Received: 7 October 2018 | Revised: 2 April 2019 | Accepted: 4 April 2019 DOI: 10.1002/mgg3.772 ORIGINAL ARTICLE TET2 missense variants in human neoplasia. A proposal of structural and functional classification Elena Bussaglia1 | Rosa Antón2 | Josep F. Nomdedéu1 | Pablo Fuentes‐Prior2 1 Hematology Department and Diagnostic Hematology Group, Barcelona, Spain Abstract 2 Molecular Bases of Disease, The Background: The human TET2 gene plays a pivotal role in the epigenetic regulation Biomedical Research Institute Sant Pau (IIB of normal and malignant hematopoiesis. Somatic TET2 mutations have been repeat- Sant Pau), Hospital de la Santa Creu i Sant edly identified in age‐related clonal hematopoiesis and in myeloid neoplasms rang- Pau, Barcelona, Spain ing from acute myeloid leukemia (AML) to myeloproliferative neoplasms. However, Correspondence there have been no attempts to systematically explore the structural and functional Josep F. Nomdedéu and Pablo Fuentes‐ consequences of the hundreds of TET2 missense variants reported to date. Prior, The Biomedical Research Institute Sant Pau (IIB Sant Pau), Hospital de la Methods: We have sequenced the TET2 gene in 189 Spanish AML patients using Santa Creu i Sant Pau, Sant Antoni Maria Sanger sequencing and NGS protocols. Next, we performed a thorough bioinfor- Claret 167, 08025 Barcelona, Spain. Email: jnomdedeu@santpau.cat (J. F. N) matics analysis of TET2 protein and of the expected impact of all reported TET2 and pfuentes@santpau.cat (P. F.‐P.) missense variants on protein structure and function, exploiting available structure‐ and‐function information as well as 3D structure prediction tools. Funding information Instituto de Salud Carlos III, Grant/Award Results: We have identified 38 TET2 allelic variants in the studied patients, includ- Number: FIS PI 10/1739 (J.F.N.) and FIS ing two frequent SNPs: p.G355D (10 cases) and p.I1762V (28 cases). Four of the de- PI 13/2729 (J.F.N.); Secretaría de Estado tected mutations are reported here for the first time: c.122C>T (p.P41L), c.4535C>G de Investigación, Desarrollo e Innovación, Grant/Award Number: SAF2014-57994-R (p.A1512G), c.4760A>G (p.D1587G), and c.5087A>T (p.Y1696F). We predict a (P.F.-P.); Agència de Gestió d'Ajuts complex multidomain architecture for the noncatalytic regions of TET2, and in par- Universitaris i de Recerca, Grant/Award ticular the presence of well‐conserved α+β globular domains immediately preceding Number: SGR383 (J.F.N.) and following the actual catalytic unit. Further, we provide a rigorous interpretation of over 430 missense SNVs that affect the TET2 catalytic domain, and we hypoth- esize explanations for ~700 additional variants found within the regulatory regions of the protein. Finally, we propose a systematic classification of all missense mutants and SNPs reported to date into three major categories (severe, moderate, and mild), based on their predicted structural and functional impact. Conclusions: The proposed classification of missense TET2 variants would help to assess their clinical impact on human neoplasia and may guide future structure‐and‐ function investigations of TET family members. KEYWORDS 5‐methylcytosine, classification of mutations, epigenetic regulation, neoplasia, TET2 This is an open access article under the terms of the Creat​ive Commo​ns Attri​bution-NonCo​mmerc​ial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. Mol Genet Genomic Med. 2019;7:e772.  wileyonlinelibrary.com/journal/mgg3 | 1 of 13 https://doi.org/10.1002/mgg3.772 2 of 13 |    BUSSAGLIA et al. 1 | IN TRO D U C T ION nearly all cancer types, most notably colorectal, lung, and skin carcinomas. (For a complete list of articles describing Methylation at the C5 position of cytosine bases to generate TET2 mutations see Table S2; see ref. (Ko et al., 2015) 5‐methylcytosine (5mC) is one of the major epigenetic mod- for a recent review of TET proteins and their role in he- ifications of mammalian genomes, with a profound impact matological cancers). TET2 mutation causes an expansion on chromatic structure and gene expression (Feng, Jacobsen, of immature hematopoietic precursors with a bias toward & Reik, 2010; Smith & Meissner, 2013). Members of the myeloid differentiation (Arends et al., 2018; Buscarlet et Ten‐Eleven‐Translocation (TET) subfamily of Fe(II)‐ and 2‐ al., 2018). oxoglutarate (2‐OG)‐dependent dioxygenases (EC 1.14.11. TET2 alterations have also been described in age‐related n2) help to revert this modification by iteratively oxidizing clonal hematopoiesis (ARCH; [Jaiswal et al., 2014]), as 5mC bases first to 5‐hydroxymethylcytosine (5hmC) (Ito et firstly demonstrated in elderly women (Busque et al., 2012). al., 2010; Tahiliani et al., 2009), and then to 5‐formylcytosine TET2 mutations may also be detected in healthy women years (5fC) and 5‐carboxy‐cytosine (5caC) (Ito et al., 2011). Since before the development of leukemia (Desai et al., 2018). both 5fC and 5caC can be removed through the base excision‐ Importantly, somatic