Leukemia (2014) 28, 2131–2138
& 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14
www.nature.com/leu
REVIEW
The emerging roles of DOT1L in leukemia and normal
development
CM McLean1,2, ID Karemaker1,2 and F van Leeuwen1
Methylation of lysines within histone proteins represents a posttranslational modification system that can have profound effects on
gene expression. An evolutionarily conserved, but poorly understood, histone methylation mark occurs on lysine 79 on histone H3
(H3K79). The H3K79 methyltransferase, DOT1L, is involved in a number of key processes ranging from gene expression to DNAdamage response and cell cycle progression. Recently, DOT1L has also been implicated in the development of mixed lineage
leukemia (MLL)-rearranged leukemia, where mistargeting of DOT1L causes aberrant H3K79 methylation at homeobox genes.
As DOT1L is essential for leukemic transformation, small-molecule inhibitors of DOT1L function are an attractive therapeutic target
for this type of leukemia. However, in order to develop safe treatments, it is necessary to also understand the biological functions of
DOT1L. Here we review the various functions of DOT1L in normal mammalian development. Dot1L knockout is embryonic lethal in
mice and is important for processes as diverse as proliferation of mouse embryonic stem cells, induced and natural reprogramming,
cardiac development and chondrogenesis. Additionally, while an important role for DOT1L in embryonic hematopoiesis is clear,
its role in postnatal hematopoiesis is less so. Establishing the precise function of DOT1L in normal adult hematopoiesis and
understanding its mode of action will aid in our understanding of the use of DOT1L as a therapeutic target in MLL-rearranged
leukemia.
Leukemia (2014) 28, 2131–2138; doi:10.1038/leu.2014.169
INTRODUCTION
The field of epigenetics studies changes in the structure of genetic
information that are stable, yet reversible, and cause a phenotype
but are not the result of alterations to the DNA sequence itself.1,2
Epigenetic modifications can occur at the level of DNA (most
notably DNA methylation) and also at the histone proteins, which
are the building blocks of the nucleosomes that package the DNA.
Epigenetic modifiers are emerging as important regulators of the
genome and as appealing targets for treatment of cancer and
other diseases that involve epigenetic mechanisms. One of the
best studied histone-modification systems is lysine methylation,
which can occur as mono-, di- or trimethylation.3 Interestingly, the
effects of histone lysine methylation are highly dependent on
context: methylation on some lysine residues is associated with
active transcription, whereas methylation on others is associated
with repressed transcription (see Figure 1).3 Two classes of histone
lysine (K) methyltransferase (KMT) proteins exist. The first
class of proteins contains an evolutionarily conserved SET
methyltransferase domain (named after Drosophila Su(var)3-9,
Enhancer of zeste [E(z)] and trithorax (trx)). The second class is
represented by a single protein, Dot1/DOT1L, which is the only
known histone KMT lacking a SET domain.4
Disruptor of telomeric silencing (Dot) 1 was identified in a yeast
screen for genes that disrupt telomeric silencing when overexpressed.5 Dot1 is the only known histone H3 lysine 79 (H3K79)
methyltransferase in both yeast and mammals, where it is called
Dot1-like (DOT1L).4,6,7 H3K79 methylation is associated with active
transcription and, interestingly, H3K79 is not located on a histone
tail, where most epigenetic modifications occur, but on the
nucleosome core (Figure 1).7 Apart from its role in telomeric
silencing and transcription, other functions of Dot1/DOT1L include
DNA repair, where H3K79 methylation is implicated in the
recruitment of Rad9/53BP1 to DNA double-strand breaks, and
cell cycle regulation, where H3K79 methylation has a role in G1–S
transition.8–12
Recently, DOT1L has been implicated in the development and
maintenance of mixed lineage leukemia (MLL)-rearranged
leukemia, where chromosomal translocations cause the MLL
(also called mixed lymphoid leukemia) gene to fuse in-frame to
one of many fusion partners. Several of these fusion partners
interact directly or indirectly with DOT1L, resulting in inappropriate recruitment of DOT1L to gene targets of these MLL fusion
proteins, such as the HoxA cluster and the homeobox gene
Meis1.13 The presence of DOT1L causes hypermethylation of
H3K79 on these genes, which induces aberrant gene expression
and contributes to leukemic transformation. MLL-rearranged
leukemia can develop into either acute myeloid leukemia or an
acute lymphoblastic leukemia, such as T-cell acute lymphoblastic
leukemia.14 These findings suggest DOT1L as a potential
therapeutic target in these leukemias and, indeed, the first
studies exploring this approach seem promising.15–17 However,
little is known about the biological functions of DOT1L in
vertebrates. The prospect of targeting DOT1L as a treatment
option for leukemia underscores the relevance of understanding
its biological functions in order to develop safe treatments. This
review discusses the functions of DOT1L in normal mammalian
development, with an emphasis on the role of DOT1L in
hematopoiesis and targeted treatment.
Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands. Correspondence: Dr F van Leeuwen, Division of Gene Regulation, Netherlands Cancer
Institute NKI, Plesmanlaan 121, Amsterdam 1066CX, The Netherlands.
E-mail: [email protected]
2
These authors contributed equally to this work.
Received 21 March 2014; revised 6 May 2014; accepted 15 May 2014; accetped article preview online 23 May 2014; advance online publication, 17 June 2014
DOT1L in leukemia and normal development
CM McLean et al
2132
indicating that progression through the cell cycle was disrupted.
Abnormal spindles were detected in proliferating cells, suggesting
that disruption of the cell cycle was caused by loss of mitotic
spindle integrity upon DOT1L depletion. However, this cell cycle
arrest did not lead to an increase in apoptosis in undifferentiated
mESCs, implying that the failure of cells to proliferate is caused by
other mechanisms.21 Likewise, after treatment with retinoic acid,
no increase in apoptosis was observed, although the proportion of
late apoptotic/necrotic cells doubled. These results suggest that
reduction of DOT1L levels has a modest effect on proliferation in
undifferentiated mESCs but severely disturbs growth rate in
mESCs after induced differentiation.
Figure 1. Overview of the commonly studied histone methylation
and ubiquitination events in the context of mammalian gene
regulation. Methylation of lysines 9 and 27 on the N-terminal tail of
histone H3, methylation of lysine 20 on the N-terminal tail of histone
H4 and ubiquitination of lysine 119 on the C-terminal tail of histone
H2A are associated with gene repression. Methylation of lysines 4
and 36 on the N-terminal tail of histone H3, methylation of lysine 79
on the core of histone H3 and ubiquitination of lysine 120 on the
C-terminal tail of histone H2B are associated with gene activation.
Crosstalk occurs between H3K79 and H2BK120: mono-ubiquitination
of H2BK120 increases methylation of H3K79.
GENERAL EMBRYONIC DEVELOPMENT
Role of DOT1L in mouse embryonic stem cells (mESCs)
A powerful model system to study mammalian embryonic
development in vitro is the mouse embryonic stem cell (mESC).
mESCs are pluripotent stem cells derived from the inner cell mass
of blastocyst-stage embryos.18,19 These cells can develop into all
three germ layers, as well as germ cells, making them a good
in vitro model for early embryonic development. Three
approaches have been used to inactivate DOT1L in mESCs and
thereby study its function: the Cre/LoxP site-specific
recombination system, knockdown by short hairpin RNA
(shRNA), or a gene trap allele.
mESCs lacking a highly conserved portion of the DOT1L
catalytic domain were generated by Zp3-Cre-mediated deletion
of exons 5 and 6 of the Dot1L gene, which results in deletion
beginning in the early stages of oocyte development and
continuing through fertilization.20 Both DOT1L heterozygous and
homozygous mutant cell lines displayed severe defects in growth
rate and exhibited G2/M arrest, as well as aneuploidy.20
Additionally, apoptosis levels were at least twice as high in the
Dot1L mutant mESCs as in wild-type cells, explaining the observed
proliferation defects.20
When shRNA interference was used to disrupt DOT1L expression in undifferentiated mESCs, proliferation was modestly
reduced and several key pluripotency genes normally enriched
for H3K79 methylation were unaffected by loss of H3K79
methylation.21 Yet, when shRNA-treated mESCs were subjected
to induced differentiation with retinoic acid, both proliferation and
differentiation were severely impaired. Similar to DOT1L knockout
cells, these cells also arrested in G2/M and displayed hyperploidy,
Leukemia (2014) 2131 – 2138
DOT1L in proliferation of non-stem cells
DOT1L is also important for proliferation of certain types of cancer
cells, which have many properties in common with mESCs,
including self-renewal, inhibited differentiation, missing or defective checkpoint controls and similar transcriptome profiles.22,23
In A549 and NCI-H1299 lung cancer cells, siRNA-mediated
knockdown of DOT1L leads to decreased growth, despite
incomplete knockdown.24 As with mESCs, these cells displayed
aneuploidy but arrested at the G1 phase of the cell cycle rather
than G2/M.24 This inconsistency is likely explained by the fact that
the G1 checkpoint is absent in mESCs.22 However, it is important
to note this is not the case with all cancer cells, as Dot1L deletion
or DOT1L inhibition selectively kills hematopoietic cells
transformed by MLL-rearrangement but has no effect on
hematopoietic cells transformed by other oncogenes.15,25–27 In
another study, cardiac-specific knockout of Dot1L was achieved by
deleting exons 5 and 6 by cardiac-specific (a-MHC (major
histocompatibility complex) driven) Cre recombinase expression.
