ADAR1 Promotes Malignant Progenitor Reprogramming in Chronic Myeloid Leukemia

ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia

Qingfei Jianga,b,c, Leslie A. Crewsa,b,c, Christian L. Barrettd, Hye-Jung Chune, Angela C. Courta,b,c, Jane M. Isquitha,b,c,f, Maria A. Zipetoa,b,c,g, Daniel J. Goffa,b,c, Mark Mindenh, Anil Sadarangania,b,c, Jessica M. Rusertc,i, Kim-Hien T. Daoj, Sheldon R. Morrisa,b, Lawrence S. B. Goldsteinc,i,k, Marco A. Marrae, Kelly A. Frazerd, and Catriona H. M. Jamiesona,b,c,1

aStem Cell Program, Department of Medicine, bMoores Cancer Center, dDivision of Genome Information Sciences, Department of Pediatrics, iDepartment of Cellular and Molecular Medicine, and kHoward Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093; eCanada’s Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3; fDepartment of Animal Science, California Polytechnic State University, San Luis Obispo, CA 93405; gDepartment of Health Sciences, University of Milano-Bicocca, 20052 Milan, Italy; hPrincess Margaret Hospital, Toronto, ON, Canada M5T 2M9; jCenter for Hematologic Malignancies, Oregon Health and Science University Knight Cancer Institute, Portland, OR 97239; and cSanford Consortium for Regenerative Medicine, La Jolla, CA 92037

Edited* by Irving L. Weissman, Stanford University, Stanford, CA, and approved November 30, 2012 (received for review July 30, 2012)

