Graphical abstract graphic file with name fx1.jpg [59]Open in a new tab Highlights * • CRISPR screening identifies BRD9 as a key regulator of tumor resistance to oHSV1 * • BRD9 inhibition enhances ICD and the antitumor activity of oHSV1 therapy * • BRD9 cooperates with RELA to regulate the expression of antiviral genes * • BRD9 is a potential biomarker for predicting clinical outcomes of oHSV1 therapy __________________________________________________________________ Guo et al. perform a genome-wide CRISPR screen and identify BRD9 as a key regulator of tumor cell resistance to oncolytic virotherapy. BRD9 inhibition enhances ICD and promotes antitumor activity in multiple glioblastoma models, suggesting that BRD9 is a vital target for improving the efficacy of oncolytic virotherapy in GBM. Introduction The successful treatment of glioblastoma multiforme (GBM), one of the most lethal malignant tumors, remains greatly challenging.[60]^1 Conventional interventions such as surgery, radiotherapy, and chemotherapy only achieve partial responses.[61]^2 The highly immunosuppressive tumor microenvironment (TME) of GBM, marked by scant infiltration of antitumor lymphocytes, curtails the effectiveness of traditional immune checkpoint blockade therapies.[62]^3^,[63]^4^,[64]^5 Oncolytic viruses, which induce tumor cell lysis and modify the TME, can stimulate immune cells through viral exposure, potential tumor antigens, and tumor cell immunogenic cell death (ICD), making it a promising approach for GBM treatment.[65]^6 Several early-phase clinical trials have demonstrated the therapeutic potential of oncolytic viruses for GBM.[66]^7^,[67]^8^,[68]^9 However, a substantial number of patients remain unresponsive to this treatment, suggesting the existence of unidentified restriction factors that inhibit the optimal efficacy of oncolytic virus therapy in GBM.[69]^5^,[70]^8^,[71]^9^,[72]^10 Here, through pooled whole-genome-wide CRISPR-Cas9 knockout screening, we identified the non-canonical BAF (ncBAF) complex as a crucial tumor-intrinsic factor that determines resistance to oncolytic viruses. Genetic or pharmacological targeting of the ncBAF-specific subunit bromodomain-containing protein 9 (BRD9) enhanced oncolytic virus replication and antitumor effectiveness, demonstrating the therapeutic potential of a combination therapy composed of oncolytic viruses and BRD9 inhibitors. Results Genome-wide CRISPR screen reveals regulators of oncolytic virus resistance To identify the genes that promote oncolytic virus therapy resistance, we selected oncolytic herpes simplex virus type 1 (oHSV1) for our study, as it is the most extensively used oncolytic virus in clinical trials for GBM.[73]^11 The neurovirulence gene ICP34.5, typically deleted in early generations of engineered oncolytic viruses,[74]^12^,[75]^13 is also crucial for countering host cell antiviral responses and enhancing viral replication.[76]^14 In our previous study, we engineered an oHSV1 by placing ICP34.5 under the control of miRNA-124, which is specifically active in neurons and often inactive in tumor cells, rather than completely removing it[77]^15 ([78]Figure S1A). We further assessed the effectiveness and safety of our oHSV1 in treating glioblastoma. Our oHSV1 selectively targeted and eradicated human glioblastoma cells (MGG4, GSC3264, and BNI21), with no detrimental impact on human normal neural progenitor cells (hNP1 and ENSA) or induced pluripotent stem cell (iPSC)-derived neurons ([79]Figure 1A). We then utilized human embryonic stem cell-derived cerebral organoids and patient-derived glioblastoma slices to assess viral infection and replication ([80]Figure 1B). Immunohistochemical anti-HSV1 staining revealed that viral replication was exclusively observed in the tumor sections ([81]Figure 1B). Notably, unlike most human glioma cells, mouse glioma cells characteristically exhibit low expression of the herpes simplex virus type 1 (HSV1) entry receptor Nectin1, which impedes oHSV1 infection.[82]^16 To address this issue and facilitate the study of oHSV1 therapy in immunocompetent mice, we overexpressed murine Nectin1 gene in mouse glioma cell lines (CT2A and GL261) for subsequent investigations ([83]Figure S1B). Furthermore, we confirmed the safety of our oncolytic virus in vivo, and immunohistochemical staining demonstrated that viral replication was restricted to tumor tissue ([84]Figure S1C). Treatment with oHSV1 did not affect mouse survival and body weight nor did it alter the morphology of major organs ([85]Figures S1D–S1F). Figure 1. [86]Figure 1 [87]Open in a new tab Genome-wide CRISPR screening reveals regulators of oncolytic virus resistance (A) Cell viability of oHSV1-treated human glioblastoma cells (MGG4, GSC3264, and BNI21) and normal neural cells (hNP1, ENSA, and iPSC-derived neuron) (0.2 MOI, 24 hpi) (n = 3). (B) Schematic illustration of cerebral organoid and patient-derived glioblastoma tumor section preparation with oHSV1 treatment. Analysis of oHSV1 replication in cerebral organoids and patient-derived glioblastoma tumor sections. After infection with