VHL synthetic lethality screens uncover CBF-β as a negative regulator of STING

Abstract

Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL, and uncover that loss of Core Binding Factor β (CBF-β) causes cell death in VHL-null ccRCC cell lines and impairs tumour establishment and growth in vivo. This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL-null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-β. Mechanistically, CBF-β loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-β at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-β in kidney cancer both selectively induces tumour cell lethality and promotes activation of type I interferon signalling.

Keywords: CBF-β; STING; VHL; clear cell renal cell carcinoma; type I interferon.

Extended Data Fig. 1.

CRISPR/Cas9 screening in ccRCC cell lines reveals VHL-associated synthetic lethality of the transcription factors CBF-β and NRF2 (a) Expression of HIF target genes in ccRCC cells and paired VHL-reconstituted cells after culture at 1% or 21% O₂ for 24 hours. n=3 biologically independent replicates. Mean ± SD. Two-way ANOVA. (b) Cumulative cell doublings from day 3 (786O screen) or day 4 (RCC4 screen) in CRISPR/Cas9 screens. Dotted circles indicate the analysed samples. (c) Efficient identification of essential genes in CRISPR/Cas9 screens. Venn diagrams of essential genes which dropout between early and late timepoints identified by BAGEL2 with FDR<0.05, compared to a reference set of Core Essential Genes. (d-f) Immunoblots of 786O Cas9 and 786O+VHL Cas9 cells transduced with sgRNAs targeting CBF-β (d), shRNAs targeting CBF-β (e), and sgRNAs targeting KEAP1 (f). sgRNA vectors were doxycycline-inducible, and cells were treated with 100 ng/ml doxycycline prior to analysis. Controls were transduced with an empty sgRNA expression vector (d,f), or a scrambled shRNA sequence (e). Immunoblots representative of 3 independent experiments. (g) KEAP1 knockout induces NRF2, but not HIF, activation. qPCR analysis of HIF targets (VEGFA and GLUT1) and NRF2 targets (NQO1 and HMOX1) in cells transduced with an sgRNA targeting KEAP1. n=3 biologically independent replicates. Mean ± SD. Two-way ANOVA.

Extended Data Fig. 2.

CBFB knockout induces cell death in VHL-deficient cells (a) Proliferation assay of ccRCC cells (RCC4, A498, RCC10 and 769P), renal proximal tubule epithelial cells (HKC8), and clonal HIF1β-deficient 786O cells and VHL-reconstituted RCC4 cells, transduced with CBF-β sg2. Cell numbers at day 4 plotted relative to control cultures transduced with an empty vector. n=4 biologically independent replicates. Mean ± SD. Unpaired t-test. (b) Representative immunoblot of transductions assayed in Extended Data Fig. 2a. n=3 biologically independent replicates. (c,d) CBFB does not regulate HIF activity in 786O Cas9 cells. 786O Cas9 and 786O+VHL Cas9 populations were transduced with CBF-β sg2 or an empty vector control and assayed by immunoblot (c), and by qPCR of HIF target genes (d). n=3 biologically independent replicates for c and d. Mean ± SD. Two-way ANOVA. (e) Rate of cell cycle progression in 786O Cas9 and 786O+VHL Cas9 cells transduced with CBF-β sg2 or an empty vector control. Cells were synchronised with a double thymidine block, and fixed for propidium iodide staining and flow cytometry every two hours following release. An asynchronous control is provided for comparison. Representative of 3 biologically independent replicates. (f) Proportion of 786O Cas9 and 786O+VHL Cas9 cells exhibiting caspase-3/7 activity following transduction with CBF-β sg2 or an empty vector control, as detected by the CellEvent Caspase-3/7 Green flow cytometry reagent. [Camp.]: cells treated with 24 hours 100 μM Camptothecin as a positive control for apoptosis. n=3 biologically independent replicates. Mean ± SD. Two-way ANOVA. (g) PARP1 cleavage in cells transduced with CBF-β sg2 or an empty vector control. Lower band on PARP1 membrane represents the cleaved form of PARP1. [Camp.]: 786O Cas9 cells treated with 10 μM Camptothecin as a positive control for apoptosis. Immunoblot representative of 3 biologically independent replicates. (h) 786O Cas9 cells transduced with CBF-β sg2 or an empty vector control, and treated for 48 hours with 50 μM Necrostatin-1s (Nec-1s), 25 μM VRT-043198, 2 μM Ferrostatin-1 or the DMSO vehicle. Cells were assayed by flow cytometry using SYTOX AADvanced dead cell stain. n=4 biologically independent replicates. Mean ± SD. Two-way ANOVA.

