Abstract 263: RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms for dynamic detection of clonal myeloproliferative neoplasm stem cell fitness

Abstract Introduction Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) derived disorders that can progress to acute myeloid leukemia (AML) at variable rates depending on macroenvironmental stressors and microenvironmental inflammatory cytokines. The total symptom score (TSS) and variant allele frequency (VAF) of mutations have proven valuable predictors of therapeutic response. Recently, we discovered inflammatory-cytokine-responsive ADAR1 RNA editing enzyme overexpression during MPN progression. However, dynamic prediction of clonal MPN stem cell fitness and progression to AML remains challenging. Methods We performed longitudinal 150-gene next-generation sequencing (NGS) analyses for 129 MPN patients. We developed single-cell RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms to quantify ADAR1 activity with a novel ADAR1 nanoluc-GFP reporter assay, whole transcriptome RNA editome analyses, and MPN stem cell replating assays. To identify novel RNA editing sites, we performed whole genome sequencing on CD34+ cells from 43 MPN patients and whole transcriptome sequencing on FACS-purified HSC and progenitor cells from 32 MPN patients and 24 non-MPN controls. Results Sequential NGS analysis was performed with a median follow-up of 958 days (range 0-4214; interquartile range (IQR) 314-1350). On average, 6 NGS analyses (range 1-19; IQR 3-9) were performed for each patient. In our cohort, 58. 9% of patients were JAK2 V617F+, while mutations in CALR and ASXL1 were observed in 11. 6% and 14. 0% of cases, respectively. In total, 44. 2% (N=57) of patients in our cohort received Inrebic, with 56% reaching the maximum dose of 400mg. Notably, most patients on Inrebic were JAK2 V617F+ (N=38, 67. 7%). In JAK2 V617F+ patients on Inrebic, 50% (n=19) experienced a reduction in the JAK2 V617F VAF from their peak value with a median drop of 16% (range 4-57). Our analyses using READAR platforms revealed increased RNA editing levels in JAK2 V617F+ patients from whole transcriptome sequencing analyses compared with JAK2 wild-type patients. Transduction of our ADAR1 nanoluc-GFP reporter into CD34+ cells from MPN patients (N=7), enabled us to quantify ADAR1 activity in an ex vivo setting. Moreover, CD34+ cells from MPN patient samples (n=7) treated in stromal co-cultures with ADAR1 inhibitor, Rebecsinib, demonstrated inhibition of self-renewal in replating assays. A significant reduction in ADAR1-GFP reporter activity was observed in MPN CD34+ cells following Rebecsinib treatment of stromal co-cultures. Also, humanized ADAR1-GFP-luciferase reporter AML mouse models showed a significant reduction in ADAR1 activity and leukemia stem cell self-renewal following Rebecsinib treatment. Conclusion These READAR platform results suggest that ADAR1 activity can be utilized to predict clonal MPN stem cell fitness and progression. Citation Format: Inge van der Werf, Larisa Balaian, Jessica Pham, Wenxue Ma, Athena Mohebbi, Emma Klacking, Antonio Ruiz, Karla Mack, John Mascarenhas, Thomas Whisenant, Ludmil Alexandrov, Sheldon Morris, Catriona Jamieson. RNA Editing ADAR1 Deaminase Activation Responsive (READAR) platforms for dynamic detection of clonal myeloproliferative neoplasm stem cell fitness abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 263.