Targeted degradation of BRD9 reverses oncogenic gene expression in synovial sarcoma

Abstract

Synovial sarcoma tumours contain a characteristic fusion protein, SS18-SSX, which drives disease development. Targeting oncogenic fusion proteins presents an attractive therapeutic opportunity. However, SS18-SSX has proven intractable for therapeutic intervention. Using a domain-focused CRISPR screen we identified the bromodomain of BRD9 as a critical functional dependency in synovial sarcoma. BRD9 is a component of SS18-SSX containing BAF complexes in synovial sarcoma cells; and integration of BRD9 into these complexes is critical for cell growth. Moreover BRD9 and SS18-SSX co-localize extensively on the synovial sarcoma genome. Remarkably, synovial sarcoma cells are highly sensitive to a novel small molecule degrader of BRD9, while other sarcoma subtypes are unaffected. Degradation of BRD9 induces downregulation of oncogenic transcriptional programs and inhibits tumour progression in vivo. We demonstrate that BRD9 supports oncogenic mechanisms underlying the SS18-SSX fusion in synovial sarcoma and highlight targeted degradation of BRD9 as a potential therapeutic opportunity in this disease.

Keywords: BRD9; SS18-SSX; SWI/SNF; biochemistry; cancer biology; chemical biology; fusion protein; human; synovial sarcoma; targeted degradation.

Figure 1.. The BRD9 bromodomain is a functional dependency in synovial sarcoma.

(A) Schematic representation of CRISPR/Cas9 based genomic screening approach. (B) Scatter plot representation of biological duplicate sgRNA screening data in synovial and Ewing’s sarcoma cell lines. Each dot denotes an individual sgRNA and axes represent log₂ fold-change in sgRNA abundance between day-3 and day-15. BRD9 bromodomain and control sgRNAs are highlighted. (C) Schematic representation of the BRD9 protein structure with sgRNA target sites indicated. (D) Negative selection based CRISPR-Cas9 mutagenesis assays. The relative GFP⁺ (sgRNA⁺) subpopulation percentage is depicted at the indicated time-points after lentiviral infection. Mean ± s.d., n = 3. (E Waterfall plot representing ‘BRD9 sensitivity’ score in a panel of cancer cell lines taken from the Project DRIVE database (ref. Lim et al., 1998) (https://oncologynibr.shinyapps.io/drive/). (F) Negative selection based CRISPR-Cas9 mutagenesis assays in bromodomain functional rescue experiments. The relative GFP⁺ (sgRNA⁺) subpopulation percentage is depicted at the indicated time-points after lentiviral infection. Mean ± s.d., n = 3.

Figure 1—figure supplement 1.. BRD9 is a specific functional dependency in synovial sarcoma.

(A) Scatter plot representation of sgRNA screening data in synovial and Ewing’s sarcoma cell lines. Each dot denotes and individual bromodomain targeting sgRNA and axes represent log₂ fold-change in sgRNA abundance between day-3 and day-15. BRD4 and BRD9 bromodomain targeted sgRNAs are indicated. Highlighted region denotes sgRNAs depleted >2 fold in SS cells and unchanged in Ewing sarcoma cells. (B) Indel quantification by TIDE (Tracking of Indels by sequence trace Decomposition) analysis 4 days after transduction with the indicated sgRNA in the indicated cell lines. Also presented are GFP FACs plots collected at time of harvesting, demonstrating the proportion of GFP+ (sgRNA+) cells in each instance. (C) Western blot analysis of the indicated proteins in a cohort of synovial, and non-synovial sarcoma cell lines. (D) Western blot analysis of the indicated proteins in four independent SS cell lines expressing a control shRNA (Renilla) or one of two independent BRD9 targeting shRNAs. (E) Negative selection-based shRNA functional assays in four independent SS cell lines. The relative GFP+ (sgRNA+) subpopulation percentage is depicted at the indicated time points after lentiviral infection. Mean ± s.d., n = 3. (F) Western blots (as in Panel c) in Ewing sarcoma (A673) and rhabdomyosarcoma (RH30) cells lines. (G) Negative selection-based shRNA assays (as in Panel e) in Ewing sarcoma (A673) and rhabdomyosarcoma (RH30) cell lines. (H) Schematic representation of the BRD9 coding region targeted by sgRNA-15. The sgRNA target sequence is highlighted in red and PAM sequence is indicated (top). The silent mutations added to render the allele insensitive to CRISPR/Cas9 targeting are indicated, and sequence of the CRISPR-resistant allele shown (bottom). (I) Western blot analysis of the indicated proteins in HSSYII cells used in function rescue experiments.