Extended Data Fig. 3.

Specific induction of ISG transcription upon CBFB knockout (a) qPCR validation of changes in ISG expression in 786O Cas9 and 786O+VHL cells upon transduction with CBF-β sg2 or an empty vector control. n=3 biologically independent replicates. Mean ± SD. Two-way ANOVA. (b,c) qPCR analysis of changes in IFIT1 expression in 786O Cas9 cells upon transduction with CBF-β sgRNA 1 (sg1) (b) or CBF-β shRNA 1 (c), relative to an appropriate empty vector or scrambled shRNA control. n=3 biologically independent replicates. Mean ± SD. Unpaired t-test. (d) qPCR analysis of OASL expression in 786O Cas9 cells upon transduction with CBF-β sg2 and overexpression vectors encoding CBF-β-FLAG (WT) or CBF-β-FLAG (N104A), or an empty vector control. n=3 biologically independent replicates. Mean ± SD. One-way ANOVA. (e) Expression of IFIT1 assayed by qPCR in A498, RCC10 and 769P ccRCC cell lines upon transduction with CBF-β sg2, normalised to equivalent cells transduced with an empty vector. n=3 biologically independent replicates. Mean ± SD. Unpaired t-test. (f) qPCR analysis of ISGs upon transduction with sgRNAs targeting CBF-β, DAAM1, DLST, or SSR4, relative to an empty vector-transduced control. n=3 biologically independent replicates. Mean ± SD. One-way ANOVA.

Extended Data Fig. 4.

ISG transcription following CBFB deletion is mediated by the STING-TBK1-IRF3 axis (a,b) 786O Cas9 cells were transduced with CBF-β sg2 and sgRNAs targeting STAT1, STAT2 or IRF9, or an empty vector. Cells were analysed by qPCR (a) or immunoblot (b). n=3 biologically independent replicates for (a) and (b). Mean ± SD. Two-way ANOVA. (c) Immunoblot of 786O Cas9 cells transduced with CBF-β sg2, a vector encoding sgRNAs targeting both STAT1 (sg1) and STAT2 (sg2), or an empty vector control. Representative of 3 biologically independent replicates. (d) CBF-β does not affect the phosphorylation status of STAT1. Cells were transduced with CBF-β sg2 or an empty vector control. Positive controls were additionally treated with 10 IU/ml IFN-β for 1 or 24 hours. Immunoblot representative of 3 biologically independent replicates. (e) Immunoblot of experimental conditions assayed in Fig. 5c. Representative of 3 biologically independent replicates. (f) Cells were transduced with CBF-β sg2 or an empty vector control. IRF3 phosphorylation was elicited in positive control cells treated with 20 μg/ml poly(I:C) for 6 hours. Immunoblot representative of 3 biologically independent replicates. (g) qPCR analysis of OASL expression in conditions described in Fig. 5f. n=3 biologically independent replicates. Mean ± SD. One-way ANOVA. (h-j) 786O Cas9 cells were transduced with sgRNAs targeting CBF-β (sg2), IRF3 (sg1), or STING (sg1), or an empty vector control, and assayed by proliferation assay (h,j), or flow cytometry with SYTOX AADvanced dead cell stain (i). n=4 biologically independent replicates. Mean ± SD. Two-way ANOVA.

