Insulator dysfunction and oncogene activation in IDH mutant gliomas

In the paper

“Genomic alterations that remove CTCF-associated boundaries allow aberrant enhancer-gene interactions and alter gene expression17.”

What the sources say

• Supported

  The Discussion section “Boundary structures are important for TAD integrity” identifies CTCF-binding clusters at the tested boundaries and reports that preserving those boundaries prevented ectopic interactions and gene misexpression. “A framework for interpreting human structural variation” states that deletions removing a boundary permit enhancer contact with genes outside the TAD, causing misexpression. (LLM, conf=0.93)

  cites [17] Lupiáñez, D. G. et al. Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell 161, 1012–1025 (2015) source

In the paper

“TET enzymes catalyze a key step in the removal of DNA methylation8,9.”

What the sources say

• Supported

  The passage headed “TET-mediated DNA demethylation: mechanisms” describes TET oxidation of 5-methylcytosine as a step in pathways that ultimately replace it with cytosine. The “Active DNA demethylation through DNA repair” passage likewise explains how TET oxidation products are removed and replaced with cytosine. Page numbers were not supplied. (LLM, conf=0.93)

  cites [8] Pastor, W. A., Aravind, L. & Rao, A. TETonic shift: biological roles of TET proteins in DNA demethylation and transcription. Nature Rev. Mol. Cell Biol. 14, 341–356 (2013) source
• Supported

  The Abstract states that TET enzymes oxidize 5-methylcytosine, advancing the understanding of DNA demethylation. The passage headed “BOX 1. Roles of 5hmC in DNA demethylation” describes TET-mediated oxidation as a step in pathways that return methylated cytosine to unmodified cytosine. (LLM, conf=0.94)

  cites [9] Kohli, R. M. & Zhang, Y. TET enzymes, TDG and the dynamics of DNA demethylation. Nature 502, 472–479 (2013) source

In the paper

“Recent studies have solidified the role of the CTCF insulator protein in creating chromatin loops and boundaries that partition such domains15.”

What the sources say

• Supported

  The Results section reports that most loop anchors bind CTCF and that many loops demarcate domains. The Discussion says loops often demarcate domains and discusses the mechanism by which CTCF and cohesin form loops, supporting the citing sentence’s broad paraphrase. (LLM, conf=0.87)

  cites [15] Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014) source

In the paper

“Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas1,2.”

What the sources say

• Partly supported

  The ‘High-frequency alterations of IDH1 in GBM’ section links IDH1 mutations to secondary GBM and substantially longer survival, supporting their clinical and prognostic relevance. The Discussion calls an activating effect a speculation and says further analysis is needed to determine the effect on enzyme activity. The paper does not establish that these mutations increase enzyme activity or are initiating events, and its findings concern GBM rather than gliomas broadly. (LLM, conf=0.90)

  cites [1] Parsons, D. W. et al. An integrated genomic analysis of human glioblastoma multiforme. Science 321, 1807–1812 (2008) sourceExperimental — accuracy not yet measured
• Partly supported

  The Results (Mutational Landscape of Lower-Grade Gliomas; page not provided) suggest that IDH mutation is an initial event in molecular progression. The Results (Clinical Characteristics and Outcomes; page not provided) and Discussion show that IDH status, together with 1p/19q status, distinguishes groups with different clinical outcomes. Our copy does not characterize the IDH mutations as gain-of-function or establish increased enzyme activity, and it presents the proposed sequence as a suggestion rather than a proven initiating event. (LLM, conf=0.86)

  cites [2] The Cancer Genome Atlas Research Network. Comprehensive, integrative genomic analysis of diffuse lower-grade gliomas. N. Engl. J. Med. 372, 2481–2498 (2015) sourceExperimental — accuracy not yet measured

In the paper

“To control for copy number changes, we first called copy number profiles from input sequencing data using CNVnator37. We then removed all regions where at least one sample had a strong deletion (<0.25), and normalized by copy number.”

