Detailed methods are provided in the online version of this paper and include the following:
CONTACT for REAGENT AND RESOURCE SHARING
EXPERIMENTAL MODEL AND SUBJECT DETAILS
- Animal Experiments
- Human Subjects
METHOD DETAILS
- Patient sample preparation
- Cell culture
- Lentiviral construct and overexpression
- ATO 3D organoid culture
- Patient derived xenograft transplantation
- Flow cytometry analysis and sorting
- RNA extraction and RT-qPCR
- Western blots
- Interferon stimulation assay
- Nanostring nCounter
- Immunofluorescence staining
- Whole RNA-sequencing
- Read alignment and gene counts
- RNA editing analysis
- Transcript and gene quantification and differential expression
- scRNA-sequencing
- Other Statistical Analysis and Reproducibility
DATA AND SOFTWARE AVAILABILITY
CONTACT for REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Qingfei Jiang ([email protected]).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Animal Experiments
All mouse studies were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego and were in compliance with federal regulations regarding the care and use of laboratory animals: Public Law 99-158, the Health Research Extension Act, and Public Law 99-198, the Animal Welfare Act which is regulated by USDA, APHIS, CFR, Title 9, Parts 1, 2, and 3. Immunocompromised Rag2-/-gc-/- mice were bred and maintained in the Sanford Consortium for Regenerative Medicine vivarium according to IACUC approved protocols of the University of California, San Diego. Neonatal mice of both sexes were used in the study. T-ALL CD34+ or CD45+ cells were injected intrahepatically into 2-3 days oldneonatal Rag2-/-gc-/- mice. Leukemic engraftment was quantified by FACS analysis-based peripheral blood screening of human CD45+ population starting from week 6 for every 2 weeks until the engraftment exceeded 1%. Mice were then humanely sacrificed, and cells were collected from hematological organs (bone marrow, spleen and thymus) for FACS analysis.
Human Subjects
Patient T-ALL samples were obtained from consenting patients at the University of California, San Diego in accordance with an approved human research protections program Institutional Review Board approved protocol (#130794) that meets the requirements as stated in 45 CFR 46.404 and 21 CFR 50.51. De-identified (IRB exempt) human cord blood samples were purchased as purified CD34+ cells from AllCells Inc or StemCell Techologies Inc. Detailed patient information can be found in Table S2.
METHOD DETAILS
Patient sample preparation.
Peripheral blood mononuclear cells (PBMC) were extracted by Ficoll density centrifugation. CD45+ or CD34+ cells were purified using magnetic columns (MACS, Miltenyi) or FACS sorted with human-specific antibody according to published methods in FACSAria II 16.
Cell culture
Jurkat human cell lines were cultured in 37oC in DMEM supplemented with 10% FBS and 2 mM L-glutamine and maintained according to ATCC protocols. MS5-DLL1 and MS5-DLL4 were maintained in high glucose DMEM with 10% FBS and 1X penicillin-streptomycin according to previous protocol 29,31. All cell lines were confirmed to be mycoplasma-free with repeated testing and authenticated by short-tandem repeat (STR) profiling. ADAR1 knockout cell line was generated by Sythego. Wildtype ADAR1 and mutant E912A were introduced into the knockout Jurkat cells by transduction of wild-type and mutant ADAR1E912A lentivirus. Stable ADAR1 expression were confirmed by RT-qPCR and western blot every 5 passages.
Lentiviral construct and overexpression.
Lentiviral vectors (pLV-shRNA-EGFP or mCherry:T2A:Puro-U6) was purchased (VectorBuilder) and wild-type and mutant ADAR1E912A (pCDH-EF1-T2A-copGFP) were produced according to published protocol 15. All lentivirus was titer by transduction of HEK293T cells and efficiency was assessed by p24 ELISA and RT-qPCR of the 5’ LTR region. Lentiviral transduction of primary T-ALL or cord blood samples was performed at a MOI of 100-200. The cells were cultured for 3-4 days in 96-well plate (2X105-5X105 cells per well) containing StemPro (Life Technologies) media supplemented with human IL-6, stem cell factor (SCF), Thrombopoietin (TPO) and FLT3 (all from R&D Systems). For Jurkat cell line, the cells were transduced at a MOI of 20-50. The gene expression was confirmed by RT-qPCR to downstream analysis. The transduced cells were then applied in in vitro flow analysis or in vivo transplant experiment.
ATO 3D organoid culture. ATO organoid experiments were performed as previously described 29. The MS5 mouse stromal cells were engineered to co-express human DLL1 NOTCH ligand and EGFP marker. MS5-DLL1 and -DLL4 were cultured up to 20 passages and the cells will be authenticated every 5 passages by flow of EGFP signal and examining the DLL1 or DLL4 expression by RT-qPCR. To generate ATOs, CD34+ cells (2-5x103 cells per ATO) were combined with MS5-DLL1/DLL4 cells at 1:20 ratio, seeded on a 0.4 mm Millicell transwell insert (EMD Millipore), and placed into 6-well plate with serum-free culture media supplemented by recombinant IL7 (50 ng/mL for T-ALL LICs, and 5 ng/mL for cord blood HSPC) and FLT3 (50 ng/mL). ATOs were cultured up to 20 weeks. The cells were harvested by adding staining media (ice-cold PBS with 2% FBS and 2 mM EDTA) to each well and pipetting to dissociate ATOs. Cells were then immunostained with antibodies (Table S1) and analyzed on a BD Aria Fusion and with FlowJo.
