Enzymes invo lved in DNA and h is tone mod if ica t ion pa thways

F^IGURE 8: Enzymes involved in DNA and histone modification pathways. The top panel depicts DNA modifications, such as DNA methylation and demethyla- tion, and the enzymes involved; the bottom panel shows histone modifications and the enzymes involved. Examples for each class of enzyme are given. 5hmC, 5-hydroxymethylcytosine; 5mC, 5-methylcytosine; BAZ1B, tyrosine protein kinase BAZ1B; BRCT, BRCT domain-containing protein; CHD, chromodomain helicase DNA-binding protein; DIDO1, death-inducer obliterator 1; DNMT, DNA methyl- transferase;HAT, histone acetyltransferase; HDAC, histone deacetylase; HMT, histone methyltransferase; KDM, lysine-specific histone demethylase; MECP2, methyl-CpG- binding protein2; PPP, serine/threonine; RPS6K, ribosomal protein S6 kinase;SAM, S-adenosyl-l-methionine; TAF3, transcriptioninitiationfactor TFIID subunit 3; TET, TET 5mC hydroxylase. From (Plaas C. et al., Nat Rev Genet, 2013)

(CpG)throughtheDNA sequence and about 70-80% of CpGs aremethylated. CpGs are not found frequently in human genome andthey are mostly present on gene promoter regions inclustersthatare called CpGislands (Weber et al., 2007). Levels of DNA methylation correlates with chromatin accessibility, andacquiredabnormal methyla- tion, which can be dueto mutationin genes controlling theDNA methylation factors (Figure 9) is a commonevent indivers diseasesincludingcancer. This resultsin tran-scriptional disorders and can beinherited by daughtercells (Jones andTakai, 2001).It iswell known that 5mCis associatedwith atranscriptionallyrepressed chromatinstate, and DNA methylation at specific genomic loci, including lineage-specific genes, can help to shapethe cellularidentity duringdevelopment (Kohli and Zhang, 2013).

The enzymes responsible forthis modification, DNMTs, are groupedintwocat- egories: de novo and maintenance DNMTs. Initiallythe DNA methylation patterns are established by the denovo DNA methyltransferases: DNMT3A, (~102 kDa), and DNMT3B,(~96 kDa), during embryonic development (Law andJacobsen, 2010). These methyl marks arethen maintained duringcell divisions throughthe action ofthe maintenance methyltransferase, DNMT1,(~183 kDa), which preferentially methy- latestheunmethylated strand ofhemimethylated DNA duringDNA replication. These mechanisms are crucial for development, sincemice deficientinDNMT3B or DNMT1, locatedrespectively on 20q11 and 19p13loci, are embryonically lethal and DNMT3A-null mice dieat 4 weeks of age(Wuand Zhang, 2010).

 

Ley and colleagues performed whole-genome sequencing onAML sampleswith normal cytogenetic andidentifiedforthefirst time somatic mutation of DNMT3A.

DNMT3A gene,located on 2p23 locus,was found mutatedin 22%,(62 of 281) ofAML cases, andthese mutatedcases were associatedwith a poor outcome(Ley et al., 2010). In another study, the direct sequencing demonstratedalso thatDNMT3A was mutated in 87(17.8%) of 489 patientswith AML (Tholet al., 2011).

Exome sequencing inM5 subtype ofAML demonstratedthat (20.5%) 23 of 112 cases presentedDNMT3A mutations andthese mutantsshowed reduced enzymaticac- tivity oraberrant affinity to histone H3 in vitro. The majority ofsomatic DNMT3A mutationsresulteither in premature truncation ofthe protein product(nonsense or

frameshift mutations), or occur at a single amino acid, R882 (60% of mutatedcases, 37 of 62 mutations) and has been shown to decrease catalyticactivity andDNA binding affinity (Gowher et al., 2006). These findings suggest acontribution of aberrantDNA methyltransferase activity tothe pathogenesis of acutemonocytic leukemia (Yan et al., 2011).

