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 β-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.
Five years ago, the demonstration that monoallelic or biallelic loss-of-function mutations and deletions recurrently target the TET2 (Ten-Eleven Translocation 2) gene in myeloid malignancies,1, 2 and the simultaneous demonstration that proteins of the TET family have a key role in the conversion of 5-methyl-cytosine (5-mC) to 5-hydroxymethyl-cytosine (5-hmC),3 have opened a large field of investigation, both in basic science and clinics. The present review summarizes our current understanding of the protein functions and regulation and discusses the significance of its deregulation in hematological malignancies as well as potential therapeutic opportunities that emerge from these studies.
The TET family of enzymes
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) gene in an acute myeloid leukemia (AML) with a t(10;11)(q22;q23) translocation, with TET2 and TET3 being identified by homology searches.4, 5 It appeared that most animals had a single TET orthologue, characterized by an amino-terminal CXXC-type zinc-finger domain and a carboxy-terminal catalytic Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenase domain inserted in a cystein-rich domain. In jawed vertebrates, the TET genes underwent triplication. TET1 and TET3 have also a CXXC domain, whereas a chromosomal inversion during vertebrate evolution split the third TET gene into distinct segments encoding the catalytic domain (the TET2 gene) and the DNA-binding CXXC domain (the CXXC4/IDAX gene). The latter is transcribed in the opposite direction and the protein exerts a regulatory function on TET2 level expression.6 The CXXC motif may be responsible for direct (TET1 and TET3) or indirect (TET2) DNA binding.6, 7 Different 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 neuronal lineages.3
α-KG dependency of TET enzymes links metabolism to epigenetics
TET enzymes are one of the homeostatic links between intracellular metabolism and epigenetic gene regulation.8 Like a number of chromatin-modifying enzymes, such as the JmjC domain-containing histone demethylases, TET dioxygenases require α-KG (also known as 2-oxoglutarate), oxygen and Fe(II) for their activity, which is enhanced in the presence of ascorbic acid.9, 10 Fe(II), 2OG-dependent dioxygenases have a common structural platform.11 Exons 7–11 of the TET2 gene encode a core made of eight anti-parallel β-strands folded into a ‘Jelly-roll’ motif that harbors the active site. The so-called ‘2-His-1-carboxylate triad’, made of three residues in the active site (two histidine and one aspartate or glutamate residues), forms a Fe(II)-binding platform. The Fe(II) metal center, locked in this triad, binds 2-oxoglutarate and O2 on the other side. α-KG, which can be derived from several sources including isocitrate and glutamic acid, is decarboxylated to succinate during the oxidation reaction.8 Somatic mutations in isocitrate dehydrogenase (IDH) enzymes, either cytosolic IDH1 or mitochondrial IDH2, which are observed in various tumors including myeloid malignancies,12 unmask a latent ability of these enzymes to produce the R enantiomer of 2-hydroxyglutarate, an ‘oncometabolite’ that inhibits 2-oxoglutarate-dependent enzymes, including TET dioxygenases (Figure 2). Potentially, TET enzymes may be also sensitive to changes in oxygen availability and susceptible to reactive oxygen species and carcinogenic metals that displace iron such as arsenic, nickel or chromium.13
TET enzymes promote DNA demethylation
Methylation at carbon atom 5 of the nucleotide cytosine (5-methyl-cytosine or 5-mC), which is the predominant epigenetic modification of DNA, and the reverse DNA demethylation process, have a profound impact on gene expression. The mechanism of cytosine methylation by DNA methyltransferases has been established for a long time. Cytosine demethylation remained enigmatic until identification of TET enzyme functions. These enzymes modify the methylation status of DNA and regulate gene transcription by catalyzing the oxidation of the 5-methyl group on 5-mC to create 5-hydroxymethyl-cytosine (5-hmC). Of note, 5-hmC levels across the genome remain low as compared with 5-mC—for example, in mouse embryonic stem cells (ESCs), there are 45 5-hmCs per 1000 5-mC.14, 15
5-hmC is a dynamic epigenetic state of DNA and the conversion of 5-mC into 5-hmC initiates demethylation that can occur in several ways16, 17 (Figure 3). First, because 5-hmC may not be recognized by DNA methyltransferase 1, the oxidation of 5-mC into 5-hmC may favor a passive demethylation that is DNA replication-dependent.17 Secondly, 5-hmC could be converted by the activation-induced deaminase/apolipoprotein B mRNA-editing enzyme complex family of cytosine demethylases into 5-hydroxymethyluracil (5-hmU), to be repaired by DNA glycosylases and the base-excision repair pathway.18, 19 The physiological importance of this second pathway in mammal cells remains controversial. Third, iterative oxidation of 5-mC and 5-hmC by TET enzymes generate 5-formylcytosine and 5-carboxylcytosine, which are recognized and excised by thymine DNA glycosylase into an abasic site. Subsequent repair by the base-excision repair pathway restores an unmodified cytosine.19, 20, 21, 22
The dynamic methylation/demethylation cycle that involves TET and thymine DNA glycosylase enzymes (TET/thymine DNA glycosylase cycle) was shown recently to occur at a large number of genomic loci across the genome.23 Interestingly, long-lived 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 factors, with limited overlap between these proteins.24, 25, 26, 27 In addition, most 5-mC-binding proteins do not recognize 5-hmC and thereby presumably dissociate from DNA when 5-mC is converted into 5-hmC.27 Thus, TET proteins may be important in fine tuning epigenetic status of the cells.
