The Ten-Eleven Translocation-2 (TET2) Gene in Hematopoiesis and Hematopoietic Diseases

SPOTLIGHT REVIEW The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases

E Solary1,2,3, OA Bernard1,3,4, A Tefferi5, F Fuks6 and W Vainchenker1,2,3

Ten-Eleven Translocation-2 (TET2) inactivation through loss-of-function mutation, deletion and IDH1/2 (Isocitrate Dehydrogenase 1 and 2) gene mutation is a common event in myeloid and lymphoid malignancies. TET2 gene mutations similar to those observed in myeloid and lymphoid malignancies also accumulate with age in otherwise healthy subjects with clonal hematopoiesis. TET2 is one of the three proteins of the TET (Ten-Eleven Translocation) family, which are evolutionarily conserved dioxygenases that catalyze the conversion of 5-methyl-cytosine (5-mC) to 5-hydroxymethyl-cytosine (5-hmC) and promote DNA demethylation. TET dioxygenases require 2-oxoglutarate, oxygen and Fe(II) for their activity, which is enhanced in the presence of ascorbic acid. TET2 is the most expressed TET gene in the hematopoietic tissue, especially in hematopoietic stem cells. In addition to their hydroxylase activity, TET proteins recruit the O-linked b-D-N-acetylglucosamine (O-GlcNAc) transferase (OGT) enzyme to chromatin, which promotes post-transcriptional modifications of histones and facilitates gene expression. The TET2 level is regulated by interaction with IDAX, originating from TET2 gene fission during evolution, and by the microRNA miR-22. TET2 has pleiotropic roles during hematopoiesis, including stem-cell self-renewal, lineage commitment and terminal differentiation of monocytes. Analysis of Tet2 knockout mice, which are viable and fertile, demonstrated that Tet2 functions as a tumor suppressor whose haploinsufficiency initiates myeloid and lymphoid transformations. This review summarizes the recently identified TET2 physiological and pathological functions and discusses how this knowledge influences our therapeutic approaches in hematological malignancies and possibly other tumor types.

Leukemia (2014) 28, 485–496; doi:10.1038/leu.2013.337 Keywords: TET2; epigenetics; DNA methylation; dioxygenase; stem cell; differentiation

INTRODUCTION zinc-finger domain and a carboxy-terminal catalytic Fe(II)- and Five years ago, the demonstration that monoallelic or biallelic loss- a-ketoglutarate (a-KG)-dependent dioxygenase domain inserted of-function mutations and deletions recurrently target the TET2 in a cystein-rich domain. In jawed vertebrates, the TET genes (Ten-Eleven Translocation 2) gene in myeloid malignancies,1,2 and underwent triplication. TET1 and TET3 have also a CXXC domain, the simultaneous demonstration that proteins of the TET family whereas a chromosomal inversion during vertebrate evolution have a key role in the conversion of 5-methyl-cytosine (5-mC) to split the third TET gene into distinct segments encoding the 5-hydroxymethyl-cytosine (5-hmC),3 have opened a large field of catalytic domain (the TET2 gene) and the DNA-binding CXXC investigation, both in basic science and clinics. The present review domain (the CXXC4/IDAX gene). The latter is transcribed in the opposite direction and the protein exerts a regulatory function on summarizes our current understanding of the protein functions SPOTLIGHT 6 and regulation and discusses the significance of its deregulation in TET2 level expression. The CXXC motif may be responsible for 6,7 hematological malignancies as well as potential therapeutic direct (TET1 and TET3) or indirect (TET2) DNA binding. Different opportunities that emerge from these studies. TET enzymes exhibit distinct expression patterns in vivo, with TET1 being mainly expressed in embryonic stem cells. TET2 and TET3 are more ubiquitous, with TET2 expression predominating in a variety of differentiated tissues, especially in hematopoietic and THE TET FAMILY OF ENZYMES neuronal lineages.3 The three enzymes of the TET family (TET1, TET2 and TET3) identified in humans are evolutionarily conserved dioxygenases (Figure 1a). The TET1 gene was initially described as a fusion partner of the MLL (myeloid/lymphoid or mixed lineage leukemia) a-KG DEPENDENCY OF TET ENZYMES LINKS METABOLISM TO gene in an acute myeloid leukemia (AML) with a t(10;11)(q22;q23) EPIGENETICS translocation, with TET2 and TET3 being identified by homology TET enzymes are one of the homeostatic links between searches.4,5 It appeared that most animals had a single TET intracellular metabolism and epigenetic gene regulation.8 Like a orthologue, characterized by an amino-terminal CXXC-type number of chromatin-modifying enzymes, such as the JmjC

1Hematology Department, Gustave Roussy, Villejuif, France; 2Inserm UMR1009, Gustave Roussy, Villejuif cedex, France; 3Faculty of Medicine, University Paris-Sud, Le Kremlin- Biceˆtre, France; 4Inserm UMR985, Gustave Roussy, Villejuif, France; 5Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA and 6Faculty of Medicine, Laboratory of Cancer Epigenetics, Universite´ Libre de Bruxelles, Brussels, Belgium. Correspondence: Professor E Solary, Inserm UMR1009, Gustave Roussy, 114 rue Edouard Vaillant, 94805 Villejuif cedex, France. E-mail: [email protected] Received 11 October 2013; accepted 14 October 2013; accepted article preview online 13 November 2013; advance online publication, 6 December 2013 The TET2 gene in hematopoiesis and hematopoietic diseases E Solary et al 486 a 1 1412/1589 1620/2138 TET1

1 28/1299 1322/2002 TET2

1 689/859 882/1660 TET3

b Chromosome 4q24

106,067,032 TET2 106,200,960 106,389,463 IDAX 105,416,058 c Exon 3 4 5 6 7 8 9 10 11

Figure 1. TET proteins and the TET2 gene. (a) Primary structure of TET proteins. The CXXC domain of TET1 and TET3 is indicated in red, the cystein-rich domains of the three proteins are in gray and the double-stranded b-helix 2-oxoglutarate and Fe(II)-dependent dioxygenase

SPOTLIGHT domains are in blue. Each of these proteins contains three Fer-binding domains and one site for 2-oxoglutarate binding in the dioxygenase domain (not shown). (b) The IDAX gene, also known as CXXC4, originates from the ancestral TET2 gene ﬁssion during vertebrate evolution and is transcribed in the opposite direction. (c) Five distinct models of Tet2 deletion in the mouse have been established. Each arrow indicates the targeted part of the gene in these models (the mouse phenotypes are described in Table 2).

