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Cited by 265Glioma Diagnosis and TreatmentChromatin Remodeling and CancerAcute Myeloid Leukemia Research

H3.3K27M Cooperates with Trp53 Loss and PDGFRA Gain in Mouse Embryonic Neural Progenitor Cells to Induce Invasive High-Grade Gliomas

Open access
Pathania, Manav, De Jay, Nicolas, Maestro, Nicola, Harutyunyan, Ashot S., Nitarska, Justyna, Pahlavan, Pirasteh, Henderson, Stephen, Mikael, Leonie G. +14 more·2017·Cancer Cell
DOI 10.1016/j.ccell.2017.09.014PMC PMC5687892
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Abstract

Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3 K27M and Trp53 loss alone are sufficient for neoplastic transformation if introduced in utero. H3.3 K27M-driven lesions are clonal, H3K27me3 depleted, Olig2 positive, highly proliferative, and diffusely spreading, thus recapitulating hallmark molecular and histopathological features of pHGG. Addition of wild-type PDGFRA decreases latency and increases tumor invasion, while ATRX knockdown is associated with more circumscribed tumors. H3.3 K27M-tumor cells serially engraft in recipient mice, and preliminary drug screening reveals mutation-specific vulnerabilities. Overall, we provide a faithful H3.3 K27M-pHGG model which enables insights into oncohistone pathogenesis and investigation of future therapies.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper reports a somatic mouse model in which in utero electroporation of H3.3K27M plus Trp53 loss is sufficient to induce invasive high-grade gliomas with 100% penetrance, and adding wild-type PDGFRA shortens latency and increases invasion. The resulting tumors recapitulate molecular and histopathological features of human H3.3K27M pediatric high-grade glioma and can be serially engrafted and used for drug screening.

Key findings

  • H3.3K27M plus Trp53 loss alone induced neoplastic transformation with 100% penetrance in both hindbrain and cortex, at 6 and 8 months respectively; no tumors formed with empty vector GFP, H3.3WT, or H3.3G34R combined with Trp53 loss.
  • Addition of wild-type PDGFRA decreased latency and increased invasion: K27M-APP mice showed ectopic proliferation at P21 versus 4 months for K27M-AP, 100% tumor penetrance at 4 and 8–10 months, and 15% Ki67+GFP+ cells in lesions; EV-APP and WT-APP had low-penetrance tumors (10–25%) at 4 months that did not increase by 8–10 months.
  • ATRX knockdown associated with more circumscribed tumors: 0% K27M-P versus 65% K27M-AP generated focal or localized cortical tumors (p<0.0001).
  • Tumors were clonal and H3K27me3-depleted; WES of 10 spatially distributed K27M-AP biopsies showed ≥98% concordance between any pair, with 7 non-synonymous exonic SNV clusters and 6 of 7 showing consistent cellular prevalence. A 430-compound kinase inhibitor screen found K27M-AP vulnerability to Vacquinol-1 (z-score 0.98) and K27M-APP vulnerability to Akti-1/2 (z-score 0.96).

Methods

  • Somatic mouse model using in utero electroporation of piggyBac transposon vectors at E12.5 (hindbrain) or E13.5 (cortex) into neural progenitor cells of C57BL/6J mice, delivering combinations of H3.3K27M, CRISPR/Cas9 Trp53 loss, ATRX shRNA, wild-type PDGFRA, or PDGFRAD842V; NOD SCID mice were used for allotransplantation.
  • Analyses included histology and immunofluorescence, RNA-seq, whole-exome sequencing, H3K27me3 ChIP-seq, cross-species comparison with human pHGG data, and a 430 small-molecule kinase inhibitor screen.
  • Statistics used mean ± SEM, unpaired two-tailed t tests, ordinary one-way ANOVA with Tukey post hoc, or Chi-squared tests, with p<0.05 considered significant.

Limitations

  • The paper states that the precise cell within the NPC lineage that can initiate tumorigenesis remains to be discovered, and that mechanisms behind de novo H3K27me3 deposition in H3.3K27M cells are unknown.
  • The paper notes the model still takes 4 months in mice to form high-grade lesions, and describes the drug screening as preliminary/proof-of-principle.
  • Evident from the text: the drug-screen hits were not validated in vivo, and the model relies on engineered embryonic mutations, so its ability to capture the full human tumor microenvironment or all human pHGG heterogeneity is not established here.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.ccell.2017.09.014

Cites

72 of 72 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 42 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    H3.3K27M-driven lesions are Olig2-positive.

