Abstract 375: A novel diagnostic assay for detection of primate-specific RNA editing events in leukemia stem cells
Notice bibliographique
Résumé
Abstract 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-expressing cells at all four sites. Conclusions These results set the stage for development of primate-specific RNA editing as a novel diagnostic strategy for clinical LSC detection and identify ADAR1 as a potential therapeutic target in LSC. These data shed new light on the mechanisms of ADAR1-mediated generation of malignant progenitors that drive therapeutic resistance, disease progression and relapse in CML and may be applicable to other CSC-driven malignancies. Citation Format: Leslie A. Crews, Qingfei Jiang, Maria A. Zipeto, Angela C. Court, Christian L. Barrett, Marco A. Marra, Kelly A. Frazer, Catriona H. M. Jamieson. A novel diagnostic assay for detection of primate-specific RNA editing events in leukemia stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 375. doi:10.1158/1538-7445.AM2014-375
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,001 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».