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GPATCH8 modulates mutant SF3B1 mis-splicing and pathogenicity in hematologic malignancies

ABSTRACT: Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, but therapeutic means to correct this mis-splicing do not exist. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SUGP1, a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mouse and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Zhuoning Li

LAB HEAD: Omar Abdel-Wahab

PROVIDER: PXD051128 | Pride | 2024-04-10

REPOSITORIES: Pride

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Synthetic Introns Identify GPATCH8 as Required for Mis-splicing by SF3B1 Mutations [TRIBE-seq]

Project description:Mutations in the RNA splicing factor SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing but therapeutic means to correct this mis-splicing do not exist. Here we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SUGP1, a G-patch protein recently implicated in SF3B1 mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1 splicing defects and was sufficient to improve hematopoiesis in SF3B1 mutant mouse and human cells. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and the therapeutic impact of correcting aberrant splicing in SF3B1 mutant cancers.

2023-09-01 | GSE242093 | GEO

Synthetic Introns Identify GPATCH8 as Required for Mis-splicing by SF3B1 Mutations [RNA-seq]

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Synthetic Introns Identify GPATCH8 as Required for Mis-splicing by SF3B1 Mutations [RNA-Seq 2]

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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

Spliceosomal disruption of the non-canonical BAF complex in cancer

Project description:SF3B1 is the most commonly mutated RNA splicing factor in cancer, but the mechanisms by which SF3B1 mutations promote malignancy are poorly understood. Here, we integrated pan-cancer RNA sequencing to identify mutant SF3B1-dependent aberrant splicing with a positive enrichment CRISPR screen to prioritize splicing alterations that functionally promote tumorigenesis. We identify that diverse, recurrent SF3B1 mutations converge on repression of BRD9, a core component of the recently described non-canonical BAF (ncBAF) complex. Mutant SF3B1 recognizes an aberrant deep intronic branchpoint within BRD9, thereby inducing inclusion of an endogenous retrovirus-derived poison exon and BRD9 mRNA degradation. BRD9 depletion causes loss of ncBAF at CTCF-bound loci and promotes melanomagenesis. We demonstrate that BRD9 is a potent tumor suppressor in uveal melanoma, such that correcting BRD9 mis-splicing in SF3B1-mutant cell lines and patient-derived melanoma xenografts with antisense oligonucleotides (ASOs) or by directly targeting its poison exon with CRISPR-directed mutagenesis profoundly suppresses tumor growth. Our results implicate disruption of ncBAF in the diverse malignancies characterized by SF3B1 mutations, identify a single aberrant splicing event which functionally contributes to the pathogenesis of SF3B1-mutant cancers, and suggest a mechanism-based therapeutic for these malignancies.

2019-06-19 | GSE124720 | GEO

Expression data from Sf3b1 heterozygous Lineage-c-Kit+Sca-1+ (LSK) bone marrow cells

Project description:Recent studies have shown that multiple components of the mRNA splicing machinery are mutated in myelodysplastic syndrome (MDS) patients. SF3B1 is frequently mutated in refractory anemia with ringed sideroblasts (RARS)-MDS patients, however, the pathophysiological role of SF3B1 mutations has not been elucidated yet. In this study, we examined the function of Sf3b1 in murine hematopoiesis. Since Sf3b1 null homozygotes died during preimplantation development, in this study, we utilized Sf3b1 heterozygous mice showing grossly normal growth. We harvested bone marrow stem/progenitor (LSK) cells from wild type (WT) and Sf3b1+/- mice (n=4) at 20 weeks old. In addition, to exclude the possibility of indirect effect from bone marrow environment, we transplanted total bone marrow cells from WT or Sf3b1+/- (CD45.2+) mice into lethally irradiated CD45.1+ recipient mice, and then harvested (CD45.2+) LSK cells from the recipients (n=5) at 9 months-post transplantation.

2013-09-20 | E-GEOD-51038 | biostudies-arrayexpress

Distinct and convergent consequences of splice factor mutations in myelodysplastic syndromes

Project description:Myelodysplastic syndromes (MDS) are characterized by recurrent somatic alterations often affecting components of RNA splicing machinery. Mutations of splice factors SF3B1, SRSF2, ZRSR2 and U2AF1 occur in >50% of MDS. To assess the impact of spliceosome mutations on splicing and to identify common pathways/genes affected by distinct mutations, we performed RNA-sequencing of 24 MDS bone marrow samples harboring spliceosome mutations (including hotspot alterations of SF3B1, SRSF2 and U2AF1; small deletions of SRSF2 and truncating mutations of ZRSR2), and devoid of other common co-occurring mutations. We uncover the landscape of splicing alterations in each splice factor mutant MDS and demonstrate that SRSF2 deletions cause highest number of splicing alterations compared with other spliceosome mutations. Although the mis-spliced events observed in different splice factor mutations were largely non-overlapping, a subset of genes, including EZH2, were aberrantly spliced in multiple mutant groups. Pathway analysis revealed that the mis-spliced genes in different mutant groups were enriched in RNA splicing and transport as well as several signaling cascades, suggesting converging biological consequences downstream of distinct spliceosome mutations.

2019-11-07 | GSE128429 | GEO

SF3B1 Degron knockdown RNA-seq

Project description:Knockdown of mutant and/or wild-type SF3B1 in MEL202 cell line by Degron knock-in, followed by RNA-seq, to identify splicing events governed by mutant SF3B1. Control: parental MEL202 cell line. Experiments: mutant-SF3B1 knockdown; wildtype-SF3B1 knockdown; mutant SF3B1 knockout. Treatments: each of these four conditions plus and minus shld.

2015-03-11 | E-GEOD-66719 | biostudies-arrayexpress

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

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

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