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Cited by 459Glioma Diagnosis and TreatmentChromatin Remodeling and CancerEpigenetics and DNA Methylation

Therapeutic targeting of polycomb and BET bromodomain proteins in diffuse intrinsic pontine gliomas

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Piunti, Andrea, Hashizume, Rintaro, Morgan, Marc A, Bartom, Elizabeth T, Horbinski, Craig M, Marshall, Stacy A, Rendleman, Emily J, Ma, Quanhong +9 more·2017·Nature Medicine
DOI 10.1038/nm.4296PMC PMC5667640
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Abstract

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

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Cites

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Claims the paper makes

0 of 38 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    In SF8628 DIPG cells, the genome-wide distribution of H3.3K27M is highly correlated with active transcription, as shown by co-localization with H3K27ac and RNA polymerase II.

    “ChIP-seq results from SF8628 human DIPG cells, containing a mutant H3F3A gene encoding H3.3K27M8, reveal a genome wide distribution of H3.3K27M that is highly correlated with active transcription, as indicated by the co-localization of acetylated H3K27 (H3K27ac) and RNA polymerase II (RNA pol II) (Fig. 1A–C and Supplementary Fig. 2A–B).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [8] Hashizume, R. et al. Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma. Nat. Med. 20, 1394–1396 (2014). source

      Not checked in the free run.

  • Findingown data

    In H3K27M mutant DIPG cells, H3K27M co-localizes with H3K27 acetylation.

    “we profile the epigenome of H3K27M mutant DIPG cells and demonstrate that H3K27M co-localizes with H3K27 acetylation (H3K27ac).”
    Quote in the paperSubstantial claim
  • Findingown data

    Pharmacologic inhibition of bromodomain proteins suppresses DIPG tumor growth in vivo.

    “Moreover, we demonstrate that pharmacologic bromodomain protein inhibition suppresses tumor growth in vivo.”
    Quote in the paperSubstantial claim
  • Findingown data

    Heterotypic H3K27M-K27ac nucleosomes co-localize with bromodomain proteins at actively transcribed genes.

    “Heterotypic H3K27M-K27ac nucleosomes co-localize with bromodomain proteins at actively transcribed genes, whereas PRC2 is excluded from the chromatin of the H3K27M occupied regions.”
    Quote in the paperSubstantial claim
  • Findingown data

    Residual PRC2 activity is needed to maintain DIPG proliferative potential by repressing neuronal differentiation and function.

    “Residual PRC2 activity is required for maintaining DIPG proliferative potential by repressing neuronal differentiation and function.”
    Quote in the paperSubstantial claim
  • May combine two claims. 32
  • Classified as probably not a substantial claim. 13

  • May combine two claims.

    Findingown data

    The correlation of H3K27M distribution with active transcription is also seen in DIPG IV cells carrying H3.1K27M and is supported by results in SF7761 cells.

    “Intriguingly, this observation is recapitulated in SU-DIPG-IV (hereafter called DIPG IV), a DIPG cell line carrying a point mutation in the HIST1H3B gene that results in the expression of H3.1K27M9, and is further supported by results obtained in SF7761, another H3F3A mutant DIPG line8 (Supplementary Figs. 3–4).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [8] Hashizume, R. et al. Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma. Nat. Med. 20, 1394–1396 (2014). source

      Not checked in the free run.

    • cites [9] Monje, M. et al. Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma. Proc. Natl. Acad. Sci. USA 108, 4453–4458 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    H3K27M localizes to H3K27ac sites present in wildtype H3.3 cells and can markedly increase H3K27ac at regions that have only modest H3K27ac in control cells.

    “H3K27M localizes to H3K27ac sites that are present in wildtype H3.3 expressing cells, but importantly it can induce a marked increase of H3K27ac at regions showing only modest H3K27ac enrichment in control cells (Supplementary Fig. 5B–C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Immunohistochemistry showed significantly lower H3K27ac levels in tumors from JQ1-treated mice than in controls, reflecting direct target inhibition in vivo.

