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Emerging Interplay of Genetics and Epigenetics in Gliomas – A New Hope for Targeted Therapy Raymund L. Yong MD, MS, FRCS(C), Nadejda M. Tsankova MD, PhD
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S1071-9091(14)00092-8 10.1016/j.spen.2014.12.004 YSPEN523
To appear in: Semin Pediatr Neurol
Cite this article as: Raymund L. Yong MD, MS, FRCS(C), Nadejda M. Tsankova MD, PhD, Emerging Interplay of Genetics and Epigenetics in Gliomas – A New Hope for Targeted Therapy, Semin Pediatr Neurol , 10.1016/j.spen.2014.12.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Emerging Interplay of Genetics and Epigenetics in Gliomas – A New Hope for Targeted Therapy
Raymund L. Yong, MD, MS, FRCS(C) * and Nadejda M. Tsankova, MD, PhD + * Department of Neurosurgery, Comprehensive Brain Tumor Program, Mount Sinai School of Medicine, New York, NY +
Departments of Pathology & Neuroscience, Friedman Brain Institute, Mount Sinai School of Medicine, New York, NY Corresponding author: Nadejda Tsankova, MD, PhD Mount Sinai School of Medicine Departments of Neuroscience / Pathology 1425 Madison Avenue (Icahn 9-20 suite E) New York City, NY 10029 Phone: 212-559-8176 / Fax: 212-849-2611 Email: [email protected]
Conflict of Interest: The authors declare no conflicts of interest.
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Abstract: Diffusely infiltrating gliomas are inherently heterogeneous tumors and there are ongoing efforts to establish a classification scheme using molecular, rather than histological, features. In less than a decade, high-throughput sequencing of gliomas has transformed the field, uncovering several pivotal, highly prevalent genetic alterations that stratify patients into different prognostic and treatment-response categories. We highlight the genetic aberrations recently discovered in isocitrate dehydrogenase, alpha thalassemia/mental retardation syndrome X-linked, death-domain associated protein, histone H3.3, and telomerase reverse transcriptase and discuss how these mutations lead to unexpected changes in the epigenetic landscape in gliomas. We describe the opportunities these discoveries might provide for the development of novel, targeted therapy aimed at reversing early epigenetic aberrations in glioma precursor cells. Finally, we discuss the challenges for effective treatment of this uniformly fatal disease posed by intratumoral heterogeneity and clonal evolution.
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Introduction Diffusely infiltrating gliomas constitute a heterogeneous group of primary glial neoplasms with diverse morphology and molecular signatures, all of which are universally fatal once they undergo anaplastic progression. We have gained deep understanding of several core biological pathways involved in their oncogenesis, including the inactivation of p53 and retinoblastoma (RB) tumor-suppressors and the activation of receptor tyrosine Ras/phosphoinositide 3-kinase signaling cascades [1]. Unfortunately, targeting these pathways for glioma treatment has been difficult. More recent molecular advances in gliomas have uncovered several novel genetic mutations that are intimately associated with changes in the tumor‟s epigenetic landscape, opening new opportunities for more selective drug discovery [2-5]. Epigenetics controls gene activity without altering the DNA sequence, but rather via the tight regulation of factors that ultimately result in the opening or closing of chromatin with corresponding gene activation and repression. A nucleosome, the functional unit of chromatin, consists of eight histones (H2A, H2B, two H3, and two H4) wrapped around ~150 base pairs of DNA. The chromatin structure is frequently remodeled through posttranslational modification of its histone moieties, which include their acetylation, methylation, phosphorylation, and ubiquitylation. Chromatin remodeling is intricately controlled through the balanced activity of „writers‟, enzymes that methylate (HMTs) or acetylate (HATs) histones; „editors‟, enzymes that demethylate (HDMs) or deacetylate (HDACs) histones; and „readers‟, effector proteins recruited to bind specific chromatin domains and facilitate functional gene activation or repression (e.g., bromodomain readers of acetylation, chromodomain readers of methylation) [6,7]. DNA methylation and demethylation, controlled respectively by DNA methyltransferase (DNMT) and demethylase (TET) enzymes, exert tight epigenetic control and are intimately related to chromatin remodeling in modulating the overall transcriptional fate of a gene. The processes of cell proliferation, differentiation, and DNA repair are regulated through a delicate balance of genetic and epigenetic interactions. Its derailment can lead to
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oncogenic transformation with constitutively open chromatin at the promoters of protooncogenes leading to their overexpression or with condensed chromatin at the promoters of tumor suppressor genes causing their permanent repression [8,9]. The purpose of this review is to highlight some of the more recently discovered genetic and epigenetic abnormalities in pediatric and adult diffusely infiltrating gliomas, and to describe how they may induce unique epigenetic states in tumor cells, leading to sustained oncogenicity. An emphasis is also given to how these genetic and epigenetic markers can be targeted for the discovery of new and better therapeutic agents. Finally, we touch on the challenges that lay ahead posed by intratumoral heterogeneity and clonal evolution.
IDH mutations and hypermethylation Whole genome analysis has enabled the discovery of novel and unexpected genetic alterations in many pathological conditions, including gliomas, some of which are intimately involved in modulating the epigenome. Perhaps the best-studied example in gliomas is the recently discovered mutations within the gene encoding isocitrate dehydrogenase (IDH) [10,11]. Mutations in IDH, most commonly within the IDH1 isoform and much less frequently within the IDH2 isoform (~1-6%), have been detected in the majority of low-grade astrocytomas (~73%), low-grade oligodendrogliomas (~87%) and low-grade oligoastrocytomas (~83%), persisting as these tumors undergo high-grade progression to WHO grade III anaplastic gliomas and WHO grade IV secondary glioblastomas (GBMs) [8,12,13]. Conversely, IDH mutations are largely absent in “de-novo” or “primary” glioblastomas; in other types of primary brain tumors (pilocytic astrocytomas, gangliocytomas, gangliogliomas, dysembryoplastic neuroepithelial tumor, pleomorphic xanthoastrocytomas, ependymomas, neurocytomas, medulloblastomas / primitive neuroectodermal tumors); as well as in glioma mimics such as tumefactive multiple sclerosis, vasculitis, encephalitis, and gliosis [13]. Since their discovery in gliomas in 2008, IDH mutations have emerged as important biomarkers for predicting longer overall survival in patients with low-grade, diffusely
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infiltrating gliomas [14]; anaplastic astrocytomas/glioblastomas [15]; and anaplastic oligodendrogliomas, even in the absence of a 1p/19q co-deletion [16]. There is also growing evidence to suggest that IDH mutations are early events in gliomagenesis [17,18]. With rare exceptions, the most prevalent IDH mutation, IDH1-R132H, is diffusely present within glioma cells [19] (Odia and Tsankova, in submission) (Fig 1). It also temporally precedes other genetic abnormalities, such as 1p/19q co-deletion in oligodendrogliomas and ATRX and TP53 mutations in astrocytomas [17,18] (Fig 2). This has led to growing excitement in the field to try and understand the downstream effects of mutant IDH1. Wild type IDH enzymes (IDH1 and IDH2)* have vital functions in regulating oxidative stress in cells under normal physiological conditions. The enzymes oxidize isocitrate to alpha-ketoglutarate ( -KG), using NADP+ as a cofactor to generate reduced NADPH during the process of catalysis. The two genes are on separate chromosomes (2q33 and 15q26, respectively) and are located in distinct subcellular locations: IDH1 is located in the cytosol and peroxisomes, while IDH2 is in the mitochondria [20]. Perhaps not surprisingly, mutant forms of IDH1/2 are associated with oxidative metabolic burden on tumor cells. Intriguingly, this new metabolic state plays a pivotal role in altering the epigenetic state of the cell. An important clue that IDH mutations modulate the epigenomic state of tumor cells came from unbiased molecular profiling analyses of a large number of gliomas [1-4]. Gene expression studies subclassified glioblastomas into four molecular categories: proneural, classical, mesenchymal, and neural [3], which have gained relevance for diffusely infiltrating gliomas in general [8]. “Proneural” tumors were found to frequently harbor mutations in IDH1/2 (along with TP53/ATRX mutations in astrocytomas or 1p/19q co-deletion and FUBP1/CIC mutations in oligodendrogliomas, whereas “classical” ones had frequent EGFR amplification and loss of chromosome 10 but wild type IDH1 status [3,21,22]. In parallel to genetic profiling, comprehensive DNA methylation analyses of gliomas showed that “proneural” tumors also distinguish themselves from the other three subgroups by exhibiting increased levels of global DNA methylation at CpG islands, commonly referred to as the
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glioma CpG island methylator phenotype (or G-CIMP) [4]. The link between IDH mutations and G-CIMP lies in the abnormal oncometabolite 2-hydroxyglutarate (2-HG), overproduced by gain-of-function IDH mutant enzymes acting on -KG [23]. Accumulation of 2-HG impairs -ketoglutarate-dependent dioxygenases, including DNA and histone demethylases [24,25]. Some of these fall in the TET family of proteins (TET 1/2), which convert DNA 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5-hmC) [25]. Impeding this essential step in demethylation of DNA effectively produces regions of CpG hypermethylation in proneural IDH1 mutant gliomas [26]. Moreover, 2-HG directly inhibits the activity of histone modifying enzymes, particularly the Jumonji domain-containing histone demethylases (JHDMs). Their inhibition results in accumulations of repressive chromatin marks, most notably H3K9 and H3K27 trimethylation [24] (Fig 2). The exact effects of increasing histone and DNA methylation levels in mutant IDH tumors are not yet clear, but the altered epigenetic landscape appears to facilitate a block in cell differentiation and a cellular state sensitized to malignant transformation by allowing the aberrant downstream expression of oncogenic gene targets [5]. This paves the way for exciting new research aimed at understanding how specific epigenetic modifications induced in IDH-mutant gliomas differentially affect distinct genes. Their elucidation will allow the use of specific small molecule inhibitors to selectively target epigenetic changes in gliomagenic cells, hopefully in their nascent state. Potential molecules to target would include mutant IDH itself, its oncogenic neometabolite 2-HG, and its downstream effectors. In just the past few years, there has been exciting progress in discovering molecules that target mutant IDH and its associated oncometabolite, with some showing efficacy for inhibiting glioma cell growth. One such example is the AGI-5198 small molecule inhibitor, detected in a high-throughput screen for selective inhibitors of IDH1-R132H. Rohle et al. recently showed that AGI-5198 can inhibit the growth of mutant (but not wild type) IDH1 glioma cells and also block the production of 2-HG [27]. Furthermore, the inhibitor induced the expression of genes associated with a differentiated glial fate. An independent small molecule high throughput screen identified the probe ML309, which could also inhibit the
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IDH1-R132H enzyme, reducing 2-HG production in a mutant glioblastoma cell line [28]. These discoveries may allow for the selective “reprogramming” of cellular metabolism in IDH1-mutant gain-of-function tumor cells but not in infiltrated normal brain cells. Other drug design strategies for inhibiting IDH1mutant gliomas have included biochemical inhibition of tumor metabolism using glutaminase inhibitors such as zaprinast, which perturbs 2-HG production but lacks the tumor specificity of AGI-5198 [29,30]. Decitabine, a non-specific DNMT inhibitor, has shown reversal of DNA methylation marks induced by IDH along with re-expression of genes associated with differentiation [31].
