Fig 2: Loss of ADAR1 impairs T-ALL LIC functions(A) Experimental setup for assessing ADAR1’s effects on T-ALL propagation in ATO and PDX models.(B) Growth curve of T-ALL samples (n = 2, patient 081 and patient 076) in ATO co-culture system. Cord blood CD34+ HSPCs (n = 2) are also shown. Error bars represent mean with SEM.(C) Human CD45+ cells were quantified by flow cytometry in non-targeting lentiviral control (shCTRL [shRNA control]) or shADAR1 conditions (n = 3 T-ALL samples).(D and E) Effects of ADAR1 knockdown on primary transplant of LICs (n = 5 mice for shCTRL and n = 6 mice for shADAR1). An example of flow cytometry showing human CD45+ engraftment in bone marrow is shown in (E).(F–I) Serial transplant assay of shCTRL and shADAR1.(F–G) The weights of spleen (F) (n = 5 mice per group) and thymus (G) (n = 6 mice per group) were measured after serial transplant.(H) Human CD45+ frequencies in the bone marrow, spleen, and thymus of two T-ALL PDX models (n = 3–7 mice per group).(I) Loss of ADAR1 is associated with improved mouse survival (patient 076, n = 7–8 mice per group). p value was determined by the Mantel-Cox log-rank test.(C–H) Error bars represent mean with SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, unpaired Student’s t test.

Fig 3: ADAR1-mediated RNA editing of SCD1 promotes its expression.a Distribution of putative A-to-I RNA editing sites (n = 6025). b-c Distribution of A-to-I RNA editing events hyper-edited in 5FU + CDDP resistant organoid lines as categorized by regions of the RNA transcript (b) and biological processes (c). d Gene ontology analysis of differentially regulated genes by comparing gastric cancer patients of intestinal subtype (TCGA-STAD) with high ADAR1 or low ADAR1 expression (stratified by median ADAR1 expression). e Putative hyper-edited genes involved in lipid metabolism from GX006, GX055 and GX060. n = 3 biologically independent samples. f-h Sequence chromatograms of the SCD1 transcript in the indicated cell groups. Dot plots represent editing levels of SCD1. i Western blot for ADAR1 and SCD1 expression in the indicated cell groups. β-actin served as a loading control. j Immunofluorescence images showing concomitant high expression of ADAR1 with SCD1. Scale bar, 20 µm. k Top 5 RNA binding proteins predicted to bind to SCD1 3’UTR A-to-I editing sites by RBPmap. Illustration of binding of KHDRBS1 on to SCD1 3’UTR and the potential effect of A-to-I editing on the binding sites on SCD1 RNA. l immunoprecipitation binding assay of KHDRBS1 in parental or resistant organoids (GX006). m Luciferase reporter assay with SCD1 3’UTR in parental or resistant organoids (GX006). n Stability of SCD1 RNA following Actinomycin D treatment (10 µg/mL) for 3, 6, or 24 hours. Lines were linear regression of the data. o Western blot for KHDRBS1 and SCD1 expression in GX006 parental and GX006 5FU + CDDP resistant organoid lines stably transfected with NTC or KHDRBS1 shRNA (clones 1 and 2). (f, h, i, j, o) n = 3 independent experiments; (g, m, n) n = 4 independent experiments; (l) n = 2 independent experiments. Significance were calculated by (e, f, l m) unpaired two-tailed student t-test; (g–h) one-way ANOVA; (n) two-way ANOVA. Data was presented as mean ± standard deviation. EV for empty vector control, WT for wild-type, MUT for catalytically-dead mutant, NTC for non-target control, ADAR1 KD1 and KD2 for shRNA knockdown (clones 1 and 2), KHDRBS1 KD1 and KD2 for shRNA knockdown (clone 1 and 2). Source data are provided as a Source Data file. Illustration for (k) was created using BioRender.com.

