Fig 2: Identification of putative ADAR1 interacting proteins by APEX2 proximity labelinga Immunoblot showing expression of the constructs used for proximity labeling in MCF-7, MDA-MB-231 and SK-BR-3. b Representative fluorescently stained gel image showing proteins purified by streptavidin-biotin pulldown following proximity labeling in MCF-7. c Volcano plot summarizing the proteins identified by mass spectrometry following streptavidin pulldown subsequent to proximity labeling. Differential abundance of all proteins in each cell line can be found in Supplementary Table 2. d Venn diagram showing overlap between enriched proteins from all three cell lines. The cut-off for enriched proteins was an FDR adjusted p-value of less than 0.05 and a Log2 fold change of greater than 0.5. e GO terms found to be overrepresented in the list of the enriched proteins. Only the top ten GO terms, by FDR, are shown for each category. All other significant GO terms are available in the Supplementary Table 3–4. f and g Venn diagrams showing overlap between the enriched proteins identified and those proteins found previously to localize to the nucleus or nucleolus in MCF-743. h Representative indirect immunofluorescence micrographs showing localization of ADAR1, fibrillarin (a nucleolar marker) and DAPI.

Fig 3: Induction of a viral mimicry phenotype upon knockdown of DHX9 and ADAR1 in MCF-7A Volcano plot showing changes in RNA expression upon knockdown of DHX9 and ADAR1 in MCF-7, a volcano plot for SK-BR-3 is in Extended Data Fig. 5f. Fold-change of RNA expression shown in a was determined using an interaction term between ADAR1 and DHX9 knockdown, volcano plots for fold change of RNA expression for single knockdown of ADAR1 or DHX9 is in Extended Data Fig. 5a–b and 5d–e. b GO terms identified by gene set enrichment analysis of the RNA-seq data in a. c-e Heatmaps and summary box plots showing RNA expression changes in MCF-7 and SK-BR-3 upon knockdown of ADAR1 and/or DHX9. Panel c shows RNA expression for core ISGs21,33, panel d shows ATF4 targets and panel e shows NF-KB targets with ISGs removed.

Fig 4: DHX9 is overexpressed in breast cancer and suppresses PKR activationa Schematic showing the domain structure of PKR, ADAR1, DHX9 and other helicases identified by proximity labeling in Fig. 1, dsRBD refers to the dsRNA Binding Domain. b Pearson and Spearman correlation coefficients for the correlation between ADAR1 expression at the RNA level and the expression of each indicated helicase at the RNA level, data from breast tumors within TCGA. Scatterplots showing the correlation between ADAR1-p110 c, or ADAR1-p150 d, and DHX9 expression in normal breast or breast tumors. e Expression of DHX9 at the RNA level in normal breast, non-TNBC or TNBC tumors. f Waterfall plot showing DHX9 dependency of breast cancer cell lines using data from DepMap, ER = estrogen receptor positive cell lines, ERRB2 = HER2 positive cell lines. g Representative immunoblot following knockdown of DHX9 with two different shRNAs in four TNBC cell lines. Immunoblots for other replicates and uncropped blots can be found in Source Data Figures. h Foci formation assay for the same cells used in g for immunoblot. i Quantification of PKR phosphorylation as determined by the immunoblot in g. Quantification of protein expression for other proteins of interest can be found in Extended Data Fig. 3a–e. Bars represent the average of at least three replicates, error bars are +/− SD. * p <0.05, ** p <0.01, *** p < 0.001. P-values determined by Dunnett’s test.

Fig 5: Rescue of PKR activation by ADAR1-p110, ADAR-p150, DHX9 and DHX9 mutantsa Schematic showing the domain structure of ADAR1 isoforms, DHX9 and mutants of DHX9, dsRBD refers to the dsRNA Binding Domain. Representative immunoblot showing the phenotype of ADAR1 and DHX9 knockdown with DHX9, DHX9K417R or dsRBD-EGFP overexpression b, or ADAR1 isoform overexpression f. Immunoblots for other replicates and uncropped blots can be found in Source Data Figures. Fold change of PKR phosphorylation at Thr-446 upon ADAR1 and DHX9 knockdown with DHX9, DHX9K417R or dsRBD-EGFP overexpression d, or ADAR1 isoform overexpression h, quantified from immunoblots in b and f respectively. Quantification of protein expression for other proteins of interest can be found in Extended Data Fig. 7b–f and 8a–f. Representative foci formation phenotype of ADAR1 and DHX9 knockdown with DHX9, DHX9K417R or dsRBD-EGFP overexpression c, or ADAR1 isoform overexpression g. Quantification of relative foci area is shown in e or i, respectively. Bars represent the average of at least three replicates, error bars are +/− SD. * p <0.05, ** p <0.01, *** p < 0.001. P-values determined by Dunnett’s test.