JANUARY 2016 x VOLUME 127, NUMBER 1 EPIGENETICS IN AML 43

Table 1. Recurrently mutated or translocated genes with epigenetic function in AML

Type of abnormalities described
Gene Epigenetic function (percentage of AML) References Remarks
DNMT3A De novo DNA methylation Mostly frameshifts; rare missense- and 7,11-16 Associated with normal karyotype;
non-sense mutations (6-36%) may be associated with poor prognosis
TET2 Conversion of 5-methylcytosine to Frameshift, nonsense and missense 7,15-17,18-21 Mutually exclusive with IDH1/2 mutations
5-hydroxymethylcytosine mutations (8-27%)
IDH1 and IDH2 Enzymes that convert isocitrate to Missense mutations (5-16% for IDH1; 7,15,16,22-24 Mutually exclusive with TET2 mutations.
a-ketoglutarate (a-KG), a cofactor 6-19% for IDH2) Mutations result in production of
for TET2 2-hydroxyglutarate, which inhibits
TET2 function
CREBBP (CBP) Histone lysine acetyltransferase Rearrangements: fusion genes 7,25 Rare
KAT6A (MYST3/MOZ) Histone lysine acetyltransferase Rearrangements: fusion genes 7,26 Rare
EP300 (p300) Histone lysine acetyltransferase Rearrangements: fusion genes 27,28 Rare
HDAC2 and HDAC3 Histone deacetylase Missense mutations 7 Rare
KMT2A (MLL/MLL1) H3K4 methyltransferase Rearrangements: fusion genes (1-10%); 7,15,16,29-32 More than 50 fusion partners reported in
partial tandem duplications (4-7%) acute leukemias
EZH2 H3K27 methyltransferase, enzymatic Mutations (2%) 7,33
component of PRC2
NSD1 H3K36 methyltransferase Rearrangement involving NUP98 (2-5%) 7,34,35
ASXL1 Recruitment of PRC2 to target loci Mostly frameshifts or nonsense 7,15,16,36,37-41 More common in elderly patients; poor
mutations (3-25%) prognosis particularly in association with
RUNX1 mutations
ASXL2 Homolog of ASXL1; function unknown Mutations (23% of AML with 42 Mutually exclusive with ASXL1 mutations
RUNX1-RUNX1T1)
JARID2 Recruitment of PRC2 to target loci Deletion in transformation of MDS 43
or MPN to AML
SUZ12 Member of PRC2 Missense mutations, insertions and 7,43,44 Sporadically mutated in progression severe
deletions congenital neutropenia to AML; deleted
in transformation MDS/MPN to AML
KDM5A (JARID1) Histone lysine demethylase Rearrangement involving NUP98 45 10% of pediatric acute megakaryoblastic
leukemia
KDM6A (UTX) Histone lysine demethylase Missense mutations 7,46 Rare

7
Sporadic mutations from TCGA extracted using COSMIC (http://cancer.sanger.ac.uk/).
MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm; PRC, polycomb repressor complex.