The molecular etiology of human progenitor reprogramming into ADAR2 are active in embryonic cell types (18), and ADAR3 self-renewing leukemia stem cells (LSC) has remained elusive. Al- may play a nonenzymatic regulatory role in RNA editing activity though DNA sequencing has uncovered spliceosome gene muta- (22). ADAR enzymes regulate fetal and adult hematopoietic stem tions that promote alternative splicing and portend leukemic cell (HSC) maintenance and stem cell responses to inflammation transformation, isoform diversity also may be generated by RNA (23–26). ADAR-mediated adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase acting on RNA (ADAR) editing in an Alu sequence containing dsRNA hairpin structures enzymes that regulate stem cell maintenance. In this study, whole- (14, 20) can generate alternative donor or acceptor splice sites transcriptome sequencing of normal, chronic phase, and serially (27, 28), alter RNA structure (21), modulate regulatory RNAs transplantable blast crisis chronic myeloid leukemia (CML) progen- and gene silencing activities (29), and introduce codon sequence itors revealed increased IFN-γ pathway gene expression in concert alterations (29). Interestingly, ADAR deregulation has been im- with BCR-ABL amplification, enhanced expression of the IFN-re- plicated in a variety of malignant cell types (30, 31). However, the sponsive ADAR1 p150 isoform, and a propensity for increased functional effects of RNA editing in leukemia have not been adenosine-to-inosine RNA editing during CML progression. Lenti- elucidated. Here we examined the role of ADAR1-mediated RNA editing in malignant reprogramming of myeloid progenitors viral overexpression experiments demonstrate that ADAR1 p150 into LSC that drive BC transformation in CML. promotes expression of the myeloid transcription factor PU.1 and Whole-transcriptome sequencing coupled with quantitative induces malignant reprogramming of myeloid progenitors. More- RT-PCR (qRT-PCR) analysis demonstrated increased IFN-re- over, enforced ADAR1 p150 expression was associated with produc- sponsive ADAR1 expression in LSC from primary BC CML pa- β tion of a misspliced form of GSK3 implicated in LSC self-renewal. tient samples as compared with CP CML and normal cord blood Finally, functional serial transplantation and shRNA studies dem- progenitors. This increase coincided with enhanced expression of MEDICAL SCIENCES onstrate that ADAR1 knockdown impaired in vivo self-renewal inflammatory pathway genes during the progression from CP to capacity of blast crisis CML progenitors. Together these data pro- BC. Moreover, ADAR1 p150 mRNA expression correlated with vide a compelling rationale for developing ADAR1-based LSC de- BCR-ABL amplification and increased A-to-I editing with dif- tection and eradication strategies. ferential expression of ADAR target genes in BC CML. Lentiviral ADAR1 p150 expression induced PU.1 expression that promotes lthough advanced malignancies are diverse in phenotype, expansion of myeloid progenitors and was associated with pro- Athey often exhibit stem-cell properties including enhanced duction of a misspliced form of glycogen synthase kinase (GSK)- survival, differentiation, quiescence, and self-renewal potential 3β that has been implicated in LSC self-renewal (7). Lentiviral ADAR1 knockdown also reduced BC LSC self-renewal capacity (1, 2). Early insights into the molecular pathogenesis of cancer − − − − stemmed from the discovery of the Philadelphia chromosome in RAG2 / γc / mice. These data shed light on the contribution + (Ph ) and its constitutively active BCR-ABL1 tyrosine kinase in of ADAR1-mediated RNA editing to malignant progenitor chronic myeloid leukemia (CML) (3–5). Tyrosine kinase inhibitor reprogramming driving leukemic progression. (TKI) therapy targeting BCR-ABL1 suppresses CML during the chronic phase (CP) of the disease in most patients able to tolerate Results long-term therapy (6). Although the CP stage of CML often can Inflammatory Mediator-Driven RNA Editing Portends Blastic Trans- be controlled for long periods of time with standard TKI thera- formation. To investigate the mechanisms driving malignant pies, subsequent genetic and epigenetic alterations promote pro- reprogramming of myeloid progenitors into LSC, we performed genitor expansion and the generation of self-renewing leukemia whole-transcriptome sequencing (RNA-seq) on FACS-purified + + − stem cells (LSC) that fuel disease progression and blast crisis (BC) CD34 CD38 Lin progenitor cells isolated from primary CML transformation along with TKI resistance (7, 8). Furthermore, TKI discontinuation usually results in CML resurgence, suggesting – that quiescent progenitors persist despite therapy (9 12). Author contributions: Q.J., M.A.M., K.A.F., and C.H.M.J. designed research; Q.J., C.L.B., Mutations in spliceosome genes and alternative splicing of H.-J.C., A.C.C., J.M.I., M.A.Z., D.J.G., A.S., and J.M.R. performed research; M.M., A.S., K.-H.T.D., coding and noncoding RNAs are emerging as important drivers L.S.B.G., M.A.M., and K.A.F. contributed new reagents/analytic tools; Q.J., L.A.C., C.L.B., of transcriptomic diversity that fuel leukemic progression and H.-J.C., A.C.C., J.M.I., M.A.Z., D.J.G., S.R.M., and C.H.M.J. analyzed data; and Q.J., L.A.C., therapeutic resistance (7, 8, 13). Moreover, previous studies re- and C.H.M.J. wrote the paper. veal extensive RNA editing in the human transcriptome (14–17), The authors declare no conflict of interest. primarily in primate-specific Alu sequences (18–20), which pro- *This Direct Submission article had a prearranged editor. motes splice isoform diversity. RNA editing activity is mediated Freely available online through the PNAS open access option. by the adenosine deaminase acting on dsRNA (ADAR) family of 1To whom correspondence should be addressed. E-mail: [email protected]. editases (21), which includes ADAR1 (also known as ADAR), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ADAR2 (ADARB1), and ADAR3 (ADARB2). ADAR1 and 1073/pnas.1213021110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1213021110 PNAS | January 15, 2013 | vol. 110 | no. 3 | 1041–1046 Downloaded by guest on September 29, 2021 Imatinib- A FGF SIGNALING G treated PATHWAY(N) NATURAL PROTEOGLYCAN KILLER 3.00 SYNDECAN-MEDIATED ROLE OF MEF2D CELL MEDIATED 2.00 SIGNALING IN CYTOTOXICITY(K) 1.00 EVENTS(N) T-CELL 0.00 APOPTOSIS(B) -1.00 PDGFR-BETA BCELL T CELL -2.00 CP-13 CP-05 CP-01 BC-17 BC-06 BC-02 CP-12 CP-04 CP-19 BC-05 BC-08 BC-09 CP-02 CB-03 BC-07 CB-02 CP-06 BC-25 SIGNALING BCELL ACTIVATION(P) CB-01 FAM107B NECTIN ACTIVATION(P) -3.00 PATHWAY(N) RECEPTOR CD70 ADHESION THROMBOXANE RPS6KA2 A2 SIGNALING DCTN4 PATHWAY(N) FARP1 SPHINGOSINE RECEPTOR PATHWAY(K) RAP1GAP2 1-PHOSPHATE SIGNALING(N) SERINC5 S1P1 NRP1 PATHWAY(N) (S1P) KIF1C PATHWAY(N) CPM TRAIL NLRC5 SIGNALING T CELL ZCCHC7 EVENTS SIGNALING LARGE RECEPTOR MXRA7 REGULATION OF PATHWAY(N) MEDIATED BY SIGNALING NEK11 PTP1B(N) NUCLEAR STAT1 PATHWAY(K) TSPAN13 SMAD2/3 TNF RECEPTOR MGAT4A SIGNALING(N) CDK9 SIGNALING IL12RB1 BCR SIGNALING PLASMA GLYPICAN 1 C4orf34 P38 MAPKPATHWAY(N) PATHWAY(N) AGPS MEMBRANE NETWORK(N) TMEM154 ESTROGEN SIGNALING OLFML2A PATHWAY(N) GLYPICAN VCAN RECEPTOR PATHWAY(N) TCR SIGNALING ANXA2 SIGNALING(N) FBXW7 REGULATION OF CLASS I PI3K IN LY96 NAIVE CD4+ T RBM47 REGULATION OF P38-ALPHA SIGNALING GALNT3 AND EVENTS(N) CELLS(N) PTPN14 CYTOPLASMIC TRIM38 AND P38-BETA(N) IL1-MEDIATED HDAC5 SIGNALING FLNB NUCLEAR TCR SIGNALING CMTM8 BMP RECEPTOR EVENTS(N) SMAD2/3 IN CLMN SIGNALING(N) ARHGAP25 SIGNALING(N) NAIVE CD8+ T CUEDC1 TGF-BETA BC Increased in CML CELLS(N) SLC27A1 RECEPTOR TCR LAPTM5 γ ALOX5 IFN- SIGNALING(N) SIGNALING(R) SLC35E3 SPATS2L IL6-MEDIATED PATHWAY(N) RNASET2 MS4A7 SIGNALING CD58 EVENTS(N) RAB31 FDR 4.0E-05 0.01 IL12-MEDIATED PLCD3 SIGNALING MEF2A CDS2 EVENTS(N) LRRFIP1 PIK3IP1 NCUBE1 ICOSLG MEF2D ADAR1 mRNA Expression ANXA6 BC HCK *P<0.05 VIL2 2 FGD6 p150 r =0.63, P=0.058 RNF125 20 **P<0.05 50 TPM4 2 S100A16 p110 r =0.42, P=0.162 FRMD4B MYO5C GUCY1A3 40 ABCA1 15 PKM2 SSBP2 SNX2 30 KIAA2018 PROM1 10 RUNX2 DUSP3 20 ANPEP TIMP2 SH3BP2 ARHGAP26 5 MAML2 10 SMAD1 TCF7L2 SVIL

ADAR1 mRNA Expression PLCB1 0 0 COQ10B ATF3 d 0.0 0.5 1.0 1.5 2.0 2.5 IRGQ Ratio of p150 to p110 mRNA Levels mRNA Ratio of p150 to p110 CP SESN1 RNF11 BCR-ABL mRNA Expression CLEC2B CML CML BC RHOC Cord Bloo RIPK2 NSUN7 RASGEF1B CNST A-to-I Editing Changes in CML BC v. CP Increased RNA Editing at ADAR Target Sites in CML BC v. CP C5orf41 DE SAMD12 5 CA5B ANKRD28 COL5A1 Sites with reduced Sites with increased BCAS4 TUFT1 ARID5B RNA editing in BC RNA editing in BC EFNA1 EIF2AK3 4 DNAJB2 C6orf141 PARD3 KIAA1598 RANBP17 MAGI3 ) UGGT2 e GSTO2 u 3 l GCNT2

va PDZD2 STXBP1

f P ( MPP7 NFIA 10 C21orf63 g TANC2 2 ELL2 -Lo RALGPS2 in CML BC in CML PBX1 Decreased GPR155 C6orf132 p=0.05 TFRC GDPD1 1 SPIRE1 TOM1L1 ARHGEF11 BACE2 NR6A1 FRRS1 LHFPL2 XKR6 0 EXT1 -6 -4 -2 0 2 4 6 FN1 Difference in average percentage of A-to-G changes PVRL1 MAP7 =2.5 fold =2.5 fold POLE2 decrease increase SOS1 FCGR2A Less edited sites More edited sites Log2 (editing fold change) BMP2K ITPKA FAM118A FHL2 SERPINE2 CML CP BLVRB Differentially Edited ADAR Target Genes TPCN1 F CML BC CDH1 100 FAM40B In Alu XRCC2 n CDC42BPA