TET-mediated histone modifications through interaction with OGT
Another mechanism that may account for TET-mediated gene activation is the recruitment of O-linked β-D-N-acetylglucosamine (O-GlcNAc) transferase (OGT) to chromatin. OGT is the enzyme that catalyzes the addition of O-GlcNAc to Ser and Thr residues of proteins. The glycosyltransferase reaction mediated by OGT and the removal of O-GlcNAc from substrates mediated by O-GlcNAcase define the O-GlcNAc cycling. This process can influence the epigenetic control of gene expression in several ways.28 O-GlcNAcylation affects the proteolysis of host cell factor 1, a component of the H3K4 methyltransferase SET1/COMPASS complex and an epigenetic cell cycle regulator. O-GlcNAcylation also regulates the function of the Polycomb and Trithorax complexes, interacts with the machineries involved in methylation, acetylation and ubiquitylation of the four core histones, and modifies RNA polymerase II. Similar to TET activation by 2-oxoglutarate, O-GlcNAc cycling links cell metabolism to higher-order chromatin organization.
All TET proteins might interact with OGT.29, 30, 31 For example, OGT interacts with the catalytic domain of TET2; depletion of TET2 from embryonic stem cells prevents the association of OGT with chromatin; overexpression of TET2 in human cells increases the 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 transcription start sites.29, 30, 31
OGT does not influence TET2 hydroxylase activity. TET2 recruits OGT to chromatin and promotes OGT activity, an effect that is independent of TET2 enzymatic activity (Figure 4). The interaction of TET2 with OGT enhances the O-GlcNAcylation of Histone 2B at Ser112, and the genes that are associated with TET2 and OGT and display O-GlcNAcylated Ser112 of Histone 2B show high levels of transcription. TET2 might help OGT to recognize chromatin substrate, and OGT-mediated O-GlcNAcylation of histone Histone 2B might contribute to TET2-dependent gene regulation.30
TET2 protein and OGT activity also promote O-GlcNAcylation of host cell factor 1 (HCF1), which is important for the integrity of SETD1A, which is the SET1/COMPASS complex, which is the main H3K4 methyltransferase complex. TET2-OGT colocalizes on chromatin at active promoters enriched for H3K4me3, and reduction of either TET2/3 or OGT activity results in a decrease in H3K4me3 and concomitant decrease in transcription—that is, global GlcNAcylation and H3K4me3 are reduced in bone marrow cells of tet2−/− mice, notably at several key regulators of hematopoiesis.29
Proteolytic regulation of TET2 expression
Whereas interaction of OGT with TET2 does not affect the 5-hmC level, IDAX—also known as CXXC4 and originating from the ancestral TET2 gene fission—interacts with the catalytic domain of TET2 and was proposed to promote the degradation of the protein, bound to genomic DNA, by caspases. According to this study,6 IDAX binds DNA sequences enriched in unmethylated CpG dinucleotides in gene promoters through its CXXC domain, which activates caspases and results in TET2 protein cleavage and degradation, without directly affecting the enzymatic activity of the dioxygenase. The TET3 expression level could be regulated in 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 protein that was involved in normal myeloid differentiation and myeloid malignancies,32 could also promote TET2 degradation. Further studies will indicate whether the altered expression of either IDAX33 or CXXC532 observed in tumor cells affects the TET2 expression level and the epigenetic control of gene expression. This mode of regulation may explain the observed tissue-specific differences in 5-hmC levels and dynamic changes observed upon differentiation, neoplastic transformation and environmental exposure. Given the role of IDAX in the regulation of the Wnt pathways,34 this also suggests a link between paracrine signals and DNA modification.