The mechanism by which mutant DNMT3A contributes to leuke-
mic transformation is not completely clear. It is hypothesized that mu-
tant DNMT3A acts as a dominant negative over wild-type DNMT3A.
AML cells with the R882H mutation have been reported to have pro-
found reduction of de novo methyltransferase activity.49 Mice trans-
planted with Dnmt3a-null hematopoietic stem cells (HSCs) in a
competitive transplantation setting do not develop myeloid malignan-
cies.50 However, when the same Dnmt3a-null HSCs are transplanted
in irradiated mice, not in a competitive setting, preleukemic disorders
are frequently observed.51 Mouse modeling experiments show that the
R882H variant, which is the most common DNMT3A mutation, is able
to drive the development of myeloproliferative disorders in vivo as
well. These data support the hypothesis that mutant DNMT3A acts as
a dominant negative.52 Mice that develop full-blown AML generally
acquire cooperating activating mutations in signaling factors such as
c-Kit.52 This is similar to what is observed in human AML patients with
DNMT3A mutations, who frequently carry concomitant FLT3-ITD
mutations. Recent work suggests that the presence of DNMT3A
R882H promotes chemoresistance, which appears to be associated
with impaired DNA damage sensing and impaired chromatin re-
modeling capacities.53
Specific patterns of genome-wide cytosine methylation are associ-
ated with distinct subtypes of AML. Using genome-wide profiling,
unique patterns of cytosine methylation are associated with AMLs
characterized by specific translocations and fusion genes (eg, RUNX1-
RUNX1T1 [AML1-ETO], PML-RARA, mutations in CEBPA, and
mutations in NPM1).8 The fact that mutations in genes encoding tran-
scription factors are associated with unique DNA methylation patterns
emphasizes the relationship between defective transcriptional regula-
tors and epigenetic alterations. In fact, evidence for a role of onco-
genic transcription factors in directing the activity of DNMTs and
changing the DNA methylation patterns has been proposed for
EVI1,54 PML-RARA55 and RUNX1-RUNX1T1.56
DNA hydroxymethylation
The oxidation of 5-methylcytosine yields 5-hydroxymethylcytosine
(5hmC). This process is catalyzed by the Ten-Eleven-Translocation
(TET)-enzymes and is dependent on a-ketoglutarate. Mutations in
TET2 are observed in 8% to 27% of patients with AML (Table 1). These
mutations are associated with reduced levels of 5hmC.57 TET2 muta-
tions confer a poor prognosis in intermediate-risk AML.17 In addition,
mutations in genes encoding the enzymes called isocitrate dehydro-
genase 1 and 2 (IDH1 and IDH2) are frequently observed in AML
(Table 1). They appear more prevalent in patients with normal cyto-
genetics. IDH proteins catalyze the conversion of isocitrate to
a-ketoglutarate. Mutations in IDH1 and IDH2 result in the production
of an aberrant metabolite, 2-hydroxyglutarate.58 This molecule has
been demonstrated to act as a competitive inhibitor of a-ketoglutarate,
resulting in the inhibition of TET2. TET2 mutations and IDH1/IDH2
mutations are mutually exclusive in AML.4 In addition, IDH1/IDH2
mutations are associated with global cytosine hypermethylation signa-
tures. Patients with TET2 mutations display overlapping hypermeth-
ylation signatures. TET2 mutations are observed in healthy aging
individuals, supporting the concept of clonal hematopoiesis that may
precede leukemogenesis.10,59 Recently, Levine and colleagues reported
that mutations in the WT1 gene are mutually exclusive with mutations
 From www.bloodjournal.org by guest on July 24, 2016. For personal use only.
44 WOUTERS and DELWEL
in IDH1/IDH2 or TET2.60 Increased global levels of 5hmC were ob-
served upon WT1 overexpression, whereas reduced 5hmC levels were
observed when WT1 was silenced. Although the mechanism of 5hmC
decrease upon WT silencing or mutation is not understood, the inves-
tigators demonstrated that WT1 physically interacts with TET2.
Decreased WT1 levels and absence of TET2 resulted in a similar
hematopoietic differentiation phenotype. The authors conclude that
TET2, IDH1/IDH2, and WT1 mutations define an AML subtype char-
acterized by dysregulated DNA hydroxymethylation.
Murine models to study the mechanism of TET2 and IDH1/2
mutations have recently been developed. Experiments performed in
conditional Tet2 knockout mice point to a role for Tet2 in the regulation
of self-renewal of hematopoietic stem cells. These mice do not develop
leukemia, but the presence of cooperating Flt3-ITD mutations does lead
to increased incidence of leukemia.61 Leukemogenesis may be depen-
dent on the downregulation of Gata2 by hypermethylation, an effect that
can be overcome by reintroduction of Gata2.61 It is predicted that mice
expressing conditional mutant Idh1 or Idh2 alleles in the presence of
Flt3-ITD or Nras mutations will develop AML in a comparable manner.
In addition to 5-methylcytosine and 5hmC, two other DNA
modifications have been recognized (ie, 5-formylcytosine and
5-carboxylcytosine).62 Similar to 5hmC, both represent oxidative der-
ivates of methylated cytosines and can be the result of the activity of
TET enzymes. The biological role of 5hmC and other derivates remains
to be established. It has been proposed that they may not merely be
intermediates toward CG demethylation, but may have distinct biolog-
ical roles, for instance in the regulation of gene expression.62
Histone acetylation
Histone acetylation involves the transfer of acetyl groups to lysine res-
idues in histone proteins. The processes of acetylation and deacetylation
are governed by histone lysine acetyltransferases (KATs) and histone
deacetylases (HDACs), respectively. Acetylation of lysine residues re-
sults in open chromatin confirmations, whereas deacetylation results
in condensed and closed chromatin.
Sporadic translocations involving KATs have been described in
myeloid malignancies, and mutations in HDACs have only rarely been
identified (Table 1). However, there is evidence that HDACs may be
aberrantly recruited by myeloid oncoproteins, such as EVI1 or PML-
RARA.63,64 These oncoproteins frequently interact with scaffold
proteins or major complexes, which recruit HDACs. This leads to
chromatin remodeling, in particular deacetylation of lysines at histone
tails near the transcription factor binding sites. These changes affect
transcription of putative target genes.65-67
