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Sample Characteristics and Quality Control

Interactions between Stromal and Myeloid compartments

Differential Expression in Isogenic Lines

Cell type-specific markers and pathways in single-cell transcriptomics

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

Supplementary Figures

Golgi Secretome Analysis in Isogenic Lines

Pathway Enrichment Analysis

Supplementary Information

Supplementary Figure 1 from Inhibition of Y-box binding protein-1 slows the growth of glioblastoma multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA methyltransferase

Supplementary Figure 2 from Inhibition of Y-box binding protein-1 slows the growth of glioblastoma multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA methyltransferase

Purpose: Oncogenic BRAF/Ras or NF1 loss can potentially trigger oncogene-induced senescence (OIS) through activation of the mitogen-activated protein kinase (MAPK) pathway. Somatic genetic abnormalities affecting this pathway occur in the majority of pilocytic astrocytomas (PA), the most prevalent brain neoplasm in children. We investigated whether OIS is induced in PA.Experimental Design: We tested expression of established senescence markers in three independent cohorts of sporadic PA. We also assessed for OIS in vitro, using forced expression of wild-type and V600E-mutant BRAF in two astrocytic cell lines: human telomerase reverse transcriptase (hTERT)-immortalized astrocytes and fetal astrocytes.Results: Our results indicate that PAs are senescent as evidenced by marked senescence-associated acidic β-galactosidase activity, low KI-67 index, and induction of p16INK4a but not p53 in the majority of 52 PA samples (46 of 52; 88.5%). Overexpression of a number of senescence-associated genes [CDKN2A (p16), CDKN1A (p21), CEBPB, GADD45A, and IGFBP7] was shown at the mRNA level in two independent PA tumor series. In vitro, sustained activation of wild-type or mutant BRAF induced OIS in both astrocytic cell lines. Loss of p16INK4a in immortalized astrocytes abrogated OIS, indicative of the role of this pathway in mediating this phenomenon in astrocytes. OIS is a mechanism of tumor suppression that restricts the progression of benign tumors. We show that it is triggered in PAs through p16INK4a pathway induction following aberrant MAPK activation.Conclusions: OIS may account for the slow growth pattern in PA, the lack of progression to higher-grade astrocytomas, and the high overall survival of affected patients. Clin Cancer Res; 17(14); 4650–60. ©2011 AACR.

Supplementary Figures S1-S3; Supplementary Tables S1-S2.

Glioblastoma multiforme (GBM) is an aggressive type of brain tumor where 5 years. In adults, GBM is the most common type of brain tumor. It is rarer in children, where it constitutes ∼15% of all brain tumors diagnosed. These tumors are often invasive, making surgical resection difficult. Further, they can be refractory to current therapies such as temozolomide. The current dogma is that temozolomide resistance rests on the expression of O6-methylguanine-DNA methyltransferase (MGMT) because it cleaves methylated DNA adducts formed by the drug. Our laboratory recently reported that another drug resistance gene known as the Y-box binding protein-1 (YB-1) is highly expressed in primary GBM but not in normal brain tissues based on the evaluation of primary tumors. We therefore questioned whether GBM depend on YB-1 for growth and/or response to temozolomide. Herein, we report that YB-1 inhibition reduced tumor cell invasion and growth in monolayer as well as in soft agar. Moreover, blocking this protein ultimately delayed tumor onset in mice. Importantly, inhibiting YB-1 enhanced temozolomide sensitivity in a manner that was independent of MGMT in models of adult and pediatric GBM. In conclusion, inhibiting YB-1 may be a novel way to improve the treatment of GBM. [Mol Cancer Ther 2009;8(12):3276–84]

Supplementary Figure Legends 1-3 from Inhibition of Y-box binding protein-1 slows the growth of glioblastoma multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA methyltransferase

Supplementary Figure 3 from Inhibition of Y-box binding protein-1 slows the growth of glioblastoma multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA methyltransferase