Other potential regulators of TET functions
In addition to OGT and IDAX, TET proteins could interact with early B-cell factor 1 that binds DNA to control its local demethylation. This interaction, which was identified by cross analysis of methylation profiles from four series of IDH-mutated cancers including AML, was confirmed by chromatin immunoprecipitation and immunoblotting;35 however, its functional significance remains unclear. This is also the case for the TET protein interaction with activation-induced deaminase that could affect their subcellular distribution.36 A systematic proteomic screen recently identified the ubiquitin ligase Uhrf2 (ubiquitin-like with PHD and ring-finger domains 2) as a protein that promotes the iterative oxidation of 5-mC by TET enzymes in mouse neuronal progenitor cells27 (Table 1). Additional partners of TET proteins have been identified, and functional analysis of these interactions may provide additional insights in their functions.
Vitamin C enhances the activity of TET enzymes
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 folding or recycling of the Fe(2+).9 In mouse embryonic stem cells, vitamin C promotes a rapid and global increase in 5-hmC, followed by the DNA demethylation of many gene promoters.10 The vitamin-C-induced changes in 5-hmC and 5-mC are TET-dependant, as they are entirely suppressed in Tet1 and Tet2 double-knockout ESCs. Regions that are resistant to vitamin-C-mediated demethylation show high levels of H3K9me3, suggesting that this mark, or readers of this mark, may have a role in protecting against Tet-mediated demethylation. Altogether, vitamin C may be a direct regulator of TET activity and DNA methylation fidelity.
An miR-22-dependent regulation of TET2
Another recently identified level of regulation of TET2 gene expression involves the microRNA miR-22.37 Alterations of this miR-22/TET2 regulatory network could be common and oncogenic in hematopoietic malignancies such as myelodysplastic syndromes (MDS, 35%) and AML (22%). Transgenic mice conditionally expressing miR-22 in the 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 malignancies. Ectopic expression of TET2 corrects the miR-22-induced phenotype, whereas miR-22 inhibition blocks proliferation in both mouse and human leukemic cells.37 According to these recent data, miR-22 may be a potent regulator of HSC maintenance and self-renewal through the modulation of TET2 expression level, and presumably other important targets.
Embryonic stem cells express TET1 and TET2
The expression of pluripotency factors and the repression of lineage differentiation genes in ESCs involve epigenetic mechanisms, including DNA methylation and histone modifications.38 Tet1 and Tet2 are expressed in mouse ESCs39 and their expression decreases upon ESC differentiation, whereas Tet3 is upregulated. The inverse changes are observed when fibroblasts are reprogrammed to generate induced-pluripotent stem cells.40, 41 Significant levels of 5-hmC have been detected in mouse ESCs, and the 5-hmC level declines during differentiation.39, 42 Tet1 is probably a major factor in ESCs, as Tet1 depletion in these cells decreases the 5-hmC level.21, 39, 43 Tet1 binds a large number of 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 to transcription start signals and is enriched in a fraction of CpG islands.38, 42, 43, 44, 45, 46 Nevertheless, how Tet1 and Tet2 regulate pluripotency remains a matter of controversy.
