Malignant A-to-I RNA editing by ADAR1 drives T cell acute lymphoblastic leukemia relapse via attenuating dsRNA sensing

Figures

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  Figure 1.. Relapsed T-ALL acquires a distinct RNA editome in contrast to non-relapsed T-ALL (A) Expression of ADAR1 and ADAR2 in T-ALL patient by RNA sequencing (n = 256). (B) Isoform expression of ADAR1 p150 and p110 between HSPCs (n = 3) and T-ALL (n = 256). (C) Quantification of ADAR1 expression in cord blood HSPCs (CD34+Lin−), T-ALL LICs (CD34+Lin−), and non-LICs (CD34−Lin+). n = 3 cord blood HSPC and 3 T-ALL samples. (D and E) Overall RNA editing between relapsed and non-relapsed patients (D) or between mortality status (E) in violin plots. (F) Comparison of RNA-editing level between relapsed and non-relapsed cohort display under-edited sites (green) and overedited sites (red) with editinglevels >0.2 and detected in >10% of patients in each group. (G) Pie chart showing RNA-editing locations in non-relapsed and relapsed T-ALL. (H) Elevated RNA-editing levels across all categories of editing locations between non-relapsed (n = 230) and relapsed groups (n = 21). Statistical analysis was calculated by unpaired Student’s t test.

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  Figure 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 <…

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  Figure 3.. ADAR1 downregulation reduces LIC stemness gene expression (A) Volcano plot depicting significantly differentially expressed genes in T-ALL LICs with ADAR1 knockdown (n = 2 samples). (B and C) Gene enrichment scores (B) and heatmap (C) of “lymphoblastic leukemia” pathway. (D) Expression of CD34, CD44, and LMO2 was quantified by RT-qPCR (n = 3, patients 070, 076, and 081) after 48 h lentiviral transduction of shADAR1. (E) Analysis of differential level of RNA editing and number of A-to-I editing sites per transcript between scramble shCTRL and ADAR1 knockdown. (F) Dandelion plot showing RNA-editing location in MYB, MAVS, and IL17RA. Each dot represents a unique RNA-editing event. Error bars represent mean with SEM. **p < 0.01 and ****p < 0.0001, unpaired Student’s t test.

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  Figure 4.. Concurrent knockdown of MDA5 and ADAR1 rescues self-renewal to various degrees in different T-ALL models (A) Experimental setup. T-ALL CD34+ cells were transduced with shCTRL, shADAR1, or shADAR1 and shMDA5 lentivirus in combination. Transduced cells were sorted based on GFP+mCherry+ (GFP for shADAR1 and mCherry for shMDA5), and serial transplant potential was measured in recipient Rag2−/−γc−/− mice. (B–D) Serial leukemia engraftment and representative bone marrow FACS plots of patient 070 (B), patient 081 (C), and patient 076 (D) were determined for shCTRL, shADAR1, and shADAR1 in combination with shMDA5 (3–8 mice per condition). (E) Images of spleen (left) and spleen weights (right) in serial transplanted mice were determined after an 8 week engraftment interval (n = 3 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001, unpaired Student’s t test.

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  Figure 5.. Basal expression of MDA5 controls dependency on the ADAR1-MDA5 axis (A and B) Expression of ADAR1 isoforms in patient 076 and patient 081 was measured by RT-qPCR and western blot in Lin−CD34+ LIC-enriched population (n = 3 independent experiments). (C) Expression of MDA5 was determined in Lin−CD34+ LIC-enriched cells of patient 076 and patient 081 (n = 3 independent experiments). (D and E) Expression of ADAR1 isoforms in three T-ALL cell lines, CUTTL1, SUP-T1, and Jurkat (n = 3 independent experiments). (F and G) MDA5 and PKR mRNA expression (F) and protein level (G) were determined in T-ALL cell lines (n = 3 independent experiments). (H) Cell counts of shRNA control, shADAR1, shMDA5, and co-knockdown of shADAR1 and shMDA5 were assessed 3 days post-lentiviral transduction. Data from three independent experiments are shown. Error bars represent mean with SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, unpaired Student’s t test.

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  Figure 6.. RNA-editing-dependent and -independent mechanisms of ADAR1 suppress aberrant dsRNA sensing (A) Western blot showing ADAR1 expression in modified Jurkat T-ALL cell lines, including wild-type (WT), ADAR1-KO, re-expressed WT ADAR1 (ADAR1WT), and ADAR1E912A by lentiviral-overexpressing vector. β-Actin was used as loading control. (B) Jurkat cells were stimulated with various concentrations of IFNβ, and the gene expression of ADAR1 was determined (n = 2 independent experiments). (C) Quantification of ADAR1 p150 and p110 isoforms upon IFNβ treatment at 10 ng/mL for 24 h (n = 3 independent experiments). (D) Cell viability was quantified in Jurkat cells upon IFNβ stimulation (n = 3 independent experiments). (E) Cell viability was quantified in Jurkat cells treated with IFNα (0.1 ng/mL), IFNβ (1 ng/mL), or IFNγ (1 ng/mL) for 48 h (n = 3 independent experiments). (F) NanoString analysis of gene expression in WT, ADAR1-KO, and ADAR1E912A-overexpressing Jurkat cells stimulated with IFNβ (1 ng/mL, 48 h) (n = 2 independent experiments). (G) Immunofluorescent staining to detect the localization of dsRNA (J2 antibody) in WT, ADAR1-KO, ADAR1WT, and ADAR1E912A Jurkat cells stimulated with IFNβ (1…