chromatin remodeling defects in pediatric and young adult glioblastoma: a tale of a variant histone...

M I N I - S Y M P O S I U M : W h e n G e n e t i c s M e e t s E p i g e n e t i c s — A N e w O p t i o n f o rT h e r a p e u t i c I n t e r v e n t i o n i n B r a i n Tu m o r s ?

Chromatin Remodeling Defects in Pediatric and Young AdultGlioblastoma: A Tale of a Variant Histone 3 TailAdam M. Fontebasso1; Xiao-Yang Liu2; Dominik Sturm3; Nada Jabado1,2,4

1 Division of Experimental Medicine, McGill University and McGill University Health Centre, Montreal, QC, Canada.2 Department of Human Genetics, McGill University and McGill University Health Centre, Montreal, QC, Canada.3 Division of Pediatric Neuro-oncology, German Cancer Research Centre (DKFZ), Heidelberg, Germany.4 Department of Pediatrics, McGill University and the McGill University Health Centre Research Institute, Montreal, QC, Canada.

Keywords

ATRX, chromatin remodeling, glioblastoma,H3F3A, pediatric, TP53, young adult.

Corresponding author:

Nada Jabado, MD, PhD, Department ofPediatrics, McGill University and McGillUniversity Health Centre, 4060 Ste-CatherineWest, PT-239, Montreal, Quebec, Canada H3Z2Z3 (E-mail: [email protected])

Received 28 December 2012Accepted 29 December 2012

doi:10.1111/bpa.12023

AbstractPrimary brain tumors occur in 8 out of 100 000 people and are the leading cause ofcancer-related death in children. Among brain tumors, high-grade astrocytomas (HGAs)including glioblastoma multiforme (GBM) are aggressive and are lethal human cancers.Despite decades of concerted therapeutic efforts, HGAs remain essentially incurable inadults and children. Recent discoveries have revolutionized our understanding of thesetumors in children and young adults. Recurrent somatic driver mutations in the tail ofhistone 3 variant 3 (H3.3), leading to amino acid substitutions at key residues, namelylysine (K) 27 (K27M) and glycine 34 (G34R/G34V), were identified as a new molecularmechanism in pediatric GBM. These mutations represent the pediatric counterpart of therecurrent mutations in isocitrate dehydrogenases (IDH) identified in young adult gliomasand provide a much-needed new pathway that can be targeted for therapeutic development.This review will provide an overview of the potential role of these mutations in alteringchromatin structure and affecting specific molecular pathways ultimately leading to gliom-agenesis. The distinct changes in chromatin structure and the specific downstream eventsinduced by each mutation need characterizing independently if progress is to be made intackling this devastating cancer.

Primary brain tumors (originating in the brain) are the leadingcause of cancer-related death in children under the age of 20, nowsurpassing leukemia, and the third leading cause of cancer-relateddeath in young adults aged 20–39 years (61). Astrocytomas are themost common primary brain tumor across the lifespan, and thehighest grade, glioblastoma multiforme (GBM), remains essen-tially incurable despite decades of concerted therapeutic efforts(14). Similar histology of pediatric and adult tumors has causedcurrent childhood GBM treatments to be fueled by adult studies,and show, across the board, little therapeutic advance. One impedi-ment to treatment is that GBM is diagnosed as a single entity bypathologists who cannot discriminate potential genetic drivers andmolecular subtypes. This impacts the design and outcome of clini-cal trials and it likely contributes to the apparent inherent resist-ance of GBM to adjuvant therapies and poor progress in improvingsurvival or the quality of life of patients and their families. Othercrucial obstacles to progress are the lack of reliable in vitro and invivo models for pediatric GBM based on the lack of identifiedgenetic drivers specific to children until very recently. Conse-quently, better stratification of patients based on tumor biology,improved identification of relevant therapeutic targets and thedesign of experimental “companion” models to test compoundsaffecting specific genetic/molecular drivers are essential for thera-peutic breakthroughs in this deadly disease. This review will

provide a short overview of gliomas across the lifespan and recentmolecular advances that led to the identification of recurrent muta-tions in H3F3A, which are genetic drivers specific to pediatricGBM, their role in chromatin remodeling and the presence of atleast six distinct molecular subgroups in GBM across ages thatneed to be tackled separately to achieve future therapeutic successin this deadly cancer.