What the sources say

• Supported

  The Discussion describes CNVnator as a method for discovering copy-number variation from sequencing read depth. The citation is attached to the use of CNVnator to call copy-number profiles; the deletion threshold and subsequent normalization are the citing authors’ own steps, not claims attributed to this source. (LLM, conf=0.93)

  cites [37] Abyzov, A., Urban, A. E., Snyder, M. & Gerstein, M. CNVnator: an approach to discover, genotype, and characterize typical and atypical CNVs from family and population genome sequencing. Genome Res. 21, 974–984 (2011) source

In the paper

“FIP1L1 encodes an RNA processing protein that is constitutively expressed in neural tissues, and particularly active in oligodendrocyte precursors, a putative glioma cell-of-origin22 (Extended Data Fig. 2a).”

What the sources say

• Supported

  Citation 22 is attached to the description of oligodendrocyte precursors as a putative glioma cell of origin. In the Introduction, under “The cells of origin of glioblastoma” (page unavailable), the review says OPCs can be considered cells of origin for Nf1- and Trp53-mutant glioblastoma. (LLM, conf=0.91)

  cites [22] Sturm, D. et al. Paediatric and adult glioblastoma: multiform (epi)genomic culprits emerge. Nature Rev. Cancer 14, 92–107 (2014) source

In the paper

“Although PDGFRA is a frequent target of genomic amplification and gain-of-function mutations in glioblastoma (15%), such alterations are rare in IDH mutant tumors23,24.”

What the sources say

• Partly supported

  The Results section on molecular subclasses associates PDGFRA amplification with the non-G-CIMP+ proneural subgroup and describes concurrent amplification in a group predominantly lacking IDH1 mutations. The Results section on somatic alterations also finds IDH1 mutations more prevalent in GBM without RTK alterations. These findings support the direction of the amplification claim in GBM, but the cited passages do not establish that PDGFRA gain-of-function mutations are rare in IDH-mutant tumors. (LLM, conf=0.84)

  cites [23] Brennan, C. W. et al. The somatic genomic landscape of glioblastoma. Cell 155, 462–477 (2013) sourceExperimental — accuracy not yet measured
• Partly supported

  The Results (Functional Annotation of Subtypes; page not available) say that most IDH1-mutant samples did not have a PDGFRA abnormality, supporting limited overlap in the glioblastomas studied. They do not establish that these alterations are rare across IDH-mutant tumors generally, or identify the observed PDGFRA mutations as gain-of-function. (LLM, conf=0.84)

  cites [24] Verhaak, R. G. et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1 . Cancer Cell 17, 98–110 (2010) sourceExperimental — accuracy not yet measured

In the paper

“Genes in top scoring domains include PDGFRA (p<10−21), an established glioma oncogene22, and other candidate regulators of gliomagenesis (Table S1).”

What the sources say

• Supported

  The Structural variations in glioblastoma section reports recurrent PDGFRA amplification in adult and paediatric glioblastoma and describes it as a suggested key oncogenic event in diffuse intrinsic pontine glioma. This supports the citing sentence’s characterization of PDGFRA as an established glioma oncogene. (LLM, conf=0.91)

  cites [22] Sturm, D. et al. Paediatric and adult glioblastoma: multiform (epi)genomic culprits emerge. Nature Rev. Cancer 14, 92–107 (2014) source

In the paper

“Insulator dysfunction may also be accompanied by promoter silencing events28,29, and by alterations to other pathways affected by 2-HG7,30.”

What the sources say

• Supported

  The citation to this paper is attached to “promoter silencing events,” not to the separate clause about other pathways affected by 2-HG. The “Promoter CpG island DNA hypermethylation” section reports that abnormal promoter methylation in cancer stabilizes transcriptional repression and causes loss of gene function. (LLM, conf=0.88)

  cites [28] Baylin, S. B. & Jones, P. A. A decade of exploring the cancer epigenome – biological and translational implications. Nature Rev. Cancer 11, 726–734 (2011) source
• ?Not checked — no full text of the sourcecites [29] Costello, J. F., Berger, M. S., Huang, H. S. & Cavenee, W. K. Silencing of p16/CDKN2 expression in human gliomas by methylation and chromatin condensation. Cancer Res. 56, 2405–2410 (1996)
• ?Not checked — no full text of the sourcecites [7] Cairns, R. A. & Mak, T. W. Oncogenic isocitrate dehydrogenase mutations: mechanisms, models, and clinical opportunities. Cancer Dis. 3, 730–741 (2013) source
• Supported