In contrast to findings in other cell types, these mice displayed
increased proliferation in the heart.28 Finally, no effect on
proliferation was observed in intestinal epithelial cells in
either the villus or the crypt, in which DOT1L was deleted
in a tissue-specific, tamoxifen-inducible manner using Villin-CreER
or Lgr5-EGFP-IRES-CreER, respectively.29 Although proliferation was
unaffected, apoptosis was increased in the intestinal crypts.29
Taken together, these results imply that the effect of DOT1L
deficiency on proliferation is cell type specific. Interestingly,
DOT1L depletion seems to have an analogous effect on mESCs
and on some types of cancer cells, which both exhibit reduced
growth and checkpoint arrest upon DOT1L depletion.30 Perhaps
this can explain the comparable effect of DOT1L deficiency on
growth and checkpoint arrest in these cell types. A short overview
of the described studies can be found in Table 1.
DOT1L in reprogramming and developmental transitions
A recent study shed new light on the role of DOT1L in
pluripotency and reprogramming of mESCs, as inhibition of
DOT1L accelerates reprogramming of somatic cells into induced
pluripotent stem cells (iPSCs).31 Adult human dermal fibroblasts
can be reprogrammed to a pluripotent state by introduction of the
transcription factors OCT4, SOX2, C-MYC and KLF4.32 This
reprogramming includes global remodeling of the epigenome,
allowing the cells to switch from a somatic differentiated state to
an undifferentiated pluripotent state. In an shRNA screen to
investigate the effect of chromatin modifiers on reprogramming,
shRNA-mediated knockdown of DOT1L increased the reprogramming efficiency in both mouse and human cells up to
fourfold.31 In fact, DOT1L inhibition could substitute for expression
of two of the four commonly used reprogramming factors, C-MYC
and KLF4, emphasizing its efficiency.31 Furthermore, inhibition of
DOT1L caused upregulation of NANOG and LIN28, two important
pluripotency factors. Chromatin immunoprecipitation sequencing
(ChIP-seq) data indicate that DOT1L deficiency enhances
reprogramming, because reduced H3K79 methylation leads to
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Table 1.
Effects of DOT1L deficiency on cell proliferation
Study (reference)
Method
Cell type
Effect of DOT1L deficiency on cell proliferation
Barry et al.
shRNA-mediated knockdown
mESCs
Jones et al.20
Zp3-Cre/loxP knockout of the
catalytic domain
siRNA-mediated knockdown
mESCs
Modest decrease in proliferation; severely decreased
proliferation upon induced differentiation
Decreased proliferation
21
Kim et al.24
Ngyuen et al.28
Ho et al.29
a-MHC-Cre/loxP knockout of the
catalytic domain
Villin-Cre/loxP knockout of the
catalytic domain
A549 and NCI-H1299
lung cancer cells
Cardiac cells in vivo
Decreased proliferation
Intestinal epithelial cells
No effect but increased apoptosis
Increased proliferation
Abbreviations: DOT1L, disruptor of telomeric silencing (Dot) 1-like; mESC, mouse embryonic stem cell; siRNA, small interfering RNA.
silencing of lineage-specific genes, as well as to upregulation of
certain pluripotency genes.31 Likewise, in a separate study, shRNA
knockdown of DOT1L resulted in severe impairment in the ability
of mESCs to differentiate into embryoid bodies, and microarray
analysis of genes regulated by DOT1L revealed an enrichment in
genes involved in differentiation.21 Taken together, these results
establish that DOT1L has an important role in maintaining
pluripotency.
Besides induced reprogramming, natural reprogramming
also exists, one example being during fertilization, when the
differentiated oocyte is reprogrammed into a totipotent state
from which the embryo can form. Interestingly, DOT1L is
associated with this reprogramming event. Before fertilization,
both H3K79 di-methylation (H3K79me2) and H3K79 tri-methylation (H3K79me3) are detected in the mouse oocyte, whereas
soon after fertilization H3K79 methylation disappears.33 During
preimplantation development, both H3K79me2 and H3K79me3
are detected at low levels, with the exception of a transient
increase in H3K79me2 at M phase. This suggests that H3K79
demethylation is involved in genome reprogramming towards a
totipotent state.33 This demethylation is remarkable as it seems to
be almost complete and it happens independently of DNA
synthesis, implying the presence of active H3K79 demethylating
factors. When a somatic cell nucleus is transplanted into an
oocyte, H3K79 methylation is also erased, suggesting that the
cytoplasm of the oocyte contains factors that are responsible for
demethylation. This is supported by controls in which a somatic
nucleus is transplanted, but not exposed to the cytoplasm, and
where no H3K79 demethylation occurs.33 Although this is
convincing evidence that the factors responsible for eliminating
H3K79 methylation are present in the cytoplasm of the oocyte,
their identity remains unknown. Thus, although it is unclear how
H3K79 demethylation is accomplished, H3K79 methylation itself
appears to be involved in reprogramming the female germline.
