ALBERT

Description: Introduction Human bone marrow aging is typified by decreased cellularity, stem cell exhaustion and myeloid lineage bias that may set the stage for development of myeloid malignancies. Secondary AML (sAML) arises from prior myelodysplastic syndromes (MDS) or myeloproliferative neoplasms (MPN), and occurs in patients with an average age of 〉65. Because of the typically advanced age of this population, patients currently have few effective treatment options available after leukemic transformation. We and others have recently identified a key role for enzymatic RNA editing activity in cancer progression, and in particular in leukemia stem cell (LSC) generation. In hematopoietic stem and progenitor cells, adenosine deaminase acting on dsRNA-1 (ADAR1) is the most abundantly expressed RNA editing gene. However, the role of abnormal RNA editing activity has not been elucidated in healthy human bone marrow aging and age-related MDS with a high risk of transforming to sAML. Therefore, we established whole transcriptome-based RNA editing signatures of benign versus malignant bone marrow progenitor cell aging, which provides novel RNA-based functionally relevant biomarkers of aging, MDS and progression to sAML. Methods Whole transcriptome sequencing (RNA-Seq) was performed on FACS-purified hematopoietic stem (CD34+CD38-Lin- HSC) and progenitor cells (CD34+CD38+Lin- HPC) from aged (average age = 65.9 y/o) versus young (average age = 25.8 y/o) adult healthy bone marrow samples, and in leukemia stem cells (LSC) from patients with sAML (average age = 71.4 y/o) and MDS (average age = 63.8 y/o). Comparative gene set enrichment analyses (GSEA) and RNA editing profiles were identified for normal and malignant progenitor cell aging. Results Aberrant RNA editing activity has recently been shown to be induced in multiple cancers, and has been implicated as a malignant reprogramming factor. Comparative whole transcriptome RNA sequencing (RNA-seq) and single nucleotide variant analyses revealed widespread increases in RNA editing rates in aged versus young HPC, and in human sAML LSC compared with age-matched normal progenitors. Moreover, RNA editing rates, represented as adenosine (A) to inosine/guanosine (G) changes at known RNA editing loci, were increased in sAML compared with MDS progenitors. The differential expression of certain sites is as high as 70%, which can be readily detected by RESS-qPCR. These data suggest that during aging niche-dependent RNA editing deregulation contributes to MDS progression to sAML. Interestingly, the highly edited loci in sAML LSC were distinct from loci that were differentially edited in aged versus young HPC, suggesting that pro-inflammatory conditions in sAML may trigger RNA editing of a unique set of transcripts, including predominantly RNA processing-related gene products and transcription factors. Notably, several loci in transcripts of APOBEC3C/D that we previously found were associated with blast crisis transformation of chronic myeloid leukemia also displayed enhanced editing in sAML LSC, but not aged versus young HPC. Conclusions Detection of aberrant RNA processing provides novel biomarkers as well as potential therapeutic targets for sAML LSC eradication with implications for treatment of a variety of human malignancies and other age-related disorders. We have identified commonly RNA-edited transcripts in multiple hematologic malignancies, which could be developed clinically and as companion diagnostic targets for LSC-targeted therapeutics. Disclosures Jamieson: CTI Biopharma: Research Funding; Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.

Description: Recent studies demonstrate the importance of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) in normal fetal and adult hematopoiesis. RNA-sequencing studies have shown that elevated levels of the ADAR1 editase has emerged as a dominant driver of cancer progression and therapeutic resistance. Specifically, the deregulation of ADAR1 promotes the transformation of chronic myeloid leukemia (CML) from chronic phase (CP) to a therapy resistant blast crisis (BC) phase. Through the regulation of mRNA and microRNA (miRNA) stability, ADAR1 plays a pivotal role in embryonic development and stem cell regulation. We have previously shown that inflammation-responsive ADAR1 heavily contributes to stem cell differentiation and self-renewal in CML disease progression. Here, we describe a novel role of ADAR1 in cell cycle regulation of BC leukemia cells through regulation of miRNA biogenesis. To investigate the role of ADAR1 in miRNA regulation, we performed miRNome miScript PCR array analysis of 1008 miRNAs in cord blood CD34+ expressing hematopoietic stem and progenitor cells (HSPCs) overexpressing ADAR1 wild type (WT) and A-to-I editing deficient ADAR1 mutant. Overall, a total of 112 miRNAs were significantly differentially expressed following ADAR1 expression with cell cycle identified as the top cellular pathway significantly targeted by miRNAs regulated by ADAR1. Notably, ADAR1 editase activity inhibits the expression of miR-26a-5p, a tumor suppressor miRNA that is frequently downregulated in BC CML. ADAR1 inhibits miR-26a-5p through direct editing of the DROSHA cleavage site of primary miR-26a-5p, preventing miR-26a-5p maturation and processing. In normal hematopoietic progenitors, ADAR1-mediated inhibition of miR-26a results in repression of cyclin-dependent kinase inhibitor 1A (CDKN1A) expression indirectly via suppression of the polycomb repressive complex, enhancer of zeste homolog 2 (EZH2), thereby accelerating cell cycle transit. However, in BC CML progenitors, decreased EZH2 and increased CDKN1A oppose the cell cycle accelerating effect of ADAR1. Moreover, we found that the miR-26a targets a different set of mRNA in BC CML compared to cord blood HSPC and has divergent roles in cell cycle regulation. Lentiviral miR-26a overexpression reduced BC leukemia stem cell (LSC) dormancy in the bone marrow and reverses the functional effects of ADAR1, including inhibition of BC cell proliferation in vivo and impaired LSC self-renewal capacity as measured by colony forming assays. Our finding reveals the effects of ADAR1 in LSC generation through impairing biogenesis of cell cycle regulatory miRNAs. The deregulation of ADAR1 contributes to the malignant reprogramming of progenitors into dormant LSCs that are resistant to therapeutic treatments. Future development of ADAR1 inhibitors may be effective in the elimination of dormant BC CML LSCs that evade tyrosine kinase inhibitors. Disclosures No relevant conflicts of interest to declare.