domain-containing histone demethylases, TET dioxygenases example, in mouse embryonic stem cells (ESCs), there are 45 require a-KG (also known as 2-oxoglutarate), oxygen and Fe(II) 5-hmCs per 1000 5-mC.14,15 for their activity, which is enhanced in the presence of ascorbic 5-hmC is a dynamic epigenetic state of DNA and the conversion acid.9,10 Fe(II), 2OG-dependent dioxygenases have a common of 5-mC into 5-hmC initiates demethylation that can occur in structural platform.11 Exons 7–11 of the TET2 gene encode a core several ways16,17 (Figure 3). First, because 5-hmC may not be made of eight anti-parallel b-strands folded into a ‘Jelly-roll’ motif recognized by DNA methyltransferase 1, the oxidation of 5-mC that harbors the active site. The so-called ‘2-His-1-carboxylate into 5-hmC may favor a passive demethylation that is DNA triad’, made of three residues in the active site (two histidine and replication-dependent.17 Secondly, 5-hmC could be converted by one aspartate or glutamate residues), forms a Fe(II)-binding the activation-induced deaminase/apolipoprotein B mRNA-editing platform. The Fe(II) metal center, locked in this triad, binds enzyme complex family of cytosine demethylases into 2-oxoglutarate and O2 on the other side. a-KG, which can be 5-hydroxymethyluracil (5-hmU), to be repaired by DNA glycosylases derived from several sources including isocitrate and glutamic and the base-excision repair pathway.18,19 The physiological acid, is decarboxylated to succinate during the oxidation reaction.8 importance of this second pathway in mammal cells remains Somatic mutations in isocitrate dehydrogenase (IDH) enzymes, controversial. Third, iterative oxidation of 5-mC and 5-hmC by TET either cytosolic IDH1 or mitochondrial IDH2, which are observed enzymes generate 5-formylcytosine and 5-carboxylcytosine, which in various tumors including myeloid malignancies,12 unmask a are recognized and excised by thymine DNA glycosylase into an latent ability of these enzymes to produce the R enantiomer abasic site. Subsequent repair by the base-excision repair pathway of 2-hydroxyglutarate, an ‘oncometabolite’ that inhibits restores an unmodified cytosine.19–22 2-oxoglutarate-dependent enzymes, including TET dioxygenases The dynamic methylation/demethylation cycle that involves TET (Figure 2). Potentially, TET enzymes may be also sensitive to and thymine DNA glycosylase enzymes (TET/thymine DNA changes in oxygen availability and susceptible to reactive oxygen glycosylase cycle) was shown recently to occur at a large number species and carcinogenic metals that displace iron such as arsenic, of genomic loci across the genome.23 Interestingly, long-lived nickel or chromium.13 5-hmC, and to a lesser extent, short-lived 5-formylcytosine and 5-carboxylcytosine may have also DNA demethylation- independent functions and serve as stable epigenetic marks that recruit specific readers, DNA repair proteins and transcription TET ENZYMES PROMOTE DNA DEMETHYLATION factors, with limited overlap between these proteins.24–27 Methylation at carbon atom 5 of the nucleotide cytosine In addition, most 5-mC-binding proteins do not recognize (5-methyl-cytosine or 5-mC), which is the predominant epigenetic 5-hmC and thereby presumably dissociate from DNA when modification of DNA, and the reverse DNA demethylation process, 5-mC is converted into 5-hmC.27 Thus, TET proteins may be have a profound impact on gene expression. The mechanism of important in fine tuning epigenetic status of the cells. cytosine methylation by DNA methyltransferases has been established for a long time. Cytosine demethylation remained enigmatic until identification of TET enzyme functions. These TET-MEDIATED HISTONE MODIFICATIONS THROUGH enzymes modify the methylation status of DNA and regulate gene INTERACTION WITH OGT transcription by catalyzing the oxidation of the 5-methyl group on Another mechanism that may account for TET-mediated gene 5-mC to create 5-hydroxymethyl-cytosine (5-hmC). Of note, 5-hmC activation is the recruitment of O-linked b-D-N-acetylglucosamine levels across the genome remain low as compared with 5-mC—for (O-GlcNAc) transferase (OGT) to chromatin. OGT is the enzyme

Leukemia (2014) 485 – 496 & 2014 Macmillan Publishers Limited The TET2 gene in hematopoiesis and hematopoietic diseases E Solary et al 487 Oxaloacétate

Malate Citrate

Fumarate Hydratase TCA cycle Isocitrate Fumarate Isocitrate Succinate . deshydrogenase deshydrogenase 2-0G-dependent enzymes Succinate 2-oxo-glutarate DNA demethylases (TETs) Histone lysine demethylases (KDMs) Prolyl Hydroxylases (PHDs) Succinyl-CoA JmjC-HDs Figure 2. The TCA cycle and alpha-ketoglutarate. a-ketoglutarate (also known as 2-oxoglutarate) is a product of the TCA cycle that, together with oxygen and Fe(II), is requested for a number of dioxygenases. Alpha-ketoglutarate dependency of TET enzymes links metabolism to epigenetics. Mutation in IDH1 or IDH2 enzymes, and possibly mutations in succinate dehydrogenase and fumarate hydratase, leads to cellular accumulation of TCA cycle intermediates with structural similarity with 2-oxoglutarate (in the case of IDH mutations, the R enantiomer of 2-hydroxyglutarate).

5-hydroxymethylcytosine Cytosine DNMT 5 methylcytosine TET

AID APOBEC

TDG TET 5-hydroxymethyluracil (5-hmU),

5-carboxylcytosine TET 5-formylcytosine

b NH2 NH2 NH2 NH2 NH2

HOH C HOOC H3C 2 OHC N

N DNMTN TETN TETN TET SPOTLIGHT 5-mC 5-mC 5-hmC 5-fC 5-caC

O O O O O N N N N N

DNA DNA DNA DNA DNA

Figure 3. The 5-hmC cycle. (a) Methylation at carbon atom 5 of the nucleotide cytosine (5-mC) is mediated by DNA methyltransferases (DNMT). TET enzymes catalyze the oxidation of the 5-methyl group on 5-mC to create 5-hmC. A passive, DNA replication-dependent demethylation can occur. 5-hmC could be converted by the activation-induced deaminase/apolipoprotein B mRNA-editing enzyme complex family of cytosine demethylases into 5-hydroxymethyluracil to be repaired by DNA glycosylases and the base-excision repair (BER) pathway. Moreover, iterative oxidation of 5-mC and 5-hmC by TET enzymes can generate 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), which are recognized and excised by thymine DNA glycosylase (TDG). (b) Iterative oxidation of 5-mC and 5-hmC by TET enzymes. that catalyzes the addition of O-GlcNAc to Ser and Thr residues of complex and an epigenetic cell cycle regulator. O-GlcNAcylation proteins. The glycosyltransferase reaction mediated by OGT and also regulates the function of the Polycomb and Trithorax the removal of O-GlcNAc from substrates mediated by O-GlcNA- complexes, interacts with the machineries involved in case define the O-GlcNAc cycling. This process can influence the methylation, acetylation and ubiquitylation of the four core epigenetic control of gene expression in several ways.28 histones, and modifies RNA polymerase II. Similar to TET O-GlcNAcylation affects the proteolysis of host cell factor 1, activation by 2-oxoglutarate, O-GlcNAc cycling links cell a component of the H3K4 methyltransferase SET1/COMPASS metabolism to higher-order chromatin organization.