TET2 mutations in normal elderly in- repair pathway (Cortellino et al., 2011; He et al., 2011; Ito et dividuals with CH raise the mortality attributed to vascular al., 2010,2011; Ko et al., 2010; Rampal et al., 2014), TET events (Jaiswal et al., 2017), most likely by promoting an ex- enzymes catalyze the first step in active DNA demethylation, acerbated atherosclerosis (Fuster et al., 2017). In this regard, and therefore play an important role in transcription regula- TET2‐deficiency results in an increased pro‐inflammatory tion. In addition, 5hmC bases function as stable, independent phenotype in murine macrophages, which may help to pro- epigenetic marks, which have been shown to accumulate for mote atherosclerosis (Abegunde, Buckstein, Wells, & Rauh, example at sites of DNA damage and to promote genome sta- 2018; Cull, Snetsinger, Buckstein, Wells, & Rauh, 2017; bility (Kafer et al., 2016). On the other hand, TET2 has been Cull, Snetsinger, & Rauh, 2016). CH also alters cardiac repair recently shown to oxidize specific mRNAs to promote infec- by inflammatory pathways (Sano et al., 2018). Also in line tion‐induced myelopoiesis (Shen et al., 2018). with the inflammatory role of TET2, patients who underwent TET2 has been shown to possess important functions that allogeneic bone marrow transplantation from donors with are independent of its enzymatic activity. For instance, TET2 CH had an increased risk of chronic graft‐versus‐host disease involvement in the resolution of inflammation depends upon (Frick et al., 2019). These findings are in line with TET2‐me- the ability of the enzyme to recruit the histone deacetylase, diated repression of inflammatory mediators such as IL‐6, as HDAC2, to specifically repress IL‐6 transcription (Zhang et mentioned above (Zhang et al., 2015). (For a recent review al., 2015). Further, both catalytic and noncatalytic activities on CH and its relevance for hematopoietic malignancies, see of TET2 are required for mast cell differentiation and prolif- [Bowman, Busque, & Levine, 2018]). eration (Montagner et al., 2016). Finally, it has been recently The TET2 gene maps to chromosome 4q24 contains 11 reported that the enzyme regulates age‐related regenerative exons, and has an open reading frame of 6009 nt. The en- decline in the aging hippocampus, with important implica- coded protein contains 2002 amino acid residues (isoform A, tions for neurogenic rejuvenation (Gontier et al., 2018). (For NM 001127208), with the actual catalytic unit located in the authoritative reviews on the function of TET proteins see e.g. C‐terminal part of the protein (Figure 1a). The three‐dimen- refs. [Pastor, Aravind, & Rao, 2013; Rasmussen & Helin, sional (3D) structure of this TET2 region and the mechanism 2016]). of 5mC oxidation have been recently characterized by X‐ray TET2 is expressed at particularly high levels in hemato- crystallography (Hu et al., 2013, 2015). The structural work poietic cells, and is critically relevant for normal hemato- revealed that the catalytic unit is comprised of two tandem poiesis. Tet2 deletion in mice models causes an enlargement cysteine‐rich modules, followed by the actual catalytic do- of the myeloid immature compartment raising the risk main (Figure 1b,c). This domain, termed double‐stranded of acquiring additional transforming mutations (Li et al., β‐helix (DSBH) for its major structural feature, contains a 2011; Moran‐Crusio et al., 2011; Quivoron et al., 2011). large, poorly conserved insertion between approximate po- Not surprisingly, numerous TET2 mutations have been de- sitions Cys1464 and Glu1841. (All numbers correspond to scribed in a wide range of human myeloid malignancies the human enzyme). Thus, the structurally characterized ranging from acute myeloid leukemia (AML) to myelopro- fragment is an artificial construct in which residues Ser1481‐ liferative neoplasms (MPN) (see e.g. [Abdel‐Wahab et al., Asn1843 are replaced by a 15‐residues‐long Gly/Ser linker, 2009; Delhommeau et al., 2009; Langemeijer et al., 2009]). and which therefore corresponds to only ≈22% of the full‐ TET2 mutations are also common in some subgroups of length protein. On the other hand, there is no structural infor- mature T‐cell lymphomas (Quivoron et al., 2011; Zang et mation available for the long N‐terminal region of the protein, al., 2017). In addition to hematological and lymphoid neo- Met1‐Pro1131. Interestingly, a second transcript of the TET2 plasms, TET2 variants have been more recently reported in gene encodes for a protein that is essentially comprised of BUSSAGLIA et al.    | 3 of 13 F I G U R E 1 Domain organization and distribution of missense variants in human TET2. (a) The domain architecture of the TET2 protein is schematically represented on top of the figure. Posttranslational modification (PTM) sites were taken from public databases, or predicted, either by similarity with the mouse enzyme (Bauer et al., 2015) or using software given in Methods. Notice that the long polypeptide stretch preceding the actual catalytic unit is predicted to contain several putative, relatively well‐conserved globular modules, separated by more variable PTM‐rich linkers. Single‐nucleotide variations reported in the human TET2 gene are given below the scheme, with the following code: missense mutations are represented by triangles, colored red if identified in hematopoietic and lymphoid neoplasms or black, if reported in solid tumors. Validated SNPs are given as circles, which are colored red and black if they have been in addition reported in cancer patients, or left empty otherwise. (b and c) Two approximately perpendicular views of the three‐dimensional structure of TET2 catalytic unit. Major structural domains are shown as ribbons, colored green (Cys‐N module), light green (Cys‐C), magenta and light pink (N‐ and C‐terminal halves of the DSBH domain, respectively). Zn2+ and Fe2+ ions are represented as gray and orange spheres, respectively; the side chains of their coordinating residues are also shown. The bound DNA oligonucleotide is shown with all its non‐hydrogen atoms, color‐coded (carbon, light pink; nitrogen, blue; oxygen, red; and phosphor, orange). Note the complex interdomain contacts, not only between the N‐ and C‐terminal halves of the catalytic domain, but also between the Zn2+‐binding domains with each other and with the DSHB module. In panel c, note the long insertion between residues Thr1463 and Gly1842 (C‐ terminal end of N‐terminal half and N‐terminal start of the C‐terminal half, respectively). DSBH, domain, termed double‐stranded β‐helix these N‐terminal residues, suggesting that it plays an import- absence of the newly described variant in the DNA from non- ant functional role(s) (isoform B, NM 017628). neoplastic samples. However, the identification of TET2 mu- As mentioned above, somatic TET2 mutations have been tations in ARCH and the presence of allele variants of TET2 repeatedly found in myeloid neoplasia. The pathogenic attribu- in nonhematologic neoplasms may complicate interpretation tion of TET2 variants to hematologic neoplasms relies on the of new variants. In silico prediction of the functional conse- earlier description in other cases, functional studies and/or the quences of TET2 variants could be clinically useful in case 4 of 13 |    BUSSAGLIA et al. these criteria are not met. However, this requires a deep knowl- 2014]) and in addition to servers PrDOS (http://prdos.hgc. edge of both the wild‐type protein and representative bona jp/; [Ishida & Kinoshita, 2007]) and DICHOT (http://idp1. fide neoplastic mutants. Rigorous pathogenicity prediction of force.cs.is.nagoya-u.ac.jp/dicho​t/; [Fukuchi, Hosoda, Homma, SNVs is limited to the structurally characterized TET2 cata- Gojobori, & Nishikawa, 2011]). Local sequence motifs were lytic unit, but has not been systematically attempted to date. predicted with ELM (http://elm.eu.org/; [Dinkel et al., 2016]) Here we report the identification and analysis of TET2 PROSITE (http://prosi​te.expasy.org/; [Sigrist et al., 2012]) and SNVs identified in 189 Spanish AML patients. Spurred by the DLocalMotif (http://bioinf.scmb.uq.edu.au:8080/dloca​lmoti​f/; lack of structural information on the noncatalytic regions of the [Mehdi, Sehgal, Kobe, Bailey, & Bodén, 2013]). Caspase cleav- enzyme, we have combined bioinformatics results and avail- age sites were predicted with CaspDB (http://caspdb.sanfo​rdbur​ able functional information into a unified model of TET2 over- nham.org/; [Kumar, van Raam, Salvesen, & Cieplak, 2014]) and all architecture. We present a rigorous interpretation of over calpain cleavage sites with CaMPDB (http://calpa​in.org/predi​ 430 missense variants that affect the TET2 catalytic domain, ct.rb?cls=subst​rate; [duVerle, Ono, Sorimachi, & Mamitsuka, and provide likely explanations for ~700 additional variants 2011]). Experimentally determined posttranslational modifi- found in the regulatory regions of the protein. Finally, we pres- cations were taken from PhosphoSitePlus (http://www.phosp​ ent a proposal of classification of missense TET2 SNVs that hosite.org/; [Hornbeck et al., 2014]) or from cited references takes into account these structural and functional data. (Bauer et al., 2015; Nakagawa et al., 2015). Secondary structure was predicted with JPred4 (http://www. compb​ io.dundee.ac.uk/jpred/​ ; [Drozdetskiy, Cole, Procter, & 2 | METHODS Barton, 2015]) and PSIPRED (http://bioinf.cs.ucl.ac.uk/psipr​ ed/; [Buchan, Minneci, Nugent, Bryson, & Jones, 2013]) as- 2.1 | Patients sisted also by the results of YASPIN (http://www.ibi.vu.nl/ Samples from patients with AML, diagnosed according to progr​ams/yaspi​ nwww/; [Lin, Simossis, Taylor, & Heringa, standard criteria (Beer et al., 2010), were analyzed for TET2 2005]) and of β‐hairpins predicted with βhairPred (http://www. mutations using Sanger sequencing and NGS protocols as re- imtech.res.in/ragha​ va/bhair​ pred/; [Kaur & Raghava, 2003]). ported elsewhere (Nomdedéu et al., 2012, 2017). The impact