    “H3.3K27M-driven lesions are clonal, H3K27me3-depleted, Olig2-positive, highly proliferative and diffusely spreading”
    • Also stated in: § H3.3K27M-mediated tumorigenesis in mice is clonal, recapitulates features of invasive human pHGG · plain
      “K27M-AP and K27M-APP tumors were also significantly enriched in cells expressing Olig2, similar to human DIPG”
    Quote in the paperSubstantial claim
  • Findingown data

    H3.3K27M-driven lesions are depleted of H3K27me3.

    “H3.3K27M-driven lesions are clonal, H3K27me3-depleted, Olig2-positive, highly proliferative and diffusely spreading”
    • Also stated in: § Tumorigenesis induced by H3.3K27M in combination with Trp53 loss and ATRX knockdown · plain
      “These lesions were positive for Ki67, Olig2, Nestin and HA; and were depleted of K27me3 signal”
    Quote in the paperSubstantial claim
  • Findingown data

    H3.3K27M-driven lesions spread diffusely.

    “H3.3K27M-driven lesions are clonal, H3K27me3-depleted, Olig2-positive, highly proliferative and diffusely spreading”
    Quote in the paperSubstantial claim
  • Findingown data

    H3.3K27M-driven lesions are clonal.

    “H3.3K27M-driven lesions are clonal”
    • Also stated in: § H3.3K27M-mediated tumorigenesis in mice is clonal, recapitulates features of invasive human pHGG · hedged
      “Altogether, these results indicate that H3.3K27M-driven tumors are clonal and derive from the originating mutations introduced during IUE.”
    Quote in the paperSubstantial claim
  • Findingown data

    H3.3K27M-driven lesions are highly proliferative.

    “H3.3K27M-driven lesions are clonal, H3K27me3-depleted, Olig2-positive, highly proliferative and diffusely spreading”
    Quote in the paperSubstantial claim
  • May combine two claims. 38
  • Classified as probably not a substantial claim. 20

  • May combine two claims.

    Findingown data

    By 9 months, K27M-AP cortical and striatal lesions progressed to fully developed high-grade glioma, with neurological signs requiring euthanasia.

    “By 9 months, the smaller proliferative lesions in K27M-AP cortex and striatum had progressed to fully developed HGG, with mice displaying neurological signs requiring euthanasia.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    K27M-APP had significantly decreased latency compared to K27M-AP, with ectopic proliferation at P21 versus 4 months.

    “showing a significantly decreased latency compared to the K27M-AP model where ectopic proliferation was only seen at 4 months”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The remaining minor cluster had cellular prevalence varying between 25% and 40%, with no variant overrepresented in any sample.

    “The cellular prevalence of the remaining minor cluster varied between 25% and 40%, with no variant belonging to this cluster overrepresented in any of the samples”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cluster M1 is strongly conserved, with 78% of genes falling into human cluster H1.

    “Cluster M1, which has the highest level of K27me3, is strongly conserved, with 78% of genes falling into human cluster H1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    20% of GFP+ cells in ectopic non-neurogenic locations were Ki67+ and Nestin+, demonstrating stem cell-like characteristics and neoplastic transformation.

    “20% of GFP+ cells were Ki67+ and Nestin+ in ectopic, non-neurogenic locations, thereby demonstrating stem cell-like characteristics (Figure 2D and 2E) and neoplastic transformation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There was a progression from the untransformed to the transformed state, with large transcriptional changes evident already at the preneoplastic stage.

    “We observed a progression from the untransformed to the transformed state, with large transcriptional changes evident already at the preneoplastic stage.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    K27M-AP tumors at 9 months were positive for Ki67, Olig2, Nestin and HA and were depleted of K27me3 signal.

    “These lesions were positive for Ki67, Olig2, Nestin and HA; and were depleted of K27me3 signal”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In K27M-APP, increased Olig2 expression was associated with extensive migration away from the electroporation site, with more Olig2+GFP+ cells in the contralateral cortex than the ipsilateral cortex.