Telomere lengthening and H3.3-ATRX-DAXX mutations Telomeres consist of a region of hexanucleotide repeats, 5 to 10 kilobases long, which cap the ends of eukaryotic chromosomes, maintaining their stability. Because telomere shortening occurs with each cell division, eventually triggering p53-dependent senescence or apoptosis, normal stem cells and malignant cells must have a mechanism of telomere maintenance to allow prolonged or unlimited cell proliferation. In many common cancers of epithelial origin, such as lung, breast, colon, and prostate carcinomas, constitutive telomerase activity in somatic stem cells is thought to increase through epigenetic dysregulation during the process of malignant transformation (reviewed in [32]). In malignancies derived from non-epithelial cells where telomerase is not constitutively active, including many primary brain tumors and certain sarcomas, telomere lengthening must occur through other means. One such mechanism, alternative lengthening of telomeres (ALT), operates in the absence of detectable telomerase activity [33]. Rather, homologous recombination resulting in telomere destabilization and length heterogeneity is observed in ALT-positive cells. A highly significant correlation between ALT and loss of nuclear expression of either of the proteins encoded by the alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) genes was first reported in a study of pancreatic neuroendocrine tumors [34]. Earlier studies had noted that a high proportion of WHO grade
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II and III astrocytomas and a lower proportion glioblastomas exhibited the ALT phenotype [22,35,36]. It was subsequently shown that gliomas harboring the ALT phenotype have loss of nuclear ATRX expression [37-40]. ATRX and DAXX form an ATP-dependent remodeling complex that localizes to telomeres and heterochromatic regions of the genome, where it has a role in histone variant H3.3 deposition in chromatin and regulating telomere homeostasis. In ATRX syndrome, germline inactivation of ATRX causes H3.3 to be lost in favor of the transcriptionally repressive histone variant macroH2A at the alpha-globin gene cluster in the subtelomeric region of chromosome 16, epigenetically silencing expression of these genes [41,42]. In addition, ATRX localization at G-rich tandem repeats near genes may assist in resolving DNA quadruplex secondary structures that are also transcriptionally repressive [43]. Thus, the specific functions of ATRX-DAXX may differ in differing chromatin contexts. ATRX mutations have been found to be highly prevalent in diffuse astrocytomas, in one study occurring at a frequency of 67% and 73% in grade II and III astrocytomas, respectively [22]. The specific somatic mutations are variable, but generally result in loss of the C-terminal DNA helicase domain of the protein via truncating frameshift mutations. ATRX mutations occur somewhat less frequently in secondary and pediatric glioblastomas; and rarely in primary glioblastomas and pure oligodendrogliomas. Furthermore, they are found almost exclusively in concert with IDH mutations, frequently with TP53 mutations, and almost never with 1p/19q co-deletion, which is the characteristic chromosomal aberration of pure oligodendrogliomas (Fig 1). Because mixed oligoastrocytomas and pure astrocytomas progress into high-grade secondary glioblastomas significantly more quickly than oligodendrogliomas, ATRX loss is emerging both as a defining diagnostic and important prognostic marker in astrocytic gliomas [22]. Given the role of ATRX in chromatin regulation, the effects of ATRX loss on the epigenome and transcriptome are of strong interest. ATRX binds to tandemly repetitive genomic regions enriched for G and C, including CpG islands near gene promoters. In a comparison of lymphoid cells expressing and lacking ATRX, four genes near or containing
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an ATRX binding target were noted to be significantly altered in expression: NME4, SLC7A5, RASA3, and GAS8 [43]. Similar studies examining differences in gene expression in IDHmutant/ATRX-intact and IDH-mutant/ATRX-lost glial progenitors will shed light on the pathogenesis of low-grade gliomas. Because ATRX also has a role in resolving abnormal DNA secondary structures, such as G-quadruplexes, it will be of considerable interest to examine how the G-CIMP-positive epigenome is affected by ATRX loss, and what impact this has on genome stability. Furthermore, the link between ATRX and G-quadruplexes presents the possibility that small molecules that bind to and stabilize these DNA structures will at least partly reconstitute ATRX function in astrocytomas. Riou et al. demonstrated the ability of one such ligand to induce senescence in immortalized ALT-positive fibroblasts [44]. The binding partner of ATRX, DAXX, is a chaperone of H3.3 [45]. In a study of 48 pediatric glioblastomas, 21 (44%) had a mutation affecting at least one of the genes encoding these proteins. Mutations in H3F3A, the gene encoding H3.3, appear to be nearly exclusive to GBMs occurring in the pediatric population or young adults, having been found to be absent in a cohort of 291 adult GBMs subjected to whole exome sequencing [2,40]. Two distinct heterozygous mutations were identified, K27M and G34R/V, each near posttranslational modification sites associated with transcriptional repression and activation, respectively. Furthermore, of 15 samples with H3F3A mutations, 9 also had mutations in ATRX and 12 in TP53. Strikingly, H3F3A mutations were never found together with IDH mutations, suggesting possibly analogous roles in reshaping the epigenome (Fig 2). K27M mutations in H3.3 and another histone variant, H3.1, tend to occur in children with brainstem and thalamic (i.e., midline) high-grade gliomas. These gliomas have also recently been found to contain mutations in ACVR1 and FGFR1, the genes encoding the type 1 activin A receptor and fibroblast growth factor receptor 1, respectively [46-49]. In contrast, G34R/V mutations in H3.3 occur more commonly in supratentorial tumors arising in young adults [37,50]. Although H3.3 comprises only 1% of total cellular H3, K27M substitutions in this histone variant dramatically decrease global levels of di- and trimethylation at H3K27, while overall levels of acetylation at H3K27 modestly increase [51].
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This effect appears to be mediated by a hydrophobic interaction between the methionine side chain and aromatic residues at the active site of the histone methyltransferase PRC2, inhibiting its activity. Non-glycine residues at position 34 of the tail of H3.3 appear to have a similar but less far-reaching inhibitory effect on another histone methyltransferase, SETD2, which normally acts at H3K36 to increase activating methylation. This suppression of methyltransferase activity contrasts with the suppression of DNA and histone demethylases due to 2-HG in IDH-mutant gliomas (Fig 2). Therefore, while compounds designed to inhibit the neo-enzymatic activity of IDH1-R132H might be predicted to be effective in treating the IDH-mutant tumors commonly found in adults, compounds with the opposite epigenetic effect, or perhaps 2-HG itself, would be predicted to reverse alterations in the H3.3-mutant tumors commonly found in children and young adults. It becomes apparent that a thorough understanding of the epigenetic imbalances extant in each molecular subtype of glioma will be required to match the most appropriate agents in preclinical and clinical testing. DAXX was mutated in 2 of 42 pediatric GBM samples analyzed by whole exome sequencing [40]. Additional tissue microarray analysis on 124 pediatric GBM samples revealed loss of DAXX staining in 6% of samples, compared to loss of ATRX in 35%. DAXX and ATRX mutations tended to be mutually exclusive of each other and of H3.3 K27M mutations, while there was a striking overlap with H3.3 G34R/V mutations. These mutational patterns may be partly explained by a requirement for intact ATRX in order for DAXX to localize to telomeres, and that many DAXX mutations occur within the C-terminal histonebinding domain of the protein, disrupting its ability to bind to and chaperone H3.3 to heterochromatin. Thus, ATRX, DAXX, and H3.3 each play complementary and essential roles in a core cellular mechanism for chromatin remodeling at telomeres and gene regulatory elements.
TERT promoter mutations Pure oligodendrogliomas and primary GBMs make up the majority of diffuse gliomas that do not exhibit the ALT phenotype. Because both these tumor types, unlike cancers of
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epithelial tissues, presumably arise from cells that are not constantly self-renewing throughout the lifespan of the organism, another mechanism of telomere lengthening is needed. A study examining 1230 tumors representing 60 tumor types demonstrated a 51.1% frequency among gliomas of somatic mutations in the telomerase reverse transcriptase (TERT) promoter gene, which encodes the catalytic subunit of telomerase [52]; these mutations had been previously described in melanoma [53,54]. TERT promoter mutations occurred in 51 of 78 tumors classified as GBM by histology, and co-occurred tightly with EGFR amplification. Likewise, TERT promoter mutations were found in 35 of 45 oligodendrogliomas diagnosed by histology, and in 98% of tumors harboring an IDH1/2 mutation and 1p19q co-deletion [55]. In contrast, no tumor exhibiting ALT/ATRX loss was found to have a TERT mutation. TERT promoter mutation status may therefore be a powerful molecular diagnostic tool, especially in helping to distinguish secondary and primary GBMs, which may portend different prognoses. The two most common TERT promoter mutations, C228T and C250T, occur in a mutually exclusive manner in gliomas and are associated with a more than six-fold increase in TERT mRNA expression over levels observed in TERT wild-type tumors [56]. Both mutations create an identical, novel 11-bp consensus-binding site for ETS family transcription factors [53,54], which explains how these heterozygous non-coding region somatic point mutations may act as drivers in gliomagenesis. The ETS family is comprised of a large number of proteins with both activating and repressing functions in the regulation of diverse gene networks important in cancer (reviewed in [57]); elucidating the specific ETS factors at play in TERT mutated gliomas will undoubtedly reveal new targets for therapy. Furthermore, both telomerase inhibition and the exploitation of telomerase-overexpressing cells hold promise as therapeutic strategies directed specifically at the self-renewing glioma stem cell population. A number of such agents, including small molecule inhibitors (e.g., BIBR1532), antisense oligonucleotides (e.g., imetelstat) [58], G-quadruplex stabilizers (e.g., telomestatin), and peptide vaccines directed against hTERT (e.g. GV1001), are actively being investigated in preclinical and early phase clinical trials (reviewed in [59]).