io 80 MPV17L tG it ST8SIA6 s n

o MAP3K5 e c p 60 PTPN13 r t GSTT1 e SLC2A10 sa GPR157 gp

e 40 v

s LRP11 A a CDRT4 b 20 DTWD2 OBSCN PTENP1 ITGB3 0 EMP2 CLEC2D .1 2 3 1 1 9 P 2 6 B 3 A S 9 3 6 6 A 1 2 L 1 S N G ST M SS S3 4 4 M S O JUB 21 ITPA ABI1 PPI C RP TD MDM2 USP4 TIALLY LYST MBD3 WTAMD LYSTTTLL CT PAICS MD GGT PAIC EX TMEM51 BEC3DSPCS BPNT RPS1 BPNT1RAB2 EEF2K SP SF3B UBECM GNL3 U APOL6 RNF213 MIR3134 CCDC84 EIF2AK2 ZNF3DCAF1P PAR- ARFGAP2 POBEC3D DCLRE1C MUC20 A APO AC011477.4AC093484. AC011477.4 BCO2 P11-368J TCF7 newgene_1971R newgene_395 newgene_3753 newgene_1971 newgene_1971 SMAGP FANK1 Increased in CML BC in CML

Fig. 1. Inflammatory mediator-driven RNA editing portends blastic transformation. (A) Significantly over-represented pathways enriched for 2,228 differentially expressed genes in BC (n = 8) versus CP (n = 8) CML progenitors. (B) Human ADAR1 p150 and p110 were analyzed in CD34+CD38+Lin− progenitors from normal cord blood (n = 8), CML CP (n = 6), and CML BC (n = 7) by qRT-PCR. Ratios of p150 to p110 were determined (overall P = 0.0067; *P < 0.05 compared with normal cord blood; **P < 0.05 compared with CML CP by one-way ANOVA with post hoc Tukey test). (C) Pearson correlation analysis of ADAR1 isoforms and BCR-ABL mRNA levels in CML BC progenitors (n = 6). (D)RNA-seq–based analysis of A-to-G (=I) changes at putative editing sites (35) in CML BC (n = 8) versus CP progenitors (n = 8) (P < 0.05 by unpaired two-tailed Student t test). For each site, the percentage of reads that contained G versus A bases was calculated for each sample. The differences between average percentages were computed between disease stages (BC–CP). Data were reported as the number of sites showing significantly different editing ratios. (E) Volcano plot analysis showing enrichment of more highly edited sites in CML BC (n = 8) compared with CP progenitors (n = 8). (F) Differential editing at ADAR target sites in CML BC (n = 8) versus CP (n = 8). All sites shown were significantly different (P < 0.05 by Student t test, Dataset S1). (G) One hundred seventy-five putative ADAR target genes were differentially expressed in CP (n = 8) versus BC (n = 8). Unsupervised hier- archical clustering (see SI Materials and Methods) separated CP and BC samples. A select subset of genes (boxes) discriminated BC from cord blood (n = 3).

1042 | www.pnas.org/cgi/doi/10.1073/pnas.1213021110 Jiang et al. Downloaded by guest on September 29, 2021 + + − patient samples (Table S1). We identified 2,228 genes that were CML CP and BC CD34 CD38 Lin progenitors was compared differentially expressed in BC and CP progenitors, and pathway with the genomic coordinates of putative A-to-I editing sites analysis demonstrated overrepresentation of inflammatory IFN- identified in a previously published dataset (35). In eight CP and related and proteoglycan-related pathways involved in hemato- eight BC CML primary patient samples, the fraction of sites that logical development (Fig. 1A) in BC as compared with CP CML. contained guanosine bases (representing inosine substitution) Among the significantly affected networks identified using compared with adenosine bases was calculated for each patient Ingenuity Pathway Analysis (IPA), IFN-γ, cytokine, and TNF sample. This comparison revealed a striking enrichment of A-to- signaling pathways (Fig. S1A) along with self-renewal and re- G changes in ADAR target sites in BC compared with CP pro- programming factors (KLF family and LEF1; Fig. S1B) were genitors (Fig. 1D), demonstrating a shift toward increased RNA found to be enriched for differentially expressed genes in BC as editing during CML progression. Furthermore, volcano plot compared with CP CML. analysis of editing at ADAR target sites revealed ∼16-fold more Analysis of inflammatory mediator splice isoform expression editing sites with significantly increased editing ratios during BC revealed up-regulation of numerous inflammation-associated re- transformation (Fig. 1E). A total of 274 sites exhibited signifi- ceptors and signaling molecules (Fig. S2A). The most significantly different editing rates (Dataset S1), and these sites were cantly up-regulated transcripts included an LSC marker, IL-3Rα located predominantly within Alu repeat sequences (Fig. 1F). (CD123), IFN receptors, and TNF receptors (TNFRSF) (Fig. S2A). These data suggest that RNA editing activity is not only cell type- Notably, transcription of the IFN-responsive isoform of ADAR1 has and context-specific but also is disease stage-specific and there- been shown to be stimulated by a variety of inflammatory molecules fore could be a harbinger of disease progression. (32, 33). Because of known functional differences between ADAR1 By examining the relative levels of ADAR target gene (35) p150 and p110 that are not evident by whole-gene expression studies, transcripts, we found that 175 of 2,593 putative ADAR target genes + + − we performed a sensitive qRT-PCR analysis in CD34 CD38 Lin were differentially expressed in BC and CP progenitors (Fig. 1G). progenitor cells from primary CML patient samples (Table S1)using Differential expression of a subset of ADAR target genes clearly splice isoform-specificprimers(Table S2). Expression levels of the distinguished BC from CP or cord blood progenitors (Fig. 1G). inflammation-responsive ADAR1 p150 isoform were increased ap- In addition, nonnegative matrix factorization, using 200 random proximately eightfold in CML BC progenitors in comparison with start sites, recapitulated these major groupings (Fig. S1H). Fi- normal cord blood (Fig. S1C), but levels of the constitutively active nally, cophenetic correlation coefficients calculated using this p110 isoform did not differ significantly among groups (Fig. S1D). human myeloid progenitor dataset, compared with a randomized Analysis of the ratio of p150 to p110 showed a relative enrich- dataset, also supported three major groupings in the data (Fig. S1I). ment of the p150 ADAR1 isoform associated with CML pro- To investigate whether there was evidence of a link between gression (Fig. 1B) and BCR-ABL amplification in BC CML aberrant RNA editing and alternative splice isoform expression in progenitors (Fig. 1C). We observed similar patterns of increased BC versus CP CML, editing ratios at ADAR target sites were ADAR1 p150 expression in the granulocyte/macrophage pro- calculated for transcripts that were differentially expressed (Fig. S2 genitor (GMP) fraction of primary CML samples (Fig. S1E), C and D). Editing ratios were found to be increased for signifi- which expands during CML progression (Fig. S1F) (34). To cantly differentially expressed transcripts (Fig. S2C). Moreover, confirm sensitivity of HSC to inflammatory mediator-induced differential isoform expression of ADAR target genes revealed ADAR1 p150 expression, qRT-PCR analysis was performed on distinct clustering of CML BC versus CP (Fig. S2D). Thus, the shift + CD34 cord blood cells treated with IFN-γ or TNF-α, and results in transcriptional patterns associated with increased RNA editing