Acetylated lysine residues in histones can be recognized by reader
proteins containing bromodomains, such as the bromodomain and extra
terminal (BET) proteins BRD2, BRD3, and BRD4. These proteins
contain 2 so-called bromodomains, which bind to acetylated histones.
BRD4 is involved in a complex including Mediator and pTEF-b, and
through this complex links histone acetylation to transcription.68,69
Although BRD4 and other BET proteins are ubiquitously present at
gene promoters and enhancers, inhibition of BET proteins results in
disproportionately large changes in expression of particular genes.70-72
Evidence is accumulating that these cell type–specific transcriptional
effects of BET inhibition can to a large extent be explained by the
association of BRD4 with exceptionally large enhancer elements,
called “super-enhancers,” which are involved in lineage-specific gene
regulation.73-76 In several types of malignancy, including AML,
disease-specific oncogenic super-enhancers, also referred to as
“stretched enhancers” or “locus control regions,” can be recognized
that drive expression of critical oncogenes, such as MYC.
BLOOD, 7 JANUARY 2016 x VOLUME 127, NUMBER 1
Histone lysine methylation
Methylation of histone lysine residues can result in mono-, di-, or
trimethylation. This is processed by lysine methyltransferases (KMTs).
Histone methylation alters the affinity of reader proteins to the meth-
ylated histones. Histone methylation can result in activation or re-
pression, the actual effect being dependent on its context.77 Marks
associated with activation include methylation of H3K4, H3H36, and
H3K79. Marks associated with silenced gene transcription include
methylation of H3K9, H3K27, and H4K20. Methylation state (eg,
mono- vs trimethylation) may have different functional consequences
in the same lysine residue.77
In AML, KMTs that are recurrently involved in translocations or are
mutated include mixed-lineage leukemia (MLL) proteins and compo-
nents of the polycomb repressor complexes (PRCs) (Table 1). KMT2A,
more frequently referred to as MLL, is a member of the family of SET
domain–containing KMTs. MLL targets H3K4 and leaves marks of
transcriptional activation. MLL is involved in translocations in approx-
imately 5% to 10% of AML.29 Partial tandem duplications of MLL are
found in 5% to 7% of de novo AML. MLL translocations result in
fusion proteins that lack the wild-type SET domain and are frequently
replaced by members of genes encoding super elongation complex
nuclear proteins, such as AF9, AF10, and ENL. This complex also
contains DOT1L, a KMT that targets H3K79. MLL-transformed AML
samples show aberrant H3K79 methylation, leading to ongoing ex-
pression of target genes, among which the homeobox genes are par-
ticularly involved.78
Enhancer of zeste 2 polycomb repressive complex 2 subunit
(EZH2) is an H3K27 methyltransferase that is part of the PRC2
polycomb repressor complex. EZH2 catalyzes di- and trimethylation
of H3K27, which results in transcriptional repression. Aberrant gain of
action of EZH2 has been associated with transcriptional repression of
critical target genes in various types of cancer. In myeloid malignancies,
however, EZH2 mutations lead to loss of function (Table 1).79,80 The
functional basis for this difference is unclear, but some preliminary
observations have been made. In a myelodysplastic syndrome (MDS)
mouse model, knockout of Ezh2 promoted the development of MDS
by impairing normal HSCs via activating inflammatory cytokine re-
sponses, while at the same time preventing the transformation to AML
via PRC1-mediated repression of Hoxa9.81 In a study focusing on
MLL-AF9 leukemia, EZH2 was not required for leukemogenesis, but
was required for tumor progression.82 A recent study highlights another
mechanism of interference with EZH2 function. Mutations in the
splicing factor gene SRSF2, frequently found in MDS, cause mis-
splicing of EZH2, resulting in nonsense-mediated decay.83
Other members of the PRC2 complex include additional sex
combs like transcriptional regulator 1 (ASXL1), Jumonji AT-rich
interactive domain 2 (JARID2), and SUZ12 polycomb repressive
complex 2 subunit (SUZ12), all of which have been implicated in the
development of AML (Table 1). Most of the mutations in these genes
result in loss of function of the complex. The PRC2-associated pro-
tein ASXL1 has an important role in the recruitment of the complex
to target loci. Similar to loss-of-function mutations in EZH2, muta-
tions in ASXL1 result in loss of PRC2-mediated H3K27 methyl-
ation. ASXL1 mutations may be associated with adverse prognosis,
an effect that seems to be correlated to the presence of RUNX1
mutations.36 Mutations in ASXL2 are frequently observed in patients
with the RUNX1-RUNX1T1 fusion gene and are mutually exclusive
with ASXL1 mutations.42
Similar to ASXL1, JARID2 recruits PRC2 to specific target loci.
Deletions of JARID2 have been reported in patients showing leukemic
transformation of chronic myeloid malignancies.43 SUZ12 has been
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BLOOD, 7 JANUARY 2016 x VOLUME 127, NUMBER 1
reported to be mutated in progression from severe congenital neutro-
penia to AML.44 Thus, in most if not all mutations found in PRC2
complex members in AML, the ultimate effect is loss of function re-
sulting in loss of H3K27 methylation. These effects are thus suggested
to cause increased expression of important target genes.
Another polycomb repressor group gene that has been linked to
AML is BMI1. BMI1 is part of the PRC1 polycomb repressor complex.
Increased BMI1 expression has been associated with poor prognosis in
AML and MDS.84,85
Several AML-related fusion proteins may interfere with normal
PRC functioning. The PML-RARA fusion protein can engage in a
complex with several PRC2 members, including SUZ12, EZH2, and
EED, thereby recruiting them to specific genomic loci.86 Another
fusion protein, PZLF-RARA, has been shown to interact with PRC1
components.87
Demethylation of histone proteins is governed by lysine demeth-
ylases (KDMs). Two classes of KDMs are known: the amine oxidases,
which include lysine-specific demethylase 1 (LSD1/KDM1A), and the
a-ketoglutarate–dependent Jumonji domain (JmjC) containing pro-
teins. LSD1 has specificity for H3K4 and H3K9 and has a role both as
a transcriptional activator as well as a transcriptional repressor, de-
pending on the cellular context.88 KDM5A (JARID1), a JmjC lysine