Figure 2.. BRD9 functions as part of SS18-SSX containing SWI/SNF complexes.

(A) Silver stains of endogenous SS18-SSX fusion protein immunoprecipitations performed on nuclear protein lysates prepared two independent synovial sarcoma cell lines. (B) Volcano plots representing fold enrichment (LFQ intensity) of proteins identified by mass spec in SS18-SSX1 or SS18-SSX2 purifications relative to IgG control purifications. Known BAF members are indicated in red. (C) Western blots analyses of the indicated proteins performed on endogenous BRD9 or IgG purifications in HSSYII cells (Input = 10% total IP material). (D) Western blots analyses of the indicated proteins performed on endogenous BRD9 or IgG purifications in SYO1 cells (Input = 10% total IP material). (E) Scatter plot representing the normalized protein abundance (IBAQ score) of proteins identified in SS18-SSX1 and SS18-SSX2 purifications. Known BAF members are indicated in red (F) High density sgRNA tiling of BRD9 in two independent SScell lines. Each bar represents the fold-change of an individual sgRNA and its target site along the BRD9 protein. (G) Negative selection based CRISPR-Cas9 mutagenesis assays in amino acid 311–345 region functional rescue experiments. The relative GFP⁺ (sgRNA⁺) subpopulation percentage is depicted at the indicated time-points after lentiviral infection. Mean ± s.d., n = 3. (H) Western blot analyses of the indicated proteins in anti-V5 purifications performed in control HSSYII cells, or HSSYII cells expressing a full-length, bromodomain deleted or amino acid 311–345 deleted BRD9.

Figure 2—figure supplement 1.. BRD9 is a component of SS18-SSX containing BAF complexes.

(A) Silver stains of anti-HA immunoprecipitations performed on nuclear protein lysates prepared from HEK293T cell lines expression GFP (control) or an SS18-SSX1 or SS18-SSX2 fusion protein. (B) Volcano plots representing fold enrichment (LFQ intensity) of proteins identified by mass spec in anti-HA-SS18-SSX1/2 purifications relative to GFP control purifications. Known BAF members are indicated in red. (C) Bar chart representing total peptide numbers identified for each of the indicated BAF complex members in mass spec analysis of ant-HA-SS18-SSX1/2 purifications. (D) Bar chart representing total peptide numbers identified for each of the indicated BAF complex members in mass spec analysis of endogenous SS18-SSX1/2 purifications. (E) Bar chart representing the fold-change in abundance of individual sgRNAs targeting the bromodomains of the indicated BAF members between day-15 and day-3 of our functional genomics screening experiments.

Figure 3.. SS18-SSX1 and BRD9 co-bind the synovial sarcoma genome.

(A) Genomic tracks showing BRD9 and SS18-SSX1 ChIP-seq signal on the 98 Mb right arm of chromosome eight in HSSYII cells. The chromosome eight ideogram is displayed above the gene tracks with the relevant region highlighted in red. (B) Pie charts representing the distribution of BRD9 and SS18-SSX1 binding sites on the synovial sarcoma genome. (C) Venn diagram overlaps of all identified BRD9 and SS18-SSX1 ChIP-seq peaks in HSSYII cells. (D) Tornado plots showing BRD9, SS18-SSX1, RNAPII and H3K27Ac ChIP-signal ±10 kb of all hg19 gene promoters in HSSYII cells. Promoters are ranked by RNAPII ChIP signal. (E) Tornado plots showing BRD9, SS18-SSX1 and H3K27Ac ChIP-signal ±10 kb of all active enhancers (defined by H327Ac) in HSSYII cells. (F) Tracks showing BRD9 and SS18-SSX1 ChIP-seq occupancy at the indicated genomic loci in HSSYII cells.

