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Topography of transcriptionally active chromatin in glioblastoma

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Science Advances  30 Apr 2021:
Vol. 7, no. 18, eabd4676
DOI: 10.1126/sciadv.abd4676
  • Fig. 1 Landscape and clustering of active regulatory elements in GBM tissues.

    (A) Unsupervised hierarchical clustering of 1107 most-varying H3K27ac peaks (mean, ≥10; median absolute deviation, >1) across 49 GBM tissues (cohort 1) and 12 normal brain tissues. Top: The clinical, mutational, and molecular data of each sample. (B) Molecular classification of GBM samples in cohort 1 based on GIE subtypes and ARE subtypes. (C) Heatmap of differential enrichments of immune cell signatures, stromal cell signatures, gene mutations, and clinical parameters among normal brain tissues and various molecular subtypes of GBM. (D) ARE clustering of an independent cohort of 46 GBM samples (cohort 2) based on the same set of 1107 variant elements that were identified from cohort 1. (E) Spearman correlation of ARE status in cohort 2 samples with each subtype centroids of cohort 1. (F) Summary of molecular GIE subtypes and ARE subtypes among cohort 2. AC, active regulatory element-based cluster; ARE, active regulatory element; CL, classical subtype; GIE, GBM-intrinsic gene expression; MES, mesenchymal subtype; PN, proneural subtype.

  • Fig. 2 Subgroup-associated SE domains and disease pathways in GBM.

    (A) Relative rank of stitched H3K27ac ChIP-seq signals in integrative subtypes of GBM samples. Representative meta-SE–associated genes and their rankings in parentheses are highlighted. (B) Pairwise comparison of similarity in meta-SE–associated genes among integrative subtypes of GBM samples. (C) Ontological enrichment of subtype-specific meta-SE–associated genes in GBM tissues. No significant pathways (Benjamini-Hochberg FDR, <0.05) were enriched in AC2-PN–specific meta-SE targets.

  • Fig. 3 GBM-promoting transcription factors preferentially engage tumor-enriched super-enhancers.

    (A) Relative rank of stitched H3K27ac ChIP-seq signals in GBM (cohort 1) and normal brain tissues. Representative meta-SE–associated genes and their rankings in parentheses are highlighted. (B) Venn diagram showing common and sample type–specific meta-SE–associated genes in GBM and normal brain tissues. (C and D) Differential deposition of H3K27ac signals across regulatory regions of EGFR, RFX2, and TGIF1. (E) Ontological analysis of GBM-enriched SE genes. (F) Association of GBM-enriched SE domains with TFs and effect of shRNA-mediated silencing of top SE-associated TFs on cell viability of U251 GBM cells and NNI-11 GBM-propagating cells. (G) Elevated expression of TFs driven by tumor-enriched SEs in GBM samples (cohort 1). Student’s t test (two tailed) was applied. Boxplots represent the 25th and 75th percentiles, with midlines indicating the median values and whiskers extended to the lowest/highest values. (H and I) Effect of shRNA-mediated silencing of either RFX2 (n = 8) or TGIF1 (n = 7) in NNI-11 cells on survival of recipient mice with intracranial xenografts. Before intracranial implantation, Western blot and qPCR analyses were used to verify effective silencing of RFX2 and TGIF1, respectively. (J) Prognostic potentials of RFX2 and TGIF1 in Rembrandt cohort of glioma patients. (K) Prognostic potentials of an SE-driven TF signature in three independent cohorts of glioma patients. Log-rank test is applied in (H) to (K). HR, hazard ratio; CI, confidence interval.

  • Fig. 4 Subgroup-associated core regulatory circuitries in GBM.

    (A) Core interconnected transcriptional regulatory circuitries in each integrative subtype of GBM tissues. Core TFs are highlighted in colored circles. Key interactive partners of core TFs are indicated in gray circles. (B) Heatmap showing response of AC1-MES core TFs to a BET protein degrader (dBET6, 500 nM) treatment in U87 cells (GSE99181; transcripts per million, > 0.1). (C) Enhanced deposition of H3K27ac signals across regulatory regions of SOX2, NR2F1, ETV6, and ETS1 in GBM tissues. (D) Pathway enrichment of recurrent TFs in two coexpressed core modules across GBM subtypes.