Evidence also points to a role for DOT1L in meiosis in the male
germline in mice. A recent study used immunofluorescence to
examine H3K79 methylation throughout meiotic prophase I in
mouse spermatocytes.34 In wild-type mice, DOT1L, H3K79me2 and
H3K79me3 levels increase steadily from pachynema through
prophase I, whereas H3K79 mono-methylation is present at low
levels. Despite the similar dynamics of DOT1L, H3K79me2 and
H3K79me3, the sub-nuclear localization of these marks differs.34
DOT1L and H3K79me3 levels significantly increase in the
heterochromatic sex body during diplonema and diakinesis, with
H3K79me3 also increasing at the centromeres. Conversely,
H3K79me2 is present throughout the chromatin, except for at
the sex body, despite the increase in DOT1L levels here.
Interestingly, patterns of the histone variants gH2AX, macroH2A,
H2A.Z and H3.3 correlated with certain aspects of DOT1L and
H3K79 methylation accumulation.34 The distribution of the H3K79
methylation marks suggests that H3K79me2 may be involved in
& 2014 Macmillan Publishers Limited
transcriptional re-activation of autosomes during pachynema,
whereas H3K79me3 may contribute to maintenance of silencing at
the sex body and the centromeres.34
An evolutionarily conserved role of DOT1L in embryonic
development
The results described above suggest that DOT1L is important in
early development in mammals, and this function seems to be
evolutionarily conserved in Drosophila. Indeed, grappa (gpp), the
Drosophila ortholog of Dot1L, is involved in fly development,
where it is required for polycomb group (Pc-G)-mediated
homeotic gene silencing.35 Unlike in mice, gpp is not involved in
early development, as H3K79 methylation is low, or even absent,
during early nuclear cleavage stages.35 Only later in development
is this epigenetic mark readily detected, which suggests that gpp
is involved in the maintenance, rather than the establishment,
phase of homeotic gene regulation.35 Consistent with
observations in Drosophila, DOT1L also has an important role
during later stages of mammalian embryonic development. The
widespread expression pattern of DOT1L later in mouse
embryonic development indicates that its function remains
important during these stages.20,36 The importance of DOT1L in
mammalian development is further emphasized by the finding
that Dot1L mutant embryos are embryonic lethal. Homozygous
mutant Dot1L embryos with Zp3-Cre/LoxP-mediated deletion of a
portion of the catalytic domain of DOT1L do not develop further
than E10.5.20 In addition, these embryos display developmental
abnormalities, including stunted growth, defective yolk sac
angiogenesis and dilation of the heart.20 Dot1L knockout by a
gene trap cassette in exon 13 of the Dot1L locus, disrupting the
nucleosome binding domain and methyltransferase activity, is also
embryonic lethal, with embryos dying between E10.5 and E13.5.37
Lethality was caused by defects in yolk sac angiogenesis,
hematopoiesis and vascular remodeling, causing severe
anemia.37 These studies demonstrate that DOT1L is required for
embryogenesis.
CARDIAC DEVELOPMENT AND FUNCTION
DOT1L is important for embryonic cardiac development
In addition to its role in general embryogenesis, DOT1L also has
tissue-specific functions. For instance, one of the developmental
abnormalities that Dot1L mutant mouse embryos display is cardiac
dilation.20 Consistent with this observation, cardiac-specific
knockout in mice causes ventricular dilation, resulting in
severely enlarged hearts.28 In addition, there is a relationship
between epigenetics and shear stress, a process which has a
morphogenetic function during zebrafish cardiac development.38
When mESCs were subjected to shear stress, this enhanced
methylation of H3K79 while simultaneously inducing
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CM McLean et al
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cardiovascular markers, implying a function for DOT1L in the
expression of these markers.39 Mice carrying a cardiac-specific
knockout of the DOT1L catalytic domain via the Cre/LoxP system
were born at Mendelian ratios but displayed cardiac dilation and
postnatal lethality.20,28 Interestingly, defects resulting from DOT1L
depletion could be rescued by postnatal expression of DOT1L.
Expression of the protein dystrophin, which is important for lateral
force transduction in the heart, is decreased upon DOT1L
depletion. The dystrophin gene is normally enriched for
methylated H3K79, suggesting a direct transcriptional regulation
of dystrophin by DOT1L.28 Together, these findings show that
DOT1L has a critical function in postnatal heart function.
Intriguingly, de novo mutations in certain histone-modifying
genes are linked to human congenital heart disease.40 Some of
these are important for ubiquitination of histone H2B lysine 120
(H2BK120; see Figure 1), a modification which enhances dimethylation of H3K79 by DOT1L.41 Although experimental
confirmation of this possible link between H3K79 methylation
and congenital heart disease via H2BK120 ubiquitination is
needed, this finding indicates that there may also be a role for
the H2BK120ub–H3K79me axis in cardiac development in humans.
