Beyond mutations: RNA editing deaminase activation as a dynamic marker to track stem cell fitness and MPN progression

Abstract Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell–derived disorders that can progress to acute myeloid leukemia (AML) at variable rates. Despite major advances in JAK2 inhibitor–based therapeutics, the main clinical challenge pertains to the prediction and prevention of the evolution of pre-cancer stem cells in MPNs into self-renewing cancer stem cells (CSCs) that drive oncogenic transformation into rapidly fatal secondary AML (sAML). Currently, diagnostic and prognostic assessments in MPNs rely largely on clinical and morphological features, often supplemented by molecular markers such as those included in MIPSS70. While total symptom score (TSS) and variant allele frequency (VAF) of driver mutations are valuable predictors of therapeutic response, they do not adequately predict disease progression. Given that MPNs are stem cell–driven disorders, we hypothesize that integrating stem cell–specific molecular biomarkers into diagnostic frameworks could improve prediction of therapeutic responses and disease evolution. Our research has revealed increased expression and activity of inflammatory cytokine–inducible RNA-editing enzymes ADAR1 and APOBEC3C in high-risk myelofibrosis derived hematopoietic stem and progenitor cells (HSPCs). We therefore aim to develop a biomarker tool incorporating ADAR1 and APOBEC3C expression, along with associated deaminase activity signatures, to predict MPN disease progression. In this study, we performed longitudinal 150-gene next-generation sequencing (NGS) analyses for 129 MPN patients with a median follow-up time of 958 days (range 0-4214). On average, 6 NGS analyses (range 1-19) were performed for each patient. Within 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. To date, we selected four patients who were treated with Inrebic, progressed to sAML, underwent stem cell transplantation, and had cryopreserved peripheral blood and bone marrow mononuclear cells stored in our biorepository. For these patients, we had 15, 17, 24, and 31 sequential time points collected between 2013 and 2025. qPCR was performed on CD34⁺ immunomagnetic bead–selected cells to measure ADAR1, APOBEC3C, STAT3, and JAK2 expression levels. During treatment, we observed dynamic changes in ADAR1, APOBEC3C, JAK2, and STAT3 expression levels. Specifically, while VAF remained stable, both lactate dehydrogenase (LDH) and ADAR1 expression levels increased. Additionally, whole transcriptome sequencing of FACS-purified hematopoietic stem cells (HSCs) revealed higher ADAR1 expression and increased A-to-I RNA editing in JAK2 V617F⁺ patients compared with JAK2 wild-type patients. These findings suggest that ADAR1 expression may serve as a dynamic marker for both disease progression and disease stratification. Further single-cell RNA sequencing studies will further delineate differences in HSCs and HPCs at sequential time points for five MPN patients that progressed to sAML.