Journal: Cell stem cell

Article Title: Reversal of malignant ADAR1 splice isoform switching with Rebecsinib.

doi: 10.1016/j.stem.2023.01.008

Figure Lengend Snippet: Figure 1. Quantification of ADAR1p150 by splice isoform RNA sequencing (RNA-seq) (A) RNA-seq-based quantification (counts per million [CPM]) of ADAR-201 (GRCh38 ENST00000368471.8, ADAR1 p110-encoding), ADAR-202 (ENST00000368474.9, ADAR1 p150- encoding), and ADAR-208 (ENST00000529168.2, ADAR1 p150-encoding 30 UTR truncated tran- scripts) was performed on FACS-purified he- matopoietic stem cells (HSCs, CD34+CD38Lin) from young bone marrow (YBM; n = 4) and aged bone marrow (ABM; n = 4) HSC, polycythemia vera (PV, n = 3), essential thrombocythemia (ET, n = 2), myelofibrosis (MF, n = 24), chronic myeloid leu- kemia (CML, n = 5), or secondary acute myeloid leukemia (sAML, n = 5). RNA-seq analyses were also performed on FACS-purified hematopoietic progenitor cells (HPCs, CD34+CD38+Lin) from primary samples, including YBM (n = 8), ABM (n = 8), PV (n = 6), ET (n = 2), MF (n = 24), CML (n = 5), and de novo (dn) AML (n = 2) and sAML (n = 13). Statistics for HSC: ADAR-201 p < 0.05 for MF, CML, and sAML versus ABM; ADAR-202 p < 0.05 for MF versus ABM; ADAR-208 differences were not significant in HSC. Statistics for HPC: ADAR- 201 p < 0.05 for PV, ET, MF, and CML versus ABM; ADAR-202 p < 0.05 for PV, ET, MF, and CML versus ABM; ADAR-208 p < 0.05 for PV, ET, and MF versus ABM. Statistical analyses were per- formed using Student’s t tests comparing MPNs and sAML versus ABM. (B) Structural diagram showing the spliceosome core complex with Rebecsinib interacting at the interface of SF3B1 and PHF5A, adapted from the spliceosome complex bound to pladienolide B.27

Article Snippet: For ADAR1 protein isoform and STAT3 phosphorylation analyses in cell lines, western blots were performed as previously described.5,8,16 Blots were probed with antibodies against ADAR1p150 (Abcam ab126745) and pan-ADAR1 (detects ADAR1p150 and p110, Cell Signaling D7E2M), along with total STAT3, phosphorylated STAT3 (Y705 D3A7, Cell Signaling), and GAPDH as a loading control (key resources table).

Techniques: RNA Sequencing

Journal: Cell stem cell

Article Title: Reversal of malignant ADAR1 splice isoform switching with Rebecsinib.

doi: 10.1016/j.stem.2023.01.008

Figure Lengend Snippet: Figure 2. Development of a lentiviral ADAR1 A-to-I RNA editing reporter (A) Schematic diagram demonstrating the synthetic RNA sequence containing an ADAR1-sensitive stop codon that, upon A-to-I editing, reads through to produce nanoluciferase and GFP proteins separated by a T2A cleavage site. (B) ADAR protein expression levels in 293T cells co-transfected with the ADAR1 nanoluciferase-GFP (nanoluc-GFP) reporter and increasing amounts of FLAG- tagged wild-type (WT) ADAR1, catalytically inactive mutant ADAR1 (E912A), or wild-type ADAR2. b-actin was used as a loading control. (C) Relative luciferase signals in 293T cells prepared as in (B). Data are represented as mean ± SEM. (D) Live-cell fluorescent imaging of GFP expression in human myeloid leukemia TF-1a cells transduced with the ADAR1 nanoluc-GFP reporter vector (lower panels) compared with untransduced controls (upper panels). (E) Detection of nanoluciferase expression via in vivo bioluminescence (IVIS) imaging of no transplant control (far left), K562-nanoluc-GFP and pCDH vector transduced, and K562-nanoluc-GFP and ADAR1 wild-type or E912A mutant transduced human leukemia cells (K562) transplanted into Rag2/gc/ mice. (F) Luminescence-based quantification of ADAR1-dependent nanoluciferase signals in CD34+ cells from primary, high-risk MF samples (*untreated patient) after in vitro transduction with the ADAR1 nanoluc-GFP reporter and treatment with vehicle control (DMSO) or Rebecsinib (72 h). Relative luciferase signals were normalized to cell viability for each condition. Data are represented as mean ± SEM. p < 0.05 Rebecsinib compared with DMSO controls by pairwise t test. (G) Intracellular flow cytometry-based quantification of STAT3 phosphorylation (expressed as mean fluorescence intensity [MFI], values within HPC populations) after in vitro treatment with vehicle control (DMSO) or Rebecsinib (1 mM, 72 h). p < 0.05 Rebecsinib compared with DMSO controls by pairwise t test. See also Figure S1.

