Mutations in the RNA Splicing Factor SF3B1 Promote Transformation through MYC Stabilization

Abstract Mutations in the RNA splicing factor SF3B1 are recurrent in CLL and myeloid neoplasms but their functional role in promoting tumorigenesis remain poorly understood. While SF3B1 mutations have been identified as promoting use of aberrant 3' splice sites (3'ss), consistent identification of mis-spliced transcripts and pathways that functionally link mutant SF3B1 to transformation remains elusive. Moreover, large-scale analyses of the impact of mutant SF3B1 on gene expression and gene regulatory networks, which may be distinct from aberrant splicing changes, remain to be performed. We therefore sought to elucidate the effects of SF3B1 mutations across hematopoietic malignancies and cancer lineages at the level of both mRNA splicing and expression. To this end, we collected RNA-seq data from 79 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations. The most frequent hotspot, K700E, was the most common mutation in CLL and breast cancers while mutations at position R625 were restricted to melanomas (Figure A, B). Regulatory network analysis of differentially expressed genes in SF3B1 mutated CLL identified MYC as the top master regulator (Figure C). MYC activation in SF3B1 mutated CLL was also verified by differential expression analyses (Figure D) and was common to SF3B1K700E mutant cancers while absent in cancers with mutations affecting R625. Taken together, these observations suggested that tumors harboring SF3B1K700E mutations activate the MYC transcriptional program. We next sought to verify the effects of c-Myc activation by mutant Sf3b1 in the B-cell lineage in vivo. We crossed Cd19-cre Sf3b1K700E/+ mice with Eμ-Myc transgenic mice to generate Cd19-cre+ control, Sf3b1K700E/+, Eμ-MycTg/+, and Sf3b1K700E/+Eμ-MycTg/+ double-mutant mice. While control or single mutant primary mice did not develop disease over one year, double-mutant mice developed a lethal B-cell malignancy. This effect was consistent in serial transplantation, where mice transplanted with double-mutant cells had shorter survival compared to single-mutant controls (Figure E). These data provide the first evidence that SF3B1 mutations contribute to tumorigenesis in vivo. To understand the molecular mechanism for MYC activation across SF3B1 mutant human and mouse cells, we analyzed RNA-seq data from CLL patients, isogenic Nalm-6 cells, and splenic B-cells from the mouse models. This revealed a significant overlap in aberrant (3'ss) events across SF3B1 mutant samples. Interestingly, mis-spliced events across mouse and human SF3B1K700E mutant samples identified aberrant 3'ss usage and decay of PPP2R5A (Figure F), a gene whose product has previously been shown to regulate c-MYC protein stability and the only gene whose aberrant splicing was most prominent in K700E compared with R625 mutant SF3B1. PPP2R5A is a subunit of the PP2A phosphatase complex that dephosphorylates Serine 62 (S62) of c-MYC, resulting in an unstable form of c-MYC that is a substrate for proteasomal degradation. Consistent with this, SF3B1K700E mutant cells exhibited dramatic increase in S62-phosphorylated c-MYC and increased stability of c-MYC protein. MYC expression, stability, and S62 phosphorylation could be abrogated in SF3B1 mutant cells by restoring PPP25RA expression. In addition to c-MYC S62 phosphorylation, PPP2R5A-containing PP2A reduced S70 phosphorylation of BCL2 (a modification important for apoptosis induction) in SF3B1 mutant cells. To functionally evaluate the importance of impaired PP2A enzymatic activity in SF3B1 mutant cells further, we assessed the therapeutic potential of the FDA-approved oral PP2A activator, FTY-720. SF3B1 mutant cells were more sensitive to FTY-720 treatment than SF3B1 WT counterparts, experiencing growth arrest at lower concentration (Figure G). Moreover, both S62-phosphorylated c-MYC and S70-phosphorylated BCL2 decreased in a dose-dependent manner upon treatment with FTY-720 (Figure H). Here through combined evaluation of the effects of the SF3B1 mutation on splicing, gene expression, and transcriptional networks across cancer types, we identify a novel mechanism by which mutant SF3B1-mediated alterations in RNA splicing contribute to activation of oncogenic MYC through effects on MYC proteolysis. Moreover, these data highlight a novel therapeutic approach targeting the impact of mutant SF3B1 on post-translational modification of MYC. Figure. Figure. Disclosures Mato: Janssen: Consultancy, Honoraria; Celgene: Consultancy; Prime Oncology: Speakers Bureau; TG Therapeutics: Research Funding; Regeneron: Research Funding; Abbvie: Consultancy; Sunesis: Honoraria, Research Funding; Acerta: Research Funding; AstraZeneca: Consultancy; Pharmacyclics: Consultancy, Honoraria, Research Funding.