    “This analysis revealed significantly lower H3K27ac levels in tumors coming from JQ1 treated mice compared to control, thus reflecting direct target inhibition in vivo (Supplementary Fig. 17A).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: H3K27ac levels in tumors serve as a valid surrogate for JQ1 target inhibition in vivo.

  • May combine two claims.

    Findingown data

    JQ1 upregulates CDKN1A (p21), TUBB3, and MAP2, which are markers of cell cycle arrest and neuronal differentiation, supporting the observed morphology change.

    “Among the genes transcriptionally upregulated we noticed CDKN1A (p21), marker of cell cycle arrest17, TUBB3 (Tuj1) and MAP2, markers of differentiation in mature neurons18, further supporting the morphology changed observed upon JQ1 treatment (Fig. 3G and Supplementary Fig. 13D–E).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [17] Galderisi, U., Jori, F.P. & Giordano, A. Cell cycle regulation and neural differentiation. Oncogene 22, 5208–5219 (2003). source

      Not checked in the free run.

    • cites [18] Gordon, J., Amini, S. & White, M.K. General overview of neuronal cell culture. Methods Mol. Biol. 1078, 1–8 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    H3K27M occupancy at active enhancers is asymmetric and shows high density over a fraction of active enhancers that also have increased BRD2/4 and active transcription marks.

    “We also found that H3K27M occupancy at active enhancers (H3K4me1 and H3K27ac positive regions) is asymmetric and characterized by the presence of high density occupancy over a fraction of active enhancers that also show increased presence of BRD2/4 and active transcription marks (Supplementary Fig. 11C–D), suggesting that H3K27M participates in the formation of super enhancer-like elements purport”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    GLI2, a crucial downstream effector of the Sonic Hedgehog pathway, is among the main downregulated transcripts and is a target of BRD2/4 and H3K27M/K27ac heterotypic nucleosomes.

    “Interestingly one of the main downregulated transcripts is GLI2, a crucial downstream effector of the Sonic Hedgehog (SHH) pathway21, that is a BRD2/4 and H3K27M/K27ac heterotypic nucleosomes target (Supplementary Fig. 16A–B).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Gupta, S., Takebe, N. & Lorusso, P. Targeting the Hedgehog pathway in cancer. Ther. Adv. Med. Oncol. 2, 237–250 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    In a DIPG xenograft mouse model, 10 days of JQ1 treatment significantly reduced tumor size by bioluminescence imaging and prolonged animal survival.

    “Remarkably, mice treated with JQ1 for 10 days exhibited significantly reduced tumor size, as indicated by bioluminescence imaging, and prolonged animal survival (Fig. 4A–B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    JQ1-treated tumors show induction of TUBB3 compared with controls, providing in vivo support for the in vitro observation that JQ1 growth inhibition is accompanied by upregulation of differentiation markers in DIPG cells.

    “Furthermore, we were also able to demonstrate induction of TUBB3 levels in JQ1 treated tumors compared to control (Fig. 4F) thus providing in vivo support for our in vitro observation that JQ1 tumor growth inhibition is accompanied by an upregulation of differentiation markers in DIPG cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SUZ12 or EED knockdown de-represses p16 expression in SF8628 cells, consistent with CDKN2A being a PRC2 target in these cells.

    “SUZ12 or EED knockdown resulted in de-repression of p16 expression in SF8628 cells, consistent with CDKN2A being a PRC2 target in these cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DIPG IV cells do not upregulate p16 after PRC2 depletion, despite the anti-proliferative effect of SUZ12 and EED knockdown.

    “In contrast, DIPG IV cells do not upregulate p16 expression upon PRC2 depletion (Supplementary Fig. 10A–B and Supplementary Fig. 9D), despite the anti-proliferative effect of SUZ12 and EED knockdown in these cells (Fig. 2A–B, Supplementary Fig. 9A–D and Supplementary Fig. 10A–B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    JQ1 treatment causes a modest but consistent increase in MYC transcripts in both SF8628 and DIPG IV cells, while MYCN is silenced by PRC2 in those cells.