The role of TET proteins in embryonic cells remains largely enigmatic
Tet enzymes were proposed initially to promote the expression of pluripotency genes. Most Tet1-bound promoters in ESCs are H3K4me3-positive, which, when isolated, is a mark of active transcription. It was proposed that, in promoters of housekeeping genes, Tet enzymes act as failsafe mechanisms that prevent aberrant methylation and maintain CpG islands free of methylation by rapidly converting 5-mC into 5-hmC.45 In promoters of pluripotency genes that become DNA-methylated during differentiation, Tet binding might ensure a timely methylation and silencing.47 Multiple links between Tet1, Tet2 and the master regulators of ESC pluripotency have been reported; however, the picture remains confusing.39, 42, 43 Analysis of Tet1-depleted ESCs suggested initially that Tet1 could promote the expression of the pluripotency gene Nanog.14 Conversely, Oct-4, another factor involved in pluripotency, was shown to regulate Tet1 and Tet2 expressions,48 and Tet1 and Tet2 proteins were shown recently to interact with Nanog.49
Actually, a number of genes are upregulated after Tet1 depletion in mouse ESCs, indicating repressive effects on gene 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), a mark of gene repression. Tet enzymes could indirectly facilitate PRC2 chromatin binding by decreasing DNA methylation levels at PRC2 target genes.43, 44, 45, 46 By preventing stable methylation of PRC2 target genes, which are enriched for genes involved in cell-fate decisions, Tet enzymes might keep the plasticity of these genes during development. Tet1 could also mediate transcriptional repression through its association with the Sin3A co-repressor complex. The recruitment of Sin3A to a subset of these genes is dependent on Tet1 expression. This repressive function of Tet1 may be independent of its enzymatic activity.43
The role of TET proteins could be slightly different in human ESCs, in which TET1 is highly expressed and TET2 is expressed at a very low level. When mammalian somatic cells are reprogrammed into induced-pluripotent stem cells, the strong induction of TET1 increases 5-hmC levels and provokes some aberrations in subtelomeric regions that distinguish induced-pluripotent stem cells from ESCs.50
A locus-specific regulatory function of TET enzymes?
Surprisingly, depletion of either Tet1 or Tet2 in ESCs induced limited changes in 5-hmC levels and DNA methylation.38, 39, 42, 43, 44, 45 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, Tet1 could regulate DNA methylation levels at certain specific genes.39, 42, 43, 45, 51 Of note, most of the transcriptional activating effects of Tet1 were also detected in the Dnmt triple knockout cells, suggesting that they could be indirect effects that do not depend on the demethylating activity of Tet enzymes.44
Tet1−/− ESCs have reduced levels of global 5-hmC and display a skewing toward trophectoderm in culture, as described with in vitro studies; however, they do not lose pluripotency, express near-physiological levels of Nanog, Oct-4 and Sox2, and are able to support organism development.51 Tet1 knockout mice are viable and fertile, although they have a smaller body size at birth.51 Tet1 and Tet2 double-knockout ESCs have also reduced levels of 5-hmC and remain pluripotent. They cause developmental defects in a fraction of chimeric embryos; however, viable and overtly normal Tet1/Tet2-deficient mice can be obtained, showing that loss of both enzymes is compatible with development, pending hypermethylation and compromised imprinting52 Altogether, Tet1, in conjunction with Tet2, probably have a locus-specific role in shaping the ESC epigenome during subsequent development but appear to be dispensable, suggesting that other pathways of DNA methylation could possibly exist.
TET2 oxygenase has a role at several steps of hematopoiesis in in vitro assays
TET2 has pleiotropic roles in hematopoiesis, including stem-cell self-renewal, lineage commitment and terminal differentiation of specific lineages. The TET2 gene is highly expressed in HSCs and in progenitor cells, and is downregulated with differentiation. One of the consequences of Tet2 depletion (or expression of a mutated Idh2 whose product affects Tet2 enzymatic activity) in primary bone marrow cells is an increase in the percentage of immature c-Kit+, Lin− cells and their replating ability, suggesting that Tet2 silencing could affect stem/progenitor cell differentiation.53, 54 Accordingly, small interfering RNA-mediated depletion of Tet2 was demonstrated to alter the pattern of transcription of homeotic (Hox) genes in pluripotent cell populations.55
RNA interference-mediated Tet2 silencing in mouse early progenitors56 as in human cord blood CD34+ cells57 decreases 5-hmC levels and allows expansion of the monocyte lineage. This latter phenotype is in line with the frequent mutations of the TET2 gene (∼50%) in chronic myelomonocytic leukemia (CMML), a disease defined by the accumulation of monocytes in the bone marrow, the peripheral blood and the spleen.58 TET2 mutations are also observed in a variety of myeloid and lymphoid malignancies in which monocytosis is not observed. Ex vivo, TET2 depletion in CD34+,CD38− promotes monocyte expansion, whereas TET2 depletion in CD34+,CD38+ cells does not, suggesting that the level at which TET2-mutated cells expand contributes to the disease phenotype, with early clonal dominance of TET2 mutations inducing a monocytosis.59, 60 An alternative hypothesis is that the primary effect of TET2 mutations is to induce the expansion of HSCs biased towards monocytic differentiation, these mutated cells being targeted by secondary genetic events that can modify the disease phenotype.