& 2014 Macmillan Publishers Limited Leukemia (2014) 485 – 496 The TET2 gene in hematopoiesis and hematopoietic diseases E Solary et al 488 All TET proteins might interact with OGT.29–31 For example, differentiation, neoplastic transformation and environmental OGT interacts with the catalytic domain of TET2; depletion of TET2 exposure. Given the role of IDAX in the regulation of the Wnt from embryonic stem cells prevents the association of OGT with pathways,34 this also suggests a link between paracrine signals chromatin; overexpression of TET2 in human cells increases the and DNA modification. level of chromatin-bound OGT; and a mutant OGT that cannot bind TET2 is not present in the chromatin fraction. TET proteins and OGT show genome-wide colocalization, especially around OTHER POTENTIAL REGULATORS OF TET FUNCTIONS transcription start sites.29–31 In addition to OGT and IDAX, TET proteins could interact with early OGT does not influence TET2 hydroxylase activity. TET2 recruits B-cell factor 1 that binds DNA to control its local demethylation. OGT to chromatin and promotes OGT activity, an effect that is This interaction, which was identified by cross analysis of independent of TET2 enzymatic activity (Figure 4). The interaction methylation profiles from four series of IDH-mutated cancers of TET2 with OGT enhances the O-GlcNAcylation of Histone 2B at including AML, was confirmed by chromatin immunoprecipitation Ser112, and the genes that are associated with TET2 and OGT and and immunoblotting;35 however, its functional significance display O-GlcNAcylated Ser112 of Histone 2B show high levels of remains unclear. This is also the case for the TET protein transcription. TET2 might help OGT to recognize chromatin interaction with activation-induced deaminase that could affect substrate, and OGT-mediated O-GlcNAcylation of histone Histone their subcellular distribution.36 A systematic proteomic screen 2B might contribute to TET2-dependent gene regulation.30 recently identified the ubiquitin ligase Uhrf2 (ubiquitin-like with TET2 protein and OGT activity also promote O-GlcNAcylation PHD and ring-finger domains 2) as a protein that promotes the of host cell factor 1 (HCF1), which is important for the integrity of iterative oxidation of 5-mC by TET enzymes in mouse neuronal SETD1A, which is the SET1/COMPASS complex, which is the main progenitor cells27 (Table 1). Additional partners of TET proteins H3K4 methyltransferase complex. TET2-OGT colocalizes on chro- have been identified, and functional analysis of these interactions matin at active promoters enriched for H3K4me3, and reduction of may provide additional insights in their functions. either TET2/3 or OGT activity results in a decrease in H3K4me3 and concomitant decrease in transcription—that is, global GlcNAcyla- / VITAMIN C ENHANCES THE ACTIVITY OF TET ENZYMES SPOTLIGHT tion and H3K4me3 are reduced in bone marrow cells of tet2 mice, notably at several key regulators of hematopoiesis.29 Another cofactor of TET enzymes is vitamin C, or ascorbic acid, a vital nutrient that stimulates the activity of all Fe(II)2-oxoglutarate dioxygenase enzymes. Vitamin C can interact with the C-terminal, catalytic domain of TET enzymes, which may promote their PROTEOLYTIC REGULATION OF TET2 EXPRESSION folding or recycling of the Fe(2 þ ).9 In mouse embryonic stem Whereas interaction of OGT with TET2 does not affect the 5-hmC cells, vitamin C promotes a rapid and global increase in 5-hmC, level, IDAX—also known as CXXC4 and originating from the followed by the DNA demethylation of many gene promoters.10 ancestral TET2 gene fission—interacts with the catalytic domain of The vitamin-C-induced changes in 5-hmC and 5-mC are TET- TET2 and was proposed to promote the degradation of the dependant, as they are entirely suppressed in Tet1 and Tet2 protein, bound to genomic DNA, by caspases. According to this double-knockout ESCs. Regions that are resistant to vitamin-C- study,6 IDAX binds DNA sequences enriched in unmethylated CpG mediated demethylation show high levels of H3K9me3, dinucleotides in gene promoters through its CXXC domain, which suggesting that this mark, or readers of this mark, may have a activates caspases and results in TET2 protein cleavage and role in protecting against Tet-mediated demethylation. degradation, without directly affecting the enzymatic activity of Altogether, vitamin C may be a direct regulator of TET activity the dioxygenase. The TET3 expression level could be regulated in and DNA methylation fidelity. a similar way through its internal CXXC domain, uncovering a general autoregulation of TET function by extrinsic and intrinsic CXXC protein domains.6 CXXC5 (also known as RINF), a related AN MIR-22-DEPENDENT REGULATION OF TET2 protein that was involved in normal myeloid differentiation and Another recently identified level of regulation of TET2 gene myeloid malignancies,32 could also promote TET2 degradation. expression involves the microRNA miR-22.37 Alterations of this Further studies will indicate whether the altered expression of miR-22/TET2 regulatory network could be common and either IDAX33 or CXXC532 observed in tumor cells affects the TET2 oncogenic in hematopoietic malignancies such as expression level and the epigenetic control of gene expression. myelodysplastic syndromes (MDS, 35%) and AML (22%). This mode of regulation may explain the observed tissue-specific Transgenic mice conditionally expressing miR-22 in the differences in 5-hmC levels and dynamic changes observed upon hematopoietic compartment in order to reproduce the overexpression observed in MDS and leukemia display reduced levels of global 5-hmC and increased hematopoietic stem-cell (HSC) self-renewal accompanied by defective differentiation, leading over time to myeloid dysplasia and hematological HCF1 malignancies. Ectopic expression of TET2 corrects the miR-22- induced phenotype, whereas miR-22 inhibition blocks SET1/ proliferation in both mouse and human leukemic cells.37 COMPASS According to these recent data, miR-22 may be a potent OGT TET2 regulator of HSC maintenance and self-renewal through the H3K4me3 H2B ser 112 O-GlcNac modulation of TET2 expression level, and presumably other important targets. Figure 4. TET-mediated histone modifications through interaction with OGT. All TET proteins might interact with OGT. TET2 was shown to recruit OGT to chromatin, an effect that is independent of its EMBRYONIC STEM CELLS EXPRESS TET1 AND TET2 enzymatic activity. So far, the interaction of TET2 with OGT was shown to enhance the O-GlcNAcylation of Histone 2B (H2B) at The expression of pluripotency factors and the repression of Ser112 and to promote O-GlcNAcylation of HCF1, which is lineage differentiation genes in ESCs involve epigenetic mechan- important for the integrity of the SET1/COMPASS complex, a H3K4 isms, including DNA methylation and histone modifications.38 Tet1 methyltransferase. and Tet2 are expressed in mouse ESCs39 and their expression

