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Differential Expression in Isogenic Lines

Glycine 34-to-tryptophan (G34W) substitutions in H3.3 arise in approximately 90% of giant cell tumor of bone (GCT). Here, we show H3.3 G34W is necessary for tumor formation. By profiling the epigenome, transcriptome, and secreted proteome of patient samples and tumor-derived cells CRISPR–Cas9-edited for H3.3 G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Single-cell transcriptomics reveals that H3.3 G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population toward an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3 G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors, which promotes neoplastic growth, pathologic recruitment of giant osteoclasts, and bone destruction.Significance:This study shows that H3.3 G34W drives GCT tumorigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3 G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumor microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT.See related commentary by Licht, p. 1794.This article is highlighted in the In This Issue feature, p. 1775

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Deregulation of the epigenome underlies oncogenesis in numerous primary brain tumours in children and young adults. In this review, we describe how recurrent mutations in isocitrate dehydrogenases or histone 3 variants (oncohistones) in gliomas, expression of the oncohistone mimic enhancer of Zeste homologs inhibiting protein (EZHIP) in a subgroup of ependymoma, and epigenetic alterations in other embryonal tumours promote oncogenicity. We review the proposed mechanisms of cellular transformation, current tumorigenesis models and their link to development. We further stress the narrow developmental windows permissive to their oncogenic potential and how this may stem from converging effects deregulating polycomb repressive complex (PRC)2 function and targets. As altered chromatin states may be reversible, improved understanding of aberrant cancer epigenomes could orient the design of effective therapies.

This paper proposes eye(I) still know!, a voice control solution for the visually impaired people. The main purpose is even though the blind cannot see they can still know where to go and what to do! Nearby 60% of total blind population across the world is present in India. In a time where no one likes to rely on anyone, this is a small effort to make the blind independent individuals. This can be achieved using wireless communication, voice recognition and image scanning. The application with the use of object identification will priorly inform about the barriers in the path. The software will use the camera of the device and scan all the obstacles with their corresponding distances from the user. This will be followed by audio instructions through audio output of the device. This will efficiently direct the user through his/her way.

Although replication repair deficiency, either by mismatch repair deficiency (MMRD) and/or loss of DNA polymerase proofreading, can cause hypermutation in cancer, microsatellite instability (MSI) is considered a hallmark of MMRD alone. By genome-wide analysis of tumors with germline and somatic deficiencies in replication repair, we reveal a novel association between loss of polymerase proofreading and MSI, especially when both components are lost. Analysis of indels in microsatellites (MS-indels) identified five distinct signatures (MS-sigs). MMRD MS-sigs are dominated by multibase losses, whereas mutant-polymerase MS-sigs contain primarily single-base gains. MS deletions in MMRD tumors depend on the original size of the MS and converge to a preferred length, providing mechanistic insight. Finally, we demonstrate that MS-sigs can be a powerful clinical tool for managing individuals with germline MMRD and replication repair–deficient cancers, as they can detect the replication repair deficiency in normal cells and predict their response to immunotherapy. Significance: Exome- and genome-wide MSI analysis reveals novel signatures that are uniquely attributed to mismatch repair and DNA polymerase. This provides new mechanistic insight into MS maintenance and can be applied clinically for diagnosis of replication repair deficiency and immunotherapy response prediction. This article is highlighted in the In This Issue feature, p. 995

Kabuki syndrome is frequently caused by loss-of-function mutations in one allele of histone 3 lysine 4 (H3K4) methyltransferase KMT2D and is associated with problems in neurological, immunological and skeletal system development. We generated heterozygous KMT2D knockout and Kabuki patient-derived cell models to investigate the role of reduced dosage of KMT2D in stem cells. We discovered chromosomal locus-specific alterations in gene expression, specifically a 110 Kb region containing Synaptotagmin 3 (SYT3), C-Type Lectin Domain Containing 11A (CLEC11A), Chromosome 19 Open Reading Frame 81 (C19ORF81) and SH3 And Multiple Ankyrin Repeat Domains 1 (SHANK1), suggesting locus-specific targeting of KMT2D. Using whole genome histone methylation mapping, we confirmed locus-specific changes in H3K4 methylation patterning coincident with regional decreases in gene expression in Kabuki cell models. Significantly reduced H3K4 peaks aligned with regions of stem cell maps of H3K27 and H3K4 methylation suggesting KMT2D haploinsufficiency impact bivalent enhancers in stem cells. Preparing the genome for subsequent differentiation cues may be of significant importance for Kabuki-related genes. This work provides a new insight into the mechanism of action of an important gene in bone and brain development and may increase our understanding of a specific function of a human disease-relevant H3K4 methyltransferase family member.