TET2 may be required at later-stage myeloid differentiation. Tet2 depletion impairs CEBPα-induced transdifferentiation of pre-B cells into macrophages; CEBPα could bind to upstream regions of Tet2 that, in turn, could activate a subset of myeloid genes through a rapid increase in their promoter hydroxy methylation.61 Mature monocytes also express TET2, and loss of DNA methylation during the differentiation of primary, post-proliferative human monocytes into dendritic cells is preceded by the local appearance of 5-hmC. The small interfering RNA-mediated knockdown of this enzyme in primary monocytes prevents active DNA demethylation, suggesting that TET2 is essential for the proper execution of this process in human monocytes.62
Tet2 is a tumor-suppressor gene
Homozygous and heterozygous mutations in the TET2 gene are recurrent events in human hematopoietic malignancies.1, 2 Most of these mutations decrease TET2 enzymatic activity by truncating the protein or affecting its catalytic activity. To determine the role of TET2 mutation in hematopoietic malignancies, several teams simultaneously developed mouse models of Tet2 deletion.54, 56, 63, 64
Analyses of these Tet2 knockout mice first enforced the evidence for a role of Tet2 in the regulation of normal hematopoiesis. All three TET family proteins are expressed in hematopoietic progenitors, and the deletion of Tet2, which does not induce an upregulation of Tet1 and Tet3, is sufficient to decrease 5-hmC content in HSCs. A decrease in LSK content in 5-hmC is observed in both Tet2–/– and Tet2+/− mice. These cells also show an increased ability to self-renew and expand, and they exhibit a competitive advantage over wild-type Tet2 HSCs for repopulating hematopoietic lineages. Tet2 loss could impair the tissue-specific pattern of the Hoxa gene expression.55 Lastly, the deletion of Tet2 allows for amplification of the monocytic lineage.54, 56, 63, 64
Tet2 deletion is sufficient to initiate myeloid and lymphoid transformations. Similar to Tet1-deficient mice, Tet2 knockout mice are viable, fertile and develop grossly normally. However, as they age, Tet2-deficient mice specifically demonstrate an increased susceptibility to myeloid and lymphoid tumors. The onset of these malignancies and the kinetics of their progression partly depend on the genetic background of the model (Table 2). In addition, Tet2+/− mice behave similar to Tet2−/− cells and develop diverse hematopoietic malignancies, although with longer latency, suggesting gene-dosage effects. Thus, TET2 haploinsufficiency, which is frequently observed in human hematological malignancies, is sufficient to change the properties of HSCs.
Tet2-deficient mice developed predominantly a CMML-like disease; however, other types of diseases, including MDS and myeloproliferative-like diseases, were observed.54, 56, 63, 64 Tet2 loss also affects lymphoid-lineage development with the expansion of an aberrant (CD19+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 induction suggests that additional genetic lesions may cooperate with Tet2 loss to induce a disease and influence the generation of distinct types of blood neoplasms,65 either in the myeloid or in the lymphoid lineage. It is appealing to assume that the loss of a TET protein could contribute to the gene-specific hypermethylation that is often observed in cancer. Aberrant methylation of CpG islands in specific gene promoters has been associated with the development of hematopoietic malignancies—for example, the silencing of p15/INK4B cell cycle regulator is observed in one-third of MDS and that of the Tif1γ (transcription intermediary factor 1 gamma) gene in 35% of human CMML.66 However, the abnormal methylation of the p15/INK4B or Tif1γ promoter does not strictly associate with TET2 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 mechanisms other than mutations, or aberrant DNA methylation at CpG islands are stochastically generated by TET mutants and occasionally provide cells with a growth advantage, leading to clonal expansion of the cells and, in combination with other genomic alterations, to disease development.
TET2 alterations in the absence of mutation
As indicated above, IDH 1 or IDH2 mutant enzymes, identified in some myeloid malignancies such as AML and CMML, induce carboxylation of glutamine-derived α-KG, with a concomitant increase in synthesis of the R enantiomer of 2-hydroxyglutarate.67 This metabolite inhibits a-KG-dependent dixoygenases, with, in the case of TET2, a decrease in the cellular 5-hmC level. Importantly, in addition to inhibiting TET enzymes, R enantiomer of 2-hydroxyglutarate is a competitive inhibitor for several other 2OG-dependent dioxygenases, including histone lysine demethylases and prolyl hydroxylases such as EGLN.68 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 to the cellular accumulation of TCA cycle intermediates with structural similarity to 2OG8 (Figure 2). The downregulation of TET (or IDH2) enzymes was shown to be the likely mechanism responsible for the loss of 5-hmC identified in melanoma cells.69, 70 At least in hematopoietic tissues, the downregulation of TET2 could result from alterations in the miR-22/TET2 regulatory network.37 Collectively, in addition to mutations, functional losses of TET2 because of additional upstream cues could have a critical role in aberrant hematopoiesis and leukemogenesis.