showed a twofold up-regulation of ADAR1 p150 (Fig. S1G). may reﬂect malignant reprogramming of progenitors in CML. MEDICAL SCIENCES Additional analysis of ADAR family gene expression in the RNA-seq data demonstrated that although total ADAR1 (ADAR) BCR-ABL Expression Enhances Inﬂammatory Mediator Gene Expression. expression was detectable in all samples, ADAR2 and ADAR3 Because qRT-PCR analyses revealed that ADAR1 p150 expres- expression was below detection thresholds (Fig. S2B). This result sion correlated with BCR-ABL levels in BC CML (Fig. 1C), we is consistent with previous reports showing that ADAR1 is the sought to investigate the potential mechanisms driving ADAR1 most highly expressed RNA-editing enzyme in tumor cells (35, inductioninBCCMLandtherelationshipbetweenADAR1 36) and suggests that ADAR1 is likely the primary functional p150 and BCR-ABL. Both qRT-PCR analyses and RNA-seq + A-to-I RNA editase in CML LSC. studies were performed with CD34 cord blood transduced with To investigate the contribution of ADAR1 up-regulation to lentivirus expressing BCR-ABL p210 tagged with GFP or vector RNA editing during CML progression, RNA-seq analysis of control (Fig. 2A and Fig. S3A). Increased phosphorylation of the

A B C 6 Differentially Expressed Genes pCDH Vector Lenti-BCR-ABL pCrkl Protein Lenti-BCR-ABL v. Vector-transduced cord blood

5 4 TNFRSF9 (CD137): Receptor- mediated inflammatory signaling P ZC3H12A: Negative regulator of 4 * =0.030

BF inflammatory processes, involved in 2 mRNA decay of interleukins 3 2

Log2 (fold change) 0 1 Lenti-BCR-ABL

(Normalized to B2M) ECM proteases or intercellular junction 0 -2 Inflammation/Potential ADAR regulation GFP AUC (Area Under the Curve) pCDH Lenti- 4 9 1 1 1 2 B 2 2 P 0 3 6 2 1 T A A R 3 .4 F9 27 . 13 23 19 0 A .1 . 2 2 Vector BCR-ABL C 11 SP C 6 1A1 B f D S 17. 27A M TJP1G W1 P A ABL1 MMP7SNX7 R PERP E S CA HSF5SCG2M1L1M1 MID1 002 C PLA FABP4C4orf F M CECR KRTR6 A E LTBP2 EPHX3 A YP EL C6or N T C C C3H1 T CYP U73166.1 O TR TM CHRNA6 Z 11-112L6C010623. C09264 AL096711.1 C092535. 11-114M5.1 P A 11-430G A A P 11-539L1011-117D22.2 R RP11-553D4. NCRN RP4-799D16P P R R R RP RP11-307C12.1

Fig. 2. BCR-ABL expression enhances inflammatory mediator gene expression. (A) Bright-field (BF) and fluorescent (GFP) microscopy showing cord blood- derived colonies transduced with lentiviral vector backbone control (pCDH) or lentivirus expressing BCR-ABL p210 and GFP. (B) Nanoproteomics analysis of + + phosphorylated (p)-Crkl levels in BCR-ABL–transduced CD34 cells from cord blood (n = 3). P < 0.05 by Student t test. (C) CD34 cord blood cells (n = 3) were transduced with lenti-BCR-ABL or vector control and processed for RNA-seq analysis. Forty-five genes were differentially expressed in BCR-ABL–expressing cells and vector controls (P < 0.05 by DESeq).

Jiang et al. PNAS | January 15, 2013 | vol. 110 | no. 3 | 1043 Downloaded by guest on September 29, 2021 BCR-ABL substrate Crkl by nanoproteomics analysis confirmed A B C functional BCR-ABL activation (Fig. 2B). These analyses sug- Lenti-ADAR1 Transduced Lenti-ADAR1 Transduced Lenti-ADAR1 Transduced Cord Blood Progenitors (2 wk) Cord Blood Progenitors (2 wk) Cord Blood Progenitors (2 wk) 40 6 4 gested that BCR-ABL may regulate ADAR1 indirectly. In support BFU-E ** r2=0.62, P<0.0001 r2=0.33, P=0.03 CFU-M * – 30 3 of this possibility, RNA-seq analyses of lenti-BCR-ABL trans- CFU-MIX 4 CFU-GM 20 2 duced cord blood compared with vector-transduced controls CFU-G 2 identified 45 differentially expressed genes (Fig. 2C). BCR and 10 1 Colony Number ABL1 were up-regulated along with inflammatory mediators such 0 0 0 PU.1 mRNA Expression PU.1 mRNA 04812 ORF control Lenti-ADAR1 0 5 10 15 Expression mRNA GATA1 as TNFRSF9 (Fig. 2C). Other differentially expressed genes in- ADAR1 mRNA Expression ADAR1 mRNA Expression cluded factors involved in ECM function or intercellular junctions D CML BC Progenitors EFCML BC Progenitors Ratio of PU.1/GATA1 Expression C fl 8 6 (Fig. 2 ). Because TNF pathways, receptors, and other in am- r2=0.78, P=0.0008 r2=0.65, P=0.005 2.0 *P=0.0018 matory mediators also were up-regulated during CML progression 6 A A A 4 1.5 (Fig. 1 and Figs. S1 and S2 ), it is conceivable that BCR-ABL 4 1.0 fl 2 regulates ADAR1 p150 through the induction of in ammatory 2 0.5 Ratio of PU.1/ GATA1 RPKM receptor expression. 0 0 0.0