    “Surprisingly, we found a modest but consistent increase of MYC transcripts in both the SF8628 and DIPG IV cell lines upon JQ1 treatment, whereas its closest homolog, MYCN, is silenced by PRC2 in those cells (Supplementary Fig. 14A–B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    In the DIPG cells studied, PRC2 is almost entirely confined to chromatin regions marked by H3K27me3, and H3K27me3 is mutually exclusive with H3K27ac.

    “In our DIPG cells, PRC2 is almost entirely confined in chromatin regions marked with H3K27me3 and this mark is mutually exclusive with H3K27ac23.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [23] Weiner, A. et al. Co-ChIP enables genome-wide mapping of histone mark co-occurrence at single-molecule resolution. Nat. Biotechnol. 34, 953–961 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Treatment with another BET inhibitor, I-BET151, and treatment of H3.1K27M DIPG IV xenografts with JQ1 both significantly impaired DIPG growth in vivo.

    “To further support our in vivo data we used another BET inhibitor (I-BET151) to treat our xenograft DIPG model (Supplementary Fig. 17B) and also tested JQ1 against H3.1K27M DIPG IV xenografts (Supplementary Fig. 17C) with both approaches showing significant impairment of DIPG growth in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    The H3K27M antibody used for ChIP-seq gave signal exclusively in cells expressing H3K27M and no enrichment in cells expressing wildtype histone.

    “ChIP-seq signal with the H3K27M antibody was detected exclusively in H3K27M expressing cells, whereas no enrichment was observed in cells expressing the wildtype counterpart (Supplementary Fig. 1B–D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    DIPG IV cells tolerate higher EPZ-6438 doses than SF8628, likely because the inhibitor is less effective in DIPG IV and higher doses are needed to de-repress CCND2 similarly to SF8628.

    “DIPG IV cells tolerate higher doses of EPZ-6438 than SF8628 (Supplementary Fig. 10C) and this likely reflects lower effectiveness of the chemical inhibitor in DIPG IV, because a common PRC2 target gene, Cyclin D2 (CCND2) (Fig 2C–F), simply requires increased doses to achieve de-repression similar to SF8628 (Supplementary Fig. 10D)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    CDKN2A is unaffected by JQ1, suggesting that the JQ1 phenotype is p16 independent.

    “CDKN2A is unaffected by JQ1 suggesting also in this case that the phenotype is p16 independent (Fig. 3G and Supplementary Fig. 13D).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    The Hedgehog pathway is implicated in DIPG pathogenesis, and the candidate DIPG cell-of-origin is proposed to be a SHH-responsive cell.

    “The Hedgehog pathway is implicated in DIPG pathogenesis as the candidate DIPG cell-of-origin is proposed to be a SHH-responsive cell9.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Monje, M. et al. Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma. Proc. Natl. Acad. Sci. USA 108, 4453–4458 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    H3K27M does not recruit or sequester PRC2 on chromatin in vivo; instead, PRC2 binding is excluded by locus-specific formation of H3K27M/K27ac heterotypic nucleosomes.

    “Collectively, these results indicate that H3K27M is not involved in PRC2 recruitment or sequestration on chromatin in vivo, but on the contrary, suggest that PRC2 binding to chromatin is excluded by locus specific formation of H3K27M/K27ac heterotypic nucleosomes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    When H3K27me3 is lost, these nucleosomes acquire H3K27ac and form H3K27M-K27ac heterotypic nucleosomes.

    “Interestingly upon loss of H3K27me3 these nucleosomes acquire H3K27ac resulting in the formation of H3K27M-K27ac heterotypic nucleosomes4 (Figure 1D and Supplementary Fig. 5B–C).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [4] Lewis, P.W. et al. Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma. Science 340, 857–861 (2013). source

      Not checked in the free run.

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

    Premisecitation

    Inhibition or activation of Hedgehog signaling negatively or positively affects DIPG neurosphere formation ability.