Canonical (H3.1/H3.2) and noncanonical (H3.3) histone 3 K27M-mutant gliomas have unique spatiotemporal distributions, partner alterations, and molecular profiles. The contribution of the cell-of-origin to these differences has been challenging to uncouple from the oncogenic reprogramming induced by the mutation. Here, we perform an integrated analysis of 116 tumors, including single-cell transcriptome and chromatin accessibility, 3D chromatin architecture and epigenomic profiles, and show that K27M-mutant gliomas faithfully maintain chromatin configuration at developmental genes consistent with anatomically distinct oligodendrocyte-precursor-like cells (OPC). H3.3K27M thalamic gliomas map to prosomere 2-derived lineages. In turn, H3.1K27M ACVR1-mutant pontine gliomas uniformly mirror early ventral NKX6-1+/SHH-dependent brainstem OPCs, while H3.3K27M gliomas frequently resemble dorsal PAX3+/BMP-dependent progenitors. Our data suggest a context-specific vulnerability in H3.1K27M-mutant SHH-dependent ventral OPCs, which rely on acquisition of ACVR1 mutations to drive aberrant BMP signaling required for oncogenesis. The unifying action of K27M mutations is to restrict H3K27me3 at PRC2 landing sites, while other epigenetic changes are mainly contingent on the cell-of-origin chromatin state and cycling rate.

Platelet-Derived Growth Factor Receptor Beta (PDGFRB) is critically implicated in development. Germline pathogenic variants (GPVs) in PDGFRB result in several distinct inherited syndromes including primary familial brain calcifications (PFBC) and infantile myofibromatosis. To date, GPVs in PDGFRB have not been identified in children with brain tumors. Here, we describe the clinicopathological features of a 9-year-old male with a medulloblastoma and an 11-year-old female with a glioma. Sequencing of the blood and tumor samples revealed the same PDGFRB c.2959C>T (p. Arg987Trp) GPV in both children. Additional fusion genes DCTN1-ALK and TRIM24-MET were also identified in the patients’ tumors through RNAseq.

Background Juvenile Pilocytic Astrocytomas (JPAs) are one of the most common pediatric brain tumors, and they are driven by aberrant activation of the mitogen-activated protein kinase (MAPK) signaling pathway. RAF-fusions are the most common genetic alterations identified in JPAs, with the prototypical KIAA1549-BRAF fusion leading to loss of BRAF’s auto-inhibitory domain and subsequent constitutive kinase activation. JPAs are highly vascular and show pervasive immune infiltration, which can lead to low tumor cell purity in clinical samples. This can result in gene fusions that are difficult to detect with conventional omics approaches including RNA-Seq. Methods To this effect, we applied RNA-Seq as well as linked-read whole-genome sequencing and in situ Hi-C as new approaches to detect and characterize low-frequency gene fusions at the genomic, transcriptomic and spatial level. Results Integration of these datasets allowed the identification and detailed characterization of two novel BRAF fusion partners, PTPRZ1 and TOP2B, in addition to the canonical fusion with partner KIAA1549. Additionally, our Hi-C datasets enabled investigations of 3D genome architecture in JPAs which showed a high level of correlation in 3D compartment annotations between JPAs compared to other pediatric tumors, and high similarity to normal adult astrocytes. We detected interactions between BRAF and its fusion partners exclusively in tumor samples containing BRAF fusions. Conclusions We demonstrate the power of integrating multi-omic datasets to identify low frequency fusions and characterize the JPA genome at high resolution. We suggest that linked-reads and Hi-C could be used in clinic for the detection and characterization of JPAs.

Pediatric low-grade gliomas (PLGGs) are the most common central nervous system (CNS) tumors in children. The current standard of care for surgically unresectable and/or progressive cases of PLGGs includes combination chemotherapy. PLGGs are molecularly characterized by alterations in the RAS/RAF/MAPK/ERK pathway in a majority of tumors. PLGGs harboring the BRAF-V600E mutation respond poorly to current chemotherapy strategies. We present a case of a two-year-old female with biopsy-proven low-grade glioma (LGG, pilocytic astrocytoma) involving the hypothalamic/optic chiasm region. At presentation, she had obstructive hydrocephalus, bitemporal hemianopia, central hypothyroidism, and right-sided hemiparesis due to the location/mass effect of the tumor. She was initially treated with chemotherapy (vincristine/carboplatin), but her tumor progressed at six weeks of treatment. She was subsequently started on dabrafenib as her tumor was positive for BRAF-V600E mutation. Dabrafenib monotherapy resulted in dramatic improvement in her clinical symptoms and near-complete resolution of tumor. Our experience and review of the literature suggest that LGGs with BRAF-V600E mutations may benefit from upfront targeted therapy in children. There is an urgent need for prospective clinical trials comparing the efficacy of upfront BRAF inhibitors versus standard chemotherapy in PLGGs with BRAF mutations.

