Targeting SWI/SNF ATPases in H3.3K27M diffuse intrinsic pontine gliomas

Abstract

Diffuse midline gliomas (DMGs) including diffuse intrinsic pontine gliomas (DIPGs) bearing lysine-to-methionine mutations in histone H3 at lysine 27 (H3K27M) are lethal childhood brain cancers. These tumors harbor a global reduction in the transcriptional repressive mark H3K27me3 accompanied by an increase in the transcriptional activation mark H3K27ac. We postulated that H3K27M mutations, in addition to altering H3K27 modifications, reprogram the master chromatin remodeling switch/sucrose nonfermentable (SWI/SNF) complex. The SWI/SNF complex can exist in two main forms termed BAF and PBAF that play central roles in neurodevelopment and cancer. Moreover, BAF antagonizes PRC2, the main enzyme catalyzing H3K27me3. We demonstrate that H3K27M gliomas show increased protein levels of the SWI/SNF complex ATPase subunits SMARCA4 and SMARCA2, and the PBAF component PBRM1. Additionally, knockdown of mutant H3K27M lowered SMARCA4 protein levels. The proteolysis targeting chimera (PROTAC) AU-15330 that simultaneously targets SMARCA4, SMARCA2, and PBRM1 for degradation exhibits cytotoxicity in H3.3K27M but not H3 wild-type cells. AU-15330 lowered chromatin accessibility measured by ATAC-Seq at nonpromoter regions and reduced global H3K27ac levels. Integrated analysis of gene expression, proteomics, and chromatin accessibility in AU-15330-treated cells demonstrated reduction in the levels of FOXO1, a key member of the forkhead family of transcription factors. Moreover, genetic or pharmacologic targeting of FOXO1 resulted in cell death in H3K27M cells. Overall, our results suggest that H3K27M up-regulates SMARCA4 levels and combined targeting of SWI/SNF ATPases in H3.3K27M can serve as a potent therapeutic strategy for these deadly childhood brain tumors.

Keywords: H3K27M mutation; SWI/SNF complex; pediatric brain cancer.

Fig. 1.

SMARCA4 protein levels are higher in H3.3K27M compared to H3WT cells. (A) Immunoblots of patient-derived H3 WT (SF188, SJGBM2, and UMPED37), H3.3G34V (KNS42), or H3.3K27M (DIPG007, DIPGXIII*p, and SF7761) cell lines probed for SMARCA4, SMARCA2, PBRM1, ARID2, BRD7, and SMARCB1; western blot for HSP90 and Ponceau staining were used as loading control. (B) Immunoblots of DIPG007 cells with or without stable knockdown (KD) of H3-3A with two independent shRNAs (sh1 or sh2) probed for H3.3K27M, H3K27me3, and H3K27ac; total H3 was probed as loading control. (C) Heatmap of normalized protein abundance (Z-score) of key components of BAF and PBAF (PBRM1, ARID2, and BRD7) SWI/SNF complex in DIPG007 H3.3K27M with or without H3-3A KD acquired by untargeted proteomics (n = 3, each). (D) Immunoblots of DIPG007 cells with or without H3-3A KDs from B probed for SMARCA4, SMARCA2, PBRM1, ARID2, BRD7, and SMARCB1. HSP90 was probed as loading control. (E) Representative images of H3K27M and H3WT (2 cases each) tumors stained for SMARCA4 by immunohistochemistry. (Scale bar, 50 μM.) (F) Matlab-based quantification of SMARCA4 (Y axis, a.u.=arbitrary units determined by number of pixels × pixel intensity; three randomly selected regions/case) in H3WT (n = 5) and H3K27M (n = 6) DMGs. Data were analyzed by two-sided, two-tailed, nonpaired t test.

Fig. 2.

H3.3K27M cell lines are sensitive to SMARCA2, SMARCA4, and PBRM1 protein degradation using the PROTAC AU-15330. (A) Heatmap of normalized protein abundance (Z-score) of key SWI/SNF complex components in untreated DIPG007 cells (n = 3) or DIPG007 cells treated with AU-15330 PROTAC (n = 4, 1 µM for 24 h) or vehicle (Veh, DMSO, n = 3), acquired by untargeted proteomics. Data were analyzed by ANOVA; P values indicated are between Veh and AU-15330-treated cells. (B) Immunoblots of H3.3K27M mutant cell lines (SF7761, BT245, and DIPG007) treated with AU-15330 (1 µM for 24 h) or Veh and probed for SMARCA4, SMARCA2, PBRM1, and SMARCB1. HSP90 was probed as loading control. (C) Immunoblots of H3.3G34V mutant (KNS42) and H3WT (SJGBM2 and SF188) cell lines treated with AU-15330 (1 µM for 24 h) or Veh and probed for SMARCA4, SMARCA2, PBRM1, and SMARCB1. HSP90 was probed as loading control. (D) Cell viability (normalized to Veh, percentage, Y axis) of H3WT (SJGBM2 and SF188), H3.3G34V mutant (KNS42), and H3.3K27M mutant (SF7761, BT245, and DIPG007) cell lines on treatment with different concentrations of AU-15330 (log concentrations, X axis) for 5 d (n = 3 for each concentration/cell line). (E) Cell viability (normalized to Veh, percentage, Y axis) of DIPG007 cells with or without for H3-3A KD with two independent shRNAs (H3-3A sh1 or sh2) treated with different concentrations of AU-15330 (log concentrations, X axis) for 5 d (n = 3 for each concentration/cell line). (F) Half maximal inhibitory concentration (IC₅₀) of AU-15330 (μM=Molar, Y axis) for H3.3K27M mutant (SF7761, BT245, and DIPG007), H3-3A KD sh1 and sh2 DIPG007, H3.3G34V mutant (KNS42), and H3WT (SJGBM2 and SF188) cells based on cell viability curves in (D) and (E).