DNMT3A mutations have also been observed in BPDCN patients, Menez et al. have assessed whole-exome sequencing (WES) on 3 BPDCNcases and onthebasis of these data,theydesigned a resequencing approach toidentify mutationsin 38selected genes in 25 BPDCN patients. They observed that half ofthetumors had mutations affecting eithertheDNA methylation or chromatin-remodelingpathways, with mutated DNMT3A,observed in BPDCNcases. DNMT3A mutations werethen correlated with a poor prognosis inthese patients(Menezeset al., 2014).

   

 TET2

   

TET2 coding gene is located on 4q24 locus and codes forTET2 protein (~220 kDa), one ofthethree proteins ofthe TET (Ten-Eleven Translocation) family,which are evo-lutionarily conserved dioxygenases that catalyze the conversion of 5-methyl-cytosine (5-mC) to 5-hydroxymethyl-cytosine (5-hmC)(Figure 9) and promoteDNA demethy- lation.In addition to theirhydroxylase activity, TET proteins recruit the O-linkedβ  - D-N-acetylglucosamine (O-GlcNAc) transferase (OGT) enzyme to chromatin, which promotes post-transcriptional modifications of histones and facilitates geneexpression (Tahiliani et al., 2009). Pathways foractive DNA demethylation includethe oxidation of 5mC by TET 5mChydroxylases.

 

Alternative mechanisms to oxidative demethylation of cytosines are based on deamination and subsequent base-excision repair (BER) ornucleotide-excision repair. These demethylation pathways arereplication-independent mechanismsallowing rapid conversions frommethylated tounmethylated states.

Duringthe development ofa neoplasm,the degree of hypomethylation of (recombinogenic sequence) repeats isanother consequence ofundermethylated DNA. These hypomethylated transposons can be translocated to other genomic regions and

cases (53%)(Jardin et al., 2011). Menez et al. by using next generationsequencing (NGS) analyzed 42 BPDCN patients and found that 36% of these patients presented TET1/2 mutations.

Also in arecent study and by usingNGS on a panel of recurrently mutated genes in AML,it has been suggestedthatan appreciable subset of BPDCNcases have TET2 mutations (Alayed et al., 2013).In this studyNGS were performed on 5 of these pa-tients andTET2 mutations were found in 4cases (80%). Thisraises the possibilitythat perturbations in DNA methylating modifying enzymes, here TET2 mutations, play a roleinthe pathogenesis of BPDCN(Alayedet al., 2013).

The major functional consequence of TET2 mutation is ahypermethylation phe-notype thus indicating thatTET2 mutated cancers should respond tohypomethylating agents(Shih et al., 2012).

   

 IDH2

   

The homodimeric enzymes, Isocytrate dehydrogenases 1 and 2 (IDHs 1 and 2), IDH1 (~235 kDa) and IDH2 (~51 kDa), both catalyzethe oxidative decarboxylation of isoc-itratetoα-ketoglutarate (α-KG) and reduceNADP⁺toNADPH, but theyhave different subcellularlocations:IDH1 inthecytoplasm and IDH2in mitochondria. Their enzy-matic activity isthenfollowed by a series of additional enzymes, whichfurther process 2-ketoglutarate to produceenergy incells. IDH1and IDH2genes arelocated on 2q33.3 and 15q26.1loci respectively. IDH1 mutations were discovered in patientswith acute myeloid leukemia(AML)through whole genome sequencing(Mardis et al., 2009,Ward et al., 2010). Sanger sequencing and SequenomMassAR- RAY platform (Sequenom) have demonstrated thatIDH2 mutations were present in angioimmunoblastic lymphoma cases (Cairnset al., 2012). Mutations are heterozygous and occurat thearginineresidue ofthe enzyme’s active site and cause bothloss of normal enzymefunction and gain of function (McKenney and Levine, 2013). IDH mutations induce a neomorphic function to produce the oncometabolite 2-hydroxyglutarate (2HG), which can

   