Posterior fossa type A (PFA) ependymomas exhibit very low H3K27 methylation and express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). Here we find that a conserved sequence in EZHIP is necessary and sufficient to inhibit PRC2 catalytic activity in vitro and in vivo. EZHIP directly contacts the active site of the EZH2 subunit in a mechanism similar to the H3 K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promotes similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at CpG islands. We find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a mechanism to explain the observed loss of H3K27me3 spreading in tumors. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors, the K27M oncohistone and the EZHIP ‘oncohistone-mimic’, that dysregulate gene silencing to promote tumorigenesis., PFA tumours express high levels of EZHIP (also known as CXORF67). Here the authors find that EZHIP directly interacts with the active site of EZH2 and is a competitive inhibitor of PRC2 and that EZHIP gives rise to H3K27me3 genomic profile similar to the K27M oncohistone.

Since the discovery of recurrent mutations in histone H3 variants in paediatric brain tumours, so-called 'oncohistones' have been identified in various cancers. While their mechanism of action remains under active investigation, several studies have shed light on how they promote genome-wide epigenetic perturbations. These findings converge on altered post-translational modifications on two key lysine (K) residues of the H3 tail, K27 and K36, which regulate several cellular processes, including those linked to cell differentiation during development. We will review how these oncohistones affect the methylation of cognate residues, but also disrupt the distribution of opposing chromatin marks, creating genome-wide epigenetic changes which participate in the oncogenic process. Ultimately, tumorigenesis is promoted through the maintenance of a progenitor state at the expense of differentiation in defined cellular and developmental contexts. As these epigenetic disruptions are reversible, improved understanding of oncohistone pathogenicity can result in needed alternative therapies.

The Zero Childhood Cancer Program’s multi-platform sequencing approach identified molecular alterations in 94% of a cohort of 247 pediatric patients with high-risk cancers, which has enabled more-precise diagnoses and alternative therapeutic recommendations.

A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.

Glycine 34-to-tryptophan (G34W) substitutions in H3.3 arise in approximately 90% of giant cell tumor of bone (GCT). Here, we show H3.3 G34W is necessary for tumor formation. By profiling the epigenome, transcriptome, and secreted proteome of patient samples and tumor-derived cells CRISPR–Cas9-edited for H3.3 G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Single-cell transcriptomics reveals that H3.3 G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population toward an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3 G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors, which promotes neoplastic growth, pathologic recruitment of giant osteoclasts, and bone destruction. Significance: This study shows that H3.3 G34W drives GCT tumorigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3 G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumor microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT. See related commentary by Licht, p. 1794. This article is highlighted in the In This Issue feature, p. 1775

Diffuse midline gliomas and posterior fossa type-A ependymomas contain the highly recurrent histone H3 K27M mutation and the H3 K27M-mimic EZHIP, respectively. In vitro, H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3K27me3 levels, however residual peaks of H3K27me3 remain at CpG islands through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with an allosterically activated form of PRC2 in vivo. The formation of H3 K27M- and EZHIP-PRC2 complexes occurs at CpG islands containing H3K27me3 and impedes PRC2 and H3K27me3 spreading. While EZHIP is not found outside of placental mammals, we find that expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved molecular mechanism. Our results highlight the mechanistic similarities between EZHIP and H3 K27M in vivo and provide mechanistic insight for the retention of residual H3K27me3 in tumors driven by these oncogenes.

International audience; Purpose: To demonstrate the effectiveness of combining retinal phenotyping and focused variant filtering from genome sequencing (GS) in identifying deep intronic disease causing variants in inherited retinal dystrophies.Methods: Affected members from three pedigrees with classical enhanced S-cone syndrome (ESCS; Pedigree 1), congenital stationary night blindness (CSNB; Pedigree 2), and achromatopsia (ACHM; Pedigree 3), respectively, underwent detailed ophthalmologic evaluation, optical coherence tomography, and electroretinography. The probands underwent panel-based genetic testing followed by GS analysis. Minigene constructs (NR2E3, GPR179 and CNGB3) and patient-derived cDNA experiments (NR2E3 and GPR179) were performed to assess the functional effect of the deep intronic variants.Results: The electrophysiological findings confirmed the clinical diagnosis of ESCS, CSNB, and ACHM in the respective pedigrees. Panel-based testing revealed heterozygous pathogenic variants in NR2E3 (NM_014249.3; c.119-2A>C; Pedigree 1) and CNGB3 (NM_019098.4; c.1148delC/p.Thr383Ilefs*13; Pedigree 3). The GS revealed heterozygous deep intronic variants in Pedigrees 1 (NR2E3; c.1100+1124G>A) and 3 (CNGB3; c.852+4751A>T), and a homozygous GPR179 variant in Pedigree 2 (NM_001004334.3; c.903+343G>A). The identified variants segregated with the phenotype in all pedigrees. All deep intronic variants were predicted to generate a splice acceptor gain causing aberrant exonization in NR2E3 [89 base pairs (bp)], GPR179 (197 bp), and CNGB3 (73 bp); splicing defects were validated through patient-derived cDNA experiments and/or minigene constructs and rescued by antisense oligonucleotide treatment.Conclusions: Deep intronic mutations contribute to missing heritability in retinal dystrophies. Combining results from phenotype-directed gene panel testing, GS, and in silico splice prediction tools can help identify these difficult-to-detect pathogenic deep intronic variants.