    “The increase in Olig2 expression was associated with extensive migration away from the electroporation site in K27M-APP cells, as more Olig2+GFP+ cells were seen in the contralateral cortex compared to the ipsilateral one”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cluster M5 has reduced K27me3 and shares 69% overlap with H3-H4.

    “M5, on the other hand, is depleted in K27me3 and shows 69% overlap with H3-H4.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    EV-APP and WT-APP brains showed low-penetrance tumors (10-25%) at 4 months, and penetrance did not increase up to 8-10 months.

    “At 4 months, EV-APP and WT-APP brains also showed low-penetrance tumors (10–25%; Table S1), but penetrance did not increase up to 8–10 months.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    WT-AP mice displayed premature differentiation, with fewer GFP+ cortical cells and a larger neuron-to-astrocyte ratio at 4 months compared to EV-AP.

    “We also noted that WT-AP mice displayed a premature differentiation effect, with fewer GFP+ cells in the cortex and a larger neuron-to-astrocyte ratio at 4 months compared to EV-AP”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    PDGFRA overexpression increased the thickness of the electroporated neocortex and the size of the ipsilateral hemisphere relative to the contralateral hemisphere.

    “It also increased the thickness of the electroporated neocortex as well as the size of the ipsilateral hemisphere in relation to the contralateral hemisphere”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based).

    Findingnone given

    H3.3K27M and Trp53 loss induce diffuse tumorigenesis in both hindbrain and forebrain.

    “H3.3K27M and Trp53 loss induce diffuse tumorigenesis in both hindbrain and forebrain”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    15% of cells in K27M-APP lesions were Ki67+GFP+ and had increased PDGFRA levels.

    “15% of cells in these lesions were Ki67+GFP+ (Figure 3D and 3E), and as expected, had increased levels of PDGFRA”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    By 9 months, K27M-APP tumors progressed with very high numbers of Ki67+Nestin+ cells and multiple necrotic foci.

    “By 9 months, K27M-APP tumors were significantly progressed, with very high numbers of Ki67+Nestin+ cells and multiple necrotic foci”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    K27M-AP tumors showed increased proliferation and contralateral infiltration.

    “and showed increased proliferation and contralateral infiltration”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ex vivo H3K27me3 levels were reduced in preneoplastic neural progenitor cells and further reduced in cells isolated from K27M-P, K27M-AP and K27M-APP tumors.

    “Ex vivo, H3K27me3 levels were reduced in preneoplastic NPCs and further reduced in cells isolated from K27M-P, K27M-AP and K27M-APP tumors”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Clonality analysis revealed 7 distinct clusters of non-synonymous exonic single nucleotide variants, and 6 of 7 clusters exhibited consistent cellular prevalence across all biopsies.

    “Clonality analysis revealed 7 distinct clusters of non-synonymous, exonic SNVs, and 6 of 7 clusters exhibit a consistent cellular prevalence across all biopsies”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cluster M1 contains top homeobox genes deregulated in the dataset (ALX4, IRX2, SIX4, POU4F1, ISL2) and some Wnt pathway genes (WNT6 and WNT10A).

    “This cluster contains the top homeobox genes deregulated in our dataset (i.e. ALX4, IRX2, SIX4, POU4F1, ISL2), as well as some of the Wnt pathway genes (i.e. WNT6 and WNT10A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    K27M-APP lesions were intermittently GFAP positive but universally depleted of MBP and K27me3 signal.

    “These lesions were intermittently positive for GFAP, but were universally depleted of MBP and K27me3 signal”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The study describes a faithful preclinical model of H3.3K27M-driven pHGG and provides a tool for studying molecular mechanisms underlying the transforming function of H3.3K27M.

    “In conclusion, we describe a faithful preclinical model of H3.3K27M-driven pHGG and provide a tool for studying molecular mechanisms underlying the transforming function of H3.3K27M.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Transformation begins during development and progresses slowly, taking 4 months in mice to advance to high-grade lesions.

    “While the process of transformation begins during development, it progresses slowly, taking 4 months in mice to advance to high-grade lesions.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    The generated tumors are clonal and derive from stem cells expressing mutant H3.3 that were targeted during development using IUE.

    “The tumors generated are clonal and derive from those stem cells expressing mutant H3.3 that were targeted during development using IUE.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    Addition of WT PDGFRA overexpression significantly decreases tumor latency and drastically promotes invasion when co-expressed with H3.3K27M.