Leukemia (2014) 485 – 496 & 2014 Macmillan Publishers Limited The TET2 gene in hematopoiesis and hematopoietic diseases E Solary et al 489 Table 1. Currently identiﬁed TET2 interacting molecules

Interactors or regulators Function, alterations Reference

Fe(II) Promotes the function of all Fe(II), 2-oxoglutarate-dependent dioxigenases 14 Oxygen Promotes the function of all Fe(II), 2-oxoglutarate-dependent dioxigenases 13 2 oxoglutarate (a-ketoglutarate) Promotes the function of TET enzymes. 2-hydroxyglutarate is a competitive inhibitor 8 0GT TET2 recruits OGT to chromatin. In turn, OGT promotes He´ K4me3 and gene transcription 29–31 IDAX (CXXC4) Binds TET2 and DNA and promotes the degradation of TET2 by caspases 6 EBF1 Identified in a systematic screen, unknown functions 35 AID Could affect the cellular distribution of TET enzymes 36 Uhrf2 Promotes the iterative oxidation of 5-mC by TET enzymes in neuronal cells 27 Vitamin C Promotes the function of all Fe(II), 2-oxoglutarate-dependent dioxigenases 9,10 miR-22 Negatively regulates Tet2 gene expression at the pre-mRNA level 37 Abbreviations: AID, Activation-induced cytidine deaminase; 5-mC, 5-methyl-cytosine; CXXC4, CXXC finger protein 4; EBF1, early B-cell factor 1; IDAX, inhibition of the Dvl and Axin complex; OGT, O-linked b-D-N-acetylglucosamine (O-GlcNAc) transferase; Tet, Ten-Eleven Translocation; Uhrf2, ubiquitin-like with PHD and ring-finger domains 2. decreases upon ESC differentiation, whereas Tet3 is upregulated. The role of TET proteins could be slightly different in human The inverse changes are observed when fibroblasts are ESCs, in which TET1 is highly expressed and TET2 is expressed at a reprogrammed to generate induced-pluripotent stem cells.40,41 very low level. When mammalian somatic cells are reprogrammed Significant levels of 5-hmC have been detected in mouse ESCs, into induced-pluripotent stem cells, the strong induction of TET1 and the 5-hmC level declines during differentiation.39,42 Tet1 is increases 5-hmC levels and provokes some aberrations in probably a major factor in ESCs, as Tet1 depletion in these cells subtelomeric regions that distinguish induced-pluripotent stem decreases the 5-hmC level.21,39,43 Tet1 binds a large number of cells from ESCs.50 genes in these cells, mostly around transcription start signals but also in gene bodies and enhancers. Most Tet1-bound promoters contain a CpG island and are unmethylated. 5-hmC also localizes A LOCUS-SPECIFIC REGULATORY FUNCTION OF TET ENZYMES? to transcription start signals and is enriched in a fraction of CpG Surprisingly, depletion of either Tet1 or Tet2 in ESCs induced islands.38,42–46 Nevertheless, how Tet1 and Tet2 regulate limited changes in 5-hmC levels and DNA methylation.38,39,42–45 pluripotency remains a matter of controversy. The main reason might be that other pathways regulating 5-hmC levels in ESCs partly compensate for the Tet depletion—for example, Tet2 may operate in Tet1-depleted ESCs. Nevertheless, THE ROLE OF TET PROTEINS IN EMBRYONIC CELLS REMAINS Tet1 could regulate DNA methylation levels at certain specific LARGELY ENIGMATIC genes.39,42,43,45,51 Of note, most of the transcriptional activating Tet enzymes were proposed initially to promote the expression of effects of Tet1 were also detected in the Dnmt triple knockout pluripotency genes. Most Tet1-bound promoters in ESCs are cells, suggesting that they could be indirect effects that do not H3K4me3-positive, which, when isolated, is a mark of active depend on the demethylating activity of Tet enzymes.44 transcription. It was proposed that, in promoters of housekeeping Tet1 / ESCs have reduced levels of global 5-hmC and display genes, Tet enzymes act as failsafe mechanisms that prevent a skewing toward trophectoderm in culture, as described with aberrant methylation and maintain CpG islands free of methylation in vitro studies; however, they do not lose pluripotency, express by rapidly converting 5-mC into 5-hmC.45 In promoters of near-physiological levels of Nanog, Oct-4 and Sox2, and are able to pluripotency genes that become DNA-methylated during support organism development.51 Tet1 knockout mice are viable differentiation, Tet binding might ensure a timely methylation and fertile, although they have a smaller body size at birth.51 Tet1 and silencing.47 Multiple links between Tet1, Tet2 and the master and Tet2 double-knockout ESCs have also reduced levels of 5-hmC regulators of ESC pluripotency have been reported; however, the and remain pluripotent. They cause developmental defects in a SPOTLIGHT picture remains confusing.39,42,43 Analysis of Tet1-depleted ESCs fraction of chimeric embryos; however, viable and overtly normal suggested initially that Tet1 could promote the expression of the Tet1/Tet2-deficient mice can be obtained, showing that loss of pluripotency gene Nanog.14 Conversely, Oct-4, another factor both enzymes is compatible with development, pending involved in pluripotency, was shown to regulate Tet1 and Tet2 hypermethylation and compromised imprinting52 Altogether, expressions,48 and Tet1 and Tet2 proteins were shown recently to Tet1, in conjunction with Tet2, probably have a locus-specific interact with Nanog.49 role in shaping the ESC epigenome during subsequent Actually, a number of genes are upregulated after Tet1 development but appear to be dispensable, suggesting that depletion in mouse ESCs, indicating repressive effects on gene other pathways of DNA methylation could possibly exist. transcription.46 A high fraction of the Tet1 and Tet2 target genes are bivalent H3K4me3/H3K27me3-positive genes, and therefore positive for binding of the polycomb repressive complex 2 (PRC2), TET2 OXYGENASE HAS A ROLE AT SEVERAL STEPS OF a mark of gene repression. Tet enzymes could indirectly facilitate HEMATOPOIESIS IN IN VITRO ASSAYS PRC2 chromatin binding by decreasing DNA methylation levels at TET2 has pleiotropic roles in hematopoiesis, including stem-cell PRC2 target genes.43–46 By preventing stable methylation of PRC2 self-renewal, lineage commitment and terminal differentiation of target genes, which are enriched for genes involved in cell-fate specific lineages. The TET2 gene is highly expressed in HSCs and in decisions, Tet enzymes might keep the plasticity of these genes progenitor cells, and is downregulated with differentiation. One of during development. Tet1 could also mediate transcriptional the consequences of Tet2 depletion (or expression of a mutated repression through its association with the Sin3A co-repressor Idh2 whose product affects Tet2 enzymatic activity) in primary complex. The recruitment of Sin3A to a subset of these genes is bone marrow cells is an increase in the percentage of immature dependent on Tet1 expression. This repressive function of Tet1 c-Kit þ , Lin cells and their replating ability, suggesting that Tet2 may be independent of its enzymatic activity.43 silencing could affect stem/progenitor cell differentiation.53,54