Patient derived xenograft transplantation
To establish T-ALL models, freshly ficolled cells were transplanted intrahepatically into neonatal Rag2-/-gc-/- mice (5x105 – 1x106 per pup) according to our preciously published methods 15,16. Bone marrow and spleen tissues were harvested after 6-20 weeks and stored in liquid nitrogen. For functional studies, CD34+ or CD45+ cells were transduced with lentiviral vectors for 2-3 days. Cells were harvested in staining media, counted, and equal numbers of cells per condition were transplanted into recipient mice (5x104 – 1x105 per pup). Transplanted mice were FACS screened for human engraftment in peripheral blood at 6-10 weeks. Once human engraftment was confirmed (>1% human CD45+ cells in peripheral blood), mice were euthanized, and single cell suspensions of hematopoietic tissues (bone marrow, spleen, and thymus) were analyzed by FACS and FlowJo.
Flow cytometry analysis and sorting
Flow staining was performed in staining media for 30 min on ice in the dark. Cells were blocked using FcR block (Biolegend, San Diego, CA) for 15 minutes before antibody staining with to a final dilution of 1:25. DAPI solution was added before analysis to exclude dead cell debris. Analysis and sorting was performed on BD Aria Fusion, Aria II or Fortessa. Sorted cells were collected into staining media filled FACS tubes or 1.7mL Eppendorf tubes. The LICs and various populations of lymphoid progenitors are evaluated by the corresponding cell surface markers (Table S3). For intracellular ADAR1 staining, cells were stained with ethidium monoazide (EMA) for 15 min in the dark and then 15 min under light, followed by cell surface staining. After washing in staining buffer, cells were fixed and permeabilizated with an intracellular buffer set (eBioscience, San Diego, CA) and intracellularly stained with an antibody against ADAR1 (Abcam, ab126745) at 1:100 dilution. Secondary antibody of Alexa488 or Alexa647 were used to amplify ADAR1 signals.
RNA extraction and quantitative real-time polymerase chain reaction.
Total RNA was isolated using RNeasy Micro kit or Mini kit (Qiagen) and the quality was determined by NanoDrop. Complementary DNA was synthesized according to published methods 16. qRT-PCR was performed in duplicate or triplicate on an CFX384 with the use of SYBR GreenER qPCR SuperMix (Invitrogen), 5 ng of template cDNA and 0.2 µM of each forward and reverse primer. Human specific HPRT primers were used as housekeeping control. Quantitative values were obtained from the cycle number (Cq value) using the Bio-Rad Maestro Software. The RT-qPCR primers are shown in Table S6.
Western blots
Cell lysate (10 mg) was mobilized onto a nitrocellulose membrane after electrophoresis on a 10% SDS- acrylamide gel. The membrane was blocked in 5% BSA/20 mM Tris-HCl for 30 min. The blot was incubated with primary antibody in 5% BSA/20 mM Tris-HCl/0.1% Tween-20 overnight at 4°C, followed by secondary HPR-linked Rabbit IgG antibody (Cell Signaling, #70745) for 2 hr at room temperature. Membrane was then incubated in SuperSignal West Femto Substrate (ThermoFisher, #34096) for chemiluminescent reading on ChemiDoc System (Bio-Rad).
Interferon stimulation assay
Jurkat cells were seeded at a density of 105 in a 12-well plate and treated with a single dose of IFNa, IFNb, or IFNg (R&D Systems) at 0.05-500 ng/mL. After 48 hours, cells were harvested and analyzed by western blot and RT-qPCR. The cell supernatants were collected and the S100A9 level was determined by an ELISA assay (Invitrogen). Cell viability was determined trypin blue assay.
Nanostring nCounter
Jurkat cells were collected after 24 hours of IFNb-stimulation and RNA was isolated (RNeasy Plus mini kit, Qiagen). The mRNA levels were directly measured using the Human CAR-T characterization panel kit with additional custom probes (Table S5) from NanoString nCounter gene expression system (NanoString). The differential expression analyses of mRNA were performed using nSolver software (NanoString) and visualized in Prism software.
Immunofluorescence staining
Cells (1-2x103) were harvested in ice-cold PBS and loaded on adhesion slides (Marienfeld Superior) by incubating for 10 min at room temperature. The slides were transferred into a coplin jar containing ice-cold PBS for 5 min and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Immunofluorescence was performed by immersing slides in PBST (1x PBS with 0.05% Tween-20). Slides were overlaid with blocking solution (2% fetal bovine serum in PBST) for 1 hour at room temperature. After washing, primary antibody was added to the slides and incubated overnight at 4C. Secondary antibody was overlaid to spotted cells for 1 hour in the dark. DAPI was added and the slides were sealed with a coverslip. Imaging was performed using a Keyence confocal microscope.