GENERAL OVERVIEW OF GLIOMASACROSS AGEGliomas are heterogeneous and are classified by the World HealthOrganization (WHO) according to the presumed cell of origin(41). WHO grade I and II gliomas are commonly referred to aslow-grade gliomas (LGG), while WHO grade III (anaplastic) andIV (GBM) tumors are regarded as high-grade gliomas (41). Tumorgrade, age at diagnosis and degree of surgical resection areregarded as the most important prognostic factors across thelifespan (35). The frequency, anatomic location, progression modeand pathologic spectrum of gliomas differ in children and adults.Oligodendroglial differentiation and progression of lower gradegliomas to higher grade tumors are rare in children, and the vastmajority of pediatric GBM occur de novo. A significant proportionof pediatric gliomas are low-grade tumors with a high prevalence

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© 2013 The Authors; Brain Pathology © 2013 International Society of Neuropathology

of grade I astrocytomas and rare grade II astrocytomas, whereas inadults, high-grade gliomas represent the vast majority of primarycentral nervous system tumors. Tumors in the optic pathway, and ininfra-tentorial locations such as the cerebellum and brainstem arethe most common localizations in children, whereas supra-tentorial tumors are more frequent in adults and include locationssuch as the thalamus and cerebral cortex. This is of importance asthe cell of origin, the microenvironment and chromatin conforma-tion will differ based on age, cell type and anatomical localizationand are, as we have shown, relevant in pathogenesis (8, 36, 40, 59,60). We and others have shown that grade I tumors, which repre-sent ~25% of all pediatric brain tumors, are characterizedby genetic alterations in the mitogen-activated protein kinase(MAPK) pathway (27, 31, 32, 53) and constitute one biologicalparadigm (13, 24, 27, 28). Grade II astrocytomas are rare in chil-dren (<5% of brain cancers) and high-grade astrocytomas (HGA,grades III and IV) account for ~10%–15% of all pediatric braintumors when combined with diffuse intrinsic pontine gliomas(DIPG), which are high-grade gliomas that occur in the brainstem,with an incidence close to 0.6/100 000 in children aged 0–19 yearsand a dismal mortality rate close to 90% at 5 years (58). Thera-peutic advances remain low and this high mortality occurs regard-less of patient’s age. This is despite numerous clinical trialscombining surgery, radiotherapy, chemotherapy and, in recentyears, the development of many therapies including temozolo-mide, and targeted agents such as receptor tyrosine kinase andangiogenesis inhibitors, suggesting that these efforts are not suf-ficient and/or adapted to counter tumor progression.

GBM TUMORS IN CHILDREN ANDYOUNG ADULTS ARE NOT A SINGLEDISEASE AND RESULT FROMMUTATIONS IN GENES AFFECTINGCHROMATIN REWIRINGThe Cancer Genome Atlas project (TCGA) and other groupsperformed genomic and epigenomic analysis and targetedsequencing of adult GBM samples (1, 7, 9, 48, 50, 68). Theyrevealed GBM in adults to be highly heterogeneous and identifiedthe crucial role for isocitrate dehydrogenase 1 or 2 (IDH) meta-bolic pathways in the genesis of secondary GBM, while EGFRamplification and gain of function mutations as well as PTENand/or CDKN2A/B loss target de novo adult GBM. Indeed, recur-rent IDH1 and 2 mutations are found in up to 80% of grade II andIII adult gliomas and secondary GBM but rarely in GBM occur-ring de novo (primary GBM) (50, 68). Pediatric GBM are mor-phologically indistinguishable from adult GBM, which promptedclinicians and investigators to view them as the same disease. Wehave, however, disproved this assumption and our group and othersdemonstrated the importance of analyzing the unique biologyof these tumors in children. Indeed, while mutations in TP53,CDKN2A and PIK3CA are common to all HGA, PTEN mutationsand EGFR amplifications occur in less than 10% of pediatric GBM(54, 55), while IDH mutations are rare in children (less than 5% ofGBM) and their frequency is higher in adolescents (56). Pediatricand adult HGA have different gene expression profiles and DNAcopy number alterations (2, 20, 23, 52, 57) including a higherproportion of amplification of PDGFRA in children, especiallyafter radiation therapy (52). Up to recently, DIPG, which occur

mainly in children aged between 5 and 10 years and have a par-ticularly grim prognosis with an overall survival rate at 2 years lessthan 10%, have been considered to be molecularly distinct fromsupra-tentorial GBM (51, 69).