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Clonal Evolution Next-generation high-throughput technologies have begun to reveal, in unprecedented detail, the complexity that exists in diffuse gliomas as a result of genomic and epigenomic heterogeneity, spatially and temporally. In applying the gene expression classification of Verhaak et al. [3] to glioblastoma fragments sampled from different regions of the same tumor, Sottoriva et al. [60] demonstrated that multiple transcriptional phenotypes coexist in the majority of patients. Further, they compared CpG methylation patterns at preselected genomic loci as a measure of the “mitotic distance” between spatially separated intratumoral samples. Using these data, phylogenies of clonal populations of tumor cells could be reconstructed, providing evidence of ongoing branching clonal evolution within gliomas. DNA methylation data have also been used to compare tumors from different individuals, identifying a handful of distinct epigenetic phenotypes (of which G-CIMP is only one), analogous to the previously recognized gene expression clusters [2,50]. It seems likely, although remains to be proven, that multiple epigenetic phenotypes also coexist in individual tumors. Adding another layer of complexity to this are the effects of therapeutic agents that kill cancer cells by damaging DNA. The two agents used as part of the standard treatment paradigm, ionizing radiation and the alkylating chemotherapy temozolomide (TMZ), are prime examples. In a study of 23 low-grade gliomas and their higher grade recurrences, Johnson et al [61] used whole exome sequencing to demonstrate a high frequency of C>T/G>A mutations at CpC and CpT dinucleotides in TMZ-treated tumors relative to those not treated, and suggested that these transitions are TMZ-induced. Furthermore, they were able to identify TMZ-associated deleterious mutations in genes coding for components of two signaling pathways associated with progression to GBM, the RB and Akt-mTOR pathways. Similarly, exposure to ionizing radiation can produce somatic mutations by inducing DNA double-strand breaks that are then misrepaired, resulting in genomic structural variants such as insertion-deletions and chromosomal translocations [62,63].
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Together, these findings suggest that DNA-damaging therapeutic agents have the potential to influence – perhaps even accelerate – the evolutionary processes already occurring spontaneously within gliomas. Unfortunately, current approaches of sequencing fragments of resected tumors in search of targetable mutations will be inadequate to characterize the neoplastic clonal populations left unresected, let alone provide information about how these cells will evolve in the face of selection pressures and mutagens during treatment. Convergent evolution, whereby different clonal populations independently acquire genetic alterations affecting the same signaling pathways [64], may offer the possibility of predicting the most probable future drivers of progression and recurrence given a particular ecosystem of clones, and hence an opportunity to subvert them. We speculate that research investigating DNA damage patterns in experimental models of different transcriptional and epigenetic tumor phenotypes will shed light on these dynamics.
Targeting glioma precursors In order to cure patients with diffusely infiltrating gliomas, we must catch the disease in its nascent state and design therapeutic targets that will eradicate its seed before it becomes widespread. To appreciate how gliomas arise, we must also expand our understanding of the early molecular alterations present in glioma precursor cells, which have been suggested to arise from neural stem cells in the subventricular zone and more committed glial progenitors found predominantly in white matter [8,65-69]. There is growing evidence that changes in the epigenetic landscape of progenitor/stem cells contribute to early stages of cancerous transformation [9,70,71]. We and others have hypothesized that some progenitor/stem cells are „„poised‟‟ in a pre-neoplastic state, where they are epigenetically primed for subsequent genetic alterations, which ultimately result in their oncogenic transformation [8,70]. Designing drugs that target genetic/epigenetic alterations in tumor cells during very early stages of gliomagenesis could effectively kill the tumor before it has a chance to fully form and infiltrate. To this end, several groups are studying the genetic and epigenetic profile of various neural/glial progenitors, using predominantly rodent
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and human cell culture models [72-74]. The ideal model of study would entail the isolation of rare and pure neural/glial precursor cells from their endogenous niche, unperturbed by artificial cell culture alterations: a challenging endeavor (Tsankova et al., in submission).
Conclusions Recent advances in glioma research reveal the presence of a sophisticated network of interactions between genetics and epigenetics in the contexts of diffuse glioma formation and its molecular substratification (Fig 2). IDH mutations in “proneural” gliomas induce hypermethylation of DNA and histones by inhibiting the actions of DNA and histone demethylases [24,26]. ATRX mutations, present in both “proneural” astrocytomas and pediatric glioblastomas, affect heterochromatin and the alternative lengthening of telomeres [40,45]. Mutations in the histone variant H3.3, seen predominantly in pediatric glioblastomas, decrease global levels of histone methylation at H3K27 and H3K36, and inhibit the activity of histone methyltransferases PRC2 and SETD2 [51] (Fig 2). The interactions of the above mutations with one another and their downstream epigenetic alterations are remarkably complex, and represent an area of active research. Their interface is dynamic and multidimensional, with the ability to change in space and time. This makes the deconvolution of specific oncogenic targets for specific gliomas tremendously challenging. Nevertheless, some clear targets do emerge, and the challenge in the next decade will be to design specific drugs that can block the genetic (and epigenetic) alterations before low-grade gliomas have undergone malignant transformation. Already, inhibitors of HDACs or DNMTs have shown activity in glioblastoma cell lines and glioma models, and have even advanced to use in clinical trials [75,76]. These drugs, however, are limited due to their nonspecific inhibition of various chromatin modifying enzymes, and their lack of distinction between neoplastic and normal cell targets. Indeed, the identification of selective IDH1R132H inhibitors that can now function in tumor-cell specific manner is a significant step forward in the discovery of drugs with superior target specificity [27,28]. Other areas for
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future exploration include inhibitors of TET enzymes; G-quadruplex stabilizers in ATRX mutant gliomas; H3F3A mutations and their effect on histone methylation through specific interaction with PRC2; and ETS factors, hTERT inhibitors, and immunotherapy in TERT mutated tumors. The use of specific small molecules to target unique enzymatic moieties within the polycomb repressor complex (EZH2 inhibitors) or bromodomains readers that selectively bind acetylated lysine histone domains (JQ1 inhibitors) are also emerging as attractive tools for therapeutic epigenetic modification, and are currently being tested in glioblastoma cell lines [74,77,78]. Several of these inhibitors show remarkably favorable toxicity profiles. Although gliomas are markedly heterogeneous, their molecular ontology is beginning to emerge. In less than a decade, genomic profiling of gliomas has transformed the field, with the discovery of several pivotal mutations that not only delineate novel means of subclassification, but also offer new hope for the discovery of highly specific drug targets to combat this deadly disease.
REFERENCES:
1.
Cancer Genome Atlas Research N. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 2008; 455: 1061-1068
2.
Brennan CW, Verhaak RG, McKenna A et al. The somatic genomic landscape of glioblastoma. Cell 2013; 155: 462-477
3.
Verhaak RG, Hoadley KA, Purdom E et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer cell 2010; 17: 98-110
4.
Noushmehr H, Weisenberger DJ, Diefes K et al. Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer cell 2010; 17: 510-522
15
5.
Kim J, DeBerardinis RJ. Cancer. Silencing a metabolic oncogene. Science 2013; 340: 558-559
6.
Jenuwein T, Allis CD. Translating the histone code. Science 2001; 293: 1074-1080
7.
Plass C, Pfister SM, Lindroth AM et al. Mutations in regulators of the epigenome and their connections to global chromatin patterns in cancer. Nature reviews Genetics 2013; 14: 765-780
8.
Tsankova NM, Canoll P. Advances in genetic and epigenetic analyses of gliomas: a neuropathological perspective. J Neurooncol 2014; 119: 481-490
9.
Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nature reviews Genetics 2006; 7: 21-33
10.
Parsons DW, Jones S, Zhang X et al. An integrated genomic analysis of human glioblastoma multiforme. Science 2008; 321: 1807-1812
11.
Yan H, Parsons DW, Jin G et al. IDH1 and IDH2 mutations in gliomas. The New England journal of medicine 2009; 360: 765-773
12.
Hartmann C, Meyer J, Balss J et al. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta neuropathologica 2009; 118: 469-474
13.
Balss J, Meyer J, Mueller W et al. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta neuropathologica 2008; 116: 597-602
14.
Houillier C, Wang X, Kaloshi G et al. IDH1 or IDH2 mutations predict longer survival and response to temozolomide in low-grade gliomas. Neurology 2010; 75: 1560-1566
15.
Hartmann C, Hentschel B, Wick W et al. Patients with IDH1 wild type anaplastic astrocytomas exhibit worse prognosis than IDH1-mutated glioblastomas, and IDH1 mutation status accounts for the unfavorable prognostic effect of higher age: implications for classification of gliomas. Acta neuropathologica 2010; 120: 707-718
16.
Cairncross JG, Wang M, Jenkins RB et al. Benefit from procarbazine, lomustine, and vincristine in oligodendroglial tumors is associated with mutation of IDH. Journal of
16
clinical oncology : official journal of the American Society of Clinical Oncology 2014; 32: 783-790 17.
Lass U, Numann A, von Eckardstein K et al. Clonal analysis in recurrent astrocytic, oligoastrocytic and oligodendroglial tumors implicates IDH1- mutation as common tumor initiating event. PloS one 2012; 7: e41298
18.
Watanabe T, Nobusawa S, Kleihues P et al. IDH1 mutations are early events in the development of astrocytomas and oligodendrogliomas. The American journal of pathology 2009; 174: 1149-1153
19.
Preusser M, Wohrer A, Stary S et al. Value and limitations of immunohistochemistry and gene sequencing for detection of the IDH1-R132H mutation in diffuse glioma biopsy specimens. Journal of neuropathology and experimental neurology 2011; 70: 715-723
20.
Horbinski C. What do we know about IDH1/2 mutations so far, and how do we use it? Acta neuropathologica 2013; 125: 621-636
21.
Bettegowda C, Agrawal N, Jiao Y et al. Mutations in CIC and FUBP1 contribute to human oligodendroglioma. Science 2011; 333: 1453-1455
22.
Jiao Y, Killela PJ, Reitman ZJ et al. Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas. Oncotarget 2012; 3: 709-722
23.
Dang L, White DW, Gross S et al. Cancer-associated IDH1 mutations produce 2hydroxyglutarate. Nature 2009; 462: 739-744
24.
Lu C, Ward PS, Kapoor GS et al. IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature 2012; 483: 474-478
25.
Figueroa ME, Abdel-Wahab O, Lu C et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer cell 2010; 18: 553-567
26.
Turcan S, Rohle D, Goenka A et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 2012; 483: 479-483
17
27.
Rohle D, Popovici-Muller J, Palaskas N et al. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science 2013; 340: 626-630
28.
