Search

BackgroundDiffuse intrinsic pontine glioma (DIPG) is associated with transcriptional dysregulation driven by H3K27 mutation. The super elongation complex (SEC) is required for transcriptional elongation through release of RNA polymerase II (Pol II). Inhibition of transcription elongation by SEC disruption can be an effective therapeutic strategy of H3K27M-mutant DIPG. Here, we tested the effect of pharmacological disruption of the SEC in H3K27M-mutant DIPG to advance understanding of the molecular mechanism and as a new therapeutic strategy for DIPG.MethodsShort hairpin RNAs (shRNAs) were used to suppress the expression of AF4/FMR2 4 (AFF4), a central SEC component, in H3K27M-mutant DIPG cells. A peptidomimetic lead compound KL-1 was used to disrupt a functional component of SEC. Cell viability assay, colony formation assay, and apoptosis assay were utilized to analyze the effects of KL-1 treatment. RNA- and ChIP-sequencing were used to determine the effects of KL-1 on gene expression and chromatin occupancy. We treated mice bearing H3K27M-mutant DIPG patient-derived xenografts (PDXs) with KL-1. Intracranial tumor growth was monitored by bioluminescence image and therapeutic response was evaluated by animal survival.ResultsDepletion of AFF4 significantly reduced the cell growth of H3K27M-mutant DIPG. KL-1 increased genome-wide Pol II occupancy and suppressed transcription involving multiple cellular processes that promote cell proliferation and differentiation of DIPG. KL-1 treatment suppressed DIPG cell growth, increased apoptosis, and prolonged animal survival with H3K27M-mutant DIPG PDXs.ConclusionsSEC disruption by KL-1 increased therapeutic benefit in vitro and in vivo, supporting a potential therapeutic activity of KL-1 in H3K27M-mutant DIPG.

Pediatric high-grade gliomas (pHGGs), including glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), are morbid brain tumors. Even with treatment survival is poor, making pHGG the number one cause of cancer death in children. Up to 80% of DIPGs harbor a somatic missense mutation in genes encoding histone H3. To investigate whether H3K27M is associated with distinct chromatin structure that alters transcription regulation, we generated the first high-resolution Hi-C maps of pHGG cell lines and tumor tissue. By integrating transcriptome (RNA-seq), enhancer landscape (ChIP-seq), genome structure (Hi-C), and chromatin accessibility (ATAC-seq) datasets from H3K27M and wild-type specimens, we identified tumor-specific enhancers and regulatory networks for known oncogenes. We identified genomic structural variations that lead to potential enhancer hijacking and gene coamplification, including A2M, JAG2, and FLRT1 Together, our results imply three-dimensional genome alterations may play a critical role in the pHGG epigenetic landscape and contribute to tumorigenesis.

Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas. This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase). Consequently, reduced H3K36me3 is observed on H3.3G34V nucleosomes relative to wild-type, contributing to genomic instability and driving a distinct gene expression signature associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3.3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3.3G34V mutant protein in pediatric glioma on H3K36me3, H3K27me3 and H3.3 enrichment in vitro. We found that the doxycycline-inducible shRNA knockdown of mutant H3F3A encoding the H3.3G34V protein resulted in loss of H3.3G34V enrichment and increased H3K36me3 enrichment throughout the genome. After knockdown, H3.3G34V enrichment was preserved at loci observed to have the greatest H3.3G34V and H3K36me3 enrichment prior to knockdown. Induced expression of mutant H3.3G34V protein in vitro was insufficient to induce genomic H3K36me3 enrichment patterns observed in H3.3G34V mutant glioma cells. We also observed strong co-enrichment of H3.3G34V and wild-type H3.3 protein, as well as greater H3K27me3 enrichment, in cells expressing H3.3G34V. Taken together, our study demonstrates the effects of H3.3G34V mutant protein on genomic H3K36me3, H3K27me3 and H3.3 enrichment patterns in isogenic cell lines.

Background Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. Methods We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). Results We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

INTRODUCTION Pediatric high-grade gliomas (pHGGs), including glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), are highly morbid brain tumors. Up to 80% of DIPGs harbor a somatic missense mutation in genes encoding Histone H3. To investigate whether the H3K27M mutant protein is associated with distinct chromatin structure affecting transcription regulation, we generated the first high-resolution Hi-C and ATAC-Seq maps of pHGG cell lines, and integrated these with tissue and cell genomic data. METHODS We generated sequencing data from patient-derived cell lines (DIPG n=6, GBM n=3, normal n=2) and frozen tissue specimens (DIPG n=1, normal brainstem n=1). Analyses included cell line RNA-Seq, ChIP-Seq (H3K27ac, H3K27me3, H3K27M) and genome-wide chromatin conformation capture (Hi-C), as well as tissue ATAC-Seq. Publicly available pediatric glioma tissue ChIP-Seq data was integrated with cell data. CRISPR knock-down of target enhancer regions was performed. RESULTS We identified tumor-specific enhancers and regulatory networks for known oncogenes in DIPG and GBM. In DIPG, FOX, SOX, STAT and SMAD families were among top H3K27Ac enriched motifs. Significant differences in Topologically Associating Domains (TADs) and DNA looping were observed at OLIG2 and MYCN in H3K27M mutant DIPG, relative to wild-type GBM and normal cells. Pharmacologic treatment targeting H3K27Ac (BET and Bromodomain inhibition) altered these 3D structures. Functional analysis of differentially enriched enhancers in DIPG implicated SOX2, SUZ12, and TRIM24 as top activated upstream regulators. Distinct genomic structural variations leading to enhancer hijacking and gene co-amplification were identified at A2M, JAG2, and FLRT1. CONCLUSION We show genome structural variations enhancer-promoter interactions that impact gene expression in pHGG in the presence and absence of the H3K27M mutation. Our results imply that tridimensional genome alterations may play a critical role in the pHGG epigenetic landscape and thereby contribute to pediatric gliomagenesis. Further studies examining the impact of the alterations is therefore underway.

