BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma

Abstract

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell-like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas.

Significance: Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1-BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma. See related commentary by Beytagh and Weiss, p. 2730. See related article by Mo et al., p. 2906.

Figure 1.

Epigenetic CRISPR screen identifies BAF complex as a novel dependency in pediatric H3K27M glioma. A, Schematic of epigenetic-focused CRISPR/Cas9-negative selection screen conducted in one H3WT-glioma and four H3.3K27M-glioma neurosphere models. B, ssGSEA of top-scoring protein complexes in four H3.3K27M-glioma models using Chronos and CERES dependency scores. C, Mean difference in ssGSEA complex scores (more negative scores indicate selective dependencies) for BAF, PRC1, PRC2, and HDAC-containing complexes in four H3.3K27M-glioma models compared with 855 human adult and pediatric cancer cell lines in the DepMap (Broad Institute). D, Mean difference in ssGSEA complex scores for BAF, PRC1, PRC2, and HDAC-containing complexes in four H3.3K27M-glioma models compared with 60 adult and pediatric brain cancer cell lines in the DepMap (Broad Institute). E, Relative cell viability across three H3.3K27M-glioma neurosphere lines following single-gene CRISPR/Cas9-mediated knockout of BAF complex genes (normalized to AAVS1-negative sgRNA control, n = 3). Three bars are shown per gene to indicate three individual sgRNAs used for knockout (sgRNA sequences are provided in Supplementary Table S5). Data are shown as mean ± SEM; ****, P < 0.0001; **, P = 0.0065. F, Dependency scores (CERES) for SMARCA4 in four H3.3K27M-glioma models compared with 23 other cancer types reported in the DepMap (Broad Institute). The median is indicated by the red dashed line, and quartiles are shown in dotted lines. Types of cancer models are displayed in descending order from least sensitive to knockout to the most sensitive (most negative median CERES score).

Figure 2.

BRG1–BAF chromatin remodeling complex regulates the OPC stem cell–like state in pediatric H3K27M glioma. A, Specificity scores of all regulons as determined by SCENIC analysis of gene regulatory networks in cycling, OPC-like, AC-like, and OC-like subpopulations of pediatric H3K27M-glioma patient tumors (n = 6). B, Relative activity of SMARCA4 regulons in cycling, OPC-like, AC-like, and OC-like subpopulations of pediatric H3K27M-glioma patient tumors (n = 6). Significance was compared between the progenitor OPC-like and the mature AC-like subpopulation of H3K27M-glioma primary tumors using a Student t test. The median is marked by the middle line within the box plot, the first and third quartiles by the upper and lower limits, and the 1.5× interquartile range by the whiskers. C, Heat maps (left) depicting BRG1, H3K27ac, and H3K27me3 ChIP-seq signals in BT869 (H3.3K27M-glioma) neurospheres at BRG1 binding sites according to their distance to transcription start sites (TSS). Regions within 1 kb of the TSS were considered TSS-proximal sites (top, n = 99), whereas all others were classified as TSS distal (bottom, n = 1,236). Each row shows 6.4-kb regions, centered on BRG1 peaks and ranked by BRG1 ChIP-seq signal intensity. Color shading corresponds to ChIP-seq read counts. Distribution of BRG1 peak counts (right) according to their distance to the TSS and genome location. D, Biological processes as determined by gene ontology (GO) analysis associated with genes detected in close proximity to BRG1 peaks in the BT869 neurosphere model. The top 15 enriched GO terms are displayed. The gene ratio refers to the ratio between the number of genes associated with BRG1 peaks present in each GO term and in all GO terms. The number of genes related to BRG1 peaks and associated with each GO term is indicated by the size of the circle, and significance is depicted by the color scale. Reg., regulation. E, Single-cell expression scores of genes defined as BRG1 bound in the BT869 neurosphere model, projected onto the scRNA-seq data of H3K27M-glioma patient tumors (n = 6). OPC-like cancer cells (high stemness score) show higher expression of BRG1-bound genes than AC-like cancer cells (low stemness score and negative lineage score) or OC-like cancer cells (low stemness score and positive lineage score). F, Representation of the enrichment of H3K27M-glioma tumor transcriptional programs in BRG1-associated genes, defined by ChIP-seq in BT869 cells, overlapping with enhancer-regulated genes identified in H3.3K27M-glioma patient tumors. The largest circle indicates a higher overlap of BRG1/enhancer-regulated genes with the OPC-like transcriptional program in H3.3K27M-glioma tumors. Red color refers to significance in overlap, as determined by a hypergeometric test. G, Gene tracks display input, BRG1, and H3K27ac ChIP-seq signal at promoters and enhancers of marker genes of the OPC-like cancer cell state (EGR1, NAV1, and ETV1).