oHSV1 (1 × 10^5 PFU, 24 hpi), the distribution of oHSV1 in the cerebral organoids and tumor sections was detected by anti-HSV1 immunohistochemistry. Scale bars, 100 μm. (C) Schematic illustration of the CRISPR screening for oHSV1 treatment in CT2A^Nectin1 cells. (D) Venn diagram showing the intersection of essential genes from the oHSV1 treatment vs. control comparison and control vs. start point comparison (left). Gene Ontology analysis of the oHSV1-specific candidate genes (right). (E) Scatterplot for the β scores of each gene in the oHSV1 treatment group versus the control group and control group versus the start point group. The selected genes (921) in control vs. the start point comparison were gated in red frame. The selected genes (402) in oHSV1 treatment vs. control comparison were gated in blue frame. The oHSV1-specific genes (351) were gated in black frame. The positive selection Nectin1 (black), canonical BAF subunits (green), non-canonical BAF subunits (red), and polybromo-associated BAF-specific subunits (blue) are highlighted. (F) Diagram showing three different variants of switch/sucrose non-fermentable complex (SWI/SNF) complexes: canonical BAF (cBAF), noncanonical BAF (ncBAF), and polybromo-associated BAF (PBAF). cBAF-specific subunits (Arid1a and Arid1b) are colored green, ncBAF-specific subunits (Brd9, Gltscr1, and Gltscr1l) are colored red, and PBAF-specific subunits (Arid2, Brd7, and Pbrm1) are colored blue. Shared components among the complexes are colored gray (D1: Smarcd1; D2: Smarcd2; D3: Smarcd3; E1: Smarce1; B1: Smarcb1; A2: Smacra2; A4: Smacra4; Ab: β-actin; C1: Smacrc1; C2: Smarcc2; A6a: Actl6a; B7: Bcl7). (G) Cell viability of oHSV1-treated CT2A^Nectin1 cells (0.2 and 0.5 MOI, 24 hpi) transfected with sgRNA targeting the indicated SWI/SNF complex-specific subunits (n = 3). Data represent mean ± SD. Unpaired two-tailed Student’s t test (A), one-way ANOVA (G). The diagrams (B and C) were created using BioRender. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. To identify genes that potentially promote resistance to oncolytic virus-mediated cytotoxicity, we performed a genome-wide CRISPR screen on mouse CT2A^Nectin1 glioma cells. The lentivirus transduced tumor cells were treated with oHSV1 or control PBS ([88]Figure 1C). The enrichment and deletion of single-guide RNAs (sgRNAs) between samples and hits identification were computed by Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) software.[89]^17 Also, we utilized the Bayesian Analysis of Gene Essentiality 2 (BAGEL2)[90]^18^,[91]^19 approach to confirm the quality of our CRISPR screening result ([92]Figures S2A–S2F). Adopting the cutoff criteria of a β score of less than −0.5 and a p value of less than 0.05, we identified 402 potential genes that may confer resistance to oHSV1 infection ([93]Figure 1D). To identify the specific genes that contribute to oHSV1 infection resistance, we excluded 51 genes that generally affect cellular proliferation in our screening data and then narrowed down the number of potential genes implicated in oHSV1-mediated cytotoxicity to 351 ([94]Figure 1D). Gene Ontology analysis of these genes highlighted several significantly enriched molecular functions and biological processing, such as chromatin binding and protein-DNA complex organization ([95]Figure 1D). Indeed, the positive selection of the HSV1 entry receptor Nectin1[96]^16 in our screening data aligns with our expectations and further attests to the robustness and reliability of our screening approach ([97]Figure 1E). Interestingly, we observed that unique subunits of the ncBAF complex, Brd9, and glioma tumor suppressor candidate region gene 1-like (Gltscr1l)[98]^20 were enriched in oHSV1-specific gene selection ([99]Figures 1E and 1F). In contrast, the unique subunits of the other two BAF complexes, canonical BAF (cBAF) complex and polybromo BAF complex, AT-rich interaction domain 1a (Arid1a), AT-rich interaction domain 1b (Arid1b), bromodomain-containing protein 7 (Brd7), polybromo 1 (Pbrm1), and AT-rich interaction domain 2 (Arid2),[100]^20 were not enriched in this selection ([101]Figures 1E and 1F). This finding suggested that the ncBAF complex may play an important role in suppressing oHSV1-mediated cytotoxicity and virus replication. We proceeded with the study by knocking out individual genes of the unique subunits of each BAF complex to examine the impact on oHSV1-mediated cytotoxicity in both mouse and human glioma models. Indeed, we found that knockout of the unique subunits of the ncBAF complex (Brd9 and Gltscr1l) could enhance oHSV1-mediated cytotoxicity, whereas knockout of the unique subunits of the cBAF and polyBAF complexes did not significantly impact oHSV1-mediated cytotoxicity ([102]Figures 1G and [103]S2G). Thus, we identified that the ncBAF complex is an important promoter of oHSV1 resistance in glioblastoma cells. BRD9 knockout enhances oHSV1-induced ICD and viral replication in glioblastoma cells To investigate the role of the ncBAF complex in resistance to oHSV1 treatment, we focused on BRD9, a unique subunit of the ncBAF complex, which also has small-molecule inhibitors.