PU.1 mRNA Expression PU.1 mRNA 04812 04812

ADAR1 mRNA Expression Expression mRNA GATA1 ADAR1 mRNA Expression CML CP CML BC ADAR1 Promotes Malignant Myeloid Progenitor Expansion and Alter- native Splicing. Lentiviral ADAR1 overexpression and shRNA G knockdown studies were performed to examine the functional consequences of ADAR1 p150 up-regulation. Transduction effi- ciency of lentiviral vectors driving human ADAR1 (p150) overexpression (lenti-ADAR1) or ADAR1-targeting shRNA (lenti- shADAR1) was validated in normal cord blood (Fig. S3 B–E). Myeloid lineage skewing was observed in colony-forming assays + + − performed with CD34 CD38 Lin cord blood progenitors transduced with lenti-ADAR1 p150, as demonstrated by an increase in the number of macrophage colonies and a corresponding decrease in the number of erythroid burst-forming unit (BFU-E) colonies (Fig. 3A). This myeloid lineage bias coincided with up-regulation of PU.1—a myeloid transcription factor—and down-regulation of GATA1—an erythroid transcription factor—in colonies from cord blood progenitors transduced with ADAR1 p150 lentivirus (Fig. 3 GATA1 B and C) and in CML BC progenitor-derived colonies (Fig. 3 D and E). RNA-seq analyses demonstrated an increase in the PU.1/ Increased in CML BC GATA1 ratio in BC versus CP progenitors (Fig. 3F). Additionally, Decreased in CML BC network analysis of RNA-seq data revealed down-regulation of genes involved in GATA1-dependent processes (Fig. 3G). Fig. 3. ADAR1 promotes malignant myeloid progenitor expansion. (A) Short-term culture of normal progenitors transduced with Lentiviral overexpression of ADAR1 p150 in FACS-purified CD34+38+Lin− lenti-ADAR1 at increasing multiplicity of infection (MOI) con- normal progenitors (n = 3) reduced erythroid (BFU-E) colony formation and firmed a positive correlation between PU.1 and ADAR1 expres- increased macrophage (M) cfu compared with vector (ORF) controls. Mix, sion, whereas the levels of GATA1 expression remained constant macrophage/erythroid colonies; GM, granulocyte/macrophage colonies; (Fig. S3F). The ADAR1-mediated myeloid lineage skewing G, granulocyte colonies. *P < 0.05 and **P < 0.01 compared with vector- recapitulates qRT-PCR data reported with aged human HSC (37) transduced controls or CP progenitors by Student t test. (B–E) Pearson cor- and the expansion of GMP (34) during progression of CML from relation analysis of HPRT-normalized ADAR1 mRNA levels and PU.1 or CP to BC (Fig. S1F). Because we previously identified pro- GATA1 in individual colonies derived from normal progenitors (n = 3) duction of a misspliced form of GSK3β lacking exons 8 and 9 (7) transduced with lenti-ADAR1 p150 (B and C) or CML BC progenitor-derived colonies (D and E). r2 values were calculated using Pearson correlation associated with GMP expansion in CML BC, and because acti- = vation of ADAR1 might promote alternative splicing (27), we analysis. (F) Ratio of PU.1 to GATA1 expression levels by RNA-seq in CP (n fi β 8) versus BC (n = 8) CML. (G) RNA-seq–based IPA network analysis of GATA1- performed splice isoform-speci c qRT-PCR for GSK3 variants associated genes in BC CML (n = 8) compared with CP (n = 8). in individual colonies derived from lenti-ADAR1–transduced cord blood or CML CP progenitors. Although GSK3β exon 8– 9del was undetectable in cord blood progenitors, in CML CP To examine the role of ADAR1 in CML progenitor survival + samples mRNA levels of this variant were increased relative to and self-renewal capacity in vivo, CD34 human BC CML pro- the exon 9del form, which represents a predominant GSK3β G genitors were transduced with lenti-shADAR1 or shRNA back- transcript in normal hematopoietic tissues (Fig. S3 ) (7). To- bone (lenti-shControl) and transplanted intrahepatically into gether, these results demonstrate that ADAR1 overexpression − − − − neonatal RAG2 / γc / mice (Fig. 4A). In addition, qRT-PCR drives hematopoietic differentiation toward the myeloid lineage, analysis was performed to confirm ADAR1 p150 knockdown. coincident with PU.1 up-regulation and alternative splicing of C GSK3β in CML progenitors. Similar to experiments in normal cord blood (Fig. S3 ), ADAR1 p150 levels before transplantation were reduced by ∼50% in ADAR1 Knockdown Impairs Malignant Myeloid Progenitor Self- CML BC progenitors transduced with lenti-shADAR1 as com- A Renewal. Previous reports demonstrate that ADAR1 mediates pared with vector controls (Fig. 4 ). mouse HSC maintenance (23, 24); however the relative effects By 10 wk after transplantation, robust leukemic engraftment of down-modulating ADAR1 expression in human LSC versus was detectable in hematopoietic tissues by FACS analysis of + B normal HSC have not been established. In support of a favorable human CD45 cells (Fig. 4 ). Similar levels of human hemato- therapeutic index for ADAR1 inhibitory strategies in CML pro- poietic cell engraftment were detected in bone marrow from genitors versus normal cord blood, shRNA knockdown and col- mice transplanted with lenti-shADAR1–transduced BC progen- ony assay experiments showed that lenti-shADAR1 knockdown itors and lenti-shControl (Fig. 4 C and D). qRT-PCR analysis in normal cord blood progenitors did not affect hematopoietic confirmed that pooled human progenitors from primary transplant- differentiation (Fig. S3H), whereas CML CP progenitors trans- recipient bone marrow maintained reduced levels of ADAR1 duced with lenti-shADAR1 produced fewer macrophage colonies p150, consistent with continued activity of the ADAR1 shRNA coupled with increased numbers of BFU-E colonies (Fig. S3I). after primary engraftment (Fig. 4A). We transplanted equal