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RUNX1 Deficiency Cooperates with SRSF2 Mutation to Further Disrupt RNA Splicing and Exacerbate Myelodysplastic Syndromes in Mouse Models

Blood ◽

10.1182/blood-2018-99-119104 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1796-1796 ◽

Cited By ~ 1

Author(s):

Yi-Jou Huang ◽

Ming Yan ◽

Jia-Yu Chen ◽

Liang Chen ◽

Eunhee Kim ◽

...

Keyword(s):

Gene Expression ◽

Differentially Expressed Genes ◽

Rna Splicing ◽

Double Mutant ◽

Mutant Mice ◽

Differentially Expressed ◽

Differential Splicing ◽

Single Mutant ◽

Mutant Cells

Abstract Myelodysplastic syndromes (MDS) and leukemias require the acquisition of multiple mutations during disease development resulting in clonal diversity and different responses. Splicing factors, transcription factors, epigenetic regulators, and cell signaling proteins are the common molecular events mutated during disease evolution and those events rarely occur alone. However, it remains unclear how the combinations of mutations in different categories may have cooperative effects in gene regulation and disease etiology. Mutations in the splicing factor SRSF2 and the transcription factor RUNX1 are closely associated in MDS patients, and their co-existence is linked to poor prognosis. To understand the functional contribution of the coexistence in vivo, we utilized Mx1-Cre based conditional knock-in Srsf2-P95H mutation (P95H/+) mice, and Mx1-Cre based Runx1 conditional knockout mice (Runx1 f/f). We crossed these two strains to establish a new mouse model with inducible double mutations (Srsf2 P95H/+ Runx1Δ/Δ). Double mutant mice showed pancytopenia with MDS features including severe leukopenia in multiple lineages, macrocytic anemia, thrombocytopenia, and dysplastic morphology in peripheral blood. Double mutant mice also displayed more dramatic skewing toward the myeloid lineage at the expense of the B cell lineage when compared to single mutant mice. In competitive bone marrow transplantation assays, SRSF2 P95H cooperated with RUNX1 deficiency to confer a competitive disadvantage in vivo. To investigate the mechanistic basis of this cooperation, differential splicing and gene expression were assessed by RNA sequencing of Lineage- c-kit+ cells isolated from WT, SRSF2 P95H, RUNX1 KO, and Double mutant bone marrow cells. Interestingly, deletion of the Runx1 gene alone resulted in significant changes to RNA splicing in 1120 genes, while the SRSF2 P95H mutation itself induced splicing changes in 935 genes. Furthermore, 2468 splice junctions in 1677 genes showed splicing changes in double mutant samples compared to wildtype controls. Among these altered splicing events, intriguingly, exon skipping was the major alteration in single and double mutants. Furthermore, the double mutant demonstrated increased aberrant splicing events when compared to the single mutants alone. We performed pathway analysis using the differentially spliced genes identified in double mutant cells. Pathways in cancer, DNA replication/repair, cell death and survival, hematological disease and inflammatory response were enriched. Splicing changes were detected in genes recurrently mutated in blood malignancies, including Fanca, Fance, Fancl, Ezh2, Atm, Gnas, Braf, Bcor, Fyn, and Wsb1 as well as in genes critical for splicing regulation, such as Srsf6, Fus, Hnrnpa2b1, and Srrm2. Gene expression analysis revealed 869 significantly differentially expressed genes in double mutant cells. Within the events in the double mutant population, 60% of the differentially expressed genes were also observed in RUNX1 single mutant cells, while only 2% of the differentially expressed genes were observed in SRSF2 single mutant cells, and 38% of the differentially expressed genes were uniquely presented in the double mutant cells. These results suggest that the gene expression program is heavily affected by loss of RUNX1 and the coexistence of an SRSF2 mutation contributes to certain synergistic effects in transcriptional regulation. Furthermore, we identified 101 genes that showed both differential splicing and expression, including Jak3, Jag2, Csf3r, Fcer1g, CD244 which are important in hematologic disorders. Together, these results suggest that the deficiency of compound RUNX1 and SRSF2 P95H mutations impairs multi-lineage hematopoiesis and exacerbates the disease phenotypes caused by single mutations alone. At the genome-wide level, loss of the transcription factor RUNX1 itself dysregulates splicing outcomes and cooperates with the splicing factor SRSF2 P95H mutation to further perturb the expression and splicing of key regulators involved in hematopoietic stem/progenitor cell development, inflammatory responses, DNA damage, and RNA splicing. Disclosures No relevant conflicts of interest to declare.

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Selective Targeting of Splicing Factor Mutant Hematopoietic Stem and Progenitor Cells Via STAT3 Inhibition

Blood ◽

10.1182/blood-2021-151338 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1509-1509

Author(s):

Kathryn S Potts ◽

Rosannah C. Cameron ◽

Noura Ghazale ◽

Varun Gupta ◽

Juan Martin Barajas ◽

...