Davis M, Pragani R, Popovici-Muller J et al. ML309: A potent inhibitor of R132H mutant IDH1 capable of reducing 2-hydroxyglutarate production in U87 MG glioblastoma cells. In, Probe Reports from the NIH Molecular Libraries Program. Bethesda (MD); 2010
29.
Seltzer MJ, Bennett BD, Joshi AD et al. Inhibition of glutaminase preferentially slows growth of glioma cells with mutant IDH1. Cancer Res 2010; 70: 8981-8987
30.
Elhammali A, Ippolito JE, Collins L et al. A high-throughput fluorimetric assay for 2hydroxyglutarate identifies Zaprinast as a glutaminase inhibitor. Cancer Discov 2014; 4: 828-839
31.
Turcan S, Fabius AW, Borodovsky A et al. Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine. Oncotarget 2013; 4: 1729-1736
32.
Zhu J, Zhao Y, Wang S. Chromatin and epigenetic regulation of the telomerase reverse transcriptase gene. Protein & cell 2010; 1: 22-32
33.
Bryan TM, Englezou A, Gupta J et al. Telomere elongation in immortal human cells without detectable telomerase activity. The EMBO journal 1995; 14: 4240-4248
34.
Jiao Y, Shi C, Edil BH et al. DAXX/ATRX, MEN1, and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors. Science 2011; 331: 11991203
35.
Henson JD, Hannay JA, McCarthy SW et al. A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas. Clinical cancer research : an official journal of the American Association for Cancer Research 2005; 11: 217-225
36.
Heaphy CM, Subhawong AP, Hong SM et al. Prevalence of the alternative lengthening of telomeres telomere maintenance mechanism in human cancer subtypes. The American journal of pathology 2011; 179: 1608-1615
18
37.
Khuong-Quang DA, Buczkowicz P, Rakopoulos P et al. K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas. Acta neuropathologica 2012; 124: 439-447
38.
Liu XY, Gerges N, Korshunov A et al. Frequent ATRX mutations and loss of expression in adult diffuse astrocytic tumors carrying IDH1/IDH2 and TP53 mutations. Acta neuropathologica 2012; 124: 615-625
39.
Kannan K, Inagaki A, Silber J et al. Whole-exome sequencing identifies ATRX mutation as a key molecular determinant in lower-grade glioma. Oncotarget 2012; 3: 1194-1203
40.
Schwartzentruber J, Korshunov A, Liu XY et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 2012; 482: 226-231
41.
Ratnakumar K, Duarte LF, LeRoy G et al. ATRX-mediated chromatin association of histone variant macroH2A1 regulates alpha-globin expression. Genes & development 2012; 26: 433-438
42.
Dhayalan A, Tamas R, Bock I et al. The ATRX-ADD domain binds to H3 tail peptides and reads the combined methylation state of K4 and K9. Human molecular genetics 2011; 20: 2195-2203
43.
Law MJ, Lower KM, Voon HP et al. ATR-X syndrome protein targets tandem repeats and influences allele-specific expression in a size-dependent manner. Cell 2010; 143: 367-378
44.
Riou JF, Guittat L, Mailliet P et al. Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands. Proceedings of the National Academy of Sciences of the United States of America 2002; 99: 2672-2677
45.
Lewis PW, Elsaesser SJ, Noh KM et al. Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres. Proceedings of the National Academy of Sciences of the United States of America 2010; 107: 14075-14080
19
46.
Wu G, Diaz AK, Paugh BS et al. The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma. Nature genetics 2014; 46: 444-450
47.
Taylor KR, Mackay A, Truffaux N et al. Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma. Nature genetics 2014; 46: 457-461
48.
Fontebasso AM, Papillon-Cavanagh S, Schwartzentruber J et al. Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma. Nature genetics 2014; 46: 462-466
49.
Buczkowicz P, Hoeman C, Rakopoulos P et al. Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations. Nature genetics 2014; 46: 451-456
50.
Sturm D, Witt H, Hovestadt V et al. Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer cell 2012; 22: 425-437
51.
Lewis PW, Muller MM, Koletsky MS et al. Inhibition of PRC2 activity by a gain-offunction H3 mutation found in pediatric glioblastoma. Science 2013; 340: 857-861
52.
Killela PJ, Reitman ZJ, Jiao Y et al. TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proceedings of the National Academy of Sciences of the United States of America 2013; 110: 6021-6026
53.
Horn S, Figl A, Rachakonda PS et al. TERT promoter mutations in familial and sporadic melanoma. Science 2013; 339: 959-961
54.
Huang FW, Hodis E, Xu MJ et al. Highly recurrent TERT promoter mutations in human melanoma. Science 2013; 339: 957-959
55.
Arita H, Narita Y, Fukushima S et al. Upregulating mutations in the TERT promoter commonly occur in adult malignant gliomas and are strongly associated with total 1p19q loss. Acta neuropathologica 2013; 126: 267-276
20
56.
Arita H, Narita Y, Takami H et al. TERT promoter mutations rather than methylation are the main mechanism for TERT upregulation in adult gliomas. Acta neuropathologica 2013; 126: 939-941
57.
Kar A, Gutierrez-Hartmann A. Molecular mechanisms of ETS transcription factormediated tumorigenesis. Critical reviews in biochemistry and molecular biology 2013; 48: 522-543
58.
Marian CO, Cho SK, McEllin BM et al. The telomerase antagonist, imetelstat, efficiently targets glioblastoma tumor-initiating cells leading to decreased proliferation and tumor growth. Clinical cancer research : an official journal of the American Association for Cancer Research 2010; 16: 154-163
59.
Ruden M, Puri N. Novel anticancer therapeutics targeting telomerase. Cancer treatment reviews 2013; 39: 444-456
60.
Sottoriva A, Spiteri I, Piccirillo SG et al. Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proceedings of the National Academy of Sciences of the United States of America 2013; 110: 4009-4014
61.
Johnson BE, Mazor T, Hong C et al. Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science 2014; 343: 189-193
62.
Mani RS, Tomlins SA, Callahan K et al. Induced chromosomal proximity and gene fusions in prostate cancer. Science 2009; 326: 1230
63.
Hakim O, Resch W, Yamane A et al. DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes. Nature 2012; 484: 69-74
64.
Gerlinger M, Rowan AJ, Horswell S et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. The New England journal of medicine 2012; 366: 883-892
65.
Sanai N, Alvarez-Buylla A, Berger MS. Neural stem cells and the origin of gliomas. The New England journal of medicine 2005; 353: 811-822
21
66.
Alcantara Llaguno S, Chen J, Kwon CH et al. Malignant astrocytomas originate from neural stem/progenitor cells in a somatic tumor suppressor mouse model. Cancer cell 2009; 15: 45-56
67.
Jacques TS, Swales A, Brzozowski MJ et al. Combinations of genetic mutations in the adult neural stem cell compartment determine brain tumour phenotypes. The EMBO journal 2010; 29: 222-235
68.
Ozawa T, Riester M, Cheng YK et al. Most human non-GCIMP glioblastoma subtypes evolve from a common proneural-like precursor glioma. Cancer cell 2014; 26: 288-300
69.
Canoll P, Goldman JE. The interface between glial progenitors and gliomas. Acta neuropathologica 2008; 116: 465-477
70.
Tsai HC, Baylin SB. Cancer epigenetics: linking basic biology to clinical medicine. Cell Res 2011; 21: 502-517
71.
Bernstein BE, Mikkelsen TS, Xie X et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 2006; 125: 315-326
72.
Auvergne RM, Sim FJ, Wang S et al. Transcriptional differences between normal and glioma-derived glial progenitor cells identify a core set of dysregulated genes. Cell reports 2013; 3: 2127-2141
73.
Yoo S, Bieda MC. Differences among brain tumor stem cell types and fetal neural stem cells in focal regions of histone modifications and DNA methylation, broad regions of modifications, and bivalent promoters. BMC genomics 2014; 15: 724
74.
Lim DA, Alvarez-Buylla A. Adult neural stem cells stake their ground. Trends in neurosciences 2014; 37: 563-571
75.
Iwamoto FM, Lamborn KR, Kuhn JG et al. A phase I/II trial of the histone deacetylase inhibitor romidepsin for adults with recurrent malignant glioma: North American Brain Tumor Consortium Study 03-03. Neuro-oncology 2011; 13: 509-516
22
76.
Friday BB, Anderson SK, Buckner J et al. Phase II trial of vorinostat in combination with bortezomib in recurrent glioblastoma: a north central cancer treatment group study. Neuro-oncology 2012; 14: 215-221
77.
Cheng Z, Gong Y, Ma Y et al. Inhibition of BET bromodomain targets genetically diverse glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research 2013; 19: 1748-1759
78.
Dror N, Mandel M, Lavie G. Unique anti-glioblastoma activities of hypericin are at the crossroad of biochemical and epigenetic events and culminate in tumor cell differentiation. PloS one 2013; 8: e73625
Figure Legends
Fig 1. Clinical neuropathological analysis of proneural gliomas Proneural gliomas include both oligodendrogliomas (top left) and diffuse astrocytomas (top right), both of which express mutant IDH1 (middle) and additionally show either 1p/19q codeletion (bottom left; red: 1p/19q probe, green: control chromosome probe) or ATRX and TP53 mutations (bottom right). The presence and extent of IDH mutation in glioma tissue is assessed by immunohistochemistry using an antibody specific for IDH1-R132H. Truncating ATRX mutations lead to loss of ATRX nuclear staining in astrocytoma cells, while TP53 mutations cause an aberrant nuclear accumulation of p53. Scale bars = 50um; all micrographs are scaled similarly.
Fig 2. Interplay between gain-of-function genetic mutations and epigenetic alterations in the pathogenesis of several diffuse glioma subtypes Gain-of-function mutations early in gliomagenesis inhibit several enzymes (PRC2, SETD2, JHDMs, TETs) key to altering the epigenetic landscape within the nucleus of a glial progenitor cell (top). Sites of mutation are indicated by lightning bolts. Then, acquired mutations in ATRX-DAXX and/or p53, or the TERT promoter (middle) immortalize the cell
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and undermine genomic integrity. This permits the accumulation and selection of additional somatic alterations, typically affecting key signaling pathways, that ultimately lead to the development of the indicated glioma subtypes, classically distinguished by histology and age of onset (bottom).
* An IDH3 enzyme also exists but no cancer-associated IDH3 mutations have been discovered to date.