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

International audience; Childhood brain tumors have suspected prenatal origins. To identify vulnerable developmental states, we generated a single-cell transcriptome atlas of >65,000 cells from embryonal pons and forebrain, two major tumor locations. We derived signatures for 191 distinct cell populations and defined the regional cellular diversity and differentiation dynamics. Projection of bulk tumor transcriptomes onto this dataset shows that WNT medulloblastomas match the rhombic lip-derived mossy fiber neuronal lineage and embryonal tumors with multilayered rosettes fully recapitulate a neuronal lineage, while group 2a/b atypical teratoid/rhabdoid tumors may originate outside the neuroectoderm. Importantly, single-cell tumor profiles reveal highly defined cell hierarchies that mirror transcriptional programs of the corresponding normal lineages. Our findings identify impaired differentiation of specific neural progenitors as a common mechanism underlying these pediatric cancers and provide a rational framework for future modeling and therapeutic interventions.

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.

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.

Pilocytic astrocytoma (PA) is the most common brain tumor in children but is rare in adults, and hence poorly studied in this age group. We investigated 222 PA and report increased aneuploidy in older patients. Aneuploid genomes were identified in 45% of adult compared with 17% of pediatric PA. Gains were non-random, favoring chromosomes 5, 7, 6 and 11 in order of frequency, and preferentially affecting non-cerebellar PA and tumors with BRAF V600E mutations and not with KIAA1549-BRAF fusions or FGFR1 mutations. Aneuploid PA differentially expressed genes involved in CNS development, the unfolded protein response, and regulators of genomic stability and the cell cycle (MDM2, PLK2),whose correlated programs were overexpressed specifically in aneuploid PA compared to other glial tumors. Thus, convergence of pathways affecting the cell cycle and genomic stability may favor aneuploidy in PA, possibly representing an additional molecular driver in older patients with this brain tumor.

Studies in pediatric high-grade astrocytomas (HGA) by our group and others have uncovered recurrent somatic mutations affecting highly conserved residues in histone 3 (H3) variants. One of these mutations leads to analogous p.Lys27Met (K27M) mutations in both H3.3 and H3.1 variants, is associated with rapid fatal outcome, and occurs specifically in HGA of the midline in children and young adults. This includes diffuse intrinsic pontine gliomas (80 %) and thalamic or spinal HGA (>90 %), which are surgically challenging locations with often limited tumor material available and critical need for specific histopathological markers. Here, we analyzed formalin-fixed paraffin-embedded tissues from 143 pediatric HGA and 297 other primary brain tumors or normal brain. Immunohistochemical staining for H3K27M was compared to tumor genotype, and also compared to H3 tri-methylated lysine 27 (H3K27me3) staining, previously shown to be drastically decreased in samples carrying this mutation. There was a 100 % concordance between genotype and immunohistochemical analysis of H3K27M in tumor samples. Mutant H3K27M was expressed in the majority of tumor cells, indicating limited intra-tumor heterogeneity for this specific mutation within the limits of our dataset. Both H3.1 and H3.3K27M mutants were recognized by this antibody while non-neoplastic elements, such as endothelial and vascular smooth muscle cells or lymphocytes, did not stain. H3K27me3 immunoreactivity was largely mutually exclusive with H3K27M positivity. These results demonstrate that mutant H3K27M can be specifically identified with high specificity and sensitivity using an H3K27M antibody and immunohistochemistry. Use of this antibody in the clinical setting will prove very useful for diagnosis, especially in the context of small biopsies in challenging midline tumors and will help orient care in the context of the extremely poor prognosis associated with this mutation. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1337-4) contains supplementary material, which is available to authorized users.

