Hyper-Editing of Cell-Cycle Regulatory and Tumor Suppressor RNA Promotes Malignant Progenitor Propagation

Abstract

Adenosine deaminase associated with RNA1 (ADAR1) deregulation contributes to therapeutic resistance in many malignancies. Here we show that ADAR1-induced hyper-editing in normal human hematopoietic progenitors impairs miR-26a maturation, which represses CDKN1A expression indirectly via EZH2, thereby accelerating cell-cycle transit. However, in blast crisis chronic myeloid leukemia progenitors, loss of EZH2 expression and increased CDKN1A oppose cell-cycle transit. Moreover, A-to-I editing of both the MDM2 regulatory microRNA and its binding site within the 3' UTR region stabilizes MDM2 transcripts, thereby enhancing blast crisis progenitor propagation. These data reveal a dual mechanism governing malignant transformation of progenitors that is predicated on hyper-editing of cell-cycle-regulatory miRNAs and the 3' UTR binding site of tumor suppressor miRNAs.

Keywords: 3′ UTR; ADAR1; RNA hyper-editing; cancer stem cell; cell cycle; chronic myeloid leukemia; epitranscriptome; leukemia; microRNAs; progenitors; self-renewal.

Figure 1.. ADAR1 regulates cell cycle in normal hematopoiesis.

(A) Representative picture of ADAR1-WT or lentiviral backbone transduced cord blood CD34⁺ cells. (B and C) Cell counts of total cell number (B), stem cells (CD34⁺CD38⁻Lin⁻), and progenitors (CD34⁺CD38⁺Lin⁻) (C) following ADAR1 WT overexpression in cord blood CD34⁺ cells (n=3). (D and E) Representative image of Ki67 immunofluorescent staining in ADAR1 WT-expressing cord blood CD34⁺ cells (D) and the corresponding calculation of Ki67⁺ cells (n=3). (F and G) Representative flow cytometry of DiR tracing in cord blood CD34⁺ cells transduced with backbone control or ADAR1 WT (F) and the corresponding calculation of GFP⁺DiR⁺ cells (G) (n=3). (H) Significant differential expressed cell cycle transcripts were determined by qRT-PCR array of 84 transcripts on cord blood HSPC (n=5) transduced with ADAR1 WT, ADAR1^E912A, or lentiviral vector control. (I) Cytoscape analysis of differentially expressed transcripts of KEGG Cell Cycle Pathway in ADAR1 WT-transduced cord blood (n=3) versus lentiviral vector control (n=3) by whole transcriptome RNA-seq. (J) RNA-seq quantification on ADAR1 WT-transduced cord blood (n=3) and lentiviral vector control (n=3) for genes corresponding to the KEGG Cell Cycle Pathway visualized in a heatmap (p<0.05, FDR <10%). (K) Representative image of ADAR1-mediated differentially expression targets in cell cycle stages. (L and M). Representative images (L) and quantification (M) of immunofluorescent staining of CDKN1A protein in ADAR1 WT-expressing CD34⁺ cells (n=3). (N) Cell cycle analysis in cord blood CD34⁺ HSPC transduced with shRNA targeting ADAR1 (shADAR1) or control shRNA (shControl) as measured by flow cytometry of Ki-67 and 7AAD (n=4). (O). CDKN1A expression measured by qRT-PCR in cord blood CD34⁺ HSPC (n=3). (P) Percentage of replated colonies in cord blood CD34⁺ HSPC transduced with shADAR1 or shControl (n=3). All graphs show mean with SEM and statistical analysis was calculated using the Student’s t-test. *p<0.05, **p<0.005, ***p<0.0005. See also Figure S1 and Table S1.

Figure 2.. Regulation of miRNome by ADAR1 in normal cord blood CD34⁺ HSPC.

(A) Top ten significantly affected pathways by differentially expressed miRNAs targeted by ADAR1 WT or ADAR1^E912A compared with lentiviral backbone. (B) Pie chart of differentially expressed miRNAs in cord blood CD34⁺ HSPC overexpression ADAR1 WT or ADAR1^E912A compared with backbone vector control (n=3-4) derived from miRNome array of 1008 miRNAs. (C and D) Volcano plot analysis derived from miRNome showing significantly differentially expressed miRNAs (p<0.005, Student’s t-test, Log₂Fold change >2) between cord blood CD34⁺ cells transduced with backbone vector control and those with lenti-ADAR1 WT (C) or between cord blood CD34⁺ cells transduced with backbone vector control and those with lenti-ADAR1^E912A (D) (n=3-4). See also Table S2.

Figure 3.. Important role of miR-26a in self-renewal capacity of normal hematopoietic progenitors