Article Snippet: For ADAR1 protein isoform and STAT3 phosphorylation analyses in cell lines, western blots were performed as previously described.5,8,16 Blots were probed with antibodies against ADAR1p150 (Abcam ab126745) and pan-ADAR1 (detects ADAR1p150 and p110, Cell Signaling D7E2M), along with total STAT3, phosphorylated STAT3 (Y705 D3A7, Cell Signaling), and GAPDH as a loading control (key resources table).

Techniques: Sequencing, Expressing, Transfection, Mutagenesis, Control, Luciferase, Imaging, Transduction, Plasmid Preparation, In Vivo, In Vitro, Cytometry, Phospho-proteomics

Journal: Cell stem cell

Article Title: Reversal of malignant ADAR1 splice isoform switching with Rebecsinib.

doi: 10.1016/j.stem.2023.01.008

Figure Lengend Snippet: Figure 5. ADAR1 expression and LSC self-renewal following Rebecsinib treatment (A) Schematic diagram showing in vivo treatment of primary patient LSC or CB-engrafted mice and sAML serial transplantation studies. (B) Flow cytometry analysis quantifying human LSC survival in sAML-engrafted mice treated with Rebecsinib Lot 1 (n = 3 sAML50261, n = 3 sAML2008-5) or Lot 2 (n = 4 sAML50261) compared with vehicle control (n = 4 sAML50261, n = 3 sAML2008-5) at 10 mg/kg twice weekly for 2 weeks (5 total doses). Data are rep- resented as mean ± SEM. p = 0.01; Rebecsinib versus vehicle control by unpaired t test. (C) Quantitative real-time PCR analyses in CD34+ cells isolated from the spleens of sAML50261 mice treated with Rebecsinib (as in A) showing decreased total ADAR1 expression by quantitative real-time PCR. Data are represented as mean ± SEM. p = 0.03; Rebecsinib versus vehicle control by unpaired t test. (D) Mean fluorescence intensity (MFI) of ADAR1p150 protein levels in human HSCs (CD45+CD34+CD38Lin) and HPCs (CD45+CD34+CD38+Lin) from the spleens of sAML50261-engrafted mice treated with Rebecsinib or vehicle. Data are represented as mean ± SEM. p = 0.046; Rebecsinib versus vehicle control HSCs (left). p = 0.009; Rebecsinib versus vehicle control HPCs (right). Statistical analyses performed using unpaired t tests. (E) Whole-transcriptome-based RNA editing analyses of previously described RNA-seq data,14 generated from CD34+ cells isolated from the spleens of sAML50261-engrafted mice treated with Rebecsinib or vehicle. Total edits, edits in unique genes, and normalized numbers of edits per million reads were calculated using RNA editing pipelines, as previously described.5

Article Snippet: For ADAR1 protein isoform and STAT3 phosphorylation analyses in cell lines, western blots were performed as previously described.5,8,16 Blots were probed with antibodies against ADAR1p150 (Abcam ab126745) and pan-ADAR1 (detects ADAR1p150 and p110, Cell Signaling D7E2M), along with total STAT3, phosphorylated STAT3 (Y705 D3A7, Cell Signaling), and GAPDH as a loading control (key resources table).

