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Patient-specific MDS-RS iPSCs define the mis-spliced transcript repertoire and chromatin landscape of SF3B1-mutant HSPCs

ABSTRACT: SF3B1 K700E is the most frequent mutation in myelodysplastic syndrome (MDS), but the mechanisms by which it drives MDS pathogenesis remain unclear. We derived a panel of 18 genetically matched SF3B1 K700E- and SF3B1 WT-induced pluripotent stem cell (iPSC) lines from patients with MDS with ring sideroblasts (MDS-RS) harboring isolated SF3B1 K700E mutations and performed RNA and ATAC sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from them. We developed a novel computational framework integrating splicing with transcript usage and gene expression analyses and derived a SF3B1 K700E splicing signature consisting of 59 splicing events linked to 34 genes, which associates with the SF3B1 mutational status of primary MDS patient cells. The chromatin landscape of SF3B1 K700E HSPCs showed increased priming toward the megakaryocyte- erythroid lineage. Transcription factor motifs enriched in chromatin regions more accessible in SF3B1 K700E cells included, unexpectedly, motifs of the TEAD family. TEAD expression and transcriptional activity were upregulated in SF3B1-mutant iPSC-HSPCs, in support of a Hippo pathway-independent role of TEAD as a potential novel transcriptional regulator of SF3B1 K700E cells. This study provides a comprehensive characterization of the transcriptional and chromatin landscape of SF3B1 K700E HSPCs and nominates novel mis-spliced genes and transcriptional programs with putative roles in MDS-RS disease biology.

ORGANISM(S): Homo sapiens

PROVIDER: GSE184246 | GEO | 2022/03/08

REPOSITORIES: GEO

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Coordinated mis-splicing of TMEM14C and ABCB7 causes ring sideroblast formation in SF3B1-mutant myelodysplastic syndrome

Project description:Purpose: Mutant SF3B1 is near universally associated with MDS-RS but the exact mechanism of how mutant-SF3B1 induces ring sideroblast formation is unclear. Methods: iPSC samples were differentiated from 5F-HPC derived from MDS-RS patient iPSCs. CD34+ progenitors were isolated using the CD34 enrichment kit (Miltenyi). CD34-CD71+GlyA- pro-erythroblasts were isolated by flow sorting on day 4. CD71+GlyA+ erythroblasts were isolated by flow sorting on day 9. Libraries with DNA fragments of ~300bp were selected using AMPure XP bead purification. Purified libraries were sequenced on an Illumina HiSeq 2000 using paired-end, 50 bp reads. Mis-splicing analysis was performed as described in Inoue et al. 2019. Results: Mis-splicing analysis reveals ~100 genes that are strongly mis-spliced (>40%) by mutant SF3B1 during erythroid differentiation. We identify that mis-splicing of TMEM14C and ABCB7 contribute to RS formation in mutant SF3B1 cells. Conclusion: Our iPS derived mutant SF3B1MDS-RS in vitro model is the first demonstration of a cell line that recapitulates mis-splicing, erythroid differentiation, and ring sideroblast formation.

2021-11-29 | GSE189546 | GEO

Transcriptome analysis of SF3B1-mutation induced changes in K562 cells

Project description:Using RNA-Seq, we determined changes to gene expression and splicing on inducible expression of SF3B1-WT and SF3B1-MUT (K700E) in K562 cells. Using RNA-Seq, we determined changes to gene expression and splicing on inducible expression of SF3B1-WT and SF3B1-MUT (K700E) in K562 cells.

2016-07-15 | E-GEOD-70959 | biostudies-arrayexpress

Chronic Lymphocytic Leukemia MEC1 SF3B1 K700E and Wild Type Cell Lines' RNA-seq after Treatment with H3B-8800 Splicing Modulator.

Project description:To study the impact of SF3B1 mutations on alternative splicing and the effect of H3B-8800 splicing modulator in wild type and SF3B1-mutant chronic lymphocytic leukemia cells, we established SF3B1 K700E MEC1 CLL isogenic cell line and carried out RNA deep sequencing in SF3B1 wild type and K700E MEC1 cell lines upon H3B-8800 treatment.

2023-08-08 | GSE190087 | GEO

Cancer-Causing Mutations in SF3B1 Alter Splicing by Disrupting Interaction with SUGP1

Project description:SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA-sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3' splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1, and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggests plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is the sole defect of mutant SF3B1 spliceosomes and, since this defect can be rescued, suggests possibilities for therapeutic intervention.

2019-07-24 | GSE128805 | GEO

RNA sequencing of bone marrow CD34+ cells from myelodysplastic syndrome patients with and without SF3B1 mutation and from healthy controls

Project description:The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndromes (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34+ cells from MDS patients with SF3B1 mutations using RNA-sequencing. Genes significantly differentially expressed at the transcript and/or exon level in SF3B1 mutant compared to wildtype cases include genes involved in MDS pathogenesis (ASXL1, CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7, SLC25A37) and RNA splicing/processing (PRPF8, HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expression/splicing in SF3B1 mutant cases. Our data indicate that SF3B1 plays a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processes/pathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link. RNA-Seq was performed to compare the transcriptome of bone marrow CD34+ cells from eight MDS patients with SF3B1 mutation, four MDS patients with no known splicing mutation and five healthy controls.

