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 & 2014 Macmillan Publishers Limited

DOT1L in leukemia and normal development CM McLean et al

2133 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 Leukemia (2014) 2131 – 2138

DOT1L in leukemia and normal development CM McLean et al

2134 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

& 2014 Macmillan Publishers Limited

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2135 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 Leukemia (2014) 2131 – 2138

DOT1L in leukemia and normal development 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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The emerging roles of DOT1L in leukemia and normal development.

Methylation of lysines within histone proteins represents a posttranslational modification system that can have profound effects on gene expression. A...
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