  • Fig. 5 SE-driven lncRNAs in GBM.

    (A) Elevated expression of SE-driven lncRNAs in GBM samples (cohort 1). Student’s t test was applied. (B) Differential deposition of H3K27ac signals across regulatory regions of LINC01094 and MIR99AHG in GBM subtypes and normal brain tissues. (C to E) Effect of shRNA-mediated silencing of LINC01094 and MIR99AHG on target expression (C) and cell viability (D), and survival of recipient mice with intracranial transplantation of NNI-11 cells (E). (F) Differential expression of LINC01094 and MIR99AHG transcripts among various human cancers. (G) Effect of BET protein degraders on the expression of LINC01094. U87 and U251 GBM cells were treated with either ZBC260 or dBET6 (200 nM, 8 hours) before harvest. (H) Effect of siRNA-mediated silencing of core TFs on the expression of LINC01094. GBM cells were transfected with indicated siRNAs (48 hours) and subjected to qPCR analysis. Either one-way ANOVA or t test was used for analysis of significance. Error bars in (C), (D), (G), and (H) represent SEM, n = 3. Log-rank test was applied for survival analysis in (E). Boxplots in (A) and (F) represent the 25th and 75th percentiles, with midlines indicating the median values and whiskers extended to the lowest/highest values. n.s., not significant.

  • Fig. 6 Analysis of GBM-enriched SEs uncovers actionable drug targets.

    (A) Engagement of GBM-enriched SE domains in druggable genome. (B) Chemical structure and biochemical activity of an MNK kinase inhibitor ETC-168. (C) Effect of ETC-168 on viability of GBM cells and GBM-propagating cells. (D and E) Effect of ETC-168 treatment on sphere formation capability (SFC) of GBM-propagating cells (i.e., NNI-11). (F) Effect of ETC-168 (5 μM) and TMZ (100 μM) on expression of downstream targets in NNI-11 cells after 24-hour treatment. (G) Effect of ETC-168 (5 μM) and temozolomide (TMZ; 100 μM) on viability of NNI-11 cells after 72-hour treatment. One-way ANOVA was used for analysis of significance. (H) Effect of ETC-168 [25 mg/kg, orally (po), twice a day (bid)] and TMZ (100 mg/kg, po, once a day (qd)] on expression of indicated proteins in orthotopic xenograft tumors (NNI-11) after 24-hour treatment. (I) Effect of ETC-168 and TMZ treatment on both survival of recipient mice with intracranial transplantation of NNI-11 cells and the tumor incidence at the end point of experiment. Log-rank test was applied for survival analysis; n = 6 or 7. (J and K) Effect of shRNA-mediated silencing of either MKNK1 or MKNK2 in NNI-11 cells on survival of recipient mice (J) and orthotopic tumor expansion 2 months after implantation (K). Log-rank test was applied for survival analysis in (J); n = 5 or 7. Error bars in (C) and (G) represent SEM; n = 3. IC50, half maximal inhibitory concentration.

Supplementary Materials

  • Supplementary Materials

    Topography of transcriptionally active chromatin in glioblastoma

    Liang Xu, Ye Chen, Yulun Huang, Edwin Sandanaraj, John S. Yu, Ruby Yu-Tong Lin, Pushkar Dakle, Xin-Yu Ke, Yuk Kien Chong, Lynnette Koh, Anand Mayakonda, Kassoum Nacro, Jeffrey Hill, Mo-Li Huang, Sigal Gery, See Wee Lim, Zhengyun Huang, Ying Xu, Jianxiang Chen, Longchuan Bai, Shaomeng Wang, Hiroaki Wakimoto, Tseng Tsai Yeo, Beng Ti Ang, Markus M�schen, Carol Tang, Tuan Zea Tan, H. Phillip Koeffler

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