    “Moreover, inhibition or activation of Hedgehog signaling negatively or positively affects DIPG neurosphere formation ability, respectively9.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Monje, M. et al. Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma. Proc. Natl. Acad. Sci. USA 108, 4453–4458 (2011). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Genes transcriptionally modulated by JQ1 have H3K27M, active transcription marks, and BRD2/4 occupancy around their promoters, indicating they are direct JQ1 targets.

    “Importantly, the genes transcriptionally modulated by JQ1 show H3K27M, active transcription marks and BRD2/4 occupancy around their promoters indicating they are direct targets affected by JQ1 treatment (Supplementary Fig. 13B–C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The work provides new molecular insights into H3K27M function in DIPG oncogenesis and identifies PRC2 and BET bromodomain proteins as potential therapeutic targets for DIPG.

    “In conclusion our work provides new important molecular insights about H3K27M function in DIPG oncogenesis and identified PRC2 and BET bromodomain proteins as potential therapeutic targets for this disease.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    HDAC inhibitors can synergize with JQ1 in a pancreatic cancer mouse model and in a murine lymphoma model, where they were suggested to induce similar transcriptional outcomes.

    “Interestingly HDAC inhibitors can synergize with JQ1 in pancreatic cancer mouse model30 and in a murine lymphoma model where they were suggested to induce similar transcriptional outcome31, thus paving the”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [30] Mazur, P.K. et al. Combined inhibition of BET family proteins and histone deacetylases as a potential epigenetics-based therapy for pancreatic ductal adenocarcinoma. Nat. Med. 21, 1163–1171 (2015). source

      Not checked in the free run.

    • cites [31] Bhadury, J. et al. BET and HDAC inhibitors induce similar genes and biological effects and synergize to kill in Myc-induced murine lymphoma. Proc. Natl. Acad. Sci. USA 111, E2721–E2730 (2014). source

      Not checked in the free run.

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

    Study design and conductown data

    In the SU-DIPG IV flank xenograft experiment, mice were randomly allocated to DMSO vehicle (n=4) and JQ1 (n=5) treatment arms.

    “Mice were randomly assigned to vehicle (DMSO, n = 4) and JQ1 (n = 5) treatment groups”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The data support a model in which H3K27M excludes PRC2 binding and thereby induces aberrant H3K27ac accumulation at H3K27M binding sites.

    “Rather, our data support a model where H3K27M excludes PRC2 binding, thus inducing aberrant accumulation of H3K27ac at H3K27M binding sites consistent with PRC2 loss of function in other systems24,25.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [24] Pasini, D. et al. Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes. Nucleic Acids Res. 38, 4958–4969 (2010). source

      Not checked in the free run.

    • cites [25] Ferrari, K.J. et al. Polycomb-dependent H3K27me1 and H3K27me2 regulate active transcription and enhancer fidelity. Mol. Cell 53, 49–62 (2014). source

      Not checked in the free run.

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

    Study design and conductnone given

    All experiments were conducted without blinding.

    “All the experiments were not conducted in blind conditions.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Abrogating the anti-differentiation effect of H3K27M/K27ac heterotypic nucleosomes with BET inhibitors such as JQ1 could decrease DIPG cell proliferation and promote terminal neuronal differentiation.

    “Abrogating the anti-differentiation effect of H3K27M/K27ac heterotypic nucleosomes through use of BET inhibitors such as JQ1 could conceivably provide the dual benefit of decreasing DIPG cell proliferation while promoting terminal neuronal differentiation of the tumor cells.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Four mice from the treatment group and one from the vehicle group in Supplementary Figure 17B and C were sacrificed for tumor-unrelated problems and excluded from the study.

    “Four mice from the treatment group and one mouse from the vehicle group in the experiments presented respectively in Supplementary Figure 17B and C were sacrificed for tumor unrelated problems and therefore excluded from the study.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    All data subjected to statistical analysis met the criteria for the appropriate statistical test, with normal distribution inferred using the empirical rule.