demethylase, is involved in a fusion protein with NUP98 in ;10% of
children with acute megakaryoblastic leukemia.45,89 KDM6A (UTX)
is another JmjC family member and can be altered by inactivating
mutations in AML (Table 1).46
Other histone modifications
In addition to the histone modifications described so far, other modi-
fications are known. Methylation of arginine residues in histones is me-
diated by the family of protein arginine methyltransferases (PRMTs), of
which 9 have been identified. Arginine methylation may be associated
both with transcriptional activation as well as with silencing.90,91
PRMT5, which symmetrically dimethylates arginine residues and is
considered to be a transcriptional repressor, is required for sustaining
normal hematopoiesis.92 Dysregulated expression of several PRMTs,
including PRMT5, has been observed in malignancies such as leuke-
mia. Tyrosine phosphorylation at histone tails has been reported, but
the mechanism by which this may occur and the function of histone
phosphorylation is poorly understood.93
Epigenetic targeted treatment of AML
Taking into account the inherent reversibility of epigenetic marks,
disordered epigenetic regulation poses the chance to use targeted drugs.
The translation of such drugs to standard clinical use is still in an early
stage. Compounds in various stages of preclinical and clinical devel-
opment are summarized in Table 2. We first describe drugs that are
currently available and then embark on approaches to identify novel
targets.
Drugs targeting disordered patterns of DNA cytosine
methylation and hydroxymethylation
Two epigenetic compounds currently approved for clinical use in
myeloid malignancies are 5-azacytidine (azacitidine) and 5-aza-29-
deoxycytidine (decitabine). Both are pyrimidine analogs that function
as inhibitors of DNA methyltransferases (Table 2). Azacitidine can be
converted into decitabine, which is incorporated into DNA. Azacitidine
itself is predominantly incorporated into RNA.106 Both drugs have been
EPIGENETICS IN AML 45
approved for the treatment of MDS as well as for AML with low blast
count. In Europe, decitabine is also approved for the treatment of elderly
AML patients with blast counts .30% who are not eligible for intensive
treatment. Azacitidine and decitabine are thought to exert their effects
by reversing aberrant DNA hypermethylation and thereby restoring
expression of critical (tumor-suppressor) genes. There are increasing
data to support the efficacy of these hypomethylating compounds in the
treatment of patients not eligible for intensive chemotherapy.107-110
There are currently no established biomarkers that predict the response
to these drugs, although it would be likely that not all patients are
equally susceptible. This hypothesis is supported by the identification
of factors that predict for response to azacitidine in MDS and methyl-
ation signatures that predict for response to decitabine in chronic
myelomonocytic leukemia, respectively.111,112 In MDS, particular
gene mutations, such as those in TET2 and DNMT3A, may predict for
better responsiveness to treatment with inhibitors of DNMTs.113 Com-
parable studies need to be carried out to dissect which AML subtypes
may benefit from treatment with DNMT inhibitors. One experimental
approach would be to test the sensitivity to these drugs in cohorts of
AML samples, taking into account subtypes that are particularly de-
fined by unique DNA-methylation signatures. We previously demon-
strated that leukemias with well-defined molecular abnormalities can be
discriminated based on these epigenetic signatures.8 Moreover, with
DNA methylation profiling we could identify AML subgroups that did
not harbor any known recurrent genetic defect. It is possible that certain
epigenetically defined AML subtypes are more responsive to DNA
methyltransferase inhibitors than others. Responsiveness studies may
be carried out in vitro using short-term assays testing cellular viability.
These experiments could provide an indication of response to the bulk
of cells to these agents. Whether the compounds will target the leukemia
stem cells needs to be established using in vivo models. Mouse models
have been generated for a number of AML subtypes carrying the distinct
mutations, which allow investigators to study the effects of hypomethy-
lating agents either alone or in combination with chemotherapy in vivo.
One should keep in mind that murine models may not fully represent the
actual human situation. Xenotransplant models in which human leukemia
cells are transplanted in immunodeficient mice provide an opportunity to
study the response of the human leukemias. The combination of these
different approaches may, at least in a subset of AML subtypes, provide
insight into responsiveness to DNMT inhibitors. Obviously the same
strategy can be applied for other newly derived compounds or com-
binations of agents, either with or without chemotherapy.
Analyzing altered DNA methylation patterns of AML cells may
help to uncover critical target genes that upon methylation have become
silenced in certain patient groups. Shih and colleagues recently dem-
onstrated how animal models can help to uncover such mechanisms.61
In their study, Tet2-null mice that also carried Flt3-ITD mutations
developed AML and expressed a unique methylation signature. Gata2
appeared to be silenced by methylation in those animals. Upon re-
expression of Gata2, the investigators reported reprogramming of the
tumor cells. It will be of interest to study the effects of DNMT inhibitors
in this model and to study reprogramming of these Tet22/2/Flt3-ITD
leukemia cells. No specific therapy for leukemias with TET2 mutations
has been developed yet; however, the observed hypermethylation sig-
nature in patients as well as in animal models suggests that in particular
in this subtype of AML hypomethylating agents may be effective.114
Further studies in xenotransplant models may help to unravel the most
optimal combination of drugs, which may also include compounds that
target uncontrolled signaling by FLT3-ITD.
As indicated, IDH1/2 mutations are also associated with a dis-
tinct methylation signature. IDH1/2 inhibitors have been devel-
oped and are already being tested in early clinical trials (Table 2).
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46 WOUTERS and DELWEL BLOOD, 7 JANUARY 2016 x VOLUME 127, NUMBER 1