Figure 3.

Genetic knockout of SMARCA4 reduces chromatin accessibility and gene expression of OPC-like markers and enriches for the AC-like subpopulation in H3K27M-glioma cells. A, Schematic showing biological assessment of SMARCA4 knockout in H3.3K27M-glioma neurospheres by ChIP-seq for histone modifications (H3K27ac and H3K27me3), assay for transposase-accessible chromatin using sequencing (ATAC-seq) for chromatin accessibility, bulk RNA sequencing (RNA-seq), and scRNA-seq for gene expression. B, Heat map depicting z-scores for chromatin accessibility of OPC-like and AC-like marker genes in AAVS1-negative sgRNA control and SMARCA4-knockout (KO) BT869 neurospheres. C, Profile plot depicting the average H3K27ac ChIP-seq signal at BRG1 binding sites (n = 1,335) in BT869 4 days after SMARCA4 knockout. The plot shows 6.4-kb regions, centered on BRG1 peaks. D, Promoter-associated H3K27ac and H3K27me3 ChIP-seq signal for genes differentially expressed [determined by bulk RNA-seq analysis, log₂(fold change [FC]) ≥ |1|] in SMARCA4-knockout cells (4 days after nucleofections). E, Profile plots (left) depicting the average H3K27ac signal at H3K27ac locations divided into positive (UP) or negative (DOWN) signal changes 4 days after SMARCA4 knockout. The plots show 6.4-kb regions, centered on H3K27ac peaks. Gene ontology (GO) analyses (right) performed on H3K27ac peaks with increased (n = 104, K27acUP) or decreased (n = 2,832, K27acDOWN) acetylation levels upon SMARC4 knockout for 4 days. The top five enriched GO terms are displayed. Pos. reg. of epidermal cell diff., positive regulation of epidermal cell differentiation.F, Heat map (left) depicting fold change (as log₂) of H3K27ac levels after SMARCA4 knockout for 4 days. Displayed are log₂ fold changes > |1|. Genes indicated on the heat map are OPC-like markers associated with decreased H3K27ac levels after SMARCA4 depletion. Gene tracks (right) displaying BRG1 ChIP-seq signal in BT869 neurospheres, as well as H3K27ac and ATAC-seq, at two OPC-like marker genes (TNR and EPN2). G, Changes in the percentage of cycling versus noncycling (left) and OPC- and AC-like cancer cell subpopulations (right) in SMARCA4-knockout and AAVS1-negative sgRNA control cells 4 days after knockout in BT869 neurospheres as determined by scRNA-seq analysis. H, Single-cell expression scores of upregulated genes in BT869 SMARCA4-knockout or AAVS1-negative sgRNA control knockout cells projected onto scRNA-seq data of pediatric H3K27M-glioma primary tumors (n = 6).

Figure 4.