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FIG 1
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FIG 2
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Emerging Interplay of Genetics and Epigenetics in Gliomas – A New Hope for Targeted Therapy Raymund L. Yong MD, MS, FRCS(C), Nadejda M. Tsankova MD, PhD
www.elsevier.com/locate/enganabound
PII: DOI: Reference:
S1071-9091(14)00092-8 10.1016/j.spen.2014.12.004 YSPEN523
To appear in: Semin Pediatr Neurol
Cite this article as: Raymund L. Yong MD, MS, FRCS(C), Nadejda M. Tsankova MD, PhD, Emerging Interplay of Genetics and Epigenetics in Gliomas – A New Hope for Targeted Therapy, Semin Pediatr Neurol , 10.1016/j.spen.2014.12.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Emerging Interplay of Genetics and Epigenetics in Gliomas – A New Hope for Targeted Therapy
Raymund L. Yong, MD, MS, FRCS(C) * and Nadejda M. Tsankova, MD, PhD + * Department of Neurosurgery, Comprehensive Brain Tumor Program, Mount Sinai School of Medicine, New York, NY +
Departments of Pathology & Neuroscience, Friedman Brain Institute, Mount Sinai School of Medicine, New York, NY Corresponding author: Nadejda Tsankova, MD, PhD Mount Sinai School of Medicine Departments of Neuroscience / Pathology 1425 Madison Avenue (Icahn 9-20 suite E) New York City, NY 10029 Phone: 212-559-8176 / Fax: 212-849-2611 Email: [email protected]
Conflict of Interest: The authors declare no conflicts of interest.
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Abstract: Diffusely infiltrating gliomas are inherently heterogeneous tumors and there are ongoing efforts to establish a classification scheme using molecular, rather than histological, features. In less than a decade, high-throughput sequencing of gliomas has transformed the field, uncovering several pivotal, highly prevalent genetic alterations that stratify patients into different prognostic and treatment-response categories. We highlight the genetic aberrations recently discovered in isocitrate dehydrogenase, alpha thalassemia/mental retardation syndrome X-linked, death-domain associated protein, histone H3.3, and telomerase reverse transcriptase and discuss how these mutations lead to unexpected changes in the epigenetic landscape in gliomas. We describe the opportunities these discoveries might provide for the development of novel, targeted therapy aimed at reversing early epigenetic aberrations in glioma precursor cells. Finally, we discuss the challenges for effective treatment of this uniformly fatal disease posed by intratumoral heterogeneity and clonal evolution.
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Introduction Diffusely infiltrating gliomas constitute a heterogeneous group of primary glial neoplasms with diverse morphology and molecular signatures, all of which are universally fatal once they undergo anaplastic progression. We have gained deep understanding of several core biological pathways involved in their oncogenesis, including the inactivation of p53 and retinoblastoma (RB) tumor-suppressors and the activation of receptor tyrosine Ras/phosphoinositide 3-kinase signaling cascades [1]. Unfortunately, targeting these pathways for glioma treatment has been difficult. More recent molecular advances in gliomas have uncovered several novel genetic mutations that are intimately associated with changes in the tumor‟s epigenetic landscape, opening new opportunities for more selective drug discovery [2-5]. Epigenetics controls gene activity without altering the DNA sequence, but rather via the tight regulation of factors that ultimately result in the opening or closing of chromatin with corresponding gene activation and repression. A nucleosome, the functional unit of chromatin, consists of eight histones (H2A, H2B, two H3, and two H4) wrapped around ~150 base pairs of DNA. The chromatin structure is frequently remodeled through posttranslational modification of its histone moieties, which include their acetylation, methylation, phosphorylation, and ubiquitylation. Chromatin remodeling is intricately controlled through the balanced activity of „writers‟, enzymes that methylate (HMTs) or acetylate (HATs) histones; „editors‟, enzymes that demethylate (HDMs) or deacetylate (HDACs) histones; and „readers‟, effector proteins recruited to bind specific chromatin domains and facilitate functional gene activation or repression (e.g., bromodomain readers of acetylation, chromodomain readers of methylation) [6,7]. DNA methylation and demethylation, controlled respectively by DNA methyltransferase (DNMT) and demethylase (TET) enzymes, exert tight epigenetic control and are intimately related to chromatin remodeling in modulating the overall transcriptional fate of a gene. The processes of cell proliferation, differentiation, and DNA repair are regulated through a delicate balance of genetic and epigenetic interactions. Its derailment can lead to
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oncogenic transformation with constitutively open chromatin at the promoters of protooncogenes leading to their overexpression or with condensed chromatin at the promoters of tumor suppressor genes causing their permanent repression [8,9]. The purpose of this review is to highlight some of the more recently discovered genetic and epigenetic abnormalities in pediatric and adult diffusely infiltrating gliomas, and to describe how they may induce unique epigenetic states in tumor cells, leading to sustained oncogenicity. An emphasis is also given to how these genetic and epigenetic markers can be targeted for the discovery of new and better therapeutic agents. Finally, we touch on the challenges that lay ahead posed by intratumoral heterogeneity and clonal evolution.
IDH mutations and hypermethylation Whole genome analysis has enabled the discovery of novel and unexpected genetic alterations in many pathological conditions, including gliomas, some of which are intimately involved in modulating the epigenome. Perhaps the best-studied example in gliomas is the recently discovered mutations within the gene encoding isocitrate dehydrogenase (IDH) [10,11]. Mutations in IDH, most commonly within the IDH1 isoform and much less frequently within the IDH2 isoform (~1-6%), have been detected in the majority of low-grade astrocytomas (~73%), low-grade oligodendrogliomas (~87%) and low-grade oligoastrocytomas (~83%), persisting as these tumors undergo high-grade progression to WHO grade III anaplastic gliomas and WHO grade IV secondary glioblastomas (GBMs) [8,12,13]. Conversely, IDH mutations are largely absent in “de-novo” or “primary” glioblastomas; in other types of primary brain tumors (pilocytic astrocytomas, gangliocytomas, gangliogliomas, dysembryoplastic neuroepithelial tumor, pleomorphic xanthoastrocytomas, ependymomas, neurocytomas, medulloblastomas / primitive neuroectodermal tumors); as well as in glioma mimics such as tumefactive multiple sclerosis, vasculitis, encephalitis, and gliosis [13]. Since their discovery in gliomas in 2008, IDH mutations have emerged as important biomarkers for predicting longer overall survival in patients with low-grade, diffusely
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infiltrating gliomas [14]; anaplastic astrocytomas/glioblastomas [15]; and anaplastic oligodendrogliomas, even in the absence of a 1p/19q co-deletion [16]. There is also growing evidence to suggest that IDH mutations are early events in gliomagenesis [17,18]. With rare exceptions, the most prevalent IDH mutation, IDH1-R132H, is diffusely present within glioma cells [19] (Odia and Tsankova, in submission) (Fig 1). It also temporally precedes other genetic abnormalities, such as 1p/19q co-deletion in oligodendrogliomas and ATRX and TP53 mutations in astrocytomas [17,18] (Fig 2). This has led to growing excitement in the field to try and understand the downstream effects of mutant IDH1. Wild type IDH enzymes (IDH1 and IDH2)* have vital functions in regulating oxidative stress in cells under normal physiological conditions. The enzymes oxidize isocitrate to alpha-ketoglutarate ( -KG), using NADP+ as a cofactor to generate reduced NADPH during the process of catalysis. The two genes are on separate chromosomes (2q33 and 15q26, respectively) and are located in distinct subcellular locations: IDH1 is located in the cytosol and peroxisomes, while IDH2 is in the mitochondria [20]. Perhaps not surprisingly, mutant forms of IDH1/2 are associated with oxidative metabolic burden on tumor cells. Intriguingly, this new metabolic state plays a pivotal role in altering the epigenetic state of the cell. An important clue that IDH mutations modulate the epigenomic state of tumor cells came from unbiased molecular profiling analyses of a large number of gliomas [1-4]. Gene expression studies subclassified glioblastomas into four molecular categories: proneural, classical, mesenchymal, and neural [3], which have gained relevance for diffusely infiltrating gliomas in general [8]. “Proneural” tumors were found to frequently harbor mutations in IDH1/2 (along with TP53/ATRX mutations in astrocytomas or 1p/19q co-deletion and FUBP1/CIC mutations in oligodendrogliomas, whereas “classical” ones had frequent EGFR amplification and loss of chromosome 10 but wild type IDH1 status [3,21,22]. In parallel to genetic profiling, comprehensive DNA methylation analyses of gliomas showed that “proneural” tumors also distinguish themselves from the other three subgroups by exhibiting increased levels of global DNA methylation at CpG islands, commonly referred to as the
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glioma CpG island methylator phenotype (or G-CIMP) [4]. The link between IDH mutations and G-CIMP lies in the abnormal oncometabolite 2-hydroxyglutarate (2-HG), overproduced by gain-of-function IDH mutant enzymes acting on -KG [23]. Accumulation of 2-HG impairs -ketoglutarate-dependent dioxygenases, including DNA and histone demethylases [24,25]. Some of these fall in the TET family of proteins (TET 1/2), which convert DNA 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5-hmC) [25]. Impeding this essential step in demethylation of DNA effectively produces regions of CpG hypermethylation in proneural IDH1 mutant gliomas [26]. Moreover, 2-HG directly inhibits the activity of histone modifying enzymes, particularly the Jumonji domain-containing histone demethylases (JHDMs). Their inhibition results in accumulations of repressive chromatin marks, most notably H3K9 and H3K27 trimethylation [24] (Fig 2). The exact effects of increasing histone and DNA methylation levels in mutant IDH tumors are not yet clear, but the altered epigenetic landscape appears to facilitate a block in cell differentiation and a cellular state sensitized to malignant transformation by allowing the aberrant downstream expression of oncogenic gene targets [5]. This paves the way for exciting new research aimed at understanding how specific epigenetic modifications induced in IDH-mutant gliomas differentially affect distinct genes. Their elucidation will allow the use of specific small molecule inhibitors to selectively target epigenetic changes in gliomagenic cells, hopefully in their nascent state. Potential molecules to target would include mutant IDH itself, its oncogenic neometabolite 2-HG, and its downstream effectors. In just the past few years, there has been exciting progress in discovering molecules that target mutant IDH and its associated oncometabolite, with some showing efficacy for inhibiting glioma cell growth. One such example is the AGI-5198 small molecule inhibitor, detected in a high-throughput screen for selective inhibitors of IDH1-R132H. Rohle et al. recently showed that AGI-5198 can inhibit the growth of mutant (but not wild type) IDH1 glioma cells and also block the production of 2-HG [27]. Furthermore, the inhibitor induced the expression of genes associated with a differentiated glial fate. An independent small molecule high throughput screen identified the probe ML309, which could also inhibit the
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IDH1-R132H enzyme, reducing 2-HG production in a mutant glioblastoma cell line [28]. These discoveries may allow for the selective “reprogramming” of cellular metabolism in IDH1-mutant gain-of-function tumor cells but not in infiltrated normal brain cells. Other drug design strategies for inhibiting IDH1mutant gliomas have included biochemical inhibition of tumor metabolism using glutaminase inhibitors such as zaprinast, which perturbs 2-HG production but lacks the tumor specificity of AGI-5198 [29,30]. Decitabine, a non-specific DNMT inhibitor, has shown reversal of DNA methylation marks induced by IDH along with re-expression of genes associated with differentiation [31].