[104]^21^,[105]^22 Knockout of BRD9 using two different sgRNAs did not impact cell proliferation in mouse or human glioblastoma cell lines ([106]Figures S3A and S3B), consistent with our CRISPR screen results. Flow cytometry analysis of Annexin V/propidium iodide (PI) staining revealed that BRD9 knockout cells exhibited increased sensitivity to oHSV1-induced cell death ([107]Figures 2A and 2B). Quantitative PCR analysis of HSV1 glycoprotein D indicated that BRD9 knockout enhanced viral replication in glioblastoma cells ([108]Figure 2C). Additionally, viral plaque assays showed a 2- to 3-fold increase in viral load in the culture medium from BRD9 knockout cells compared to controls ([109]Figures 2D and 2E). Previous studies have shown that oHSV1 induces ICD in tumor cells, releasing damage-associated molecular patterns such as surface-exposed calreticulin (CRT), secreted ATP, and high-mobility group protein B1 (HMGB1), which activate dendritic cells (DCs) and T cells to stimulate an antitumor immune response.[110]^23^,[111]^24 We assessed these ICD markers and found that BRD9 knockout elevated CRT exposure, increased extracellular ATP levels, and enhanced HMGB1 release in response to oHSV1 ([112]Figures 2F–2I). This suggests that BRD9 knockout amplifies oHSV1-induced ICD in glioblastoma cells. Furthermore, we found that BRD9 knockout enhanced the antigen cross-presentation of CT2A^Nectin1-OVA-B2m^−/− tumor cells by DCs, as evidenced by increased OT-1 proliferation and activation ([113]Figures S3C and S3D). Further examination showed that BRD9 ablation did not affect viral binding or entry abilities for host cells ([114]Figures S3E–S3H). In summary, our findings suggest that disrupting the ncBAF complex through BRD9 knockout promotes viral replication and enhances oHSV1-induced cell death, particularly ICD, leading to an increased antitumor immune response. Figure 2. [115]Figure 2 [116]Open in a new tab Knockout of BRD9 enhances the sensitivity of glioblastoma cells to oHSV1-mediated ICD and potentiates viral replication (A) Flow cytometry analysis of oHSV1-treated control or Brd9-deficient CT2A^Nectin1 cells subjected to PI/Annexin V staining for cell death analysis (PI+) (n = 3). (B) Flow cytometry analysis of oHSV1-treated control or BRD9-deficient MGG4 cells subjected to PI/Annexin V staining for cell death analysis (PI+) (n = 3). (C) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in oHSV1-treated control or BRD9-deficient glioma cells (CT2A^Nectin1 and MGG4). GAPDH transcript normalization (n = 3). (D) Schematic illustration of the plaque formation assay in Vero cells. (E) Plaque formation assay using culture medium from oHSV1-treated control or BRD9-deficient glioma cells (CT2A^Nectin1 and MGG4). Left: representative images of crystal violet-stained Vero cell plaques treated with different culture medium (n = 3). (F) Calreticulin (CRT) exposure analysis of oHSV1-treated control or Brd9-deficient CT2A^Nectin1 cells (n = 3). (G) Calreticulin (CRT) exposure analysis of oHSV1-treated control or BRD9-deficient MGG4 cells (n = 3). (H) Extracellular ATP level analysis in control or BRD9-deficient glioma cells (CT2A^Nectin1 and MGG4) treated with oHSV1 (n = 3). (I) Extracellular HMGB1-level analysis in control or BRD9-deficient glioma cells (CT2A^Nectin1 and MGG4) treated with oHSV1 (n = 3). Data represent mean ± SD. Two-way ANOVA (A, B, F, G, H, and I), one-way ANOVA (C and E). The diagram (D) was created using BioRender. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. Brd9 knockout enhances the antitumor activity of oHSV1 therapy in vivo We then investigated whether ablation of BRD9 enhances the therapeutic potency of oHSV1 in vivo. We intracranially implanted Brd9 knockout or control CT2A^Nectin1 and GL261^Nectin1 mouse glioma cells into immunocompetent mice (C57BL/6J) followed by oHSV1 treatment. We observed markedly enhanced survival outcomes in oHSV1-treated mice bearing Brd9 knockout tumors ([117]Figures 3A, 3B, [118]S4A, and S4B). However, without oHSV1 treatment, there was no significant difference in survival between control tumor cell-bearing mice and Brd9 knockout tumor cell-bearing mice ([119]Figures 3A, 3B, [120]S4A, and S4B). Moreover, we intracranially implanted BRD9 knockout or control human patient-derived GBM cells (MGG4) into NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice, followed by treatment with oHSV1. Our results demonstrated prolonged survival in oHSV1-treated mice with BRD9-knockout tumors ([121]Figure S4C). These results align with our in vitro data, confirming that BRD9 ablation enhances oHSV1 therapeutic efficacy without directly affecting tumor cell proliferation. Immunohistochemical staining with an anti-HSV1 antibody and quantitative real-time PCR for HSV1 genomic DNA further confirmed that Brd9 knockout enhanced HSV1 replication in vivo ([122]Figures 3C and [123]S4D–S4F). Figure 3. [124]Figure 3 [125]Open in a new tab Knockout of Brd9 increases the efficacy of oHSV1 therapy in mouse glioma models and augments the