Keyword(s):

Research Funding ◽

Synthetic Lethality ◽

K562 Cells ◽

Splicing Factor ◽

Publicly Traded ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Mutant Cells ◽

Stat3 Inhibition

Abstract Myelodysplastic syndrome (MDS) is a bone marrow failure disorder driven by dysfunction of hematopoietic stem and progenitor cells (HSPCs). Patient sequencing studies over the last decade have revealed that mutations in splicing machinery predominate in MDS, thus selective targeting of these cells is therapeutically attractive. STAT3 inhibition has been explored previously as a means to eradicate HSPCs in MDS. While efficacy was demonstrated in a subset of samples, the underlying mechanism for this selectivity remains unknown. We examined RNAseq of MDS CD34+ HSPCs with splicing factor mutations versus wildtype, finding alternative splicing and differential expression of STAT3 pathway components. Functionally, we explored if STAT3 signaling represents a novel vulnerability in SF3B1 mutant HSPCs using a multi-model approach of in vivo zebrafish and mouse systems, and in vitro assays of CRISPR-engineered human leukemia K562 cells and primary MDS samples. Utilizing the small molecule STAT3 inhibitor STATTIC, we found that human cells carrying MDS-associated SF3B1 point mutations had heightened sensitivity to STAT3 inhibition compared to wildtype controls. To evaluate the activity of STAT3 inhibition in vivo, we utilized an Mx1-cre conditional knock-in mouse model of mutant SF3B1 (Sf3b1+/K700E). We demonstrated that in vivo STATTIC treatment selectively depleted Sf3b1 mutant cells over wildtype in vivo. RNAseq of sf3b1 homozygous mutantzebrafish cells revealed conserved dysregulation of STAT3 pathway splicing and target expression. Diminishing Stat3 (via morpholino knockdown, stable mutants, or STATTIC treatment) decreased HSPCs in sf3b1 heterozygotes but not wildtype embryos, demonstrating synthetic lethality between Sf3b1 and Stat3. Our data indicate that SF3B1 heterozygosity, regardless of the type of mutation, confers a heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Critically, our data indicate that SF3B1-mutant cells can be selectively killed in vivo while sparing wildtype cells. We sought to rescue HSPCs in sf3b1 homozygous mutant zebrafish, however overexpression of ligands Osm and Il6 or wildtype Stat3 was insufficient. Instead, overexpression of constitutively-active Stat3 partially restored HSPCs, indicating that functional Stat3 signaling downstream of Sf3b1 is critical for HSPC formation. To investigate the specificity of the synthetic lethality for SF3B1, we assessed the STAT3 synthetic lethal interaction with other mutated splicing factors in MDS. Similar to SF3B1, we demonstrated STAT3 synthetic lethality with U2AF1 and SRSF2 heterozygosity in zebrafish and human cells. RNA-sequencing analysis of STATTIC-treated K562 cells revealed an exacerbation of splicing alterations upon STAT3 inhibition that was more pronounced in SF3B1+/K666N cells compared to wildtype. Even more strikingly, we demonstrated that constitutive activation of STAT3 could partially reverse defective splicing in zebrafish sf3b1 homozygous mutant cells. Mechanistically, these data strongly support coordinated splicing dysfunction as the underlying cause for STAT3-SF3B1 synthetic lethality. Together, we demonstrated a conserved and selective synthetic lethal interaction between STAT3 function and splicing factor defects that represents a novel liability for mutant HSPCs with important implications for MDS treatment. Disclosures Shastri: Guidepoint: Consultancy; Kymera Therapeutics: Research Funding; Onclive: Honoraria; GLC: Consultancy. Verma: Curis: Research Funding; BMS: Research Funding; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; Novartis: Consultancy; Acceleron: Consultancy; Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company.

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Identification of Recurrent Alternative RNA Splicing in Adverse-Risk Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-129537 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 457-457

Author(s):

Govardhan Anande ◽

Ashwin Unnikrishnan ◽

Nandan Deshpande ◽

Sylvain Mareschal ◽

Aarif M. N. Batcha ◽

...