Background: Patients with advanced, disabling multiple sclerosis (MS) have few effective treatment options. Little is known about the role that patients and their care providers want their neurologist to fill in this situation. Objective: To better understand the role that patients with disabling MS and their care providers want their neurologist to have in their care. Methods: In this exploratory qualitative study, we conducted semi-structured interviews with 29 participants (19 patients with severe disability due to MS and 10 care providers). Interview transcripts were analyzed using inductive thematic analysis. Results: Participants identified three main roles for their neurologist: a source of hope for therapeutic advances, an educator about the disease and its management, and a source of support. Conclusion: Despite sustaining a level of disability that may be refractory to standard medical therapy, patients with disabling MS and care providers continue to value certain roles of their neurologist. The neurologist’s role as a source of hope and support in particular has not received enough attention in the literature.

Supplemental material, MSJ845107_appendix_2 for The neurologist’s role in disabling multiple sclerosis: A qualitative study of patient and care provider perspectives by Jean-Pierre R Falet, Shriya Deshmukh, Abrar Al-Jassim, Gregory Sigler, Melanie Babinski and Fraser Moore in Multiple Sclerosis Journal

Subcutaneous panniculitis-like T cell lymphoma (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation that adversely affects survival1,2. T cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants altering highly conserved residues of TIM-3, c.245A>G (p.Tyr82Cys) and c.291A>G (p.Ile97Met), each with specific geographic distribution. The variant encoding p.Tyr82Cys TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while p.Ile97Met TIM-3 occurs in patients with European ancestry. Both variants induce protein misfolding and abrogate TIM-3’s plasma membrane expression, leading to persistent immune activation and increased production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, promoting HLH and SPTCL. Our findings highlight HLH–SPTCL as a new genetic entity and identify mutations causing TIM-3 alterations as a causative genetic defect in SPTCL. While HLH–SPTCL patients with mutant TIM-3 benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here, we show that 50% of G34R/V-tumours (n=95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. While considered gliomas, G34R/V-tumours actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V-mutations impair neuronal differentiation. The lineage-of-origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V-tumours harbour dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell-fate specification. G34R/V may become dispensable for tumour maintenance, while mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumours. G34R/V-gliomas are neuronal malignancies, where interneuron progenitors are stalled in differentiation by G34R/V-mutations, and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signalling.

Background: Pediatric glioblastomas (pGBM) are aggressive and lethal brain tumors with few effective treatment options. Since the discovery of recurrent histone H3 mutations in pGBMs, multiple groups have sought to uncover the mechanism by which these mutations drive tumorigenesis. One striking aspect of histone H3 mutations is their tumor location and age-specificity: the K27M mutation occurs in midline brain structures in infants and young children, whereas the G34R/V mutations occur in the cerebral hemisphere of adolescents. Efforts to elucidate the mechanism underlying K27M tumorigenesis have centered on K27M-mediated inhibition of the PRC2 complex and the resulting drastic loss of the repressive H3K27me3 mark in these tumors. Studies have shown that G34 mutations affect methylation of the adjacent K36 residue, resulting in a local decrease of the active H3K36me3 mark on the mutant histone. It remains uncertain whether the G34R/V mutations promote global changes in the epigenome that drive tumorigenesis. Methods: To investigate the epigenetic consequences of the G34R mutation, we generated an isogenic in vitro model using CRISPR/Cas9 to correct the G34R mutation in a pGBM-derived cell line. We then comprehensively characterized and compared the epigenome and transcriptome of CRISPR-unedited clones (carrying the G34R mutation) and CRISPR-edited (wild-type) clones. Using ChIP-seq, we profiled H3K36me3 and H3K27me3 marks to study G34R-mediated regulation of these antagonistic marks. Results: ChIP-seq profiling revealed that CRISPR editing of the G34R mutation had minimal effects on total levels or genomic distribution and profile of H3K36me3 and H3K27me3 marks. In contrast, we previously showed that CRISPR editing of the K27M mutation in pGBM cell lines rescued total H3K27me3 levels and genomic distribution to that of histone wild-type pGBMs. In agreement with their similar epigenetic profiles, there were very few transcriptomic differences between G34R and wildtype cells. Conclusions: Our results suggest that the epigenome in G34R-mutant pGBM cells may be less dynamic or amenable to epigenetic perturbation from removal of the initial driver oncomutation than K27M-mutant tumors. These findings underscore that different strategies may be required to target and treat G34R and K27M-mutant pGBM tumors. Citation Format: Shriya Deshmukh, Carol C.L. Chen, Amira Ouanouki, Nada Jabado. Epigenetic changes mediated by H3.3 G34R mutation in a CRISPR-edited pediatric glioblastoma cell line [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B09.