(A) Lentiviral construct for human primary (pri-) miR-26a expression. (B) The expression of mature miR-26a was determined by qRT-PCR in cord blood CD34⁺ HSPC transduced with pri-miR-26a lentivirus (n=3). (C and D) The total colony number (C) and the percentage of replated colonies in cord blood CD34⁺ cells overexpressing hsa-miR-26a or the backbone control (n=3). (E) Expression of LIN28B in cord blood CD34⁺ HSPC overexpressing hsa-miR-26a or the backbone control (n=3). (F) Representative cell cycle flow analysis of CB CD34⁺ HSPC (n=3) transduced with either backbone or hsa-miR-26a overexpression lentivirus. (G) Flow cytometry analysis of cell cycle transit in cord blood CD34⁺ HSPC overexpressing hsa-miR-26a or the backbone control (n=3). (H) Expression of CDKN1A mRNA level was determined by qRT-PCR in cord blood CD34⁺ HSPC overexpressing hsa-miR-26a or the backbone control (n=3). (I) EZH2 expression evaluated by RNA-seq in cord blood CD34⁺ cells transduced with backbone control of ADAR1 WT (n=3). (J and K). Examination of EZH2 expression in cord blood CD34⁺ cells with either ADAR1 knockdown by shRNA (J) or overexpression of hsa-miR-26a (K) (n=3). (L) Expression of primary (pri-), precursor (pre-) and mature miR-26a transcripts was measured by qRT-PCR in cord blood CD34⁺ HSPCs transduced with backbone, ADAR1 WT, or ADAR1^E912A (n=3). (M) The A-to-I RNA editing efficiency was determined by TOPO sequencing of blood CD34⁺ cells overexpressing backbone, ADAR1 WT, or ADAR1^E912A (n=3). 20.4% of colonies examined contain A-to-G change at the DROSHA cleavage site. The arrow pointed to the A-to-G mutation site, reverse sequenced as T-to-C change. (N) Confirmation of lentiviral constructs of “unedited” and “edited” pri-miR-26a. The arrow points to the A-to-I (G) mutation site. (O) HEK293T cells were transfected with “unedited” or “edited” pri-miR-26a lentivirus, and the mature miR-26a expression was determined by qRT-PCR (n=5 experiments). (P) Cell cycle flow analysis of G₀ population in HEK293T cells transduced with ADAR1 WT in combination with “unedited” or “edited” miR-26a (n=3 experimental triplicate). All graphs show mean with SEM and statistical analysis was calculated using the Student’s t-test. *p<0.05, **p<0.005, ***p<0.0005. See also Figure S2.

Figure 4.. Reduced miR-26a enhances self-renewal capacity of CML Progenitors.

(A) Expression of mature miR-26a in CML CP (n=3) and CML BC (n=3) CD34⁺ cells measured by qRT-PCR (n=3). (B) miR-26a expression in CML CP CD34⁺ cells transduced with backbone control or ADAR1 WT examined by miRNA PCR array (n=3). (C) Validation of miR-26a expression in CP CML CD34⁺ cells transduced with backbone control or ADAR1 WT lentivirus by qRT-PCR (n=3). (D and E) Number of colonies formed in primary colony-formation assay (D) and percentage of secondary colonies formed after replating primary colonies (E) in BC CML CD34⁺ cells transduced with backbone control or hsa-miR-26a (n=3). (F) Experimental design of in vivo xenograft mouse studies. (G) Percentage of granulocyte macrophage progenitors (GMP) engraftment in the bone marrow or spleen of Rag2^−/−γc^−/− mice transplanted with BC CD34⁺ cells overexpressing control or hsa-miR-26a (n=3 mice per group). (H) Cell cycle flow analysis of backbone or lenti-miR-26a transduced CD34⁺ cells isolated from engrafted BC bone marrow (n=3 mice per group). (I) RNA-seq analysis of CDKN1A and EZH2 expression in CP (n=7) and BC CML (n=6). (J) Expression of EZH2, CDKN1A, and LIN28B in BC CD34⁺ cells transduced with backbone control or hsa-miR-26a (n=3). (K) RNA-seq analysis of the expression of MYC in CP (n=7) and BC CML (n=6). All graphs show mean with SEM and statistical analysis was calculated using the Student’s t-test. *p<0.05, **p<0.005, ***p<0.0005. See also Figure S3.

Figure 5.. Differential A-to-I RNA editing in 3’UTR regions of normal HSPCs and BC LSC.

(A and B) Volcano plot showing the A-to-I(G) editome of cell cycle genes in ADAR1 WT-transduced cord blood CD34⁺ cells compared with lentiviral vector controls (n=3) (A) and in CP progenitors (n=7) compared with BC counterparts (n=6) (B). (C) A-to-I RNA editing of MDM2 3’UTR in individual CP (n=7) and BC (n=6) samples. The predicted miRNA binding sites within MDM2 3’UTR using miRcode transcriptome-wide miRNA target prediction tool were shown (Jeggari et al., 2012). (D) Relative miRNA expression determined by miRNA qPCR array of 84 miRNAs in CML CP CD34⁺ cells transduced with backbone or ADAR1 WT (n=3). (E) Relative miRNA expression in normal aged-matched (>55 year old) CD34⁺ cells (n=4) and BC CML CD34⁺ cells (n=3). (F and G) The expression of MDM2/p53 pathway transcripts, MDM2 (F) and TP53 (G) in progenitor population of normal peripheral blood (NPB), CML CP (n=7), and CML BC (n=6) determined by RNA-seq. (H) MDM2 expression in HEK293T cells transduced with ADAR1 WT, ADAR1^E912A alone or in combination with miR-155 (n=3 experiments). (I) Structure of “wt” or “edited” MDM2 3’UTR reporter construct with A-to-(I)G changes introduced at miRNA targeting sites (highlighted in red). The genomic loci of A-to-(I)G changes were also indicated with arrows. (J) The MDM2 3’UTR reporters were transfected into HEK293T cells and then challenged with miR-155 overexpressing lentivirus (n=3 experimental triplicate). The miRNA targeting efficiency was measured as the relative luciferase activity (GLuc/SEAP ratio). (K) MDM2 and LIN28B expression in BC CML CD34⁺ cells with shControl or shADAR1 (n=3). All graphs show mean with SEM and statistical analysis was calculated using the Student’s t-test. *p<0.05, **p<0.005, ***p<0.0005. See also Figure S4 and Table S3.

Figure 6.

Summary of A-to-I RNA editing function in normal HSPCs and BC LSC.