corresponding immune cell response (A and B) GBM mouse models were established by intracranial injection of 2 × 10^4 control or Brd9-deficient mouse glioma cells (CT2A^Nectin1 and GL261^Nectin1) into C57BL/6J mice. 3 days later, the mice were intratumorally injected with 6 × 10^5 PFU of oHSV1 or mock treatment. (A) Survival curve of control or Brd9-deficient CT2A^Nectin1 tumor-bearing mice treated with oHSV1 or mock treatment (n = 6). (B) Survival curve of control or Brd9-deficient GL261^Nectin1 tumor-bearing mice treated with oHSV1 or mock treatment (n = 6). (C) Analysis of oHSV1 replication in the brains of control or Brd9-deficient CT2A^Nectin1 tumor-bearing mice. The samples were collected 2 days after intratumoral injection with 6 × 10^5 PFU of oHSV1; the distribution of oHSV1 in the tumors was detected via anti-HSV1 immunohistochemistry. Scale bars, left, 100 μm; right, 50 μm; (n = 3). (D) Flow cytometry analysis of the percentages of CD3^+ T cells and cDC1s (CD11c+MHCII+XCR1+) in the control or Brd9-deficient CT2A^Nectin1 tumor samples within oHSV1 treatment (n = 3 in mock group and n = 4 in oHSV1 treatment group). (E) scRNA-seq analysis of CD45^+ cells from oHSV1-treated control or Brd9-deficient CT2A^Nectin1 tumor-bearing mice. The CD45^+ tumor-infiltrating leukocytes were sorted with anti-mouse CD45 magnetic beads and combined in equal numbers within the same groups for 10× Genomics scRNA-seq (n = 4 mice per group). Uniform manifold approximation and projection (UMAP) plot of the scRNA-seq data depicting the different immune cell subsets (left). The proportion of cells in each cluster in the oHSV1-treated Brd9 deficiency group vs. the oHSV1-treated control group (right). Positive values indicate an increase in cluster occupancy following Brd9 deficiency. (F) UMAP plot of T cell subclusters (left). The proportion of cells in each subcluster in the oHSV1-treated Brd9-deficient group vs. the oHSV1-treated control group (right). Positive values indicate an increase in cluster occupancy following Brd9 deficiency. (G) Characterization of clusters with functional T cell markers. Dot plot shows the average expression levels and cell expression proportions of selected T cell marker genes (top); larger dots indicate a higher proportion of cells with expression, and red versus blue indicates higher expression. Heatmap shows the scaled expression of T cell functional markers (bottom). (H) Flow cytometry analysis of the percentages of CD8^+ T cells and CD8^+ T cells expressing functional markers (CD69 and GZMB) in the control or Brd9-deficient CT2A^Nectin1 tumor samples within oHSV1 treatment (n = 3 in mock group and n = 4 in oHSV1 treatment group). (I) Flow cytometry analysis of the percentage of central memory CD8^+ T cells (TCM, identified as CD44^+ and CD62L+ in CD8^+ cells) in the control or Brd9-deficient CT2A^Nectin1 tumor samples within oHSV1 treatment (n = 4). Data represent mean ± SD. Log rank test (A and B), unpaired two-tailed Student’s t test (C), two-way ANOVA (D, H, and I). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. Oncolytic virotherapy not only exerts direct cytotoxic effects on tumor cells but also elicits antitumor immune responses within the TME.[126]^6^,[127]^25 To investigate the differences in the TME between Brd9-knockout and control tumors after oHSV1 treatment, we performed both flow cytometry and single-cell RNA sequencing (scRNA-seq). Flow cytometry data revealed that Brd9 ablation promoted the fraction of T cells and type 1 conventional DCs (cDC1s), as well as reduction of tumor-associated microglia in oHSV1-treated tumors ([128]Figures 3D and [129]S4G). Insights from scRNA-seq further confirmed that Brd9 knockout increased the infiltration of immune cells critical for the antitumor immune response, including T cells, cDC1s, and natural killer cells ([130]Figures 3E and [131]S4H). T cells play a crucial role in the antitumor immune response within oncolytic virotherapy. We observed an increased percentage of T cells in both flow cytometry and scRNA-seq data. Further analysis of T cell subsets revealed that three CD8^+ T cell clusters were enriched in Brd9-knockout tumors treated with oHSV1 ([132]Figure 3F). Among these clusters, cluster C2 exhibited high expression of stem-like T cell markers, including Lef1 and Tcf7. Clusters C3 and C1 expressed cytotoxic markers such as Gzmb and Prf1, with cluster C1 also displaying cell proliferation markers (Mki67, Top2a, Stmn1, and Tuba1b) ([133]Figure 3G). Flow cytometry analysis confirmed the upregulation of both CD8^+ T cell infiltration and the T cell activation marker CD69, as well as the cytotoxicity marker GZMB, on infiltrating CD8^+ T cells in oHSV1-treated Brd9-knockout tumors ([134]Figure 3H). We also observed an increased frequency of central memory-like CD8^+ T cells (defined as CD44^+CD62L+ in CD8^+ T cells) in Brd9-knockout tumors ([135]Figure 3I). To further investigate the role of CD8^+ T cells in oHSV1 therapy, we conducted a CD8^+ T cell depletion experiment. Depletion of CD8^+ T cells markedly diminished the survival outcomes associated with oHSV1, highlighting the critical role of CD8^+ T cells in the immune response to oHSV1 ([136]Figures S4I and S4J). BRD9 cooperates with RELA to regulate the expression of antiviral genes To further explore the underlying molecular mechanism, we performed bulk RNA sequencing (RNA-seq) to analyze the differences in the transcriptome between BRD9 knockout and control samples in MGG4 cells after oHSV1 treatment ([137]Figure 4A). Pathway enrichment analysis identified significant suppression of the nuclear factor κB (NF-κB) pathway in BRD9-knockout samples ([138]Figure 4B), which is a pathway essential for the antiviral response.