1044 | www.pnas.org/cgi/doi/10.1073/pnas.1213021110 Jiang et al. Downloaded by guest on September 29, 2021 A -/- -/- B Control Bone Marrow Fig. 4. ADAR1 knockdown impairs malignant myeloid shADAR1 Lentiviral RAG2 c Engraftment analysis (FACS) 250K 250K FACS, qPCR CML BC transduction (48 hrs), 200K 200K progenitor self-renewal. (A) Diagrammatic scheme of qPCR and transplant 10 weeks b c b d Progenitors 150K 150K in vivo experimental design. Before and after primary 0.19 68 100K 100K FSC-A 50K 50K transplantation, qRT-PCR was performed in human 0K 0K Human Progenitors - 0102 103 104 105 0102 103 104 105 ﬁ CML BC Progenitors - Engraftment CD45 progenitors, con rming ADAR1 knockdown. BM, bone Before Transplant 1° Transplant After Primary Transplant 1.5 1.5 analysis Spleen Liver 250K 250K marrow. (B) Representative FACS plots showing human 2° Transplant of + e f 200K 200K 1.0 1.0 BM Progenitors CD45 engraftment in hematopoietic organs of primary 150K 150K − − − − 5.01 0.74 / / 100K 100K γ

FSC-A RAG2 c transplant recipients or untransplanted 0.5 0.5 50K 50K ADAR1 p150 ADAR1 p150 mRNA Expression mRNA mRNA Expression mRNA 0K 0K control mouse bone marrow. (C and D) FACS analysis of 0.0 0.0 0102 103 104 105 0102 103 104 105 + shControl shADAR1 shControl shADAR1 CD45 CD45 human cell engraftment and progenitor cells in C D E F the bone marrow of mice transplanted with BC pro- Primary Engraftment in Bone Marrow Primary Engraftment in Bone Marrow Serial Engraftment in Bone Marrow Serial Engraftment in Bone Marrow CD45+ Human Cells CD34+CD38+ Progenitors CD45+ Human Cells CD34+CD38+ Progenitors genitors transduced with lenti-shControl (n = 3) or lenti- P =0.38 *P 100 =0.011 shADAR1 (n = 5). (E and F) FACS analysis of human cells 100 100 20 P P=0.27 =0.056 = 75 75 75 in mouse bone marrow (shControl n 7 and shADAR1 15 + n = 8) after serial transplantation of BM CD34 cells 50 50 50 10 pooled from primary transplant-recipient mice. *P < 25 25 25 5 CD45+ Frequency CD45+ Frequency 0.05 compared with vector-transduced controls by Stu- 0 0 0 0 CD34+38+Lin- Frequency shControl shADAR1 shControl shADAR1 shControl shADAR1 CD34+38+Lin- Frequency shControl shADAR1 dent t test.

+ numbers of human CD34 progenitors derived from the bone hematopoietic progenitors to inflammatory stimuli that drive marrow of primary recipients of lenti-shControl or lenti-shADAR1– ADAR1 expression and promote CML LSC self-renewal. transduced cells into secondary recipient mice, and the bone Whole transcriptome-based RNA editing analyses revealed + marrow was analyzed for human cell engraftment and CD34 that CML progression is accompanied by differential RNA + − CD38 Lin progenitors by FACS (Fig. 4 E and F). Notably, editing. Recent in-depth transcriptomic studies have shown that serial engraftment potential of shADAR1-transduced CML BC gene products with predicted A-to-I editing events are signifi- progenitors was reduced considerably (∼20%) compared with cantly enriched in cancer-related pathways (35). The present shControls (∼50%) (Fig. 4E). Although leukemic burden was study provides further evidence supporting a role for ADAR- not diminished significantly, the LSC self-renewal capacity was directed RNA editing in splice isoform diversity in cancer and irrevocably reduced by ADAR1 knockdown, suggesting that demonstrates the activation of inflammation-associated RNA ADAR1 plays a pivotal role in the propagation of leukemia editing during the progression of hematologic malignancies. driven by self-renewing malignant progenitors. Our results suggest that ADAR1 p150 up-regulation may con- + tribute to blastic transformation of CML driven by the CD34 + − Discussion CD38 Lin progenitor and GMP subpopulations. Because Activation of IFN-responsive ADAR1 p150 in primary CML ADAR-mediated RNA editing occurs primarily in primate- progenitors correlated with increased A-to-I RNA editing follow- specific Alu repeat sequences (14, 19, 20), the activation of ing blastic transformation. Full transcriptome RNA-seq analyses RNA editases in human cells may fuel species-specificmalig- suggest that up-regulation of ADAR1 p150 in BC CML may be nant reprogramming of progenitors to adopt a more primitive related to the activation of inflammatory pathways such as cyto- stem cell fate under pathological conditions. MEDICAL SCIENCES kines and TNF in advanced disease. Hematopoietic progenitor Myeloid lineage skewing was observed in response to enforced studies demonstrated that lentivirally enforced ADAR1 p150 ex- ADAR1 p150 expression, concomitant with PU.1 activation. pression promotes human myeloid differentiation fate. Lentiviral- Although a previous study in an in vitro model implicated PU.1 shRNA knockdown of ADAR1 in a xenotransplantation model in the regulation of murine ADAR1 expression (48), cell type- impaired self-renewal capacity of BC LSC. Together, these data and niche-specific stimuli may have differential effects on RNA suggest that an inflammatory mediator-driven isoform switch fa- editing, and our studies in human cells, in which 90% of RNA voring ADAR1 p150 expression drives expansion of malignant editing occurs in primate-specific Alu sequences, suggest that the progenitors and contributes to CML progression. converse may occur also. It is conceivable that ADAR1 directly ADAR-mediated RNA editing can regulate myriad molecular controls PU.1 through RNA editing-dependent effects or via processes including RNA interference (29), microRNA function potential transcriptional regulation related to its Z-DNA binding (38), and RNA stability, localization, nuclear retention, degra- function (41, 49). In support of the former possibility, DARNED dation, and alternative splicing (27, 39–41). Moreover, high levels (50), a database compiling RNA editing sites from multiple of ADAR-mediated RNA editing activity may reflect a reversion publications, reports that transcripts of Spi1 (a gene encoding to a primitive transcriptional program typical of embryonic stem PU.1; chromosome 11, start: 47379732, end: 47400127) showed cells (18). A previous study demonstrated that ADAR1 was evidence of A-to-I RNA editing at 28 sites (14). Future gene- among the top 5% of genes expressed in the mutational evolution expression analyses and identification of de novo RNA editing of lobular breast cancer (36), indicating that activation of ADARs sites through analysis of whole-genome and transcriptome DNA- may correlate with disease progression in multiple malignant cell RNA differences (15) in normal HSC harboring enforced types (31). Although previous studies have shown ADAR1 p110 ADAR1 p150 expression and LSC will be necessary to dissect up-regulation in a murine leukemia model (42) and in pediatric further the link between ADAR1 activation PU.1 expression. acute leukemias (43), it should be emphasized that human he- In addition to up-regulation of PU.1 in response to ADAR1 matopoietic tissues undergo dramatic changes during aging (44, p150 overexpression, we also detected production of a misspliced 45) that are caused in part by increased inflammation and ge- GSK3β variant in colonies derived from CML CP progenitors nomic instability (46). transduced with lentiviral ADAR1 p150. We have shown pre- Our data suggest that inflammatory cues may facilitate selec- viously that this particular form of GSK3β harbors reduced active up-regulation of the IFN-responsive ADAR1 p150 isoform in tivity and promotes LSC self-renewal via activation of β-catenin hematologic malignancies. Using whole-transcriptome sequencing (7). In support of a role for ADAR1 in CML LSC self-renewal, analysis, we found that inflammatory signaling receptors were dif- we showed in humanized CML xenograft mouse models that ferentially expressed in BCR-ABL–expressing cord blood. Inflam- ADAR1 knockdown reduced the potential of CML BC LSC for matory cytokines such as IFN, TNF-α, and other interleukins have serial transplantation. Given that these CML BC progenitors beenshowntostimulateADAR1p150expression(32,33),andIFN expressed significantly higher levels of ADAR1 p150 than did regulates HSC quiescence (47). Together, BCR-ABL–mediated normal cord blood progenitors, we expect that LSC are rela- up-regulation of inflammatory pathway receptors could sensitize tively more dependent on IFN-responsive ADAR1 activity (24).

