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1
Warnasooriya, Chandani, Callen F. Feeney, Kholiswa M. Laird, Dmitri N. Ermolenko, and Clara L. Kielkopf. "A splice site-sensing conformational switch in U2AF2 is modulated by U2AF1 and its recurrent myelodysplasia-associated mutation." Nucleic Acids Research 48, no. 10 (April 28, 2020): 5695–709. http://dx.doi.org/10.1093/nar/gkaa293.
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Abstract An essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3′ splice sites. U2AF1 frequently acquires an S34F-encoding mutation among patients with myelodysplastic syndromes (MDS). The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Förster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.
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Biancon, Giulia, Poorval Joshi, Torben Hunck, Yimeng Gao, Valentina Botti, Ashley Qin, Mukhtar Sadykov, et al. "U2AF1 Driver Mutations in Hematopoietic Disorders Alter but Do Not Abrogate RNA Binding and Enlighten Structural Dependencies of the U2AF-RNA Complex." Blood 134, Supplement_1 (November 13, 2019): 1230. http://dx.doi.org/10.1182/blood-2019-130759.
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Among genetic aberrations responsible for ineffective hematopoiesis in myelodysplastic syndromes (MDS) and acute myeloid leukemia, somatic mutations in splicing factors such as U2AF1 are of significant interest as they are recurrent, mutually exclusive and early occurring. U2AF1 participates in mRNA splicing through the recognition of the intronic 3' splice site, forming the U2AF complex as a heterodimer with U2AF2. Heterozygous hotspot mutations at S34 or Q157, in the two U2AF1 zinc fingers respectively, result in sequence dependent aberrant splicing, suggestive of altered RNA binding. The mechanism by which these mutations alter U2AF1-U2AF2-RNA interactions has to date not been elucidated, yet understanding the structure-function relationship is critical to devise novel therapeutic strategies that either aim to correct or exploit RNA binding and splicing defects. To address this issue, we profiled the transcriptome of HEL erythroleukemic cell lines expressing wild-type (WT) and mutant U2AF1. U2AF1 S34F and Q157R mutants induced widespread alterations in splicing patterns of 3250 and 1791 genes respectively, with an overlap of 23.8% genes. On the other hand, we observed only minor alterations in gene expression levels. Meta-analysis and comparison with published RNA sequencing datasets on U2AF1 mutants revealed both conserved and unique splicing changes, with a strong enrichment for genes involved in cell cycle (P=6.7E-15) and DNA repair (P=2.6E-5). Confirming previous literature, U2AF1 S34F preferentially leads to the exclusion of exons preceded by 3' splice sites bearing an intronic UAG motif, while U2AF1 Q157R preferentially excludes exons starting with the AGA motif (Figure 1A). Collectively, the S34F mutation has a stronger effect on splicing, ultimately decreasing the global translation state of cells. To understand how mutations eventually result in the observed splicing alterations, we also profiled with unprecedented resolution the RNA interactome of the physiological and pathological U2AF heterodimer. We first performed enhanced crosslinking immunoprecipitation (eCLIP) on U2AF1 WT, U2AF1 mutants and U2AF2. Comparison of U2AF1 and U2AF2 binding profiles revealed a high degree of similarity, suggesting that they mostly bind to RNA as a tight dimer. Only by performing fractionated eCLIP on U2AF1 we were able to isolate, at the molecular level, the individual contributions of the U2AF components in the recognition of the 3' splice site. In particular, we deconvolved the U2AF2 signal, insisting on the polypyrimidine region, and the U2AF1 signal, peaking on the AG dinucleotide at the intronic end (Figure 1B). Importantly, the S34F mutant displays an aberrant binding profile, with a specific peak on the nucleotide in position -3, matching the sequence specificity previously observed in aberrant splicing events (Figure 1A-B). Systematic analysis of bound junctions suggests a complex model where the S34F mutation does not simply abrogate the ability of U2AF1 to bind splicing junctions ending with the UAG sequence, but rather alters the conformation of the U2AF complex bound to RNA, resulting in a differential ability to effectively recruit the U2 complex. To confirm this model, we identified and validated a set of gain-of-function splice junctions in genes contributing to hemopoiesis and cell cycle, characterized by increased binding of U2AF1 S34F mutant and parallel decreased binding of U2AF2. In summary, we identified novel RNA sequence and structure determinants of U2AF complex conformation, uncovered by the binding alterations induced by the U2AF1 S34F mutation. Our data further dissect the complexities of post-transcriptional regulation and provide the basis for development of U2AF directed cancer therapies. Disclosures Hunck: B**hringer-Ingelheim Foundation.: Other: During my stay in the Halene Lab I was founded by an MD fellowship.
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Yang, Chao-Yie, Xinrui Yuan, Mona Kazemi Sabzvar, Arda Durmaz, Krishnapriya Chinnaswamy, Jeanne Stuckey, Seth J. Corey, Jaroslaw P. Maciejewski, and Valeria Visconte. "Small-Molecule U2 Auxiliary Factor Homology Motif (UHM) Domain Inhibitors Cause Splicing Pattern Changes in U2AF1 Mutant Leukemia Cells and Induce Sub-G1 Cell Cycle Arrest." Blood 142, Supplement 1 (November 28, 2023): 115. http://dx.doi.org/10.1182/blood-2023-190365.
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Mutations in factors involved in the spliceosomal pathway have emerged to be an important contributor to the development of myeloid neoplasms including MDS and secondary AML (sAML). A set of frequently mutated genes encoding RNA splicing factors including SF3B1, U2AF1, SRSF2, and ZRSR2 or haploinsufficiency of LUC7L2 have beenreported, but pharmacological intervention targeting these genes remains limited and under-developed. Combination of mutations of these splicing factors are rarely found in patients indicating that multiple defects in a spliceosome pathway may be deleterious. Heterozygous mutation in U2AF1 found in MDS leads to the acquisition of the neomorphic mutant U2AF1 (U2AF1 wt) that relies on wild-type U2AF1 (U2AF1 wt) to survive and proliferate. To support our hypothesis, we undertook a discovery campaign to identify small-molecule compounds targeting U2AF1. By recognizing the challenge of discovering compounds selectively targeting the mutant zinc-finger domains in U2AF1 found clinically, we devised a tractable intervention strategy to inhibit the UHM domain of U2AF1 (U2AF1-UHM). U2AF1-UHM binds with U2AF2 to form a protein complex (U2AF1/U2AF2) that recognizes the 3' splice site in mRNA processing. U2AF1-UHM inhibitors may decrease the binding between U2AF1 and U2AF2 and abrogate U2AF1/U2AF2 functions to change expression patterns of protein isoforms. To identify hit compounds for development, we performed screening of ~3000 fragment molecules against U2AF1-UHM using the thermal shift assay. Two of the top hits were derivatives of the same chemical scaffold. We verified that both hits inhibited the binding of U2AF1-UHM and U2AF2-ULM (U2AF ligand motifs) in our Homogenous Time-Resolved Fluorescence (HTRF) assay. We next evaluated the top hit, SF-1-8, in our K562-U2AF1 S34F mutant cell line and obtained an IC50 at the micromolar range. In contrast, SF-1-8 had no activity in K562-U2AF1 wt cell line and bone marrow cells obtained from healthy individuals. Based on SF-1-8, we performed chemical modifications to develop structure-activity relationship (SAR) of SF-1-8 and obtained SF-1-50 that was two-fold more potent than SF-1-8. To assess the selectivity of SF-1-8 and analogs to other UHM containing proteins, we further determined the IC50 values of our SF-1-8 analogs to RBM39-UHM, SPF45-UHM, and PUF60-UHM to construct the selectivity profiles of our compounds. The selectivity profiles of SF-1-8 and SF-1-50 showed they were selectively more effective to U2AF1-UHM than RBM39-UHM, SPF45-UHM, and PUF60-UHM. We then studied pathways impacted by these inhibitors in K562-U2AF1 S34F cells by performing RNA-seq in K562-U2AF1 S34F cells treated with SF-1-8 at 5 uM and control. We found that 36 and 63 genes were significantly (p <0.002) up- and down-regulated respectively by SF-1-8 in K562-U2AF1 S34F cells. Downregulated genes included CBL, CBLL1, and a subset of collagen genes ( COL1, COL3, COL5) and upregulated genes included ATF3 and BCL2. When analyzing the transcript changes affected by SF-1-8, we identified substantial protein isoform changes in genes involved with proteosome, endocytosis, apoptosis, extracellular matrix/cell adhesion, histones, and stress response. Primary effects from changes of protein isoform patterns ameliorated the restoration of the trans-Golgi network ( ERGIC3, COPB2), secretory pathway ( RUSC1, AP4E1) and impairment of clathrin mediated endocytosis and endosome-lysosome transport ( CBL, PICALM, VAMP7, ASAP1). NKM-1 cell line was previously characterized to carry U2AF1 mutation. We also found SF-1-50 caused accumulation of sub-G1 cells in NKM-1 cells in a dose-dependent manner. In summary, SF-1-8 caused disruption of extra- and intracellular protein transport in K562-U2AF1 S34F cells and represented a new class of small-molecule inhibitors to target U2AF1-UHM. Further optimization of this class of compounds will allow us to develop effective chemical probes for study in the U2af1 murine models and assess the potential of U2AF1 as a therapeutic target in MDS and sAML.
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Biancon, Giulia, Poorval Joshi, Torben Hunck, Josh Zimmer, Yimeng Gao, Martin Machyna, Valentina Botti, et al. "High-Resolution Binding Atlas of U2AF1 Mutants Uncovers New Complexity in Splicing Alterations and Kinetics in Myeloid Malignancies." Blood 136, Supplement 1 (November 5, 2020): 3–4. http://dx.doi.org/10.1182/blood-2020-142854.