Keyword(s):

Alternative Splicing ◽

Board Of Directors ◽

Rna Splicing ◽

Myeloid Leukemia ◽

Research Funding ◽

Splicing Factor ◽

Rna Seq ◽

Advisory Committees ◽

Alternatively Spliced ◽

Acute Myeloid

RNA splicing is a fundamental biological process that generates protein diversity from a finite set of genes. Recurrent somatic mutations of genes involved in RNA splicing are present at high frequency in Myelodysplasia (up to 70%) but less so in Acute Myeloid Leukemia (AML; less than 20%). To investigate whether there were aberrant and recurrent RNA splicing events in the AML transcriptome that were associated with poor prognosis in the absence of splicing factor mutations, we developed a bioinformatics pipeline to systematically annotate and quantify alternative splicing events from RNA-sequencing data (Fig A). We first analysed publicly available RNA-seq data from The Cancer Genome Atlas (TCGA, n=170). We focussed on non-M3 AML patients with no splicing factor mutations (based on reported genomic sequencing and verified by re-analysis of RNA-seq data from all patients) who had received intensive chemotherapy. We segregated these patients based on their European Leukaemia Net (ELN) risk classification and identified 1290 alternatively spliced events impacting 910 genes that were significantly different (FDR<0.05) between all ELNAdv (n=41) versus all ELNFav patients (n=21, Fig B). The majority were exon skipping events (716 events, 62%, Fig B-C), followed by intron retention (201 events, 15.6%, Fig B). We next used RNA-seq data from a second non-M3 AML patient cohort (ClinSeq- Sweden; ELNAdv, n=75 and ELNFav, n=47), detecting 2507 events mapping to 1566 genes. Comparing across the two cohorts, 222 shared genes were detected to be affected by alternative splicing (Fig D). Ingenuity pathway analysis associated these genes with pathways related to protein translation. In order to prioritise those alternatively spliced events most likely to have a deleterious function, we developed an analytical framework to predict their impact on protein structure (Fig E). 87 alternatively spliced events, 25.81% of the commonly shared splicing events, relating to 78 genes (35.13% of all genes) were predicted to directly alter highly conserved protein domains within the affected genes, leading to either a complete (~25%, Fig E) or a partial loss of a domain (20%, Fig E). These in silico predictions are likely to be an underestimate of the true impact, as splicing alterations mapping to poorly annotated domains or affecting the tertiary structure of proteins would be missed. A number of splicing factors themselves were differentially spliced, with the alternative splicing predicted to have functional consequences. This was exemplified by hnRNPA1, a factor with well-established roles in splicing, is itself alternatively spliced in patients and predicted to be deleterious. Consistent with this, motif scanning analyses indicated that a number of mis-spliced transcripts had hnRNPA1 binding motifs (Fig F). To assess the impact of these alternatively spliced events (that were predicted to also disrupt highly conserved protein domains) on the transcriptome, we simultaneously quantified differential gene expression. IPA analysis of the 602 genes that were differentially expressed between ELNAdv and ELNFav patients and shared between both TCGA and ClinSeq cohorts indicated that they were associated with pathways (Fig G) that were distinct from those associated with aberrantly spliced genes (Fig D). A number of pathways related to inflammation were enriched amongst the genes observed to be upregulated in ELNAdv patients (Fig G). Network analyses integrating the alternatively spliced genes with differentially expressed genes revealed strong interactions (Fig H), indicating functional associations between these biological events. Given these strong network interactions, we investigated the potential prognostic significance of these alternatively spliced events. To this end, we utilised machine-learning methods to derive a "splicing signature" of four mis-spliced genes with a predictive capacity equivalent to the ELN (Fig I). The splicing signature further refined existing risk prediction algorithms to improve the classification of patients (Fig J). Taken together, we report the presence of extensive deregulation of RNA splicing in AML patients even in the absence of splicing factor mutations. Many of these events were shared in patients with adverse outcomes and their impact on the AML transcriptome points towards vulnerabilities that could be targeted. Figure Disclosures Unnikrishnan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lehmann:TEVA: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Abbive: Membership on an entity's Board of Directors or advisory committees. Pimanda:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Harbors Frequent Splicesosome Mutations That Cause Aberrant RNA Splicing Affecting Genes Critical in pDC Differentiation and Function

Blood ◽

10.1182/blood.v128.22.738.738 ◽

2016 ◽

Vol 128 (22) ◽

pp. 738-738 ◽

Cited By ~ 4

Author(s):

Katsuhiro Togami ◽

Vikas Madan ◽

Jia Li ◽

Alexandra-Chloe Villani ◽

Siranush Sarkizova ◽

...