Extended Data Fig. 5.

Absence of overt mitochondrial dysfunction or genomic DNA damage following CBF-β loss (a,b) 786O Cas9 cells were transduced with CBF-β sg2 or an empty vector, and analysed by flow cytometry with TMRM and MitoTracker Green FM (a) or MitoSOX Red (b) staining to assay mitochondrial membrane potential, total mitochondrial mass, and mitochondrial superoxide generation, respectively. Empty vector-transduced cells treated for 30 minutes prior to staining with inhibitors of oxidative phosphorylation, either 1 μM FCCP (a) or 20 μM Antimycin A (b), served as controls for the efficacy of staining. Representative of 3 (a) or 4 (b) biologically independent replicates. (c) 786O Cas9 cells with CBF-β depleted or overexpressed were analysed by Mito Stress Test using a Seahorse XF analyser to identify the basal level of respiration, compared to cells transduced with an empty lentiviral vector. OCR: oxygen consumption rate. n=2 (CBF-β-FLAG) or 3 (control and CBF-β sg2) biologically independent replicates. Mean ± SD. (d) CBF-β loss does not cause overt DNA damage. 786O Cas9 cells were transduced with CBF-β sg2 or an empty vector, and a population of empty vector-transduced controls were treated with 5 μM Cisplatin for 24 hours prior to analysis. Cells were stained with propidium iodide, and assessed for levels of Ser139 phosphorylation of histone 2A.X (γ-H2A.X) by intracellular antibody staining and flow cytometry. Empty vector controls were either stained fully or with the secondary antibody alone. Representative of 4 biologically independent replicates. (e) Immunoblot of 786O Cas9 cells in the same conditions as described in Fig. 6e, following transfection with 0.5 μg/ml HT-DNA for 6 hours or having been left untransfected. Representative of 4 biologically independent replicates.

Extended Data Fig. 6.

Direct transcriptional repression of STING by CBF-β/RUNX across kidney cell lines (a-c) The expression of STING and downstream ISGs is induced by CBFB knockout in renal proximal tubule epithelial (HKC8), and ccRCC (RCC4 and RCC10) cell lines. qPCR analysis of HKC8 Cas9 (a), RCC4 Cas9 (b), and RCC10 Cas9 (c) cells transduced with CBF-β sg2 or an empty vector (EV), and either transfected with 0.5 μg/ml HT-DNA for 6 hours or left untransfected. n=3 biologically independent replicates. Mean ± SD. Two-way ANOVA (IFIT1, OASL and IFNB1), or unpaired t-test (CGAS and STING). (d) Putative RUNX binding sites at the STING locus on chromosome 5. RUNX1 ChIP-Seq data from K562 cells, and human kidney candidate cis-regulatory element (cCRE) annotations were extracted from ENCODE files ENCFF985UVT and ENCFF657KYN, and visualised in IGV together with STING transcripts derived from the Ensembl genome browser. Primer pairs used for ChIP-qPCR in Fig. 6e were designed around RUNX consensus motifs (5’-YGYGGTY-3’) in the reverse strand, which are indicated as dashes.

Fig. 1.