    “All the data where a statistical analysis was reported meet the criteria to use the appropriate statistical test, (for the normal distribution of data, the empirical rule was used to infer the distribution).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    PRC2 activity is redirected during neuronal differentiation of mouse embryonic stem cells, suggesting that external signals shape PRC2 recruitment and repress distinct gene sets at specific developmental stages.

    “PRC2 activity is redirected during the neuronal differentiation of mouse embryonic stem cells, suggesting that external signals shape PRC2 recruitment, resulting in the repression of distinct gene sets at specific developmental stages26.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] Mohn, F. et al. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30, 755–766 (2008). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The data support the possibility that locus-specific reduction of PRC2 activity or binding by H3K27M could promote DIPG formation, while overall PRC2 function is essential for maintaining the oncogenic status of these tumors.

    “Therefore a logical conclusion from our data supports the possibility that while a locus specific reduction of PRC2 activity/binding by H3K27M could promote DIPG formation the overall PRC2 function is essential for the maintenance of their oncogenic status.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Heterotypic H3K27M/K27ac nucleosomes may impede PRC2 repressive activity at a critical subset of genes that maintain an undifferentiated state while confining PRC2 to genes that should be activated during differentiation.

    “Heterotypic H3K27M/K27ac nucleosomes may on one hand create an impediment for PRC2 repressive activity at a critical subset of genes involved in the maintenance of an undifferentiated state while on the other hand confining PRC2 to genes that should be activated during the differentiation process.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    In the SF8628 pontine xenograft experiment, mice were randomly allocated to OraPlus vehicle (n=9) and I-BET (n=5) treatment arms.

    “Mice were randomly assigned to vehicle (OraPlus, n = 9) and I-BET (n = 5) treatment groups”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Together with other genetic alterations in DIPG, this would increase proliferation potential and decrease differentiation ability, two hallmarks of cancer.

    “This, in cooperation with other genetic alterations characterizing DIPG (e.g. TP53, PDGFRA, ACVR1, etc.), would increase the proliferation potential and decrease the differentiation ability, two events widely known to be hallmarks of cancer27,28.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [27] Hanahan, D. & Weinberg, R.A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011). source

      Not checked in the free run.

    • cites [28] Jones, C. & Baker, S.J. Unique genetic and epigenetic mechanisms driving paediatric diffuse high-grade glioma. Nat. Rev. Cancer 14, 651–661 (2014). source

      Not checked in the free run.

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

    Study design and conductnone given

    In each multi-panel figure, all images were acquired with identical confocal settings and processed identically in NIS Elements.

    “All images within each multi-panel figure were taken with the same confocal settings, and were processed in NIS Elements the exact same way.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The primary pediatric human glioma cell lines were obtained in accordance with institutionally approved protocols at each institution.

    “Primary pediatric human glioma cell lines, SF8628, SF7761 and SU-DIPG-IV, pcGBM2, SF9402 and SF9427 were obtained from UCSF medical center and Stanford University, and in accord with institutionally approved protocol at each institution.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    For the in vivo therapy-response experiment, ten mice were randomly allocated to DMSO vehicle (n=9) and JQ1 (n=9) treatment arms.

    “For the in vivo therapy-response analysis, ten mice were randomly assigned to vehicle (DMSO, n = 9) and JQ1 (n = 9) treatment groups”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Two control mice in Figure 4A died immediately after vehicle administration ended and were excluded from the survival study.

    “Two mice from the control group in Figure 4A died immediately after the end of the vehicle administration therefore they were excluded from the survival study.”
    Quote in the paperNot a substantial claim
38 of 38

  • The paper relies on this without support.

    The DIPG cell lines and xenograft models used are adequate representatives of human DIPG for drawing conclusions about disease biology and therapy.

    “ChIP-seq results from SF8628 human DIPG cells, containing a mutant H3F3A gene encoding H3.3K27M8, reveal a genome wide distribution of H3.3K27M that is highly correlated with active transcription”
    “we employed a xenograft mouse model of DIPG based on injection of SF8628 cells into the brainstem of athymic mice8.”

  • The paper relies on this without support.