Keyword(s):

Dendritic Cell ◽

Rna Splicing ◽

Intron Retention ◽

Plasmacytoid Dendritic Cell ◽

Splicing Factor ◽

Splicing Factors ◽

Rna Seq ◽

Aberrant Splicing ◽

Recurrent Mutations ◽

Cell Neoplasm

Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive malignancy thought to result from transformation of plasmacytoid dendritic cells (pDCs). Clinical outcomes are poor and pathogenesis is unclear. To better understand BPDCN genomics and disease mechanisms, we performed whole exome- (12 BPDCNs), targeted DNA- (additional 12 BPDCNs), bulk whole transcriptome RNA- (12 BPDCNs and 6 BPDCN patient-derived xenografts [PDXs]), and single cell RNA-sequencing (scRNA-seq) compared to normal DCs. We observed RNA splicing factor mutations in 16/24 cases (7 ZRSR2, 6 SRSF2, 1 each SF3B1, U2AF1, SF3A2, SF3B4). Additional recurrent alterations were in genes known to be mutated in other blood cancers: TET2, ASXL1, TP53, GNB1, NRAS, IDH2, ETV6, DNMT3A, and RUNX1. From exome sequencing we also discovered recurrent mutations in CRIPAK (6/12 cases), NEFH (4/12), HNF1A (2/12), PAX3 (2/12), and SSC5D (2/12) that may be unique to BPDCN. ZRSR2 is notable among the recurrently mutated splicing factors in hematologic malignancies in that all mutations are loss-of-function (e.g., nonsense, frameshift). Of note, BPDCN is very male predominant, ZRSR2 is located on chrX and all mutations are in males. ZRSR2 plays a critical role in "minor" or U12-type intron splicing (only 0.3% of all introns). Thus, we hypothesized that mis-splicing, possibly of U12 genes, contributes to BPDCN pathogenesis. Using RNA-seq, we measured aberrant splicing in BPDCN. Intron retention was the most frequent abnormality in ZRSR2 mutant BPDCNs and PDXs compared to non-mutant cases. ZRSR2 mutant intron retention predominantly affected U12 introns (patients: 29.4% of retained introns, P<0.0001; PDX: 94%, P<0.0001). To test if ZRSR2 loss directly causes U12 intron retention in otherwise isogenic cells, we performed ZRSR2 knockdown using doxycycline-inducible shRNAs in the BPDCN cell line, CAL1, which has no known splicing factor mutation. RNA-seq was performed 0, 2, and 7 days after addition of doxycycline in 3 independent clones each of control or ZRSR2 knockdown. Consistent with what we observed in primary BPDCN, intron retention events were higher in ZRSR2 compared to control shRNA cells after 7 days of doxycycline (mean 885.7 vs 122.7 events, P=0.041). Aberrant intron retention after ZRSR2 knockdown largely involved U12 introns (30/732 U12 vs 37/207,344 U2 introns, P<0.0001). SRSF2 and SF3B1 mutations in BPDCN were at hotspots seen in other cancers: SRSF2 P95H/L/R and SF3B1 K666N, mutants that induce specific types of aberrant splicing (Kim, Ca Cell 2015; Darman, Cell Rep 2015). Mutant BPDCNs demonstrated the same aberrations: SRSF2, exon inclusion/exclusion based on CCNG/GGNG exonic splicing enhancer motifs; SF3B1, aberrant 3' splice site recognition. We hypothesized that aberrant splicing may affect RNAs important for pDC development or function. To further define genes uniquely important in BPDCN, we performed scRNA-seq on 4 BPDCNs and on DCs from healthy donors. By principal component analysis, BPDCNs were more similar to pDCs than to conventional DCs (cDCs) or other HLA-DR+ cells. However, several critical genes for pDC function had markedly lower expression in BPDCN including the transcription factors IRF4 and IRF7. Next we determined which genes were commonly mis-spliced in splicing factor mutant BPDCNs. Strikingly, this list included genes already known to be important in driving DC biology or identified in our scRNA-seq as being differentially expressed between BPDCN and healthy pDCs, including IRF7, IRF8, IKZF1, FLT3, and DERL3. To determine if splicing factor mutations affect DC function, we stimulated ZRSR2 knockdown or control CAL1 cells with Toll-like receptor (TLR) 7, 8, and 9 agonists (R848 or CpG oligo). ZRSR2 knockdown inhibited upregulation of the CD80 costimulatory molecule and aggregation of CAL1 cells, suggesting impairment in activation. Using mouse conditional knock-in bone marrow in ex vivo multipotent progenitor assays, DC differentiation induced by FLT3 ligand was biased toward pDCs and away from cDCs in SRSF2 P95H mutant compared to wild-type cells. However, cDC and monocyte differentiation in the presence of GM-CSF was not affected. In conclusion, splicing factors are frequently mutated in BPDCN and lead to specific splicing defects. Splicing factor mutations may promote BPDCN by affecting pathways important in DC maturation or activation, which could contribute to transformation. Disclosures Seiler: H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Lane:Stemline Therapeutics: Research Funding; N-of-1: Consultancy.

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The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing

Nature Communications ◽

10.1038/s41467-021-23505-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Evi Goulielmaki ◽

Maria Tsekrekou ◽

Nikos Batsiotos ◽

Mariana Ascensão-Ferreira ◽

Eleftheria Ledaki ◽

...

