Fig 2: 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 knockout128. n = 3 biologically independent replicates. Adaptive multi-level split Monte-Carlo method129. 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 Padj = 0.01. Pink dots highlight genes represented within the Hallmark INTERFERON_ALPHA_RESPONSE set. Number of genes upregulated or downregulated (determined by the log2(Fold Change) and Padj cut-off values above) are specified. Data points represent the mean value for each gene from 3 biologically independent replicates. Two-sided Wald test (c), Benjamini-Hochberg-adjusted moderated t-test (d). 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 two-sided 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 h. Representative of 3 biologically independent replicates. h Analysis of single-cell transcriptomic data from patients with ccRCC48. The average expression and the percentage of cells that express type I interferon genes are shown for the principal cell types within the tumour micro-environment. Source data are provided as a Source Data file.

Fig 3: CBF-β loss causes cell death in VHL-null ccRCCs and impairs tumour growth in vivo.a Relative CBF-β protein expression in ccRCC samples and matched normal kidney tissue within the CPTAC ccRCC Discovery dataset126,127. n = 84 individuals. Mean denoted by horizontal bar. Paired two-sided t-test. b, c Kaplan–Meier survival analysis of TCGA (b) and CPTAC (c) ccRCC data7,126,127, comparing tumours in the highest and lowest quartiles of CBFB mRNA (b) or CBF-β protein (c) expression. n = 130 (b) or 27 (c) individuals per group. Log-rank test. d Cells transduced with CBF-β sg2 or an empty vector control analysed by clonogenic assay. Representative image of crystal violet-stained colonies. n = 4 biologically independent replicates. Mean ± SD. Two-way ANOVA. e Proportion of cells in the indicated cell cycle phases in asynchronous cell populations transduced with CBF-β sg2 or a control, determined by BrdU incorporation and propidium iodide staining. n = 4 biologically independent replicates. Mean ± SD. f SYTOX AADvanced cell death assay of 786O Cas9 and 786O + VHL Cas9 transduced with CBF-β sg2 or a control. n = 3 biologically independent replicates. Mean ± SD. Two-way ANOVA. g LDH in culture supernatants in cells transduced with CBF-β sg2 or a control. n = 3 biologically independent replicates. Mean ± SD. Two-way ANOVA. h Tumour volume following subcutaneous injection of 786O Cas9 cells transduced with sgRNAs targeting CBF-β or LacZ into NSG mice. Tumours in this model show an initial growth before regressing and finally adopting a sustained growth trajectory after 21 days124. n = 6 mice per group. Curves connect mean value for each timepoint. Two-sided Mann–Whitney U test of tumour volumes at day 28 post-injection. i–m Xenograft model of luciferase-expressing 786O Cas9 cells injected orthotopically into the left kidneys of NSG mice, using doxycycline treatment to induce 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 (i). Quantification of the bioluminescence of primary tumours following doxycycline treatment (j, k). Representative and quantified bioluminescence of isolated lungs harvested from mice at day 56 post-doxycycline treatment (l, m). n = 10 mice per group, of which 1 died between days 42 and 56 in the LacZ and CBF-β sg2 groups. Mean ± SEM. One-way ANOVA. Source data are provided as a Source Data file.

Fig 4: 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 h. 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. Guides targeting EGFP serve as negative controls. 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. Curves connect mean value for each timepoint. 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. Source data are provided as a Source Data file.

Fig 5: RUNX/CBF-β proteins directly inhibit STING transcription.a Chromatin binding profile of CBF-β at STING and IFIT1. ChIP-Seq analysis of 786O Cas9 cells expressing the CBF-β-FLAG overexpression plasmid or an empty vector. Mean normalised reads across all replicates displayed using the IGV genome browser. Arrows indicate the directionality of the gene of interest. RUNX consensus motifs (5’-YGYGGTY-3’) are denoted below each track. n = 3 (empty vector) or 2 (CBF-β-FLAG) biologically independent replicates. b Chromatin binding of CBF-β at the STING locus in SAOS2 cells. ChIP-Seq data obtained and reanalysed from Shin et al. (GSE76937)75, and mean normalised reads across all replicates displayed using the IGV genome browser. n = 1. c, d Chromatin binding of CBF-β at various sites in the STING gene, assayed by ChIP-qPCR analysis of 786O Cas9 cells transduced with CBF-β sg2 or an empty vector (d). Primer locations are illustrated in (c). Immunoprecipitation of CBF-β was achieved using Cell Signaling Technology antibody Cat# 62184 (primer sets 1 and 2), or Diagenode antibody Cat#C15310002 (primer sets 3 and 4). n = 5 (primer set 1) or 3 (primer sets 2, 3 and 4) biologically independent replicates. Mean ± SD. Unpaired two-sided t-test. e qPCR analysis of OASL and STING mRNA expression in 786O Cas9 cells transduced with vectors encoding CBF-β sg2, GFP-tagged RUNX1, RUNX2, or empty vector controls. n = 3 biologically independent replicates. Mean ± SD. Two-way ANOVA. f Immunoblots of 786O Cas9 cells in the experimental conditions assayed in Fig. 7e. Representative of 3 biologically independent replicates. 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 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 h 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. Source data are provided as a Source Data file.