Telomere lengthening and H3.3-ATRX-DAXX mutations Telomeres consist of a region of hexanucleotide repeats, 5 to 10 kilobases long, which cap the ends of eukaryotic chromosomes, maintaining their stability. Because telomere shortening occurs with each cell division, eventually triggering p53-dependent senescence or apoptosis, normal stem cells and malignant cells must have a mechanism of telomere maintenance to allow prolonged or unlimited cell proliferation. In many common cancers of epithelial origin, such as lung, breast, colon, and prostate carcinomas, constitutive telomerase activity in somatic stem cells is thought to increase through epigenetic dysregulation during the process of malignant transformation (reviewed in [32]). In malignancies derived from non-epithelial cells where telomerase is not constitutively active, including many primary brain tumors and certain sarcomas, telomere lengthening must occur through other means. One such mechanism, alternative lengthening of telomeres (ALT), operates in the absence of detectable telomerase activity [33]. Rather, homologous recombination resulting in telomere destabilization and length heterogeneity is observed in ALT-positive cells. A highly significant correlation between ALT and loss of nuclear expression of either of the proteins encoded by the alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) genes was first reported in a study of pancreatic neuroendocrine tumors [34]. Earlier studies had noted that a high proportion of WHO grade
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II and III astrocytomas and a lower proportion glioblastomas exhibited the ALT phenotype [22,35,36]. It was subsequently shown that gliomas harboring the ALT phenotype have loss of nuclear ATRX expression [37-40]. ATRX and DAXX form an ATP-dependent remodeling complex that localizes to telomeres and heterochromatic regions of the genome, where it has a role in histone variant H3.3 deposition in chromatin and regulating telomere homeostasis. In ATRX syndrome, germline inactivation of ATRX causes H3.3 to be lost in favor of the transcriptionally repressive histone variant macroH2A at the alpha-globin gene cluster in the subtelomeric region of chromosome 16, epigenetically silencing expression of these genes [41,42]. In addition, ATRX localization at G-rich tandem repeats near genes may assist in resolving DNA quadruplex secondary structures that are also transcriptionally repressive [43]. Thus, the specific functions of ATRX-DAXX may differ in differing chromatin contexts. ATRX mutations have been found to be highly prevalent in diffuse astrocytomas, in one study occurring at a frequency of 67% and 73% in grade II and III astrocytomas, respectively [22]. The specific somatic mutations are variable, but generally result in loss of the C-terminal DNA helicase domain of the protein via truncating frameshift mutations. ATRX mutations occur somewhat less frequently in secondary and pediatric glioblastomas; and rarely in primary glioblastomas and pure oligodendrogliomas. Furthermore, they are found almost exclusively in concert with IDH mutations, frequently with TP53 mutations, and almost never with 1p/19q co-deletion, which is the characteristic chromosomal aberration of pure oligodendrogliomas (Fig 1). Because mixed oligoastrocytomas and pure astrocytomas progress into high-grade secondary glioblastomas significantly more quickly than oligodendrogliomas, ATRX loss is emerging both as a defining diagnostic and important prognostic marker in astrocytic gliomas [22]. Given the role of ATRX in chromatin regulation, the effects of ATRX loss on the epigenome and transcriptome are of strong interest. ATRX binds to tandemly repetitive genomic regions enriched for G and C, including CpG islands near gene promoters. In a comparison of lymphoid cells expressing and lacking ATRX, four genes near or containing
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an ATRX binding target were noted to be significantly altered in expression: NME4, SLC7A5, RASA3, and GAS8 [43]. Similar studies examining differences in gene expression in IDHmutant/ATRX-intact and IDH-mutant/ATRX-lost glial progenitors will shed light on the pathogenesis of low-grade gliomas. Because ATRX also has a role in resolving abnormal DNA secondary structures, such as G-quadruplexes, it will be of considerable interest to examine how the G-CIMP-positive epigenome is affected by ATRX loss, and what impact this has on genome stability. Furthermore, the link between ATRX and G-quadruplexes presents the possibility that small molecules that bind to and stabilize these DNA structures will at least partly reconstitute ATRX function in astrocytomas. Riou et al. demonstrated the ability of one such ligand to induce senescence in immortalized ALT-positive fibroblasts [44]. The binding partner of ATRX, DAXX, is a chaperone of H3.3 [45]. In a study of 48 pediatric glioblastomas, 21 (44%) had a mutation affecting at least one of the genes encoding these proteins. Mutations in H3F3A, the gene encoding H3.3, appear to be nearly exclusive to GBMs occurring in the pediatric population or young adults, having been found to be absent in a cohort of 291 adult GBMs subjected to whole exome sequencing [2,40]. Two distinct heterozygous mutations were identified, K27M and G34R/V, each near posttranslational modification sites associated with transcriptional repression and activation, respectively. Furthermore, of 15 samples with H3F3A mutations, 9 also had mutations in ATRX and 12 in TP53. Strikingly, H3F3A mutations were never found together with IDH mutations, suggesting possibly analogous roles in reshaping the epigenome (Fig 2). K27M mutations in H3.3 and another histone variant, H3.1, tend to occur in children with brainstem and thalamic (i.e., midline) high-grade gliomas. These gliomas have also recently been found to contain mutations in ACVR1 and FGFR1, the genes encoding the type 1 activin A receptor and fibroblast growth factor receptor 1, respectively [46-49]. In contrast, G34R/V mutations in H3.3 occur more commonly in supratentorial tumors arising in young adults [37,50]. Although H3.3 comprises only 1% of total cellular H3, K27M substitutions in this histone variant dramatically decrease global levels of di- and trimethylation at H3K27, while overall levels of acetylation at H3K27 modestly increase [51].
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This effect appears to be mediated by a hydrophobic interaction between the methionine side chain and aromatic residues at the active site of the histone methyltransferase PRC2, inhibiting its activity. Non-glycine residues at position 34 of the tail of H3.3 appear to have a similar but less far-reaching inhibitory effect on another histone methyltransferase, SETD2, which normally acts at H3K36 to increase activating methylation. This suppression of methyltransferase activity contrasts with the suppression of DNA and histone demethylases due to 2-HG in IDH-mutant gliomas (Fig 2). Therefore, while compounds designed to inhibit the neo-enzymatic activity of IDH1-R132H might be predicted to be effective in treating the IDH-mutant tumors commonly found in adults, compounds with the opposite epigenetic effect, or perhaps 2-HG itself, would be predicted to reverse alterations in the H3.3-mutant tumors commonly found in children and young adults. It becomes apparent that a thorough understanding of the epigenetic imbalances extant in each molecular subtype of glioma will be required to match the most appropriate agents in preclinical and clinical testing. DAXX was mutated in 2 of 42 pediatric GBM samples analyzed by whole exome sequencing [40]. Additional tissue microarray analysis on 124 pediatric GBM samples revealed loss of DAXX staining in 6% of samples, compared to loss of ATRX in 35%. DAXX and ATRX mutations tended to be mutually exclusive of each other and of H3.3 K27M mutations, while there was a striking overlap with H3.3 G34R/V mutations. These mutational patterns may be partly explained by a requirement for intact ATRX in order for DAXX to localize to telomeres, and that many DAXX mutations occur within the C-terminal histonebinding domain of the protein, disrupting its ability to bind to and chaperone H3.3 to heterochromatin. Thus, ATRX, DAXX, and H3.3 each play complementary and essential roles in a core cellular mechanism for chromatin remodeling at telomeres and gene regulatory elements.
TERT promoter mutations Pure oligodendrogliomas and primary GBMs make up the majority of diffuse gliomas that do not exhibit the ALT phenotype. Because both these tumor types, unlike cancers of
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epithelial tissues, presumably arise from cells that are not constantly self-renewing throughout the lifespan of the organism, another mechanism of telomere lengthening is needed. A study examining 1230 tumors representing 60 tumor types demonstrated a 51.1% frequency among gliomas of somatic mutations in the telomerase reverse transcriptase (TERT) promoter gene, which encodes the catalytic subunit of telomerase [52]; these mutations had been previously described in melanoma [53,54]. TERT promoter mutations occurred in 51 of 78 tumors classified as GBM by histology, and co-occurred tightly with EGFR amplification. Likewise, TERT promoter mutations were found in 35 of 45 oligodendrogliomas diagnosed by histology, and in 98% of tumors harboring an IDH1/2 mutation and 1p19q co-deletion [55]. In contrast, no tumor exhibiting ALT/ATRX loss was found to have a TERT mutation. TERT promoter mutation status may therefore be a powerful molecular diagnostic tool, especially in helping to distinguish secondary and primary GBMs, which may portend different prognoses. The two most common TERT promoter mutations, C228T and C250T, occur in a mutually exclusive manner in gliomas and are associated with a more than six-fold increase in TERT mRNA expression over levels observed in TERT wild-type tumors [56]. Both mutations create an identical, novel 11-bp consensus-binding site for ETS family transcription factors [53,54], which explains how these heterozygous non-coding region somatic point mutations may act as drivers in gliomagenesis. The ETS family is comprised of a large number of proteins with both activating and repressing functions in the regulation of diverse gene networks important in cancer (reviewed in [57]); elucidating the specific ETS factors at play in TERT mutated gliomas will undoubtedly reveal new targets for therapy. Furthermore, both telomerase inhibition and the exploitation of telomerase-overexpressing cells hold promise as therapeutic strategies directed specifically at the self-renewing glioma stem cell population. A number of such agents, including small molecule inhibitors (e.g., BIBR1532), antisense oligonucleotides (e.g., imetelstat) [58], G-quadruplex stabilizers (e.g., telomestatin), and peptide vaccines directed against hTERT (e.g. GV1001), are actively being investigated in preclinical and early phase clinical trials (reviewed in [59]).