Techniques: Expressing, In Vivo, Transplantation Assay, Flow Cytometry, Control, Real-time Polymerase Chain Reaction, Isolation, RNA Sequencing, Generated

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Rescue of Adar Δ7-9 by Mavs −/− , Ifih1 −/− and Mavs −/− ; Adarb1 −/− ; Gria2 R/R . ( A) Post-natal survival curves of Adar Δ7-9 ; Mavs −/− , Adar1 Δ7-9 ; Ifih1 −/− and Adar Δ7-9 ; Mavs −/− ; Adarb1 −/− ; Gria2 R/R mice. Expression levels of 5 Interferon stimulated genes measured by qPCR in three different tissues of ( B ) Adar Δ7-9 ; Mavs −/− ( C ) Adar Δ7-9 ; Ifih1 −/− and ( D ) Adar Δ7-9 ; Mavs −/− ; Adarb1 −/− ; Gria2 R/R mice.

Article Snippet: We therefore tested commercial monoclonal anti Adar antibody 15.8.6 (Santa Cruz) on HeK293 cells that were transfected with the above mentioned FLAG-tagged ADAR1 p110, p150, p110 Δ7-9 and p150 Δ7-9 constructs.

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: ADAR1 variants without exons 7–9 can be expressed in cells. ( A ) Illustration of the p150 and p110 isoforms of ADAR1 indicating the deleted parts of the Adar Δ7-9 allele (red box). The truncation affects only the third double-stranded RNA binding domain (dsRBD), the embedded nuclear localization signal (NLS) and the deaminase domain. Z-DNA binding domains (Zα and Zβ), the nuclear export signal (NES), dsRBD 1 and 2 as well as a C-terminal NLS motif are unaffected. Amplicons tested for detecting truncation are indicated. ( B ) RT-PCR analysis of wildtype and Adar Δ7-9 transcript in primary MEFs isolated at E11.5. ( C ) Fluorescence microscopy images of N-terminally FLAG-tagged and C-terminally fused with 2A-eGFP versions of ADAR1 (p110, p110 Δ7-9 , p150 and p150 Δ7-9 ) transfected into HeLa cells. DAPI shows nuclear DNA and FLAG-tagged fusions are visualized in the mCherry channel. ( D ) Western blot analysis of whole-cell extracts of HeLa cells transfected as in (C) using FLAG antibody to visualize fusion proteins (upper panel). eGFP was used for normalization (lower panel). ( E ) Detection of transfected constructs used in (C) with monoclonal anti-ADAR1 antibody 15.8.6 by western blotting. ( F ) Detection of ADAR1 in wt and ADAR1 Δ7-9 -derived MEFs after IFN induction using a polyclonal antibody directed ADAR1 p150. ( G ) Detection of ADAR1 in wt and ADAR1 Δ7-9 -derived MEFs after IFN induction using a monoclonal pan-anti ADAR1 antibody. Arrowhead marks size of predicted p150 Δ7-9 protein and asterisk depicts the size of the p110 Δ7-9 protein. ( H ) Detection of ADAR1 in wt and ADAR1 Δ7-9 brain lysates. tubulin marks detection of the lower part of the blot with an anti-tubulin antibody. (I) Quantification of ADAR p110 in three independent blots of cell lysates indicates ∼20% expression of p110 Δ7-9 protein

Article Snippet: We therefore tested commercial monoclonal anti Adar antibody 15.8.6 (Santa Cruz) on HeK293 cells that were transfected with the above mentioned FLAG-tagged ADAR1 p110, p150, p110 Δ7-9 and p150 Δ7-9 constructs.