& 2014 Macmillan Publishers Limited Leukemia (2014) 485 – 496 The TET2 gene in hematopoiesis and hematopoietic diseases E Solary et al 490 Accordingly, small interfering RNA-mediated depletion of Tet2 was these mutations decrease TET2 enzymatic activity by truncating demonstrated to alter the pattern of transcription of homeotic the protein or affecting its catalytic activity. To determine the role (Hox) genes in pluripotent cell populations.55 of TET2 mutation in hematopoietic malignancies, several teams RNA interference-mediated Tet2 silencing in mouse early simultaneously developed mouse models of Tet2 deletion.54,56,63,64 progenitors56 as in human cord blood CD34 þ cells57 decreases Analyses of these Tet2 knockout mice first enforced the 5-hmC levels and allows expansion of the monocyte lineage. This evidence for a role of Tet2 in the regulation of normal latter phenotype is in line with the frequent mutations of the TET2 hematopoiesis. All three TET family proteins are expressed in gene (B50%) in chronic myelomonocytic leukemia (CMML), hematopoietic progenitors, and the deletion of Tet2, which does a disease defined by the accumulation of monocytes in the not induce an upregulation of Tet1 and Tet3, is sufficient to bone marrow, the peripheral blood and the spleen.58 TET2 decrease 5-hmC content in HSCs. A decrease in LSK content in mutations are also observed in a variety of myeloid and 5-hmC is observed in both Tet2–/– and Tet2 þ / mice. These cells lymphoid malignancies in which monocytosis is not observed. also show an increased ability to self-renew and expand, and they Ex vivo, TET2 depletion in CD34 þ ,CD38 promotes monocyte exhibit a competitive advantage over wild-type Tet2 HSCs for expansion, whereas TET2 depletion in CD34 þ ,CD38 þ cells does repopulating hematopoietic lineages. Tet2 loss could impair the not, suggesting that the level at which TET2-mutated cells expand tissue-specific pattern of the Hoxa gene expression.55 Lastly, the contributes to the disease phenotype, with early clonal deletion of Tet2 allows for amplification of the monocytic dominance of TET2 mutations inducing a monocytosis.59,60 lineage.54,56,63,64 An alternative hypothesis is that the primary effect of TET2 Tet2 deletion is sufficient to initiate myeloid and lymphoid mutations is to induce the expansion of HSCs biased towards transformations. Similar to Tet1-deficient mice, Tet2 knockout mice monocytic differentiation, these mutated cells being targeted by are viable, fertile and develop grossly normally. However, as they secondary genetic events that can modify the disease phenotype. age, Tet2-deficient mice specifically demonstrate an increased TET2 may be required at later-stage myeloid differentiation. susceptibility to myeloid and lymphoid tumors. The onset of these Tet2 depletion impairs CEBPa-induced transdifferentiation of malignancies and the kinetics of their progression partly depend pre-B cells into macrophages; CEBPa couldbindtoupstream on the genetic background of the model (Table 2). In addition, þ / /

SPOTLIGHT regions of Tet2 that, in turn, could activate a subset of myeloid Tet2 mice behave similar to Tet2 cells and develop genes through a rapid increase in their promoter hydroxy diverse hematopoietic malignancies, although with longer latency, methylation.61 Mature monocytes also express TET2, and loss suggesting gene-dosage effects. Thus, TET2 haploinsufficiency, of DNA methylation during the differentiation of primary, which is frequently observed in human hematological malignan- post-proliferative human monocytes into dendritic cells is cies, is sufficient to change the properties of HSCs. preceded by the local appearance of 5-hmC. The small Tet2-deficient mice developed predominantly a CMML-like interfering RNA-mediated knockdown of this enzyme in disease; however, other types of diseases, including MDS and primary monocytes prevents active DNA demethylation, myeloproliferative-like diseases, were observed.54,56,63,64 Tet2 loss suggesting that TET2 is essential for the proper execution of also affects lymphoid-lineage development with the expansion of this process in human monocytes.62 an aberrant (CD19 þ B220low) lymphoid population, a decrease in B-cell lineages in the bone marrow and an increase in CD4 CD8 T-cell progenitors in the thymus.64 The kinetics of disease TET2 IS A TUMOR-SUPPRESSOR GENE induction suggests that additional genetic lesions may Homozygous and heterozygous mutations in the TET2 gene are cooperate with Tet2 loss to induce a disease and influence the recurrent events in human hematopoietic malignancies.1,2 Most of generation of distinct types of blood neoplasms,65 either in the

Table 2. Animal models of Tet2 deletions

Quivoron et al.64 Moran-Cruzio et al.54 Ko et al.6 Li et al.63

Modelsa Exon 9 (gene trap). Exons Exon 3 (conditional Exons 8–10 Tet2 disruption 6 bp 10–11 (conditional deletion) (conditional upstream of the deletion) deletion) transcription start Reproduction Expected Mendelian Expected Expected Expected Mendelian ratios ratios Mendelian ratios Mendelian ratios Development Normal growth and organ Normal growth and Normal growth Normal growth and organ development organ development and organ development development Tet1, Tet2 levels Unchanged Unchanged Unchanged Unchanged 5-hmC levels Decreased Decreased Decreased Decreased Bone marrow Lin-,c-Kit þ Increased, serial Increased Increased replating Bone marrow LSK CD150 þ (CD48 ) Increased Increased Increased Increased Bone marrow progenitors Increased CMP, MEP Increased CMP Increased CMP Increased GMP Lymphocyte lineages Altered, T and B Unchanged Unchanged Extramedullary hematopoiesis Spleen, Liver Spleen, 20 weeks Spleen 12 weeks Spleen, 2–4 months Repopulation capability in vivo Increased Increased Increased Increased Diseases CMML-like CMML-like (70%) Accumulation of CMML-like and spectrum of CD115 þ , F4/80 þ myeloid malignancies cells Disease tranplantability Yes Yes Yes Abbreviations: CMML, chronic myelomonocytic leukemia; CMP, common myeloid progenitor; LSK, lineage-negative, Sca1-positive, c-kit-negative cells; MEP, megakaryocytic erythroid progenitor; Tet, Ten-Eleven Translocation. Five independent animal models of Tet2 deletion demonstrated very similar features, except for lymphocyte lineage abnormalities. aSee Figure 1b.