Figure 3—figure supplement 1.. BRD9 and SS18-SSX1 co-localise genome-wide.

(A) Schematic representation of the CRISPR/Cas9 mediated targeting of a 3xHA epitope tag to the C-termini of the BRD9 and SS18-SSX1 loci. (B) Pie charts representing the proportion and total number of BRD9 and SS18-SSX1 ChIP-seq peaks occurring in each of the three indicated genomic regions. (C) Scatter plot representing the correlation of BRD9 and SS18-SSX1 ChIP-seq signal within all identified BRD9 binding peaks. (D) Venn diagram overlap of all BRD9 and SS18-SSX1 target genes identified in this study, with SS18-SSX1 targets identified in the work of Banito et al. (Banito et al., 2018).

Figure 4.. Complete ablation of BRD9 function by targeted protein degradation.

(A) Cellular viability dose-response data in the indicated panel of cell lines treated with the BRD9 bromodomain inhibitors BI7273 (top) or I-BRD9 (bottom). Mean ± s.d., n = 3. (B) Tornado plots and meta-tracks representing BRD9 ChIP-Rx signal in control (DMSO) and BI7273 treated (24 hr) HSSYII cells. (C) ChIP-qPCR analysis of 3xHA epitope tagged full-length BRD9, Δ311–345 BRD9 or Δ bromodomain BRD9 at the indicated gene promoters in HSSYII cells. Mean ± s.d., n = 3. (D) Chemical structure of our BRD9 degrader compound dBRD9-A. (E) Selectivity of phage-displayed bromodomain displacement by dBRD9-A (Bromoscan). (F) Western blot analysis of the indicated proteins, in two independent synovial sarcoma cell lines following treatment with dBRD9-A at 100 nM for 6–72 hr. (G) Waterfall plot representing changes in BRD9 occupancy at BRD9 peak regions in ChIP-Rx experiments of BI7273 (10 μM) (left panel) or dBRD9-A (100 nM) (right panel) treated HSSYII cells following 24 hr treatment. (H) Tracks showing BRD9 ChIP-seq occupancy on the 98 Mb right arm of chromosome eight after DMSO or 100 nM dBRD9-A treatment. The chromosome eight ideogram is displayed above the gene tracks with the relevant region highlighted in red. (I) Cellular viability dose-response data in HSSYII and SYO1 cells treated with dBRD9-A or the BRD9 bromodomain inhibitors BI7273 or I-BRD9. Mean ± s.d., n = 3.

Figure 4—figure supplement 1.. Transcriptional regulation by BRD9 in SS cells.

(A) Tornado plots showing BRD9-WT and BRD9-N216A ChIP-signal signal within all BRD9 peaks regions. Regions are ranked by change in ChIP-signal (BRD9-N216A/BRD9-WT). (B) Genomic tracks showing BRD9-WT and BRD9-N216A ChIP-seq signal at the indicated locus in HSSYII cells. (C) Western blot analysis of the indicated proteins in wildtype or CRBN -/- HEK293T cells treated with 100 nM DBRD9-A for 0, 24 or 48 hr. (D) Western blot analysis of the indicated proteins in HSSYII cells treated with increasing doses of dBRD9-A (100 nM, 500 nM) for 6 hr following pre-treatment of cells for 12 hr with DMSO or BI7273 (5 μM). (E) Cellular viability dose-response data in SS (HSSYII and SYO1), Ewing sarcoma (A673) and rhabdomyosarcoma (RH30) cell lines treated with dBRD9-A. Mean ± s.d. n = 3. (F) Western blot analysis of the indicated proteins in Ewing (A673) and rhabdomyosarcoma (RH30) cell lines treated with dBRD9-A at 100 nM for 6 to 72 hr. (G) Western blot analyses of the indicated proteins in two independent synovial sarcoma cell lines treated for the specified time course with BI7273 (left panels) at 5 μM or dBRD9-A (right panels) at 100 nM. (H) Fold-change of all known BAF members identified by mass spectrometry in SS18-SSX1 purifications performed in HSSYII cells treated with DMSO or dBRD9-A for 24 hr.