of selected SNVs was assessed through metaserv- ers PredictSNP (http://losch​midt.chemi.muni.cz/predi​ctsnp/​; [Bendl et al., 2014]) and Meta‐SNP (http://snps.biofo​ld.org/me- 2.2 | DNA extraction and sequencing ta-snp; [Capriotti, Altman, & Bromberg, 2013]). Threading and Genomic DNA was extracted from bone marrow aspirates or modeling were performed with RaptorX (http://rapto​rx.uchic​ from peripheral blood of healthy donors and patients using ago.edu/; [Källberg et al., 2012]) or Phyre2 (http://www.sbg.bio. standard methods, and analyzed for mutations in NPM1, ic.ac.uk/phyre​2/; [Kelley, Mezulis, Yates, Wass, & Sternberg, FLT3, CEBPA, and MLL genes using well‐established proto- 2015]), and models were finally refined with ModRefiner cols (Munoz et al., 2003, 2001, 2003; Nomdedéu et al., 2012). (https​://zhang​lab.ccmb.med.umich.edu/ModRe​finer/​; [Xu & For mutational analysis of the TET2 gene, DNA was ampli- Zhang, 2011]). The impact of point variants on protein struc- fied using primers spanning the entire coding region and PCR ture and function was assessed with PROVEAN (http://prove​ conditions reported elsewhere (Delhommeau et al., 2009). an.jcvi.org/, ref. [Choi, Sims, Murphy, Miller, & Chan, 2012]), PCR products were purified with ExoSAP‐IT (Mouradov et PolyPhen‐2 (http://genet​ics.bwh.harva​rd.edu/pph2/index.shtml​, al., 2014), and sequenced using the BigDyeTM Terminator ref. [Adzhubei et al., 2010]), and CUPSAT (http://cupsat.tu-bs. Cycle Sequencing kit. Sequence analysis was performed on de/; ref. [Parthiban, Gromiha, & Schomburg, 2006]). Structure an ABI PRISM‐3100 Genetic Analyzer. In 20 cases, a tar- figures were prepared with PyMol (www.pymol.org). geted NGS approach that allowed a mean coverage of 88% of the coding TET2 sequence was also used, and variations were confirmed by Sanger sequencing (Nomdedéu et al., 2017). 3 | RESULTS AND DISCUSSION 3.1 | Novel TET2 mutations in Spanish 2.3 | Bioinformatics analysis neoplasia patients Missense mutations were retrieved from the COSMIC database Thirty‐eight TET2 allelic variants were identified in 189 stud- (http://cancer.sanger.ac.uk/cosmi​c/gene/analy​sis?ln=TET2) or ied AML patients, including two frequent SNPs: p.G355D (10 extracted from published work. Validated SNPs were taken from cases) and p.I1762V (28 cases) (Table S1). Four of the de- the NCBI database (http://www.ncbi.nlm.nih.gov/snp). For the tected mutations are reported here for the first time: c.122C>T identification of disordered regions, protein sequences were sub- (p.P41L), c.4535C>G (p.A1512G), c.4760A>G (p.D1587G), mitted to the DisMeta meta server (http://www-nmr.cabm.rutge​ and c.5087A>T (p.Y1696F) (Figure S1). Further, in 100 pa- rs.edu/bioin​forma​tics/disor​der/; [Huang, Acton, & Montelione, tients, we excluded the presence of additional variations BUSSAGLIA et al.    | 5 of 13 affecting residues that bind Zn2+ ions in TET2 catalytic unit. respectively) affect the large N‐terminal, uncharacterized Finally, none of the following missense mutations or SNPs region of the protein (Met1‐Pro1131) or the long loop was detected in 100 healthy controls: p.Y867H, p.T1270A, inserted within the catalytic unit (Cys1464‐Asn1843). p.R1359P, p.A1512G, and p.P1723S. Although both stretches are commonly described as in- trinsically disordered regions (IDRs), it seems likely that such large, relatively well‐conserved polypeptides are 3.2 | Predicted domain organization of relevant for TET2 structure and function. This prompted TET2 protein us to integrate the results of several bioinformatics tools A growing number of missense mutations and validated into a working model of full‐length TET2 architecture SNPs in TET2 (summarized in Table S2 and Table S3, (Figure 1a). (a) (c) (b) F I G U R E 2 Relevance of the non‐catalytic regions of TET2 protein for enzyme function. Focus is on experimentally verified or predicted roles of residues within the Gly93‐Gln116 TET2 stretch. (a) Close‐up of human p300 catalytic region, highlighting pockets that could accommodate the substrate lysine residue (Lys100; ref. [Zhang et al., 2017]) in TET2, as well as surrounding residues. p300 secondary structure elements are colored pink, and the CoA cofactor is shown with all its nonhydrogen atoms to the bottom‐left, color‐coded (carbon, green; oxygen, red; nitrogen, blue; phosphor, orange and sulfur, yellow). Well‐ordered PEG molecules bound close to the active site in the reported crystal structure (PDB code 4PZR, [Maksimoska, Segura‐Peña, Cole, & Marmorstein, 2014]) are also shown color‐coded (carbon, orange; oxygen, red). Molecule PEG1 overlays with the incoming lysine residue to be acetylated, while PEG2 was proposed to mimic a substrate residue with a small side chain, ideally glycine, flanking the substrate lysine. In TET2, however, Lys110 is flanked by residues with bulkier side chains, and the path of the TET2 peptide in the actual p300·TET2 complex might differ from that with canonical substrates, and/or it would require some