Jiang et al. PNAS | January 15, 2013 | vol. 110 | no. 3 | 1045 Downloaded by guest on September 29, 2021 Together these data suggest that inflammation-dependent acti- primary samples or normal cord blood were processed for RNA-Seq, qRT-PCR, vation of the ADAR1 p150 editase promotes CML progression. and for in vitro and xenotransplantation studies. Detailed methods are Aberrant ADAR1 activation in CML endows myeloid progeni- available in SI Materials and Methods. tors with self-renewal capacity leading to LSC generation. Thus, ACKNOWLEDGMENTS. We thank Wenxue Ma, Alice Shih, and Heather Leu inhibition of ADAR1 activity could represent an effective strategy (all of Moores Cancer Center, University of California at San Diego) for as- to prevent LSC-driven relapse in CML while sparing normal HSC sistance with in vivo experiments; Sa Li (BC Cancer Agency) for performing populations. Furthermore, ADAR1-mediated RNA editing acti- SNV analysis; Jonathan Lee (Moores Cancer Center, University of California vation could prove to be a diagnostic and prognostic indicator of at San Diego) for assistance with figures; Guanming Wu for providing the mapping information of pathway-to-gene relationships; and the Genome disease progression with important implications for other cancer Sciences Centre Library Construction, Sequencing, and Bioinformatics teams stem cell-driven malignancies. of the BC Cancer Agency. This work was supported by California Institute for Regenerative Medicine (CIRM) Grants RN2-00910-1 (to C.H.M.J.) and DR1- Materials and Methods 01430 (to C.H.M.J.), by CIRM Training Grant TG2-01154 (to Q.J.), by CIRM Sci- entific Excellence through Exploration and Development (SEED) Grant RS1- Primary samples from CML patients were obtained from consenting patients 00228-1, by a Cancer Stem Cell Consortium with funding from the Government at the University of California San Diego, Stanford University, and the Uni- of Canada through Genome Canada and the Ontario Genomics Institute (OGI- versity of Toronto Health Network. FACS-purified progenitor cells from 047), and through the Canadian Institutes of Health Research (CSC-105367).