Table 2. Examples of epigenetic targeted therapy in AML in various stages of development

Class of epigenetic
regulator Target Compound Phase of development
DNA DNMTs Azacitidine Approved (see text)
methyltransferase Decitabine Approved (see text)
Rationally designed novel inhibitors Preclinical and clinical94,95
Regulator of IDH1, IDH2 Inhibitors of mutant IDH1/2 Clinical trials ongoing with compounds including IDH305 (ClinicalTrials.gov
methylation identifier: NCT02381886; targeted at IDH1 R132 mutation), AG-221
(NCT01915498; targeted at mutant IDH2), AG-120 (NCT02074839;
targeted at mutant IDH1)
Histone lysine CREBBP (CBP) CREBBP inhibitor Preclinical96
acetyltransferase EP300 (p300) EP300 inhibitor Preclinical97
Histone deacetylase HDACs HDAC inhibitors Several clinical trials ongoing, often in combination with other treatment
modalities (eg, with DNMT inhibitors [examples ClinicalTrials.gov
identifiers NCT01617226 and NCT00867672], conventional
chemotherapy [example NCT01802333], or in conjunction with allogeneic
stem cell transplantation [examples NCT01451268 and http://www.hovon.
nl/studies/studies-per-ziektebeeld/aml.html?
action5showstudie&studie_id5104&categorie_id54])
Histone acetyl Bromodomain BET inhibitors Several clinical trials ongoing with compounds including OTX-015
reader containing proteins (ClinicalTrials.gov identifier: NCT01713582), CPI-0610 (NCT02158858),
(BET proteins) TEN-010 (NCT02308761), GSK525762 (NCT01943851)
Histone lysine EZH2 EZH2 inhibitors Preclinical98,99
methyltransferase MLL-complexes DOT1L inhibitors Clinical trial with compounds including EPZ-5676 (ClinicalTrials.gov
identifier: NCT01684150)
Inhibitors of MLL-Menin interface Preclinical100
Inhibitors of MLL-LEDGF interface Preclinical101
Histone lysine LSD1 LSD1 inhibitors Clinical trials with compounds including GSK2879552 (ClinicalTrials.gov
demethylase identifier: NCT02177812) and tranylcypromine in combination with
tretinoine (NCT02261779)
Jumonji family of Small molecular inhibitors competitive for Preclinical102,103
KDMs 2-oxoglutarate
Histone arginine PRMTs PRMT inhibitors Preclinical104,105
methyltransferase