rearrangements of residues surrounding the p300 active site for binding in a productive conformation. On the other hand, the shallow negatively charged pocket on p300 termed “P2 site” (top right in this panel) might accommodate Lys113; this interaction is critical for effective substrate binding (Liu et al., 2008). (b) 3D model of TET2 peptide Leu107‐Lys113 with acetylated Lys110 bound to HDAC1. The model has been generated according to the crystal structure of human HDAC1 bound to a substrate mimic based on the sequence of the histone H4 tail (5ICN, [Watson et al., 2016]). HDAC1 secondary structure elements are colored purple. Only side chains of HDAC1 residues that coordinate the catalytic Zn2+ ion are shown. TET2 peptide is green, with all side chains shown color‐coded. Note that the side chain of Lys113 is in the position to engage in electrostatic interactions with the bound inositol hexaphosphate (I6P) molecule, a physiologically relevant activator of HDACs (Watson et al., 2016). (c) 3D model of the Gly93‐Gln116 polypeptide of human TET2 bound to the nuclear importer, importin‐α (IMP‐α). The model has been developed based on the structure of human IMP‐α bound to CPB80 (Dias, Wilson, Rojas, Ambrosio, & Cerione, 2009). The IMP‐α helices are colored lime green, and the TET2 fragment in shown with all is non‐hydrogen atoms, color‐coded (carbon, orange; oxygen, red and nitrogen, blue). Note that the TET2 polypeptide runs in an extended, approximately antiparallel direction to the IMP‐α superhelix 6 of 13 |    BUSSAGLIA et al. Most notably, the results of our analyses strongly sug- activity, the sequences immediately preceding and following gest that TET2 contains several globular domains in addi- the catalytic unit (residues Ala1045‐Pro1131 and Lys1924‐ tion to the catalytic unit, which might adopt definite 3D Ile2002 in human TET2, respectively) are highly conserved structures upon binding for example to cognate modules in from reptiles to humans and have previously unrecognized specific coregulators. These domains are linked by IDRs counterparts in TET3 (Figure S5a,b), pointing to important that are targets of various posttranslational modifications conserved functions. The results of secondary structure pre- (PTMs; Figure 1a), and which appear to be functionally diction algorithms suggest that these regions might also fold relevant. For instance, residue Lys110 has been recently into small α+β domains. Of note, we have been able to re- reported to be acetylated by p300/CBP, which in turn re- combinantly express most of these regions in E. coli at high sults in DNMT1 binding, enhanced protein stability, and yields. The recombinant proteins are soluble in physiological higher catalytic activity in vivo (Zhang et al., 2017). (See conditions and could be straightforwardly purified without Figure 2a for a close‐up of p300 catalytic region, highlight- signs of degradation, indicating the presence of well‐folded ing pockets that accommodate the substrate lysine residue, globular domains (manuscript in preparation). as well as surrounding residues). These findings explain the negative impact of mutant p.Lys110Arg, identified in 3.3 | Genotype–phenotype correlation: a patient with myelodysplastic syndrome (Papaemmanuil a proposed classification of TET2 et al., 2013). Alternatively, mutations that interfere with missense mutations the deacetylation of AcLys100 by HDAC1/2 might result in a hyperactive TET2 enzyme. (For a model of the Leu107‐ In Table S2 and Table S3, we present a brief analysis of all Lys113 TET2 peptide bound to HDAC1, see Figure 2b). missense mutants and validated SNPs of human TET2 re- Further, a well‐conserved bipartite nuclear localization sig- ported to date, respectively. In each case, sequence conserva- nal is predicted between residues Gly93 and Asp115, and tion, impact on experimentally confirmed or predicted PTMs a 3D model of the putative complex between this TET2 and likely structural implications are considered. Considering stretch and the nuclear transport receptor, importin‐α, is the available results of structure and function studies (Hu et given in Figure 2c. A sequence alignment around this NLS al., 2013, 2015) and bioinformatics data summarized in these is given in Figure S2a and details of the putative TET2— tables and Figure 1a, we propose a hypothetical but testable importin‐α interaction interface are presented in Figures classification of the pathogenicity of missense SNVs iden- S2b–e. Alternatively, the Gly93‐Asp115 peptide might in- tified in the human TET2 gene into three major categories teract with the TPR‐repeat domain of another known TET2 (Table 1). interactor, O‐linked GlcNAc transferase (Chen, Chen, Bian, Fujiki, & Yu, 2013; Deplus et al., 2013; Vella et al., 2013), which shares structural similarities and a common bind- 3.3.1 | Severe mutations ing mode with importin‐α (Jinek et al., 2004). These inter- Among them, we distinguish between (a) exchanges that actions might be regulated by phosphorylation