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Spliceosomal gene mutations function as drivers of hematologic malignancies and other cancers with an occurrence of more than 50% in myelodysplastic syndromes and secondary acute myeloid leukemia. Hotspot mutations S34F and Q157R in the two zinc finger domains of the splicing factor U2AF1, forming with U2AF2 the U2AF complex that recognizes 3' splice site (3'SS) of U2 introns, alter exon usage in a sequence-specific manner. However, how pathological U2AF1 mutations disrupt ordered splicing, from binding to recruitment of cooperating RNA binding proteins and ultimately splicing kinetics, is still not known at the molecular level. To obtain unique insights into in vivo RNA binding mechanisms, we performed fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA sequencing (freCLIP-seq) on human erythroleukemia (HEL) cells expressing wild-type (WT) or mutant (S34F, Q157R) U2AF1. Transcriptome-wide analysis of binding at single nucleotide resolution in light and heavy fractions, corresponding respectively to U2AF1 only and U2AF complex, allowed to: i) deconvolute U2AF1 signal peaking over the AG dinucleotide at the intronic end of the 3'SS region, and U2AF2 signal sitting on the adjacent polypyrimidine tract (PPT); ii) identify conformational changes in mutant U2AF1 binding with a novel peak in position -3 of the 3'SS region for S34F and in position +1 for Q157R. Alternative splicing analysis on newly collected RNA-seq data showed that less included exons present higher probability of U in position -3 for S34F and A in position +1 for Q157R, pinpointing a match with nucleotide positions affected by aberrant binding in freCLIP-seq. In both U2AF1 mutants, aberrant binding and splicing mechanisms affected genes involved in mRNA processing and transport (P-value<0.01) highlighting the involvement of U2AF1 mutations in the dysregulation of these key biological processes. We then performed a combined analysis of differential binding and aberrant splicing in U2AF1 mutants vs WT considering 4 categories: ">inclusion/>binding", "<inclusion/<binding", "<inclusion/>binding", ">inclusion/<binding". The first 2 categories correspond to the loss-of-function binding model suggested in literature to explain the splicing outcome of U2AF1 mutations: U2AF1 mutants bind certain splicing junctions with less affinity, leading to their exclusion. The last 2 categories represent a non-canonical gain-of-function model where increased mutant U2AF1 binding results in the impairment of the splicing machinery. Surprisingly, while Q157R mainly exhibited a loss-of-function mechanism where ineffective splicing is related to absence of binding ("<inclusion/<binding", 51.1%), S34F mostly follows a gain-of-function mechanism affecting splicing progression by an increased, yet skewed, binding. The most represented category was, indeed, "<inclusion/>binding" with 123 events out of 309 (Figure 1A). Moreover, differential binding was not dependent on specific nucleotides in position -3: events characterized by increased S34F binding (Figure 1B, top), as well as events characterized by decreased S34F binding (Figure 1B, bottom), showed -3U in less included exons or -3C in more included exons. The binding analysis across the 4 categories showed that increased S34F binding was associated with reduced U2AF2 binding (Figure 1C, top) particularly in less included exons, while decreased S34F binding was associated with increased U2AF2 binding (Figure 1C, bottom) especially in more included exons. Finally, analysis of branch point and splice junction features revealed that PPT strength influences the splicing outcome with "<inclusion/>binding" category characterized by a weak PPT that impairs U2AF2 binding in the presence of skewed U2AF1 S34F binding (Figure 1D). Additionally, transcriptome-wide RNA kinetics analysis by TimeLapse-seq demonstrated that U2AF1 S34F and Q157R, compared to WT, globally decrease synthesis of aberrantly spliced and bound 3'SS regions. Of note, this shutdown effect was particularly evident in the downstream exons pointing towards a role of U2AF1 mutations in a widespread alteration of RNA synthesis and splicing dynamics. Collectively, these results disclose novel molecular mechanisms of pathogenic U2AF1 mutations in the context of myeloid malignancies and provide the basis for the development of effective U2AF1 directed therapeutic strategies. Disclosures Hunck: Boehringer Ingelheim Fonds: Other: MD Fellowship.
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Yuan, Xinrui, Mona Kazemi Sabzvar, Amol D. Patil, Arda Durmaz, Jaroslaw Maciejewski, Valeria Visconte, and Chao-Yie Yang. "Targeting Poly(U) Binding Splicing Factor 60 (PUF60): A Small-Molecule Inhibitor Shows Anti-Leukemic Activity and Impacts Cell Cycle in Leukemia Models." Blood 144, Supplement 1 (November 5, 2024): 7470. https://doi.org/10.1182/blood-2024-210241.
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The emerging role of altered RNA splicing in the development of myeloid neoplasms including MDS and AML has received wide attention. Facilitating genes, including the frequently mutated splicing factors, SF3B1, U2AF1, and SRSF2, have been reported to play a key role in leukemogenesis. Pharmacological intervention targeting cells harboring these mutations has been pursued, but limited agents have been reported. Rare events of more than one hit in splicing factors were found in MDS and AML patients' samples. This suggests that inhibiting another target in the splicing process may overcome the threshold of survival in these vulnerable cells carrying splicing factor mutations and induce toxicity. The concept has been demonstrated in the case of leukemia cells carrying U2AF1 mutations being vulnerable to inhibitors modulating wild-type SF3B1 function. The primary function of U2AF1 is to form a complex with U2AF2 for 3‘ splice site (SS) definition in RNA splicing and lower frequency U2AF2 mutations are also detected in MDS patients, underlying the significance of dysfunctional 3‘ SS definition as a potential facilitator of leukemogenesis. Here, we hypothesize that compounds inhibiting proteins participating in 3' SS selection may disrupt isoform patterns in leukemia cells. These agents may act alone or serve as a second hit in the RNA processing to invoke additional vulnerabilities to leukemia cells carrying the most frequent splicing factor mutations. Definition of 3' SS by U2AF2/U2AF1 is mediated by the recognition of the polypyridine tract (PPT) by U2AF2 and the binding of U2AF1 to the conserved AG dinucleotides at the 3' SS. Like U2AF2, PUF60 contains two zinc-finger domains, recognizing the polyuridine tract preceding 3' SS, and a U2 homology motif (UHM) domain that engages in protein-protein interaction with partner proteins including U2AF2, SF1, and SF3B1. Different from the U2AF1/U2AF2 complex, PUF60 does not have a protein domain to bind to the exon intron boundary junction at the 3' SS. PUF60 has been reported to enhance the splicing activity of U2AF1/U2AF2, but also directly regulates alternative splicing of a subset of genes. A recent finding indicated that knockout of PUF60 leads to alternative splicing switching of CDC25 from isoform A to isoform C to induce cell cycle arrest at G2/M in solid tumors. Leukemia patients carrying PUF60 overexpression have also been found to have less progression free survival (https://ualcan.path.uab.edu/cgi-bin/TCGA-survival1.pl?genenam=PUF60&ctype=LAML) compared to patients with low expression of PUF60. To support our hypothesis, we undertook an inhibitor development campaign to discover small-molecule compounds selectively targeting PUF60. Based on a relatively less selective and weak inhibitor to the UHM domain, SF-153, we conducted chemical modifications and obtained a PUF60 inhibitor, SF2-69, with >15-fold selectivity against U2AF1, RBM39, SPF45, and U2AF2. We found SF2-69 had an IC50 value of ~3 mM in NKM-1 and K562 leukemia cell lines. In the cell cycle analysis, we found SF2-69 increased S and subG1 cell population in NKM-1 at 24h, but induced G1 arrest in K562 at 48h in a dose dependent manner. In comparison, E7820, an RBM39 degrader, increased G2/M phase in both NKM-1 and K562. RNA-seq analysis of NKM-1 treated with SF2-69 after 24h revealed that SF2-69 upregulated a set of genes participating in cholesterol biosynthesis, NME1-NME2 (a tumor suppressor kinase) and downregulated PLK1, a kinase that phosphorylates and activates CDC25C in G2/M transition. Because K562 is p53-null and NKM-1 has wild-type p53, SF2-69 likely induced p53-dependent and p53-independent responses in NKM-1 and K562 cells, respectively. In summary, SF2-69 is a new selective PUF60 inhibitor which disrupts cell cycle progression in leukemia cell lines by modulating p53-mediated responses. Upregulation of cholesterol biosynthesis may be a feedback loop to counteract the inhibition of PUF60 by SF2-69. Further optimization of SF2-69 will allow us to develop more effective and selective chemical probes for studying the role of PUF60 overexpression in leukemia and the potential use of PUF60 inhibitors in MDS and AML cells carrying splicing factor mutations.
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Akef, Abdalla, Kathy McGraw, Steven D. Cappell, and Daniel R. Larson. "Ribosome biogenesis is a downstream effector of the oncogenic U2AF1-S34F mutation." PLOS Biology 18, no. 11 (November 2, 2020): e3000920. http://dx.doi.org/10.1371/journal.pbio.3000920.
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U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) forms a heterodimeric complex with U2AF2 that is primarily responsible for 3ʹ splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). The U2AF heterodimer also has a noncanonical function as a translational regulator. Here, we report that the U2AF1-S34F mutation results in specific misregulation of the translation initiation and ribosome biogenesis machinery. The net result is an increase in mRNA translation at the single-cell level. Among the translationally up-regulated targets of U2AF1-S34F is Nucleophosmin 1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of the U2AF1-S34F mutant cells and causes ribosomal RNA (rRNA) processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1, and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation that recapitulates translational misregulation in myeloid disease.
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Maji, Debanjana, Eliezra Glasser, Steven Henderson, Justin Galardi, Mary J. Pulvino, Jermaine L. Jenkins, and Clara L. Kielkopf. "Representative cancer-associated U2AF2 mutations alter RNA interactions and splicing." Journal of Biological Chemistry 295, no. 50 (October 5, 2020): 17148–57. http://dx.doi.org/10.1074/jbc.ra120.015339.
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High-throughput sequencing of hematologic malignancies and other cancers has revealed recurrent mis-sense mutations of genes encoding pre-mRNA splicing factors. The essential splicing factor U2AF2 recognizes a polypyrimidine-tract splice-site signal and initiates spliceosome assembly. Here, we investigate representative, acquired U2AF2 mutations, namely N196K or G301D amino acid substitutions associated with leukemia or solid tumors, respectively. We determined crystal structures of the wild-type (WT) compared with N196K- or G301D-substituted U2AF2 proteins, each bound to a prototypical AdML polypyrimidine tract, at 1.5, 1.4, or 1.7 Å resolutions. The N196K residue appears to stabilize the open conformation of U2AF2 with an inter-RNA recognition motif hydrogen bond, in agreement with an increased apparent RNA-binding affinity of the N196K-substituted protein. The G301D residue remains in a similar position as the WT residue, where unfavorable proximity to the RNA phosphodiester could explain the decreased RNA-binding affinity of the G301D-substituted protein. We found that expression of the G301D-substituted U2AF2 protein reduces splicing of a minigene transcript carrying prototypical splice sites. We further show that expression of either N196K- or G301D-substituted U2AF2 can subtly alter splicing of representative endogenous transcripts, despite the presence of endogenous, WT U2AF2 such as would be present in cancer cells. Altogether, our results demonstrate that acquired U2AF2 mutations such as N196K and G301D are capable of dysregulating gene expression for neoplastic transformation.
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Grammatikakis, Ioannis, Amit Behera, Corrine Corrina R Hartford, Erica C. Pehrsson, XiaoLing Li, Yongmei Zhao, Biraj Shrethsa, et al. "Abstract A013: Molecular mechanisms of intron retention in Long Non-Coding RNAs." Molecular Cancer Therapeutics 23, no. 11_Supplement (November 14, 2024): A013. http://dx.doi.org/10.1158/1538-8514.rnadrivers24-a013.