TET2 mutations accumulate with aging
It had been postulated that the biased hematopoietic differentiation toward the myeloid lineage could explain the increasing incidence of myeloid malignancies observed in aging, otherwise healthy people.1, 2 By sequencing the DNA of neutrophils (PMNs), somatic TET2 mutations, analogous to the inactivating mutations observed in patients with myeloid malignancies, were identified in 10/182 (5.5%) elderly individuals, and in none of 96 younger adults. Clinical follow-up for seven of the TET2 mutant subjects for ⩾5 years after mutational analysis identified the occurrence of a JAK2V617F-positive essential thrombocythemia in one case.71 The appearance of TET2 mutation in hematopoietic cells of aging people could account for the absence of TET2 mutation in juvenile myelomonocytic leukemia,72 the low prevalence (<5%) of TET2 mutations in pediatric AML73 and the age-associated increase in the prevalence of TET2 mutations in adult AMLs.74 An alternative hypothesis is that a unique mutation is sufficient for the emergence of a malignant clone in children hematopoiesis, whereas mutations that induce a clonal hematopoiesis are needed in older subjects for disease expression. Whatever the explanation, these observations support an initiating role for TET2 mutation in the pathogenesis of age-associated hematological cancers, also supported by analysis of the clonal architecture in CMML, showing that TET2 mutations are often the first detected oncogenic event in this disease.59, 75, 76 The age-dependent decrease in global 5-hmC levels observed in myeloid cells suggests that it may be an important feature in the aging hematopoietic system. The potential risk of 5-hmC decrease and TET2 mutations now deserves to be tested prospectively in large cohorts of aging individuals in order to detect acquired states that predispose to leukemogenesis. The working hypothesis is that these mutations may increase the HSC fitness whose transformation will be fixed by the accumulation of additional genetic or epigenetic alterations.
TET2 mutations in myeloid malignancies
Somatic alterations in TET2, including deletions and missense, nonsense and frameshift mutations, were identified initially in 10–26% of MPN1 and MDS1, 2 patients. These TET2 gene alterations 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 consistent with the improved function of Tet2-deleted murine HSPCs.54, 56, 63, 64 The TET2 gene has been subsequently sequenced in series of patients with different subtypes of myeloid neoplasm to delineate the frequency of TET2 mutations (Table 3). Two distinct TET2 mutations were identified in a number of cases—for example, in ∼25% of TET2-mutated MDS—77 indicating that biallelic loss of TET2 is frequently part of the disease evolution.
Acquired somatic alterations in TET2 were identified in 2–20% of classical MPNs, including polycythemia vera, essential thrombocytosis and primary myelofibrosis.78, 79 These mutations, which can be an early genetic event in the MPN course, have no clear prognostic impact—that is, they do not increase the risk of leukemic transformation. In some cases, however, TET2 mutations appear only when the disease progresses to an acute phase.80, 81 In chronic myeloid leukemia, TET2 mutations were associated in most cases with acute blastic transformation.82
TET2 alteration was also the most prevalent genetic abnormality (25–35%) identified in MDS.77, 83, 84, 85 Larger series failed to identify a strong association of TET2 alteration with clinical phenotype, risk scores or overall survival.77 Nevertheless, in higher risk MDS and AML with low blast count, the TET2 status may predict a better response to the demethylating agent azacitidine.85 As already indicated, the highest rate of TET2 genetic alterations was 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 mutation as one of the earliest event in the accumulation of genetic alterations that lead to the leukemic clone expansion.59, 61 TET2 mutations are less frequent in other MPN/MDS, although they can be associated to SF3B1 and JAK2 mutations in refractory anemia with ring sideroblasts and thrombocytosis.88 Analysis of large cohorts failed to demonstrate any prognostic impact of TET2 mutations in CMML.58