Genome-wide CRISPR/Cas9 screening reveals synthetic viability interactors of VHL (a) HIF-α levels in ccRCC cells and paired VHL-reconstituted cells after culture at either 1% or 21% O₂ for 24 hours. Immunoblot representative of 3 independent experiments. (b) Schematic of CRISPR/Cas9 screen design. Paired VHL-null and -reconstituted cell lines were transduced with the genome-wide TKOv3 sgRNA library. Cells were passaged in parallel for several weeks, and genomic DNA extracted at early, intermediate and late timepoints for the identification of sgRNA abundance by Next Generation Sequencing (NGS). sgRNAs targeting synthetic lethal interactors of VHL are selectively depleted in the VHL-null 786O Cas9 and RCC4 Cas9 backgrounds. (c) Pairwise analysis of the late timepoints of 786O Cas9 vs. 786O+VHL Cas9 cells (x-axis) and RCC4 Cas9 vs. RCC4+VHL Cas9 cells (y-axis) from CRISPR/Cas9 screens. A higher Bayes Factor calculated by BAGEL2 indicates a more robust synthetic lethal relationship between the given gene and VHL. Orange lines denote FDR<0.05. (d) Normalised sgRNA counts from CRISPR/Cas9 screens. (e) Competitive growth assay method. Transduced fluorophore-expressing 786O and 786O+VHL cells are mixed 1:1, passaged and harvested for flow cytometry analysis. 100 ng/ml doxycycline is added to experiments involving inducible sgRNAs to initiate gene editing. (f,g) Validation of functional interactions between VHL and CBFB (f), or between VHL and KEAP1 and NRF2 (g), by competitive growth assay using doxycycline-inducible sgRNAs or constitutively-expressed shRNAs. n=3 biologically independent replicates. One-way ANOVA based on Area Under the Curve compared to empty vector or scrambled control: CBF-β sgRNA 1 P<0.0001; CBF-β sgRNA 2 P<0.0001; CBF-β shRNA 1 P=0.14; CBF-β shRNA 2 p=0.0034; CBF-β shRNA 3 P=0.042; KEAP1 sgRNA 1 P=0.42; KEAP1 sgRNA 2 P=0.11; NRF2 sgRNA 1 P=0.0002; NRF2 sgRNA 2 P<0.0001.

Fig. 2.

CBF-β loss causes cell death in VHL-null ccRCCs and impairs tumour growth in vivo (a) High expression of CBFB in kidney cancer is associated with poor outcomes. Kaplan-Meier survival analysis of TCGA data for ccRCC, comparing tumours in the highest and lowest quartiles of CBFB mRNA expression. n=130 patients for each group. Log-rank test. (b) Proliferation assay of 786O Cas9 and 786O+VHL Cas9 cells transduced with an sgRNA targeting CBF-β (CBF-β sg2), or an empty vector control. n=4 biologically independent replicates. Two-way ANOVA of cell number at day 4. (c) Cells were transduced with CBF-β sg2 or an empty vector control and analysed by clonogenic assay. Representative image of colonies after staining with 0.5% crystal violet solution. n=4 biologically independent replicates. Mean ± SD. Two-way ANOVA. (d) Proportion of cells in G0/G1, S, and G2/M cell cycle phases in asynchronous 786O Cas9 and 786O+VHL Cas9 populations transduced with CBF-β sg2 or an empty vector control, determined by BrdU incorporation and propidium iodide staining. n=4 biologically independent replicates. Mean ± SD. (e) SYTOX AADvanced cell death assay, indicating the proportion of 786O Cas9 and 786O+VHL Cas9 cells stained positive following transduction with CBF-β sg2 or an empty vector control. n=3 biologically independent replicates. Mean ± SD. Two-way ANOVA. (f) LDH cytotoxicity assay, quantifying the amount of LDH in culture supernatant in 786O Cas9 and 786O+VHL Cas9 cells transduced with CBF-β sg2 or an empty vector control. Background absorbance at 680 nm was subtracted from the 490 nm signal, and readings normalised to a cell-free unconditioned media control. n=3 biologically independent replicates. Mean ± SD. Two-way ANOVA. (g) CBF-β is required for tumour establishment in vivo. Tumour area 28 days after subcutaneous injection of 786O Cas9 cells transduced with sgRNAs targeting CBF-β or LacZ into NSG mice. n=6 mice per group. Mann-Whitney U test. (h-n) Xenograft model of luciferase-expressing 786O Cas9 cells injected orthotopically into the left kidneys of NSG mice, using doxycycline treatment to induce the expression of sgRNAs targeting CBF-β (sg1 and sg2) or LacZ as a control. Representative bioluminescence imaging of mice at day 0 and day 56 after the initiation of doxycycline treatment (h). Quantification of the bioluminescence of primary tumours following doxycycline treatment (i,j). Left kidneys following euthanasia at day 56 post-doxycycline treatment, with tumour mass quantified by the subtraction of right kidney mass from left kidney mass (k,l). Representative and quantified bioluminescence of isolated lungs harvested from mice at day 56 post-doxycycline treatment (m,n). n=10 mice per group, of which 1 died between day 42 and day 56 post-treatment in each of the LacZ and CBF-β sg2 groups. Mean ± SEM (i) or SD (l). One-way ANOVA.