2014-11-22 | E-GEOD-63569 | biostudies-arrayexpress

Targeting the EIF2AK1 signaling pathway rescues red blood cell production in SF3B1-mutant myelodysplastic syndromes with ringed sideroblasts

Project description:SF3B1 mutations, which occur in 20% of patients with myelodysplastic syndromes (MDS), are the hallmarks of a specific MDS subtype, MDS with ringed sideroblasts (MDS-RS), which is characterized by the accumulation of erythroid precursors in the bone marrow and affects the elderly population. Here, using single-cell technologies and functional validation studies of primary SF3B1-mutant MDS-RS samples (MDS-RS-3-bis, MDS-RS-13, and MDS-RS-14), we show that SF3B1 mutations lead to the activation of the EIF2AK1 pathway in response to heme deficiency and that targeting this pathway rescues aberrant erythroid differentiation and enables the red blood cell maturation of MDS-RS erythroblasts. These data support the development of EIF2AK1 inhibitors to overcome transfusion dependency in patients with SF3B1-mutant MDS-RS with impaired red blood cell production. MACS-purified CD34+ cells were subjected to a 3-phase in vitro culture as described previously (Huang et al., 2020a). Cells were maintained in phase 1 from the day of collection until day 8, at which time the cells underwent ribonucleoprotein-based gene editing and were transitioned to phase 2 medium. On day 13 of culture, the cells were transitioned to phase 3 medium. Samples for Western blot and cytospin analyses, and sample for flow cytometry were harvested on day 13 or 15 of culture, respectively.

2022-07-19 | GSE202722 | GEO

Transcriptome analysis of SF3B1-mutation induced changes in K562 cells

Project description:Using RNA-Seq, we determined changes to gene expression and splicing on inducible expression of SF3B1-WT and SF3B1-MUT (K700E) in K562 cells.

2016-07-15 | GSE70959 | GEO

Ringsideroblasts in acute myeloid leukemia are associated with adverse risk and result from an abberant heme-metabolism gene program

Project description:Ringsideroblasts(RS) emerge as result of aberrant erythroid differentiation leading to excessive mitochondrial iron accumulation. This feature is characteristic for myelodysplastic syndromes with mutations in spliceosome gene SF3B1. However, RS can also be observed in patients diagnosed with acute myeloid leukemia (AML). The objective of this study was to characterize the presence of RS in a cohort of 109 AML and 17 high-risk MDS patients. Clinically, RS-AML is enriched for ELN adverse-risk (55%). In line with this finding, 35% of all cases had complex cytogenetic aberrancies and TP53 was most recurrently mutated in this cohort (42%), followed by DNMT3A (29%), RUNX1 (21%) and ASXL1 (19%). In contrast to RS-MDS, the incidence of SF3B1 mutations was low (8%). Whole exome sequencing and SNP array analysis on a subset of patients did not uncover one single defect underlying the RS phenotype. Shared genetic defects between erythroblasts and total mononuclear cell fraction indicate common ancestry for the erythroid lineage and the myeloid blast cells in RS-AML patients. Gene expression analysis by performing RNA sequencing on CD34+ AML cells revealed differential gene expression between RS-AML and a separate AML cohort, including genes involved in megakaryocytic/erythroid differentiation and mRNA splicing. Furthermore, several heme-metabolism-related genes were found upregulated in RS-AML, as was observed in SF3B1mut MDS. These results demonstrate that erythroblasts share ancestry with malignant myeloid blast cells in RS-AML. The genetic background of RS-AML differs from that of RS-MDS, however downstream effector pathways may be comparable, providing a possible explanation for presence of RS in AML.

2020-03-17 | GSE127861 | GEO

EIF2AK1-mediated signaling drives SF3B1 mutation-induced arrest in terminal erythroid differentiation in myelodysplastic syndrome with ringed sideroblasts

2022-07-30 | GSE188367 | GEO

RNA-seq analysis in SF3B1 5’UTR WT and A88G MOLM-13 cells

Project description:SF3B1 is the most mutated splicing factor (SF) in myelodysplastic syndromes (MDS), clonal hematopoietic disorders with variable risk of leukemic transformation. Although the tumorigenic effects of SF3B1 mutations have been defined, the role of “non-mutated” SF3B1 in cancer remains largely unresolved. Here we identify a conserved epitranscriptomic program that steers SF3B1 translation to counteract leukemogenesis. Our analysis of human and murine pre-leukemic MDS cells reveals a remarkable SF3B1 protein increase. Selective inhibition of SF3B1 upregulation accelerates MDS-to-leukemia progression in vivo. Mechanistically, ALKBH5-driven m6A demethylation within the SF3B1 5’ UTR fine-tunes SF3B1 translation to direct splicing of central DNA repair and epigenetic regulators during transformation. Loss of 5’ UTR m6A increases SF3B1 abundance, genome stability and delays leukemia progression in vivo, supporting integrative analysis of SF3B1 molecular signatures in humans that may predict tumor mutational burden and poor prognosis. These findings highlight a post-transcriptional gene expression nexus that unveils unanticipated SF3B1-dependent cancer vulnerabilities.

2023-03-21 | GSE189584 | GEO

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