The prevalence of TET2 mutations is higher in secondary than in de novo AMLs.89, 90, 91, 92, 93 Genetic alterations of TET2 could be associated with adverse outcome in cytogenetically defined subgroups of AML patients,89, 93 and thus could be integrated to the list of parameters that guide therapeutic choices in this disease. TET2 mutations were identified in ∼20% of mastocytosis, mostly in aggressive forms of the disease, with a demonstrated oncogenic cooperation with KITD816V in mast cells.94, 95 More recently, TET2 mutations were found in ∼30% of patients with a blastic plasmacytoid dendritic cell neoplasm.96, 97
IDH2 and IDH1 mutations complement TET2 mutations
In myeloid malignancies, TET2 mutations are mutually exclusive with somatic heterozygous mutations at highly conserved positions in IDH1 (R132) and IDH2 (R140, R172). In addition, IDH1 and IDH2 mutations are mutually exclusive from one another.11 These mutations are observed in 5–20% of AMLs, most commonly in adult patients with cytogenetically normal AML, and in 5–20% of chronic myeloid malignancies in which their appearance is 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, 99, 100 A cell-permeable (R) enantiomer of 2-hydroxyglutarate was shown to affect hematopoietic differentiation,101 and targeted inhibition of mutant IDH2 can restore leukemic cell differentiation.102, 103 Thus, IDH mutations and TET2 loss-of-function alterations may converge on a shared mechanism of hematopoietic transformation characterized by impaired hydroxymethylation, global hypermethylation of DNA and deregulated cell differentiation.100 IDH mutant leukemic cells accumulate high levels of (R)2-hydroxyglutarate, which can also be detected in elevated quantities in the sera, and may serve as a biomarker of IDH1/2 mutation, either to detect the mutated cases or to follow the residual disease upon therapy.104
Combination of TET2 mutations with various alterations in other epigenetic genes
Mutations in the DNA-methylating enzyme DNA methyltransferase 3A, and in components of the PRC2, including the catalytic subunit EZH2 (Enhancer of Zest homolog 2) and the associated proteins EED (Embryonic Ectoderm Development), SUZ12 (Suppressor of Zeste 2) and JARID2 (Jumonji, AT-Rich Interactive Domain 2), can be associated with the TET2 gene deletions or mutations in myeloid malignancies.105 PRC2 directly binds to gene promoters or is recruited by transcription factors to catalyze the trimethylation of lysine 27 of histone H3 (H3K27me3), a prototypical repressive histone mark. Mutations in the H3K27me3 demethylase UTX can also be detected in combination with TET2 alterations.105 Lastly, mutation in ASXL1 (Additional sex combs-like 1), a component of the Polycomb Repressive Deubiquitinase complex and a recruiter of PRC2 to some gene promoters,106 is a recurrent event in myeloid malignancies in which it is strongly and independently associated with a poor clinical outcome (Figure 5).58 TET2 loss-of-function may synergize with these other gene mutations to have an impact on the epigenetic landscape and promote malignant transformation or disease progression.
TET2 mutations in lymphoid malignancies
Two observations led to the identification of TET2 somatic mutations in human B and T lymphomas. First, Tet2-deficient mice demonstrate an altered T- and B-cell lineage development.64 Secondly, some patients with a TET2-mutated myeloid malignancy develop a lymphoma. TET2 mutations were identified in mostly mature B-cell (2%) and T-cell (11.9%) lymphomas.67, 107, 108 In addition, they were observed in ∼33% of angioimmunoblastic T-cell lymphoma, an aggressive neoplasm of CD4+ T lymphocytes.67, 109 In this latter disease, TET2 mutations are frequently associated with DNA methyltransferase 3A mutations,108 and IDH2 mutations are identified in 20–45% of cases.109 AML/MDS arising secondary to lymphoma was demonstrated to carry the same TET2 mutation as the previous lymphoma, indicating a common cell of origin. More recently, TET2 mutations were identified in mantle cell lymphomas110 and in diffuse large B-cell lymphoma (12%) in which they were associated with an altered DNA methylation pattern of genes involved in hematopoietic development111 (Table 1).