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Abstract Intron retention (IR) is a form of alternative splicing in which an intron that should be spliced out from a precursor transcript, is retained in the mature RNA after the splicing is completed. Although there is emerging evidence of widespread IR in protein-coding genes and long noncoding RNAs (lncRNAs), the underlying molecular mechanisms remain largely unclear. Here, we report the discovery of novel transcripts from the p53-induced lncRNA PURPL, in which intron 2 is retained. To determine the molecular mechanism(s) of IR in PURPL, we conducted a CRISPR-based screen in 3 different cell lines. For this purpose, we utilized a genome-wide guide RNA library expressing a reporter minigene containing the sequence of PURPL intron 2 and its flanking exons. Considering the Percent Intron Retention as readout, we unexpectedly identified proteins of the basal splicing machinery as potential promoting factors of PURPL IR, including the U2-Auxiliary Factor 2 (U2AF2) splicing factor which was one of the top hits of the screen. We next analyzed ENCODE eCLIP-seq datasets to identify RNA binding proteins that could regulate intron 2 and decided to focus on U2AF2 which showed the highest number of binding sites and strongest eCLIP signals within PURPL intron 2 sequence. We validated the effect of U2AF2 by knocking it down, confirming that U2AF2 is a positive regulator of PURPL intron 2 retention as opposed to its canonical function. We also identified the RNA Binding Protein SON as a splicing regulator that acts as an antagonist of U2AF2 in regulating PURPL intron 2 retention. To determine the global impact of U2AF2 knockdown, we performed Iso-Seq and RNA-seq upon U2AF2 depletion. We found that although U2AF2 predominantly acts to promote the splicing of introns in most cellular transcripts, it promotes intron retention in a subset of transcripts. One of these targets is MALAT1, a lncRNA known to play role in splicing by interacting with splicing factors in nuclear speckles. Interestingly, U2AF2 depletion results in MALAT1 exclusion from nuclear speckles. We are currently in the process of characterizing the effect of U2AF2 in the function of the two lncRNAs. These data provide mechanistic insights on PURPL and MALAT1 splicing and function and reveal a previously unrecognized non-canonical function of U2AF2 in promoting intron retention. Citation Format: Ioannis Grammatikakis, Amit Behera, Corrine Corrina R Hartford, Erica C Pehrsson, XiaoLing Li, Yongmei Zhao, Biraj Shrethsa, Tayvia Brownmiller, Natasha J Caplen, Kannanganattu V Prasanth, Thomas Gonatopoulos-Pournatzis, Ashish Lal. Molecular mechanisms of intron retention in Long Non-Coding RNAs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A013.
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Kang, Hyun-Seo, Carolina Sánchez-Rico, Stefanie Ebersberger, F. X. Reymond Sutandy, Anke Busch, Thomas Welte, Ralf Stehle, et al. "An autoinhibitory intramolecular interaction proof-reads RNA recognition by the essential splicing factor U2AF2." Proceedings of the National Academy of Sciences 117, no. 13 (March 18, 2020): 7140–49. http://dx.doi.org/10.1073/pnas.1913483117.
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The recognition of cis-regulatory RNA motifs in human transcripts by RNA binding proteins (RBPs) is essential for gene regulation. The molecular features that determine RBP specificity are often poorly understood. Here, we combined NMR structural biology with high-throughput iCLIP approaches to identify a regulatory mechanism for U2AF2 RNA recognition. We found that the intrinsically disordered linker region connecting the two RNA recognition motif (RRM) domains of U2AF2 mediates autoinhibitory intramolecular interactions to reduce nonproductive binding to weak Py-tract RNAs. This proofreading favors binding of U2AF2 at stronger Py-tracts, as required to define 3′ splice sites at early stages of spliceosome assembly. Mutations that impair the linker autoinhibition enhance the affinity for weak Py-tracts result in promiscuous binding of U2AF2 along mRNAs and impact on splicing fidelity. Our findings highlight an important role of intrinsically disordered linkers to modulate RNA interactions of multidomain RBPs.
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Biancon, Giulia, Poorval Joshi, Joshua T. Zimmer, Torben Hunck, Yimeng Gao, Mark D. Lessard, Edward Courchaine, et al. "U2AF1 Mutations Enhance Stress Granule Response in Myeloid Malignancies." Blood 138, Supplement 1 (November 5, 2021): 321. http://dx.doi.org/10.1182/blood-2021-149618.
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Abstract Somatic mutations in splicing factor genes are drivers of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The splicing factors U2AF1 and U2AF2 form the U2AF heterodimer that is critical in the 3' splice site (3'SS) recognition and in the recruitment of U2 small nuclear ribonucleoproteins for the activation of the spliceosome complex. U2AF1 carries hotspot mutations in its two RNA binding motifs; yet the molecular mechanisms affecting the splicing process and promoting clonal advantage remain unclear, albeit necessary to develop effective targeted therapies. We applied a multi-omics approach comparing the activities of two U2AF1 mutants (S34F and Q157R) in MDS/AML cell lines and primary samples. Using a novel approach of fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA-sequencing (freCLIP-seq), we mapped transcriptome-wide binding at nucleotide resolution and we identified conformational changes in mutant vs wild-type U2AF1 binding. Specifically, we observed an emergent peak in position -3 of the 3'SS for the S34F mutant and in position +1 for the Q157R mutant, matching the critical positions observed by differential splicing analysis on RNA-seq data. Altered U2AF1-RNA binding compromised U2AF2-RNA interactions, resulting predominantly in exon exclusion and intron retention. Combined binding-splicing analysis showed that while the Q157R mutant mainly exhibits loss of binding, the S34F mutant follows a gain-of-binding pattern, where splicing progression appears impaired by increased mutant binding. Functional analysis of genes affected by both binding and splicing alterations revealed that U2AF1 mutants alter RNA granule biology, affecting in particular stress granule-enriched transcripts and proteins. Stress granules are membrane-less cytoplasmic assemblies of RNAs and RNA binding proteins that improve cellular adaptation in response to stress conditions. Increased stress granule formation has been linked to tumorigenesis as a strategy exploited by cancer cells to regulate gene expression and signal transduction, enhancing their fitness under stress. To probe how aberrant binding and splicing of stress granule components affected stress granule biology, we assessed stress granule formation in U2AF1 mutant vs wild-type cells at steady state and after stress induction with sodium arsenite treatment. Immunofluorescent staining followed by confocal imaging demonstrated that U2AF1 mutations enhance stress granule formation upon arsenite stress in both cell lines and primary samples. RNA turnover analysis by TimeLapse-seq confirmed that U2AF1 S34F and Q157R mutations promote stability/synthesis of transcripts that are enriched in stress granules and determine degradation/shutdown of transcripts that are depleted in stress granules, providing a molecular explanation for the increase in stress granules observed by imaging. Finally, we were able to corroborate our observations by single-cell RNA-seq in patient-derived U2AF1-mutant MDS blasts, establishing the causal link between U2AF1 mutations and upregulation of stress granule components. Collectively, this multi-omics analysis identified biological processes directly influenced by mutant U2AF1 binding and splicing, laying the foundation for a new paradigm where splicing factor mutations enhance stress granule formation by acting on the availability of their RNA and protein components. The enhanced formation of stress granules potentially fosters the stress adaptation of U2AF1-mutant cells, contributing to their clonal advantage in MDS/AML. Stress granule perturbations may therefore represent a novel therapeutic vulnerability in U2AF1-mutant MDS/AML patients and possibly in patients carrying other splicing factor mutations. Disclosures Hunck: Boehringer Ingelheim: Other: Fellowship.
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Adamia, Sophia, Christian Bach, Patrick M. Pilarski, Martha Wadleigh, David P. Steensma, Gabriela Motyckova, Daniel J. DeAngelo, Richard M. Stone, Daniel G. Tenen, and James D. Griffin. "Aberrant Splicing In Patients With AML Is Associated With Over- Expression Of Specific Splicing Factors." Blood 122, no. 21 (November 15, 2013): 3749. http://dx.doi.org/10.1182/blood.v122.21.3749.3749.
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Abstract Pre-mRNA processing, referred to as alternative RNA splicing (AS), is a critical determinant of protein diversity. AS produces multiple transcripts and, as a result, multiple proteins from a single gene. Recently, frequent mutations in splicing factor genes have been reported in myelodysplasia (MDS) and chronic lymphocytic leukemia (CLL), and less frequently in AML. However, in previous studies, we found that aberrant patterns of splicing were common in cells from 66 AML patients compared to 10 normal donors (NDs), more common than could be explained by mutations in splicing factor genes. Here, we evaluated expression levels of 24 core splicing factors (SFs), which are involved in splicing reactions, in cells from 30 AML patients compared to 10 NDs. Among these SFs we identified three, U2AF2, PTBP, and SFRS12 that were significantly (P<0.001) upregulated in AML samples. Of the 30 patients 65% , 75%, and 25% had increased levels of U2AF2, PTBP, and SFRS12, respectively. We detected increased expression of U2AF2 and PTBP at the protein level in several patient samples as well where sufficient protein was available for immunoblotting. Expression of the SFRS12 protein was not evaluated. We asked if overexpression of U2AF2 or PTBP altered splicing by overexpressing cDNAs encoding these splicing factors in HEK293T cells. To test this hypothesis HEK293T cells stably expressing U2AF2 and PTBPs were transfected with mini-genes derived from two genes, FLT3 and CD13, which we previously found commonly mis-spliced in AML. Overexpression of PTBP but not U2AF2 induced FLT3 and CD13 mini-gene splicing. This study suggest that overexpression of the PTBPs induce FLT3 and CD13 splicing. We also evaluated growth and cell proliferation effects of the U2AF2 and PTBPs. The HEK293T cells expressing U2AF2 formed colonies 11 days later after seeding, while cells expressing PTBPs formed foci in 5 days. Interestingly, overexpression of PTBP, and also U2AF2 to a lesser extent, accelerated growth and colony formation of HEK293T cells in MethoCult. These results suggest that PTBPs may increase cell proliferation and enhance anchorage-independent cell growth. Currently, we are investigating growth and cell proliferation effects of the U2AF2 and PTBPs in AML patient samples and cell lines, and in murine leukemia models. In a preliminary study, PTBP was overexpressed in murine marrow LSK cells, with or without the MLL-AF9 oncogene, and these cells were transplanted into irradiated recipient mice. Reconstitution was monitored measuring donor-specific myeloid and lymphocyte populations. Overexpression of PTPB by itself did not result in the development of leukemia, but was associated with shortened survival in mice co-expressing MLL-AF9 in stem cells. Using RNA-seq analysis we are evaluating effects of PTBP overexpression on genome-wide splicing in the samples obtained from in vivo studies. Results obtained from this will be presented. Taken together, the data suggest that overexpression of splicing factor genes may result in altered splicing, and can accelerate malignant cell growth in vitro and possibly in vivo. Disclosures: Griffin: Novartis: Research Funding; Janssen: Research Funding.
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Zhang, Xiaoqing, Matias A. Bustos, Rebecca Gross, Romela Irene Ramos, Teh-Ling Takeshima, Gordon B. Mills, Qiang Yu, and Dave S. Hoon. "Abstract 3233: Interleukin enhancer-binding factor 2 amplification controls mRNA stability and enhances DNA damage response in metastatic melanoma." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3233. http://dx.doi.org/10.1158/1538-7445.am2022-3233.