Introduction Pediatric high-grade gliomas (pHGGs), including glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), are highly morbid brain tumors. Up to 80% of DIPGs harbor a somatic missense mutation in genes encoding Histone H3. To investigate whether the H3K27M mutant protein is associated with distinct chromatin structure affecting transcription regulation, we generated the first high-resolution Hi-C and ATAC-Seq maps of pHGG cell lines, and integrated these with tissue and cell genomic data. Methods We generated sequencing data from patient-derived cell lines (DIPG n=6, GBM n=3, normal n=2) and frozen tissue specimens (DIPG n=1, normal brainstem n=1). Analyses included cell line RNA-Seq, ChIP-Seq (H3K27ac, H3K27me3, H3K27M) and genome-wide chromatin conformation capture (Hi-C), as well as tissue ATAC-Seq. Publicly available pediatric glioma tissue ChIP-Seq data was integrated with cell data. Results We identified tumor-specific enhancers and regulatory networks for known oncogenes in DIPG and GBM. In DIPG, FOX, SOX, STAT and SMAD families were among top H3K27Ac enriched motifs. Significant differences in Topologically Associating Domains (TADs) and DNA looping were observed at OLIG2 and MYCN in H3K27M mutant DIPG, relative to wild-type GBM and normal cells. Pharmacologic treatment targeting H3K27Ac (BET and Bromodomain inhibition) altered these 3D structures. Functional analysis of differentially enriched enhancers in DIPG implicated SOX2, SUZ12, and TRIM24 as top activated upstream regulators. Distinct genomic structural variations leading to enhancer hijacking and gene co-amplification were identified at A2M, JAG2, and FLRT1. Conclusion We show genome structural variations enhancer-promoter interactions that impact gene expression in pHGG in the presence and absence of the H3K27M mutation. Our results imply that tridimensional genome alterations may play a critical role in the pHGG epigenetic landscape and thereby contribute to pediatric gliomagenesis. Further studies examining the impact of the alterations, including CRISPR knock-down of target enhancer regions, is therefore underway.

BACKGROUND Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas (pHGGs). This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase). Consequently, reduced H3K36me is observed on H3G34V nucleosomes relative to wild-type, contributing to genomic instability and driving a distinct gene expression signature associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3G34V mutation in pediatric glioma on genomic H3K36me3 enrichment in vitro. METHODS Doxycycline-inducible short hairpin RNA (shRNA) against H3F3A was delivered via lentivirus to established H3G34V mutant pediatric glioma cell line KNS42, and H3G34V introduced into H3.3 wild type normal human astrocytes (NHA). Transfections were confirmed by western blot, fluorescent imaging, and flow cytometry, with resulting H3.3WT and H3K36me3 expression determined by western blot. H3.3WT, H3K36me3, and H3G34V ChIP-Seq was performed to evaluate effects of H3G34V mutation on genomic enrichment patterns. RESULTS Complete knockdown of H3G34V was achieved with DOX-induced shRNA, with no change in total H3.3, suggesting disproportionate allelic frequency of genes encoding H3.3 (H3F3A and H3F3B). Modest increase in H3K36me3 expression occurred after H3F3A-knockdown from KNS42, suggesting H3G34V alone impacts observed H3K36me3 levels. H3G34V knock-in to NHAs was verified via western blot. Distinct H3K36me3 genomic enrichment was observed with H3G34V on ChIP-Seq. CONCLUSIONS We demonstrate that DOX-inducible knockdown of H3F3A in an H3G34V mutant pediatric glioma cell line, and H3G34V mutation transduction in wild-type NHAs, directly impacts H3K36me3 expression level. Further evaluation by ChIP-Seq analysis for restoration of wild-type genomic H3K36me3 enrichment patterns with H3G34V knockdown, and mutant H3K36me3 patterns with H3G34V transduction, is currently underway.

Diffuse intrinsic pontine glioma (DIPG), an infiltrative, high grade glioma (HGG) affecting young children, has the highest mortality rate of all pediatric cancers. Despite treatment, average survival is less than twelve months, and five-year survival under 5%. We previously detected increased expression of Tenascin-C (TNC) protein in DIPG cerebrospinal fluid and tumor tissue relative to normal specimens. TNC is an extracellular matrix (ECM) glycoprotein that mediates cell-matrix interactions, guides migrating neurons during normal brain development and is thought to maintain the periventricular stem cell niche in the developing brain. Tumor TNC expression is reported in adult glioma and other cancers. However, the pattern and effects of TNC expression in DIPG has not been previously explored. Here, we characterize TNC expression in patient derived pediatric supratentorial HGG (n = 3) and DIPG (n = 6) cell lines, as well as pediatric glioma tumor (n = 50) and normal brain tissue specimens (n = 3). We found tumor specific TNC gene and protein overexpression that directly correlated with higher tumor grade (WHO III and IV, p = 0.05), H3K27 M mutation (p = 0.012), shorter progression free survival (p = 0.034), and poorer overall survival (0.041) in association with these factors. TNC knockdown via lentiviral shRNA transfection of HGG (n = 1) and DIPG (n = 3) cell lines resulted in decreased cell proliferation, migration, and invasion in vitro (p