Techniques: RNA Binding Assay, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Fluorescence, Microscopy, Transfection, Western Blot, Construct, Derivative Assay, Expressing

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Phenotypic analysis of Adar Δ7-9 ; Mavs −/− mice at P15. ( A ) Dot plot showing reduced body weight of Adar Δ7-9 ; Mavs −/− compared to Adar +/+ ; Mavs −/− mice ( n = 11 per genotype). ( B ) Normalization of spleen (left) and kidney (right) weight to total body weight. A specific effect is seen on spleen, but not the kidney of Adar Δ7-9 ; Mavs −/− mice (n = 7 Adar +/+ ; Mavs −/− and n = 9 Adar Δ7-9 ; Mavs −/− ). ( C ) Dot plots showing reduced splenocytes (left, n = 6/genotype) and bone marrow cells (right, n = 8/genotype) in Adar Δ7-9 ; Mavs −/− compared to Adar +/+ ; Mavs −/− mice. Horizontal bars in (A) – (C) represent the mean; *** P < 0.001; ns = non-significant as determined by unpaired Student's t test. ( D ) Three mice per genotype were analyzed histologically at day 15 post-partum. Analyzed organs were spleen, small intestine, colon and kidney. Note the diminished cellularity of the spleen and the underdeveloped germinal center (dotted circle) in Adar Δ7-9 ; Mavs −/− mice. In the small intestine and to a lower extent in the colon, goblet cell (denoted by arrowhead) number is reduced in in Adar Δ7-9 ; Mavs −/− mice. Shown are representative sections in 8× magnification (20× in inlays) of spleen, small intestine and colon and 2× magnification (8× in inlays) of kidney with scale bars indicated.

Article Snippet: We therefore tested commercial monoclonal anti Adar antibody 15.8.6 (Santa Cruz) on HeK293 cells that were transfected with the above mentioned FLAG-tagged ADAR1 p110, p150, p110 Δ7-9 and p150 Δ7-9 constructs.

Techniques:

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Altered B cell and neutrophil numbers in P15 Adar Δ7-9 ; Mavs −/− mice. ( A and B ) Immature and mature B cells in bone marrow (upper panels) and spleen (lower panels) were analyzed by flow cytometry. (A) Representative flow plots of bone marrow (upper panel) and spleen (lower panel) from Adar1 +/+ ; Mavs −/− (left panels) and Adar Δ7-9 ; Mavs −/− (right panels) animals, subgated for living NK1.1 − cells. Numbers next to the outlined areas indicate percentages within this population. (B) Dot plots showing mature (B220 + CD19 + ) and immature (B220 + CD19 − ) B cells in the bone marrow (upper panel) and spleen (lower panel). Percentages of living cells (left panels) were used to determine total cell numbers (right panels). ( C and D ) Neutrophils and inflammatory monocytes were analyzed in bone marrow and spleen of Adar1 +/+ ; Mavs −/− and Adar Δ7-9 ; Mavs −/− animals. (C) Representative flow plots of subgated for living, CD11b + cells. Numbers next to the outlined areas indicate percentages within the CD11b + population. (D) Dot plots showing neutrophils (Ly6G + Ly6C lo ) and inflammatory monocytes (Ly6G − Ly6C hi ). Percentages of living cells (left panel) were used to determine total cell numbers (right panel). n = 3 mice/genotype; horizontal bars in (C) and (D) represent the mean; * P < 0.05, ** P < 0.01 and *** P < 0.001 determined by unpaired Student's t test.

Article Snippet: We therefore tested commercial monoclonal anti Adar antibody 15.8.6 (Santa Cruz) on HeK293 cells that were transfected with the above mentioned FLAG-tagged ADAR1 p110, p150, p110 Δ7-9 and p150 Δ7-9 constructs.