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Abstract Background: 1q21.3 amplification is frequently observed in metastatic melanoma. Interleukin enhancer-binding factor 2 (ILF2) is in the 1q21.3 amplified region. The functional role of ILF2 as well as its contribution in promoting an aggressive phenotype in cutaneous metastatic melanoma is unknown. Methods: In silico analyses were performed using the TCGA SKCM dataset with clinical annotations. The results were validated in three melanoma cohort microarray datasets from the GEO database. Melanoma tissues mRNA levels were assessed by RNA in situ hybridization and protein levels were analyzed by immunohistochemistry. Four stable metastatic melanoma cell lines were established for in vitro ILF2 functional characterization. Results: Our results showed that the ILF2 copy number variation (CNV) is positively correlated with ILF2 mRNA expression (r=0.68, p<0.0001). Additionally, ILF2 mRNA expression significantly increased with melanoma progression (p<0.0001) and is significantly associated with poor overall survival in metastatic melanoma patients (p=0.026). In vitro, ILF2 overexpression (ILF2-OV) enhances cellular proliferation, whereas ILF2 knockdown decreases cellular proliferation by blocking the cell cycle. Mechanistically, we demonstrated the interaction between ILF2 and the splicing factor U2AF2. Reinforcing their functions as a complex, the U2AF2 knockdown reverses the cellular proliferation effects mediated by ILF2-OV. The levels of U2AF2 mRNA and protein levels increased during melanoma progression. Stage IIIB-C melanoma patients with high ILF2-U2AF2 expression showed significantly shorter overall survival (p=0.024). Functional assays showed that enhanced ILF2/U2AF2 expression promotes a more efficient DNA-damage repair by increasing RAD50 and ATM mRNA expression. Paradoxically, metastatic melanoma cell lines with ILF2-OV were more sensitive to ATM inhibitors. Conclusion: Our study uncovered that the ILF2 and U2AF2 complex may have biological implications in controlling the mRNA stability of RAD50 and ATM in metastatic melanoma. Clinically, ILF2 amplification is associated with melanoma progression, triggers a functional downstream pathway in metastatic melanoma that promotes DNA damage repair, and increases the sensitivity to ATM inhibitors. Citation Format: Xiaoqing Zhang, Matias A. Bustos, Rebecca Gross, Romela Irene Ramos, Teh-Ling Takeshima, Gordon B. Mills, Qiang Yu, Dave S. Hoon. Interleukin enhancer-binding factor 2 amplification controls mRNA stability and enhances DNA damage response in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3233.
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Lin, Chien-Ling, Allison J. Taggart, Kian Huat Lim, Kamil J. Cygan, Luciana Ferraris, Robbert Creton, Yen-Tsung Huang, and William G. Fairbrother. "RNA structure replaces the need for U2AF2 in splicing." Genome Research 26, no. 1 (November 13, 2015): 12–23. http://dx.doi.org/10.1101/gr.181008.114.
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Okeyo-Owuor, Theresa, Brian S. White, Dipika Mohan, Malachi Griffith, Matthew J. Walter, and Timothy Graubert. "Allele-Specific Effects Of U2AF1 Mutations On Alternative Splicing." Blood 122, no. 21 (November 15, 2013): 2748. http://dx.doi.org/10.1182/blood.v122.21.2748.2748.
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Abstract Four independent groups, including ours, recently discovered recurrent mutations in core components of the pre-mRNA splicing complex (the “spliceosome”) in myelodysplastic syndrome (MDS) patient samples using next-generation sequencing approaches. We previously identified mutations in U2AF1 affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of patients with de novo MDS. Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how these mutations affect splicing or contribute to MDS. To determine the effects of S34 and Q157 mutations on U2AF1 splicing activity, we utilized GH1 and FMR1 minigene assays, which we have previously used to assess alternative splicing. Here, we report that recurrent mutations in U2AF1 have allele-specific effects on alternative splicing for both GH1 and FMR1 minigenes when transiently transfected in 293T cells. The U2AF1 S34F mutant allele yielded the most significant increase in alternative splicing activity for both GH1 and FMR1 (GH1: p<0.01; FMR1: p<0.001; n>3 biological replicates), compared to wildtype (WT). The S34Y allele modestly enhanced expression of the alternative isoforms for both minigenes (GH1: p<0.05; FMR1: p<0.01). Conversely, we saw a reduction in expression of the alternative isoforms in cells expressing the Q157R and Q157P alleles compared with WT (p<0.01) or the S34 alleles (p<0.001). In cells expressing the S34F/Q157R mutant (in which both the S34F and Q157R mutations occur on one allele, discovered in one patient with MDS), GH1 and FMR1 splicing was indistinguishable from WT. We then assessed the effect of the S34F mutation on U2AF1 sub-cellular localization and protein interaction within the spliceosome. Using fluorescence immunocytochemistry in transfected 293T cells, we found that S34F U2AF1 localized normally within the nuclear speckles and co-localized with U2AF2 and SRSF2. However, immunoprecipitation of epitope-tagged constructs transfected in K562 cells demonstrated that S34F U2AF1 had reduced direct interaction with U2AF2, compared to WT U2AF1. We next performed RNA-seq to comprehensively determine the effects of the S34F mutation on pre-mRNA splicing and gene expression. We transfected primary human CD34+ cells (derived from cord blood) with constructs expressing either WT or S34F U2AF1 (3 biological replicates for each condition) and sorted for transfected (GFP positive) cells after 24 hours. cDNA libraries depleted of ribosomal RNA were sequenced on the Illumina platform. Transient overexpression of WT vs. S34F U2AF1 did not significantly alter global gene expression profiles (by Cufflnks FPKM) in an unsupervised analysis. Analyses of splice junctions using ALEXA-Seq and EdgeR revealed significant differences in the abundance of known splice junctions (S34F>WT: 104, S34F<WT: 188; FDR<0.05, fold change >4). We also detected expression differences at novel splice junctions resulting from use of either known or novel alternative splice acceptor and/or donor sites (S34F>WT: 135, S34F<WT: 229). Both the known and novel junctions reflect alterations in canonical splicing, exon skipping or retention, intron retention and cryptic site usage. We selected junctions with higher expression in S34F samples for validation in CD34+ cells using RT-PCR and gel electrophoresis. 16/27 genes with known junctions and 10/10 with novel junctions validated (p<0.05; n=3 biological replicates). 9 of the validated changes were tested in primary clinical samples by RT-PCR (n=6 MDS bone marrow with S34F U2AF1 vs. n=6 MDS controls with no U2AF1 splicing mutations) and 5 were confirmed (p<0.01), including alternative splicing involving known (DEK, SERPIN8B, KIAA1033, IFI44) and novel (ABI1) junctions. We conclude that the S34F mutation affects U2AF1 function, leading to aberrant alternative splicing of target genes. Whether alternative splicing perturbs hematopoiesis and contributes to MDS pathogenesis is not yet known. Disclosures: No relevant conflicts of interest to declare.
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Schott, Geraldine, Gaddiel Galarza-Muñoz, Noe Trevino, Xiaoting Chen, Matthew T. Weirauch, Simon G. Gregory, Shelton S. Bradrick, and Mariano A. Garcia-Blanco. "U2AF2 binds IL7R exon 6 ectopically and represses its inclusion." RNA 27, no. 5 (February 10, 2021): 571–83. http://dx.doi.org/10.1261/rna.078279.120.
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Love, Sierra L., and Aaron A. Hoskins. "Stuck on UUUU: New splicing inhibitors enhance U2AF2-RNA binding." Cell Chemical Biology 28, no. 8 (August 2021): 1106–8. http://dx.doi.org/10.1016/j.chembiol.2021.07.021.
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Howard, Jonathan M., Hai Lin, Andrew J. Wallace, Garam Kim, Jolene M. Draper, Maximilian Haeussler, Sol Katzman, Masoud Toloue, Yunlong Liu, and Jeremy R. Sanford. "HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo." Genome Research 28, no. 5 (April 12, 2018): 689–98. http://dx.doi.org/10.1101/gr.229062.117.
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Kralovicova, Jana, Ivana Borovska, Monika Kubickova, Peter J. Lukavsky, and Igor Vorechovsky. "Cancer-Associated Substitutions in RNA Recognition Motifs of PUF60 and U2AF65 Reveal Residues Required for Correct Folding and 3′ Splice-Site Selection." Cancers 12, no. 7 (July 11, 2020): 1865. http://dx.doi.org/10.3390/cancers12071865.
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U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3′ splice sites (3′ss). Both proteins preferentially bind uridine-rich sequences upstream of 3′ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3′ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3′ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3′ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity.
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Fang, Li, Ting Ye, and Yanmei An. "Circular RNA FOXP1 Induced by ZNF263 Upregulates U2AF2 Expression to Accelerate Renal Cell Carcinoma Tumorigenesis and Warburg Effect through Sponging miR-423-5p." Journal of Immunology Research 2021 (September 3, 2021): 1–16. http://dx.doi.org/10.1155/2021/8050993.
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Renal cell carcinoma (RCC), as one of the most common malignant tumors in the urinary system, is featured with high morbidity and mortality. Although the improvement of clinical intervention, such as surgery technology, chemotherapy, and radiotherapy, has been made, the outcomes of RCC patients are still poor. Novel targets for RCC treatment are urgently needed. Recently, circRNA has been in-depth studied and is considered as a promising direction for gene target therapy. In this study, we explored the function of circFOXP1 in RCC progression and its underlying mechanisms. Firstly, we demonstrated the characterization and expression of circFOXP1 in RCC tissues and cells. Next, by conducting a serial experiment, we found that downregulated circFOXP1 inhibited cell proliferation, migration, invasion, and the Warburg effect. Next, our experiments found that circFOXP1 upregulated U2AF2 expression via sponging miR-423-5p in RCC cells. Moreover, we found that ZNF263 induced circFOXP1 expression in RCC cells. To sum up, our study partially demonstrated that the novel ZNF263/circFOXP1/miR-423-5p/U2AF2 axis has a role in RCC progression. Our results might provide a new direction for RCC therapeutic target exploring.
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Braish, Julie, Prithviraj Bose, Naveen Pemmaraju, Sherry Pierce, Koji Sasaki, Keyur P. Patel, Hagop Kantarjian, and Lucia Masarova. "Deeper Insight into Splicing Mutations in Myelofibrosis." Blood 142, Supplement 1 (November 28, 2023): 4580. http://dx.doi.org/10.1182/blood-2023-189168.
