Abstract 375: A novel diagnostic assay for detection of primate-specific RNA editing events in leukemia stem cells

• Published in: Cancer research (Chicago, Ill.) Ill.), 2014-10, Vol.74 (19_Supplement), p.375-375
• Main Authors: Crews, Leslie A., Jiang, Qingfei, Zipeto, Maria A., Court, Angela C., Barrett, Christian L., Marra, Marco A., Frazer, Kelly A., Jamieson, Catriona H. M.
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2014-375

Introduction The adenosine deaminase acting on RNA (ADAR) family of RNA editases has been linked to the pathogenesis of diverse malignancies, including leukemia, breast cancer and hepatocellular carcinoma. We previously showed that human leukemia stem cells (LSC) from blast crisis (BC) chronic myeloid leukemia (CML) patients harbor increased ADAR1 expression compared with normal and chronic phase (CP) progenitors. Whole transcriptome RNA sequencing (RNA-Seq) revealed increased adenosine to inosine (A-to-I) RNA editing during CML progression concentrated within primate specific Alu-containing transcripts. However, detection of RNA editing by RNA-Seq in rare cell populations can be technically challenging, costly and requires PCR validation. Thus, the objectives of this study were to validate RNA editing of a subset of these LSC-associated transcripts in the context of lentivirally enforced ADAR1 expression, and to develop an RNA editing reporter reporter assay in human leukemia cells and a qPCR-based diagnostic test to rapidly detect CSC-associated aberrant RNA editing. Methods The BCR-ABL+ human leukemia cell line K562 was stably transduced with lentiviral human ADAR1 or vector. FACS-purified K562-ADAR1 cells were transfected with a luciferase-based reporter vector to confirm RNA editing activity. Two genes, MDM2 and APOBEC3D, were selected from our previous RNA-Seq studies of BC progenitors (Jiang et al, 2013). Targeted sequencing was performed on high fidelity PCR products using primers flanking each of 2 editing sites in each gene. RNA editing-specific qPCR primers were designed for each editing site using an allele-specific strategy that detects cDNA containing either an A or G(I) representing an RNA editing event. Both targeted sequencing and qPCR were used to detect RNA editing in K562-ADAR1 and primary cord blood-derived hematopoietic stem cells (HSC) lentivirally transduced with ADAR1. Results Lentivirally enforced ADAR1 expression promoted RNA editing activity as measured by luciferase reporter activity. Increased A-to-I changes in MDM2 and APOBEC3D were confirmed by targeted sequencing. In independent experiments, RNA editing site-specific qRT-PCR accurately detected RNA editing in K562-ADAR1 cells (n=3) and in primary HSC overexpressing ADAR1 (n=4). Site-specific primers distinguished G(I) bases at RNA editing sites in cDNA and as predicted gave no signal in gDNA. Relative A-to-I RNA editing ratios were increased by 2 to 3 fold in ADAR1-expressi