Genetic knockout of SMARCA4 reduces tumorigenicity of H3K27M-glioma PDX models. A, Quantifications of MRI in PDX mice bearing tumors with either AAVS1-negative sgRNA control or SMARCA4 knockout in BT869 cells (H3.3K27M-glioma, biopsy-derived, n = 10 per group) at 110 and 117 days after injections. B, Quantifications of in vivo bioluminescence measurements in PDX mice bearing tumors with either AAVS1-negative sgRNA control or SMARCA4 knockout in SU-DIPGXIIIP* cells (H3.3K27M-glioma, autopsy-derived, n = 8 per group) until 93 days after injections. C, Kaplan–Meier survival curves of BT869 PDX mice following SMARCA4 knockout compared with AAVS1 controls. The median survival of SMARCA4-knockout mice was 144 days after injection compared with 115 days in AAVS1 controls; ****, P < 0.0001 (n = 10 mice per group). D, Kaplan–Meier survival curves of SU-DIPGXIIIP* PDX mice after the loss of SMARCA4 compared with AAVS1 controls. Median survival of SMARCA4-knockout mice was 75.5 days after injection compared with 52.5 days in AAVS1 controls; **, P = 0.0014 (n = 8 mice per group). E, Representative hematoxylin and eosin (H&E) staining in AAVS1 control and SMARCA4 knockout BT869 PDX tumors collected at end-stage disease. The number of days indicated in the figure refers to the survival endpoint for each mouse from the date of tumor cell injections. F, Representative H&E staining in AAVS1 control and SMARCA4-knockout SU-DIPGXIIIP* PDX tumors collected at end-stage disease. The number of days indicated in the figure refers to the survival endpoint for each mouse from the date of tumor cell injections.G, Representative immunostaining for nuclei (DAPI), oncohistone (H3K27M), and AC-like marker (GFAP) in AAVS1 control and SMARCA4-knockout (KO) BT869 PDX tumors collected at the end-stage disease. H, Representative immunostaining for nuclei (DAPI), oncohistone (H3K27M), and AC-like marker (SOX9) in AAVS1 control and SMARCA4-knockout BT869 PDX tumors collected at end-stage disease. I, Quantification of immunofluorescence staining for GFAP in AAVS1 control (n = 4) and SMARCA4-knockout (n = 4) BT869 PDX tumors collected at end-stage disease (**, P = 0.002, unpaired t test). J, Quantification of immunofluorescence staining for SOX9 in AAVS1 control (n = 4) and SMARCA4-knockout (n = 4) BT869 PDX tumors collected at end-stage disease (****, P < 0.0001, unpaired t test).

Figure 5.

BAF complex ATPase inhibition and degradation are novel therapeutic strategies in pediatric H3K27M-glioma. A, Heat map of IC₅₀ values comparing small-molecule inhibitors and a degrader targeting BAF complex members, and its regulators (BRG1/BRM inhibitors: Compounds 11, 12, 14, PFI-3; CARM1 inhibitors: CARM1 inhibitor, TP064; BRD9 inhibitor: I-BRD9; and BRD9 degrader: dBRD9-13) in H3.3K27M (n = 5), H3.1K27M (n = 1), and H3WT (n = 3) pediatric glioma neurosphere models and nonmalignant cell lines (n = 2, NHA-hTERT: immortalized normal human astrocytes, and Oli Neu: immortalized normal mouse OPCs). B, PRISM analysis of 694 cancer cell lines representing 23 lineages (Broad Institute), treated with a BRG1/BRM inhibitor (Compound 11) at an 8-point dose curve (3-fold dilution, with a maximum of 10 μmol/L) for 5 days. The black dashed line represents the mean AUC computed over cell lines of all lineages. Cancer lineages below this line represent those sensitive to BRG1/BRM inhibition by Compound 11. C, Chemical structures of BRG1/BRM inhibitors (Compounds 11, 12, and 14) and a novel BRG1/BRM degrader (JQ-dS-4). D, Log₂ fold change (FC) of differential proteins (left) as assessed by SILAC of DMSO control (light isotope labeled) and 1 μmol/L JQ-dS-4 (heavy isotope labeled)–treated BT869 H3.3K27M-glioma neurospheres (2 days of treatment). Heat map (right) of BAF complex proteins (with encoding genes shown in parentheses) depleted upon JQ-dS-4 treatment in BT869 neurospheres. E, Immunoblot for BRG1 and BRM protein levels in BT869, HSJD-DIPG007, and SU-DIPGXIIIP* neurospheres treated with novel BRG1/BRM degraders (AU-15330 and JQ-dS-4) at indicated doses and time points. Cleaved PARP was used as a marker for apoptosis. Total H3 and GAPDH served as loading controls. F, Heat map of IC₅₀ values comparing two BRG1/BRM degraders (JQ-dS-4 and AU-15330) in H3.3K27M (n = 5), H3.1K27M (n = 1), and H3WT (n = 3) pediatric glioma neurosphere models and nonmalignant cell lines (n = 2, NHA-hTERT: immortalized normal human astrocytes, and Oli Neu: immortalized normal mouse OPCs). G, Dose–response curves for BRG1/BRM degraders (AU-15330 and JQ-dS-4) in H3.3K27M (n = 5), H3.1K27M (n = 1), and H3WT (n = 3) pediatric glioma neurosphere models and nonmalignant cell lines (n = 2, NHA-hTERT: immortalized normal human astrocytes, and Oli Neu: immortalized normal mouse OPCs).

Figure 6.