[139]^26 Gene set enrichment analysis also demonstrated that genes downregulated in the BRD9 knockout group were highly enriched in the NF-κB signaling pathway ([140]Figure 4C). To further validate our findings, we examined the expression of several NF-κB signaling downstream antiviral response genes, including BST2, ISG20, and OPTN,[141]^27^,[142]^28^,[143]^29 via quantitative real-time PCR. Consistent with the RNA-seq results, BRD9 knockout prevented the increase in the expression of these genes following oHSV1 infection ([144]Figures 4D and [145]S5A). To investigate whether BRD9 directly regulates the expression of these genes, we performed BRD9 chromatin immunoprecipitation sequencing (ChIP-seq). Approximately 60% of the genes downregulated by BRD9 knockout, according to the RNA-seq data, were found to be directly bound by BRD9 ([146]Figure 4E). These BRD9-bound genes were also enriched in the NF-κB signaling pathway ([147]Figure 4F). Driven by the consistent pathway enrichment observed in both RNA-seq and ChIP-seq data, we hypothesized that BRD9 might modulate antiviral-related genes via interaction with RELA, the key transcription factor in the NF-κB signaling pathway. Our ChIP-seq data confirmed the binding of BRD9 with RELA ([148]Figure 4G), particularly for specific antiviral genes ([149]Figures 4H and [150]S5B). Subsequently, coimmunoprecipitation (coIP) was employed to further substantiate the physical interaction between BRD9 and RELA ([151]Figure 4I). Interestingly, our CRISPR screen data further revealed that RELA knockout increased tumor cell sensitivity to oHSV1 infection, a phenotype consistent with that of BRD9 knockout ([152]Figure 4J). Further validation confirmed that either RELA knockout or treatment with the RELA inhibitor SC75741 enhanced oHSV1-mediated cell death and increased viral replication ([153]Figures 4K–4M, [154]S5C, and S5D). RELA knockout or inhibition also reduced the expression of the same antiviral response genes, including BST2, ISG20, and OPTN, as those regulated by BRD9 ([155]Figures 5N and [156]S5E). In summary, our results suggest that BRD9 interacts with RELA and upregulates the expression of downstream antiviral genes. Figure 4. [157]Figure 4 [158]Open in a new tab BRD9 interacts with RELA to regulate antiviral gene expression (A) Volcano plot illustrating differential gene expression between oHSV1-treated control MGG4 cells and oHSV1-treated BRD9 knockout MGG4 cells. Genes with log2(fold change) > 1 and false discovery rate (FDR) < 0.05 are marked in red. Genes with log2(fold change) < −1 and FDR < 0.05 are marked in blue. Other genes are marked in gray. (B) Pathway enrichment analysis of genes downregulated in BRD9 knockout MGG4 cells following oHSV1 treatment. (C) Gene set enrichment analysis (GSEA) of TNFA signaling via NFKB pathway in oHSV1-treated BRD9 knockout MGG4 group vs. oHSV1-treated control MGG4 group. (D) Quantitative real-time PCR analysis of antiviral gene (BST2, ISG20, and OPTN) expression in control and BRD9-deficient MGG4 cells upon oHSV1 treatment. GAPDH transcript normalization (n = 3). (E) Pie chart representing BRD9 binding to genes that were significantly downregulated in BRD9 knockout cells after oHSV1 infection, as determined by BRD9 ChIP-seq. (F) Pathway enrichment analysis of BRD9 binding to genes that were significantly downregulated in BRD9 knockout cells after oHSV1 treatment. (G) Histogram representation of ChIP read density (±1 kb) at BRD9 and RELA common binding sites in control and BRD9 knockout MGG4 cells. (H) Genome browser tracks of BRD9 ChIP-seq signals and RELA ChIP-seq signals ([159]GSM2394420) at BST2, ISG20, and OPTN loci. (I) CoIP analysis of the interaction between RELA and BRD9 in nuclear extracts from MGG4 cells. (J) Scatterplot for the β scores of each gene in the oHSV1 treatment vs. the control comparison and the control vs. start point comparison. Rela (green) and Brd9 (red) are highlighted. (K) Western blot analysis of the RELA knockout efficiency in MGG4 cells. (L) Flow cytometry analysis of cell death in oHSV1-treated control or RELA-deficient MGG4 cells, as indicated by PI/Annexin V staining (PI+) (n = 3). (M) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in control or RELA-deficient MGG4 cells following oHSV1 treatment. GAPDH transcript normalization (n = 3). (N) Quantitative real-time PCR analysis of antiviral genes (BST2, ISG20, and OPTN) in control or RELA-deficient MGG4 cells following oHSV1 treatment. GAPDH transcript normalization (n = 3). Data represent mean ± SD. Two-way ANOVA (D, L, and N) and unpaired Student’s t test (M). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. Figure 5. [160]Figure 5 [161]Open in a new tab Pharmaceutically targeting BRD9 enhances the antitumor effect of oHSV1 in vitro (A) Flow cytometry analysis of oHSV1-treated control or IBRD9-pretreated (1 μM, 24 h) CT2A^Nectin1 and MGG4 cells subjected to PI/Annexin V staining for cell death analysis (PI+) (n = 3). (B) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in control or IBRD9-pretreated (1 μM, 24 h) CT2A^Nectin1 and MGG4 cells treated with oHSV1. GAPDH/Gapdh transcript normalization (n = 3). (C) Plaque formation assay of oHSV1-treated control or IBRD9-pretreated (1 μM, 24 h) CT2A^Nectin1 and MGG4 cell culture medium. The virus titer was determined after 48 h (n = 3). (D) Calreticulin (CRT) exposure analysis of control or IBRD9-pretreated (1 μM, 24 h) CT2A^Nectin1 cells treated with oHSV1 (n = 3). (E) Calreticulin (CRT) exposure analysis of control or IBRD9-pretreated (1 μM, 24 h) MGG4 cells treated with oHSV1 (n = 3). (F) Extracellular ATP level analysis in control or IBRD9-pretreated (1 μM, 24 h) CT2A^Nectin1 and MGG4 cells treated with oHSV1 (n = 3). (G) Extracellular HMGB1 level analysis in control or IBRD9-pretreated (1 μM, 24 h) CT2A^Nectin1 and MGG4 cells treated with oHSV1 (n = 3). (H) In vitro co-culture proliferation experiments with OT-I CD8^+ T cells and cDC1s generated from WT mice in the IBRD9- and oHSV1-treated CT2A^Nectin1-OVA-B2m^−/− cells (n = 3). (I) Schematic of human glioblastoma-derived organoid processing and verification of oHSV1-mediated killing by PI staining, 3D cell titer assays, and ICD marker analysis. (J) PI staining of IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1. Scale bars, 300 μm. (K) 3D cell viability assay of IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 (n = 3). (L) Extracellular ATP-level analysis in control or IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 (n = 3). (M) Extracellular HMGB1-level analysis in control or IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 (n = 3). (N) Schematic of human glioblastoma-derived tumor slice processing and verification of oHSV1 replication by anti-HSV1 staining and quantitative real-time PCR. (O) Representative immunohistochemistry images of HSV1 staining in IBRD9-pretreated or control human glioblastoma-derived tumor slices. Scale bars, 50 μm. (P) Analysis of oHSV1 replication in IBRD9-pretreated (2 μM, 24 h) or control human glioblastoma-derived tumor slices. After oHSV1 treatment, the distribution of oHSV1 in the sections was detected by anti-HSV1 immunohistochemistry (n = 3). (Q) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in control or IBRD9-pretreated (2 μM, 24 h) human glioblastoma-derived tumor sections treated with oHSV1. GAPDH transcript normalization (n = 3). Data represent mean ± SD. Two-way ANOVA (A, D, E, F, G, H, K, L, and M), unpaired two-tailed Student’s t test (B, C, P, and Q). The diagrams (I and N) were created using BioRender. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. Pharmaceutically targeting BRD9 enhances the antitumor effect of oHSV1 We then investigated the potential of pharmaceutically targeting BRD9 to augment the antitumor efficacy of oHSV1. Several BRD9 inhibitors or proteolysis-targeting chimera degraders have been developed recently, including IBRD9 and CFT8634.[162]^21^,[163]^22 We then treated human and mouse glioblastoma cells with IBRD9, followed by treatment with oHSV1. We found that BRD9 inhibition increased oHSV1-induced cytotoxicity in both cell types ([164]Figure 5A). Moreover, BRD9 inhibition enhanced virus replication and increased virus production ([165]Figures 5B and 5C). Furthermore, IBRD9 treatment also augmented oHSV1-induced ICD in glioblastoma cells, indicated by surface-exposed CRT, extracellular ATP, and HMGB1 release ([166]Figures 5D–5G). These findings suggest that BRD9 inhibition amplifies oHSV1-induced cell death, particularly ICD, and promotes viral replication in tumor cells. We next performed the cross-presentation experiment to further determine whether IBRD9 could improve the antitumor immune response in combination with oHSV1 treatment. Our findings indicate that inhibition of BRD9 enhances antigen cross-presentation by DCs of CT2A^Nectin1-OVA-B2m^−/− tumor cells, as evidenced by increased OT-1 proliferation ([167]Figure 5H). To bridge the gap between in vitro cell lines and the complexities of primary tumor tissues, we employed glioblastoma patient-derived organoids and primary tumor slices to investigate the impact of BRD9 inhibition on oHSV1-mediated cytotoxicity and virus replication in these ex vivo models.