Lys-27-Met mutations in histone 3 genes (H3K27M) characterize a subgroup of deadly gliomas and decrease genome-wide H3K27 trimethylation. Here we use primary H3K27M tumor lines and isogenic CRISPR-edited controls to assess H3K27M effects in vitro and in vivo. We find that whereas H3K27me3 and H3K27me2 are normally deposited by PRC2 across broad regions, their deposition is severely reduced in H3.3K27M cells. H3K27me3 is unable to spread from large unmethylated CpG islands, while H3K27me2 can be deposited outside these PRC2 high-affinity sites but to levels corresponding to H3K27me3 deposition in wild-type cells. Our findings indicate that PRC2 recruitment and propagation on chromatin are seemingly unaffected by K27M, which mostly impairs spread of the repressive marks it catalyzes, especially H3K27me3. Genome-wide loss of H3K27me3 and me2 deposition has limited transcriptomic consequences, preferentially affecting lowly-expressed genes regulating neurogenesis. Removal of H3K27M restores H3K27me2/me3 spread, impairs cell proliferation, and completely abolishes their capacity to form tumors in mice., Lysine27-to-methionine mutations in histone H3 genes (H3K27M) occur in a subgroup of gliomas and decrease genome-wide H3K27 trimethylation. Here the authors utilise primary H3K27M tumour lines and isogenic CRISPR-edited controls and show that H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3.

High-grade gliomas (HGG) defined by histone 3 K27M driver mutations exhibit global loss of H3K27 trimethylation and reciprocal gain of H3K27 acetylation, respectively shaping repressive and active chromatin landscapes. We generated tumor-derived isogenic models bearing this mutation and show that it leads to pervasive H3K27ac deposition across the genome. In turn, active enhancers and promoters are not created de novo and instead reflect the epigenomic landscape of the cell of origin. H3K27ac is enriched at repeat elements, resulting in their increased expression, which in turn can be further amplified by DNA demethylation and histone deacetylase inhibitors providing an exquisite therapeutic vulnerability. These agents may therefore modulate anti-tumor immune responses as a therapeutic modality for this untreatable disease.

DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in

In the version of this article originally published, the main-text sentence “In three patients of European ancestry, we identified the germline variant encoding p.Ile97Met in TIM-3, which was homozygous in two (P12 and P13) and heterozygous in one (P15) in the germline but with no TIM-3 plasma membrane expression in the tumor” misstated the identifiers of the two homozygous individuals, which should have been P13 and P14. The error has been corrected in the HTML, PDF and print versions of the paper.