Leukemia (2014) 485 – 496 & 2014 Macmillan Publishers Limited The TET2 gene in hematopoiesis and hematopoietic diseases E Solary et al 491 myeloid or in the lymphoid lineage. It is appealing to assume that TET2 mutations are often the first detected oncogenic event that the loss of a TET protein could contribute to the gene- in this disease.59,75,76 The age-dependent decrease in global specific hypermethylation that is often observed in cancer. 5-hmC levels observed in myeloid cells suggests that it may be an Aberrant methylation of CpG islands in specific gene promoters important feature in the aging hematopoietic system. The potential has been associated with the development of hematopoietic risk of 5-hmC decrease and TET2 mutations now deserves to be malignancies—for example, the silencing of p15/INK4B cell cycle tested prospectively in large cohorts of aging individuals in order to regulator is observed in one-third of MDS and that of the Tif1g detect acquired states that predispose to leukemogenesis. (transcription intermediary factor 1 gamma) gene in 35% of The working hypothesis is that these mutations may increase the human CMML.66 However, the abnormal methylation of the HSC fitness whose transformation will be fixed by the accumulation p15/INK4B or Tif1g promoter does not strictly associate with TET2 of additional genetic or epigenetic alterations. mutations in human diseases. Either the loss of TET2 activity is not responsible for the accumulation of DNA methylation at these specific promoters, or TET protein functions can be altered by TET2 MUTATIONS IN MYELOID MALIGNANCIES mechanisms other than mutations, or aberrant DNA methylation at CpG islands are stochastically generated by TET mutants and Somatic alterations in TET2, including deletions and missense, nonsense and frameshift mutations, were identified initially in occasionally provide cells with a growth advantage, leading to 1 1,2 clonal expansion of the cells and, in combination with other 10–26% of MPN and MDS patients. These TET2 gene alterations genomic alterations, to disease development. were rapidly shown to result in a marked reduction in global levels of 5-hmC, indicating that TET2 function was altered.15 TET2-mutated MPN samples also exhibited improved engraftment,1 TET2 ALTERATIONS IN THE ABSENCE OF MUTATION consistent with the improved function of Tet2-deleted murine 54,56,63,64 As indicated above, IDH 1 or IDH2 mutant enzymes, identified in HSPCs. The TET2 gene has been subsequently sequenced in some myeloid malignancies such as AML and CMML, induce series of patients with different subtypes of myeloid neoplasm to carboxylation of glutamine-derived a-KG, with a concomitant delineate the frequency of TET2 mutations (Table 3). Two distinct increase in synthesis of the R enantiomer of 2-hydroxyglutarate.67 TET2 mutations were identified in a number of cases—for example, B 77 This metabolite inhibits a-KG-dependent dixoygenases, with, in 25% of TET2-mutated MDS— indicating that biallelic loss of in the case of TET2, a decrease in the cellular 5-hmC level. TET2 is frequently part of the disease evolution. Importantly, in addition to inhibiting TET enzymes, R enantiomer Acquired somatic alterations in TET2 were identified in 2–20% of classical MPNs, including polycythemia vera, essential thrombo- of 2-hydroxyglutarate is a competitive inhibitor for several other 78,79 2OG-dependent dioxygenases, including histone lysine cytosis and primary myelofibrosis. These mutations, which can demethylases and prolyl hydroxylases such as EGLN.68 be an early genetic event in the MPN course, have no clear Theoritically, TET dioxygenases could be altered also by mutations in other key enzymes of the TCA cycle, mainly succinate dehydrogenase (SDH) and fumarate hydratase, leading Table 3. Prevalence of TET2 mutations in healthy donors and in to the cellular accumulation of TCA cycle intermediates with hematopoietic malignancies structural similarity to 2OG8 (Figure 2). The downregulation of TET (or IDH2) enzymes was shown to be the likely mechanism 69,70 Cohorts Prevalence References responsible for the loss of 5-hmC identified in melanoma cells. (%) At least in hematopoietic tissues, the downregulation of TET2 could result from alterations in the miR-22/TET2 regulatory Healthy donors, age 460 years 5.5 71 network.37 Collectively, in addition to mutations, functional Healthy donors, age o60 years 0 71 losses of TET2 because of additional upstream cues could have a critical role in aberrant hematopoiesis and leukemogenesis. Myeloid malignancies Pediatric de novo acute myeloid 4 73 leukemia 12,75,89,90,93 TET2 MUTATIONS ACCUMULATE WITH AGING Adult de novo acute myeloid 12–17 leukemia It had been postulated that the biased hematopoietic differentia- Secondary acute myeloid leukemia 24–32 91,92 SPOTLIGHT tion toward the myeloid lineage could explain the increasing Polycythemia vera and essential 2–10 1,78 incidence of myeloid malignancies observed in aging, otherwise thrombocytosis healthy people.1,2 By sequencing the DNA of neutrophils (PMNs), Primary myelofibrosis 10-20 1,79 82 somatic TET2 mutations, analogous to the inactivating mutations Chronic myeloid leukemia 2 All myelodysplastic syndromes 19–26 1,2,77,83–85,87 observed in patients with myeloid malignancies, were identified in 88 10/182 (5.5%) elderly individuals, and in none of 96 younger Refractory anemia, ring sideroblasts 10–26 and thrombocytosis adults. Clinical follow-up for seven of the TET2 mutant subjects for Chronic myelomonocytic leukemia 50–60 58,83,86,87 X5 years after mutational analysis identified the occurrence of a 70 71 Juveline myelomonocytic leukemia o1 JAK2V617F-positive essential thrombocythemia in one case. Systemic mastocytosis 20–40 94,95 The appearance of TET2 mutation in hematopoietic cells of Blastic plasmacytoid dendritic cell 30 96,97 aging people could account for the absence of TET2 mutation in neoplasms juvenile myelomonocytic leukemia,72 the low prevalence (o5%) of TET2 mutations in pediatric AML73 and the age-associated Lymphoid malignancies 74 Mature B-cell lymphomas 2 64 increase in the prevalence of TET2 mutations in adult AMLs. An 64,107 alternative hypothesis is that a unique mutation is sufficient for Mature T-cell lymphomas 12 Angioimmunoblastic T-cell 33 64,108,109 the emergence of a malignant clone in children hematopoiesis, lymphomas whereas mutations that induce a clonal hematopoiesis are needed Mantle cell lymphomas 4 110 in older subjects for disease expression. Whatever the explanation, Diffuse large B-cell lymphomas 12 111 these observations support an initiating role for TET2 mutation in the pathogenesis of age-associated hematological cancers, also Abbreviations: MPN, myeloproliferative neoplasms; Tet, Ten-Eleven supported by analysis of the clonal architecture in CMML, showing Translocation.