Fig. 3.

AU-15330 reduces chromatin accessibility at nonpromoter regions in H3.3K27M mutant cells. (A) Heat maps of peak intensity (plotted as ± 2.0 kb from peak center) and overall peak representation of nonpromoter regions and promoters (plotted as ± 2.0 kb from center, transcriptional start site). (B) Overall peak representation of nonpromoter and promoter regions from 3A. (C) Immunoblots of H3.3K27M mutant cell lines (SF7761, DIPG007, and BT245) treated with AU-15330 (1 µM for 24 h) or Veh and probed for H3K27Ac. Total H3 was probed as loading control. (D) Heatmap of differentially expressed genes (Left, RNA-Seq) and proteins (Right, untargeted proteomics) in DIPG007 cells treated with AU-15330 (1 µM for 24 h) or Veh. Red = up-regulated genes, yellow = up-regulated proteins, and blue = down-regulated genes or proteins (RNA-Seq n = 2/condition; proteomics: n = 3 for Veh and n = 4 for AU-15330). (E) Venn diagram depicting overlap of genes with lowered chromatin accessibility (ATAC-Seq), lowered expression by RNA-Seq, and proteomics in DIPG007 cells treated with AU-15330 versus Veh. (F) Pathway analysis of 161 genes from Venn diagram. Red bars indicate pathways related to development.

Fig. 4.

Chromatin accessibility, gene expression, and protein abundance of FOXO1 are lowered by AU-15330 PROTAC. (A) Venn diagram depicting overlap of 161 down-regulated genes from Fig. 3F, with motif analysis of genes with lowered chromatin accessibility by ATAC-Seq, and all known human transcription factor genes. FOXO1 motif is indicated. (B) Hi-C heatmap (Left) depicting a topologically associating domain (TAD) between the FOXO1 promoter and ATAC-Seq peaks 1 and 2 associated with the FOXO1 locus in DIPG007 cells treated with AU-15330 (1 µM for 24 h, tan, n = 2) or Veh (blue, n = 2). H3K27ac (pale blue) track from DIPG007 shows localizations of peaks 1 and 2 with H3K27ac-enriched FOXO1 enhancer sites (pale blue boxes). Magnified FOXO1 locus (rectangle, right) is illustrated. (C and D) Representation (C) and quantification (Y axis, a.u. = arbitrary units normalized to Veh) (D) of ATAC-Seq peak tracks/chromatin accessibility at the FOXO1 promoter and at peak 1 and 2 associated with the FOXO1 locus from (B). (E) FOXO1 mRNA and protein levels (a.u. = arbitrary units normalized to Veh) of DIPG007 cells treated with or without AU-15330 by RNA-Seq (n = 2/condition) and proteomics (n = 3 for Veh; n = 4 for AU-15330). (F) Immunoblots of H3WT (SF188 and SJGBM2), H3.3G34V mutant (KNS42), and H3.3K27M mutant cell lines (SF7761, DIPGXIII*P, BT245, and DIPG007) probed for FOXO1, RHOB, and RHOA/B/C. HSP90 was probed as loading control. (G) Immunoblots of H3.3K27M mutant cell lines (SF7761, BT245, and DIPG007) treated with AU-15330 (1 µM for 24 h) or Veh probed for FOXO1, RHOB, and RHOA/B/C. HSP90 was probed as loading control. Arrows indicate FOXO1 and RHOB. (H) Cell viability assessment (normalized to Veh, percentage, Y axis) of H3.3K27M mutant cell lines DIPG007 with (n=16) or without (n = 8) FOXO1 knock down and DIPG007 (n = 4) and DIPGXIII*p (n = 8) cells treated with the FOXO1 inhibitor AS-1842856 (2, 6, or 10 µM for 48 h) or Veh.