BACKGROUND: Mutations in genes encoding Histone protein isoforms H3.3 (H3F3A and H3F3A) and H3.1 (HIST1H3B) are detected at high frequency in pediatric high-grade glioma (pHGG). Up to 50% of cortical pHGGs bear a point mutation in H3F3A, resulting in replacement of glycine 34 with arginine or valine (G34R/V). H3G34V mutation is known to impair SETD2 activity (H3K36-specific trimethyltransferase). Reduced K36me3 is observed on nucleosomes bearing H3G34V protein relative to wild-type, contributing to genomic instability and driving a distinct gene expression signature associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3G34V mutant protein alone. We therefore set to elucidate the effect of H3G34V mutant protein on genomic H3K36me3 enrichment in vitro. METHODS: A doxycycline-inducible short hairpin RNA (shRNA) against G34V mutant H3F3A was delivered via lentivirus to an established G34V mutant pediatric glioma cell line (KNS42). H3G34V mutation was introduced into H3.3 wild type normal human astrocytes (NHA). Mutation knockdown / knockin was confirmed by sequencing and western blot. H3.3WT, H3K36me3, and H3G34V ChIP-Seq was performed to determine relative genomic distribution in transfected and unperturbed KNS42 cells and NHAs, and also compared to a H3WT glioma cell line. RESULTS: shRNA-induced knockdown of H3G34V restored the pattern of genomic H3K36me3 enrichment to that observed in H3.3 wild-type glioma cells and NHAs. In turn, introducing H3G34V to NHAs was sufficient to drive the distinct K36 methylation profile seen in H3G34V glioma cells. H3G34V mutation was also associated with a global increase in overall H3.3WT genomic enrichment, representing a heightened state of active transcription relative to wild-types. CONCLUSIONS: Our study is the first to demonstrate H3G34V mutation is sufficient to induce differential H3K36me3 enrichment patterns associated with genomic instability and increased transcription. Gene expression analysis is currently underway to further evaluate these effects on transcription regulation.

BACKGROUND Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas. This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase), resulting in reduced H3K36me on H3G34V nucleosomes relative to wild-type. This contributes to genomic instability and drives distinct gene expressions associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3G34V on genomic H3K36me3 enrichment in vitro. METHODS Doxycycline-inducible short hairpin RNA (shRNA) against H3F3A was delivered via lentivirus to established H3G34V mutant pediatric glioma cell line KNS42, and H3G34V introduced into H3.3 wild type normal human astrocytes (NHA). Transfections were confirmed by western blot, fluorescent imaging, and flow cytometry, with resulting H3.3WT and H3K36me3 expression determined by western blot. H3.3WT, H3K36me3, and H3G34V ChIP-Seq was performed to evaluate genomic enrichment. RESULTS Complete knockdown of H3G34V was achieved with DOX-induced shRNA, with no change in total H3.3, suggesting disproportionate allelic frequency of genes encoding H3.3 (H3F3A and H3F3B). Modest increase in H3K36me3 occurred after H3F3A-knockdown from KNS42, suggesting H3G34V alone impacts observed H3K36me3 levels. Distinct H3K36me3 genomic enrichment was observed with H3G34V knock-in. CONCLUSIONS We demonstrate that DOX-inducible knockdown of H3F3A in an H3G34V mutant pediatric glioma cells and H3G34V mutation transduction in wild-type astrocytes affects H3K36me3 expression. Further evaluation by ChIP-Seq analysis for restoration of wild-type genomic H3K36me3 enrichment patterns with H3G34V knockdown, and mutant H3K36me3 patterns with H3G34V transduction, is currently underway.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) has the highest mortality of all pediatric solid tumors. Histone H3 mutation (H3K27M) occurs in 80%, altering chromatin structure and function. To characterize the effects of H3K27M as an oncogenic driver, we analyzed the transcriptome and epigenome in DIPG cell lines. METHODS: Transcriptomes (RNA-Seq) and genomic enrichment of Histone proteins (ChIP-Seq) were characterized in H3K27M DIPG (n=7), wild-type pediatric high-grade glioma (n=3), neural stem cells (n=1) and astrocyte cell lines (n=1). Reads were aligned (UCSC hg19, Tophat), normalized and quantified (HTSeq), and formatted \ for peak calling (SICER) and genomic enrichment mapping (Ensembl). Differential expression and enrichment patterns were identified (edgeR) then analyzed for biological relevance (TopGO, IPA). Epigenetic therapeutic targets were studied in vitro and in vivo. RESULTS: Unsupervised clustering revealed two DIPG subgroups, distinct from control and H3 wild-type lines. Increased EZH2 (p