    H3K27ac levels in tumors serve as a valid surrogate for JQ1 target inhibition in vivo.

    “we performed an immunohistochemistry analysis for H3K27ac on tumors coming from JQ1 treated and control mice as a surrogate marker for JQ1 target inhibition.”
2 of 2

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?

  • Such examples have been mapped in the context of multiple myeloma and diffuse intrinsic pontine glioma (DIPG).

    “Such examples have been mapped in the context of multiple myeloma 51 and diffuse intrinsic pontine glioma (DIPG) 52 .”

    From How many human proteoforms are there? (2018) · cites this paper as [52]

  • Lower sub-cytotoxic doses of DNMT inhibitors have received regulatory approval for the treatment of myelodysplastic syndrome (MDS) and AML as agents with demonstrated efficacy for patients unfit for intensive chemotherapy.

    “Lower sub-cytotoxic doses have received regulatory approval for the treatment of myelodysplastic syndrome (MDS) and AML as agents with demonstrated efficacy for patients unfit for intensive chemotherapy 122-124 .”

    From The roles of DNA, RNA and histone methylation in ageing and cancer (2019) · cites this paper as [124]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Different I-BET compounds are named for the in vivo experiment

    Major

    The results identify the BET inhibitor used for the xenograft experiment as I-BET151, while the corresponding methods identify it as I-BET-762.

  • Treatment-group counts exceed the stated total

    Minor

    The stated groups contain 9 mice each, for 18 mice in total, not 10. The two additional mice described separately do not resolve this discrepancy.

    Reported 10 · recomputed 18

2 of 2

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Authors
Piunti, A., Hashizume, R., Morgan, M. A., Bartom, E. T., Horbinski, C. M., Marshall, S. A., Rendleman, E. J., Ma, Q., Takahashi, Y.-H., Woodfin, A. R., Misharin, A. V., Abshiru, N. A., Lulla, R. R., Saratsis, A. M., Kelleher, N. L., James, C. D., & Shilatifard, A.
Affiliations
  • Northwestern University (US)
  • Lurie Children's Hospital (US)
Corresponding author
  • James, C David
  • Shilatifard, Ali
ORCID
Venue
Nature Medicine
Publisher
Springer Science and Business Media LLC
Published
2017-02-27
Volume
23
Issue
4
Pages
493-500
ISSN
  • 1078-8956 (ISSN-L)
  • 1546-170X
DOI
10.1038/nm.4296
PMC
PMC5667640
OpenAlex
W2592743985
Cited by
445
References
46
Citations by year
2026: 23 · 2025: 26 · 2024: 47 · 2023: 56 · 2022: 51 · 2021: 53 · 2020: 73 · 2019: 58 · 2018: 30 · 2017: 25 · 2016: 1 · 2014: 1
FWCI
29.22
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Medicine (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health (KL2TR001424)
  • National Cancer Institute (R35CA197569, P30CA060553)
  • National Institute of General Medical Sciences (P41GM108569)
  • National Institute of Neurological Disorders and Stroke (R01NS093079, K08NS097624)
  • National Center for Advancing Translational Sciences
Topics
  • Health Sciences › Medicine › Genetics › Glioma Diagnosis and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
Keywords
diffuse intrinsic pontine glioma, polycomb repressive complex 2, BET inhibitors, H3K27 acetylation, H3K27me3, epigenetic regulation, tumor cell proliferation, neuronal differentiation, pediatric brainstem tumors, epigenetic profiling
MeSH terms
Acetylation, Animals, Azepines, Chromatin, Glioma, Histones, Humans, Methylation, Mutation, Nucleosomes, Triazoles, Gene Expression Regulation, Neoplastic, RNA-Binding Proteins, Brain Stem Neoplasms, Protein Transport, Xenograft Model Antitumor Assays, Histone Code, Cell Line, Tumor, Cell Proliferation, Mice, Neurogenesis, Epigenomics, Molecular Targeted Therapy, Polycomb Repressive Complex 2

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 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

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.

What counts as a claim ›How we measure confidence ›How we check this ›

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