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Aberrant splicing events by RNA sequencing. (a) Number of alternatively splicing events in U2af1 mut/+, Tet2 −/− and U2af1 mut/+ Tet2 −/− mice. (b) Venn diagram showing the overlap of genes with alternative spliced events among comparison groups. (c) DNA damage accumulation in WT (n = 10), U2af1 mut/+ (n = 4) and U2af1 mut/+ Tet2 −/− (n = 6) represented as percentage of γ-H2AX+ cells in LSK, determined by flow cytometry. (d) Validation of aberrant splicing events identified
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Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involv...
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Context 1
... component analysis showed segregation of the genotypes, with U2af1 mut/+ samples being more heterogeneous ( Figure S3). Using pair-wise comparisons against WT control, we identified 1578 differentially spliced events in U2af1 mut/+ , 1211 in Tet2 −/− and 1242 in U2af1 mut/+ Tet2 −/− (p value < 0.01, FDR < 0.01) (Figure 5a and Supplementary Table S4). This analysis showed that U2af1 samples exhibit predominantly exon skipping events, which is in line with previously published results for mutated U2AF1 [4,16], and that 152 alternative spliced genes were common among the different pair-wise contrasts (Figure 5b). ...
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... pair-wise comparisons against WT control, we identified 1578 differentially spliced events in U2af1 mut/+ , 1211 in Tet2 −/− and 1242 in U2af1 mut/+ Tet2 −/− (p value < 0.01, FDR < 0.01) (Figure 5a and Supplementary Table S4). This analysis showed that U2af1 samples exhibit predominantly exon skipping events, which is in line with previously published results for mutated U2AF1 [4,16], and that 152 alternative spliced genes were common among the different pair-wise contrasts (Figure 5b). ...
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... seek downstream biological consequences, we performed enrichment pathway analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted with the genes significantly mis-spliced in U2af1 mut/+ , Tet2 −/− and U2af1 mut/+ Tet2 −/− mice compared to WT control mice, highlighting enrichment in cell processes, such as DNA damage, DNA repair and cell cycle, chromatin regulator and kinase pathway ( FigureS S4, S5, S6). Of relevance, KEGG functional annotation in U2af1 mut/+ Tet2 −/− group distributed misspliced genes in pathways similar to those found for U2af1 mut/+ . ...
Context 4
... validate this further, we determined the phosphorylation status of histone γ-H2AX at Ser 139 in LSK cells as being a sensitive marker of DNA injury [33] and the first step in recruiting and localizing DNA repair proteins [34]. We found no differences in phosphorylated γ-H2AX in LSK cells from U2af1 mut/+ and U2af1 mut/+ Tet2 −/− compared to WT controls (Figure 5c). Next, we validated selected aberrant splicing events using RT-PCR in c-kit + cells. ...
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Citations
... It has also been observed that the effects of different mutations may vary on different exons; for example, the U2AF1 S34 mutation prefers C or A more frequently in the-3 position when analysed in AML patients, whereas the Q157 mutation, involving the first zinc finger, shows preferential recognition of G over A in the +1 position. By combining comprehensive data from RNA sequencing and U2AF1 mutations with 3' SS recognition ability, a clearer understanding of the mechanism by which U2AF1 mutations affect splicing in patients with MDS can be established (10,12,35,36). ...
U2 small nuclear RNA auxiliary factor 1 (U2AF1) is a multifunctional protein that plays a crucial role in the regulation of RNA splicing during eukaryotic gene expression. U2AF1 belongs to the SR family of splicing factors and is involved in the removal of introns from mRNAs and exon-exon binding. Mutations in U2AF1 are frequently observed in myelodysplastic syndrome, primary myelofibrosis, chronic myelomonocytic leukaemia, hairy cell leukaemia and other solid tumours, particularly in lung, pancreatic, and ovarian carcinomas. Therefore, targeting U2AF1 for therapeutic interventions may be a viable strategy for treating malignant diseases. In the present review, the pathogenic mechanisms associated with U2AF1 in different malignant diseases were summarized, and the potential of related targeting agents was discussed. Additionally, the feasibility of natural product-based therapies directed against U2AF1 was explored.
... The heterozygous U2af1 mut/+ mice generated by the CRISPR/Cas9 system exhibited aberrant RNA splicing and a defective reconstitution capacity of HSPC in transplantation assays. U2af1/Tet2 double mutant mice had increased monogranulocytic precursors, but did not succumb to MDS [60]. Briefly, these studies using different cell and mouse models strongly prove that transgenic and knock-in of the U2AF1 S34F mutant may lead to severe defects in HSCs and hematopoiesis. ...
