Abstract 2625: Role of base deaminase and splicing deregulation in pediatric B cell acute lymphoblastic leukemia stem cell persistence

Abstract Introduction: B cell acute lymphoblastic leukemia (B ALL) is the most common childhood malignancy with more than ninety percent cure rates and current research efforts are directed towards development of curative therapy in patients who relapse. Leukemia stem cell (LSC) persistence contributes to relapse. Whole genome sequencing and RNA sequencing (RNA seq) have revealed APOBEC3C and ADAR1 base deaminase induced splicing alterations in LSCs in both adult and pediatric acute myeloid leukemia. Through our study we strive to determine the role of base deamination deregulation in LSC persistence in pediatric B ALL. Methods: We designed a longitudinal early relapse detection study by collecting bone marrow aspirates and/or peripheral blood samples from patients ages 1 to 25 years diagnosed with B ALL at University of California, San Diego 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 were used as controls (Van Der Werf, ⋯Jamieson. Cell Reports Medicine 2023, March cover). Mononuclear cells were isolated followed by CD34+ cell selection. Fluorescence activated cell sorting purification of HSCs (CD34+CD38-Lin-) and HPCs (CD34+CD38+Lin-) was followed by RNA extraction (Qiagen), library preparation, sequencing (Illumina Hi Seq) and RMATS as well as A to I editome analysis. Results: To date, twelve B ALL patients have been enrolled in the study, with 67 percent belonging to high risk and 33 percent to standard risk NCI subgroups, and 7 patients available for analysis. RNA seq analysis was performed on purified HSCs and HPCs from three patients. 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 and 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 prime splice site (A5SS) as well as alternative 3 prime 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: Preliminary data suggests 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 nanoluciferase GFP reporter and splicing reporter assays in combination with functional survival and self renewal assays with the goal of developing base deaminase targeted strategies to predict and prevent pediatric B ALL relapse. Citation Format: Mrinal Joshirao, Inge Van Der Werf, Jessica Pham, Antonio W. Ruiz, Neha Katragadda, Emma E. Klacking, Elsa Molina, Ludmil Alexandrov, Thomas Whisenant, Catriona Jamieson. Role of base deaminase and splicing deregulation in pediatric B cell acute lymphoblastic leukemia stem cell persistence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2625.