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Introduction: Prognostically adverse mutations in patients with primary myelofibrosis (MF) include ASXL1, EZH2 IDH1, IDH2, and two types of splicing mutations U2AF1 Q157 and SRSF2 (as in MIPSS70.v2 score). The other splicing mutations (SPM), such as SF3B1 or ZRSR2, do not appear to have impact on prognosis. In patients with MF secondary to polycythemia vera or essential thrombocytopenia (PPV-MF, PET-MF), the role of SPM is less known, and they are not included in the MYSEC-PM score. Objective: We aimed to evaluate the role of SPM on the outcome of patients with PPV/PET-MF, and their interplay with other HMR on prognosis of all MF patients from our center. Methods: We retrospectively reviewed medical charts of 114 patients with MF from our institution who had SPM on next generation sequencing (51+ gene myeloid panel). We assessed impact of high-risk SPM (HR_spl: U2AF1 Q157; SRSF2) and remaining SPM (low risk, LR_spl: SF3B1, PRPF40B, U2AF2, ZRSR2) on the outcome of MF patients. Separate analysis was carried per disease phenotype (MF vs PET/PPV-MF), and presence of the other high-risk molecular mutations (HMR: ASXL1, IDH1/2, EZH2). Descriptive statistics was used for demographic variables; Kaplan-Meier curve with log-rank test was used for overall survival (OS), calculated from the time of presentation. Results: Table summarizes clinical characteristics of all patients. The most common SPM were SRSF2 and SF3B1 (41% each), followed by U2AF1 (17.5%) and ZRSR2 (11%). 6 patients had more than one SPM. The distribution of SPM types did not statistically differ between PMF and PET/PPV-MF patients, albeit numerically, more patients with PMF had SRSF2 and PET/PPV-MF had SF3B1 (table). 77% of all patients had higher DIPSS score; 85% of PMF patients had higher MIPSS70.v2 score and 36% of PET/PPV-MF had higher MYSEC-PM. HMR mutations were detected in 77 (68%) patients: ASXL1 (65%), EZH2 (19%), IDH1/2 (16%). ASXL1 co-occurred more frequently with SF3B1 in PET/PPV-MF patients (75% vs 25%), and with SRSF2 (62% vs 38%) and U2AF1 (89% vs 11%) in PMF patients. The median OS (months [95% CI]) according to SPM: SRSF2, U2AF1, SF3B1 and others (U2AF2 in 2 patients, PRPF40B in 1 patient and ZRSR2 in 13 patients) was as follows: 46 [23-70], not reached, 130 [104-156], unreached, respectively. The median OS (months [95% CI]) per HR_spl (n = 61) and LR_spl (n = 67) was 51 [26-76] vs 130 [101-159], p < 0.01, HR 0.31, 95% CI 0.13-0.74. Sub-analysis for PMF (n = 86) and PET/PPV-MF (n = 28) showed comparable results, median OS for HR_spl and LR_spl in PMF of 51 vs 109 months and for PET/PPV-MF of 46 and 130 months, respectively. The median OS (months [95% CI] of HR_spl with and without HMR and LR_spl with and without HMR as shown in Figure was 46 [20-72] and 51 [21-81] vs 109 [0.5-221] and 130 [95-165], respectively. Two years survival for patients with HR_spl and LR_spl with and without HMR was at 69% vs 85% and 87% vs 95%, respectively. Median OS (months; 95% CI]) of each individual SPM without and with HMR was 51 [not estimated] and 46 [11-82] for SRSF2; unreached for all groups in U2AF1 and ZRSR2, respectively; 130 [98-161] and 109 [10-220] for SF3B1. Conclusions: Various SPM seem to have similar effect on survival of PET/PPV-MF and PMF patients. PET/PPV-MF patients have more low risk SPM (SF3B1) and the co-occurrence of HMR does not appear to impact the outcome. Among the high-risk SPM, SRSF2 has the worst prognostic role irrespective of concurrent HMR. Further validation of our data, including association with received therapy, is ongoing and will be presented at the conference.
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Douet-Guilbert, Nathalie, Benoît Soubise, Delphine G. Bernard, and Marie-Bérengère Troadec. "Cytogenetic and Genetic Abnormalities with Diagnostic Value in Myelodysplastic Syndromes (MDS): Focus on the Pre-Messenger RNA Splicing Process." Diagnostics 12, no. 7 (July 7, 2022): 1658. http://dx.doi.org/10.3390/diagnostics12071658.
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Myelodysplastic syndromes (MDS) are considered to be diseases associated with splicing defects. A large number of genes involved in the pre-messenger RNA splicing process are mutated in MDS. Deletion of 5q and 7q are of diagnostic value, and those chromosome regions bear the numbers of splicing genes potentially deleted in del(5q) and del(7q)/-7 MDS. In this review, we present the splicing genes already known or suspected to be implicated in MDS pathogenesis. First, we focus on the splicing genes located on chromosome 5 (HNRNPA0, RBM27, RBM22, SLU7, DDX41), chromosome 7 (LUC7L2), and on the SF3B1 gene since both chromosome aberrations and the SF3B1 mutation are the only genetic abnormalities in splicing genes with clear diagnostic values. Then, we present and discuss other splicing genes that are showing a prognostic interest (SRSF2, U2AF1, ZRSR2, U2AF2, and PRPF8). Finally, we discuss the haploinsufficiency of splicing genes, especially from chromosomes 5 and 7, the important amplifier process of splicing defects, and the cumulative and synergistic effect of splicing genes defects in the MDS pathogenesis. At the time, when many authors suggest including the sequencing of some splicing genes to improve the diagnosis and the prognosis of MDS, a better understanding of these cooperative defects is needed.
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Adamia, Sophia, Hervé Avet-Loiseau, Jana Jakubikova, Suzan Lazo-Kallanian, John Daley, Laurence Lode, Ilene Galinsky, Richard M. Stone, and James D. Griffin. "Genome-Wide Aberrant Splicing in Patients with Acute Myelold Leukemia (AML) Is Associated with Altered Expression of Splicing Factors." Blood 120, no. 21 (November 16, 2012): 652. http://dx.doi.org/10.1182/blood.v120.21.652.652.
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Abstract Abstract 652 Long-term survival of patients with acute myeloid leukemia (AML) is poor, and new forms of therapy are needed. Many genetic lesions have been identified and studied, and most patients have chromosome translocations or other mutations that promote self-renewal of leukemic stem cells, block differentiation, enhance growth, and block apoptosis. Only a few of these mutations result in druggable targets (e.g., PML-RARa, Kit, PDGFR, FLT3 for instance). In addition to genetic lesions, epigenetic abnormalities have been shown to be very common in AML, and provide opportunities for novel treatments. Using genome-wide approaches to identify alternative splicing, we have recently shown that AML cells have a high level of aberrantly regulated genome-wide alternative splicing (AS) as a frequent epigenetic event. By comparing samples from 62 AML patients with 10 normal donors (NDs) we identified 428 genes differentially spliced in AML. A list of differentially spliced genes includes 50 oncogenes and 52 tumor suppressor genes, as well as genes encoding proteins involved in cell proliferation and differentiation, and apoptosis. We evaluated splicing event frequency in AML compared to NDs and we observed that on average 527 (range 137–1657) genes were identified as differentially spliced in any given patient, out of 62 analyzed. Also, we found that any given differentially spliced gene, of the 3,108 detected, were spliced on average in 26 (range 1–54) AML patients. Thus, splicing aberrations are highly recurrent in AML patients. To identify the causes of aberrant splicing in AML, we evaluated transcript levels of the 24 major splicing factors (SFs) that are involved in the first and second splicing transesterification reactions. These splicing factors are important proteins involved in spliceosomalassembly. Expression levels of these SFs were evaluated in 20 AML patients exhibiting high levels of AS. Quantitative RT-PCR analysis showed significant (up to 30 fold) upregulation of U2AF2 (P<2.00E-07), PTBP (P=3.00E-04) and SFRS12 (P=0.002) SF transcript levels in AML patient samples compared to CD34+ cells from NDs. In preliminary studies, we also detected elevated expression of U2AF2 and PTBP proteins in several patient samples. These results suggest the intriguing possibility that aberrant splicing in AML may be the result of alterations of these SFs. To test this hypothesis we generated stably transfected HEK293 cell lines overexpressing U2AF2 or PTBP. We have developed a synthetic semi-quantitative splicing assay to evaluate the effects of overexpression of these SFs. We have obtained a minigene cassette of the p53 inducible PIG3 gene based on previous splicing studies. The minigene cassette was cloned between RFP (red fluorescent prtoein) and GFP (green fluorescent prtoein) in such a way that translation of the normally spliced transcript results in expression of RFP and GFP, while aberrant splicing results in the expression of RFP only. Production of a similar minigene cassette that includes exons/introns of a gene that is subjected to aberrant splicing in AML (NOTCH2, FLT3 and CD13) is in progress. In studies, completed so far, with the PIG3 minigene cassette construct transiently transfected into the HEK293 cells lines, overexpression of PTBP increased aberrant splicing of the PIG3 minigene. Similar studies testing the effects of elevated levels of U2AF2 and PTBP on NOTCH2 and other genes misspliced in AML (such as FLT3 and CD13) will be presented. Our results indicate that aberrant splicing could be an important event in AML, and development of an in vitro, synthetic splicing assay will enable us to better understand the underlying causes of this process in AML. Disclosures: No relevant conflicts of interest to declare.
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Zhang, P., S. Feng, G. Liu, H. Wang, A. Fu, H. Zhu, Q. Ren, et al. "CD82 suppresses CD44 alternative splicing-dependent melanoma metastasis by mediating U2AF2 ubiquitination and degradation." Oncogene 35, no. 38 (April 4, 2016): 5056–69. http://dx.doi.org/10.1038/onc.2016.67.
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Glasser, Eliezra, Anant A. Agrawal, Jermaine L. Jenkins, and Clara L. Kielkopf. "Cancer-Associated Mutations Mapped on High-Resolution Structures of the U2AF2 RNA Recognition Motifs." Biochemistry 56, no. 36 (September 2017): 4757–61. http://dx.doi.org/10.1021/acs.biochem.7b00551.
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Kielkopf, Clara L., Callen F. Feeney, Rakesh Chatrikhi, Andrew MacRae, Gerorgios Alachouzos, Zackary Falls, Kholiswa M. Laird, et al. "A synthetic molecule stalls pre-mRNA splicing by enhancing cancer-relevant U2AF2–RNA complexes." Acta Crystallographica Section A Foundations and Advances 75, a1 (July 20, 2019): a103. http://dx.doi.org/10.1107/s0108767319098969.
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Whisenant, Thomas C. "Gene expression profiling of U2AF2 dependent RNA-protein interactions during CD4 + T cell activation." Genomics Data 11 (March 2017): 77–80. http://dx.doi.org/10.1016/j.gdata.2016.12.006.
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Kashlakova, A. I., E. N. Parovichnikova, B. V. Biderman, Y. V. Sidorova, Y. A. Chabaeva, V. V. Troitskaya, I. A. Lukianova, et al. "Next-generation sequencing-based molecular genetic profiling in adults with acute myeloid leukaemia." Russian journal of hematology and transfusiology 65, no. 4 (December 10, 2020): 444–59. http://dx.doi.org/10.35754/0234-5730-2020-65-4-444-459.
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Introduction. Acute myeloid leukaemia (AML) is associated with multiple driver mutations, which prognostic value remains understudied.Aim. Assessment of the frequency of mutations in various genes and their impact on acute myeloid leukaemia outcome in adults.Materials and methods. The study included 90 adult patients with newly diagnosed AML; 76 were aged under 60, 14 were 60 and more years old. Patients under 60 had chemotherapy (CT) “7+3” as induction, the elder cohort had variant low-dose CT with hypomethylating agents. The molecular genetic status of patients was determined using next-generation sequencing; the in-house gene panel included ASXL1, BCOR, DNMT3, FLT3, IDH1, IDH2, PIGA, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53 and U2AF2.Results. Nucleotide substitutions were identified in genes DNMT3, TET2, TP53, SETBP1, BCOR, RUNX1, IDH2, IDH1, FLT3, U2AF2, SF3B1 in 57.8 % of the patients (n = 52), with 17.8 % (n = 16) having compound mutations in two or three genes. Treatment efficacy and long-term outcomes were assessed against age, ELN-2017 risk groups and mutations in genes TP53, RUNX1, IDH1, IDH2 and DNMT3. In the long term, a reliable variation was revealed in the overall survival (OS) rate with respect to mutations in genes TP53 and RUNX1. Patients with mutant TP53 had 30 % OS, those with the intact gene — 53.4 % (p = 0.0037). Similar results were obtained with RUNX1: mutations marked 20 % OS, intact patients had 54% OS (p = 0.0466).Conclusion. Mutations in genes FLT3-ITD, NPM1 and CEBPA are proxy to AML. However, a more accurate prognosis and optimal choice of therapy require detailed molecular profiling due to genetic heterogeneity of AML patients.