Mutations of spliceosome genes have been frequently identified in myeloid malignancies with the large-scale application of advanced sequencing technology. U2 small nuclear RNA auxiliary factor 1 (U2AF1), an essential component of U2AF heterodimer, plays a pivotal role in the pre-mRNA splicing processes to generate functional mRNAs. Over the past few decades, the mutation landscape of U2AF1 (most frequently involved S34 and Q157 hotspots) has been drawn in multiple cancers, particularly in myeloid malignancies. As a recognized early driver of myelodysplastic syndromes (MDSs), U2AF1 mutates most frequently in MDS, followed by acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs). Here, for the first time, we summarize the research progress of U2AF1 mutations in myeloid malignancies, including the correlations between U2AF1 mutations with clinical and genetic characteristics, prognosis, and the leukemic transformation of patients. We also summarize the adverse effects of U2AF1 mutations on hematopoietic function, and the alterations in downstream alternative gene splicing and biological pathways, thus providing comprehensive insights into the roles of U2AF1 mutations in the myeloid malignancy pathogenesis. U2AF1 mutations are expected to be potential novel molecular markers for myeloid malignancies, especially for risk stratification, prognosis assessment, and a therapeutic target of MDS patients.
... To simplify nomenclature, we referred to all of them with the female genotypes. To generate animals carrying alterations in both Zrsr2 and Tet2, Zrsr2 mutant mice were crossed with a Tet2 KO line previously described [20]. Both female and male double mutant animals were used for experiments. ...
RNA splicing and epigenetic gene mutations are the most frequent genetic lesions found in patients with myelodysplastic neoplasm (MDS). About 25% of patients present concomitant mutations in such pathways, suggesting a cooperative role in MDS pathogenesis. Importantly, mutations in the splicing factor ZRSR2 frequently associate with alterations in the epigenetic regulator TET2 . However, the impact of these concurrent mutations in hematopoiesis and MDS remains unclear. Using CRISPR/Cas9 genetically engineered mice, we demonstrate that Zrsr2 m/m Tet2 −/− promote MDS with reduced penetrance. Animals presented peripheral blood cytopenia, splenomegaly, extramedullary hematopoiesis, and multi-lineage dysplasia, signs consistent with MDS. We identified a myelo-erythroid differentiation block accompanied by an expansion of LT-HSC and MPP2 progenitors. Transplanted animals presented a similar phenotype, thus indicating that alterations were cell-autonomous. Whole-transcriptome analysis in HSPC revealed key alterations in ribosome, inflammation, and migration/motility processes. Moreover, we found the MAPK pathway as the most affected target by mRNA aberrant splicing. Collectively, this study shows that concomitant Zrsr2 mutation and Tet2 loss are sufficient to initiate MDS in mice. Understanding this mechanistic interplay will be crucial for the identification of novel therapeutic targets in the spliceosome/epigenetic MDS subgroup.
... These mutations occur in genes encoding SFs entailed in the early steps of SPL assembly, including the SF3B1 [13][14][15][16][17][18], U2AF1, U2AF2, ZRSR2 [14,[19][20][21], and SRSF2 [22]. SF3B1 mutations are primarily heterozygous and likely to be gain/changeof-function mutations; however, it is unclear whether U2AF1 mutations result in a gain-of or loss-of-function of the protein [23], whereas the ZRSR2 mutations are predicted to be loss-offunction mutations [24]. Additionally, somatic mutations of the SPL helicase Brr2 and the core SPL protein Prp8 are linked to retinitis pigmentosa [25]. ...
Introduction
RNA splicing is a pivotal step of eukaryotic gene expression during which the introns are excised from the precursor (pre-)RNA and the exons are joined together to form mature RNA products (i.e a protein-coding mRNA or long non-coding (lnc)RNAs). The spliceosome, a complex ribonucleoprotein machine, performs pre-RNA splicing with extreme precision. Deregulated splicing is linked to cancer, genetic and neurodegenerative diseases. Hence, the discovery of small-molecules targeting core spliceosome components represents an appealing therapeutic opportunity.
Area Covered
Several atomic-level structures of the spliceosome and distinct splicing-modulators bound to its protein/RNA components have been solved. Here, we review recent advances in the discovery of small-molecule splicing-modulators, discuss opportunities and challenges for their therapeutic applicability and showcase how structural data and/or all-atom simulations can illuminate key facets of their mechanism, thus contributing to future drug-discovery campaigns.