CHONDROGENESIS
In mouse developing limbs, DOT1L is strongly expressed during
chondrogenesis, the process of cartilage development.42
Furthermore, in vitro, short-hairpin microRNA (shmiRNA)-mediated
knockdown of DOT1L in mouse chondrogenic ATDC5 cells leads to
impaired chondrogenesis, as demonstrated by a reduction of the
proteoglycan and collagen content, as well as decreased
mineralization.42 Although this strongly suggests that DOT1L is
involved in chondrogenesis, the underlying mechanisms remain
unclear. As mRNA expression of several Wnt target genes is
downregulated in knockdown cells, it was proposed that DOT1L
acts through Wnt signaling to stimulate chondrogenesis.42
However, as discussed below, the role of DOT1L in Wnt
signaling is still under debate.
HEMATOPOIESIS
Hematopoiesis is the formation of new blood cells and takes place
in the bone marrow, where the hematopoietic stem cells (HSCs)
reside. HSCs can give rise to progenitors of the three hematopoietic lineages: the lymphoid, myeloid, and erythroid lineages.
These progenitors subsequently develop into a number of
differentiated cell types, as illustrated in Figure 2.
MLL-rearranged leukemia
Hematopoiesis occurs both in the embryo and the adult organism
where it is needed to increase or to retain, respectively,
homeostatic levels of all types of blood cells in the body.
However, a large number of mutations in hematopoietic
precursors can result in the accumulation of poorly differentiated
Figure 2. Overview of hematopoietic developmental lineages. Hematopoiesis results in the formation of lymphoid, myeloid and erythroid
differentiated cell types from a common stem cell progenitor.
Leukemia (2014) 2131 – 2138
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blood cells, a process referred to as leukemic transformation.43
Many mutations are known to contribute to leukemic
transformation, one of which is translocation of the MLL gene,
causing an in-frame fusion of part of MLL to 1 of 470 known
fusion partners (reviewed in Barry et al.44). Numerous fusion
partners interact directly or indirectly with DOT1L, leading to
mistargeting of DOT1L and aberrant H3K79 methylation.13
Furthermore, DOT1L enzymatic activity is required for this class
of leukemic transformations, making DOT1L an emerging
therapeutic target.26,44 However, as DOT1L is implicated in
normal hematopoiesis, a potential problem is that inhibiting
DOT1L in leukemia may disturb the normal hematopoietic
process.
Germline inactivation of DOT1L
Several DOT1L knockout models have provided important
information about the role of DOT1L in embryonic hematopoiesis.
Embryos carrying the Dot1L gene trap allele (as discussed above)
die between E10.5 and E13.5 and have a much paler complexion
than wild-type embryos, resulting from a near absence of
erythrocytes.37 Also, the yolk sacs of the knockout embryos
contain less blood and exhibit vascular remodeling defects,
leading to abnormal caliber of the blood vessels. Yolk sac
angiogenesis defects and pale complexion were also found in
embryos carrying a Zp3-Cre/LoxP-mediated knockout of Dot1L.20
This suggests an important function for DOT1L in embryonic
hematopoiesis.37 Indeed, erythroid progenitor cells, but not
myeloid progenitors, display proliferation defects when assessed
through quantitative colony-forming unit assays. This defect in
erythropoiesis in Dot1L knockout embryos is caused by a
reduction in the number of progenitor cells, resulting from both
arrest in G0/G1 phase and increased apoptosis.37 These findings
imply that DOT1L is required for proper cell cycle progression and
survival of erythroid progenitor cells during development. The
transcription factor GATA2 is downregulated in the Dot1L
knockout cells, while there is an accompanying upregulation of
PU.1 (see Figure 2).37 These two transcription factors are known to
constitute a differentiation switch: high levels of GATA2 and low
levels of PU.1 direct hematopoietic progenitor cells to the
erythroid lineage, whereas low levels of GATA2 and high levels
of PU.1 stimulate differentiation along the myeloid lineage.45
DOT1L depletion causes this switch to be continuously on myeloid
differentiation, leading to a deficit in erythroid progenitor cells.37
These results demonstrate that DOT1L is essential for embryonic
erythropoiesis but probably not myelopoiesis.