Using next-generation sequencing technology, we recently iden-tified a new molecular mechanism driving GBM in children,namely two recurrent heterozygous somatic mutations in H3F3A,which encodes the replication-independent histone 3 variant H3.3(59). These mutations lead to amino acid changes in key residues(K27M, G34R/G34V) and were identified in 35% of supra-tentorial pediatric and young adult HGA (59). This is the firstreport to identify mutations in a regulatory histone in humans andthese mutations are the pediatric counterpart of the recurrent IDHmutations identified in young adult GBM, which indirectly affectthese histone marks (50, 68). H3.3 mutations significantly over-lapped with mutations in TP53 and in ATRX (a-thalassemia/mental retardation syndrome-X-linked) (59) and less frequentlyin the ATRX heterodimer DAXX, which encodes subunits of achromatin remodeling complex required for H3.3 incorporation atpericentric heterochromatin and telomeres (16, 39). Using an inde-pendent cohort of ~790 gliomas across age, group and grade, wefurther showed H3.3 mutations to be specific to high-grade tumors,to be prevalent in children (incidence <3.4% in adult HGA) and tobe mutually exclusive with IDH mutations (59, 62). We furtheridentified that K27M-H3.3 characterize 71% of brainstem high-grade gliomas (DIPG) (34) and 80% of thalamic pediatric GBM(62). G34R/V-H3.3 in HGA and K27M-H3.3 in HGA and DIPGwere independently identified by another group, which also iden-tified K27M in the related canonical histone H3.1 in 18% of DIPG(67). H3F3A and ATRX/DAXX were not part of the close to ~600genes initially sequenced in adult GBM by the adult consortiaincluding TCGA. This and their relative specificity to childrenexplain why these mutations were not previously identified despitetheir staggering frequency. Rather than selecting and sequencinggenes thought to be important, the use of unbiased genome-widesequencing methods to cover all protein-coding genes in samplesfrom diseased and normal tissue, and the enrichment in pediatricsamples helped in identifying these mutations. Next-generationsequencing technologies have currently become the method ofchoice to interrogate a tumor genome based on coverage andcost-effectiveness. Remarkably, H3F3A mutations seem for thetime being specific to pediatric HGA and have not yet been iden-tified in other pediatric tumors including other brain tumors orleukemia (17) or in available datasets on adult cancers. However,one needs to bear in mind that most of these samples are from adultcohorts and that possibly enrichment of a specific rare tumorsubset may still reveal presence of these mutations that mayotherwise be overlooked even in children.

H3F3A AND IDH MUTATIONS ARISE INDISTINCT ANATOMICAL LOCATIONSOF THE BRAIN AND MAY ARISE FROMDISTINCT DEVELOPMENTAL TIMEPOINTS AND CELLULAR ORIGINSOur findings indicate neuroanatomical and age specificities of themutation type and the combination of mutations in this settingrelative to older age GBM and point to a developmental defect atthe origin of pediatric and young adult HGA (Figure 1) (34, 59,

Fontebasso et al Chromatin Remodeling Defects in Pediatric and Young Adult Glioblastoma