Expert Opinion
This review highlights the potential of modulating pre-RNA splicing with small-molecules, and anticipates how the synergy of computer and wet-lab experiments will enrich our understanding of splicing regulation/deregulation mechanisms. This information will aid future structure-based drug-discovery efforts aimed to expand the currently limited portfolio of selective splicing-modulators.
... Mutation of Tet2 alone was associated with splicing abnormalities, including both U2 and U12 IR, and in some genes we observed additivity between Tet2 and Zrsr2 mutations to promote increased IR (Fig. 7E). These data are consistent with existing evidence that mutations in TET2 and other epigenetic modifiers can themselves promote splicing abnormalities and, in concert with splicing factor mutations, produce cooperative effects on splicing and hematopoiesis (33,(45)(46)(47). In vivo, Zrsr2/Tet2-mutant pDCs had impaired activation after systemic exposure to R848 (Fig. 7F). ...
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive, male-biased (>3:1 M:F) hematologic malignancy in which some patients have bone marrow involvement at diagnosis (50%) and most have tumor formation in the skin (~90%), often preceding marrow disease. The prognosis is poor (median survival of 12-24 months) and there is unmet need for biological insight. TET2, ASXL1, and RNA splicing genes (SRSF2, SF3B1, and ZRSR2) are recurrently mutated in BPDCN. The X chromosome gene ZRSR2 was the most frequently mutated spliceosome gene reported in a prior BPDCN cohort (7 of 24, 29.2%; Taylor, ASH 2013). Our goal was to define the functional consequences of ZRSR2 mutations in BPDCN.
First, we confirmed the frequency of ZRSR2 mutations in a larger cohort from the US and Europe; we found ZRSR2 mutations in 24 of 93 (25.8%). Notably, ZRSR2 mutations were almost exclusively in males (23/73 males vs 1/20 females, P=0.019). Next, we compared the global mutation pattern to 30 predefined signatures from >7000 cancers in COSMIC. Analysis of all somatic single nucleotide variants in 11 tumor-normal pairs using whole exome sequencing (tumor was sorted BPDCN cells from marrow) revealed that BPDCN had an ultraviolet (UV)-induced mutation signature (score >0.25 in 6/11 or 55%; Figure 1A). For comparison, we detected the UV signature in melanoma but not in AML from The Cancer Genome Atlas. These data suggest that mutations acquired in the skin stage of BPDCN evolution are retained in subsequent leukemic disease.
Next, we performed RNA-sequencing from sorted BPDCN and normal plasmacytoid dendritic cells (pDCs). Differentially expressed genes between BPDCN and pDCs (BCL2, MYB, IRF4, CEP70, IGLL1, GZMB) were similar to those that distinguish BPDCNs from pDCs by bulk and single cell RNA-sequencing. By gene set enrichment analysis (GSEA), BPDCNs were enriched for overexpression of MYC/E2F targets and PI3K/AKT/MTORC1 signaling pathway-associated genes. BPDCNs transcriptomes were also enriched for gene sets associated with RNA splicing machinery and RNA nonsense mediated decay (NMD).
To link RNA splicing with functional consequences of ZRSR2 mutations, we generated ZRSR2-knockout BPDCN cells (CAL1) using CRISPR/Cas9. This models primary tumors because ZRSR2-mutant BPDCNs have complete loss of ZRSR2 protein. Activation marker (CD80) upregulation and type 1 interferon secretion after Toll-like receptor (TLR) stimulation with lipopolysaccharide (LPS) or R848 were reduced in ZRSR2-deficient cells. We found similar defective cytokine production in stimulated primary BPDCN cells compared to normal pDCs. After activation, normal pDCs undergo apoptosis in a negative feedback process. In contrast, ZRSR2-knockout, but not control cells, were protected from TLR activation-induced apoptosis. Reexpression of wild-type ZRSR2 in knockout cells restored activation-induced apoptosis (Figure 1B).