of residue impair binding of the essential cofactors, 2‐OG and/or Fe2+ Ser99 by AMP‐activated kinase, which has been recently (Figure 3a) and (b) mutations that would have an impor- shown to stabilize TET2 protein. This PTM thus links hy- tant impact on the global 3D structure of the TET2 catalytic perglycemia and diabetes to cancer through modification unit (e.g., by disrupting major internal H‐bonded networks, of the 5hmC landscape (Wu et al., 2018). We also note that Figure 3b, or by introducing polar/charged residues in the the highly variable Asp297‐Thr395 linker is a likely major densely packed hydrophobic core, Figure 3c). It is conceiv- target of cysteine proteinases that regulate TET2 activity: able that type Ib mutations would have a more severe impact caspases (Ko et al., 2013) and calpains (Wang & Zhang, on TET2 protein function. 2014). Removal of the N‐terminal region and concomitant loss of binding sites for the important interactors men- tioned above might explain, at least partly, the regulatory 3.3.2 | Intermediate mutations role of these proteinases. This category comprises (a) replacements of core residues of Furthermore, threading algorithms could identify accu- the catalytic unit with a limited impact on its structure and/or rate templates for some of the previously unforeseen globular stability (Figure 3d), as well as (b) mutations that would dis- domains, in particular for the Thr396‐Gln574 and Gln866‐ rupt the 3D structure of regulatory domains. Also included Lys1044 stretches. (The low, previously unappreciated se- in this class are nonconservative exchanges of strictly con- quence similarity between these two stretches is shown in served exposed residues that might weaken TET2 interac- Figure S3, 3D models for these regions are given in Figure tions with either (c) substrate DNA (Figure 3e), (d) with S4a,b, and model quality is illustrated in Figure S4c,d, re- other protein domains or with other proteins (Figure 3f), or spectively). Perhaps more relevant for TET2 enzymatic that (e) would interfere with conserved PTMs. BUSSAGLIA et al.    | 7 of 13 TABLE 1 A proposed classification of TET2 missense mutations Mutant class Mutation impact Examples Ia Impairment of enzyme activity, but overall protein structure p.R1261C/H/L/G, p.S1284F, p.H1881R/Q/N, essentially conserved. (Replacement of Fe2+/2‐OG ligands, or p.R1896M/S/G, p.S1898F major steric clashes with these side chains) Ib Severely compromised 3D structure and/or stability of the p.R1176G/T, p.L1229R, p.C1289Y/F, p.L1322Q, catalytic unit. (Introduction of polar or charged residues in p.L1329Q/R, p.R1359C/H/P, p.H1380Y/R/N/P, the hydrophobic core, disruption of major internal H‐bonded p.V1864E networks, replacement of Zn2+ ligands) IIa Creation of small cavities in protein core, destabilization of p.E1207Q/D, p.L1231P, p.W1233G, p.Y1245S/C, major secondary structure elements and/or of partially buried p.E1879Q, p.I1897S, p.M1907K H‐bonded networks IIb Impairment of the 3D structure of noncatalytic domains p.A215V, p.V238F, p.A241V, p.P463L, p.L1065F, p.K1094I, p.T1985I IIc Impaired recognition and processing of substrate DNA p.W1291R, p.K1299E/N, p.R1302G, p.S1303R/N, p.H1904R/Q, p.K1905E IId Impaired docking of other TET2 domains and/or of interacting p.R96C, p.E1206K, p.E12434G, p.D1314G, proteins (e.g., IDAX, importin‐α, OGT) p.E1320A IIe Altered posttranslational modification of strictly conserved resi- p.K110R, p.K1339R dues with functional implications IIIa Conservative mutations of buried residues within the catalytic p.I1175V, p.I1195V, p.V1199I, p.L1229I, unit, with only minimal effect on the protein stability p.F1287L, p.L1332F, p.L1360M, p.A1919V IIIb Conservative mutations of exposed, not strictly conserved resi- p.S1204C, p.K1243R, p.L1312V, p.E1357Q, dues within the catalytic unit p.E1405Q IIIc Mutations of nonconserved residues within the noncatalytic p.P23S, p.K53E, p.S334F, p.S585L, p.A727T, regions p.S1497P residues that are part of internal, complex H‐bonded networks 3.3.3 | Mutations with mild to (e.g., p.Arg1161Ser, p.Arg1176Gly/Thr) or that coordinate negligible effects Zn2+ ions (e.g., Cys1221Arg, Cys1289Ser/Phe) are predicted This category includes (a) conservative exchanges of buried as “stabilizing” by CUPSAT, even though loss of these inter- residues within the catalytic unit, which would thus be fully actions in the mutant proteins are quite likely to compromise tolerated (Figure 3g), (b) mutations that either directly or in- overall folding and stability. In conclusion, neither simple directly affect nonconserved PTMs (Figure 3h), and (c) other analysis of residue conservation nor “blind” consideration of replacements of nonconserved residues within the noncata- structural features allow for a detailed classification of TET2 lytic modules of the enzyme. variants, and we propose that our scheme should be used in- We compared our structure‐ and function based classifi- stead for predicting the likely impact of point mutations