Session: 603. Lymphoid Oncogenesis: Basic: Poster I
METHODS: We designed a longitudinal early relapse detection study by collecting bone marrow aspirates and/or peripheral blood samples from patients ages 1-25 years diagnosed with B-ALL at the University of California, San Diego (UCSD) according to Institutional Review Board guidelines. Timepoints include diagnosis, end of induction cycle and/or subsequent cycle until they achieve remission or relapse. Cord blood and pediatric non-leukemic (pNL) samples from lymphoma patients without bone marrow involvement were used as controls (Van Der Werf, ...Jamieson. Cell Reports Medicine 2023, March cover). Mononuclear cells were isolated via Ficoll density gradient centrifugation followed by immunomagnetic bead-mediated CD34+ cell selection. Fluorescence-activated cell sorting (FACS) purification of HSCs (CD34+CD38-Lin-) and HPCs (CD34+CD38+Lin-) was followed by RNA extraction (Qiagen) followed by library preparation, sequencing (Illumina Hi-Seq) and RMATS as well as A-to-I editome analysis.
RESULTS: To date, ten B-ALL patients have been enrolled in the study, with 60% belonging to high risk (HR) and 40% to standard risk (SR) NCI subgroups, and 7 patients available for analysis. Whole transcriptome RNA sequencing (RNA-seq) analysis was performed on purified HSCs and HPCs from three patients to investigate differential gene expression. While there was a trend toward APOBEC3C overexpression in B-ALL versus pNL control HSCs, there was a statistically significant upregulation of ADAR1 in B-ALL HSCs and HPCs. Moreover, we detected ADARp150 splice isoform overexpression in B-ALL HPCs. Furthermore, RMATS differential splicing analysis detected alternative splicing deregulation, including exon skipping (ES), intron retention (IR), and alternative 5’ splice site (A5SS) as well as alternative 3’ splice site (A3SS) usage. With more than 2000 differential events identified, ES was the most frequent event in our HSC and HPC RNA-seq analyses, which has been linked to increased adenosine-to inosine (A-to-I) splice acceptor site editing by ADAR1p150.
CONCLUSIONS: Together, RNA splicing deregulation and ADAR1p150 splice isoform-mediated A-to-I RNA editing activity may contribute to LSC persistence following therapy. To validate our findings, we will perform lentiviral ADAR1-nanoluc-GFP reporter and splicing reporter assays in combination with functional survival and self-renewal assays with the ultimate aim of developing base deaminase-targeted strategies to predict and prevent pediatric B-ALL relapse.
Disclosures: Jamieson: Aspera Biomedicines: Other: co-founder; Impact Biomedicines: Other: co-founded; Forty Seven Inc.: Patents & Royalties.