ADAR1 promotes malignant progenitor reprogramming in chronic myeloid leukemia

Abstract

The molecular etiology of human progenitor reprogramming into self-renewing leukemia stem cells (LSC) has remained elusive. Although DNA sequencing has uncovered spliceosome gene mutations that promote alternative splicing and portend leukemic transformation, isoform diversity also may be generated by RNA editing mediated by adenosine deaminase acting on RNA (ADAR) enzymes that regulate stem cell maintenance. In this study, whole-transcriptome sequencing of normal, chronic phase, and serially transplantable blast crisis chronic myeloid leukemia (CML) progenitors revealed increased IFN-γ pathway gene expression in concert with BCR-ABL amplification, enhanced expression of the IFN-responsive ADAR1 p150 isoform, and a propensity for increased adenosine-to-inosine RNA editing during CML progression. Lentiviral overexpression experiments demonstrate that ADAR1 p150 promotes expression of the myeloid transcription factor PU.1 and induces malignant reprogramming of myeloid progenitors. Moreover, enforced ADAR1 p150 expression was associated with production of a misspliced form of GSK3β implicated in LSC self-renewal. Finally, functional serial transplantation and shRNA studies demonstrate that ADAR1 knockdown impaired in vivo self-renewal capacity of blast crisis CML progenitors. Together these data provide a compelling rationale for developing ADAR1-based LSC detection and eradication strategies.

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 hierarchical clustering (see SI Materials and Methods) separated CP and BC samples. A select subset of genes (boxes) discriminated BC from cord blood (n = 3).

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).

Fig. 3.

ADAR1 promotes malignant myeloid progenitor expansion. (A) Lentiviral overexpression of ADAR1 p150 in FACS-purified CD34⁺38⁺Lin⁻ normal progenitors (n = 3) reduced erythroid (BFU-E) colony formation and increased macrophage (M) cfu compared with vector (ORF) controls. Mix, macrophage/erythroid colonies; GM, granulocyte/macrophage colonies; G, granulocyte colonies. *P < 0.05 and **P < 0.01 compared with vector-transduced controls or CP progenitors by Student t test. (B–E) Pearson correlation analysis of HPRT-normalized ADAR1 mRNA levels and PU.1 or GATA1 in individual colonies derived from normal progenitors (n = 3) transduced with lenti-ADAR1 p150 (B and C) or CML BC progenitor-derived colonies (D and E). r² values were calculated using Pearson correlation analysis. (F) Ratio of PU.1 to GATA1 expression levels by RNA-seq in CP (n = 8) versus BC (n = 8) CML. (G) RNA-seq–based IPA network analysis of GATA1-associated genes in BC CML (n = 8) compared with CP (n = 8).

Fig. 4.

ADAR1 knockdown impairs malignant myeloid progenitor self-renewal. (A) Diagrammatic scheme of in vivo experimental design. Before and after primary transplantation, qRT-PCR was performed in human progenitors, confirming ADAR1 knockdown. BM, bone marrow. (B) Representative FACS plots showing human CD45⁺ engraftment in hematopoietic organs of primary RAG2^−/−γc^−/− transplant recipients or untransplanted control mouse bone marrow. (C and D) FACS analysis of CD45⁺ human cell engraftment and progenitor cells in the bone marrow of mice transplanted with BC progenitors transduced with lenti-shControl (n = 3) or lenti-shADAR1 (n = 5). (E and F) FACS analysis of human cells in mouse bone marrow (shControl n = 7 and shADAR1 n = 8) after serial transplantation of BM CD34⁺ cells pooled from primary transplant-recipient mice. *P < 0.05 compared with vector-transduced controls by Student t test.