Introduction: Pediatric glioblastomas (pGBM) are malignant brain tumors associated with a dismal prognosis. A subset of pGBMs carry mutations of either the Lysine 27 or Glycine 34 (G34) amino acid residues of histone 3 variant genes. The same G34 residue is also mutated in 85-95% of giant cell tumors of bone (GCTs), albeit to Tryptophan (G34W) in GCTs rather than to Arginine or Valine (G34R/V) as in pGBMs. The G34 mutation is predicted to impede access of histone methyltransferases like SetD2 to the nearby Lysine 36 residue, thereby altering the epigenome and transcriptome. Methods: To elucidate the tumorigenic effect of G34 mutations, we used the gene-editing technology CRISPR/Cas9 to correct the G34W mutation to wild-type in 2 GCT cell lines. We then investigated CRISPR-edited cell lines using functional assays, proteomic, epigenomic, and transcriptomic analyses. Results: Correction of the G34W mutation to wild-type in CRISPR-edited GCT cells results in phenotypic and functional changes suggestive of reduced tumorigenicity. By mass spectrometry, G34W-mutant GCT cell lines display decreased level of Lysine 36 trimethylation (H3K36me3) on the mutant G34-peptide, similar to G34-mutated pGBM cell lines. However, unlike pGBMs, GCTs display increased levels of Lysine 36 dimethylation (H3K36me2) on the mutant G34W-peptide. Ongoing Experiments and Analyses: We are currently comparing the level and distribution of multiple histone marks by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) of CRISPR-edited wild-type clones relative to the parent G34W-mutant GCT line. We are also assessing differential gene expression by RNA-Seq and characterizing the methylation signature by 850K DNA methylation array of G34W-mutant GCTs and edited clones. Conclusion: The G34W-mutation clearly has an impact on tumorigenic potential, as evidenced by in vitro functional assays. The G34W-mutant peptide of GCT cell lines features a distinct profile of post-translational histone modifications compared to G34R- or G34V-mutant peptides of pGBM cell lines. Further investigation could elucidate the epigenetic mechanism(s) through which the G34W mutation confers its tumorigenic properties. Citation Format: Shriya Deshmukh, Sima Khazaei, Dylan Marchione, Ashot Harutyunyan, Benjamin Garcia, Nada Jabado. Characterizing the epigenetic effects of the histone 3.3 G34W mutation in giant cell tumors of bone [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A39.

Background: Glioblastoma is a grade IV malignant brain tumor with poor prognosis and rapid disease progression. Recurrent somatic mutations in histone H3 genes have been identified in the majority of pediatric glioblastoma cases. The K27M mutation in H3.1 and H3.3 histones globally inhibits lysine methylation at the K27 position, whereas H3.3 G34R/V possibly affects histone lysine methylation at the K36 position. H3 K27M mutation has been shown to dramatically decrease the total levels of H3K27me3 and H3K27me2 marks and increase H3K27ac levels. However, the effect of H3 K27M on global epigenomic changes is not fully characterized. Furthermore, standard profiling of histone marks by chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq) is not quantitative, a significant caveat when global levels of histone marks change so drastically. Methods: We assembled a collection of H3 K27M mutant and wild-type cell lines derived from the glioblastoma patients. The epigenomes of these cell lines were comprehensively characterized by profiling for six histone marks (H3K4me1, H3K4me3, H3K27ac, H3K27me3, H3K36me2, H3K36me3) using ChIP-seq. In addition, we derived isogenic cell lines overexpressing H3.3 K27M, as well as cell lines with knockin or knockout of the K27M mutation using the CRISPR/Cas9 genome editing system. These cell lines were profiled for H3K27me3 mark by ChIP-seq. We used a modified ChIP-seq protocol, chromatin immunoprecipitation with exogenous reference genome (ChIP-Rx), which allows quantitation of histone mark abundance by normalization to proportions of added Drosophila chromatin in the ChIP reaction. RNA sequencing was performed on both primary and isogenic cell lines. Results: The most striking difference we observed between H3 K27M and wild-type cells was in H3K27me3 mark. Using ChIP-Rx, we observe significantly lower levels of H3K27me3 mark in H3 K27M cell lines, both in primary cells and isogenic contexts. Despite very low total levels of H3K27me3 mark, K27M mutant cells display enrichment of the mark in certain regions, at comparable levels to wild-type cell lines. Using our isogenic cell line models, we show that K27M mutation is indeed responsible for those genome-wide changes in the epigenome. Correlating H3K27me3 distribution with transcriptome data, we show that expression changes mainly among the genes that are lowly expressed in these cells. Pathway analysis of differentially expressed genes shows enrichment for neural development and differentiation that suggests links to disease pathogenesis. Conclusions: Despite the fact that primary cell lines have different origins and a variety of additional driver mutations, their epigenomes appears to be remarkably similar, due to being shaped predominantly by the effects of histone mutations, as demonstrated in isogenic cell line systems. Global changes in H3K27me3 levels and distribution in H3 K27M mutant cells lead to specific changes in gene expression. The changes induced by K27M mutations also appear to be specific to the cell type and/or developmental context of origin. This may help better understand the effect they have in reshaping the epigenome to promote oncogenesis. Citation Format: Ashot S. Harutyunyan, Brian Krug, Simon Papillon-Cavanagh, Haifen Chen, Shriya Deshmukh, Warren A. Cheung, Rui Li, Jad Belle, Denise Bechet, Nicolas De Jay, Michele Zeinieh, Tenzin Gayden, Caterina Russo, Leonie Mikael, Damien Faury, Claudia Kleinman, Tomi Pastinen, Jacek Majewski, Nada Jabado. Identification of epigenomic changes induced by H3 K27M mutation in glioblastoma using patient-derived and CRISPR/Cas9 edited cell lines [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B44.