Acute myeloid leukemia (AML) develops in a step-wise manner from pre-leukemic clonal expansion to full-blown disease driven by aberrant epigenetic changes. Indeed, regulators of the epigenome such as DNMT3A, TET2, IDH1/2, EZH2 and ASXL1 are often mutated in pre-leukemia and myeloid malignancies. We and others identified K27M/I mutations in histone H3 in AML (Boileau et al. Nat Commun, 2019; Lehnertz et al. Blood, 2017). We demonstrated that K27 mutations are found in pre-leukemic hematopoietic stem cells (HSCs), are enriched in secondary AML, expand the functional human HSC pool and increase leukemic aggressiveness. Transcriptomic and epigenomic analysis determined that K27 mutations alter gene expression through a global decrease in promoter H3K27 tri-methylation and a gene-specific increase in H3K27 acetylation in leukemic cells (Boileau et al. Nat Commun, 2019). Here, we have analyzed the effects of the K27M mutation on HSCs at the single-cell level to understand its role in pre-leukemic clonal expansion. Healthy CD34+CD38- human cord blood cells were transduced with HIST1H3H WT or K27M and injected intrafemorally into sub-lethally irradiated NSG mice. After 14 weeks, bone marrow cells from the femur were collected and sorted for CD34+ transduced (GFP+) cells. Single-cell transcriptomics were performed by generating gene expression libraries from ~8,000 CD34+ cells using the 10X Genomics technology and sequenced using HiSeq4000. We have performed initial clustering and dimensionality reduction (t-SNE and UMAP) and identified 10 and 11 distinct clusters in the WT and K27M samples, respectively. Gene sets distinguishing the individual clusters have been determined. Using published gene lists for primitive hematopoietic cell types, the clusters have been assigned to specific cell types such as HSC, granulocyte-monocyte progenitors (GMP), common myeloid progenitors (CMP), multi-lymphoid progenitors (MLP) and megakaryocyte-erythroid progenitors (MEP) (Laurenti et al. Nat Immunol, 2013). Preliminary joint clustering analysis indicates the presence of two distinct clusters for the WT and K27M samples that were both assigned as "HSCs" in individual clustering. Further analysis to identify the differences in the clusters and cell populations between WT and K27M samples is being performed and will be presented at this meeting. Overall, this single-cell transcriptomic analysis will aid in determining the mechanism of action of the K27M mutant histone in pre-leukemic HSC clonal expansion. In addition, we will be performing similar single-cell analysis on HSCs expressing mutant ASXL1 as a comparison. Further understanding of the role of mutations in epigenetic regulators, such as histone H3 and ASXL1, in pre-leukemic clonal hematopoiesis will provide valuable insight on how to better prevent and treat AML and other myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

Recurrent mutations affecting the histone H3.3 residues Lys27 or indirectly Lys36 are frequent drivers of pediatric high-grade gliomas (over 30 % of HGGs). To identify additional driver mutations in HGGs, we investigated a cohort of 60 pediatric HGGs using whole-exome sequencing (WES) and compared them to 543 exomes from non-cancer control samples. We identified mutations in SETD2, a H3K36 trimethyltransferase, in 15 % of pediatric HGGs, a result that was genome-wide significant (FDR = 0.029). Most SETD2 alterations were truncating mutations. Sequencing the gene in this cohort and another validation cohort (123 gliomas from all ages and grades) showed SETD2 mutations to be specific to high-grade tumors affecting 15 % of pediatric HGGs (11/73) and 8 % of adult HGGs (5/65) while no SETD2 mutations were identified in low-grade diffuse gliomas (0/45). Furthermore, SETD2 mutations were mutually exclusive with H3F3A mutations in HGGs (P = 0.0492) while they partly overlapped with IDH1 mutations (4/14), and SETD2-mutant tumors were found exclusively in the cerebral hemispheres (P = 0.0055). SETD2 is the only H3K36 trimethyltransferase in humans, and SETD2-mutant tumors showed a substantial decrease in H3K36me3 levels (P

Dysembryoplastic neuroepithelial tumor (DNET) is a benign brain tumor associated with intractable drug-resistant epilepsy. In order to identify underlying genetic alterations and molecular mechanisms, we examined three family members affected by multinodular DNETs as well as 100 sporadic tumors from 96 patients, which had been referred to us as DNETs. We performed whole-exome sequencing on 46 tumors and targeted sequencing for hotspot FGFR1 mutations and BRAF p.V600E was used on the remaining samples. FISH, copy number variation assays and Sanger sequencing were used to validate the findings. By whole-exome sequencing of the familial cases, we identified a novel germline FGFR1 mutation, p.R661P. Somatic activating FGFR1 mutations (p.N546K or p.K656E) were observed in the tumor samples and further evidence for functional relevance was obtained by in silico modeling. The FGFR1 p.K656E mutation was confirmed to be in cis with the germline p.R661P variant. In 43 sporadic cases, in which the diagnosis of DNET could be confirmed on central blinded neuropathology review, FGFR1 alterations were also frequent and mainly comprised intragenic tyrosine kinase FGFR1 duplication and multiple mutants in cis (25/43; 58.1 %) while BRAF p.V600E alterations were absent (0/43). In contrast, in 53 cases, in which the diagnosis of DNET was not confirmed, FGFR1 alterations were less common (10/53; 19 %; p