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Galardi, Justin W., Victoria N. Bela, Nazish N. Jeffery, Eliezra Glasser, Sarah Loerch, Jermaine L. Jenkins, Mary J. Pulvino, and Clara L. Kielkopf. "A UHM-ULM interface contributes to the pre-mRNA splicing functions of U2AF2 and SF3B1." Biophysical Journal 121, no. 3 (February 2022): 451a. http://dx.doi.org/10.1016/j.bpj.2021.11.513.
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Maji, Debanjana, Eliezra Glasser, Jermaine L. Jenkins, and Clara L. Kielkopf. "Cancer-associated mutations of the pre-mRNA splicing factor U2AF2 alter splice site signal recognition." Acta Crystallographica Section A Foundations and Advances 74, a1 (July 20, 2018): a232. http://dx.doi.org/10.1107/s0108767318097672.
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Chatrikhi, Rakesh, Callen F. Feeney, Mary J. Pulvino, Georgios Alachouzos, Andrew J. MacRae, Zackary Falls, Sumit Rai, et al. "A synthetic small molecule stalls pre-mRNA splicing by promoting an early-stage U2AF2-RNA complex." Cell Chemical Biology 28, no. 8 (August 2021): 1145–57. http://dx.doi.org/10.1016/j.chembiol.2021.02.007.
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Butler, Emily G., Debanjana Maji, Mary J. Pulvino, Jermaine L. Jenkins, and Clara L. Kielkopf. "Representative cancer-associated U2AF2 mutations occurring at the inter-RRM interface alter RNA interactions and splicing." Biophysical Journal 121, no. 3 (February 2022): 480a. http://dx.doi.org/10.1016/j.bpj.2021.11.388.
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Sutandy, F. X. Reymond, Stefanie Ebersberger, Lu Huang, Anke Busch, Maximilian Bach, Hyun-Seo Kang, Jörg Fallmann, et al. "In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors." Genome Research 28, no. 5 (April 11, 2018): 699–713. http://dx.doi.org/10.1101/gr.229757.117.
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Mohamed, Aminetou Mint, Morgan Thenoz, Catherine Koering, Pierre Mallinjoud, Didier Auboeuf, Francoise Solly, Meyling Cheok, et al. "DEK and WT1 Affect Alternative Splicing of Genes Involved in Hematopoietic Cell Lineage and Resistance to Chemotherapy in Acute Myeloid Leukemia Cells." Blood 120, no. 21 (November 16, 2012): 2392. http://dx.doi.org/10.1182/blood.v120.21.2392.2392.
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Abstract Abstract 2392 In humans, the majority of all protein-coding transcripts contain introns that are removed by mRNA splicing carried out by spliceosomes. Mutations in the spliceosome machinery have recently been identified using whole-exome/genome technologies in myelodysplastic syndromes (MDS) and in acute myeloid leukemia (AML). In MDS the frequency of somatic spliceosomal mutations (SSM) range from 1–3% for U2AF1 in RARS/RCMD-RS to more than 70% for SF3B1 in ARSI. These values are significantly lower in AML whereas AML cells cumulate numerous splicing defects. Beside SSMs, one can propose that alternative splicing (AS) might be disturbed by other processes such as abnormal protein-protein interactions. DEK and WT1 are 2 oncogenes overexpressed in most patients with AML. They physiologically influence AS through physical interactions with the heterodimer U2AF1/U2AF2 involved in the recognition of splice acceptor site by the splicing machinery. It is therefore possible that the leukemogenic overexpression of DEK or WT1 might deregulate AS in AML cells, even in the absence of SSM. Here we show that DEK and WT1 affect AS in AML cells. Exon expression profiling was performed in triplicate with MOLM13, KASUMI and KG1 AML cells stably knocked down or not for DEK and WT1 through shRNA. The efficiency of shRNA-mediated silencing was confirmed by western blot and total RNA was analyzed using the Exon microarray platform GeneChip Human Exon 1.0 ST (Affymetrix). Microarray data were cross-compared between cell lines and only statistically significant modifications (p<0.05) shared by MOLM13, KASUMI and KG1 cells were selected. DEK and WT1 knock-down induced the transcriptional deregulation of 1613 (813 up) and 3280 (998 up) genes in AML cell lines, respectively. AS events were selected and annotated with fasterDB database (http://fasterdb.com/faster/home.pl) for genes displaying either no or low (<2) differential transcription. With this approach, differential expression of DEK coincided with changes in 1049 AS events over 934 genes. Those were distributed in 4 alternative acceptor sites (ACC), 222 first exons (AFE), 257 last exons (ALE), 539 spliced exon (ASE), 6 deletions (DEL), 21 donor sites (DON). Differential expression of WT1 led to modifying 1371 alternative splicing entities over 1198 genes. Those were distributed in 6 ACC, 385 AFE, 343 ALE, 590 ASE, 13 DEL, and 34 DON. Genes with AS events were then sorted based on gene function with DAVID bioinformatics resources version 6.7 (http://david.abcc.ncifcrf.gov/). The results indicated that, in both DEK- and WT1-dependent assays, a large subset of genes were related to hematopoietic cell lineage followed by other functional categories such as calcium signaling, ATP-binding cassette (ABC) transporters, and focal adhesion pathways that have been previously reported to be affected in AML cells and involved in resistance to chemotherapy. Differential expression of WT1 modified AS of CD3E, CD9, CSF1R, CSF3R, CR2, GP1BA, ITGA1, ITGA3, ITGB3, IL1A and IL6 while that of DEK led to modulate AS of CD19, CD1d, CD36, CD3G, CSF1R, CR2, ITGA1, ITGA2B, ITGA4, ITGA6, IL7, HLA-DRB5, and MME. Microarray data were validated by exon specific RT-PCR. Exon expression profiling of fresh AML bone marrow samples with or without U2AFs mutations and various levels of DEK or WT1 expression is currently in progress and will be presented. In conclusion in AML cells, DEK and WT1 overexpression affects AS of numerous key genes involved in hematologic differentiation, leukemogenesis and resistance to chemotherapy. These posttranscriptional effects occur in the absence of transcriptional change and therefore highlight hitherto unknown phenotypic alterations having putative diagnostic, prognostic and therapeutic interests. DEK and WT1 are overexpressed in more than 80% of AML and target AS via U2AFs that are mutated in only 0–8% of cases. Accordingly present results strongly suggest that other factors than SSM divert AS in AML. Disclosures: No relevant conflicts of interest to declare.
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Shepard, Jeremiah, Martin Reick, Sara Olson, and Brenton R. Graveley. "Characterization of U2AF6, a Splicing Factor Related to U2AF35." Molecular and Cellular Biology 22, no. 1 (January 1, 2002): 221–30. http://dx.doi.org/10.1128/mcb.22.1.221-230.2002.
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ABSTRACT The essential splicing factor U2AF (U2 auxiliary factor) is a heterodimer composed of 65-kDa (U2AF65) and 35-kDa (U2AF35) subunits. U2AF35 has multiple functions in pre-mRNA splicing. First, U2AF35 has been shown to function by directly interacting with the AG at the 3′ splice site. Second, U2AF35 is thought to play a role in the recruitment of U2AF65 by serine-arginine-rich (SR) proteins in enhancer-dependent splicing. It has been proposed that the physical interaction between the arginine-serine-rich (RS) domain of U2AF35 and SR proteins is important for this activity. However, other data suggest that this may not be the case. Here, we report the identification of a mammalian gene that encodes a 26-kDa protein bearing strong sequence similarity to U2AF35, designated U2AF26. The N-terminal 187 amino acids of U2AF35 and U2AF26 are nearly identical. However, the C-terminal domain of U2AF26 lacks many characteristics of the U2AF35 RS domain and, therefore, might be incapable of interacting with SR proteins. We show that U2AF26 can associate with U2AF65 and can functionally substitute for U2AF35 in both constitutive and enhancer-dependent splicing, demonstrating that the RS domain of the small U2AF subunit is not required for splicing enhancer function. Finally, we show that U2AF26 functions by enhancing the binding of U2AF65 to weak 3′ splice sites. These studies identify U2AF26 as a mammalian splicing factor and demonstrate that distinct U2AF complexes can participate in pre-mRNA splicing. Based on its sequence and functional similarity to U2AF35, U2AF26 may play a role in regulating alternative splicing.
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Gault, Christine M., Federico Martin, Wenbin Mei, Fang Bai, Joseph B. Black, W. Brad Barbazuk, and A. Mark Settles. "Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development." Proceedings of the National Academy of Sciences 114, no. 11 (February 27, 2017): E2195—E2204. http://dx.doi.org/10.1073/pnas.1616173114.
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RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants. The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2. ZRSR2 mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects. Maize rgh3 mutants have aberrant endosperm cell differentiation and proliferation. We found that most U12-type introns are retained or misspliced in rgh3. Genes affected in rgh3 and ZRSR2 mutants identify cell cycle and protein glycosylation as common pathways disrupted. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. The rgh3 mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates.
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Kohlmann, Alexander, Sandra Weissmann, Ulrike Schoeck, Vera Grossmann, Wolfgang Kern, Claudia Haferlach, Susanne Schnittger, and Torsten Haferlach. "First Results of a 31-Gene Panel Targeted to Investigate Myeloid Malignancies by Next-Generation Amplicon Deep-Sequencing." Blood 120, no. 21 (November 16, 2012): 883. http://dx.doi.org/10.1182/blood.v120.21.883.883.
