Sanford Stem Cell Institute, Division of Regenerative Medicine, Department of Medicine, Moores Cancer Center, University of California, San Diego, United States
Abstract: Inflammatory cytokine-inducible APOBEC3 cytosine deaminases evolved to protect human genomic integrity by restricting retroviral integration and retrotransposition during stress responses. However, in cancer, APOBEC3-induced mutagenesis drives genomic instability, facilitating tumor initiation and progression in inflammatory microenvironments. By performing whole genome and whole transcriptome sequencing analyses of myeloproliferative neoplasm (MPN) derived hematopoietic stem and progenitor (HSPCs) compared to age-matched controls, we discovered that APOBEC3C overexpression, C-to-T mutagenesis and IL-6ST upregulation, which induces ADAR1p150 splice isoform switching, correlates with MPN progression. 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, F, G, or pCDH lentiviral backbone controls for whole genome and whole transcriptome analyses. Following lentiviral APOBEC3B, APOBEC3C, and APOBEC3G transduction of CD34+ cells, gene set enrichment analysis (GSEA) revealed splicing pathway alterations along with differential splicing typified by increased exon skipping. Subsequently, CD34+ cells derived from aged normal bone marrow (ABM), were transduced with APOBEC3C for whole genome and whole transcriptome analyses, coupled with functional assays to assess the effects of APOBEC3C overexpression on RNA splicing, which further confirmed splicing pathway alterations. Moreover, we observed that lentiviral APOBEC3C overexpression in ABM-derived CD34+ cells resulted in differential exon usage using splicing reporter assays. Specifically, upon confirmation of APOBEC3C overexpression, we seeded CD34+ derived ABM cells into our 3D biosensing nanobioreactor system to analyze differential exon usage using confocal fluorescence microscopy along with FACS analyses. Together, these findings suggest that detection of APOBEC3-mediated splicing deregulation may inform the development of diagnostic and therapeutic strategies aimed at preventing splicing-mediated MPN progression.