F^IGURE 9: Associated mutations in the DNA methylation and demethylation path- way. The process of DNA methylation and the enzymes involved are shown. En- zymes affected by somatic mutations in myeloid leukaemia, are marked with an asterisk. AID, activation-induced cytidine deaminase; BER, base-excision repair; DNMT3A, DNA methyltransferase 3A; IDH, isocitrate dehydrogenase; NADP+, nicotinamide adenine dinucleotide phosphate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; TDG, thymine DNA glycosylase, 5fC (5-formylcytosine), 5caC (5-carboxylcytosine). From (Shih et al., Nat RevCancer, 2012)

   

alter the epigeneticlandscape. IDH mutations are most common in patientswith cyto- genetically normal AML (CN-AML), andthe prevalence ofIDH1 and IDH2 missense mutations among patients with AML is between 5% and 20%(McKenney andLevine, 2013). By using WES, Menezet al. analyzed atotal of 25 BPDCN patients andreported (1/25) 4% and (3 of 25) 12% of these cases were mutated forIDH1 and IDH2 genes respectively (Menezeset al., 2014).

2.2.2.6 Mutations affecting histone-modulatingfactors MLL

Mixed lineage leukemia (MLL) gene is located at chromosome 11, band q23 and en- codesa histone methyltransferase that directly binds DNA and positively regulates genetranscription, and particularly regulates theexpression ofhomeobox (HOX) genes. MLL is a member ofTrithorax group(TrxG) of proteins andisknown because ofits involvementinleukemia(Schuettengruberet al., 2011).

MLL (~180 kDa) isa large multi-domain protein, ubiquitously expressed in hematopoietic cells including stem and progenitor populations. Rearrangements of MLL,the human homolog oftheDrosophila gene trithorax, are associatedwith aggres-sive lymphoid and myeloid acuteleukemiasin both children and adults.MLL mutations arefound as afusion proteinthat has no H3K4methylation function(Krivtsov and Arm-strong, 2007). MLL fusions are highlyefficient oncoproteinsthattransform hematopo i-etic progenitors and causeaggressive leukemia(Slany, 2009). Approximately 10% of human acute leukemiasharbor MLL translocations andMLL isalso affected by abnor- malities such as partial tandem duplications (PTDs) ofexons encodingthe Nterminus of MLL in about 10% of normalcytogenetic AML cases (Muntean andHess, 2012).

 

Depending onthe fusion partner, theyseem to eitheraffect chromatin-associated processes or, morefrequently, specifically stimulatetranscriptionalelongation (Mae th-ner et al., 2013). “Eleven NineteenLeukemia” protein (ENL) originallydiscovered as an MLL fusion partnerinthe recurrenttranslocationt(11;19) (Maethneret al., 2013). ENL belongs toa protein complex called EAP as “elongation assisting proteins”.It has been demonstrated that EAP has also the capacity toaccommodate MLL fusion proteins. Thereforthe constitutive recruitment ofEAP toMLL targetlociis respons i-ble for persistent targettranscriptionthrough stimulation oftranscriptionalelongation. This mechanism resists differentiation stimuli andtherefore causes a maturation arrest (Mueller et al., 2009). TheMLL rearrangements have beenalso reportedin a BPDCN case with MLL-ENL fusion gene, suggesting itsimplicationinthedevelopment ofthis disease (Toya et al., 2012). Two BPDCN cases with 11q23 abnormalities were also

reportedin another study on 5cases ofDC malignancy, which carriedt(9;11)(p21; q23)

A model predictingtherecruitment of PRC2 to chromatin has been proposed: first the interaction of JARID2 and AEBP2 with DNA, next the interaction of the histone chaperones RbAp46 or 48 with histonesH3 or H4,thentheinteraction ofEED with the product of PRC2 catalysis, H3K27me3 andtheinteraction of PRC2 components with a long non codingRNA (ncRNA). The binding specificity could be modulated bythevar i-ation inthe composition ofthe PRC2 holoenzyme and posttranslational modifications (PTMs) of itscomponents. The ncRNA isthought to bea major player inthe cell- specificrecruitment of PRC2(Figure 10). A large pool oflongncRNA may functionto directthe complex to definedtargetgenes andthistargetingmay not necessarily entail linear base pairingwith target sequences,but insteadthetertiary structure oftheRNA may bethekey to specifictarget generecognition(Margueron andReinberg, 2011).