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Abstract Abstract 883 Introduction: Massively parallel next-generation sequencing data have changed the landscape of molecular mutations in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The number of molecular markers used to characterize myeloid malignancies continues to constantly increase. As such, physicians and laboratories face a great unmet need to test panels of genes at a high level of sensitivity and throughput. Methods: We developed a sensitive next-generation deep-sequencing assay for routine diagnostics. In total, 31 genes with relevance in myeloid malignancies providing both favorable and adverse molecular prognostic information were chosen: ASXL1, BCOR, BCORL1, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KDM6A, KIT, KRAS, NOTCH1, NPM1, NRAS, PHF6, PRPF40B, PTPN11, RUNX1, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2, and ZRSR2. Targets of interest comprised either complete coding gene regions or hotspots. In summary, 1,375 amplicons were designed with a median length of 175 bp (range 109–194 bp), representing a total target sequence of 140.35 kb. The sequencing library was constructed starting off 2.2 μg genomic DNA per patient using a singleplex microdroplet-based assay (RainDance, Lexington, MA). Sequencing data was generated using the MiSeq instrument (Illumina, San Diego, CA) loading 4 patients per run. Using the 300 cycles sequencing-by-synthesis chemistry in median 6.099 millions of paired-end reads were generated per run. This resulted in a median coverage per gene of 1,766 reads (range 992-2,432). The total turn-around time of the analysis with this assay was less than 4 days. 49 clinically well-annotated patients harboring myeloid malignancies were analyzed during the evaluation phase. These included 9 acute myeloid leukemia (AML), 9 myelodysplastic syndrome (MDS), 13 chronic myelomonocytic leukemia (CMML), and 18 mixed phenotype acute leukemia, T/myeloid (MPAL-TM) cases. The median age was 69 years (range: 23 – 90 years). Results: In median, the coverage per amplicon harboring a mutation was 2,095-fold, thus enabling a sensitive detection of variants. In total, 146 mutations in 28 of the 31 genes were detected in 47/49 patients with a range of 1–7 mutations per case (median: 3). According to chromosome banding analysis 31/49 cases presented with a normal karyotype. In 30/31 cases with a normal karyotype at least one molecular mutation was observed using this screening panel. 42/146 mutations were detected with a clone size <20%, thus being detected only due to the higher sensitivity of this technique in comparison to direct capillary Sanger sequencing. In this cohort, the most frequently mutated genes were RUNX1 (14/49), DNMT3A (14/49), SRSF2 (11/49), ASXL1 (9/49), and TET2 (9/49). The mutation types comprised 97 missense, 17 duplications, 24 deletions, 5 insertions and 3 insertion/deletions alterations. Novel variants were verified using direct capillary Sanger sequencing (n=19) or sensitive amplicon deep-sequencing (n=65) (454 Life Sciences, Branford, CT). With respect to the technical limit of detecting larger insertions or deletions both a 27-bp insertion (RUNX1, p.Thr121delins9) and a 23-bp deletion variant (ASXL1, p.Glu635ArgfsX15) were successfully sequenced. The highest number of mutations was observed for CMML patients (mean of 3.6 per case; CMML vs remainder: P=0.201). Also, in CMML patients we observed the highest frequency of mutations in major splicing machinery genes such as SF1, SF3A1, SF3B1, SRSF2, U2AF1, U2AF2, and ZRSR2 (11/13 CMML, 84.6% vs 14/36 remainder cases, 38.9%; P<0.001). Importantly, a number of patients (39/49) was detected to harbor mutations in genes reported to be associated with decreased overall survival, both in AML (e.g. TP53, RUNX1, ASXL1, DNMT3A, IDH1, or TET2) and low- or intermediate-1 IPSS risk categories in MDS (e.g. ASXL1, EZH2, ETV6, RUNX1, TP53). As such, detecting these adverse somatic alterations may influence the course of therapy for these patients. Conclusion: We here demonstrated that microdroplet-based sample preparation enabled to target 31 candidate genes for next-generation sequencing in myeloid malignancies in a routine diagnostic environment. This approach provides the potential to screen for prognostically relevant mutations in a fast and comprehensive way providing actionable information suitable to guide therapy. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Schoeck:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
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Chen, Xiaofeng, Dongling Cai, Hao Li, Qipeng Wei, Xi Li, Zhuangxun Han, Jinjun Liang, et al. "Exosomal U2AF2 derived from human bone marrow mesenchymal stem cells attenuates the intervertebral disc degeneration through circ_0036763/miR-583/ACAN axis." Regenerative Therapy 25 (March 2024): 344–54. http://dx.doi.org/10.1016/j.reth.2024.01.006.
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Zhao, Yangjing, Weili Cai, Ye Hua, Xiaochen Yang, and Jingdong Zhou. "The Biological and Clinical Consequences of RNA Splicing Factor U2AF1 Mutation in Myeloid Malignancies." Cancers 14, no. 18 (September 10, 2022): 4406. http://dx.doi.org/10.3390/cancers14184406.
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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.
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Boddu, Prajwal, Abhishek Gupta, Rahul Roy, barbara De La Pena Avalos, Anne Olazabal Herrero, Joshua Zimmer, Matthew Simon, et al. "Impaired Early Spliceosome Complex Assembly Underlies Gene Body Elongation Transcription Defect in SF3B1K700E." Blood 142, Supplement 1 (November 28, 2023): 714. http://dx.doi.org/10.1182/blood-2023-187303.
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Introduction: Recurrent mutations in splicing factors (SFs) such as SF3B1, U2AF1 and SRSF2 are common in clonal myeloid disorders. These mutations are typically hemizygous, exclusive to each other, and non-synonymous, pointing to neomorphic functions. Molecular mechanisms connecting the mutations to disease pathogenesis remain unclear. Previous studies predominantly focused on the well-established role of splicing factors in pre-mRNA splicing, seeking to explain how mutations in these SFs leading to changes in the abundance of key regulatory genes through their effects on alternative splicing. These studies are limited by inconsistent and relatively low isoform changes across mutations and independent datasets. They also don't explain the mutual exclusivity of these mutations. In this study, we explored a novel mechanism that link SF mutations to disease biology: dysregulation of RNA Polymerase II (Pol2) transcriptional kinetics arising from impaired spliceosome assembly. Methods: We generated inducible isogenic K562 cell lines that express single mutant alleles (SF3B1 K700E and U2AF1 S34F,and corresponding wild-type controls). A novel combinatory approach of AAV-intron trap with CRISPR/Cas9 (Boddu et al, Comms Biol, 2021) was utilized for gene editing. RNAPII transcription kinetics changes were assessed, at an early post-induction time point of 4 days (Fig1A), using ChIP-seq, and nascent-transcriptome assays (including GRO-seq and transient transcriptome-time lapse sequencing or TT-TL seq). Spliceosome assembly kinetics were assayed with non-denaturing gels. Co-transcriptional splicing efficiency (CoSE) was assessed using nascent long-read sequencing (LRS) (Reimer, Mol Cell 2021). Key findings in K562 cell lines were validated in CD34+ progenitor populations from MDS-derived patient samples (and healthy volunteer controls). Results: ChIP-seq for Pol 2 (both total (Fig1B) and elongation-specific Ser2P (Fig1C) showed a redistribution of Pol2 into gene-bodies, suggesting a failure of Pol2 elongation in SF3B1 K700E. This Pol2 redistribution was dependent on gene length and intron number (long, multi-intron genes most affected). GRO-seq confirmed the ChIP-seq results (Fig1D). Additionally, spike-in normalized TT-seq showed significantly reduced nascent transcription in gene bodies in SF3B1 K700E (Fig 1E), consistent with a gene-body elongation rate defect. A similar redistribution of Pol2 into gene bodies was noted by ChIP-seq and TT-seq in U2AF1S34F, pointing to a convergent transcriptional response among distinct SF mutations. Genome-wide CoSE was noted to be lower in SF3B1 K700Eindicating an associated splicing assembly defect (Fig1F). Changes to transcription due to SF3B1 mutations were similarly observed, using low-input Pol2 CUT&RUN, in CD34+ progenitor populations isolated from human MDS patient samples (Fig1G). Of pathologic relevance, this transcriptional dysregulation led to generation of R-loops, DNA damage, and S-phase arrest. Given close coordination of splicing and transcription, and role of SF3B1 and U2AF1 in early spliceosome formation, we speculated if mutant SF3B1 leads to defective assembly of early spliceosomes. Compared to wild-type protein, SF3B1 K700E was noted to have significantly impaired interactions with U2AF1, U2AF2, HTATSF1 (Fig1H). We then determined how spliceosome assembly kinetics of SF3B1 K700E differed from those of SF3B1 WTusing an in vitro splicing assay. The transition from E- to A- complex intermediates was reduced in SF3B1 K700E(Fig1I). Furthermore, disrupting the early pre-spliceosome assembly (through loss of DDX46 or HTATSF1 using both siRNA and inducible shRNA (Fig1J) knockdown systems) resulted in a transcriptional response similar to SF3B1 K700E. Based on recent cryo-EM structures of Pol II-U1 snRNP interactions (Zhang et al, Science, 2021), we speculate that the impaired transition to A/B complex in SF3B1 K700E prevents the release of U1 snRNP and release of Pol2, leading to intron-predominant defective Pol II elongation (Fig1K). Conclusion: Here, we found that SF3B1 and U2AF1 mutations impair Pol2 transcription elongation along gene bodies, leading to pathologic R-loops and replication stress. This elongation results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1.
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Vandermeulen, Charlotte, Tina O’Grady, Jerome Wayet, Bartimee Galvan, Sibusiso Maseko, Majid Cherkaoui, Alice Desbuleux, et al. "The HTLV-1 viral oncoproteins Tax and HBZ reprogram the cellular mRNA splicing landscape." PLOS Pathogens 17, no. 9 (September 20, 2021): e1009919. http://dx.doi.org/10.1371/journal.ppat.1009919.
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Viral infections are known to hijack the transcription and translation of the host cell. However, the extent to which viral proteins coordinate these perturbations remains unclear. Here we used a model system, the human T-cell leukemia virus type 1 (HTLV-1), and systematically analyzed the transcriptome and interactome of key effectors oncoviral proteins Tax and HBZ. We showed that Tax and HBZ target distinct but also common transcription factors. Unexpectedly, we also uncovered a large set of interactions with RNA-binding proteins, including the U2 auxiliary factor large subunit (U2AF2), a key cellular regulator of pre-mRNA splicing. We discovered that Tax and HBZ perturb the splicing landscape by altering cassette exons in opposing manners, with Tax inducing exon inclusion while HBZ induces exon exclusion. Among Tax- and HBZ-dependent splicing changes, we identify events that are also altered in Adult T cell leukemia/lymphoma (ATLL) samples from two independent patient cohorts, and in well-known cancer census genes. Our interactome mapping approach, applicable to other viral oncogenes, has identified spliceosome perturbation as a novel mechanism coordinated by Tax and HBZ to reprogram the transcriptome.
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Ozygała, Aleksandra, Joanna Rokosz-Mierzwa, Paulina Widz, Paulina Skowera, Mateusz Wiliński, Borys Styka, and Monika Lejman. "Biological Markers of Myeloproliferative Neoplasms in Children, Adolescents and Young Adults." Cancers 16, no. 23 (December 8, 2024): 4114. https://doi.org/10.3390/cancers16234114.
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Myeloproliferative neoplasms (MPNs) are clonal hematopoietic cancers characterized by hyperproliferation of the myeloid lineages. These clonal marrow disorders are extremely rare in pediatric patients. MPN is reported to occur 100 times more frequently in adults, and thus research is primarily focused on this patient group. At present, modern diagnostic techniques, primarily genetic, facilitate the identification of the biology of these diseases. The key genes are JAK2, MPL, and CALR, namely, driver mutations, which are present in approximately 90% of patients with suspected MPN. Moreover, there are more than 20 other mutations that affect the development of these hematological malignancies, as evidenced by a review of the literature. The pathogenic mechanism of MPNs is characterized by the dysregulation of the JAK/STAT signaling pathway (JAK2, MPL, CALR), DNA methylation (TET2, DNMT3A, IDH1/2), chromatin structure (ASXL1, EZH2), and splicing (SF3B1, U2AF2, SRSF2). Although rare, myeloproliferative neoplasms can involve young patients and pose unique challenges for clinicians in diagnosis and therapy. The paper aims to review the biological markers of MPNs in pediatric populations—a particular group of patients that has been poorly studied due to the low frequency of MPN diagnosis.
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Soucek, Sharon, Yi Zeng, Deepti L. Bellur, Megan Bergkessel, Kevin J. Morris, Qiudong Deng, Duc Duong, et al. "Evolutionarily Conserved Polyadenosine RNA Binding Protein Nab2 Cooperates with Splicing Machinery To Regulate the Fate of Pre-mRNA." Molecular and Cellular Biology 36, no. 21 (August 15, 2016): 2697–714. http://dx.doi.org/10.1128/mcb.00402-16.