The outcomes of these studies are pending, but preliminary data are
promising.115 It was shown that AGI-6780, a selective inhibitor of the
IDH2 R140Q mutation, acts by binding an allosteric site located within
the dimer interface and induces differentiation of AML cell lines in
vitro.116 Idh1/2-mutant animal models have been developed as well,
providing another opportunity to study the effects of the specific agents
at a molecular level. There is also experimental evidence that these
mutations may be targeted by alternative modes of action, such as
BCL2 inhibitors.117 In addition, IDH1 R132H mutations result in a
tumor-specific potential neoantigen that may offer a possibility of
development of vaccination strategies that induce mutation-specific
T-cell responses.118
Drugs targeting disordered patterns of histone acetylation
HDAC inhibitors were among the first epigenetic drugs developed, and
several have been tested in clinical trials for malignancies, including
AML.119 Responses to HDAC inhibitors as a single agent in AML or
MDS appear to be modest.120 More recent work has therefore focused
on combining these compounds with other drugs. A phase 2 study
testing a combination of vorinostat with idarubicin and cytarabine sug-
gested that the addition of an HDAC inhibitor to standard chemother-
apy may lead to improved response rates in patients with newly
diagnosed AML and MDS.121 More recently, combinations of DNMT
inhibitors and HDAC inhibitors have been tested.122-124 In a phase 2
randomized trial, combination of the HDAC inhibitor valproic acid and
low-dose decitabine did not lead to improved outcome in MDS and
AML patients.125 Moreover, in another phase 2 study, the HDAC
inhibitor entinostat appeared even to be antagonistic when administered
concurrently with azacitidine.126
It will be challenging to find the optimal combinations of HDAC
inhibitors with other agents. It is also likely that timing of treatment (eg,
concurrent vs subsequent administration) may affect responses—an
aspect that will in fact be crucial for other epigenetic drugs as well.
Answering these questions will require proper in vivo models as
discussed earlier. It is also possible that targeting altered histone
acetylation or the consequences thereof has to be carried out in a
completely different manner. This is supported by recent findings
involving the targeting of transcription elongation factors such as
bromodomains proteins using small molecules. Bromodomain pro-
teins, including BET proteins, exert their function by binding acetylated
lysine residues in histone proteins, such as H3K27. There are to date no
known mutations in bromodomain proteins in AML. However, BET
inhibitors showed therapeutic effects in various AML mouse models as
well as in cell lines and small series of primary samples.9,70,127,128 BET
inhibitors may be effective in AML associated with MLL transloca-
tions.70 Furthermore, it was recently demonstrated that in AML with
chromosome 3q abnormalities, a subtype with an adverse prognosis,
the leukemic translocation results in the formation of a novel super-
enhancer that drives overexpression of the EVI1 proto-oncogene.9
Indeed, AML t(3;3) or inv(3;3) cell lines appear sensitive to the BET
inhibitor JQ1. Not surprisingly, given the large molecular heterogeneity
among AML, previous studies have shown that BET inhibitors are not
uniformly active against all leukemia cell lines and patient samples.129
BET inhibitors have entered phase 1/2 clinical trials (Table 2).130
Studies in larger series will be needed to identify molecular features
linked to response to these drugs and to further clarify the mechanisms
underlying the efficacy of BET inhibition in AML and its target genes
and proteins. An important question that needs to be answered is how
 From www.bloodjournal.org by guest on July 24, 2016. For personal use only.
BLOOD, 7 JANUARY 2016 x VOLUME 127, NUMBER 1
to identify factors that differ between normal super-enhancers,
which direct normal cellular functions, and oncogenic super-
enhancers, which are unique to particular types of malignancy.
Insights into these differences would enable the development of
more selective treatment approaches. A recent study describes
phthalimide conjugation to the BET inhibitor JQ1 as a modified
method to BET inhibition. In this approach, the BET inhibitor is used
to target BRD4, whereas the appended phthalimide moiety results in
hijacking of the cereblon E3 ubiquitin ligase complex, resulting in
targeted BRD4 degradation.131
Drugs targeting disordered patterns of histone methylation
The histone demethylase LSD1 is highly expressed in hematopoietic
cells and is specifically required for the terminal differentiation of
myeloid cells.132 LSD1 may be overexpressed in AML and appears to
show a higher level of expression in less differentiated subtypes of the
disease. Recent reports have highlighted a potential application of
LSD1 inhibition in AML, and early clinical trials have been initiated
(Table 2).133,134 MLL translocations result in fusion proteins that lack
the wild-type SET domain and instead frequently recruit another lysine
methyltransferase, DOT1L, which targets H3K79. DOT1L can be
targeted by small-molecule inhibition.78,135 The first clinical trials in-
volving these compounds have been started and results have yet to
follow (Table 2).
Together, single epigenetic drugs such as inhibitors of BET pro-
teins, DOT1L, mutant IDH1/2, or DNMTs may show some significant
effects. However, these responses willmostlikely be partial or transient.
Therefore, these novel compounds, derived from functional epige-
nomics, need to be further studied in representative models in com-
bination with other drugs that target complementary pathways or with
chemotherapy. Molecular and epigenetic studies are essential for a
better understanding of epigenetic changes that lead to leukemic trans-
formation, and these investigations can lead to novel drugs that target
those lesions. We therefore believe that further searching for novel
targets and molecules that recognize those elements will be crucial.
How to identify novel targets for
epigenetic treatment?
As was pointed out, the application of targeted epigenetic treatment is
still in an early phase. Still, there are already multiple examples of AML
with unique epigenetic alterations, identified using integrated genetic
and epigenetic analysis, that may be matched to appropriate compounds
(Figure 1). A number of strategies can be applied to identify novel tar-
gets and to match those to appropriate modes of targeted treatment.
1. A first strategy is to apply high-throughput compound screening
approaches. Numerous compounds and small molecules are cur-
rently available that interfere with particular known epigenetic
processes. Large-scale titration sensitivity experiments of these
molecules in representative series of primary AML specimens in
vitro, for instance using tritiated thymidine- or MTT assays, will