Whole RNA-sequencing
Samples with RNA integrity numbers (RIN) ≥7 will be processed using SMART cDNA synthesis and NEBNext paired-end DNA Sample Prep Kit to prepare libraries. RNA-sequencing were performed on NovaSeq 6000 S4 with 150bp paired-end reads. T-ALL RNA sequencing dataset were obtained from data generated by the Therapeutically Applicable Research to Generate Effective Treatments (https://ocg.cancer.gov/programs/target) initiative, phs000464. The data used for this analysis are available at https://portal.gdc.cancer.gov/projects." The minimal reads per sample was 50M to ensure optimal RNA editing calling.
Whole RNA-seq Read alignment and gene counts
Reads were aligned using STAR's two-pass alignment method, using the GRCh38.84 reference genome and corresponding Ensembl GTF 85,86. STAR was used to output a sorted genome-coordinate based BAM file, as well as a transcriptome-coordinate based BAM file 87. STAR also was used to output the number of reads aligned to each gene for gene expression quantification. STAR settings were based on those used for the ENCODE STAR-RSEM pipeline. The infer_experiment.py script from the RSeQC package was used to confirm the strandedness option corresponding to the correct read counts 88,89 and to confirm the forward strand probability for input to RSEM. The total reads per million (TPM) 90 over the total collapsed exonic regions represent the ‘gene’ expression level.
RNA editing analysis
Coordinates from the DARNED and RADAR databases were combined and converted to GRCh38 using Crossmap 91-93. The resulting coordinates were used as input to the REDItoolKnown.py script from the REDItools package to determine the number of A, C, G, and T base calls at each coordinate 94. Only coordinates with coverage greater than or equal to 5 in all samples for a given comparison were reported. The percentage of bases called as G at bases with reference A was reported. Coordinates with a percentage G of 0 in all samples for a given sample were not reported. Using percentage G at a coordinate as an input metric, the mean percentage G in each group, the log2 fold change of percentage G of one group versus another, the p values, and minus log10 p values by both the Wilcox and student t-tests were recorded for each coordinate similar to published methods 14. Coordinates were annotated with the name of the closest gene using bedtools closest and bedtools intersect 95. The coordinates annotated with the names of genes in the KEGG cell cycle gene set were recorded.
Transcript and gene quantification and differential expression
The transcriptome-coordinate based BAM from the read alignment step was input to RSEM, using settings based on the ENCODE STAR-RSEM pipeline 89. RSEM was provided the GRCh38.84 reference genome and corresponding Ensembl GTF for its transcriptome reference. RSEM was used to provide TPM and expected counts for genes and transcripts. For genes, the gene count data generated by STAR in the alignment step was used as input to EdgeR 87,96. For transcripts, the expected counts data from RSEM was used as input to EdgeR. Only features with a minimum CPM of 0.5 (in at least half the samples in the comparison) as measured by EdgeR were submitted to EdgeR's differential expression, to yield log2 fold change, p value, and FDR for each feature for the comparison. The threshold for significant genes and transcripts was set at a p value less than 0.05 and an FDR less than 0.10. Heatmaps visualize the log2(TPM+1) transformed TPM quantity from RSEM for each feature and were generated using GENE-E with default settings for a row and column clustered heatmap and dendrogram.
scRNA-sequencing and processing
Single cell was barcoded using a 10X Chromium Controller (10x Genomics) and sequencing libraries were prepared with reagents from a Chromium Single Cell 3’ kit. All sequencing was performed on Illumina NovaSeq. Low-quality cells were removed using these quality control criteria ( <20% genomic reads and/or < 1000 genes were detected per barcode). Statistical analysis of scRNA-seq data was carried out using core functions of R programming language and the library Seurat 97. After reading the raw 10X data, filtering cells for fraction of mitochondrially-encoded genes (< 20%) and for number of genes detected per cell (>1500 in shCTRL cells and >500 in shADAR1 cells), we obtained 4890 shCTRL cells and 1049 shADAR1 cells for further analysis. Cells from these two samples were integrated using SCTransform 98. The two-dimensional UMAP and clustering was done using Seurat’s functions. Differential expression (DE) analysis between clusters and/or genotypes was done using the bootstrap algorithm of Pollard and van der Laan 99 using SCTransform-ed expression levels. Significance of each gene in each comparison was expressed as local false discovery rate lfdr using Efron’s Empirical Bayes approach 100. Genes in a DE comparison were subject to Gene Set Enrichment Analysis 101 using a fast implementation fgsea 102. UMAP plots, violin plots and heatmaps are standard functions of Seurat.
Other Statistical Analysis and Reproducibility
All experiments were performed with at least three biological or experimental replicates, with specific number of replicates stated in the figure legends. Unless otherwise stated, the statistical analyses were performed using GraphPad Prism (v7.0) and statistical significance were determined at p value < 0.05, with specific statistical test stated in the figure legends.
DATA AND SOFTWARE AVAILABILITY
The RNA-sequencing dataset used in this study will be upload to the GEO with a special password for editors and reviewers. The data will be made publicly available upon acceptance for publication.