Keyword(s):

Dna Damage ◽

Rna Splicing ◽

Excision Repair ◽

Intron Retention ◽

Dna Lesions ◽

Splicing Factor ◽

Loop Formation ◽

Rna Targets ◽

Underlying Mechanisms

AbstractRNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.

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RNA Splicing Defects in Cancer-Linked Genes Indicate Mutation or Focal Gene Deletion and Are Associated with TKI Resistance in CML

Blood ◽

10.1182/blood-2019-123180 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 662-662

Author(s):

Naranie Shanmuganathan ◽

Daniel Thomson ◽

Carol Wadham ◽

Verity A Saunders ◽

Nur Hezrin Shahrin ◽

...

Keyword(s):

Splice Site ◽

Rna Splicing ◽

Research Funding ◽

Cancer Genes ◽

Aberrant Splicing ◽

Cryptic Exon ◽

Large Deletions ◽

Tki Resistance ◽

Splice Junctions ◽

Runx1 Mutation

Background Mutated cancer genes in patients (pts) with TKI failure and blast crisis (BC) CML have recently been described. RUNX1 mutations, namely single nucleotide variants (SNVs) and indels, were the most frequently detected besides BCR-ABL1 [reviewed in Branford, Kim Leuk 2019]. They were found in ~18% of pts, although splice variants were rarely described. RNA splicing events were associated with focal deletion of IKZF1 and RUNX1 in TKI resistant pts that were identified by copy number analysis and RNAseq [Branford Blood 2018]. Novel splicing associated with mutation of cancer genes is an unexplored area of study in resistance. RNA sequencing can assess the functional effect of splice site variants, which lead to splicing errors due to the use of alternative or cryptic splice sites and cause alterations to protein function. Aim We determined whether novel splicing can identify cancer genes with potential altered function. Methods RNAseq analysis was performed for 48 pts at diagnosis and 33 at BC using a protocol that preserved intron-retaining precursor RNA. Coverage of intron-exon borders was sufficient to detect intronic splice region variants. The STAR aligner was used to bioinformatically collate unannotated RNA splice junctions. 54 cancer genes were assessed and aberrant splice events were filtered based on the number of samples in which a splice junction occurred. Manual inspection of the splice junctions was performed using the Integrative Genomics Viewer. This approach identified previously verified aberrant splicing associated with IKZF1 and RUNX1 deletions. Results Ten previously undetected novel splice junctions were revealed in 9/33 pts (27%) in BC within key tumor suppressor genes CDKN2A/B (5), RB1 (1), ATM (1), and RUNX1 (3). The aberrant splicing pattern of CDKN2A and RB1 (Fig A/B) in 6 pts suggested large deletions, as previously described in our cohort with IKZF1 and RUNX1 deletions. Breakpoints associated with deletions ranging from 53 to 181 Kb were detected in the 5 pts with CDKN2A aberrant splicing. Similarly, a 90 Kb deletion of exons 18-27 of the RB1 gene led to the aberrant splicing. The pts transformed to lymphoid BC (median 5 months). 4 of these 6 pts were tested at diagnosis and the deletions were not detected, indicating they were gained at resistance. The aberrant splicing patterns of ATM and RUNX1 did not predict large deletions. These were related to somatic SNVs at canonical splice sites in ATM and in 2 of the pts with RUNX1 aberrant splicing. A splice acceptor site SNV in ATM resulted in skipping of exon 61 (Fig C) and protein truncation. This novel SNV has not been reported in any population or somatic variant database. Two pts in myeloid BC at 28 and 48 months after diagnosis had an identical somatic RUNX1 mutation at the canonical splice donor site of exon 5. The SNV was not detectable prior to imatinib treatment in both pts. The splice site SNV led to activation of a cryptic splice site within exon 5 in both pts (Fig D), which predicted premature termination. While this mutation is novel, an adjacent intronic SNV occurs in familial platelet disorder, leading to activation of the same cryptic splice site. The atypical RUNX1 splicing of the 3rd patient was associated with retention of 55 bp of intron 6 as a cryptic exon (Fig E), leading to protein truncation. A deep intronic SNV identified at lymphoid BC at 6 months of imatinib was detected near the cryptic exon by RNAseq and verified as somatic by DNA Sanger sequencing. This was predicted to activate cryptic RNA splicing elements and lead to intron sequence retention in a RUNX1 transcript. We sequenced the diagnosis sample using an RNA-based gene panel method under development that provides enhanced sensitivity of variant detection. The same pattern of atypical splicing was observed and the intronic SNV was present at low level. The RUNX1 mutation at diagnosis may have contributed to early BC. To our knowledge this is the first report of a RUNX1 truncating variant in CML involving a cryptic exon. Conclusion Enhanced bioinformatic analysis of RNAseq data has revealed a high proportion of pts with truncating mutations in cancer genes indicated by novel RNA splicing (27% pts in BC). Using RNA-based sequencing allows an evaluation of the potential functional effect of variants that are not apparent by DNA-based mutation analysis. We suggest that future studies include RNA sequencing to detect a broader spectrum of mutations associated with TKI resistance. Disclosures Shanmuganathan: Gilead: Other: Travel Support; Janssen: Other: Travel Support; Amgen: Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Novartis: Honoraria, Other: Travel Support. Yeung:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. Scott:Celgene: Honoraria. Hughes:Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. Branford:Cepheid: Consultancy, Honoraria; Qiagen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau.