Figure 5.. BRD9 degradation blocks synovial sarcoma tumour progression and oncogenic transcription.

(A) Relative changes in cell cycle dynamics in two independent SS cell lines treated with dBRD9-A for 3/6/9 days at 100 nM. Mean ±s.d., n = 3 (B) Relative changes in Annexin-V positive cells in two independent SS cell lines treated with dBRD9-A for 3/6/9 days at 100 nM. Mean ± s.d., n = 3 (C) Tumour progression in a subcutaneous xenograft model of SS in control vehicle treated mice, and mice treated at 50 mg/kg dBRD9-A once daily for 24 days. Mean ± SEM, five mice per treatment group. P value is from 2way ANOVA (D) Western blot analysis of the indicated proteins in protein lysates derived from tumour tissue from two independent mice per treatment group as in panel c. (E) Volcano plot representing gene expression changes in HSSYII cells treated with dBRD9-A at 100 nM for 6 hr. The number of genes, the expression of which changes >1.5 fold up or down are indicated. (F) H3K27Ac ChIP-seq signal (rpm/bp) at all enhancer regions in HSSYII cell. Enhancers are ranked by increasing H3K27Ac signal. (G) Box plot representations of changes in gene expression amongst genes associated with typical enhancers and genes associated with super enhancers. P values are from Welch’s two-tailed t-tests. ***p≤0.001. (H) Heat map representing changes in gene expression amongst all super enhancer associated genes in HSSYII cells following 6 hr dBRD9-A treatment at 100 nM, or HSSYII cells following infection with two independent SS18-SSX1 shRNAs for 96 hr. (I) Tracks showing BRD9 and SS18-SSX1 ChIP-seq occupancy at the indicated genomic loci in DMSO and dBRD9-A treated cells. Also shown is H3K27Ac ChIP-seq signal in untreated cells.

Figure 5—figure supplement 1.. Targeted degradation of BRD9.

(A) Schematic representation of wildtype BRD9 and BRD7, and the chimeric BRD9 bromodomain swap containing the BRD7 bromodomain (top panel). Western blot analysis of the indicated proteins in HSSYII cells expressing vector (control), BRD9-WT or BRD9 containing the bromodomain of BRD7 (BRD9-BD7) treated with dBRD9-A at 100 nM for 6 hr (bottom panel). (B) Growth assays of HSSYII cells (as in panel a) cultured in the presence of dBRD9-A at 100 nM for a total of 9 days. (C) Mouse weight measurements in vehicle control and dBRD9-A treated mice. Mean ±s.d., n = 5. (D) Complete blood counts (CBCs) performed on vehicle control and dBRD9-A treated mice. Measurements were taken on day-23 of the 24 day treatment experiment. Mean ±s.d., n = 5. (E) Box plot representation of the relative abundance of BRD9 and SS18-SSX1 ChIP-seq signal at promoter, typical enhancer and super enhancer elements. P values are from Welch’s two-tailed t-tests. ***p≤0.001. (F) Box plot representations of changes in SS18-SSX1 occupancy at active promoters, typical enhancers and super enhancers comparing DMSO and dBRD9-A treated HSSYII cells. P values are from Welch’s two-tailed t-tests. *p≤0.05, ***p≤0.001. (G) Venn diagram representing the proportion of overlap between all up/down-regulated genes (±1.5 fold) in dBRD9-A treated cells at 6 hr post-treatment with all direct SS18-SSX target genes.

Chemical structure 1.. 4-bromo-2-methyl-2,7-naphthyridin-1(2H)-one.

Chemical structure 2. tert-butyl 2-((4-bromo-2,6-dimethoxybenzyl)(methyl)amino)acetate.

Chemical structure 3.. tert-butyl 2-((2,6-dimethoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)(methyl)amino)acetate.

Chemical structure 4.. tert-butyl 2-((2,6-dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)(methyl)amino)acetate.

Chemical structure 5.. 2-((2,6-dimethoxy-4-(2-methyl-1-oxo-1,2-dihydro-2,7-naphthyridin-4-yl)benzyl)(methyl)amino)acetic acid.

Chemical structure 6.. 4-((8-aminooctyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4).

Chemical structure 7.. dBRD9-A.