    “On the other hand, the addition of WT PDGFRA overexpression significantly decreases tumor latency while drastically promoting invasion when co-expressed with H3.3K27M.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    Clonal inference with PyClone produced a final set of 336 variants grouped into 10 clusters; after removing clusters containing a single SNV, 7 clusters remained.

    “Using PyClone (v0.13.0) (DOI:10.1038/nmeth.2883) to infer the clonal structure of the tumor, we obtained a final set of 336 variants that were clustered into 10 clusters. The number of clusters was reduced to 7 after discarding the clusters consisting of a single SNV (Figure S5).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites DOI:10.1038/nmeth.2883

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The study generated a preclinical model of H3.3K27M oncohistone-driven neoplastic transformation that can be engrafted and recapitulates histologic features of human pHGG.

    “In this study, we generate a preclinical model of H3.3K27M oncohistone-driven neoplastic transformation, from which cells can be engrafted in recipient animals and that recapitulates histologic features of human pHGG.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: There are currently no in vivo models simulating H3.3K27M-driven brain tumorigenesis.

  • May combine two claims.

    Findingown data

    The mouse tumors show characteristic invasive nature, migration along blood vessels, colonization of the contralateral hemisphere, upregulation of Olig2, and scattered GFAP expression.

    “This includes 1) the characteristic invasive nature of these tumors, 2) migration along blood vessels, 3) colonization of the contralateral hemisphere, 4) upregulation of Olig2, an established diagnostic marker for H3.3K27M pHGG (Sturm et al., 2012), which positively correlates with the extent of invasion of transformed cells in vivo, and 5) scattered GFAP expression is in tumor samples.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Sturm et al., 2012 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    G34R/V mutations in H3.3 occur in 36% of pediatric high-grade gliomas in the cerebral hemispheres.

    “while mutations of glycine 34 to arginine or valine (G34R/V) in H3.3 occur in 36% of pHGG in the cerebral hemispheres”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Fontebasso et al., 2013 source

      Not checked in the free run.

    • cites Fontebasso et al., 2014 source

      Not checked in the free run.

    • cites Khuong-Quang et al., 2012 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Other in vivo H3.3K27M-DIPG models required constitutive PDGF-B ligand, which is not present in pediatric high-grade glioma, and could induce tumors even with wild-type H3.3.

    “Other attempts to model H3.3K27M-DIPG in vivo have required constitutive expression of the PDGF-B ligand, which is not present in pHGG and can induce tumors even with wild-type (WT) H3.3”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Cordero et al., 2017 source

      Not checked in the free run.

    • cites Hennika et al., 2017 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Human embryonic stem cell-derived neural progenitor cells transduced with H3.3K27M, p53 shRNA and mutant PDGFRAD842V transformed only after months of in vitro passaging and produced low-grade glioma, not high-grade glioma, when transplanted into immunocompromised mice.

    “A recent study reported that transduction of human embryonic stem cell-derived NPCs with H3.3K27M, p53 shRNA and constitutively active, mutant PDGFRAD842V led to neoplastic transformation, but only via in vitro passaging over several months (Funato et al., 2014). Furthermore, when transplanted into recipient immunocompromised mice, these genetically modified NPCs gave rise exclusively to low-grade”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Funato et al., 2014 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Alterations affecting the p53 pathway occur in about 87% of H3.3K27M tumors.

    “These include alterations affecting the p53 pathway in ~87% of H3.3K27M tumors”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Fontebasso and Jabado, 2015

      Not checked in the free run.

    • cites Fontebasso et al., 2014 source

      Not checked in the free run.

    • cites Nikbakht et al., 2016 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    H3.3K27M has gain-of-function activity that inhibits PRC2, leading to a global reduction in H3K27me3 levels in primary tumors and tumor-derived primary cells.

    “H3.3K27M has been shown to have gain-of-function activity inhibiting the Polycomb Repressive Complex 2 (PRC2) (Bender et al., 2013; Lewis et al., 2013), which in turn leads to a global reduction in H3K27me3 levels in primary tumors and tumor-derived primary cells.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Bender et al., 2013 source

      Not checked in the free run.

