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Long-read sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations in cancer (SF3B1 & U2AF2 iCLIP)

ABSTRACT: Alternative splicing plays a critical role in generating transcriptome diversity, and aberrant splicing is frequently observed in cancer. Mutations in the splicing factor SF3B1 are particularly common in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS), with different survival prognoses. We applied long-read sequencing for the investigation of the SF3B1 mutation effect on the transcriptome of MDS and CLL patients, as well as isogenic cell lines. Our results revealed that SF3B1 mutation effect was common across the different diseases and specifically altered the usage of 3’ alternative splice sites within short proximity to the canonical splice sites. Based on computational simulations, the most common K700E mutation led to destabilization of the SF3B1-mRNA binding. Furthermore, we combined the full isoform information with genome-wide SF3B1-RNA binding maps to predict the functional consequences of the aberrant splicing and gain mechanistic insights into the role of mutated SF3B1 in splicing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE247658 | GEO | 2024/08/19

REPOSITORIES: GEO

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

Project description: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.

2022-03-08 | GSE184246 | GEO

Novel SF3B1 Deletion Mutations Result in Aberrant RNA Splicing in CLL Patients

Project description:Recurrent mutations in RNA splicing factors SF3B1, U2AF1, and SRSF2 have been reported in hematologic cancers including myelodysplastic syndromes (MDS) and chronic lymphocytic leukemia (CLL). However, SF3B1 is the only splicing associated gene to be found mutated in CLL and has been shown to induce aberrant splicing. To investigate if any other genomic aberration caused similar transcriptome changes, we clustered RNASeq samples based on an alternative 3’ splice site (ss) pattern previously identified in SF3B1-mutant CLL patients. Out of 215 samples, we identified 37 (17%) with alternative 3’ ss usage, the majority of which harbored known SF3B1 hotspot mutations. Interestingly, 3 patient samples carried previously unreported in-frame deletions in SF3B1 around K700, the most frequent mutation hotspot. To study the functional effects of these deletions, we used various minigenes demonstrating that recognition of canonical 3’ ss and alternative branchsite are required for aberrant splicing, as observed for SF3B1 p.K700E. The common mechanism of action of these deletions and substitutions result in similar sensitivity of primary cells towards splicing inhibitor E7107. Altogether, these data demonstrate that novel SF3B1 in-frame deletion events identified in CLL result in aberrant splicing, a common biomarker in spliceosome-mutant cancers.

2017-06-27 | GSE95352 | GEO

Engineering oncogenic hotspot mutations on SF3B1 via CRISPR-directed PRECIS mutagenesis

Project description:To engineer novel SF3B1 mutant cell lines, we developed a methodology called PRECIS to install the pathogenic K700E mutation. We used this method to engineer CLL cell lines HG-3 and MEC-1 with the SF3B1 K700E mutation and then carried out RNA-seq.

2024-08-28 | GSE231387 | 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

Transcriptome analysis of chronic lymphoid leukemia reveals isoform regulation associated with mutations in SF3B1

Project description:Several DNA sequencing studies of chronic lymphocytic leukemia (CLL) revealed that the splicing factor SF3B1 accumulated somatic point mutations in about 10 percent of the patients. In most cases the mutations were located in the genomic regions coding for the C-terminal HEAT-repeat domain and in many cases, the mutations gave rise to specific amino acid substitutions. Here, we aimed to investigate differential usage of exons associated with the mutation K700E of SF3B1 in CLL tumuor cells. We generated RNA-Seq transcriptome data from two patients with mutations in SF3B1, two patient without mutations in SF3B1 and from healthy donors. We report interesting examples and possible consequences of the alternative exon usage in these genes. This dataset contains only the files resulting from the processing the RNA sequencing raw data. This avoids potential patient identifiability, but ensures the full reproducibility of the results described in the publication.

2013-12-27 | E-MTAB-2025 | biostudies-arrayexpress

A genetic murine model of CLL based on B cell-restricted expression of Sf3b1 mutation and Atm deletion

Project description:The RNA splicing factor SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its functional role in the pathogenesis of this disease has not been firmly established. Here, we show that conditional expression of heterozygous Sf3b1-K700E mutation in mouse B lineage cells disrupts pre-mRNA splicing, alters B-cell development and function, and induces a state of cellular senescence. B-cell restricted expression of this mutation combined with Atm deletion led to the overcoming of cellular senescence, together with enhanced genome instability and the development of clonal B220+CD5+ CLL cells in elderly mice at low penetrance. Mice with CLL-like disease were found to have amplifications of chromosomes 15 and 17. Integrated transcriptome and proteome analysis of the CLL-like cells revealed coordinated dysregulation of multiple CLL-associated cellular processes. This included an unexpected signature of deregulated B-cell receptor (BCR) signaling, which we could also identify in SF3B1-mutated CLL samples from two independent patient cohorts. Notably, human CLLs harboring SF3B1 mutations exhibited greater sensitivity and altered response kinetics to BTK kinase ibrutinib. Our genetically faithful murine model of CLL thus reveals fresh insights regarding the impact of SF3B1 mutation on CLL pathogenesis and suggests a system for identifying vulnerabilities related to this mutation that can be further exploited for the treatment of CLLs with this common mutation.

2018-11-20 | GSE122668 | 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

Cancer-associated SF3B1-K700E mutation controls immune responses by regulating Treg function via aberrant Anapc13 splicing

Project description:Recurrent somatic mutations in spliceosome factor 3b subunit 1 (SF3B1) are identified in hematopoietic cell-derived malignancies, with SF3B1-K700E being the most common one. Here we show that Treg specific expression of SF3B1-K700E (Sf3b1K700Efl/+/Foxp3YFP-Cre) results in spontaneous autoimmune phenotypes including enlarged spleen and tissue infiltration with IFN-γ-producing CD4+ T cells. CD4+ T cells from Sf3b1K700Efl/+/Foxp3YFP-Cre mice display defective Treg differentiation and inhibitory function, which is demonstrated by failed prevention of adoptive transfer colitis by Sf3b1K700Efl/+/Foxp3YFP-Cre Tregs. Mechanically, SF3B1-K700E induces an aberrant splicing event that results in reduced expression of a cell proliferation regulator Anapc13 due to insertion a 231 bp DNA fragment to the 5’ untranslated region (UTR). Forced expression of Anapc13 gene restores the differentiation and ability of Sf3b1K700Efl/+/Foxp3YFP-Cre Tregs to prevent adoptive transfer colitis. Our results thus highlight the impact of a hematological malignancy-associated SF3B1 mutation on immune responses and indicate a potential relationship between splicing factor mutation-dysregulated immune responses and cancer development.

2024-06-01 | GSE267436 | GEO

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