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62). Indeed, K27M-H3.3 mutations characterize midline GBM andoccur in younger children with brainstem and thalamic tumors(70%–80% of all HGA cases in these regions, median age 10.5years, range 5–23 years) (34, 59, 62). They overlap with TP53mutations in 80% of cases regardless of location, while ATRXmutations occur in only 50% of cases and favor older children andthalamic location (only 20% of DIPG are mutant for ATRX) (34).G34R/V-H3.3 mutations are mainly found in HGA located withinthe cerebral hemispheres (34, 59, 62). They occur in patientsaround the threshold between the adolescent and adult populations(median age 18 years, range 9–42 years) and almost always overlapwith mutant TP53/ATRX. IDH alterations affect younger adults(median age 40 years, range 13–71 years) and occur in the cortex,mainly in the frontal lobes suggesting that these tumors also arisefrom a neural precursor population that is spatially and temporallyrestricted in the brain (38). IDH alterations are gain of function,heterozygous, somatic mutations and have been shown to beinitiating events in gliomas (50, 65, 66). Intriguingly, they alsoseem to require association with other genetic events to achievefull-blown tumorigenesis. They are associated with two mutuallyexclusive genetic alterations, TP53 alterations and 1p19q co-deletions (5, 49) that respectively characterize astrocytic and oli-godendroglial IDH-mutant gliomas. We (34, 40, 59) and others(29, 33) recently showed that mutations in ATRX characterize IDH-and TP53-mutant astrocytomas in young adults with low- andhigh-grade tumors. Furthermore, ATRX alterations are specific toastrocytic tumors and are mutually exclusive with CIC mutationsand loss of 1p19q, which characterize oligodendrogliomas. ATRXinactivating mutations thus characterize older children with GBMand adult IDH-mutant gliomas of the astrocytic lineage arguing forthe importance of an H3F3A & ATRX & TP53 or an ATRX &IDH1/2 & TP53 mutant phenotype in their early development andprogression and have now been found in pediatric and adult GBM(25, 59), older patients with neuroblastomas (10, 45) and in

pancreatic neuroendocrine tumors (29). Mutations in ATRX/DAXXmay interfere with H3.3 incorporation at these loci, thus compro-mising the structural integrity of the chromosome. Supportiveof this hypothesis is the presence of alternative lengthening oftelomeres (ALT), a telomerase-independent telomere maintenancemechanism, in tumors with ATRX mutations (25, 59). ALT seemsto be promoted by loss of ATRX that may increase the rate ofhomologous recombination at telomeres and pericentric hetero-chromatin and thus lead to increased lengthening of the telomeresthrough an alternate mechanism to increased telomerase activity,providing cells with the capacity for unlimited cellular prolifera-tion. Based on the role of ATRX-DAXX in H3.3 deposition inspecific areas of the chromatin, in telomere maintenance and ingenomic stability (6, 26), these findings further support the role ofchromatin modifications in the genesis of HGA in children andyoung adults.

ALTERATIONS IN THE HISTONE CODEUNDERLIE PEDIATRIC AND YOUNGADULT GBM AND INDUCE DEFECTS INCHROMATIN REMODELINGVirtually all DNA processes are regulated by the histone code,including replication and repair, regulation of gene expression andcentromere and telomere maintenance (11). Accordingly, muta-tions in genes affecting histone post-translational modifications(PTMs) are increasingly described in cancer (11). Histonespackage and organize DNA at the level of the fundamental unit ofchromatin, the nucleosome. Nucleosomes can be modulated by alarge variety of covalent PTMs mostly occurring in the N-terminaltails of histones, and also by the incorporation of histone variants(18, 21, 22). H3.3 is a universal, replication-independent histonepredominantly incorporated into transcription sites and associatedwith active and open chromatin [reviewed in (18, 63)]. H3.3 not

Figure 1. Neuroanatomical and age specificity of isocitrate dehydroge-nase (IDH), K27M-H3.3 and G34R/V-H3.3 mutations in high-grade astro-cytomas (HGA). K27M-H3.3 or H3.1 (yellow stars) occur mainly inbrainstem HGA and K27M-H3.3 mainly thalamic HGA (70%–80% of allGBM in these locations). This H3.3 mutation is inconsistently associatedwith ATRX mutations. G34R/V-H3.3 occur mainly in the cerebral hemi-spheres similar to IDH mutations which have been previously shown to

have a predilection for the frontal cortex. Both H3.3 mutations aresignificantly associated with ATRX mutations (purple filled circles) andTP53 mutations (not represented here for clarity issues). IDH and H3.3mutations represent distinct disease entities that possibly arise fromseparate cellular origins as the result of largely non-overlapping sets ofmolecular events. Note: the size of the shape illustrating each mutationis approximately proportional to the % identified in (34, 59, 62).