Fig. 3.

Post-transcriptional regulation of RUNX1 and RUNX2 by CBF-β (a) Combined loss of RUNX1 and RUNX2 functionally mimics CBFB deletion. Competitive growth assay of cells transduced with sgRNAs targeting CBF-β, RUNX1, RUNX2, or both RUNX1 and RUNX2. n=3 biologically independent replicates. One-way ANOVA based on Area Under the Curve compared to empty vector control. CBF-β sgRNA 2 P=0.0003; RUNX1 sgRNA P=0.31; RUNX2 sgRNA P=0.12; RUNX1 sgRNA + RUNX2 sgRNA P=0.0024. (b) Proliferation assay of 786O Cas9 cells transduced with sgRNAs targeting CBF-β or RUNX1, RUNX2 and RUNX3 in combination, or an empty vector control. n=4 biologically independent replicates. Mean ± SD. One-way ANOVA. (c) CBF-β loss depletes RUNX protein levels. Immunoblot of cells transduced with sgRNAs targeting CBF-β, RUNX1, RUNX2, or both RUNX1 and RUNX2. Representative of 3 biologically independent replicates. (d) Immunoblot of 786O Cas9 and 786O+VHL Cas9 cells transduced with CBF-β sg2 or an empty vector control. Representative of 3 biologically independent replicates. (e) Cells were transduced with overexpression vectors encoding CBF-β-FLAG (WT), CBF-β-FLAG (N104A), or an empty vector. FLAG-tagged CBF-β and endogenous RUNX2 were immunoprecipitated (IP) from cell lysates as indicated. Immunoblots representative of 3 biologically independent replicates. (f) RUNX-binding mutant of CBF-β restores 786O Cas9 cell growth to the same level as wild-type (WT) CBF-β following CBFB knockout. Competitive growth assay of cells transduced with CBF-β sg2 and overexpression vectors encoding CBF-β-FLAG (WT), CBF-β-FLAG (N104A), or an empty vector control. n=3 biologically independent replicates. One-way ANOVA based on Area Under the Curve compared to empty sgRNA vector control: CBF-β sgRNA 2 + empty vector: P=0.0002; CBF-β sgRNA 2 + CBF-β-FLAG (WT) P=0.066; CBF-β sgRNA 2 + CBF-β-FLAG (N104A) P=0.017. One-way ANOVA based on Area Under the Curve compared to CBF-β sgRNA 2: CBF-β sgRNA 2 + CBF-β-FLAG (WT) P=0.0048; CBF-β sgRNA 2 + CBF-β-FLAG (N104A) P=0.017. (g) Proliferation assay of 786O Cas9 cells transduced with CBF-β sg2 and overexpression vectors encoding CBF-β-FLAG (WT) or CBF-β-FLAG (N104A), or an empty vector control. n=4 biologically independent replicates. Mean ± SD. One-way ANOVA. (h,i) Regulation of RUNX by CBF-β is post-transcriptional. 786O Cas9 cells were transduced with CBF-β sg2 and overexpression vectors encoding CBF-β-FLAG (WT) or CBF-β-FLAG (N104A), or an empty vector control. RUNX protein and mRNA levels were analysed by immunoblot (h) and qPCR (i). n=3 biologically independent replicates. Mean ± SD. One-way ANOVA.

Fig. 4.