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Clonal Evolution Next-generation high-throughput technologies have begun to reveal, in unprecedented detail, the complexity that exists in diffuse gliomas as a result of genomic and epigenomic heterogeneity, spatially and temporally. In applying the gene expression classification of Verhaak et al. [3] to glioblastoma fragments sampled from different regions of the same tumor, Sottoriva et al. [60] demonstrated that multiple transcriptional phenotypes coexist in the majority of patients. Further, they compared CpG methylation patterns at preselected genomic loci as a measure of the “mitotic distance” between spatially separated intratumoral samples. Using these data, phylogenies of clonal populations of tumor cells could be reconstructed, providing evidence of ongoing branching clonal evolution within gliomas. DNA methylation data have also been used to compare tumors from different individuals, identifying a handful of distinct epigenetic phenotypes (of which G-CIMP is only one), analogous to the previously recognized gene expression clusters [2,50]. It seems likely, although remains to be proven, that multiple epigenetic phenotypes also coexist in individual tumors. Adding another layer of complexity to this are the effects of therapeutic agents that kill cancer cells by damaging DNA. The two agents used as part of the standard treatment paradigm, ionizing radiation and the alkylating chemotherapy temozolomide (TMZ), are prime examples. In a study of 23 low-grade gliomas and their higher grade recurrences, Johnson et al [61] used whole exome sequencing to demonstrate a high frequency of C>T/G>A mutations at CpC and CpT dinucleotides in TMZ-treated tumors relative to those not treated, and suggested that these transitions are TMZ-induced. Furthermore, they were able to identify TMZ-associated deleterious mutations in genes coding for components of two signaling pathways associated with progression to GBM, the RB and Akt-mTOR pathways. Similarly, exposure to ionizing radiation can produce somatic mutations by inducing DNA double-strand breaks that are then misrepaired, resulting in genomic structural variants such as insertion-deletions and chromosomal translocations [62,63].
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Together, these findings suggest that DNA-damaging therapeutic agents have the potential to influence – perhaps even accelerate – the evolutionary processes already occurring spontaneously within gliomas. Unfortunately, current approaches of sequencing fragments of resected tumors in search of targetable mutations will be inadequate to characterize the neoplastic clonal populations left unresected, let alone provide information about how these cells will evolve in the face of selection pressures and mutagens during treatment. Convergent evolution, whereby different clonal populations independently acquire genetic alterations affecting the same signaling pathways [64], may offer the possibility of predicting the most probable future drivers of progression and recurrence given a particular ecosystem of clones, and hence an opportunity to subvert them. We speculate that research investigating DNA damage patterns in experimental models of different transcriptional and epigenetic tumor phenotypes will shed light on these dynamics.
Targeting glioma precursors In order to cure patients with diffusely infiltrating gliomas, we must catch the disease in its nascent state and design therapeutic targets that will eradicate its seed before it becomes widespread. To appreciate how gliomas arise, we must also expand our understanding of the early molecular alterations present in glioma precursor cells, which have been suggested to arise from neural stem cells in the subventricular zone and more committed glial progenitors found predominantly in white matter [8,65-69]. There is growing evidence that changes in the epigenetic landscape of progenitor/stem cells contribute to early stages of cancerous transformation [9,70,71]. We and others have hypothesized that some progenitor/stem cells are „„poised‟‟ in a pre-neoplastic state, where they are epigenetically primed for subsequent genetic alterations, which ultimately result in their oncogenic transformation [8,70]. Designing drugs that target genetic/epigenetic alterations in tumor cells during very early stages of gliomagenesis could effectively kill the tumor before it has a chance to fully form and infiltrate. To this end, several groups are studying the genetic and epigenetic profile of various neural/glial progenitors, using predominantly rodent
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and human cell culture models [72-74]. The ideal model of study would entail the isolation of rare and pure neural/glial precursor cells from their endogenous niche, unperturbed by artificial cell culture alterations: a challenging endeavor (Tsankova et al., in submission).
Conclusions Recent advances in glioma research reveal the presence of a sophisticated network of interactions between genetics and epigenetics in the contexts of diffuse glioma formation and its molecular substratification (Fig 2). IDH mutations in “proneural” gliomas induce hypermethylation of DNA and histones by inhibiting the actions of DNA and histone demethylases [24,26]. ATRX mutations, present in both “proneural” astrocytomas and pediatric glioblastomas, affect heterochromatin and the alternative lengthening of telomeres [40,45]. Mutations in the histone variant H3.3, seen predominantly in pediatric glioblastomas, decrease global levels of histone methylation at H3K27 and H3K36, and inhibit the activity of histone methyltransferases PRC2 and SETD2 [51] (Fig 2). The interactions of the above mutations with one another and their downstream epigenetic alterations are remarkably complex, and represent an area of active research. Their interface is dynamic and multidimensional, with the ability to change in space and time. This makes the deconvolution of specific oncogenic targets for specific gliomas tremendously challenging. Nevertheless, some clear targets do emerge, and the challenge in the next decade will be to design specific drugs that can block the genetic (and epigenetic) alterations before low-grade gliomas have undergone malignant transformation. Already, inhibitors of HDACs or DNMTs have shown activity in glioblastoma cell lines and glioma models, and have even advanced to use in clinical trials [75,76]. These drugs, however, are limited due to their nonspecific inhibition of various chromatin modifying enzymes, and their lack of distinction between neoplastic and normal cell targets. Indeed, the identification of selective IDH1R132H inhibitors that can now function in tumor-cell specific manner is a significant step forward in the discovery of drugs with superior target specificity [27,28]. Other areas for
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future exploration include inhibitors of TET enzymes; G-quadruplex stabilizers in ATRX mutant gliomas; H3F3A mutations and their effect on histone methylation through specific interaction with PRC2; and ETS factors, hTERT inhibitors, and immunotherapy in TERT mutated tumors. The use of specific small molecules to target unique enzymatic moieties within the polycomb repressor complex (EZH2 inhibitors) or bromodomains readers that selectively bind acetylated lysine histone domains (JQ1 inhibitors) are also emerging as attractive tools for therapeutic epigenetic modification, and are currently being tested in glioblastoma cell lines [74,77,78]. Several of these inhibitors show remarkably favorable toxicity profiles. Although gliomas are markedly heterogeneous, their molecular ontology is beginning to emerge. In less than a decade, genomic profiling of gliomas has transformed the field, with the discovery of several pivotal mutations that not only delineate novel means of subclassification, but also offer new hope for the discovery of highly specific drug targets to combat this deadly disease.
REFERENCES:
1.
Cancer Genome Atlas Research N. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 2008; 455: 1061-1068
2.
Brennan CW, Verhaak RG, McKenna A et al. The somatic genomic landscape of glioblastoma. Cell 2013; 155: 462-477
3.
Verhaak RG, Hoadley KA, Purdom E et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer cell 2010; 17: 98-110
4.
Noushmehr H, Weisenberger DJ, Diefes K et al. Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer cell 2010; 17: 510-522
15
5.
Kim J, DeBerardinis RJ. Cancer. Silencing a metabolic oncogene. Science 2013; 340: 558-559
6.
Jenuwein T, Allis CD. Translating the histone code. Science 2001; 293: 1074-1080
7.
Plass C, Pfister SM, Lindroth AM et al. Mutations in regulators of the epigenome and their connections to global chromatin patterns in cancer. Nature reviews Genetics 2013; 14: 765-780
8.
Tsankova NM, Canoll P. Advances in genetic and epigenetic analyses of gliomas: a neuropathological perspective. J Neurooncol 2014; 119: 481-490
9.
Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nature reviews Genetics 2006; 7: 21-33
10.
Parsons DW, Jones S, Zhang X et al. An integrated genomic analysis of human glioblastoma multiforme. Science 2008; 321: 1807-1812
11.
Yan H, Parsons DW, Jin G et al. IDH1 and IDH2 mutations in gliomas. The New England journal of medicine 2009; 360: 765-773
12.
Hartmann C, Meyer J, Balss J et al. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta neuropathologica 2009; 118: 469-474
13.
Balss J, Meyer J, Mueller W et al. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta neuropathologica 2008; 116: 597-602
14.
Houillier C, Wang X, Kaloshi G et al. IDH1 or IDH2 mutations predict longer survival and response to temozolomide in low-grade gliomas. Neurology 2010; 75: 1560-1566
15.
Hartmann C, Hentschel B, Wick W et al. Patients with IDH1 wild type anaplastic astrocytomas exhibit worse prognosis than IDH1-mutated glioblastomas, and IDH1 mutation status accounts for the unfavorable prognostic effect of higher age: implications for classification of gliomas. Acta neuropathologica 2010; 120: 707-718
16.
Cairncross JG, Wang M, Jenkins RB et al. Benefit from procarbazine, lomustine, and vincristine in oligodendroglial tumors is associated with mutation of IDH. Journal of
16
clinical oncology : official journal of the American Society of Clinical Oncology 2014; 32: 783-790 17.
Lass U, Numann A, von Eckardstein K et al. Clonal analysis in recurrent astrocytic, oligoastrocytic and oligodendroglial tumors implicates IDH1- mutation as common tumor initiating event. PloS one 2012; 7: e41298
18.
Watanabe T, Nobusawa S, Kleihues P et al. IDH1 mutations are early events in the development of astrocytomas and oligodendrogliomas. The American journal of pathology 2009; 174: 1149-1153
19.
Preusser M, Wohrer A, Stary S et al. Value and limitations of immunohistochemistry and gene sequencing for detection of the IDH1-R132H mutation in diffuse glioma biopsy specimens. Journal of neuropathology and experimental neurology 2011; 70: 715-723
20.
Horbinski C. What do we know about IDH1/2 mutations so far, and how do we use it? Acta neuropathologica 2013; 125: 621-636
21.
Bettegowda C, Agrawal N, Jiao Y et al. Mutations in CIC and FUBP1 contribute to human oligodendroglioma. Science 2011; 333: 1453-1455
22.
Jiao Y, Killela PJ, Reitman ZJ et al. Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas. Oncotarget 2012; 3: 709-722
23.
Dang L, White DW, Gross S et al. Cancer-associated IDH1 mutations produce 2hydroxyglutarate. Nature 2009; 462: 739-744
24.
Lu C, Ward PS, Kapoor GS et al. IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature 2012; 483: 474-478
25.
Figueroa ME, Abdel-Wahab O, Lu C et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer cell 2010; 18: 553-567
26.
Turcan S, Rohle D, Goenka A et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 2012; 483: 479-483
17
27.
Rohle D, Popovici-Muller J, Palaskas N et al. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science 2013; 340: 626-630
28.