Pharmacologic targeting of the BAF complex in vitro and in vivo mimics the biological effects of SMARCA4 depletion in H3K27M glioma. A, Schematic showing biological assessment of BRG1/BRM ATPase inhibitor– and degrader–treated H3.3K27M-glioma neurospheres for changes in chromatin accessibility by ATAC-seq, gene expression by bulk RNA sequencing (RNA-seq), and in vivo efficacy of chemical compounds in subcutaneous xenograft mouse models. B, Profile plot (top) depicting average ATAC-seq signal at BRG1 binding sites (n = 1,335) in BT869 (H3.3K27M-glioma) neurospheres after 24 hours of treatment with 1 μmol/L of BRG1/BRM inhibitors [Compound 11 (C11), Compound 14 (C14)] and degraders [JQ-dS-4 (JQ), AU-15330 (AU)] and DMSO controls. The plot shows 6.4-kb regions, centered on BRG1 peaks. Gene tracks (bottom) exemplify changes in chromatin accessibility observed at BRG1 binding sites. Displayed are BRG1 ChIP-seq signal in control BT869 cells as well as ATAC-seq data on the same cells after 24 or 48 hours of treatment with 1 μmol/L of BRG1/BRM inhibitors or degraders and DMSO controls. C, Biological processes (gene ontology analysis) enriched in regions with decreased accessibility in BT869 neurosphere cells treated for 24 hours with 1 μmol/L of Compound (Cmpd) 14, Compound 11, JQ-dS-4, or AU-15330 as determined by ATAC-seq. The number of genes is indicated by the size of the circle, and significance is depicted by the color scale. Reg., regulation of. D, Overlap of genes with reduced chromatin accessibility in drug-treated BT869 neurospheres (as determined by bulk ATAC-seq) with single-cell transcriptional metaprograms in H3K27M glioma. BT869 cells were treated with 1 μmol/L of either Compound 11 (C11), Compound 14 (C14), JQ-dS-4 (JQ), or AU-15330 (AU) for 24 hours. The color scale indicates the number of overlapping genes. E, A heat map showing z-scores of gene expression of OPC-like metaprogram marker genes with statistically significant changes upon Compound 11 or 14 treatments in BT869 neurospheres after 24 hours. Colored bars indicate z-scores of gene expression. rep, replicate.F, Tumor volume measurements (top) in BRG1/BRM inhibitor Compound 14 (20 mg/kg i.p. daily)– or BRG1/BRM degrader JQ-dS-4 (50 mg/kg i.p. daily)–treated mice bearing BT869 (H3.3K27M-glioma) subcutaneous tumors as compared with vehicle controls (n = 10 mice per group). Data are shown as mean ± SEM, P < 0.0001 (unpaired t test comparing vehicle and Compound 14 groups). Kaplan–Meier survival curves (bottom) of mice treated with either Compound 14 (20 mg/kg i.p. daily) or JQ-dS-4 (50 mg/kg i.p. daily) for 60 days in a subcutaneous BT869 xenograft model (n = 10 mice per group). The P value was calculated using the log-rank (Mantel–Cox) test. Gray bars show the duration of treatment. G, Tumor volume measurements (top) in BRG1/BRM inhibitor Compound 14 (20 mg/kg i.p. daily)–treated mice bearing SU-DIPGXIIIP* (H3.3K27M) subcutaneous tumors as compared with vehicle controls (n = 8 mice per group). Data are shown as mean ± SEM, P < 0.0001 (unpaired t test). Gray bar shows the duration of the treatment. Tumor volume (bottom) measured after 30 days of Compound 14 treatment compared with vehicle control in mice bearing SU-DIPGXIIIP* (H3.3K27M) subcutaneous tumors. P = 0.0017 (unpaired t test). H, Tumor volume measurements (top) in BRG1/BRM inhibitor Compound 14 (20 mg/kg i.p. daily)–treated mice bearing HSJD-GBM001 (H3WT) subcutaneous tumors as compared with vehicle controls (n = 10 mice per group). Data are shown as mean ± SEM, P = 0.578 (unpaired t test). Kaplan–Meier survival curves (bottom) of mice treated with Compound 14 (20 mg/kg i.p. daily) for 30 days in a subcutaneous xenograft model of HSJD-GBM001 (n = 10 mice per group). The P value was calculated using the log-rank (Mantel–Cox) test. Gray bars show the duration of treatment.