Purpose: Gliomas with isocitrate dehydrogenase 1 mutations (IDH1mut) are less aggressive than IDH1 wild-type (IDH1wt) gliomas and have global genomic hypermethylation. Yet it is unclear how specific hypermethylation events contribute to the IDH1mut phenotype. Previously, we showed that the gene encoding the procoagulant tissue factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas, correlating with greatly reduced thrombosis in patients with IDH1mut glioma. Because TF also increases the aggressiveness of many cancers, the current study explored the contribution of TF suppression to the reduced malignancy of IDH1mut gliomas. Experimental Design: TF expression was manipulated in patient-derived IDH1mut and IDH1wt glioma cells, followed by evaluation of in vitro and in vivo behavior and analyses of cell signaling pathways. Results: A demethylating agent, decitabine, increased F3 transcription and TF-dependent coagulative activity in IDH1mut cells, but not in IDH1wt cells. TF induction enhanced the proliferation, invasion, and colony formation of IDH1mut cells, and increased the intracranial engraftment of IDH1mut GBM164 from 0% to 100% (P = 0.0001). Conversely, TF knockdown doubled the median survival of mice engrafted with IDH1wt/EGFRvIIIamp GBM6, and caused complete regression of IDH1wt/EGFRamp GBM12 (P = 0.001). In vitro and in vivo effects were linked to activation of receptor tyrosine kinases (RTK) by TF through a Src-dependent intracellular pathway, even when extracellular RTK stimulation was blocked. TF stimulated invasion predominately through upregulation of β-catenin. Conclusions: These data show that TF suppression is a component of IDH1mut glioma behavior, and that it may therefore be an attractive target against IDH1wt gliomas.

// Sridevi Yadavilli 1 , Joseph Scafidi 2 , Oren J. Becher 3 , Amanda M. Saratsis 4 , Rebecca L. Hiner 5 , Madhuri Kambhampati 1 , Santi Mariarita 6 , Tobey J. MacDonald 7 , Kari-Elise Codispoti 8 , Suresh N. Magge 9 , Jyoti K. Jaiswal 1,10 , Roger J. Packer 11 and Javad Nazarian 1,10 1 Research Center for Genetic Medicine, Children’s National Health System, Washington, DC, USA 2 Department of Neurology and Center for Neuroscience Research, Children’s National Health System, Washington, DC, USA 3 Department of Pediatrics and Pathology, Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, USA 4 Division of Neurosurgery, Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago, IL, USA 5 Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, NY, USA 6 Department of Pathology and Lab Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 7 Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA 8 Department of Pathology, Children’s National Health System, Washington, DC, USA 9 Division of Neurosurgery, Children’s National Health System, Washington, DC, USA 10 Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, DC, USA 11 Brain Tumor Institute, Center for Neuroscience and Behavioral Medicine, Children’s National Health System, Washington, DC, USA Correspondence to: Javad Nazarian, email: // Keywords : DIPG, NG2, PDGF, histone 3, glioma Received : March 04, 2015 Accepted : March 11, 2015 Published : March 30, 2015 Abstract Diffuse intrinsic pontine gliomas (DIPGs) have a dismal prognosis and are poorly understood brain cancers. Receptor tyrosine kinases stabilized by neuron-glial antigen 2 (NG2) protein are known to induce gliomagenesis. Here, we investigated NG2 expression in a cohort of DIPG specimens (n= 50) . We demonstrate NG2 expression in the majority of DIPG specimens tested and determine that tumors harboring histone 3.3 mutation express the highest NG2 levels. We further demonstrate that microRNA 129-2 (miR129-2) is downregulated and hypermethylated in human DIPGs, resulting in the increased expression of NG2. Treatment with 5-Azacytidine, a methyltransferase inhibitor, results in NG2 downregulation in DIPG primary tumor cells in vitro . NG2 expression is altered (symmetric segregation) in mitotic human DIPG and mouse tumor cells. These mitotic cells co-express oligodendrocyte (Olig2) and astrocyte (glial fibrillary acidic protein, GFAP) markers, indicating lack of terminal differentiation. NG2 knockdown retards cellular migration in vitro, while NG2 expressing neurospheres are highly tumorigenic in vivo, resulting in rapid growth of pontine tumors. NG2 expression is targetable in vivo using miR129-2 indicating a potential avenue for therapeutic interventions. This data implicates NG2 as a molecule of interest in DIPGs especially those with H3.3 mutation.

Posterior fossa ependymomas (EPN_PF) in children comprise two morphologically identical, but biologically distinct tumor entities. Group-A (EPN_PFA) tumors have a poor prognosis and require intensive therapy. In contrast, group-B tumors (EPN_PFB) exhibit excellent prognosis and the current consensus opinion recommends future clinical trials to test the possibility of treatment de-escalation in these patients. Therefore, distinguishing these two tumor subtypes is critical. EPN_PFA and EPN_PFB can be distinguished based on DNA methylation signatures, but these assays are not routinely available. We have previously shown that a subset of poorly prognostic childhood EPN_PF exhibits global reduction in H3K27me3. Therefore, we set out to determine whether a simple immunohistochemical assay for H3K27me3 could be used to segregate EPN_PFA from EPN_PFB tumors. We assembled a cohort of 230 childhood ependymomas and H3K27me3 immunohistochemistry was assessed as positive or negative in a blinded manner. H3K27me3 staining results were compared with DNA methylation-based subgroup information available in 112 samples [EPN_PFA (n=72) and EPN_PFB tumors (n=40)]. H3K27me3 staining was globally reduced in EPN_PFA tumors and immunohistochemistry showed 99% sensitivity and 100% specificity in segregating EPN_PFA from EPN_PFB tumors. Moreover, H3K27me3 immunostaining was sufficient to delineate patients with worse prognosis in two independent, non-overlapping cohorts (n=133 and n=97). In conclusion, immunohistochemical evaluation of H3K27me3 global reduction is an economic, easily available and readily adaptable method for defining high-risk EPN_PFA from low-risk posterior fossa EPN_PFB tumors to inform prognosis and to enable the design of future clinical trials.