CBF-β loss induces an Interferon Stimulated Gene (ISG) signature (a) 786O Cas9 and 786O+VHL Cas9 cells were analysed by RNA sequencing and tandem mass tag (TMT)-labelled LC-MS to assess global changes in mRNA and protein upon CBF-β depletion relative to empty vector-transduced cells. (b) Gene Set Enrichment Analysis of Hallmark gene sets from RNA sequencing analysis of differential gene expression in 786O Cas9 cells following CBFB knockout. n=3 biologically independent replicates. (c,d) Volcano plots of RNA sequencing (c) and mass spectrometry (d) data in the conditions outlined in (a). Orange lines indicate log₂(Fold Change) ± 0.5, and P_adj=0.01. Pink dots highlight genes represented within the Hallmark INTERFERON_ALPHA_RESPONSE. Number of genes upregulated or downregulated (determined by the log₂(Fold Change) and P_adj cut-off values above) are specified. Data points represent the mean value for each gene from 3 biologically independent replicates. (e) IFIT1 is specifically upregulated by CBF-β loss. qPCR analysis of IFIT1 expression in 786O Cas9 cells upon transduction with CBF-β sg2 and overexpression vectors encoding CBF-β-FLAG (WT) or CBF-β-FLAG (N104A), or an empty vector control. n=3 biologically independent replicates. Mean ± SD. One-way ANOVA. (f) qPCR analysis of ISGs in clonal 786O HIF1β knockout Cas9 cells upon CBF-β sg2 transduction, relative to empty vector-transduced controls. n=3 biologically independent replicates. Mean ± SD. Unpaired t-test. (g) Immunoblot of ISGs in 786O Cas9 and 786O+VHL Cas9 cells transduced with CBF-β sg2 or an empty vector control. Controls were treated with 10 IU/ml IFN-β for 1 or 24 hours. Representative of 3 biologically independent replicates. (h) Analysis of single cell transcriptomic data from patients with ccRCC. The average expression and the percentage of cells that express type I interferon genes is shown for the principal cell types within the tumour micro-environment.

Fig. 5.

CBFB loss induces a STING-TBK1-IRF3 cell-intrinsic ISG response (a) Schematic of the type I IFN signalling pathway. Aberrant or foreign nucleic acids are detected by pattern recognition receptors (PRRs), which signal through adaptor proteins to stimulate TBK1-mediated phosphorylation of IRF3. IRF3 heterodimers translocate to the nucleus to trigger transcription of IFNs and a subset of ISGs. Secreted IFNs act through IFNAR receptors to promote STAT1- and STAT2-mediated ISG expression. (b) qPCR analysis of IFIT1 expression in 786O Cas9 cells transduced with a vector encoding sgRNAs targeting both STAT1 (sg1) and STAT2 (sg2), or an empty vector control. Cells were additionally transduced with CBF-β sg2, or treated with 10 IU/ml IFN-β for 6 hours. n=3 biologically independent replicates. Mean ± SD. Two-way ANOVA. (c) ISG expression is dependent on IRF3. qPCR analysis of cells transduced with sgRNAs targeting CBF-β (sg2), IRF3 and IRF7, or an empty vector control. n=3 biologically independent replicates. Mean ± SD. Two-way ANOVA. (d) Cells were transduced with CBF-β sg2 or an empty vector control, and treated with 5 μM GSK8612 for 24 hours (to inhibit TBK1), as indicated. Immunoblot representative of 3 biologically independent replicates. (e) 786O Cas9 cells were transduced with CBF-β sg2 or an empty vector control, treated for 24 hours with the indicated doses of the TBK1 inhibitor GSK8612, the NF-κB inhibitor BAY 11–7085, or the DMSO vehicle, and analysed by qPCR. n=3 biologically independent replicates. Mean ± SD. One-way ANOVA. (f) CBF-β loss specifically activates STING. qPCR analysis of cells transduced with CBF-β sg2 alone or in combination with sgRNAs targeting the PRR adaptors TRIF, MAVS and STING, compared to an empty vector-transduced control. n=3 biologically independent replicates. Mean ± SD. One-way ANOVA. (g) Immunoblot of 786O Cas9 cells transduced with sgRNAs targeting CBF-β (sg2) and STING (sg1), or an empty vector control. Representative of 3 biologically independent replicates. (h) Dose-response relationship between IFN-β treatment and cell proliferation over 72 hours in 786O Cas9 cells and proximal tubule HK2 Cas9 cells. n=6 biologically independent replicates. Mean ± SD.

