Abstract 111: Detection of clonal hematopoietic APOBEC3-mediated splicing deregulation

Abstract Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC)-derived disorders that often arise as a result of JAK2/STAT3 signal activating mutations. In the setting of microenvironmental inflammatory cytokines, MPNs also acquire splicing alterations that are associated with progression to rapidly lethal secondary acute myeloid leukemia (sAML) (reviewed in Jamieson, Weisman. NEJM. 2023). By performing whole genome and RNA sequencing analyses of MPN hematopoietic stem and progenitor populations (HSPCs), we discovered that inflammatory cytokine inducible APOBEC3C upregulation was associated with IL6ST and ADAR1p150 splice isoform overexpressing progenitor proliferation and progression to sAML (Jiang⋯Jamieson. Cell Reports. 2021). Thus, we sought to investigate the role of APOBEC3 activation as a driver of clonal hematopoietic progenitor splicing deregulation and propagation. We cloned human APOBEC3 family genes into pCDH lentiviral vectors and transduced cord blood derived immunomagnetic bead selected CD34+ cells with APOBEC3A, B, C, D, G or H, or pCDH lentiviral backbone controls for whole genome and whole transcriptome analyses. Additionally, CD34+ cells derived from aged normal bone marrow (ABM), collected according to IRB approved protocols, were transduced with APOBEC3C for whole genome and whole transcriptome analyses, coupled with functional assays to assess the effects of APOBEC3 overexpression on RNA splicing and editing deregulation. Following lentiviral APOBEC3B, APOBEC3C, and APOBEC3G transduction of CD34+ cells, gene set enrichment analysis (GSEA) revealed splicing pathway alterations. Significantly altered APOBEC3-responsive pathways involved genes crucial for hematopoiesis, including ZRSR2, U2AF1, and SRSF2. Additional analysis of RNA sequencing data of CD34+ HSPCs derived from aged normal bone marrow overexpressing APOBEC3C, and CD34+ HSPCs derived from MPNs further confirmed these splicing pathway alterations and revealed a correlation with APOBEC expression levels. Moreover, we observed that lentiviral APOBEC3C wild-type overexpression in ABM-derived CD34+ cells resulted in differential exon usage using splicing reporter assays. Together, these results suggest that detection of APOBEC3-mediated splicing deregulation may inform the development of diagnostic and therapeutic strategies aimed at preventing splicing-mediated MPN progression. Citation Format: Inge van der Werf, Jane Isquith, Emma Klacking, Jessica Pham, Thomas Whisenant, Ludmil Alexandrov, Catriona Jamieson. Detection of clonal hematopoietic APOBEC3-mediated splicing deregulation [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 111.