Recent discovery of somatic histone gene mutations, resulting in replacement of lysine 27 by methionine (K27M) in the encoded histone H3.3 proteins, in diffuse intrinsic pontine glioma (DIPG) has dramatically improved our understanding of disease pathogenesis, and stimulated the development of novel therapeutic approaches targeting epigenetic regulators for disease treatment. K27M mutant DIPG shows a dramatic reduction in global methylation at K27 residues on all 16 H3 proteins. This reduction in H3K27 methylation is believed to modify cellular gene expression in a way that favors tumor development. We have shown that inhibition of the H3K27 demethylase JMJD3 acts to restore K27 methylation in DIPG cells, while demonstrating potent anti-tumor activity, in vitro and in vivo. In addition to H3K27 methylation, H3K27 can also be acetylated (K27ac), which requires bromo- and extra-terminal domain (BET) protein activity. We have recently identified H3K27M-K27ac nucleosomes co-localize with BET bromodomain proteins at actively transcribed genes, whereas a polycomb repressive complex 2 (PRC2) is excluded from these regions, demonstrating that H3K27M and PRC2 occupy distinct chromatin regions in DIPG cells. Despite a major loss of H3K27 methylation, PRC2 activity is still detected in H3K27M DIPG cells, and the residual PRC2 activity is required to maintain DIPG proliferative potential by repressing neuronal differentiation and function. Small molecule inhibitor of EZH2 inhibited cell growth through the upregulation of gene that are normally repressed by PRC2 in DIPG. We also tested the anti-tumor activity of BET bromodomain containing protein 4 (BRD4) inhibitor, JQ1, and EZH2 inhibitor, GSK126, in our human DIPG xenograft model, and JQ1 and GSK126 treatment results in a significant delay of tumor progression and prolonged animal survival. Our findings suggest EZH2 inhibition could be a potential combinatorial strategy with BRD4 inhibition in treating K27M DIPG, that could counteract the development of resistance to single-agent epigenetic therapy.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is a highly morbid pediatric brainstem tumor. Tenascin-C (TNC) is an extracellular matrix protein associated with NOTCH pathway activation, and is expressed during normal brain development by oligodendroglial cells (OPCs), the purported DIPG cell of origin. We previously reported TNC overexpression in DIPG tissue and CSF, associated with Histone H3K27M mutation. We therefore investigated the pattern and effect of TNC expression using pediatric glioma primary cell lines. METHODS: To determine the molecular effects of altered TNC expression in DIPG in vitro, we evaluated endogenous TNC expression in DIPG cell lines (n=3) and compared to TNC suppression and over-expression via vector-mediated shRNA knockdown and cDNA amplification, respectively. Effects of genetic manipulations were confirmed by western blot and qPCR. Cell proliferation, migration and adhesion were compared, as well as patterns of gene expression (RNA-Seq) across cell lines. Luciferase-expressing DIPG cells modified with TNC shRNA were used to create mouse intracranial xenografts. Animal survival and tumor growth was compared between groups, and TNC expression confirmed by immunohistochemistry. RESULTS: shRNA knockdown achieved up to 80% reduction in TNC expression, inhibiting cell proliferation and migration on functional analysis compared to control DIPG lines. Distinct pathways of gene expression favoring dedifferentiation and stem-like state correlated with higher TNC expression. Anaylsis cDNA and in vivo studies is currently underway. CONCLUSIONS: Here, we report the pattern and effect of modulated TNC expression in pediatric brainstem glioma (DIPG). Our findings suggest TNC may play a role in cell proliferation, migration, and maintaining stem-ness, possibly contributing to brainstem gliomagenesis. Further in vitro and in vivo studies exploring the mechanism of overexpression and effects of targeting TNC expression in DIPG are planned.

Diffuse midline gliomas (including diffuse intrinsic pontine glioma, DIPG) are highly morbid glial neoplasms of the thalamus or brainstem that typically arise in young children and are not surgically resectable. These tumors are characterized by a high rate of histone H3 mutation, resulting in replacement of lysine 27 with methionine (K27M) in genes encoding H3 variants H3.3 (H3F3A) and H3.1 (HIST1H3B). Detection of these gain-of-function mutations has clinical utility, as they are associated with distinct tumor biology and clinical outcomes. Given the paucity of tumor tissue available for molecular analysis and relative morbidity of midline tumor biopsy, CSF-derived tumor DNA from patients with diffuse midline glioma may serve as a viable alternative for clinical detection of histone H3 mutation. We demonstrate the feasibility of two strategies to detect H3 mutations in CSF-derived tumor DNA from children with brain tumors (n = 11) via either targeted Sanger sequencing of H3F3A and HIST1H3B, or H3F3A c.83 A > T detection via nested PCR with mutation-specific primers. Of the six CSF specimens from children with diffuse midline glioma in our cohort, tumor DNA sufficient in quantity and quality for analysis was isolated from five (83%), with H3.3K27M detected in four (66.7%). In addition, H3.3G34V was identified in tumor DNA from a patient with supratentorial glioblastoma. Test sensitivity (87.5%) and specificity (100%) was validated via immunohistochemical staining and Sanger sequencing in available matched tumor tissue specimens (n = 8). Our results indicate that histone H3 gene mutation is detectable in CSF-derived tumor DNA from children with brain tumors, including diffuse midline glioma, and suggest the feasibility of “liquid biopsy” in lieu of, or to complement, tissue diagnosis, which may prove valuable for stratification to targeted therapies and monitoring treatment response. Electronic supplementary material The online version of this article (doi:10.1186/s40478-017-0436-6) contains supplementary material, which is available to authorized users.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise. A heterozygous point mutation of histone H3 occurs in more than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M). Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3), and this is hypothesized to be a driving event of DIPG oncogenesis. Despite a major loss of H3K27me3, PRC2 activity is still detected in DIPG cells positive for H3K27M. To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis, we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation (H3K27ac). In accordance with previous biochemical data, the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes, whereas PRC2 is excluded from these regions; this suggests that H3K27M does not sequester PRC2 on chromatin. Residual PRC2 activity is required to maintain DIPG proliferative potential, by repressing neuronal differentiation and function. Finally, to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells, we performed treatments in vivo with BET bromodomain inhibitors and demonstrate that they efficiently inhibit tumor progression, thus identifying this class of compounds as potential therapeutics in DIPG.