  This source is cited for the second clause, concerning other pathways affected by 2-HG, not for promoter silencing. In the supplied passages labeled Abstract, the paper identifies additional 2-HG targets, including TET2 and histone demethylases, and proposes that alterations in other 2-HG-affected enzymes may contribute to epigenetic changes. No page number is supplied. (LLM, conf=0.90)

  cites [30] Koivunen, P. et al. Transformation by the (R)-enantiomer of 2-hydroxyglutarate linked to EGLN activation. Nature 483, 484–488 (2012) source

In the paper

“L1CAM (p<10−8), a regulator of the glioma stem cells and tumor growth39”

What the sources say

• Supported

  The Abstract reports that L1CAM knockdown inhibited growth in CD133+ glioma stem cells and suppressed tumor growth in glioma xenografts. The Results and Discussion section also describes L1CAM's role in maintaining CD133+ glioblastoma cell growth. The p-value appears to belong to the citing paper's analysis, not the claim attributed to this source. (LLM, conf=0.94)

  cites [39] Bao, S. et al. Targeting cancer stem cells through L1CAM suppresses glioma growth. Cancer Res. 68, 6043–6048 (2008) source

In the paper

“Conversely, disruption of chromosomal topology and oncogene insulation may be more generally relevant to methylator phenotypes observed in colorectal and renal cell carcinomas, leukemia and other malignancies28.”

What the sources say

• Partly supported

  In “Promoter CpG island DNA hypermethylation” (page unavailable), the paper describes methylation of an H19 insulator, loss of CTCF binding, and IGF2 activation in pre-neoplastic kidneys, with a link to colon cancer progression. In “Genetics meets epigenetics” (page unavailable), it discusses methylator phenotypes in gliomas and increased DNA hypermethylation in leukaemia. It does not link disrupted chromosomal topology or oncogene insulation to methylator phenotypes across the named cancers, and its kidney example concerns Wilms’ tumour rather than renal cell carcinoma. (LLM, conf=0.84)

  cites [28] Baylin, S. B. & Jones, P. A. A decade of exploring the cancer epigenome – biological and translational implications. Nature Rev. Cancer 11, 726–734 (2011) sourceExperimental — accuracy not yet measured

In the paper

“Although PDGFRA is a frequent target of genomic amplification and gain-of-function mutations in glioblastoma (15%), such alterations are rare in IDH mutant tumors23,24.”

What the sources say

• Supported

  The Results section (‘The landscape of somatic alterations in glioblastoma’) reports PDGFRA alterations in 13.1% of glioblastomas and specifies focal amplification and/or mutation. That supports the paraphrase’s approximate 15% figure. (LLM, conf=0.92)

  cites [23] Brennan, C. W. et al. The somatic genomic landscape of glioblastoma. Cell 155, 462–477 (2013) source
• Partly supported

  The Results (Functional Annotation of Subtypes; page unavailable) report PDGFRA amplification across glioblastoma subtypes and PDGFRA mutations in some Proneural tumors. They do not give an approximately 15% frequency or establish that the mutations are gain-of-function; the authors say the point mutations may disrupt ligand interaction. (LLM, conf=0.86)

  cites [24] Verhaak, R. G. et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1 . Cancer Cell 17, 98–110 (2010) sourceExperimental — accuracy not yet measured

In the paper

“BT142 G-CIMP status was confirmed by evaluating LINE methylation with the “Global DNA Methylation Assay – LINE-1″ kit (Active Motif), as described36, and by methylation-sensitive restriction digests.”

What the sources say

• Partly supported

  The “Levels of 5m-dC, 5hm-dC and LINE1 Methylation” section describes evaluating LINE-1 methylation, but by pyrosequencing, not with the named Active Motif kit. The cited paper does not report confirming BT142 G-CIMP status or using methylation-sensitive restriction digests for that purpose. No page number is available in our copy. (LLM, conf=0.82)

  cites [36] Lai, R. K. et al. Genome-wide methylation analyses in glioblastoma multiforme. PLoS ONE 9, e89376 (2014) sourceExperimental — accuracy not yet measured

In the paper

“In support of an enhancer identity, the element is enriched for H3 lysine 4 mono-methylation (H3K4me1), but lacks H3K4me3, and contains conserved motifs bound by the glioma master transcription factors, OLIG2 and SOX2.”