    • cites Lewis et al., 2013 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Oncohistones are found almost exclusively in pediatric high-grade gliomas and are mutually exclusive with IDH mutations found in young adult gliomas.

    “These “oncohistones” are found almost exclusively in pHGG and are mutually exclusive with the recurrent mutations in isocitrate dehydrogenase enzymes (IDH) identified in young adult gliomas”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Yan et al., 2009 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Loss-of-function mutations in ATRX occur with increasing frequency in older patients.

    “loss-of-function mutations in ATRX, which occur with increasing frequency in older patients”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Fontebasso et al., 2014 source

      Not checked in the free run.

    • cites Khuong-Quang et al., 2012 source

      Not checked in the free run.

    • cites Nikbakht et al., 2016 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Addition of wild-type PDGFRA shortens the latency of tumorigenesis and acts synergistically with H3.3K27M to transform neural progenitor cells in vivo.

    “Thus, addition of WT PDGFRA shortens the latency of tumorigenesis and acts synergistically with H3.3K27M to transform NPCs in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    H3.3K27M-driven tumors are clonal and derive from the originating mutations introduced during in utero electroporation.

    “Altogether, these results indicate that H3.3K27M-driven tumors are clonal and derive from the originating mutations introduced during IUE.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In H3.3K27M DIPG, ATRX loss is rare, less than 10%, and subclonal.

    “This is in contrast to H3.3K27M DIPG where ATRX loss is rare (less than 10%) and subclonal (Nikbakht et al., 2016), potentially in accordance with our model.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Nikbakht et al., 2016 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation

    The incubation period of this model is in accordance with most other autochthonous models of medulloblastoma or adult glioma and may represent a fundamental aspect of brain cancer modeling in mice.

    “However, this incubation period is in accordance with most, if not all, other autochthonous models of medulloblastoma or adult glioma and may represent a fundamental aspect of brain cancer modeling in mice (Alcantara Llaguno et al., 2009; Hill et al., 2015).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Alcantara Llaguno et al., 2009 source

      Not checked in the free run.

    • cites Hill et al., 2015 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    In WES samples, positions with coverage greater than 1000 were treated as sequencing artefacts and excluded.

    “For WES samples, positions where coverage is greater than 1000 were considered to be sequencing artefacts and thus ignored.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The DiffBind package was modified so that libraries could be normalized using their respective ChIP-Rx scaling factors.

    “based on the peak calls using the R Bioconductor package DiffBind (v2.5.4), which was modified to allow libraries to be normalized according to their respective ChIP-Rx scaling factors.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    The residual H3K27me3 deposition in H3.3K27M cells may result from residual EZH2 activity or continued EZH1-containing complex activity.

    “Active deposition of the H3K27me3 mark at selected sites remains possible in H3.3K27M cells (Figure 8E; Figure S8D), possibly by residual EZH2 activity, as recently suggested (Mohammad et al., 2017), or by continued activity of EZH1-containing complexes (Shen et al., 2008).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Mohammad et al., 2017 source

      Not checked in the free run.

    • cites Shen et al., 2008 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    A control shRNA vector lacking homology to any known mouse and human genes was used.

    “A control (ctl) shRNA vector was constructed using a scrambled shRNA sequence (5’-CCAACTCTTGTTTCAGCCG-3’) lacking homology to any known mouse and human genes as described previously (Singh et al., 2010).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Singh et al., 2010

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Immunoblots were performed at least three times to confirm reproducibility.

    “Signal detection and quantification was performed using an Odyssey infrared detection instrument (LI-COR) and immunoblots were performed at least thrice to confirm reproducibility.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Kinase inhibitor screening plates were prepared in triplicate.

    “Plates were prepared in triplicate.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    GFP+Ki67+Nestin+ and GFP+Olig2+ cells were counted manually in a blinded manner.

    “GFP+Ki67+Nestin+ and GFP+Olig2+ cells in cortex and striatum were manually counted using the multi-point tool in ImageJ in a blinded manner.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Two ATRX shRNA vectors with equivalent knockdown efficiencies were used.

    “Two ATRX shRNA vectors with equivalent knockdown efficiencies were obtained by cloning the following sequences into the short hairpin RNA construct PBmU6pro (kind gift from J. Loturco), cut with BbsI/XbaI: 5’-TTCATTTACATTCTCATCCGTG-3’; 5’-TCATTTACATTCTCATCCG-3’.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Multiple filters were applied to remove likely false positive variant calls from WES or RNA-seq samples, retaining only variants meeting thresholds for coverage, alternative nucleotide count, SNV ratio, indel ratio, variant quality, and mapping quality.