1. Jamieson C (2009) The MLLgnant consequences of reverting to an embryonic tran- 25. Wang Q (2011) RNA editing catalyzed by ADAR1 and its function in mammalian cells. scriptional program. Cell Stem Cell 4(2):97–98. Biochemistry (Mosc) 76(8):900–911. 2. Eppert K, et al. (2011) Stem cell gene expression programs influence clinical outcome 26. Wang Q, Khillan J, Gadue P, Nishikura K (2000) Requirement of the RNA editing in human leukemia. Nat Med 17(9):1086–1093. deaminase ADAR1 gene for embryonic erythropoiesis. Science 290(5497):1765–1768. 3. Ben-Neriah Y, Daley GQ, Mes-Masson AM, Witte ON, Baltimore D (1986) The chronic 27. Rueter SM, Dawson TR, Emeson RB (1999) Regulation of alternative splicing by RNA myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid editing. Nature 399(6731):75–80. gene. Science 233(4760):212–214. 28. Lev-Maor G, et al. (2007) RNA-editing-mediated exon evolution. Genome Biol 8(2): 4. Rowley JD (1973) Letter: A new consistent chromosomal abnormality in chronic my- R29. elogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. 29. Nishikura K (2006) Editor meets silencer: Crosstalk between RNA editing and RNA Nature 243(5405):290–293. interference. Nat Rev Mol Cell Biol 7(12):919–931. 5. Shtivelman E, Lifshitz B, Gale RP, Canaani E (1985) Fused transcript of abl and bcr 30. Galeano F, Tomaselli S, Locatelli F, Gallo A (2012) A-to-I RNA editing: The “ADAR” side genes in chronic myelogenous leukaemia. Nature 315(6020):550–554. of human cancer. Semin Cell Dev Biol 23(3):244–250. 6. Hughes TP, et al.; International Randomised Study of Interferon versus STI571 (IRIS) 31. Cenci C, et al. (2008) Down-regulation of RNA editing in pediatric astrocytomas: Study Group (2003) Frequency of major molecular responses to imatinib or interferon ADAR2 editing activity inhibits cell migration and proliferation. J Biol Chem 283(11): alfa plus cytarabine in newly diagnosed chronic myeloid leukemia. N Engl J Med 349 7251–7260. – (15):1423 1432. 32. Meltzer M, et al. (2010) The RNA editor gene ADAR1 is induced in myoblasts by in- β 7. Abrahamsson AE, et al. (2009) GSK3 missplicing contributes to leukemia stem cell flammatory ligands and buffers stress response. Clin Transl Sci 3(3):73–80. – generation. Proc Natl Acad Sci USA 106(10):3925 3929. 33. Yang JH, et al. (2003) Widespread inosine-containing mRNA in lymphocytes regulated 8. Perrotti D, Jamieson C, Goldman J, Skorski T (2010) Chronic myeloid leukemia: by ADAR1 in response to inflammation. Immunology 109(1):15–23. – Mechanisms of blastic transformation. J Clin Invest 120(7):2254 2264. 34. Jamieson CH, et al. (2004) Granulocyte-macrophage progenitors as candidate leuke- 9. Copland M, et al. (2006) Dasatinib (BMS-354825) targets an earlier progenitor pop- mic stem cells in blast-crisis CML. N Engl J Med 351(7):657–667. ulation than imatinib in primary CML but does not eliminate the quiescent fraction. 35. Bahn JH, et al. (2012) Accurate identification of A-to-I RNA editing in human by Blood 107(11):4532–4539. transcriptome sequencing. Genome Res 22(1):142–150. 10. Jamieson CH, Barroga CF, Vainchenker WP (2008) Miscreant myeloproliferative dis- 36. Shah SP, et al. (2009) Mutational evolution in a lobular breast tumour profiled at order stem cells. Leukemia 22(11):2011–2019. single nucleotide resolution. Nature 461(7265):809–813. 11. Jørgensen HG, Allan EK, Jordanides NE, Mountford JC, Holyoake TL (2007) Nilotinib 37. Pang WW, et al. (2011) Human bone marrow hematopoietic stem cells are increased exerts equipotent antiproliferative effects to imatinib and does not induce apoptosis in frequency and myeloid-biased with age. Proc Natl Acad Sci USA 108(50):20012– in CD34+ CML cells. Blood 109(9):4016–4019. 20017. 12. Huang X, Cortes J, Kantarjian H (2012) Estimations of the increasing prevalence and 38. Yang W, et al. (2006) Modulation of microRNA processing and expression through plateau prevalence of chronic myeloid leukemia in the era of tyrosine kinase inhibitor RNA editing by ADAR deaminases. Nat Struct Mol Biol 13(1):13–21. therapy. Cancer 118(12):3123–3127. 39. Heale BS, Keegan LP, O’Connell MA (2009) ADARs have effects beyond RNA editing. 13. Perrotti D, Neviani P (2007) From mRNA metabolism to cancer therapy: Chronic my- Cell Cycle 8(24):4011–4012. elogenous leukemia shows the way. Clin Cancer Res 13(6):1638–1642. 40. Heale BS, et al. (2009) Editing independent effects of ADARs on the miRNA/siRNA 14. Kim DD, et al. (2004) Widespread RNA editing of embedded alu elements in the pathways. EMBO J 28(20):3145–3156. human transcriptome. Genome Res 14(9):1719–1725. 41. Herbert A, Wagner S, Nickerson JA (2002) Induction of protein translation by 15. Ramaswami G, et al. (2012) Accurate identification of human Alu and non-Alu RNA editing sites. Nat Methods 9(6):579–581. ADAR1 within living cell nuclei is not dependent on RNA editing. Mol Cell 10(5): – 16. Peng Z, et al. (2012) Comprehensive analysis of RNA-Seq data reveals extensive RNA 1235 1246. editing in a human transcriptome. Nat Biotechnol 30(3):253–260. 42. Ma CH, et al. (2011) [Expression of ADAR1 isoforms in murine acute T-ALL leukemia – 17. Levanon EY, et al. (2004) Systematic identification of abundant A-to-I editing sites in model]. Zhongguo Shi Yan Xue Ye Xue Za Zhi 19(3):566 569. the human transcriptome. Nat Biotechnol 22(8):1001–1005. 43. Ma CH, et al. (2011) Abnormal expression of ADAR1 isoforms in Chinese pediatric – 18. Osenberg S, et al. (2010) Alu sequences in undifferentiated human embryonic stem acute leukemias. Biochem Biophys Res Commun 406(2):245 251. cells display high levels of A-to-I RNA editing. PLoS ONE 5(6):e11173. 44. Morrison SJ, Wandycz AM, Akashi K, Globerson A, Weissman IL (1996) The aging of – 19. Eisenberg E, et al. (2005) Is abundant A-to-I RNA editing primate-specific? Trends hematopoietic stem cells. Nat Med 2(9):1011 1016. Genet 21(2):77–81. 45. Wang LD, Wagers AJ (2011) Dynamic niches in the origination and differentiation of 20. Athanasiadis A, Rich A, Maas S (2004) Widespread A-to-I RNA editing of Alu-con- haematopoietic stem cells. Nat Rev Mol Cell Biol 12(10):643–655. taining mRNAs in the human transcriptome. PLoS Biol 2(12):e391. 46. Chambers SM, et al. (2007) Aging hematopoietic stem cells decline in function and 21. Bass BL, Weintraub H (1988) An unwinding activity that covalently modifies its dou- exhibit epigenetic dysregulation. PLoS Biol 5(8):e201. ble-stranded RNA substrate. Cell 55(6):1089–1098. 47. Essers MA, et al. (2009) IFNalpha activates dormant haematopoietic stem cells in vivo. 22. Chen CX, et al. (2000) A third member of the RNA-specific adenosine deaminase gene Nature 458(7240):904–908. family, ADAR3, contains both single- and double-stranded RNA binding domains. 48. Wu T, Zhao Y, Hao Z, Zhao H, Wang W (2009) Involvement of PU.1 in mouse adar-1 RNA 6(5):755–767. gene transcription induced by high-dose esiRNA. Int J Biol Macromol 45(2):157–162. 23. Hartner JC, Walkley CR, Lu J, Orkin SH (2009) ADAR1 is essential for the maintenance 49. Herbert A, et al. (1997) A Z-DNA binding domain present in the human editing en- of hematopoiesis and suppression of interferon signaling. Nat Immunol 10(1):109– zyme, double-stranded RNA adenosine deaminase. Proc Natl Acad Sci USA 94(16): 115. 8421–8426. 24. XuFeng R, et al. (2009) ADAR1 is required for hematopoietic progenitor cell survival 50. Kiran A, Baranov PV (2010) DARNED: A DAtabase of RNa EDiting in humans. via RNA editing. Proc Natl Acad Sci USA 106(42):17763–17768. Bioinformatics 26(14):1772–1776.

1046 | www.pnas.org/cgi/doi/10.1073/pnas.1213021110 Jiang et al. Downloaded by guest on September 29, 2021