Mutations in chromatin machinery define pediatric high-grade gliomas; efforts to define and target their functions are under way., Pediatric central nervous system tumors are the most common solid tumor of childhood. Of these, approximately one-third are gliomas that exhibit diverse biological behaviors in the unique context of the developing nervous system. Although low-grade gliomas predominate and have favorable outcomes, up to 20% of pediatric gliomas are high-grade. These tumors are a major contributor to cancer-related morbidity and mortality in infants, children, and adolescents, with long-term survival rates of only 10 to 15%. The recent discovery of somatic oncogenic mutations affecting chromatin regulation in pediatric high-grade glioma has markedly improved our understanding of disease pathogenesis, and these findings have stimulated the development of novel therapeutic approaches targeting epigenetic regulators for disease treatment. We review the current perspective on pediatric high-grade glioma genetics and epigenetics, and discuss the emerging and experimental therapeutics targeting the unique molecular abnormalities present in these deadly childhood brain tumors.

INTRODUCTION: Pediatric high-grade glioma (HGG), including H3K27M diffuse midline glioma, has the highest mortality of pediatric solid tumors. Recurrent Histone H3 mutations result in methionine for lysine substitution (H3K27M) in 80% of diffuse midline gliomas, or valine for glycine/arginine substitution (G34V/R) in 50% of hemispheric HGGs. These mutations alter chromatin structure and are associated with distinct patterns of gene expression and poorer response to therapy. To elucidate the mechanism by which these mutations affect chromatin function, we characterized genomic deposition of Histone H3 proteoforms in a large cohort of rare pediatric glioma cell lines. METHODS: H3K27M DIPG (n=6), H3G34V (n=1) and wild-type pediatric high-grade glioma cells (n=2), neural stem cells (n=1) and astrocytes (n=1) were analyzed for genomic deposition patterns of H3.3, H3K27M, H3G34V, H3K27me3, H3K27Ac, H4Kme1, and H4Kme3. Extracted chromatins were sonicated to produce DNA fragments for ChIP. RNA was extracted for whole transcriptome profiling. DNA/RNA libraries were prepared using the KAPA HTP Library Preparation Kit and sequenced (ChIP-and RNA-Seq, Illumina NextSeq 500). Genomic enrichments and gene expressions were determined, quantified, and analyzed for biological relevance. RESULTS: Distinct genomic enrichment of H3 proteoforms was observed between mutant and wild type cell lines, corresponding with respective gene expression levels. Differential co-localization of H3 post-translational marks with mutant vs. wild type H3 protein was also observed, consistent with our prior description of mutant heterotypic nucleosomes. Location of specific marks (promoter, gene body) suggests altered transcription factor recruitment and function may result in observed patterns of gene expression. CONCLUSION: We present the largest known epigenetic analysis of pediatric glioma cell lines to date, indicating distinct patterns of Histone H3 enrichment and gene expression in H3K27M and H3G34V/R mutant lines. These data provide insight into the mechanisms by which H3 mutations impact pediatric glioma biology, which may inform novel therapeutic approaches.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is the most deadly solid tumor of childhood. Histone 3 mutation occurs in 80% of DIPGs, causing global transcriptional changes. Tenascin-C (TNC) is an extracellular matrix protein expressed during normal brain development by oligodendroglial progenitor cells (OPCs), the purported DIPG cell of origin. TNC is highly expressed in adult glioma, contributing to local invasion and poor survival. We report increased TNC in tumor tissue and cerebrospinal fluid (CSF) from children with high-grade glioma, including DIPG, and characterize TNC expression in relation to H3K27M mutation and DNA methylation status. METHODS: Tumor tissue collected intraoperatively or post-mortem from children with brainstem (DIPG, n = 14), supratentorial (n = 7), and cerebellar astrocytoma (n = 2), and CSF from DIPG (n = 9) and supratentorial astrocytoma (n = 17) was subjected to MS/MS proteomic analysis. Tissue gene expression, DNA methylation, H3F3A and HIST1H3B sequencing was performed. TNC expression was validated via Western blot and immunohistochemistry. Data integration was performed with Genome Studio, Partek and Ingenuity Pathway Analysis. RESULTS: TNC expression was significantly increased in 75% of gliomas compared to normal tissue, including all DIPG specimens tested (fold change >2, p < 0.05). Secreted TNC was detected in 7/9 DIPG CSF specimens (77.8%). Tumor-specific TNC expression was confirmed with Western blot and tissue immunohistochemistry. TNC expression correlated with tumor grade and was associated with Notch pathway activation, H3K27M mutation, and TNC promoter hypomethylation. CONCLUSIONS: We report increased TNC expression in tissue and CSF of pediatric high-grade gliomas, including DIPGs, associated with promoter hypomethylation and H3K27M mutation. Given the effect of TNC on OPC proliferation, migration and differentiation, TNC could serve as a clinical biomarker of disease and rational therapeutic target for a substantial subgroup of DIPG patients. Studies exploring the mechanism of TNC overexpression and effects of targeting TNC expression in DIPG are currently underway.