    “Finally, we applied multiple levels of filters to remove likely false positive variant calls on WES or RNA-seq samples, retaining only variants with coverage ≥ 10 reads, alternative nucleotide count ≥ 3, SNV ratio ≥ 5%, indel ratio ≥ 15%, variant quality ≥ and mapping quality ≥ 15.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    From the filtered putative SNVs from 10 WES samples of one K27M-AP tumour, only non-synonymous exonic variants were retained as the most reliable.

    “From the filtered list of putative SNVs obtained for each of the 10 WES samples derived from spatial biopsies of one K27M-AP tumour, we discarded all the but the most reliable SNVs, that is, non-synonymous variants occurring in exonic regions.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All mice were housed, bred and subjected to procedures according to institutional, AWERB and UK Home Office guidelines.

    “All mice were housed, bred and subjected to listed procedures according to institutional, Animal Welfare and Ethical Review Body (AWERB) and UK Home Office guidelines (Project licence 70/8240, 70/7428 and 80/2325).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    In adult HGG, ATRX loss associates with IDH mutations and TP53 loss and is associated with improved prognosis.

    “Furthermore, in adult HGG, ATRX loss associates with IDH mutations and TP53 loss and is also associated with improved prognosis (Pekmezci et al., 2017; Wiestler et al., 2013), potentially due to better resection of focal tumors during surgery.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Pekmezci et al., 2017 source

      Not checked in the free run.

    • cites Wiestler et al., 2013 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Peaks that were significantly increased in tumor samples relative to controls were manually inspected using IGV.

    “Peaks significantly increased (FDR < 0.05) in tumor samples with respect to controls were inspected manually using IGV and reported in Figure S9D.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Cell counting and fluorescent intensity analyses were carried out on images from a minimum of 3 mice per condition.

    “All cell counting and fluorescent intensity analyses were carried out on images from a minimum of 3 mice per condition.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The H3.3K27M mutation arises early in actively dividing neural precursors during CNS development and produces a tumorigenic event by disrupting normal neurogenesis.

    “Therefore, our data support an embryonic origin of H3.3K27M–driven tumors in mice, suggesting that the H3.3K27M mutation arises early in actively dividing neural precursors during CNS development where it produces a tumorigenic event by disrupting normal neurogenesis.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Only one-to-one ortholog associations were used to obtain a conservative measure of agreement between human and mouse H3K27me3 cluster gene assignments.

    “Finally, we considered only one-to-one associations in order to obtain a conservative measure of agreement between cross-species H3K27me3 cluster gene assignment.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Smedley et al., 2015 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    A large proportion of thalamic H3.3K27M tumors and the majority of cortical H3.3G34R/V tumors are more focal at initial presentation and display loss of ATRX.

    “We have reported elsewhere that a large proportion of thalamic H3.3K27M tumors, and the majority of cortical H3.3G34R/V tumors, are more focal at initial presentation and display loss of ATRX (Fontebasso et al., 2014).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Fontebasso et al., 2014 source

      Not checked in the free run.

55 of 55

  • The paper relies on this without support.

    H3.3K27M tumors are predominantly located in the brain midline and pons.

    “Given that H3.3K27M tumors are predominantly found in the brain midline and pons, we used piggyBac IUE to target NPCs in the lower rhombic lip of the developing hindbrain”

  • The paper relies on this without support.

    The lower rhombic lip of the developing hindbrain contributes to the pontine nuclei as development proceeds.

    “we used piggyBac IUE to target NPCs in the lower rhombic lip of the developing hindbrain, which contributes to the pontine nuclei as development proceeds (Figure 1A).”

  • The paper relies on this without support.

    NOD-SCID recipient mice permit engraftment of mouse tumor cells without immune rejection.

    “K27M-P, K27M-AP and K27M-APP tumor cells were injected into the striatum of NOD-SCID mice, and brains were collected 45–70 days after transplantation.”

  • The paper relies on this without support.

    Human orthologs of mouse differentially expressed genes preserve the regulatory and expression relationships needed for cross-species comparison.

