Isogenic MDS-RS Patient-Derived iPSCs Define the Mis-Spliced Transcript Repertoire and Chromatin Landscape of SF3B1-Mutant Hematopoietic Stem/Progenitor Cells

• Published in: Blood 2021, Vol.138 (Supplement 1), p.147-147
• Main Authors: Asimomitis, George, Deslauriers, André G., Kotini, Andriana G., Bernard, Elsa, Esposito, Davide, Olszewska, Malgorzata, Spyrou, Nikolaos, Arango Ossa, Juan, Mortera-Blanco, Teresa, Koche, Richard, Aaronson, Stuart, Hellström-Lindberg, Eva, Papaemmanuil, Elli, Papapetrou, Eirini P.
• Format: Article
• Language: English
• Subjects: Medicin och hälsovetenskap
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2021-149830

▪ The somatic hotspot mutation SF3B1K700E is characteristically found in myelodysplastic syndrome with ring sideroblasts (MDS-RS) and frequently occurs as an isolated mutation. However, our understanding of how this mutation drives MDS pathogenesis remains limited. To explore the downstream consequences of the SF3B1K700E mutation and its role in disease pathogenesis, we generated a panel of isogenic SF3B1K700E and SF3B1WT induced pluripotent stem cell (iPSC) lines from 3 MDS-RS patients with isolated SF3B1K700E mutation (3 SF3B1K700E and 3 SF3B1WT lines per patient, total 18). Upon hematopoietic differentiation, SF3B1K700E cells exhibited lower growth and colony-forming ability, compared to SF3B1WT cells, recapitulating hallmark phenotypes of MDS cells. To investigate the effects of the SF3B1K700E mutation on the transcriptome and chromatin landscape, we performed RNA- and ATAC- sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from the panel of the 18 isogenic SF3B1K700E and SF3B1WT iPSC lines. Principal component analysis (PCA) and hierarchical clustering based on gene expression grouped the iPSC lines primarily by genotype (SF3B1K700E vs SF3B1WT) and secondarily by genetic background. To assess the impact of the SF3B1K700E mutation at the exon, transcript and gene level, we developed an analytical framework integrating differential splicing with differential transcript usage and differential gene expression analyses. We thus discovered 59 splicing events linked to 34 genes (most statistically significant events that also mapped to differentially used transcripts and differentially expressed genes). This SF3B1K700E splicing signature includes genes previously reported as mis-spliced in SF3B1K700E cells (e.g BRD9, ABCB7), as well as novel genes. We tested this signature against a published dataset of primary MDS patient samples (Pellagatti et al.). PCA based on the inclusion level of the splicing events of our signature separated SF3B1-mutated MDS patients from patients without splicing factor mutations (SF-WT) or healthy individuals. Furthermore, it identified one patient erroneously annotated as SF-WT that clustered together with the SF3B1-mutated patients, who had a, previously overlooked, 6bp in-frame deletion spanning the K700E hotspot. By comparing the chromatin accessibility profiles of SF3B1K700E and SF3B1WT iPSC-HSPCs to those defined in primary human cell types along the hematopoietic hierarchy (Corces et al.