These data suggested that ZRSR2-mutant BPDCNs have defects downstream of TLR stimulation. By RNA-sequencing, we found that IRF7 mRNA was mis-spliced in all ZRSR2- (2/2), SRSF2- (4/4), and SF3B1- (1/1) mutant BPDCNs compared to those with no mutated splicing gene (4/4). IRF7 (interferon regulatory factor 7) is a transcription factor activated by TLR signaling that is important for pDC activation and apoptosis. The IRF7 mRNA transcript contains a "weak intron" (intron 4) that is subject to intron retention, which leads to NMD and reduced IRF7 protein level in stimulated dendritic cells (Luke, Mol Cell 2019). IRF7 intron 4 was mis-spliced in ZRSR2-, SRSF2-, and SF3B1-mutant BPDCNs. ZRSR2-knockout CAL1 cells had severely impaired ability to upregulate IRF7 after LPS stimulation, which was partially rescued by reepxression of wild-type ZRSR2 (Figure 1C). Expression of constitutively activated IRF7 inhibited growth of both ZRSR2-knockout and control cells, confirming that the inability to activate IRF7 is important for the effect of ZRSR2 loss on TLR agonist-induced growth inhibition.
In conclusion, male-biased ZRSR2 mutations are frequent in BPDCN and impair pDC activation and apoptosis, at least in part via TLR-IRF7. These data may explain why BPDCNs have an impaired activation state (Bierd, BCJ 2019). They also suggest that splicing factor mutations affect cell type-specific pathways to promote transformation, underscoring the importance of studying cancer genes in relevant contexts.
Figure
Disclosures
Griffin: Moderna Therapeutics: Consultancy. Ghandi:Monte Rosa Therapeutics: Consultancy; Cambridge Data Science LLC: Current Employment, Current equity holder in private company. Seiler:Remix Therapeutics: Current Employment. Konopleva:Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Eli Lilly: Research Funding; Genentech: Consultancy, Research Funding; Agios: Research Funding; Rafael Pharmaceutical: Research Funding; Sanofi: Research Funding; AbbVie: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding; Calithera: Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; Cellectis: Research Funding; Ablynx: Research Funding; Kisoji: Consultancy; Stemline Therapeutics: Consultancy, Research Funding. Pemmaraju:Cellectis: Research Funding; Daiichi Sankyo: Research Funding; DAVA Oncology: Honoraria; Plexxikon: Research Funding; Blueprint Medicines: Honoraria; Incyte Corporation: Honoraria; SagerStrong Foundation: Other: Grant Support; Celgene: Honoraria; Pacylex Pharmaceuticals: Consultancy; Affymetrix: Other: Grant Support, Research Funding; MustangBio: Honoraria; Roche Diagnostics: Honoraria; Novartis: Honoraria, Research Funding; LFB Biotechnologies: Honoraria; Stemline Therapeutics: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Samus Therapeutics: Research Funding. Abdel-Wahab:H3 Biomedicine Inc.: Consultancy, Research Funding; Merck: Consultancy; Janssen: Consultancy; Envisagenics Inc.: Current equity holder in private company. Lane:Qiagen: Consultancy; Abbvie: Research Funding; Stemline Therapeutics: Research Funding.
... Mutation of Tet2 alone was associated with splicing abnormalities, including both U2 and U12 IR, and in some genes we observed additivity between Tet2 and Zrsr2 mutations to promote increased IR (Fig. 7E). These data are consistent with existing evidence that mutations in TET2 and other epigenetic modifiers can themselves promote splicing abnormalities and, in concert with splicing factor mutations, produce cooperative effects on splicing and hematopoiesis (33,(45)(46)(47). In vivo, Zrsr2/Tet2-mutant pDCs had impaired activation after systemic exposure to R848 (Fig. 7F). ...