Introduction: Diffuse intrinsic pontine glioma (DIPG) is among the most devastating childhood cancers, and virtually all patients die within two years after diagnosis. Since these tumors occur in the brainstem, there are no surgical options for providing relief to patients. The only therapeutic modality routinely used in the treatment of DIPG is radiation. Radiation often provides transient relief from DIPG symptoms, as cancer recurrence and patient demise are inevitable. Therefore, identifying efficacious therapeutics to enhance radiation effect is desperately needed. We have recently shown that inhibition of the histone H3K27 demethylase JMJD3 acts to restore H3K27 methylation in DIPG harboring H3K27M mutation, while demonstrating potent anti-tumor activity, both in vitro and in vivo. Our gene expression profiling of K27M DIPG treated with GSK-J4, a JMJD3 demethylase inhibitor, revealed several decreases in the genes encoding proteins involved in DNA double-strand break (DSB) repair. Our aim is to investigate the hypothesis that GSK-J4 may inhibit radiation-induced DNA repair by chromatin modification, in association with analysis of therapeutic combination of GSK-J4 and radiation. METHODS: We evaluated DNA damage repair via quantitative-PCR, immunocytochemistry of DSB markers γH2AX and 53BP1, western blotting and DNA repair assay. MNase digestion and FAIRE-seq was used to analyze nucleosome assembly and transcriptional activity in cellular chromatin. Assessment of DIPG response to radiation therapy (RT) in combination with GSK-J4 was performed using clonogenic survival assays. In vivo response to radiation monotherapy and combination of RT + GSK-J4 were measured by bioluminescence imaging and animal survival studies. RESULTS: qPCR results showed that GSK-J4 significantly reduces DNA DSB repair genes such as PARP1, PARP2, SMARCB1 and BRCA1, in irradiated DIPG cells. Immunocytochemistry results support that GSK-J4 sustains high levels of γH2AX and 53BP1 in irradiated DIPG cells, thereby inhibiting DNA DSB repair. Mnase assay indicated that GSK-J4 causes chromatin compaction which prevents DNA DSB repair. We are currently conducting clonogenic assays to assess long term effect of GSK-J4 on radiated cells as well as western blotting, FAIRE-seq and in vivo studies, the results of which will be reported at the meeting.

BACKGROUND Radiation therapy is the most commonly used postsurgical treatment for primary malignant brain tumors. Consequently, investigating the efficacy of chemotherapeutics combined with radiation for treating malignant brain tumors is of high clinical relevance. In this study, we examined the cyclin-dependent kinase 4/6 inhibitor palbociclib, when used in combination with radiation for treating human atypical teratoid rhabdoid tumor (ATRT) as well as glioblastoma (GBM). METHODS Evaluation of treatment antitumor activity in vitro was based upon results from cell proliferation assays, clonogenicity assays, flow cytometry, and immunocytochemistry for DNA double-strand break repair. Interpretation of treatment antitumor activity in vivo was based upon bioluminescence imaging, animal subject survival analysis, and staining of tumor sections for markers of proliferation and apoptosis. RESULTS For each of the retinoblastoma protein (RB)-proficient tumor models examined (2 ATRTs and 2 GBMs), one or more of the combination therapy regimens significantly (P < .05) outperformed both monotherapies with respect to animal subject survival benefit. Among the combination therapy regimens, concurrent palbociclib and radiation treatment and palbociclib treatment following radiation consistently outperformed the sequence in which radiation followed palbociclib treatment. In vitro investigation revealed that the concurrent use of palbociclib with radiation, as well as palbociclib following radiation, inhibited DNA double-strand break repair and promoted increased tumor cell apoptosis. CONCLUSIONS Our results support further investigation and possible clinical translation of palbociclib as an adjuvant to radiation therapy for patients with malignant brain tumors that retain RB expression.