Inducible and conditional inactivation of DOT1L
In addition to its role in embryonic hematopoiesis, the role of
DOT1L in adult hematopoiesis has been investigated. Two studies
used similar methods to generate inducible Dot1L knockout mice
by crossing R26-Cre-ER mice with mice in which part of the Dot1L
locus was floxed. Inducible, rather than germline, knockout mice
were used as Dot1L knockout is embryonic lethal.20,28 Subsequent
administration of tamoxifen caused either excision of exon 2 with
a downstream frameshift27 or of exons 5 and 6, which contain part
of the catalytic domain46 of the Dot1L gene. In one study,
homozygous knockout mice died 8–12 weeks after the initial
tamoxifen treatment, confirming that DOT1L is also essential
postnatally.27 Death was caused by severe anemia and
hypocellularity in the bone marrow, which led to depletion of
functional HSCs as well as multiple types of progenitor cells
(common lymphoid, megakaryocyte, granulocyte macrophage
and erythroid).27 These results were largely confirmed in a second
mouse model in which one allele of Dot1L was constitutively
inactivated, while knockout of the other allele could be induced
by tamoxifen.46 Although the age at which these mice died
was not reported, they displayed gross anemia and general
& 2014 Macmillan Publishers Limited
hypocellularity in the bone marrow, as well as bleeding and brain
hemorrhaging. These defects were also caused by depletion of
HSCs, various progenitor cell populations (granulocyte/monocyte,
megakaryocyte/erythrocyte and common myeloid) and terminally
differentiated cells from the myeloid lineage.46 In mixed bone
marrow transplantation experiments, all assessed lineages showed
minimal contribution of Dot1L knockout cells as compared with
wild type, indicating that bone marrow failure upon DOT1L
depletion is cell autonomous.27,46 These studies show an essential
role for DOT1L in maintaining adult hematopoiesis, indicating that
inhibition of DOT1L as a treatment for leukemia can potentially
have serious side effects.
In contrast, a third study reported slightly different findings.
The knockout mouse model in this study was generated by
crossing Vav-Cre mice with mice in which exon 5 of Dot1L
was floxed, deleting a portion of the catalytic domain.26 VAV is
expressed specifically in the hematopoietic compartment
beginning in embryonic development,47 facilitating the
generation of hematopoiesis-specific homozygous Dot1L
knockout mice. Although Dot1L deletion was not complete, as
evidenced by residual H3K79me2 in Dot1L knockout mice, these
hematopoietic Dot1L knockout mice did display anemia with
hypocellularity in the bone marrow. However, DOT1L depletion
did not cause a total loss of myeloid or lymphoid development.
Several progenitor cell populations were shown to be moderately
to severely affected by loss of DOT1L (granulocyte/macrophage
and common myeloid), but megakaryocyte/erythroid progenitors
were less affected.26 In addition, peripheral Dot1L / blood
leukocytes could be isolated from the Dot1L knockout mice,
implying that their development was not severely disrupted. Thus,
specific loss of DOT1L in the hematopoietic lineage leads to
impaired hematopoiesis, but as some hematopoietic activity
remains, DOT1L is not essential for all hematopoietic cells.
Intriguingly, different conclusions can be reached about the role
of DOT1L in hematopoiesis and, therefore, its suitability as a drug
target in leukemia. These differences may be caused by the
experimental models employed or efficiency of deletion. Ubiquitous knockout of Dot1L leads to defects in embryonic erythropoiesis and adult hematopoiesis,27,37,46 whereas knockout of Dot1L
only in hematopoietic cells does not affect all adult
hematopoiesis.26 Perhaps ubiquitous loss of DOT1L has side
effects which influence processes in addition to hematopoiesis,
resulting in a more severe phenotype. Nevertheless, taking all
studies into account, it appears that DOT1L has an important
function in maintaining normal hematopoiesis.
SMALL-MOLECULE INHIBITORS OF DOT1L AS A TREATMENT
FOR LEUKEMIA
Given the important role of DOT1L in MLL-rearranged leukemia,
several studies have investigated the potential of DOT1L inhibitors
as a treatment regimen. A good indication that inhibition of
DOT1L can be a treatment for those types of leukemia in which
DOT1L is mistargeted is that global gene expression in MLL-AF9
rearranged leukemia cells does not change dramatically upon
knockout of Dot1L. Indeed, although many genes were affected,
downregulated genes were enriched for MLL-AF9 targets,
indicating that inhibition of DOT1L particularly affects genes
associated with MLL translocation.26
DOT1L small-molecule inhibitors as a treatment for leukemia
Currently, three compounds (EPZ004777, EPZ-5676 and SGC0946)
exist that display great specificity for DOT1L over other histone
methyltransferases.15,17,48 These compounds exert their function
by competing with S-adenosyl methionine, the cofactor needed
for the methyltransferase activity of DOT1L.48 In MLL-rearranged
leukemia cells, all three compounds cause downregulation of MLL
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CM McLean et al
2136
fusion target genes, such as HoxA9 and Meis1, demonstrating that
they can reverse the effects of aberrant H3K79 methylation.
Moreover, these compounds inhibit proliferation specifically in
MLL-rearranged leukemia cells by stimulating apoptosis, while
proliferation in non-MLL-rearranged cells is less affected.15
Another recent study confirmed these findings in slightly
different cell types.16 Importantly, EPZ004777 and EPZ-5676 are
able to exert their function in vivo in mice and rats, respectively.