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Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations

Blood ◽

10.1182/blood-2018-04-843771 ◽

2018 ◽

Vol 132 (12) ◽

pp. 1225-1240 ◽

Cited By ~ 56

Author(s):

Andrea Pellagatti ◽

Richard N. Armstrong ◽

Violetta Steeples ◽

Eshita Sharma ◽

Emmanouela Repapi ◽

...

Keyword(s):

Focal Adhesion ◽

Rna Splicing ◽

Splicing Factor ◽

Rna Seq ◽

Poor Survival ◽

Cd34 Cells ◽

Key Points ◽

Clinical Associations

Key Points RNA-seq analysis of CD34+ cells identifies novel aberrantly spliced genes and dysregulated pathways in splicing factor mutant MDS. Aberrantly spliced isoforms predict MDS survival and implicate dysregulation of focal adhesion and exosomes as drivers of poor survival.

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Evidence for reprogramming of monocytes into reparative alveolar macrophages in vivo by targeting PDE4b

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00145.2021 ◽

2021 ◽

Author(s):

Ian Rochford ◽

Jagdish Chandra Joshi ◽

Rayees Sheikh ◽

Mumtaz Anwar ◽

Md Zahid Akhter ◽

...

Keyword(s):

Lung Injury ◽

Alveolar Macrophages ◽

Lung Edema ◽

Rna Seq ◽

Activation Of Transcription ◽

The Impact ◽

Camp Levels ◽

Phosphodiesterase 4B ◽

Lung Vascular Permeability

Increased lung vascular permeability and neutrophilic inflammation are hallmarks of acute lung injury. Alveolar macrophages (AMϕ), the predominant sentinel cell type in the airspace, die in massive numbers while fending off pathogens. Recent studies indicate that the AMϕ pool is replenished by airspace-recruited monocytes, but the mechanisms instructing the conversion of recruited monocytes into reparative AMϕ remain elusive. Cyclic AMP (cAMP) is a vascular barrier protective and immunosuppressive second messenger in the lung. Here, we subjected mice expressing GFP under the control of the Lysozyme-M promoter (LysM-GFP mice) to the LPS model of rapidly resolving lung injury to address the impact of mechanisms determining cAMP levels in AMϕ and regulation of mobilization of the reparative AMϕ-pool. RNA-seq analysis of flow-sorted Mϕ identified phosphodiesterase 4b (PDE4b) as the top LPS-responsive cAMP-regulating gene. We observed that PDE4b expression markedly increased at the time of peak injury (4 h) and then decreased to below the basal level during the resolution phase (24 h). Activation of transcription factor NFATc2 was required for transcription of PDE4b in Mϕ. Inhibition of PDE4 activity at the time of peak injury, using i.t. rolipram, increased cAMP levels, augmented the reparative AMϕ pool, and resolved lung injury. This response was not seen following conditional depletion of monocytes, thus establishing airspace-recruited PDE4b-sensitive monocytes as the source of reparative AMϕ. Interestingly, adoptive transfer of rolipram-educated AMϕ into injured mice resolved lung edema. We propose suppression of PDE4b as an effective approach to promote reparative AMϕ generation from monocytes for lung repair.

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In Silico Analysis of a Highly Mutated Gene in Cancer Provides Insight into Abnormal mRNA Splicing: Splicing Factor 3B Subunit 1K700E Mutant

Biomolecules ◽

10.3390/biom10050680 ◽

2020 ◽

Vol 10 (5) ◽

pp. 680 ◽

Cited By ~ 2

Author(s):

Asmaa Samy ◽

Baris Suzek ◽

Mehmet Ozdemir ◽

Ozge Sensoy

Keyword(s):