Diffuse intrinsic pontine glioma (DIPG) is a leading cause of brain tumor-related death in children. DIPG is not surgically resectable, resulting in a paucity of tissue available for molecular studies. As such, tumor biology is poorly understood, and, currently, there are no effective treatments. In the absence of frozen tumor specimens, body fluids--such as cerebrospinal fluid (CSF), serum, and urine--can serve as more readily accessible vehicles for detecting tumor-secreted proteins. We analyzed a total of 76 specimens, including CSF, serum, urine, and normal and tumor brainstem tissue. Protein profiling of CSF from patients with DIPG was generated by mass spectrometry using an LTQ-Orbitrap-XL and database search using the Sequest algorithm. Quantitative and statistical analyses were performed with ProteoIQ and Partek Genomics Suite. A total of 528 unique proteins were identified, 71% of which are known secreted proteins. CSF proteomic analysis revealed selective upregulation of Cyclophillin A (CypA) and dimethylarginase 1 (DDAH1) in DIPG (n = 10), compared with controls (n = 4). Protein expression was further validated with Western blot analysis and immunohistochemical assays using CSF, brain tissue, serum, and urine from DIPG and control specimens. Immunohistochemical staining showed selective upregulation of secreted but not cytosolic CypA and DDAH1 in patients with DIPG. In this study, we present the first comprehensive protein profile of CSF specimens from patients with DIPG to demonstrate selective expression of tumor proteins potentially involved in brainstem gliomagenesis. Detection of secreted CypA and DDAH1 in serum and urine has potential clinical application, with implications for assessing treatment response and detecting tumor recurrence in patients with DIPG.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is a rare but uniformly fatal cancer of the brain, and often harbors oncogenic H3F3A gene mutations, resulting in replacement of lysine 27 by methionine (K27M) in the encoded histone H3.3 protein. Despite being just one of 32 genes that encode histone H3 peptides in a diploid cell, this mutation causes substantial reduction in histone H3K27 methylation in cellular chromatin, and we have recently shown that inhibition of the histone H3K27 demethylase JMJD3 acts to restore H3K27 methylation in DIPG cells, while demonstrating potent anti-tumor activity, both in cell culture and in xenograft models of DIPG. Our expression profiling of K27M DIPG treated with GSKJ4 histone H3K27 demethylase inhibitor revealed decreased transcripts from several genes whose encoded proteins are known to be involved with DNA double-strand break (DSB) repair. These results provide a basis for considering the possibility of GSKJ4 acting as a radiation enhancer, and we investigate this possibility, in association with analysis of therapeutic combination of GSKJ4 and radiation. METHOD: We examine cell growth, colony formation, and DNA DSB repair in association with GSKJ4 monotherapy and combined with radiotherapy (RT) in vitro. We use bioluminescence imaging (BLI), immunohistochemical analysis of tumors, and evaluation of survival benefit from the monotherapies and combination therapies in DIPG xenograft models. RESULTS: Mice received brainstem injection of K27M DIPG cells and are treated with vehicle, GSKJ4 (100mg/kg), RT (1Gy x 10 times), and combination of GSKJ4 + RT. BLI shows that brainstem tumor growth is inhibited by either GSKJ4 or RT monotherapy. Although this study is still ongoing, combination therapy with GSKJ4 + RT shows further reduction of tumor growth rate relative to either monotherapy. We are currently examining the effect of GSKJ4 on radiation-induced DNA DSB repair that will be reported at the meeting.

10037 Background: Diffuse intrinsic pontine glioma (DIPG) is the most deadly solid tumor of childhood. Histone 3 mutation occurs in up to 80% of DIPGs, causing global epigenetic aberration. Tenasci...

Introduction: Diffuse Intrinsic Pontine Glioma (DIPG) is a highly morbid form of pediatric brainstem glioma. Molecular characterization is limited due to lack of tissue. Recent investigations suggest possible molecular subtypes may account for the historical poor response to therapy. We previously generated protein profiles of CSF and formalin fixed DIPG tumor specimens to characterize patterns of protein expression. Here, we present the first comprehensive tissue proteome of fresh frozen DIPG tumor specimens (n=16) and normal brain tissue (n=10). We characterize differential protein expression in DIPG tumor specimens, and compare these to gene expression and DNA methylation profiles of the same tissue. Methods: Normal brain and tumor tissue was collected intraoperatively or post-mortem. Extracted total tissue protein was quantified by mass spectrometry (MS/MS) via LTQ-Orbitrap-XL and database search using the Sequest algorithm. Gene expression profiles were detected using whole-genome Human HT-4 v12 Gene Expression Bead Chips. DNA methylation profiles were characterized after bisulphite conversion using Infinium HumanMethylation 450K BeadChip arrays. Quantitative and statistical analysis was performed with Genome Studio, ProteoIQ, and Partek Genomics Suite. Functional analysis was performed using Ingenuity Pathways Analysis. Gene and protein expression was validated via western blot and immunohistochemical staining of tumor and normal brain tissue. Results: 1,918 differentially expressed genes were detected in DIPG tumor tissue (ANOVA, p2 or 2), with differential ShH pathway (GLI1 z-score -0.626 vs. 2.254) and protein expression COL1A2, LMNA, MAP4, NES, NRCAM, STMN1, and TNC between subgroups (ANOVA, p2 or 2). Conclusions: We present the first comprehensive protein profile of DIPG fresh frozen tumor tissue and correlate to tissue gene expression profiles, which suggest differential activity of SHh pathway. This may in part be explained by differential methylation patterns of key genes. Proteomic analysis of DIPG tumor tissue reveals protein expression profiles reflective of this differential pathway activation, and hence may be useful tool for elucidating mechanisms of brainstem gliomagenesis in what likely is a heterogeneous tumor population. Biomarkers identified through proteomic analysis may in turn serve to more accurately diagnose patients with DIPG and measure response to therapy. Citation Format: Amanda Saratsis, Sridevi Yadavilli, Madhuri Kambhampati, Eric Raabe, Suresh Magge, Javad Nazarian. Four dimensional molecular analysis of pediatric diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C30.