Administration of EPZ004777 leads to a modestly increased
survival in a mouse xenograft model of MLL-rearranged
leukemia.15,16 These results seem encouraging for the use of
DOT1L inhibitors as a treatment for MLL-rearranged leukemia.
However, when healthy mice were treated with EPZ004777 for 2
weeks, a mild-to-moderate decrease of several hematopoietic
progenitor cell populations could be detected, indicating that
some hematopoietic side effects occur.15 Additionally, the
pharmacokinetic properties of EPZ004777 are not ideal for
in vivo application, diminishing its potential as a treatment for
MLL-rearranged leukemia. Nonetheless, EPZ-5676 demonstrates
improved pharmacokinetic properties, and complete tumor
regression was observed in a rat xenograft model.17 Moreover,
most of the tumors showed little to no re-growth for over 30 days,
following a treatment period of 14–21 days, suggesting that tumor
regression was sustained. In addition to its improved pharmacokinetic properties, EPZ-5676 seems to have the same, or better,
features for treating leukemia as EPZ004777, making it a
promising new drug.
Effects of DOT1L small-molecule inhibitors on normal
hematopoiesis
Although initial results with DOT1L inhibitors as a treatment for
leukemia are encouraging, some points warrant further investigation. For example, the average white blood cell count significantly
increased in healthy mice treated with EPZ004777 compared with
untreated mice. The observed increase in the number of
neutrophils, monocytes and lymphocytes points to a stimulating
effect on cell numbers in the myeloid and lymphoid hematopoietic lineages.15 This observation is opposite of the finding that
general hematopoietic cell levels go down upon DOT1L
depletion.27,46 Indeed, Daigle et al. mention that the origin of
this effect is unclear.15 Perhaps the increase in myeloid cells can
be explained by the fact that knockout of Dot1L stimulates the
GATA2/PU.1 differentiation switch to be continuously on myeloid
differentiation.37 It would be interesting to measure the levels of
these two transcription factors upon treatment with DOT1L
inhibitors to see whether this effect, which has so far only been
described in embryos, also occurs in adult mice.
Although Dot1L knockout mice die of anemia and hypocellularity in the bone marrow, healthy mice seem to tolerate
treatment with EPZ004777 well, despite some negative effects on
the hematopoietic system.15,27 Several factors could contribute to
these different outcomes. For example, inhibition of DOT1L may
selectively inhibit proliferation of MLL-rearranged leukemia cells
in vitro and in vivo.15,17 It is known that cancer cells are often
dependent on different factors than healthy cells,49 which could
explain why MLL-rearranged leukemia cells are more sensitive to
inhibition of DOT1L. Another possible explanation may be that
EPZ004777 and EPZ-5676 cause incomplete inhibition of DOT1L
and therefore result in incomplete removal of H3K79 methylation,
leading to smaller effects on hematopoiesis than in knockout
mouse models. Indeed, residual H3K79me2 is seen by western
blotting upon treatment with EPZ004777.15 It is also possible that
DOT1L harbors a methyltransferase-independent function, which
would allow for a less severe phenotype in inhibitor-based versus
knockout approaches, in which expression of the DOT1L protein is
reduced or eliminated. For example, yeast Dot1 induces chromatin
rearrangements through mechanisms independent of histone
Leukemia (2014) 2131 – 2138
methylation,50 and mouse DOT1L affects the formation of
pericentromeric heterochromatin and association with RNA
polymerase II, independent of its methyltransferase activity.51,52
It will be interesting to see whether corresponding concentrations
of EPZ004777 or EPZ-5676 in humans inhibit DOT1L function
enough to treat leukemia but not so much as to result in
hematopoietic side effects. EPZ-5676 is currently in clinical trials,
so these data are forthcoming.
DOT1L–H3K79 METHYLATION: MECHANISMS OF ACTION
Although DOT1L has a role in a variety of developmental
processes, the mechanism by which DOT1L acts in each of these
systems is largely unknown. For example, H3K79 methylation and
DOT1L occupancy show very strong correlations with gene
transcription rate in fly and mammals.10,52,53 However, in
mouse adrenal cells, DOT1L and H3K79 methylation have been
linked to transcriptional repression.54 Furthermore, studies
in Caenorhabditis elegans suggest that DOT1/H3K79 methylation
may promote RNA polymerase pausing.55 Hence, we have much
to learn about the consequences of DOT1 binding at chromatin.
One major limitation to this mechanistic understanding is that the
molecular signal that the H3K79 methyl mark elicits is poorly
understood. H3K79 methylation may act by affecting the structure
of the nucleosome surface directly,56 or it may act through
additional ‘reader’ proteins that recognize the H3K79 methyl mark
and produce downstream effects. One recently reported example
of a DOT1L reader is the survival motor neuron (SMN) protein.
