Mutations in the splicing factor SF3B1 are linked to frequent emergence of HLA-DRlow/neg monocytes in lower-risk myelodysplastic neoplasms

Recurrent mutations in splicing factors, in particular the splicing factor 3B subunit 1 (SF3B1), are commonly seen in lower-risk (LR) myelodysplastic neoplasms (MDS) and result in various aberrantly spliced transcripts. The cell type-specific changes and their contribution to immune dysregulation in MDS remain vaguely understood. In this study, we performed RNA sequencing on classical monocytes isolated from patients with an isolated SF3B1K700E hotspot mutation. Our analysis revealed downregulated expression of genes involved in inflammatory cytokine signaling, as well as differential mRNA splicing of several genes involved in the regulation of the defense response and cytokine signaling, mRNA metabolism, apoptotic signaling, and mitotic cell cycle. Overall design: Classical monocytes (CD14+CD16-) were magnetically isolated from viably frozen peripheral blood mononuclear cells (PBMCs) of treatment-naïve LR-MDS patients (n = 3 SF3B1K700E, n = 3 SF3B1wt) and hematologically healthy donors (HD, n = 3) as reference, yielding routinely >95% CD14+CD16- monocytes based on flow cytometric assessment. Monocytes were cultured/rested for 4 hours (195000 – 500000 cells per 96-well in 250 µl RPMI 1640/PenStrep/5% heat-inactivated human serum), followed by RNA isolation with TRIzol and Direct-zol™ RNA Microprep, mRNA library preparations, and sequencing reactions.