[168]^30^,[169]^31 Surgical samples obtained from patients with glioblastoma were used to generate organoids, which were subsequently treated with IBRD9 and oHSV1 ([170]Figure 5I). PI staining and luminescent-based organoid viability assays revealed enhanced cytotoxicity upon combination treatment with IBRD9 and oHSV1 in GBM organoids ([171]Figures 5J and 5K). Moreover, the release of ATP and HMGB1 demonstrated that treatment with IBRD9 and HSV1 enhanced the HSV1-induced ICD in human glioblastoma organoids ([172]Figures 5L and 5M). Then, we utilized patient-derived human glioblastoma slices to evaluate the function of IBRD9 in viral infection ([173]Figure 5N). Treatment with IBRD9 and oHSV1 enhanced virus replication in human glioblastoma slices, as evidenced by anti-HSV1 staining and HSV1 glycoprotein D qPCR results ([174]Figures 5O–5Q). In our in vivo tumor assessments, IBRD9 treatment alone did not exert a notable impact on mouse survival. Intriguingly, combination therapy using oHSV1 and IBRD9 significantly enhanced mouse survival outcomes ([175]Figures 6A and 6B). Additionally, analysis of T cell proportions and functional markers (CD69 and GZMB) in combination treatment with IBRD9 and oHSV1 revealed higher fraction of CD8^+ T cells and functional marker expression in tumors from IBRD9 and oHSV1 dual-treated mice than in those from mice treated solely with oHSV1 ([176]Figure S6A). The efficacy of combining immune checkpoint blockade (ICB) with the oncolytic virus has been well documented, leading to corresponding clinical trials.[177]^25^,[178]^32 In our experiments, triple combination therapy involving IBRD9, oHSV1, and ICB using anti-mouse programmed cell death protein 1 (PD-1) and anti-mouse cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) exhibited markedly superior antitumor effects compared to any mono- or dual-combination therapy ([179]Figures 6A and 6B). Remarkably, this triple therapy regimen almost entirely eradicated the tumors ([180]Figures 6A and 6B). To model the inevitable tumor recurrence observed in patients with GBM and assess the antitumor efficacy of central memory-like T cells, we rechallenged long-term survivor mice that had received triple combination therapy 60 days after the initial tumor implantation. Age-matched control mice succumbed to the two mouse tumor models within 1 month, whereas long-term survivors exhibited significant protection against tumor rechallenge, with 4 of 5 mice achieving tumor regression in the CT2A^Nectin1 mouse model ([181]Figure 6C) or complete tumor regression in the GL261^Nectin1 mouse model ([182]Figure 6D). Figure 6. [183]Figure 6 [184]Open in a new tab Pharmaceutically targeting BRD9 enhances the antitumor effect of oHSV1 in vivo, and BRD9 expression is associated with poor clinical outcome in cancer patients treated with oHSV1 (A) Survival curve of IBRD9, oHSV1, and ICB (the anti-mouse PD-1 antibody and the anti-mouse CTLA4 antibody) combination therapy in CT2A^Nectin1 tumor-bearing mice (n = 5). (B) Survival curve of IBRD9, oHSV1, and ICB (the anti-mouse PD-1 antibody and the anti-mouse CTLA4 antibody) combination therapy in GL261^Nectin1 tumor-bearing mice (n = 5). (C) Survival curve of long-term survivor mice and age-matched control mice challenged with CT2A^Nectin1 cells (n = 5). (D) Survival curve of long-term survivor mice and age-matched control mice challenged with GL261^Nectin1 cells (n = 5). (E) Kaplan-Meier analysis showing the PFS of patients with glioblastoma treated with oHSV1 subdivided by the expression of BRD9 (high-expression patients: n = 7; low-expression patients: n = 6). HR, hazard ratio. (F) Analysis of BRD9 expression in liver cancer and pancreatic cancer biopsy sections from patients participating in an oHSV1 clinical trial. Representative immunohistochemistry images of BRD9 staining (oHSV1 response: SD, stable disease; oHSV1 nonresponse: PD, progressive disease). Scale bars, 50 μm. (G) BRD9-stained sections were quantified by H-score (SD: n = 14; PD: n = 13). Data represent mean ± SD. Unpaired two-tailed Student’s t test (G) and log rank test (A–E). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. To further elucidate the clinical implications of our findings, we analyzed RNA-seq data and progression-free survival (PFS) data obtained from patients with glioblastoma enrolled in a recently published oHSV1 clinical trial for patients with GBM[185]^9 ([186]NCT03152318). Notably, lower expression levels of BRD9 mRNA significantly correlated with prolonged PFS ([187]Figure 6E). Beyond glioblastoma, immunohistochemistry analysis of BRD9 protein levels in liver and pancreatic cancer samples from an oHSV1 clinical trial further demonstrated that reduced BRD9 expression was associated with improved clinical outcomes[188]^33 ([189]NCT04806464) ([190]Figures 6F and 6G). These findings suggest that BRD9 may serve not only as a therapeutic target for combination therapy with oncolytic viruses, but also as a prognostic biomarker for predicting treatment outcomes in oncolytic virotherapy. Discussion Despite the considerable advances and promising outcomes in oncolytic virus therapy for cancer treatment, its overall response rate and antitumor efficacy have been limited.[191]^10 One of the major hurdles lies in tumor-intrinsic factors that determine resistance to oncolytic viruses.