provide compound response signatures of each AML sample.
AML patients may be classified based on these response signa-
tures (Figure 2). Because epigenetic changes in response to spe-
cific compounds may need several rounds of cell cycle, several
time points should be assessed if possible (eg, 3, 7, and 14 days
of culture). These experiments should be carried out under well-
defined conditions using appropriate combinations of hematopoi-
etic growth stimuli. Based on the outcome of these experiments,
EPIGENETICS IN AML 47
specific epigenetic modulators may be predicted to be targetable in
certain AML subtypes. Biochemical, molecular, and cell biological
studies should be applied to uncover mechanisms of action of the
identified compounds. An example of a compound that was iden-
tified to target a specific AML subset is the BET inhibitor JQ1.
BET inhibitors in general show a higher affinity for super-
enhancers compared with normal (small) enhancers. Molecular and
biochemical analyses revealed that JQ1 particularly targets the
oncogenic super-enhancer that causes overexpression of the EVI1
transforming gene.9 The effects of BET inhibitors on leukemic
stem cells of super-enhancer–driven EVI1 AMLs should be tested
in mouse models or in xenotransplant models.
A complementary approach involves the use of genetic tools
such as shRNA libraries or CRISPR/Cas9 technology targeting
epigenetic regulators. These studies may be carried out in vitro
as well as in vivo. Using an inducible shRNA library targeting
approximately 250 epigenetic modulators in MLL-fusion mouse
AML cells, Zuber and colleagues identified Brd4 as a potential
target for treatment in vivo.127 These studies pointed to the
potential usage of BET inhibitors for this specific AML subtype.
As one may anticipate based on the partial response of leukemia
cells to particular drugs, a third approach to identify novel targets
would be to combine the use of selected compounds (eg, BET
inhibitors) with genetic screening approaches such as shRNA.
2. An alternative route would be to start with detailed maps of genetic
and epigenetic marks in particular AML subtypes. This way, one
could prioritize certain drugs to be tested in distinct subtypes of the
disease. For instance, TET2 mutations have been associated with
increased levels of cytosine methylation. This may be a rationale
for specifically testing DNMT inhibitors in those patients
(Figure 1). Indeed, patients with TET2 mutations may be more sus-
ceptible to azacitidine than AML patients in general.114 Similarly,
one could compile genome-wide maps of super-enhancers by
applying ChIP-sequencing using antibodies directed against BRD4
and H3K27. Identifying AML cases with particular super-enhancer
profiles could provide leads to test BET inhibitors specifically in
subsets of the disease. By combining genetic mapping studies with
screening studies using extensive libraries of compounds or genetic
libraries, it should be possible to match particular drugs to patient
subgroups and to identify biomarkers.
3. A third approach is to use chemical-based engineering tech-
niques to design novel molecules predicted to specifically interfere
with certain epigenetic processes. These types of structural ap-
proaches heavily rely on chemical methods such as crystallogra-
phy, frequently in combination with computer-based modeling.
This could result in the development of inhibitors with a relatively
high affinity for mutated proteins, as has been done for IDH1/2
mutations.116 Alternatively, the development of BET inhibitors and
derivates illustrates that also inhibitors of wild-type proteins may
be applicable in certain situations.131,136
Optimal applications of epigenetic therapy
Despite great progress in the understanding of the molecular basis
of AML in the past years, treatment of the disease has essentially not
shown major changes in the past decades. This review highlights the
potential of compounds targeting epigenetic regulators in AML.
An important question is what the role of such compounds should be
in clinical practice. There are several challenges that may hamper the
clinical introduction of novel targeted therapies in general.137 Some of
 From www.bloodjournal.org by guest on July 24, 2016. For personal use only.
48 WOUTERS and DELWEL
these challenges include the inherent problems in the translation of
preclinical findings to the clinic, the presence of multiple coactive
deregulated pathways in the disease, and questions related to the
optimal design of clinical trials (how to best select patients to
include and which end points to address). In particular, the testing
of novel targeted treatment in an isolated fashion may be problem-
atic and may in fact underestimate the effectiveness of these novel
compounds. It is reasonable to assume that combination therapy
Figure 2. Epigenetic modifier screen for human AML.
Numerous compounds and small molecules are currently
available that interfere with particular known epigenetic
processes. Large-scale titration sensitivity experiments of
these molecules in primary AML specimens in vitro can
provide compound response signatures of each AML
sample (A). Using those assays, AML subtypes may be
uncovered that uniformly respond to specific epigenetic
compounds (B). The molecular, epigenetic, and biological
effects to these AML cells may subsequently be studied in
vitro and in vivo (C). The outcomes of such experiments
should lead to the translation to clinical trials in human
patients (D).
BLOOD, 7 JANUARY 2016 x VOLUME 127, NUMBER 1
Figure 1. Examples of integrated genetic and epi-
genetic analysis of human AML leading to potential
epigenetic treatment. The integrated analysis of gene
mutations, DNA methylation, and histone modifications
(A) has revealed multiple AML subtypes with specific
mutations in association with specific epigenetic alter-
ations. Four examples are indicated (B). Mutations in
IDH1/2 and TET2 result in aberrant patterns of DNA
cytosine methylation and hydroxymethylation (C),
whereas rearrangements affecting the EVI and MLL
loci result in alterations of chromatin marks (C). For
each of these 4 examples, certain epigenetic inhibitors
have been designed or are predicted to be active, of-
fering the potential to translate these findings to clinical
trials in human patients (D). The various inhibitors are
discussed in more detail in the text.
will be key, which may involve either combination with standard
chemotherapy or with other targeted therapies that are predicted to
target parallel critical pathways.
Together, epigenetic targeted treatment holds great promise. At the
same time, there are various challenges that will have to be dealt with.
In the first place, many of the types of targeted treatment that will be
applied will not be mutation-specific, but will rather target particular
epigenetic regulating systems that are also active in nontransformed
 From www.bloodjournal.org by guest on July 24, 2016. For personal use only.