Davis M, Pragani R, Popovici-Muller J et al. ML309: A potent inhibitor of R132H mutant IDH1 capable of reducing 2-hydroxyglutarate production in U87 MG glioblastoma cells. In, Probe Reports from the NIH Molecular Libraries Program. Bethesda (MD); 2010
29.
Seltzer MJ, Bennett BD, Joshi AD et al. Inhibition of glutaminase preferentially slows growth of glioma cells with mutant IDH1. Cancer Res 2010; 70: 8981-8987
30.
Elhammali A, Ippolito JE, Collins L et al. A high-throughput fluorimetric assay for 2hydroxyglutarate identifies Zaprinast as a glutaminase inhibitor. Cancer Discov 2014; 4: 828-839
31.
Turcan S, Fabius AW, Borodovsky A et al. Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine. Oncotarget 2013; 4: 1729-1736
32.
Zhu J, Zhao Y, Wang S. Chromatin and epigenetic regulation of the telomerase reverse transcriptase gene. Protein & cell 2010; 1: 22-32
33.
Bryan TM, Englezou A, Gupta J et al. Telomere elongation in immortal human cells without detectable telomerase activity. The EMBO journal 1995; 14: 4240-4248
34.
Jiao Y, Shi C, Edil BH et al. DAXX/ATRX, MEN1, and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors. Science 2011; 331: 11991203
35.
Henson JD, Hannay JA, McCarthy SW et al. A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas. Clinical cancer research : an official journal of the American Association for Cancer Research 2005; 11: 217-225
36.
Heaphy CM, Subhawong AP, Hong SM et al. Prevalence of the alternative lengthening of telomeres telomere maintenance mechanism in human cancer subtypes. The American journal of pathology 2011; 179: 1608-1615
18
37.
Khuong-Quang DA, Buczkowicz P, Rakopoulos P et al. K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas. Acta neuropathologica 2012; 124: 439-447
38.
Liu XY, Gerges N, Korshunov A et al. Frequent ATRX mutations and loss of expression in adult diffuse astrocytic tumors carrying IDH1/IDH2 and TP53 mutations. Acta neuropathologica 2012; 124: 615-625
39.
Kannan K, Inagaki A, Silber J et al. Whole-exome sequencing identifies ATRX mutation as a key molecular determinant in lower-grade glioma. Oncotarget 2012; 3: 1194-1203
40.
Schwartzentruber J, Korshunov A, Liu XY et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature 2012; 482: 226-231
41.
Ratnakumar K, Duarte LF, LeRoy G et al. ATRX-mediated chromatin association of histone variant macroH2A1 regulates alpha-globin expression. Genes & development 2012; 26: 433-438
42.
Dhayalan A, Tamas R, Bock I et al. The ATRX-ADD domain binds to H3 tail peptides and reads the combined methylation state of K4 and K9. Human molecular genetics 2011; 20: 2195-2203
43.
Law MJ, Lower KM, Voon HP et al. ATR-X syndrome protein targets tandem repeats and influences allele-specific expression in a size-dependent manner. Cell 2010; 143: 367-378
44.
Riou JF, Guittat L, Mailliet P et al. Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands. Proceedings of the National Academy of Sciences of the United States of America 2002; 99: 2672-2677
45.
Lewis PW, Elsaesser SJ, Noh KM et al. Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres. Proceedings of the National Academy of Sciences of the United States of America 2010; 107: 14075-14080
19
46.
Wu G, Diaz AK, Paugh BS et al. The genomic landscape of diffuse intrinsic pontine glioma and pediatric non-brainstem high-grade glioma. Nature genetics 2014; 46: 444-450
47.
Taylor KR, Mackay A, Truffaux N et al. Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma. Nature genetics 2014; 46: 457-461
48.
Fontebasso AM, Papillon-Cavanagh S, Schwartzentruber J et al. Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma. Nature genetics 2014; 46: 462-466
49.
Buczkowicz P, Hoeman C, Rakopoulos P et al. Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations. Nature genetics 2014; 46: 451-456
50.
Sturm D, Witt H, Hovestadt V et al. Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer cell 2012; 22: 425-437
51.
Lewis PW, Muller MM, Koletsky MS et al. Inhibition of PRC2 activity by a gain-offunction H3 mutation found in pediatric glioblastoma. Science 2013; 340: 857-861
52.
Killela PJ, Reitman ZJ, Jiao Y et al. TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal. Proceedings of the National Academy of Sciences of the United States of America 2013; 110: 6021-6026
53.
Horn S, Figl A, Rachakonda PS et al. TERT promoter mutations in familial and sporadic melanoma. Science 2013; 339: 959-961
54.
Huang FW, Hodis E, Xu MJ et al. Highly recurrent TERT promoter mutations in human melanoma. Science 2013; 339: 957-959
55.
Arita H, Narita Y, Fukushima S et al. Upregulating mutations in the TERT promoter commonly occur in adult malignant gliomas and are strongly associated with total 1p19q loss. Acta neuropathologica 2013; 126: 267-276
20
56.
Arita H, Narita Y, Takami H et al. TERT promoter mutations rather than methylation are the main mechanism for TERT upregulation in adult gliomas. Acta neuropathologica 2013; 126: 939-941
57.
Kar A, Gutierrez-Hartmann A. Molecular mechanisms of ETS transcription factormediated tumorigenesis. Critical reviews in biochemistry and molecular biology 2013; 48: 522-543
58.
Marian CO, Cho SK, McEllin BM et al. The telomerase antagonist, imetelstat, efficiently targets glioblastoma tumor-initiating cells leading to decreased proliferation and tumor growth. Clinical cancer research : an official journal of the American Association for Cancer Research 2010; 16: 154-163
59.
Ruden M, Puri N. Novel anticancer therapeutics targeting telomerase. Cancer treatment reviews 2013; 39: 444-456
60.
Sottoriva A, Spiteri I, Piccirillo SG et al. Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proceedings of the National Academy of Sciences of the United States of America 2013; 110: 4009-4014
61.
Johnson BE, Mazor T, Hong C et al. Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma. Science 2014; 343: 189-193
62.
Mani RS, Tomlins SA, Callahan K et al. Induced chromosomal proximity and gene fusions in prostate cancer. Science 2009; 326: 1230
63.
Hakim O, Resch W, Yamane A et al. DNA damage defines sites of recurrent chromosomal translocations in B lymphocytes. Nature 2012; 484: 69-74
64.
Gerlinger M, Rowan AJ, Horswell S et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. The New England journal of medicine 2012; 366: 883-892
65.
Sanai N, Alvarez-Buylla A, Berger MS. Neural stem cells and the origin of gliomas. The New England journal of medicine 2005; 353: 811-822
21
66.
Alcantara Llaguno S, Chen J, Kwon CH et al. Malignant astrocytomas originate from neural stem/progenitor cells in a somatic tumor suppressor mouse model. Cancer cell 2009; 15: 45-56
67.
Jacques TS, Swales A, Brzozowski MJ et al. Combinations of genetic mutations in the adult neural stem cell compartment determine brain tumour phenotypes. The EMBO journal 2010; 29: 222-235
68.
Ozawa T, Riester M, Cheng YK et al. Most human non-GCIMP glioblastoma subtypes evolve from a common proneural-like precursor glioma. Cancer cell 2014; 26: 288-300
69.
Canoll P, Goldman JE. The interface between glial progenitors and gliomas. Acta neuropathologica 2008; 116: 465-477
70.
Tsai HC, Baylin SB. Cancer epigenetics: linking basic biology to clinical medicine. Cell Res 2011; 21: 502-517
71.
Bernstein BE, Mikkelsen TS, Xie X et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 2006; 125: 315-326
72.
Auvergne RM, Sim FJ, Wang S et al. Transcriptional differences between normal and glioma-derived glial progenitor cells identify a core set of dysregulated genes. Cell reports 2013; 3: 2127-2141
73.
Yoo S, Bieda MC. Differences among brain tumor stem cell types and fetal neural stem cells in focal regions of histone modifications and DNA methylation, broad regions of modifications, and bivalent promoters. BMC genomics 2014; 15: 724
74.
Lim DA, Alvarez-Buylla A. Adult neural stem cells stake their ground. Trends in neurosciences 2014; 37: 563-571
75.
Iwamoto FM, Lamborn KR, Kuhn JG et al. A phase I/II trial of the histone deacetylase inhibitor romidepsin for adults with recurrent malignant glioma: North American Brain Tumor Consortium Study 03-03. Neuro-oncology 2011; 13: 509-516
22
76.
Friday BB, Anderson SK, Buckner J et al. Phase II trial of vorinostat in combination with bortezomib in recurrent glioblastoma: a north central cancer treatment group study. Neuro-oncology 2012; 14: 215-221
77.
Cheng Z, Gong Y, Ma Y et al. Inhibition of BET bromodomain targets genetically diverse glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research 2013; 19: 1748-1759
78.
Dror N, Mandel M, Lavie G. Unique anti-glioblastoma activities of hypericin are at the crossroad of biochemical and epigenetic events and culminate in tumor cell differentiation. PloS one 2013; 8: e73625
Figure Legends
Fig 1. Clinical neuropathological analysis of proneural gliomas Proneural gliomas include both oligodendrogliomas (top left) and diffuse astrocytomas (top right), both of which express mutant IDH1 (middle) and additionally show either 1p/19q codeletion (bottom left; red: 1p/19q probe, green: control chromosome probe) or ATRX and TP53 mutations (bottom right). The presence and extent of IDH mutation in glioma tissue is assessed by immunohistochemistry using an antibody specific for IDH1-R132H. Truncating ATRX mutations lead to loss of ATRX nuclear staining in astrocytoma cells, while TP53 mutations cause an aberrant nuclear accumulation of p53. Scale bars = 50um; all micrographs are scaled similarly.
Fig 2. Interplay between gain-of-function genetic mutations and epigenetic alterations in the pathogenesis of several diffuse glioma subtypes Gain-of-function mutations early in gliomagenesis inhibit several enzymes (PRC2, SETD2, JHDMs, TETs) key to altering the epigenetic landscape within the nucleus of a glial progenitor cell (top). Sites of mutation are indicated by lightning bolts. Then, acquired mutations in ATRX-DAXX and/or p53, or the TERT promoter (middle) immortalize the cell
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and undermine genomic integrity. This permits the accumulation and selection of additional somatic alterations, typically affecting key signaling pathways, that ultimately lead to the development of the indicated glioma subtypes, classically distinguished by histology and age of onset (bottom).
* An IDH3 enzyme also exists but no cancer-associated IDH3 mutations have been discovered to date.
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FIG 1
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FIG 2
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