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only functions as a neutral replacement histone, but in additionparticipates in the epigenetic transmission of active chromatinstates and is associated with chromatin assembly factors in large-scale replication-independent chromatin remodeling mechanisms(37, 42). Its role in histone replacement at active genes and pro-moters is conserved in the single histone H3 present in yeast,indicating its importance throughout evolution (18). This histonevariant is actively loaded in the developing brain, replacing otherresident histones 3.1 or 3.2 (4). Several PTMs regulate histonefunction in the nucleosome. Reversible methylation of severalhistone lysine residues is mediated by distinct histone methyltrans-ferases or demethylases to specific histone Lys (K) or Arg residues.Polycomb repressor complex 2 (PRC2) recruitment is associatedwith methylation at K27 that represses transcription and cell dif-ferentiation (3). Gain of function mutations in EZH2, the mainK27 trimethyltransferase, has been identified in leukemias andlymphomas (44, 46, 70), and overexpression of this gene was alsoidentified in many cancers including medulloblastoma (30, 47).Thus, altered methylation or loss of acetylation of K27 is a poten-tial driver of gliomagenesis. Methylation of K36 has been widelyassociated with active chromatin but also with transcriptionalrepression, alternative splicing, DNA replication and repair, DNAmethylation and imprinting, and the transmission of memory ofgene expression from parents to offspring [reviewed in (4)].H3.3K36 methylation is potentially disrupted by the G34R/V-H3.3mutation. Indeed, glycine 34 of histone H3 (H3G34) lies in closeproximity to lysine 36 (H3K36), a residue that regulates transcrip-tional elongation. The G34R/V-H3.3 mutation may impact theability of histone-modifying complexes to methylate or acetylateH3K36, thereby altering the transcription of several target genes.Strikingly, our gene expression analysis revealed different geneexpression patterns between samples with the K27M-H3.3 muta-tion vs. samples with the G34R/V-H3.3 mutation, suggesting thateach mutation favors the expression of a specific genetic program.IDH mutations that give a neomorphic function to these enzymesenable the generation of high quantities of 2-hydroxyglutarate(15), an oncometabolite. This oncometabolite, in turn, competi-tively inhibits the activity of histone demethylases (43), affectingchromatin structure through alteration of histone PTMs. Interest-ingly, the oncometabolite produced by IDH1 mutations impairshistone demethylation, affecting UTX (H3K27me) and KDM4A/JMJD2A (H3K36/K9me) resulting in a block to cell differentiation(12), further supporting a role in gliomas for altered methylation ofthese residues.

INTEGRATED EPIGENETIC ANDGENETIC ANALYSIS IDENTIFIES ATLEAST SIX BIOLOGICALLY DISTINCTGBM SUBGROUPS ACROSS AGESAltogether, findings from several groups converge to indicate thatalterations of the histone code are at the origin of pediatric andyoung adult HGA. DNA methylation patterns are better correlatedwith histone lysine methylation patterns than with the underlyinggenome sequence context (19) and, in recent years, a distinctglioma-CpG-island methylator phenotype (G-CIMP) was identi-fied and found to correlate with IDH1-mutant gliomas (48, 64). Ourinvestigation of the genome-wide DNA methylation patterns usingthe Illumina 450k methylation array in a cohort of 210 GBM from

children and adult patients (62) sub-classified GBM into at least sixdistinct epigenetic groups, indistinguishable by histological appear-ance, but correlating with molecular genetic factors as well as keyclinical variables such as patient age and tumor location. Indeed,IDH1 mutations characterized, as expected, a mutation-definedsubgroup which included pediatric IDH-mutant GBM, while eachH3F3A mutation defined an epigenetic subgroup of GBM with adistinct global methylation pattern. The last three epigenetic sub-groups were enriched for hallmark genetic events of adult GBMand/or established transcriptomic signatures. In line with reportsof PDGFRA copy number alterations being more prevalent inchildhood high-grade gliomas (2, 52, 57), an epigenetic cluster, wenamed receptor tyrosine kinase (RTK) I “PDGFRA,” harbored aproportion of pediatric patients (median age 36 years, range 8–74 years). A mesenchymal cluster displayed a widespread agedistribution (median age 47, range 2–85 years), while an RTK II“Classic” cluster was mostly composed of older adults (medianage 58, range 36–81 years) patients. Interestingly, the two H3F3Amutations gave rise to GBMs with differential regulation of tran-scription factors—OLIG1, OLIG2 and FOXG1, which may reflectdifferent cellular origins. Last, our results demonstrate differentialsurvival for each of these six subgroups, with worse prognosis andrapid death for K27M tumors, which behave like DIPG regardlessof their localization within the brain, and a slightly improvedsurvival for IDH-mutant GBM which show the longest overallsurvival followed by G34R/V-H3.3 GBM, while wild-type tumorsfor these mutations have the expected bad prognosis seen in GBM.