BLOOD, 7 JANUARY 2016 x VOLUME 127, NUMBER 1 EPIGENETICS IN AML 49

cells, posing the risk of toxicity. Furthermore, the action of epigenetic

marks and regulators is often cell type–specific, the actual result being
dependent on particular protein-protein interactions, which also carries
risks of off-target toxicity. Along the same lines, many chromatin-
modifying enzymes have more diverse functions, including a role in
modifying nonhistone substrates. The identification of biomarkers that
can predict the susceptibility to certain epigenetic compounds will be
essential for a proper choice of epigenetic treatment of the distinct AML
subtypes.138 An additional challenge in the treatment of AML that has
emerged is the concept of clones and subclones.139,140 Targeted therapy
may be successful in eradicating some subclones, but may not be able
to eradicate subclones that do not harbor the particular epigenetic
abnormality. In this respect, it is promising to note that recent work has
indicated that many of the mutations that can be found in clonal
hematopoiesis are in epigenetic regulators such as TET2 and DNTM3A.
These mutations seem to enhance the self-renewal capacity of hema-
topoietic progenitor cells and may predispose individuals to the devel-
opment of leukemia. Targeting such early lesions may eradicate the
founding clones. Still, it is most likely that the combination of
several types of treatment (eg, epigenetic therapy in combination
with conventional chemotherapy) will have the best potential to be
successful.
Acknowledgments
The authors thank Egied Simons for help with preparing the figures
and Dr Peter Valk for critically reading the manuscript. They
apologize to authors whose work could not be cited due to space
limitations.
B.J.W. is a recipient of a Fellowship Award by the Dutch Cancer
Society (K.W.F.) and a Special Fellowship Award by the Leukemia
& Lymphoma Society.
Authorship
Contribution: B.J.W. and R.D. wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Ruud Delwel, Erasmus Medical Center, De-
partment of Hematology, P.O. Box 2040, 3000 CA Rotterdam, The
Netherlands; e-mail: h.delwel@erasmusmc.nl.

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2016 127: 42-52

doi:10.1182/blood-2015-07-604512 originally published
online December 10, 2015

Epigenetics and approaches to targeted epigenetic therapy in acute

myeloid leukemia
Bas J. Wouters and Ruud Delwel

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