SMN recognizes and interacts with methylated H3K79 in HeLa
cells.57 SMN is needed for the health and survival of motor
neurons, and deficient SMN protein levels leads to spinal muscular
atrophy.58 The finding that the SMN protein recognizes
methylated H3K79 indicates that there may be an epigenetic
dimension to spinal muscular atrophy and that DOT1L may be
important for motor neuron function.57 Future research should
determine the specific interplay and whether this function of
DOT1L is confirmed in vivo. Several other key questions need
further attention. For example, very little is known about how
DOT1L activity is regulated. It is also unknown whether and how
H3K79me can be removed; does it involve demethylases or other
mechanisms such as histone–protein turnover? Finally, DOT1L
may act on multiple substrates. A recent study showed that
human DOT1L methylates the androgen receptor and thereby
regulates its activity on chromatin.59
A possible mechanism through which DOT1L influences
development is canonical Wnt signaling, an important developmental signaling pathway. Knockdown of the DOT1L ortholog,
GPP, in Drosophila causes reduced expression of several Wnt
target genes, including senseless, suggesting that GPP is required
for the expression of target genes that require a high level of Wnt
signaling.60 This result was confirmed by two additional studies. In
one study, expression of several Wnt target genes was decreased
in a mouse chondrocyte cell line in which DOT1L was knocked
down through shmiRNAs, as compared with the control.42 A
second study found that morpholino-mediated knockdown of
DOT1L in zebrafish caused reduced expression of Wnt target
genes in vivo.61 However, in all three studies only a select group of
Wnt target genes was examined, so the extent to which Wnt
signaling is affected by loss of DOT1L is still unknown. Also, these
studies were not conducted in native mammalian tissues, so it is
possible that conclusions drawn from these experiments may not
be indicative of the involvement of DOT1L in Wnt signaling in
mammalian development. Indeed, a recent study showed that the
mRNA levels of Wnt target genes were not affected in Dot1L
knockout mouse crypt epithelium cells, suggesting that DOT1L is
not essential for Wnt signaling.29 These contradictory results could
reflect differences in methods used or could indicate that the role
of DOT1L in Wnt signaling is cell type specific.
& 2014 Macmillan Publishers Limited
DOT1L in leukemia and normal development
CM McLean et al
2137
It is likely that DOT1L has many more functions in development
than those discussed here. For instance, DOT1L appears to have a
role in metamorphosis in the frog species Xenopus tropicalis.62 As
Xenopus metamorphosis is thought to be a good model system to
study mammalian postembryonic development,63 Xenopus may
prove a valuable model to further investigate the functions of
DOT1L in development. Also, in Drosophila, a model system widely
used to study DOT1L, a new function of DOT1L was found. A
partial loss-of-function mutation of GPP, the fly ortholog of DOT1L,
led to decreased lifespan, indicating that GPP is needed for normal
lifespan in Drosophila.64 It is currently unknown whether DOT1L
has a similar function in mammals and is worth investigating using
the existing inhibitors and mouse models.
CONCLUDING REMARKS
The DOT1L protein is a conserved epigenetic writer responsible for
placing methyl marks on H3K79 and is ubiquitously expressed. As
such, DOT1L is expected to have numerous roles in development.
Indeed, in general embryogenesis, DOT1L is important for
proliferation of mESCs, as well as lung cancer cells. Dot1L
knockout mouse embryos are embryonic lethal and show a
variety of developmental deficiencies. Although this is strong
evidence for an essential role for DOT1L in general embryogenesis, the underlying mechanisms remain elusive. In addition to its
role in general embryonic development, specific functions of
DOT1L have been established. For instance, DOT1L has an
essential role in chondrogenesis, cardiac development and
postnatal cardiac function in mice and possibly in humans. DOT1L
also has an important function in induced reprogramming and is
associated with natural germline reprogramming after fertilization.
However, similar to general embryogenesis, the underlying
mechanisms remain unclear here as well. Highly relevant to
leukemia, DOT1L has an important function in hematopoiesis.
However, studies using small-molecule inhibitors to block DOT1L
function show that DOT1L inhibition does not cause obvious
hematopoietic defects. Further research, such as the ongoing
clinical trials with EPZ-5676, is required to establish
whether DOT1L is a suitable target for treating MLL-rearranged
leukemia. A full understanding of the biological functions of
DOT1L will also require understanding the molecular consequences of writing a small methyl mark on the surface of the
nucleosome core.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
We thank the members of the van Leeuwen lab for discussions and Heinz Jacobs for
critical reading of the manuscript. CMM and FvL were supported by the Dutch Cancer
Society (KWF 2009-4511).
AUTHOR CONTRIBUTIONS
All authors have contributed to writing the manuscript. All authors have read
and approved the final version of the manuscript.
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