   

F^IGURE 10: Schematic representation of thePRC2 holoenzyme at chromatin. Puta- tiveinteractionswith either DNA or histones thatcould explain PRC2 recruitment are highlighted. From (Raphae¨l Margueron and Danny Reinberg, Nat Rev,2011)

       

  PRC2 catalytic unit: EZH2

   

EZH2 gene located on 7q36 locus, encodes for aprotein (~95 kDa), which is the ca t-alytic subunit ofthe PRC2. EZH2 is a highlyconserved histonemethyltransferase that methylates lysine 27 of histone 3. It containsconserved regions includingcysteine-rich

amino acid stretch thatprecedes the carboxy-terminalSET domainwith HMTase ac- tivity (Xiao, 2011).EZH2-mediated methylation is a potentialindependent mechanism forepigeneticsilencing oftumorsuppressor genes in cancer. Oncogenic activity of overexpressed EZH2 has been shown in a wide range of cancers suchas those of the prostate and breast(Deb et al., 2013,Yu et al., 2007).

EZH2 has been reported to harborvarious heterozygous mutationsat tyrosine 641 (Y641) inthe C-terminalSET domain (Morinet al., 2010) resultingin again of func-tion. Althoughinitially reportedto be aloss-of-function mutation, subsequent studies have shown that the Y641 mutation actually results in a gain of function (Yap et al., 2011). These mutants have enhanced catalyticefficiency for dimethylated H3K27me2, comparing tothewild type showing substrate preference forunmethylated H3K27 and monomethylated H3K27me1, therefore both thewild type and the Y641 mutantscan work togethertoincreasethelevels of H3K27me3.It has been reportedthat suchmu- tations arefoundin 7% offollicular lymphomas and 22% of germinal center B-cell and diffuse large B-cell lymphomas(DLBCLs)(Tan et al., 2014).

Unlike in B-cell lymphomas,EZH2 mutations in myeloid malignancies are inac-tivating/hypomorphic (Khan et al., 2013).Inarecent study, Khan et al. studiedthe mutational status by Sanger sequencing in myeloid malignancies and foundEZH2 mu- tations in 8% of cases (n=469cases examined). In addition, they observed reduced EZH2 expression in 78% ofcases with hemizygous deletion (-7/del7qcases involving EZH2 locus), and 41% ofcases with diploid chromosome 7(Khanet al., 2013).

Another study suggestedthat in patientswith myeloid disorders,EZH2 mutations were found most commonly in patientswith myelodysplastic/myeloproliferative neo-plasms(27 out of 219 individuals, or 12%) andinthosewith myelofibrosis(4 out of 30 individuals, or 13%)(Ernstet al., 2010).

In additiontoitsroleas atranscriptionalrepressor, several studieshave shown that EZH2 may also functionintarget geneactivation. It has beenrecentlyfoundthat a sub-set ofEZH2-bound genes did not bindthe PRC2subunit SUZ12 or display H3K27me3. Many ofthese genes weredown regulated uponEZH2 knockdown, suggestingthatthe role of EZH2 as an activator was independent ofthe PRC2complex (Xuet al., 2012).

They found that the oncogenic function of EZH2 in resistant prostate cancer cells is independent ofitsroleas atranscriptionalrepressor, but itinvolvesthe ability ofEZH2 toact as acoactivator for criticaltranscriptionfactors suchas androgenreceptor.

   

  PRC2 accessorysub-unit: ASXL1  

 

The Additional sex combslike 1(Asxl1) gene,located on 20q11locus, is 1 of 3mam- malian homologs oftheAdditional sex combs(Asx) gene of Drosophila.Asx genes are unusual because they belongto bothPcG genes, which silence their targets, andtrxG

The Additional sex combslike 1(Asxl1) gene,located on 20q11locus, is 1 of 3mam- malian homologs oftheAdditional sex combs(Asx) gene of Drosophila.Asx genes are unusual because they belongto bothPcG genes, which silence their targets, andtrxG

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