Gliomas are the most common primary brain tumors in children and adults. We recently identified frequent alterations in chromatin remodelling pathways including recurrent mutations in H3F3A and mutations in ATRX (α-thalassemia/mental-retardation-syndrome-X-linked) in pediatric and young adult glioblastoma (GBM, WHO grade IV astrocytoma). H3F3A mutations were specific to pediatric high-grade gliomas and identified in only 3.4 % of adult GBM. Using sequencing and/or immunohistochemical analyses, we investigated ATRX alterations (mutation/loss of expression) and their association with TP53 and IDH1 or IDH2 mutations in 140 adult WHO grade II, III and IV gliomas, 17 pediatric WHO grade II and III astrocytomas and 34 pilocytic astrocytomas. In adults, ATRX aberrations were detected in 33 % of grade II and 46 % of grade III gliomas, as well as in 80 % of secondary and 7 % of primary GBMs. They were absent in the 17 grade II and III astrocytomas in children, and the 34 pilocytic astrocytomas. ATRX alterations closely overlapped with mutations in IDH1/2 (p < 0.0001) and TP53 (p < 0.0001) in samples across all WHO grades. They were prevalent in astrocytomas and oligoastrocytomas, but were absent in oligodendrogliomas (p < 0.0001). No significant association of ATRX mutation/loss of expression and alternative lengthening of telomeres was identified in our cohort. In summary, our data show that ATRX alterations are frequent in adult diffuse gliomas and are specific to astrocytic tumors carrying IDH1/2 and TP53 mutations. Combined alteration of these genes may contribute to drive the neoplastic growth in a major subset of diffuse astrocytomas in adults.

Pediatric glioblastomas (GBM) including diffuse intrinsic pontine gliomas (DIPG) are devastating brain tumors with no effective therapy. Here, we investigated clinical and biological impacts of histone H3.3 mutations. Forty-two DIPGs were tested for H3.3 mutations. Wild-type versus mutated (K27M-H3.3) subgroups were compared for HIST1H3B, IDH, ATRX and TP53 mutations, copy number alterations and clinical outcome. K27M-H3.3 occurred in 71 %, TP53 mutations in 77 % and ATRX mutations in 9 % of DIPGs. ATRX mutations were more frequent in older children (p

Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

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.

Background: Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location. Methods and results: Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53 – 66%) and does not occur in other LGA subtypes (0 of 27) or NF1-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30 – 80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16 – 62.5%) and is rare in hemispheric JPA (1 of 7 – 14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation. Conclusions and interpretation: Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway.

Resistance to drug is a major cause of treatment failure in pediatric brain cancer. The multidrug resistance (MDR) phenotype can be mediated by the superfamily of adenosine triphosphate-binding cassette (ABC) transporters. The dynamics of expression of the MDR genes after exposure to chemotherapy, especially the comparison between pediatric brain tumors of different histology, is poorly described. To compare the expression profiles of the multidrug resistance genes ABCB1, ABCC1, and ABCG2 in different neuroepithelial pediatric brain tumor cell lines prior and following short-term culture with vinblastine. Immortalized lineages from pilocytic astrocytoma (R286), anaplasic astrocytoma (UW467), glioblastoma (SF188), and medulloblastoma (UW3) were exposed to vinblastine sulphate at different schedules (10 and 60 nM for 24 and 72 h). Relative amounts of mRNA expression were analyzed by real-time quantitative polymerase chain reaction. Protein expression was assessed by immunohistochemistry for ABCB1, ABCC1, and ABCG2. mRNA expression of ABCB1 increased together with augmenting concentration and time of exposure to vinblastine for R286, UW467, and UW3 cell lines. Interestingly, ABCB1 levels of expression diminished in SF188. Following chemotherapy, mRNA expression of ABCC1 decreased in all cell lines other than glioblastoma. ABCG2 expression was influenced by vinblastine only for UW3. The mRNA levels showed consistent association to protein expression in the selected sets of cell lines analyzed. The pediatric glioblastoma cell line SF188 shows different pattern of expression of multidrug resistance genes when exposed to vinblastine. These preliminary findings may be useful in determining novel strategies of treatment for neuroepithelial pediatric brain tumors.