[192]^34^,[193]^35 By leveraging a comprehensive whole-genome CRISPR-Cas9 knockout screen, we discovered that the ncBAF complex is a pivotal regulator of such resistance. Genetic or pharmacological disruption of BRD9, a unique subunit of the ncBAF complex, markedly augments the replication and antitumor potency of oncolytic viruses. These findings are further supported by clinical data demonstrating an inverse correlation between BRD9 expression and oncolytic virus treatment success. Consequently, our findings identify BRD9 as a potential predictive biomarker for the clinical outcomes of oncolytic virotherapy and demonstrate that combination therapies incorporating oncolytic viruses and BRD9 inhibitors hold significant promise for advancing the development of effective therapeutic strategies in glioblastoma. Several existing studies have aimed to enhance oncolytic virus infection and replication within tumor cells by modifying the virus structure, thereby improving antitumor efficacy.[194]^36^,[195]^37^,[196]^38^,[197]^39 In contrast, we achieved significant enhancement of oncolytic virus replication within tumor cells by simply treating them with BRD9 inhibitors. This approach mitigates the potential toxic side effects associated with prolonged virus activation. The immunosuppressive TME of GBM tumors also plays a pivotal role in limiting the antitumor effect of oncolytic viruses.[198]^40 Notably, previous studies have shown that BRD9 supports regulatory T (Treg) cell function by upregulating Foxp3 expression.[199]^41 These findings indicate that combining a BRD9 inhibitor with oncolytic virus therapy may provide a dual advantage: enhancing CD8^+ T cell activation while attenuating Treg cell function, thereby reshaping the immunosuppressive TME to support a more robust antitumor immune response. Our research mainly employed GBM as our experimental model for evaluating oncolytic virus therapy; the translational implications of our findings extend beyond brain tumors. Interestingly, we found that clinical trial data on oncolytic virus therapy for liver and pancreatic cancers also reveal a consistent inverse correlation between BRD9 expression and therapeutic response rates. This observation underscores the potential of BRD9 as a target for combination therapies across a variety of tumor types. In our study, we harnessed oncolytic viruses derived from HSV1. In addition to HSV1, other oncolytic viruses, such as adenovirus and poliovirus, are currently being investigated in clinical trials for glioblastoma treatment and have demonstrated promising outcomes.[200]^42^,[201]^43 It has been reported that BRD9 can enhance the interferon (IFN) response genes expression, subsequently inhibiting the replication of various viruses.[202]^44 Taking together, our study identified a different molecular mechanism by which BRD9 modulates oncolytic virus resistance, specifically through the NF-κB pathway, rather than the previously reported IFN signaling, which is often inactive or downregulated in many types of cancers including glioblastoma.[203]^45^,[204]^46^,[205]^47 Hence, targeting BRD9 could potentially enhance the therapeutic effects of various oncolytic viruses in a wide range of cancer types, a prospect that warrants further exploration in future studies. Limitations of the study In the current study, we focused solely on glioblastoma tumor models to assess the therapeutic efficacy of oncolytic viruses. Future studies should include a broader array of tumor models to determine whether the enhancement of oncolytic viruses via BRD9 inhibition holds true across different tumor types. Moreover, our study primarily employed an immunocompetent mouse model, which may not fully replicate the complexities of human cancer. Incorporating humanized mouse models with patient-derived xenografts in future research would improve the translational relevance and applicability of these findings. Future studies will strengthen the translational relevance of this finding. Resource availability Lead contact Further information and request for resources and reagents should be directed to and will be fulfilled by the lead contact, Qi Xie (xieqi@westlake.edu.cn). Materials availability This study did not generate new unique reagents. Data and code availability * • Bulk RNA-seq and scRNA-seq data reported in this paper have been deposited into the National Center for Biotechnology Information Sequence Read Archive under accession number [206]PRJNA1066491. BRD9 ChIP-seq data can be downloaded from the Gene Expression Omnibus website, accession number GEO: [207]GSE260886 . Gene expression and survival data of the phase 1 trial of patients with glioblastoma treated with CAN-3110 (aka rQNestin34.5v.2) were downloaded from the dbGap (phs003378.v1.p1). ChIP-seq data for RELA used in this study were retrieved from [208]GSM2394420. * • The custom code used for this paper is available on GitHub at the following address: [209]https://github.com/Dragonlongzhilin/OV_BRD9. * • Any additional information required to reanalyze the data reported in this work paper is available from the [210]lead contact upon request. Acknowledgments