& 2014 Macmillan Publishers Limited Leukemia (2014) 485 – 496 The TET2 gene in hematopoiesis and hematopoietic diseases E Solary et al 492 prognostic impact—that is, they do not increase the risk of or is recruited by transcription factors to catalyze the trimethylation leukemic transformation. In some cases, however, TET2 mutations of lysine 27 of histone H3 (H3K27me3), a prototypical repressive appear only when the disease progresses to an acute phase.80,81 histone mark. Mutations in the H3K27me3 demethylase UTX can In chronic myeloid leukemia, TET2 mutations were associated in also be detected in combination with TET2 alterations.105 Lastly, most cases with acute blastic transformation.82 mutation in ASXL1 (Additional sex combs-like 1), a component of TET2 alteration was also the most prevalent genetic abnormality the Polycomb Repressive Deubiquitinase complex and a recruiter (25–35%) identified in MDS.77,83–85 Larger series failed to identify a of PRC2 to some gene promoters,106 is a recurrent event in strong association of TET2 alteration with clinical phenotype, risk myeloid malignancies in which it is strongly and independently scores or overall survival.77 Nevertheless, in higher risk MDS and associated with a poor clinical outcome (Figure 5).58 TET2 loss-of- AML with low blast count, the TET2 status may predict a better function may synergize with these other gene mutations to have response to the demethylating agent azacitidine.85 As already an impact on the epigenetic landscape and promote malignant indicated, the highest rate of TET2 genetic alterations was transformation or disease progression. observed in CMML in which a heterozygous or homozygous mutation was identified in 50–60% of this overlapping MPN/ MDS.58,86,87 Single-cell-derived colony genotyping identified TET2 TET2 MUTATIONS IN LYMPHOID MALIGNANCIES mutation as one of the earliest event in the accumulation of Two observations led to the identification of TET2 somatic 59,61 genetic alterations that lead to the leukemic clone expansion. mutations in human B and T lymphomas. First, Tet2-deficient TET2 mutations are less frequent in other MPN/MDS, although mice demonstrate an altered T- and B-cell lineage development.64 they can be associated to SF3B1 and JAK2 mutations in refractory Secondly, some patients with a TET2-mutated myeloid malignancy 88 anemia with ring sideroblasts and thrombocytosis. Analysis of develop a lymphoma. TET2 mutations were identified in mostly large cohorts failed to demonstrate any prognostic impact of TET2 mature B-cell (2%) and T-cell (11.9%) lymphomas.67,107,108 58 mutations in CMML. In addition, they were observed in B33% of angioimmunoblastic The prevalence of TET2 mutations is higher in secondary than in T-cell lymphoma, an aggressive neoplasm of CD4 þ 89–93 de novo AMLs. Genetic alterations of TET2 could be associated T lymphocytes.67,109 In this latter disease, TET2 mutations are 108 SPOTLIGHT with adverse outcome in cytogenetically defined subgroups of frequently associated with DNA methyltransferase 3A mutations, 89,93 AML patients, and thus could be integrated to the list of and IDH2 mutations are identified in 20–45% of cases.109 AML/MDS parameters that guide therapeutic choices in this disease. TET2 arising secondary to lymphoma was demonstrated to carry the mutations were identified in B20% of mastocytosis, mostly in same TET2 mutation as the previous lymphoma, indicating a aggressive forms of the disease, with a demonstrated oncogenic common cell of origin. More recently, TET2 mutations were 94,95 cooperation with KITD816V in mast cells. More recently, TET2 identified in mantle cell lymphomas110 and in diffuse large B-cell mutations were found in B30% of patients with a blastic lymphoma (12%) in which they were associated with an altered 96,97 plasmacytoid dendritic cell neoplasm. DNA methylation pattern of genes involved in hematopoietic development111 (Table 1). IDH2 AND IDH1 MUTATIONS COMPLEMENT TET2 MUTATIONS In myeloid malignancies, TET2 mutations are mutually exclusive TET2 ALTERED EXPRESSION IN NON-HEMATOLOGICAL with somatic heterozygous mutations at highly conserved MALIGNANCIES positions in IDH1 (R132) and IDH2 (R140, R172). In addition, IDH1 11 TET2 mutations were detected in a small number of solid and IDH2 mutations are mutually exclusive from one another. tumors—that is, were recently proposed to define a subset of These mutations are observed in 5–20% of AMLs, most commonly metastatic tumors in castration-resistant prostate cancers.112 Most in adult patients with cytogenetically normal AML, and in 5–20% importantly, the levels of 5-hmC are dramatically reduced in a of chronic myeloid malignancies in which their appearance is variety of human solid tumors compared with the matched usually associated with transformation to AML, implying a role for IDH1/2 mutation, which can predate clinical evidence of overt transformation, in the progression to secondary leukemia.98–100 A cell-permeable (R) enantiomer of 2-hydroxyglutarate was shown 101 to affect hematopoietic differentiation, and targeted inhibition EED 102,103 of mutant IDH2 can restore leukemic cell differentiation. EZH2 Thus, IDH mutations and TET2 loss-of-function alterations may converge on a shared mechanism of hematopoietic SUZ12 transformation characterized by impaired hydroxymethylation, JARID2 global hypermethylation of DNA and deregulated cell 100 differentiation. IDH mutant leukemic cells accumulate high DNA H3K27me3 H3K27me1 levels of (R)2-hydroxyglutarate, which can also be detected in elevated quantities in the sera, and may serve as a biomarker of UTX IDH1/2 mutation, either to detect the mutated cases or to follow the residual disease upon therapy.104 Figure 5. The PRC2. PRC2, made of a catalytic subunit EZH2 (Enhancer of Zest homolog 2) and several associated proteins, COMBINATION OF TET2 MUTATIONS WITH VARIOUS including EED (Embryonic Ectoderm Development), SUZ12 ALTERATIONS IN OTHER EPIGENETIC GENES (Suppressor of Zeste 2) and JARID2 (Jumonji, AT-Rich Interactive Mutations in the DNA-methylating enzyme DNA methyltransferase Domain 2), catalyze the trimethylation of lysine 27 of histone H3 3A, and in components of the PRC2, including the catalytic subunit (H3K27me3), a prototypical repressive histone mark. The H3K27me3 demethylase UTX catalyzes the formation of H3K27me1. In addition, EZH2 (Enhancer of Zest homolog 2) and the associated proteins ASXL1 (Additional sex combs-like 1) could mediate the recruitment EED (Embryonic Ectoderm Development), SUZ12 (Suppressor of of PRC2 to some gene promoters (not shown). Mutations in Zeste 2) and JARID2 (Jumonji, AT-Rich Interactive Domain 2), can these components of the epigenetic machinery have been be associated with the TET2 gene deletions or mutations in found to be associated to TET2 mutations in various myeloid myeloid malignancies.105 PRC2 directly binds to gene promoters malignancies.