Fig 4: Both non-enzymatic and catalytic functions of ADAR1-p150 may be important to prevent cell lethality in cancer cell lines. a GFP control, WT ADAR1-p150, E912A ADAR1-p150, or WT ADAR1-p110 proteins were overexpressed in HCC366 (right) or NCI-H1650 cells (left) prior to transduction with lentivirus that co-expressed Cas9 and sgRNAs targeting GFP or ADAR. Protein lysates were collected 6 days after GFP or ADAR KO and were probed with antibodies against ADAR1, phospho-PKR, total PKR, and β-Actin (loading control) in HCC366 (left) or NCI-H1650 cells (right). The fold change in the phospho-PKR to total PKR ratio (P-PKR/Total PKR) relative to the corresponding GFP sgRNA control is shown in each lane (n = 2). b Cell viability of GFP control or ADAR1-overexpressing HCC366 cells was assessed by crystal violet staining 11–13 days after GFP or ADAR KO (using ADAR sg2) with CRISPR-Cas9. A representative image of crystal violet staining (left) and quantitation of cell viability (right) from two independent biological replicates are shown. Cell viability values were normalized to the GFP sg2 control within each pair of isogenic cell lines. c Cell viability of GFP-overexpressing (control) or ADAR1-overexpressing NCI-H1650 cells was assessed by crystal violet staining 13–16 days after GFP or ADAR KO (using ADAR sg2) with CRISPR-Cas9. A representative image of crystal violet staining (left) and quantitation of cell viability (right) from two independent biological replicates are shown. Cell viability values were normalized to the GFP sg2 control within each pair of isogenic cell lines. d Model of the pathways that mediate cell lethality and interferon-induced interferon production after ADAR deletion in cancer cell lines. Error bars represent standard deviation in all graphs

Fig 5: High expression of ISGs in cancer cell lines is predictive of sensitivity to ADAR deletion. aZ-scores representing the degree of cell lethality after ADAR knockdown in lung cancer cell lines included in published genome-scale loss-of-function screens9. Z-scores represent the number of standard deviations from the mean for each data point. b Cell viability was assessed by ATP bioluminescence 11 days after GFP or ADAR KO with CRISPR-Cas9. ATP bioluminescence values were normalized to the GFP sg1 control within each cell line. Three independent biological replicates were performed for each cell line. *p = 0.0054, **p = 0.0008, and ***p < 0.0001 as calculated by the Kruskal–Wallis test. c Immunoblots showing protein levels of ISGs and β-actin (loading control) in ADAR KO-sensitive and KO-insensitive cancer cell lines (n = 3). d Spontaneous IFN-β secretion by ADAR KO-sensitive and KO-insensitive cancer cell lines as measured by ELISA 24 h after replacement of culture media. Technical replicates from one representative experiment are shown (n = 2). e Cell viability was assessed by ATP bioluminescence 11 days after GFP or ADAR KO with CRISPR-Cas9 in additional lung cancer cell lines. ATP bioluminescence values were normalized to the GFP sg1 control within each cell line (n = 1). f Cell viability of control or ADAR1-deficient A549 cells was assessed by ATP bioluminescence 3 days after vehicle or IFN-I treatment (10 ng/mL). ATP bioluminescence values were normalized to the GFP sg1 control within each treatment group. Three independent biological replicates were performed. Two-way ANOVA showed a significant interaction between ADAR KO and IFN-I treatment (*p < 0.0001, degrees of freedom = 8, F-ratio = 10.51). Dunnett’s multiple comparisons post-test showed a significant difference between vehicle and IFN-I treatment groups and between control and ADAR KO groups (*p < 0.0001). g Cell viability of control or IFNAR1-deficient HCC366 cells was assessed by crystal violet staining 11–13 days after GFP or ADAR KO with CRISPR-Cas9. A representative image of crystal violet staining (left) and quantitation of cell viability (right) from two independent biological replicates are shown. Cell viability values were normalized to the GFP sg2 control #2 within each group of isogenic cells. Error bars represent standard deviation in all graphs