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive malignancy previously known as blastic natural killer cell lymphoma, CD4+CD56+ hematodermic neoplasm, or agranular CD4+ NK cell leukemia. BPDCN has recently been classified as the malignant counterpart of plasmacytoid dendritic cells (pDCs), the most common dendritic cell subset in peripheral blood. Clinical outcomes in BPDCN are poor, with median survival of less than 12 months. The pathogenesis and genetic changes associated with pDC transformation are largely unknown, and the optimal treatment for this disease is unclear. Loss of the CDKN2A locus on 9p21 is the most common copy number alteration, and the sole targeted sequencing effort reported to date focused only on somatic mutations in TP53 and TET2 (Jardin et al. Br J Haem 2011). The goal of this study was to characterize the genetics of BPDCN by next-generation sequencing of all exons of 219 genes known to be recurrently mutated in hematologic malignancies. We sequenced a discovery cohort of seven adult patients with BPDCN, and confirmed the somatic status of single nucleotide variants (SNVs) and insertion-deletions (InDels) not present in dbSNP using paired germline tissue where available. All cases met World Health Organization criteria for pathological diagnosis of BPDCN. We confirmed the presence of TET2 (4 of 7 patients) and TP53 (1 of 7) mutations in BPDCN, and noted that the overall mutational spectrum was overlapping with previously sequenced hematologic neoplasms. Specifically, we observed mutations in ASXL1 (in 2 patients: K586* and an InDel causing a frameshift at S795), IDH2 (R140Q), KRAS (G13D), ABL1 (T315I), ARID1A (R1721*), GNA13 (E313*), U2AF1 (Q157L), and SRSF2 (P95L) that have been reported in myeloid and mature B cell neoplasms. Also of interest was an IRF8 R404W mutation; IRF8 is a transcription factor that drives pDC development and germline loss of IRF8 in humans is associated with dendritic cell deficiency. The most striking finding was the presence of premature stop, frame shift, and splice site mutations in the splicing factor ZRSR2 on chromosome Xp22.1 in 4 of 7 (57.1%) BPDCNs. ZRSR2 mutations were present in 71-84% of sequence reads at their respective locations, consistent with homo/hemizygous alterations in a dominant clone. Xenografting of one BPDCN that harbored a ZRSR2 premature stop mutation resulted in leukemia engraftment that retained the ZRSR2 mutation. From a validation cohort of 32 additional adult and pediatric BPDCNs, ZRSR2 mutations were present in 11 cases, for a total of 15 of 39 (38.5%) patients. Ten of 15 mutations were premature stop, frame shift, or splice site, while the remaining were missense variants. ZRSR2 is recurrently mutated in MDS and AML but at a much lower frequency (1-5%), and ZRSR2 mutations have not been described as characteristic of any other malignancy. Thus, ZRSR2 mutations are approximately 10-fold more prevalent among BPDCNs as compared to MDS or AML, indicating a unique association between BDPCN and ZRSR2 mutation. BPDCN is predominantly a disease of the male sex, both in previous studies and in our cohort (28 males among 36 patients, 77.8%). In a prior report (Yoshida et al. Nature 2012), 12 of 12 cases of MDS with ZRSR2 nonsense and frame shift mutations were male. All 10 cases of BPDCN with ZRSR2 nonsense, frame shift, and splice site mutations in our cohort were male (P=0.076 by two-sided Fisher’s exact test). Thus, we hypothesize that BPDCN may be more common in males because of a gene dosage effect related to the chr.X location of ZRSR2. There was also a trend toward an association between ZRSR2 loss-of-function mutation and age ≥65 (P=0.068). There was no statistically significant difference in overall survival between patients with and without mutations in ZRSR2, although we were limited by the small cohort size and the heterogeneity of therapies received. We conclude that loss of ZRSR2 function is specifically associated with pDC transformation and leukemogenesis, as well as male sex and older age. Further studies to confirm these findings in additional cohorts and define the mechanism linking ZRSR2 mutation with BPDCN are underway.
Disclosures
DeAngelo: Ariad, Novartis, BMS: Consultancy. Neuberg:Synta Pharmaceuticals: Trust owns stock; I am a Trustee Other.
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive leukemia of plasmacytoid dendritic cells (pDC). BPDCN occurs at least three times more frequently in men than in women, but the reasons for this sex bias are unknown. Here, studying genomics of primary BPDCN and modeling disease-associated mutations, we link acquired alterations in RNA splicing to abnormal pDC development and inflammatory response through Toll-like receptors. Loss-of-function mutations in ZRSR2, an X chromosome gene encoding a splicing factor, are enriched in BPDCN, and nearly all mutations occur in males. ZRSR2 mutation impairs pDC activation and apoptosis after inflammatory stimuli, associated with intron retention and inability to upregulate the transcription factor IRF7. In vivo, BPDCN-associated mutations promote pDC expansion and signatures of decreased activation. These data support a model in which male-biased mutations in hematopoietic progenitors alter pDC function and confer protection from apoptosis, which may impair immunity and predispose to leukemic transformation.
Significance
Sex bias in cancer is well recognized, but the underlying mechanisms are incompletely defined. We connect X chromosome mutations in ZRSR2 to an extremely male-predominant leukemia. Aberrant RNA splicing induced by ZRSR2 mutation impairs dendritic cell inflammatory signaling, interferon production, and apoptosis, revealing a sex- and lineage-related tumor suppressor pathway.
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