CONCLUSIONS AND FUTUREPERSPECTIVESCurrently, there are no effective treatments for pediatric HGAwhich carry the highest rate of morbidity and mortality in child-hood cancer. Collectively, recent findings have changed the land-scape for this disease, providing at last relevant targets that willdrive new trial models based on well-characterized gene subsets,thus providing hope for future change in the management andoutcome of this deadly group of cancers. Defects in chromatinremodeling are central in the genesis of pediatric and young adultHGA, and age and brain-location specific defects in chromatinstructure underlie the genesis of these tumors. This provides alaunching point for the identification of diagnostics and therapiesspecific to the molecular subtypes in children and young adults.Although histologically indistinguishable, there are at least sixdistinct disease entities that may arise from separate cell types oforigin as the result of largely non-overlapping sets of molecularevents. The development of the normal brain is a very dynamic andcomplex process, involving numerous extracellular factors that arepresent at precise times in specific brain locations. This is of majorsignificance as the human brain continues to develop post-natally,reaching completion in the early to mid-20s. What is unique aboutthe developing brain that enables K27M-H3.3 mutations to betumorigenic mainly in children and G34R/V-H3.3 and IDH muta-tions to affect mainly adolescents and younger adults? Why theneed for ATRX mutations that seem to be mutation (G34R/V-H3.3and IDH more than K27M-H3.3), and/or location (cortex andthalamus more than the brainstem) and/or age (older more thanyoung children) specific? Why the need for added TP53 muta-tions? Is it possible that the different extracellular factors in the


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developing brain tissue contribute to the transformation of cellswith K27M-H3.3 and G34R/V-H3.3 mutations, allowing them toform tumors? The timing, the contribution and the geneticprogram that are altered by these mutations/combinations of muta-tions need further investigation to help address and better targettheir effects in HGA. Further dissection of the effects downstreamof each H3F3A mutation in primary tumors and the generation ofmuch needed “in vitro” and “in vivo” models of these mutationsand their interaction(s) with ATRX and TP53 alterations are thuswarranted. This will help us better tackle this cancer as uncoveringwhat they induce on the epigenetic/genetic level has major poten-tial to improve our understanding of HGA, brain development andpotentially other diseases affecting the epigenome. Optimal clini-cal management should account for the distinction between theseGBM disease subtypes, and as routine histopathology is unable todistinguish these genetic subgroups, use of molecular tools wouldresult in better stratification of patients and enable better therapeu-tic choices as they become available.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGMENTSThis work was funded in part by Genome Canada and the Cana-dian Institute for Health Research (CIHR) with co-funding fromGenome BC, Genome Quebec, CIHR-ICR (Institute for CancerResearch) and C17, through the Genome Canada/CIHR jointATID Competition (project title: The Canadian Paediatric CancerGenome Consortium: Translating next generation sequencingtechnologies into improved therapies for high-risk childhoodcancer) and the PedBrain project contributing to the InternationalCancer Genome Consortium funded by the German Cancer Aid(109252). AMF and XYL are the recipient of studentship awardsfrom the CIHR. NJ is the recipient of a Chercheur Clinicien Awardfrom Fonds de Recherche en Santé du Québec and a “Next Gen-eration Champion of Genetics” award from the Canadian GeneCure Foundation.

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chromatin remodeling defects in pediatric and young adult glioblastoma: a tale of a variant histone...

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