in cation with tools routinely used to predict the impact of mis- the TET2 gene. sense variants, PROVEAN and PolyPhen, and also with an Of note, the mutation pattern along the TET2 sequence algorithm that predicts changes in structural stability upon is strikingly different between solid and hematologic neo- point mutations, CUPSAT. The results of these analyses for plasms (Figure 1a). However, it remains to be investigated all SNVs that map to the TET2 catalytic unit are presented in whether TET2 variants that belong to different categories are the Table S4. As can be seen, type I mutations, for instance, unevenly distributed in myeloid neoplasia, solid tumors and are usually found among the most deleterious by PROVEAN tumor‐infiltrating lymphocytes, and whether they would have (values much lower than the threshold between “neutral” different effects on atherogenesis (Bowman et al., 2018). and “deleterious” replacements, −2.50). Type Ia/Ib vari- ants are also commonly predicted as “probably damaging” by PolyPhen. There are, however, a large number of variants 3.4 | Clinical significance of TET2 mutations that are also predicted as deleterious/probably damaging, for Previous attempts to use the TET2 gene as a predictor of clini- which careful inspection of the deposited 3D structures sug- cal variables including disease‐free survival (DFS) and overall gests at most a limited impact on the structure and/or func- survival (OS) have led to contradictory results. For instance, tion of the enzyme. On the other hand, some replacements of the presence of TET2 mutated was reported as a neutral (Bejar 8 of 13 |    BUSSAGLIA et al. (a) (b) (c) (d) (e) (f) (g) (h) et al., 2011) or even favorable prognostic biomarker in MDS risk” or “intermediate risk” than to “adverse risk” categories (Kosmider, Gelsi‐Boyer, Cheok, et al., 2009), and in chronic according to the European LeukemiaNet (ELN) classification. myelomonocytic leukemia (CMML) (Grossmann et al., 2011; Of note, TET2 mutations were quite rarely found in isolation Kohlmann et al., 2010). Further, TET2 mutation status did not in AML patients, indicating that they are not sufficient for influence OS or DFS in a cohort of cytogenetically normal overt leukemia (Papaemmanuil et al., 2016). Also along these AML patients younger than 60 years (Damm et al., 2014), and lines, the presence of mutant TET2 did not affect survival or TET2 gene status was not significantly correlated with DFS or leukemic transformation in patients with polycythemia vera OS in de novo AML (Nibourel et al., 2010). In addition, in a or primary myelofibrosis (Tefferi, 2010; Tefferi et al., 2009), large cohort of younger adult patients with AML, TET2 vari- and TET2‐mutated MPN patients were not cytogenetically dif- ants did not impact the response to induction therapy and clin- ferent from their TET2‐wild‐type counterparts (Hussein et al., ical outcome (Gaidzik et al., 2012). Finally, in a more recent 2010). Finally, the results of a recent meta‐analysis suggest study of an even larger cohort of 1,540 AML patients, TET2 that TET2 mutations may not impact prognosis on OS of pa- mutations were much more frequently associated to “favorable tients with MDS (Guo, Zhang, Zou, Fan, & Lyu, 2017). BUSSAGLIA et al.    | 9 of 13 F I G U R E 3 Representative examples of mutations affecting the structure and function of human TET2 catalytic unit. (a) Close‐up of TET2 catalytic site. Shown in the picture are only cofactors Fe2+ (orange sphere) and 2‐OG (color‐coded: carbon, pink; nitrogen, red; oxygen, blue), as well as the side chains of residues directly involved in their coordination (color‐coded as the 2‐OG cofactor, but with carbon atoms green). Note that several TET2 missense mutations affect these residues (e.g., p.Arg1261Cys/His/Leu/Gly, p.His1881Arg/Glu/Asn; class Ia mutants). (b and c), representative examples of proposed type Ib variants. (b) Mutations likely to disrupt the overall 3D structure of the catalytic unit. Shown is a close‐up around residue Arg1359. Note the intricate network of strong H‐bonds centered on its side chain, which directly donates H‐bonds to the carbonyl oxygen from Met1907 (C‐terminal half of the DSBH domain), but is also connected through water molecules to the carbonyls of Gln1348 and Arg1366 (N‐terminal half). Note also that the residue immediately preceding Arg1359 is a Zn2+ ligand. Therefore, mutants p.Arg1359Cys/ His/Pro would most likely result in the collapse of the whole 3D structure. (c) Representative example of a mutation introducing a polar or charged residue in the densely packed hydrophobic core. Replacement of Val1864 by a glutamate, as in p.Val1864Glu, would result in major clashes of the mutant Glu1864 carboxylate with the side chains of aliphatic residues shown in the picture and/or with main chain carbonyl oxygen atoms of these and/or other residues. (d) Close‐up around residue Ile1897. Only side chains of residues that make at least one vdW contact with one of the atoms of the Ile1897 