TET2 altered expression in non-hematological malignancies
TET2 mutations were detected in a small number of solid tumors—that is, were recently proposed to define a subset of metastatic tumors in castration-resistant prostate cancers.112 Most importantly, the levels of 5-hmC are dramatically reduced in a variety of human solid tumors compared with the matched surrounding normal tissues,113 and this 5-hmC decrease, which could be an epigenetic hallmark of tumor development, is associated with a downregulation of TET or IDH2 genes.69, 71 Interestingly, re-introduction of an active TET2 or IDH2 was shown to suppress melanoma growth and to increase survival in animal models, indicating a 5-hmC-mediated suppression of melanoma progression.69
Clinical implications of TET2 alterations
Targeting aberrant DNA methylation using hypomethylating agents is an alternative to conventional chemotherapy. As they alter the methylation pattern of leukemic cells, TET2 gene alterations were anticipated to predict an increased response to hypomethylating agents and this predictive effect was explored in several cohorts of high-risk MDS, AML with low blast counts and severe CMML patients.58, 85, 114, 115, 116 In some of these studies, the TET2 status appeared as an independent genetic predictor of response to azacitidine, although not conferring a survival advantage.85, 114 Several confounding variables could interfere with these analyses—for example, the TET2 gene downregulation as a consequence of miR-22 overexpression would affect the epigenetic landscape of the cell in a similar manner to the loss-of-function mutations in TET2.37 The impact of TET2 (and IDH) mutations on clinical outcome,93 which deserves to be tested on larger series of homogeneously treated patients, will never reach the strong and independent negative impact of ASXL1 mutations identified in CMML58 and other myeloid malignancies.79, 117 Conversely, treatment with demethylating agents might be considered in patients with angioimmunoblastic T-cell lymphoma or peripheral T-cell lymphoma who have mutations in the TET2 gene.
Specifically targeting the consequences of TET2 loss-of-function is a more challenging issue. Inhibition of the EGLN family of prolyl hydroxylases, initially developed to antagonize the oncogenic effect of (R)-2-hydroxyglurate in IDH2-mutated cells (Figure 2), demonstrated an effect on TET2-mutated cells.68 Several other epigenetic therapies are currently developed, including inhibitors of BET bromodomains,118 small molecule inhibitors of mutant IDH enzymes101 and others.119 Ongoing studies will indicate whether and how some of these molecules could be of interest in the treatment of TET2-mutated tumors.
Altogether, a scheme has emerged in which, in many hematopoietic malignancies, acquired TET2 disruption alters the epigenetic landscape with decreased 5-hmC and global hypermethylation of the DNA, resulting in the expansion of hematopoietic stem and progenitor cells. The occurrence of additional gene mutations in early hematopoietic progenitors—leading to perturbations in epigenetic programming, or pre-mRNA splicing, or cell signaling—can promote the outcome of a myeloid or a lymphoid disorder. In some patients, a myeloid and a lymphoid malignancy develop sequentially on a unique TET2-mutated background through diverse combinations of additional gene mutations. Thus, TET2 mutations are acquired in HSPCs and the disease emergence is dictated by the presence of additional disease allele, either myeloid (for example, JAK2 or FLT3) or lymphoid (for example, MYD88 or JAK3). The order of appearance of mutations could matter—for example, early clonal dominance of TET2 mutations, as observed in CMML, could promote monocytosis,58 whereas their appearance after the mutations in the spliceosome machinery, as observed in MDS, would not induce monocytosis120 (Figure 6). Several unanswered biological and clinical questions will guide future investigations, including the importance of TET2 gene-deregulated expression in diseases in which this gene is not deleted or mutated, the potential impact of a decreased TET2 enzymatic activity on specific target genes (if any), the significance of TET2 alterations in blood cells of otherwise healthy subjects and the possibility to target the consequences of decreased TET2 activity in a transformed cell. Hopefully, the answer to these questions will ultimately generate new therapeutic approaches in hematological and possibly other malignancies.
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ES, OAB and WV are heading teams independently supported by the Ligue Nationale Contre le Cancer (Equipes labellisées) and receive grants from Inserm, Agence National de la Recherche and Institut National du Cancer. We are grateful to all members of Inserm UMR895 and UMR1009 who were involved in cited works or provided helpful comments. We apologize to all colleagues whose work was not cited because of space restrictions.
The authors declare no conflict of interest.
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Solary, E., Bernard, O., Tefferi, A. et al. The Ten-Eleven Translocation-2 (TET2) gene in hematopoiesis and hematopoietic diseases. Leukemia 28, 485–496 (2014). https://doi.org/10.1038/leu.2013.337
- DNA methylation
- stem cell
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