Purpose Pediatric glioblastoma (pGBM) is a rare, but devastating brain tumor. In contrast to GBM in adults (aGBM), little is known about the mechanisms underlying its development. Our aim is to gain insight into the molecular pathways of pGBM. Materials and Methods Thirty-two pGBM and seven aGBM samples were investigated using biochemical and transcriptional profiling. Ras and Akt pathway activation was assessed through the phosphorylation of downstream effectors, and gene expression profiles were generated using the University Health Network Human 19K cDNA arrays. Results were validated using real-time polymerase chain reaction and immunohistochemistry and compared with existing data sets on aGBM. Results There are at least two subsets of pGBM. One subset, associated with Ras and Akt pathway activation, has very poor prognosis and exhibits increased expression of genes related to proliferation and to a neural stem-cell phenotype, similar to findings in aggressive aGBM. This subset was still molecularly distinguishable from aGBM after unsupervised and supervised analysis of expression profiles. A second subset, with better prognosis, is not associated with activation of Akt and Ras pathways, may originate from astroglial progenitors, and does not express gene signatures and markers shown to be associated with long-term survival in aGBM. Both subsets of pGBM show overexpression of Y-box-protein-1 that may help drive oncogenesis in this tumor. Conclusion Our work, the first study of gene expression profiles in pGBM, provides valuable insight into active pathways and targets in a cancer with minimal survival, and suggests that these tumors cannot be understood exclusively through studies of aGBM.

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly paediatric brain tumours where needle biopsies help guide diagnosis and targeted therapies. To address spatial heterogeneity, here we analyse 134 specimens from various neuroanatomical structures of whole autopsy brains from nine DIPG patients. Evolutionary reconstruction indicates histone 3 (H3) K27M—including H3.2K27M—mutations potentially arise first and are invariably associated with specific, high-fidelity obligate partners throughout the tumour and its spread, from diagnosis to end-stage disease, suggesting mutual need for tumorigenesis. These H3K27M ubiquitously-associated mutations involve alterations in TP53 cell-cycle (TP53/PPM1D) or specific growth factor pathways (ACVR1/PIK3R1). Later oncogenic alterations arise in sub-clones and often affect the PI3K pathway. Our findings are consistent with early tumour spread outside the brainstem including the cerebrum. The spatial and temporal homogeneity of main driver mutations in DIPG implies they will be captured by limited biopsies and emphasizes the need to develop therapies specifically targeting obligate oncohistone partnerships., Diffuse Intrinsic Pontine Gliomas are diagnosed by sampling a small portion of the tumour. Here, using multiple samples from tumours, the authors analyse the spatial and temporal distribution of driver mutations revealing that H3K27M mutations arise first in tumorigenesis followed by a specific invariable sequence of driver mutations, which are homogeneously distributed across the tumour mass.

Invasion of brain tumor cells has made primary malignant brain neoplasms among the most recalcitrant to therapeutic strategies. We tested whether the secreted protein Slit2, which guides the projection of axons and developing neurons, could modulate brain tumor cell invasion. Slit2 inhibited the invasion of medulloblastoma cells in a variety of in vitro models. The effect of Slit2 was inhibited by the Robo ectodomain. Time-lapse videomicroscopy indicated that Slit2 reduced medulloblastoma invasion rate without affecting cell direction or proliferation. Both medulloblastoma and glioma tumors express Robo1 and Slit2, but only medulloblastoma invasion is inhibited by recombinant Slit2 protein. Downregulation of activated Cdc42 may contribute to this differential response. Our findings reinforce the concept that neurodevelopmental cues such as Slit2 may provide insights into brain tumor invasion.