Techniques: Flow Cytometry

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Increased apoptosis in B cells and neutrophils in P15 Adar Δ7-9 ; Mavs −/− mice. The rate of apoptotic B cells and neutrophils was determined by flow cytometric analysis of Annexin-V and 7-AAD stained cells. ( A ) Bone marrow cells were stained and subgated for neutrophils (CD11b + Ly6G + Ly6C lo , upper panels) and B cells (CD19 + B220 + ). Left panels show representative flow plots. Numbers within the quadrants indicate percentage within the population. Right dot plots show percentages of early apoptotic (Annexin-V + 7-AAD − ), late apoptotic (Annexin-V + 7-AAD + ) and the sum of both. ( B ) Analysis of splenocytes performed as described in (A). n = 3 mice/genotype; horizontal bars in dot plots represent the mean; * P < 0.05 and ** P < 0.01 determined by unpaired Student's t test.

Article Snippet: We therefore tested commercial monoclonal anti Adar antibody 15.8.6 (Santa Cruz) on HeK293 cells that were transfected with the above mentioned FLAG-tagged ADAR1 p110, p150, p110 Δ7-9 and p150 Δ7-9 constructs.

Techniques: Staining

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Differentially expressed genes of Adar Δ7-9 ; Mavs −/− liver, bone marrow and cortex. ( A ) Volcano plots showing differentially expressed genes in liver, bone marrow and cortex of P15 mice. Genes with significantly altered representation(p-value < 0.001) are marked in color. ( B ) Go-terms enriched for significant differentially expressed genes for liver and bone-marrow. ( C ) Common differentially expressed genes of liver, bone-marrow and cortex.

Article Snippet: We therefore tested commercial monoclonal anti Adar antibody 15.8.6 (Santa Cruz) on HeK293 cells that were transfected with the above mentioned FLAG-tagged ADAR1 p110, p150, p110 Δ7-9 and p150 Δ7-9 constructs.

Techniques:

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Adar Δ7-9 ; Mavs −/− show altered ratios of 40S and 60S ribosomal sub-units. ( A ) Polysome profiling of P15 Adar Δ7-9 ; Mavs −/− and Mavs −/− liver (upper). RNA from fraction 7–12 loaded on a gel was stained with EtBr to confirm accumulation of 60S particles (lower). ( B ) Polysome profiling of HEK293T cells over-expressing Flag-Rps3a3 compared to mock transfected cells (upper). RNA from fraction 14–23 loaded on EtBr stained gel to confirm ribosomal peaks (lower). ( C ) Western blot of selected fractions from (B) showing incorporation of Flag-Rps3a3 in ribosomal subunits. ( D ) Expression of the pseudogene, Rps3a3 in wildtype mice in regions of craniofacial tissue at E8.5, E9.5 and E10.5 Data from .

Article Snippet: We therefore tested commercial monoclonal anti Adar antibody 15.8.6 (Santa Cruz) on HeK293 cells that were transfected with the above mentioned FLAG-tagged ADAR1 p110, p150, p110 Δ7-9 and p150 Δ7-9 constructs.

Techniques: Staining, Expressing, Transfection, Western Blot

Journal: Nucleic Acids Research

Article Title: An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype

doi: 10.1093/nar/gkaa025

Figure Lengend Snippet: Read coverage over Rps3a1 and Rps3a3 in rescued Adar Δ7-9 , Adar Δ2-13 and Adar E861A/E861A RNA-Seq read coverage over the UTR and coding regions of Rps3a1 and Rps3a3 in livers of P15 Adar Δ7-9 ; Mavs −/− and Mavs −/− , in E15.5 Adar1 Δ2-13 ; Mavs −/− , E15.5 Adar1 +/+ ; Mavs −/− , E11.5 Adar1 Δ2-13 ; Mavs +/+ , adult Adar1 E861A/E861A ; Ifih1 −/− and adult Adar1 +/+ ; Ifih1 −/− . Red lines in the graphs indicate the mismatches between Rps3a1 and Rps3a3 .

Article Snippet: We therefore tested commercial monoclonal anti Adar antibody 15.8.6 (Santa Cruz) on HeK293 cells that were transfected with the above mentioned FLAG-tagged ADAR1 p110, p150, p110 Δ7-9 and p150 Δ7-9 constructs.

Techniques: RNA Sequencing Assay