    “we derived gene signatures based on the human orthologs of differentially expressed genes in the mouse models (Table S5).”

  • The paper relies on this without support.

    There is a lack of preclinical in vivo models that fully recapitulate human tumor genetics and histopathology, which prevents identification and validation of high-confidence treatment strategies.

    “This is in part due to a lack of preclinical in vivo models that fully recapitulate the genetic and histopathological features of human tumors, thereby preventing the identification and validation of high-confidence treatment strategies.”

  • The paper relies on this without support.

    There are currently no in vivo models simulating H3.3K27M-driven brain tumorigenesis.

    “For pHGG in particular, there are currently no in vivo models simulating H3.3K27M-driven brain tumorigenesis.”
6 of 6

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Aberrant upregulation of PDGFRA and loss of wild type p53 activity corresponded to more aggressive glioblastoma tumors.

    “Aberrant upregulation of PDGFRA and loss of wt-p53 activity corresponded to more aggressive GBM tumors.”

    From The p53 Pathway in Glioblastoma (2018) · cites this paper as [92]

    The Results report that H3.3K27M and Trp53 loss induced diffuse tumors in mice, while adding PDGFRA overexpression shortened tumor latency and increased invasion. The Discussion likewise describes these effects in H3.3K27M-bearing mouse tumors modeled on pediatric high-grade glioma. The claim omits that essential H3.3K27M and mouse-model context and presents the finding broadly as applying to GBM tumors. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

Cites

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Cited by

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Related

Authors
Pathania, M., De Jay, N., Maestro, N., Harutyunyan, A. S., Nitarska, J., Pahlavan, P., Henderson, S., Mikael, L. G., Richard-Londt, A., Zhang, Y., Costa, J. R., Hébert, S., Khazaei, S., Ibrahim, N. S., Herrero, J., Riccio, A., Albrecht, S., Ketteler, R., Brandner, S., Kleinman, C. L., Jabado, N., & Salomoni, P.
Affiliations
  • University College London (GB)
  • McGill University (CA)
  • MRC Laboratory for Molecular Cell Biology (GB)
  • German Center for Neurodegenerative Diseases (DE)
  • UCL Queen Square Institute of Neurology (GB)
  • Jewish General Hospital (CA)
Corresponding author
  • Jabado, Nada
  • Salomoni, Paolo
ORCID
Venue
Cancer Cell
Publisher
Elsevier BV
Published
2017-11
Volume
32
Issue
5
Pages
684-700.e9
ISSN
1535-6108 (ISSN-L)
DOI
10.1016/j.ccell.2017.09.014
PMC
PMC5687892
OpenAlex
W2765199703
Cited by
264
References
72
Citations by year
2026: 23 · 2025: 23 · 2024: 26 · 2023: 33 · 2022: 37 · 2021: 39 · 2020: 30 · 2019: 36 · 2018: 15 · 2017: 2
FWCI
12.96
Type
journal article
Language
en
Open access
Licence
Locations
  • Cancer Cell (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • UCL Discovery (University College London) (submittedVersion)
  • DZNE Pub (submittedVersion)
Funders
  • European Research Council (GA 616744)
  • US NIH (P01CA196539)
  • Canadian Institutes of Health Research (MOP 286756)
  • Fonds de Recherche du Québec - Santé
  • UK Medical Research Council (MC_U12266B)
  • Brain Tumour Charity
  • Biomedical Research Council
Topics
  • Health Sciences › Medicine › Genetics › Glioma Diagnosis and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
  • Health Sciences › Medicine › Hematology › Acute Myeloid Leukemia Research
Keywords
pediatric high-grade glioma, p53 deficiency, PDGFRA amplification, oncohistones, tumor invasion, ATRX, Olig2, H3K27me3, tumor engraftment, drug screening, glioma pathogenesis
MeSH terms
X-linked Nuclear Protein, Animals, Brain, Cell Transformation, Neoplastic, Glioma, Histones, Humans, Mutation, Neoplasm Invasiveness, Gene Expression Regulation, Neoplastic, Tumor Suppressor Protein p53, Receptor, Platelet-Derived Growth Factor alpha, RNA Interference, Mice, Embryonic Stem Cells, Neural Stem Cells, Neoplasm Grading

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-08.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-08.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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