Leukemia (2014) 485 – 496 & 2014 Macmillan Publishers Limited The TET2 gene in hematopoiesis and hematopoietic diseases E Solary et al 493 surrounding normal tissues,113 and this 5-hmC decrease, which oncogenic effect of (R)-2-hydroxyglurate in IDH2-mutated cells could be an epigenetic hallmark of tumor development, is (Figure 2), demonstrated an effect on TET2-mutated cells.68 associated with a downregulation of TET or IDH2 genes.69,71 Several other epigenetic therapies are currently developed, Interestingly, re-introduction of an active TET2 or IDH2 was shown including inhibitors of BET bromodomains,118 small molecule to suppress melanoma growth and to increase survival in animal inhibitors of mutant IDH enzymes101 and others.119 Ongoing models, indicating a 5-hmC-mediated suppression of melanoma studies will indicate whether and how some of these progression.69 molecules could be of interest in the treatment of TET2- mutated tumors.

CLINICAL IMPLICATIONS OF TET2 ALTERATIONS Targeting aberrant DNA methylation using hypomethylating CONCLUSIONS agents is an alternative to conventional chemotherapy. As they Altogether, a scheme has emerged in which, in many hemato- alter the methylation pattern of leukemic cells, TET2 gene poietic malignancies, acquired TET2 disruption alters the alterations were anticipated to predict an increased response to epigenetic landscape with decreased 5-hmC and global hyper- hypomethylating agents and this predictive effect was explored in methylation of the DNA, resulting in the expansion of hemato- several cohorts of high-risk MDS, AML with low blast counts and poietic stem and progenitor cells. The occurrence of additional severe CMML patients.58,85,114–116 In some of these studies, the gene mutations in early hematopoietic progenitors—leading to TET2 status appeared as an independent genetic predictor of perturbations in epigenetic programming, or pre-mRNA splicing, response to azacitidine, although not conferring a survival or cell signaling—can promote the outcome of a myeloid or a advantage.85,114 Several confounding variables could interfere lymphoid disorder. In some patients, a myeloid and a lymphoid with these analyses—for example, the TET2 gene downregulation malignancy develop sequentially on a unique TET2-mutated as a consequence of miR-22 overexpression would affect the background through diverse combinations of additional gene epigenetic landscape of the cell in a similar manner to the loss-of- mutations. Thus, TET2 mutations are acquired in HSPCs and the function mutations in TET2.37 The impact of TET2 (and IDH) disease emergence is dictated by the presence of additional mutations on clinical outcome,93 which deserves to be tested on disease allele, either myeloid (for example, JAK2 or FLT3)or larger series of homogeneously treated patients, will never reach lymphoid (for example, MYD88 or JAK3). The order of appearance the strong and independent negative impact of ASXL1 mutations of mutations could matter—for example, early clonal dominance identified in CMML58 and other myeloid malignancies.79,117 of TET2 mutations, as observed in CMML, could promote Conversely, treatment with demethylating agents might be monocytosis,58 whereas their appearance after the mutations in considered in patients with angioimmunoblastic T-cell the spliceosome machinery, as observed in MDS, would not lymphoma or peripheral T-cell lymphoma who have mutations induce monocytosis120 (Figure 6). Several unanswered biological in the TET2 gene. and clinical questions will guide future investigations, including Specifically targeting the consequences of TET2 loss-of-function the importance of TET2 gene-deregulated expression in diseases is a more challenging issue. Inhibition of the EGLN family of in which this gene is not deleted or mutated, the potential impact prolyl hydroxylases, initially developed to antagonize the of a decreased TET2 enzymatic activity on specific target genes

AML

FLT3 SPOTLIGHT AML TET2 CTCF

JAK2 MF JAK2 PV ASXL1

SRSF2

RAS CMML

Figure 6. TET2 mutations as background mutations for various malignancies. TET2 mutations may appear with age in hematopoietic stem and progenitor cells of otherwise healthy subjects.71 The disease emergence will be dictated by the accumulation of additional mutations. In the ﬁrst example,76 mutations in CTCF, then in FLT3, induce an AML. In the second example,71,80 mutation in JAK2 generates a myeloproliferative neoplasm—for example, an essential thrombocythemia, which can transform into secondary myeloﬁbrosis due to an additional ASXL1 mutation. In the third example,59 mutations in SRSF2, then in KRAS generate a chronic myelomonocytic leukemia in its proliferative form.

& 2014 Macmillan Publishers Limited Leukemia (2014) 485 – 496 The TET2 gene in hematopoiesis and hematopoietic diseases E Solary et al 494 (if any), the significance of TET2 alterations in blood cells of 19 Ito S, Shen L, Dai Q, Wu SC, Collins LB, Swenberg JA et al. Tet proteins can otherwise healthy subjects and the possibility to target the convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science consequences of decreased TET2 activity in a transformed cell. 2011; 333: 1300–1303. Hopefully, the answer to these questions will ultimately generate 20 He YF, Li BZ, Li Z, Liu P, Wang Y, Tang Q et al. Tet-mediated formation of new therapeutic approaches in hematological and possibly other 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 2011; malignancies. 333: 1303–1307. 21 Maiti A, Drohat AC, Thymine DNA. glycosylase can rapidly excise 5-formylcytosine and 5 carboxylcytosine: potential implications for active demethylation of CpG CONFLICT OF INTEREST sites. J Biol Chem 2011; 286: 35334–35338. 22 Cortellino S, Xu J, Sannai M, Moore R, Caretti E, Cigliano A et al. Thymine DNA The authors declare no conflict of interest. glycosylase is essential for active DNA demethylation by linked deamination- base excision repair. Cell 2011; 146: 67–79. 23 Shen L, Wu H, Diep D, Yamaguchi S, D’Alessio AC, Fung HL et al. Genome-wide ACKNOWLEDGEMENTS analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. ES, OAB and WV are heading teams independently supported by the Ligue Nationale Cell 2013; 153: 692–706. Contre le Cancer (Equipes labelliseés) and receive grants from Inserm, Agence 24 Frauer C, Hoffmann T, Bultmann S, Casa V, Cardoso MC, Antes I et al. 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