In the paper

“This analysis highlighted 203 domain boundaries (Fig. 2a; Table S1; see Methods).”

In the paper

“This is associated with reduced CTCF binding and a global deregulation of boundary elements that partition topological domains.”

In the paper

“Consistently, 3C reveals a strong interaction between FIP1L1 enhancer and PDGFRA promoter that is specific to the mutant line (Fig. 3i), and PDGFRA is highly expressed.”

In the paper

“The human genome is organized into topological domains that represent discrete structural and regulatory units12.”

What the sources say

• ?Not checked — no full text of the sourcecites [12] Bickmore, W. A. & van Steensel, B. Genome architecture: domain organization of interphase chromosomes. Cell 152, 1270–1284 (2013) source

In the paper

“Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation.”

In the paper

“These results directly implicate DNA hyper-methylation in compromising CTCF binding, boundary function and oncogene insulation in IDH mutant tumors.”

In the paper

“Cultures were confirmed to be mycoplasma-free via PCR methods.”

In the paper

“CHD4 (p<10−32), a driver of glioblastoma tumor initiation38”

What the sources say

• ?Not checked — no full text of the sourcecites [38] Chudnovsky, Y. et al. ZFHX4 interacts with the NuRD core member CHD4 and regulates the glioblastoma tumor-initiating cell state. Cell Rep. 6, 313–324 (2014) source

In the paper

“Notably, CTCF knock-down has been shown to increase cross-boundary interactions and decrease intra-domain interactions21.”

What the sources say

• ?Not checked — no full text of the sourcecites [21] Zuin, J. et al. Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells. Proc. Natl Acad. Sci. USA 111, 996–1001 (2014) source

In the paper

“To ensure robustness to noise from lowly expressed genes, we first filtered out 6,476 genes expressed < 1 TPM in more than half of the samples (keeping 14,055 genes).”

In the paper

“Data was normalized across days using an ATP standard curve.”

In the paper

“We conservatively estimate that CTCF motif disruption causes a ~3-fold increase in PDGFRA expression, given that DNA level analysis indicates that <50% of insulator CRISPR cells were successfully edited.”

In the paper

“Sonication of tumor specimens and gliomaspheres was calibrated such that DNA was sheared to between 400bp and 2000bp.”

In the paper

“Single live cells were selected for analysis via side and forward scatter, and viable cells were selected by lack of an unstained channel (APC) autofluorescence.”

In the paper

“In conclusion, we present a novel epigenetic mechanism by which gain-of-function IDH mutations induce PDGFRA expression and thereby promote fitness in a subset of gliomas.”

In the paper

“To account for batch effects and difference in ChIP efficiency, we quantile normalized each dataset.”

In the paper

“To ensure robustness to cell type-specific boundaries, we repeated the analysis with GM12878, K562 and NHEK defined boundaries.”

In the paper

“In order to avoid possible saturation, reads were downsampled to 5% reads collapsed as PCR duplicates, or 5 million fragments.”

In the paper

“To ensure robustness, we also repeated the analysis using boundaries defined from HiC data for K562 and NHEK.”

In the paper

“PCR signal from tumor and gliomasphere 3C was normalized to digestion efficiency and BAC primer signal.”

In the paper

“We confirmed IDH1 mutant status of GSC119 by RNA-seq (82 out of 148 reads overlapping the relevant position in the transcript correspond the mutant allele).”

In the paper

“Normalization was also verified by β-actin, F: 5′-AGA AAA TCT GGC ACC ACA CC -3′, R: 5′-AGA GGC GTA CAG GGA TAG CA -3′.”

In the paper

“For sequence analysis, identical reads were collapsed to a single paired-end read in order to avoid PCR duplicates.”

In the paper

“All samples were acquired with Institutional Review Board approval, and were deidentified prior to receipt.”

In the paper

“For correlation analysis, patients with copy number variation in either gene were excluded from the analysis to control for effects of co-amplification.”

In the paper

“The observation that genetic disruption of this CTCF boundary element induces PDGFRA expression and enhances proliferation provides strong support for our model that epigenetic disruption of this element offers similar growth advantage to IDH mutant gliomas.”

In the paper

“Although disruption of this single boundary confers a growth advantage, it is unlikely to be the only mediator of IDH mutations in gliomas.”