side chain are shown, color‐coded according to the domain they belong to. Note that replacement of the aliphatic side chain by a shorter, polar serine, although disfavored, would not be expected to cause major structural rearrangements, as the mutant Ser1897 side chain would not clash with any of the surrounding core residues (class IIa mutation). Note in addition the somehow polar environment created by the nearby Thr1249 side chain. (e) Close‐up of the substrate DNA‐binding site; the side chains of some of the residues mutated in cancer patients are shown with all of their nonhydrogen atoms. Replacement of single residues, such as in mutants p.Arg1302Gly or p.Lys1905Glu, is likely to affect the rate of DNA oxidation, but would not be expected to completely abolish processing of the 5mC residue (class IIc variants). (f) Close‐up showing exposed, well‐conserved residues that are mutated in some cancer patients (e.g., p.Leu1312Val, p.Asp1314Gly, p.Glu1320Ala). Their relative proximity to the bound DNA oligonucleotide suggests that they might form a binding site for a TET2 cofactor such as IDAX/CXXC4, which recruits TET2 to DNA (Ko et al., 2013) (type IId mutants). (g) Close‐up around Ser1189‐Arg1201 TET2 stretch. Only a few side chains of interacting residues are shown for simplicity. Some TET2 missense variants that affect residues within this sequence are likely to be fully tolerated without any important rearrangements of TET2 protein structure (e.g., p.Ile1195Val, p.Val1199Ile; class IIIa). (h) Close‐up showing not conserved, exposed TET2 residues. Missense variants of these residues that introduce physicochemically related residues, commonly found in TET2 from other species (e.g., p.Ser1204Cys, p.Lys1243Arg, p.Glu1405Gln) are unlikely to have any impact on TET2 function (class III variants). DSBH, domain, termed double‐stranded β‐helix In striking contrast, TET2 mutations have been associ- from treatment with hypomethylating drugs, as suggested in ated with decreased OS in de novo AML patients with in- recent studies (Bejar et al., 2014). In this regard, a much bet- termediate‐risk cytogenetics compared to TET2‐wild‐type ter response to the DNA hypomethylating agent, azacitidine patients (Abdel‐Wahab et al., 2009), and other authors have has been reported in AML and MDS patients carrying TET2 corroborated this association between TET2 mutations and mutations than in wild‐type patients (82% vs. 45% response poor prognosis in AML (Aslanyan et al., 2014; Metzeler et rate, including hematological improvement; [Itzykson et al., al., 2011; Ohgami et al., 2015). Further, TET2 gene muta- 2011]). tion has been reported to negatively impact patient survival The understanding of the structural and functional conse- in CMML (Kosmider, Gelsi‐Boyer, Ciudad, et al., 2009) as quences of TET2 mutations may also be useful for the optimal well as in the most common peripheral T‐cell lymphomas design of advanced immunotherapeutic approaches. It has (Lemonnier et al., 2012). Furthermore, the nonepigenetic been recently reported the cure of a patient with chronic lym- effects of TET2 variants may also be of some relevance in phocytic leukemia carrying the class IIa p.Glu1879Gln vari- hematologic neoplasms, as it has been recently shown for ant in one TET2 allele, after disruption of the second TET2 other TET family members (Khoueiry et al., 2017). allele in its T cells through lentiviral integration (Fraietta Notwithstanding other differences in the molecular pa- et al., 2018). Altogether, consideration of the structure and thologies of various hematological neoplasms, we consider function impact of TET2 SNVs might have important impli- that the apparently contradictory results listed above may cations for predicting the prognosis of leukemia patients and be due, in part, to studying patient cohorts in which TET2 their stratification in future clinical trials. mutations have largely different structural/functional conse- quences. Therefore, establishment of a scoring system and ACKNOWLEDGMENTS patient stratification according to our proposed classification might be helpful in future studies aimed at establishing the We thank a large number of colleagues for answering our clinical relevance of TET2 variants. In particular, we might questions regarding TET2 mutants identified in their laborato- expect a significant correlation between the presence of se- ries. This work was supported by grants SAF2014‐57994‐R, vere TET2 mutations (classes Ia/Ib) and a poor disease out- AGAUR 2014‐SGR‐01214 (P.F.‐P.), FIS PI 10/1739, come. Furthermore, these patients could benefit the most 13/2729, and AGAUR SGR383 (J.N.). 10 of 13 |    BUSSAGLIA et al. CONFLICT OF INTEREST mutations in myelodysplastic syndromes. New England Journal of Medicine, 364(26), 2496–2506. https​://doi.org/10.1056/NEJMo​ None of the authors declare any conflict of interest. a1013343 Bendl, J., Stourac, J., Salanda, O., Pavelka, A., Wieben, E. 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