Rna Splicing ◽

In Silico Analysis ◽

Therapy Resistance ◽

Mrna Splicing ◽

Splicing Factor ◽

Cancer Types ◽

Dynamics Simulations ◽

Aberrant Rna ◽

The Impact ◽

Splicing Machinery

Cancer is the second leading cause of death worldwide. The etiology of the disease has remained elusive, but mutations causing aberrant RNA splicing have been considered one of the significant factors in various cancer types. The association of aberrant RNA splicing with drug/therapy resistance further increases the importance of these mutations. In this work, the impact of the splicing factor 3B subunit 1 (SF3B1) K700E mutation, a highly prevalent mutation in various cancer types, is investigated through molecular dynamics simulations. Based on our results, K700E mutation increases flexibility of the mutant SF3B1. Consequently, this mutation leads to i) disruption of interaction of pre-mRNA with SF3B1 and p14, thus preventing proper alignment of mRNA and causing usage of abnormal 3’ splice site, and ii) disruption of communication in critical regions participating in interactions with other proteins in pre-mRNA splicing machinery. We anticipate that this study enhances our understanding of the mechanism of functional abnormalities associated with splicing machinery, thereby, increasing possibility for designing effective therapies to combat cancer at an earlier stage.

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Dysregulation of Splicing in Multiple Myeloma: The Splicing Factor SRSF1 Supports MM Cell Proliferation Via Splicing Control

Blood ◽

10.1182/blood-2018-99-118845 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4500-4500

Author(s):

Mariateresa Fulciniti ◽

Michael A Lopez ◽

Anil Aktas Samur ◽

Eugenio Morelli ◽

Hervé Avet-Loiseau ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Alternative Splicing ◽

Board Of Directors ◽

Research Funding ◽

Splicing Factor ◽

Splicing Factors ◽

Rna Seq ◽

Advisory Committees ◽

Disease Biology

Abstract Gene expression profile has provided interesting insights into the disease biology, helped develop new risk stratification, and identify novel druggable targets in multiple myeloma (MM). However, there is significant impact of alternative pre-mRNA splicing (AS) as one of the key transcriptome modifier. These spliced variants increases the transcriptomic complexity and its misregulation affect disease behavior impacting therapeutic consideration in various disease processes including cancer. Our large well annotated deep RNA sequencing data from purified MM cells data from 420 newly-diagnosed patients treated homogeneously have identified 1534 genes with one or more splicing events observed in at least 10% or more patients. Median alternative splicing event per patient was 595 (range 223 - 2735). These observed global alternative splicing events in MM involves aberrant splicing of critical growth and survival genes affects the disease biology as well as overall survival. Moreover, the decrease of cell viability observed in a large panel of MM cell lines after inhibition of splicing at the pre-mRNA complex and stalling at the A complex confirmed that MM cells are exquisitely sensitive to pharmacological inhibition of splicing. Based on these data, we further focused on understanding the molecular mechanisms driving aberrant alternative splicing in MM. An increasing body of evidence indicates that altered expression of regulatory splicing factors (SF) can have oncogenic properties by impacting AS of cancer-associated genes. We used our large RNA-seq dataset to create a genome wide global alterations map of SF and identified several splicing factors significantly dysregulated in MM compared to normal plasma cells with impact on clinical outcome. The splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1), regulating initiation of spliceosome assembly, was selected for further evaluation, as its impact on clinical outcome was confirmed in two additional independent myeloma datasets. In gain-of (GOF) studies enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells; and conversely, in loss-of function (LOF) studies, downregulation of SRSF1, using stable or doxy-inducible shRNA systems significantly inhibited MM cell proliferation and survival over time. We utilized SRSF1 mutants to dissect the mechanisms involved in the SRSF1-mediated MM growth induction, and observed that the growth promoting effect of SRSF1 in MM cells was mainly due to its splicing activity. We next investigated the impact of SRSF1 on allelic isoforms of specific gene targets by RNA-seq in LOF and confirmed in GOF studies. Splicing profiles showed widespread changes in AS induced by SRSF1 knock down. The most recurrent splicing events were skipped exon (SE) and alternative first (AF) exon splicing as compared to control cells. SE splice events were primarily upregulated and AF splice events were evenly upregulated and downregulated. Genes in which splicing events in these categories occurred mostly did not show significant difference in overall gene expression level when compared to control, following SRSF1 depletion. When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affects genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells. We have observed increased levels of SRSF phosphorylation, which regulates it's subcellular localization and activity, in MM cell lines and primary patient MM cells compared to normal donor PBMCs. Moreover, we evaluated the chemical compound TG003, an inhibitor of Cdc2-like kinase (CLK) 1 and 4 that regulate splicing by fine-tuning the phosphorylation of SR proteins. Treatment with TG003 decreased SRSF1 phosphorylation preventing the spliceosome assembly and inducing a dose dependent inhibition of MM cell viability. In conclusions, here we provide mechanistic insights into myeloma-related splicing dysregulation and establish SRSF1 as a tumor promoting gene with therapeutic potential. Disclosures Avet-Loiseau: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:OncoPep: Other: Board of director.

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