Pediatric midline high-grade astrocytomas (mHGAs) are incurable with few treatment targets identified. Most tumors harbor mutations encoding p.Lys27Met in histone H3 variants. In 40 treatment-naive mHGAs, 39 analyzed by whole-exome sequencing, we find additional somatic mutations specific to tumor location. Gain-of-function mutations in ACVR1 occur in tumors of the pons in conjunction with histone H3.1 p.Lys27Met substitution, whereas FGFR1 mutations or fusions occur in thalamic tumors associated with histone H3.3 p.Lys27Met substitution. Hyperactivation of the bone morphogenetic protein (BMP)-ACVR1 developmental pathway in mHGAs harboring ACVR1 mutations led to increased levels of phosphorylated SMAD1, SMAD5 and SMAD8 and upregulation of BMP downstream early-response genes in tumor cells. Global DNA methylation profiles were significantly associated with the p.Lys27Met alteration, regardless of the mutant histone H3 variant and irrespective of tumor location, supporting the role of this substitution in driving the epigenetic phenotype. This work considerably expands the number of potential treatment targets and further justifies pretreatment biopsy in pediatric mHGA as a means to orient therapeutic efforts in this disease.

Pilocytic astrocytoma, the most common childhood brain tumor, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression and often becoming a chronic disease with substantial morbidities. Here we describe whole-genome sequencing of 96 pilocytic astrocytomas, with matched RNA sequencing (n = 73), conducted by the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. We identified recurrent activating mutations in FGFR1 and PTPN11 and new NTRK2 fusion genes in non-cerebellar tumors. New BRAF-activating changes were also observed. MAPK pathway alterations affected all tumors analyzed, with no other significant mutations identified, indicating that pilocytic astrocytoma is predominantly a single-pathway disease. Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in the NF1 gene. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.

Background: Dysembryoplastic neuroepithelial tumors (DNETs) are benign developmental brain tumors associated with intractable, drug-resistant epilepsy. Distinguishing this entity from other low-grade ganglioneuronal tumors is challenging for neuro-pathologists. We set out to identify the genetic causes of DNETs and to clarify the molecular mechanisms underlying this condition. Experimental procedures: We collected a family with three individuals with seizures and multinodular DNETs together with 100 sporadic tumors from 96 persons referred to us as DNETs. Whole-exome sequencing was performed on 46 tumours and targeted sequencing for hotspot FGFR1 mutations and BRAFp.V600E was used on the remaining samples. Blind neuropathology review and molecular characterization were performed. FISH, Copy Number Variation assays and Sanger sequencing were used to validate the findings. Supporting evidence for functional defects was obtained by in silico modelling of novel FGFR1p.R661P variant. Functional impact of this and other FGFR1 mutations were assessed using Flow Cytometry and β-galactosidase staining in HEK293 cells. Results: We identified a novel germline FGFR1 mutation, p.R661P, in a father and his two children with DNETs. Somatic activating FGFR1 mutations (p.N546K or p.K656E) were observed in cis in the tumors with the germline mutation. Pathology review distinguished DNETs (WHO grade I) (45%) from DNET-like tumors (55%). FGFR1 alterations, mainly intragenic tyrosine kinase FGFR1 duplication and multiple mutants in cis, characterized DNETs (25/43;58.1%) whereas FGFR1 mutations (10/53;19%) (p < 0.0001) and hotspot BRAFp.V600E (12/53;22.6%) (p < 0.001) were identified in DNET-like tumors. Phospho-ERK overexpression in FGFR1p.R661P and p.N546K mutant cells support enhanced MAPK/ERK activation in this condition. Conclusions: This study identifies constitutional and somatic FGFR1 alterations and hotspot BRAFV600E as key events in DNETs and DNET-like tumors respectively. The final common effect of these alterations appears to be a balanced level of signalling that results in benign rather than malignant tumors. The integrated pathology and molecular characterization performed here reveals the key role of the MAP-Kinase pathway in these epileptogenic low-grade glioneuronal tumors, pointing the way towards existing targeted therapies. Citation Format: Barbara Rivera, Tenzin Gayden, Jian Zhang, Javad Nadaf, Talia Boshari, Damien Faury, Michele Zeinieh, Romeo Blanc, David Burk, Somayyeh Fahiminiya, Eric Bareke, Ulrich Schueller, Camelia M. Monoranu, Ronald Sträter, Kornelius Kerl, Thomas Niederstadt, Gerhard Kurlemann, Benjamin Ellezam, Zuzanna Michalak, Maria Thom, Paul Lockhart, Richard Leventer, Milou Ohm, Duncan McGregor, David Jones, Jason Karamchandani, Celia Greenwood, Albert Berghuis, Susanne Bens, Reiner Siebert, Magdalena Zakrzewska, Pawel Liberski, Krzysztof Zakrzewski, Sanjay Sisodiya, Werner Paulus, Steffen Albrecht, Martin Hasselblatt, Nada Jabado, William D. Foulkes, Jacek Majewski. FGFR1 abnormalities in seizure-associated familial and sporadic dysembryoplastic neuroepithelial tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-019.

SummaryGlioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

Graft-versus-host disease is uncommon in autologous hematopoietic cell transplantation (HCT) and is typically brief and mild. We report unusual, protracted, and severe Omenn syndrome-like autoaggression following autologous HCT. We identified a profound FOXP3 + regulatory T cell defect that coincided with hyperinflammatory T cell responses which were reversible with rapamycin in vitro .

Purpose: Oncogenic BRAF/Ras or NF1 loss can potentially trigger oncogene-induced senescence (OIS) through activation of the mitogen-activated protein kinase (MAPK) pathway. Somatic genetic abnormalities affecting this pathway occur in the majority of pilocytic astrocytomas (PA), the most prevalent brain neoplasm in children. We investigated whether OIS is induced in PA. Experimental Design: We tested expression of established senescence markers in three independent cohorts of sporadic PA. We also assessed for OIS in vitro, using forced expression of wild-type and V600E-mutant BRAF in two astrocytic cell lines: human telomerase reverse transcriptase (hTERT)-immortalized astrocytes and fetal astrocytes. Results: Our results indicate that PAs are senescent as evidenced by marked senescence-associated acidic β-galactosidase activity, low KI-67 index, and induction of p16INK4a but not p53 in the majority of 52 PA samples (46 of 52; 88.5%). Overexpression of a number of senescence-associated genes [CDKN2A (p16), CDKN1A (p21), CEBPB, GADD45A, and IGFBP7] was shown at the mRNA level in two independent PA tumor series. In vitro, sustained activation of wild-type or mutant BRAF induced OIS in both astrocytic cell lines. Loss of p16INK4a in immortalized astrocytes abrogated OIS, indicative of the role of this pathway in mediating this phenomenon in astrocytes. OIS is a mechanism of tumor suppression that restricts the progression of benign tumors. We show that it is triggered in PAs through p16INK4a pathway induction following aberrant MAPK activation. Conclusions: OIS may account for the slow growth pattern in PA, the lack of progression to higher-grade astrocytomas, and the high overall survival of affected patients. Clin Cancer Res; 17(14); 4650–60. ©2011 AACR.

Available data on genetic events in pediatric grade IV astrocytomas (glioblastoma [pGBM]) are scarce. This has traditionally been a major impediment in understanding the pathogenesis of this tumor and in developing ways for more effective management. Our aim is to chart DNA copy number aberrations (CNAs) and get insight into genetic pathways involved in pGBM. Using the Illumina Infinium Human-1 bead-chip-array (100K single-nucleotide polymorphisms [SNPs]), we genotyped 18 pediatric and 6 adult GBMs. Results were compared to BAC-array profiles harvested on 16 of the same pGBM, to an independent data set of 9 pediatric high-grade astrocytomas (HGAs) analyzed on Affymetrix 250K-SNP arrays, and to existing data sets on HGAs. CNAs were additionally validated by real-time qPCR in a set of genes in pGBM. Our results identify with nonrandom clustering of CNAs in several novel, previously not reported, genomic regions, suggesting that alterations in tumor suppressors and genes involved in the regulation of RNA processing and the cell cycle are major events in the pathogenesis of pGBM. Most regions were distinct from CNAs in aGBMs and show an unexpectedly low frequency of genetic amplification and homozygous deletions and a high frequency of loss of heterozygosity for a high-grade I rapidly dividing tumor. This first, complete, high-resolution profiling of the tumor cell genome fills an important gap in studies on pGBM. It ultimately guides the mapping of oncogenic networks unique to pGBM, identification of the related therapeutic predictors and targets, and development of more effective therapies. It further shows that, despite commonalities in a few CNAs, pGBM and aGBMs are two different diseases.