Fig. 6.

CBF-β represses STING to tune the type I interferon response (a) cGAS-STING signalling axis. cGAS is activated by exogenous or mislocalised double stranded DNA (dsDNA), leading to cGAMP production which triggers a series of phosphorylation events involving STING, TBK1 and IRF3. (b) CBF-β regulates STING transcriptionally. qPCR analysis of CGAS and STING expression in 786O Cas9 cells transduced with CBF-β sg2, a wild-type CBF-β-FLAG overexpression construct, or an empty lentiviral vector. n=3 biologically independent replicates. Mean ± SD. One-way ANOVA. (c) 786O Cas9 cells were transduced with an sgRNA targeting STING, or an empty vector control, and transfected 6 hours prior to analysis with 0.5 μg/ml herring testis DNA (HT-DNA) as indicated. Additional controls (indicated by [+]) were treated for 6 hours with 0.5 μg/ml HT-DNA, in the absence of the Lipofectamine 2000 transfection reagent. qPCR analysis of IFIT1 mRNA expression. n=3 biologically independent replicates. Mean ± SD. Two-way ANOVA. (d) CBF-β controls the magnitude of the ISG response to dsDNA transfection. qPCR analysis of 786O Cas9 cells transduced with CBF-β sg2, a wild-type CBF-β-FLAG overexpression construct, or an empty vector, and either transfected with 0.5 μg/ml HT-DNA for 6 hours or left untransfected. n=3 biologically independent replicates. Mean ± SD. Two-way ANOVA. (e) qPCR analysis of 786O Cas9 cells transduced with sgRNAs targeting CBF-β (sg2) and cGAS, or an empty vector. n=4 biologically independent replicates. Mean ± SD. Two-way ANOVA. (f) Chromatin binding of CBF-β and RUNX2 at various sites in the STING gene, by ChIP-qPCR analysis of 786O Cas9 cells transduced with CBF-β sg2 or an empty vector. Primer locations are illustrated in Extended Data Fig. 6d. n=5 (primers 1 and 2), and n=3 (primers 3 and 4) biologically independent replicates. Mean ± SD. Unpaired t-test. (g) Subcellular fractionation following transduction of 786O Cas9 cells with an HIV GFP-Vif expression construct, or a GFP-only control. Cyt.: cytoplasmic fraction. N.: nucleoplasmic fraction. Chr.: chromatin-associated fraction. Immunoblot representative of 2 biologically independent replicates. (h,i) 786O Cas9 cells were transduced with vectors encoding CBF-β sg2, GFP-Vif, or a GFP-only control, as indicated, and either transfected with 0.5 μg/ml HT-DNA for 6 hours or left untransfected. qPCR analysis of IFIT1, CGAS and STING mRNA expression (h), and immunoblot analysis of ISG15 levels (i). n=4 biologically independent replicates. Mean ± SD. Two-way ANOVA.

Fig. 7.

Model of regulation of STING expression by CBF-β/RUNX In basal conditions, CBF-β acts as a rheostat to fine-tune cGAS-STING pathway activity. In the absence of CBF-β/RUNX nuclear translocation, as occurs with sequestration of CBF-β by the lentiviral protein Vif, STING is abundantly expressed, thus amplifying the response to cGAS activation to stimulate signalling through TBK1 and IRF3, ultimately inducing an IFN/ISG-dependent antiviral state. Conversely, CBF-β and RUNX levels are elevated in ccRCC, permitting cell growth. Additionally, CBF-β and RUNX binding at the STING locus suppresses IFN signalling and likely promotes immune escape.