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Numerous RNA binding proteins are deposited onto an mRNA transcript to modulate posttranscriptional processing events ensuring proper mRNA maturation. Defining the interplay between RNA binding proteins that couple mRNA biogenesis events is crucial for understanding how gene expression is regulated. To explore how RNA binding proteins control mRNA processing, we investigated a role for the evolutionarily conserved polyadenosine RNA binding protein, Nab2, in mRNA maturation within the nucleus. This study reveals thatnab2mutant cells accumulate intron-containing pre-mRNAin vivo. We extend this analysis to identify genetic interactions between mutant alleles ofnab2and genes encoding a splicing factor,MUD2, and RNA exosome,RRP6, within vivoconsequences of altered pre-mRNA splicing and poly(A) tail length control. As further evidence linking Nab2 proteins to splicing, an unbiased proteomic analysis of vertebrate Nab2, ZC3H14, identifies physical interactions with numerous components of the spliceosome. We validated the interaction between ZC3H14 and U2AF2/U2AF65. Taking all the findings into consideration, we present a model where Nab2/ZC3H14 interacts with spliceosome components to allow proper coupling of splicing with subsequent mRNA processing steps contributing to a kinetic proofreading step that allows properly processed mRNA to exit the nucleus and escape Rrp6-dependent degradation.
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Wang, S. Y., J. L. Huo, Y. W. Miao, W. M. Cheng, and Y. Z. Zeng. "Complementary DNA cloning, sequence analysis, and tissue transcription profile of a novel U2AF2 gene from the Chinese Banna mini-pig inbred line." Genetics and Molecular Research 12, no. 2 (2013): 925–34. http://dx.doi.org/10.4238/2013.april.2.9.
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Li, Jing, Dongdong Cheng, Miaoxin Zhu, Huajian Yu, Zhen Pan, Lei Liu, Qin Geng, Hongyu Pan, Mingxia Yan, and Ming Yao. "OTUB2 stabilizes U2AF2 to promote the Warburg effect and tumorigenesis via the AKT/mTOR signaling pathway in non-small cell lung cancer." Theranostics 9, no. 1 (2019): 179–95. http://dx.doi.org/10.7150/thno.29545.
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Przychodzen, Bartlomiej P., Hideki Makishima, Andres Jerez, Richard A. Padgett, and Jaroslaw P. Maciejewski. "Downstream Consequences of U2AF1 Mutations in MDS Are a Result of a Specific Misplicing Patterns Due to Faulty Recognition of 3'acceptor Splice Site." Blood 120, no. 21 (November 16, 2012): 3517. http://dx.doi.org/10.1182/blood.v120.21.3517.3517.
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Abstract Abstract 3517 Recently, various spliceosomal mutations have been identified in myeloid neoplasms, in particular MDS, MDS/MPN and sAML evolving from these conditions. While functionally related, because they all affect the spliceosomal machinery, they are present in various clinical contexts. Thus, the downstream consequences of individual mutations may be diverse and involve likely specific oncogenic pathways. Because of an altered splicing pattern they create, spliceosomal mutations can be best referred as to “change of function mutations”. It is likely that phenotypic differences between various splicing gene mutants are a result of specific missplicing patterns. To investigate this issue further we chose to focus our study on U2AF1 mutations and compare the splicing patterns and target sequences to other spliceosomal mutations. We set our experiments to delineate what are the molecular determinants of misplicing and how mutant-specific splicing patterns are generated. We hypothesized that mutant, U2AF1 proteins misplice pre-mRNA at specific recognition sequences e.g., in the acceptor splice sites, resulting in different exon usage of a group of genes. We have focused on mutations in U2AF1: to indentify cases for further investigation we have screened 536 patients with myeloid malignancies and have identified U2AF1 mutations in 8% of MDS/MPN, 7% of sAML and 10% of MDS, of which majority carried high risk prognostic features. The most common mutation was S34Y (47%) followed by Q157P (29%). Additionally, we screened these patients for other spliceosomal mutations (SF3B1, U2AF26, SRSF2) in order to compare exon usage ratios between each of them. For these analyzes next generation sequencing data was available on 270 patients, the rest being sequenced manually. In order to elucidate variations in splicing and expression patterns associated with U2AF1 mutation on a global level, patients with complementary RNAseq and exome sequencing were selected. Patients were divided into subgroups according to a mutational status (U2AF1, SF3B1, SRSF2, or U2AF26 mutant etc. or spliceosomal WT, N=197). Using T-test (p<.0001) and absolute, average difference in exon inclusion ratio (≥±15%) we tested 201,837 exons in 17,097 genes and identified 35 exons in 35 genes with significantly different splicing pattern between mutants and spliceosomal WT cases; 77% of differentially spliced exons had low exon usage (exon skipping) in U2AF1 mutant cases (remaining had a higher frequency of exon usage (exon retention) in mutant cases). Thus, a total of 35 genes were prioritized for further investigation. Target analysis was performed on U2AF1 mutant (n=6) and WT (n=14) carriers. Within the genes exhibiting strong, differential, exon usage pattern we identified genes involved in different stages of mitosis, including CEP164, EHMT1, WAC and ATR or members of a gene group involved in RNA processing (STRAP, PPWD1, PABPC4 and UPF3B). As an illustrative example, PTBP1 gene, responsible for mediation of polypyrimidine-U2 complex interaction, was found to have a higher frequency of exon skipping. This finding suggests a possible augmentation of the effect of U2AF1 mutation by disturbing interaction between mutated U2AF1 and pre-mRNA polypyrimidine binding site. Interestingly, 25/35 of misspliced genes were already reported to harbor somatic mutations in various tumor tissues. RNAseq results were confirmed on an independent U2AF1 mutant/WT patient cohort using RT-PCR of total cellular RNA. U2AF1 missplicing pattern was highly specific as determined by comparison of RNA deep sequencing results of SF3B1, U2AF26 and SRSF2. Finally, in order to explain differences in exon usage between U2AF1 mutants and WT, we analyzed sequences surrounding 3' and 5' of all exons of each mutant case and disease controls. Interestingly, we found that -3 position (intron) around 3' splice site had a higher frequency of thymine (83%) in exons that were found to have a higher exon skipping ratio than in unaffected intron/exon junctions with a typical U2AF1 splice site motif. Such a misplicing restriction was not observed in any of the other group of spliceosomal mutations tested. In sum, our results show that characteristic features of misplicing created by spliceosomal mutations are related to the variant recognition sequences present in specific pre-mRNAs and thus creating characteristic downstream effects. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.
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Lu, Sydney X., Eric Wang, Alessandro Pastore, Chen Xufeng, Jochen Imig, Stanley C. Lee, Yohana Ghebrechristos, et al. "Therapeutic Targeting of an RNA Splicing Factor Network for the Treatment of Myeloid Neoplasms." Blood 132, Supplement 1 (November 29, 2018): 427. http://dx.doi.org/10.1182/blood-2018-99-111430.
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Abstract RNA-binding proteins (RBPs) regulate many aspects of transcription and translation in a cell- and tissue-specific manner and are frequently dysregulated in malignancy. We systematically evaluated RBPs preferentially required in acute myeloid leukemia (AML) over other forms of cancer or normal hematopoietic precursors using a CRISPR/Cas9 domain-based, loss-of-function screen targeting 490 classical RBPs with 2,900 sgRNAs (Fig. A). This screen was performed in cells lines representing AML, T-cell acute lymphoblastic leukemia (T-ALL), and lung adenocarcinoma (LUAD) and revealed multiple RBPs preferentially required for AML survival, but not for T-ALL or LUAD survival. We identified genes encoding 21 RBPs that were >3-fold depleted in AML cells and significantly overexpressed in AML patient samples versus normal adult CD34+ precursors (p-value < 0.05; Fig. B). Amongst RBPs required and upregulated in AML was RBM39, an RBP described to be involved in a number of cellular processes and to interact with key splicing proteins SF3B1 and U2AF2. Genetic ablation of Rbm39 in mouse MLL-AF9 leukemia cells dramatically delayed AML development and progression (Fig. C). In parallel, it has recently been described that a class of clinically-validated anti-cancer sulfonamide compounds (including indisulam and E7820) mediate RBM39 degradation as their dominant cellular mechanism of action. This occurs via novel interactions with the DCAF15 adapter protein of the CUL4/Ddb1 ubiquitin ligase complex with RBM39 as a neo-substrate. Treatment of MOLM-13 cells xenografted into mice with indisulam conferred significant anti-leukemic effects and improved overall survival (Fig. D). To explore the mechanism of RBM39 dependence in AML, we performed proteomic analyses of RBM39 interacting proteins in MOLM-13 cells as well as transcriptome-wide analysis of RBM39 RNA binding by enhanced UV cross-linking and immunoprecipitation (eCLIP) in the same cells. RBM39 physically interacted with an entire network of RBPs identified by our CRISPR screen as crucial for AML cell survival in addition to interacting with the core SF3b splicing complex. Further, anti-RBM39 eCLIP revealed RBM39 binding to exonic regions and most enriched at exon/intron borders at 5' and 3' splice sites of pre-mRNA (Fig. E), suggesting a prominent role of RBM39 in regulating splicing. Consistent with this, RNA-sequencing of AML cells following RBM39 deletion revealed significant effects of RBM39 loss on RNA splicing, most prominently causing increased cassette exon skipping (Fig. F). Recent studies suggest that myeloid leukemias with mutations in RNA splicing factors are sensitized to pharmacologic perturbation of RNA splicing. Analysis of the effects of RBM39 degrading compounds over a panel of 18 AML cells revealed that leukemia cells bearing splicing factor mutations or with high DCAF15 expression were the most sensitive to treatment (Fig. G). Genetic introduction of SF3B1, SRSF2, or U2AF1 hotspot mutations in K562 or NALM6 cells resulted in a 20-50% reduction in IC50 in response to sulfonamides. We next performed RNA sequencing of isogenic K562 cells with or without knockin of SF3B1K700E and SRSF2P95H mutations into the endogenous loci, and treated at the IC50 of E7820 or E7107, a small molecule that inhibits the SF3b core spliceosome complex. Treatment with either drug dramatically increased cassette exon skipping events and intron retention relative to DMSO control, with greater effects in splicing mutant cells. However, at equipotent doses, E7820 markedly increased mis-splicing compared with E7107. Furthermore, E7820 treatment resulted in mis-splicing of a number of RBP targets identified in our CRISPR screen as being required for AML survival, including SUPT6H, hnRNPH, and SRSF10, as well as RBM3 and U2AF2, consistent with previous observations (Fig. H). Here through systematic evaluation of RBPs across several cancers, we identify RBPs specifically required in AML. In so doing we identify a network of functionally and physically interacting RBPs upregulated in AML over normal precursors. Genetic or pharmacologic elimination one such RBP, RBM39, led to aberrant splicing of multiple members of this RBP network as well as of transcriptional regulators required for AML survival. These data suggest important clinical potential for anti-cancer sulfonamide treatment in splicing mutant myeloid leukemias. Disclosures Uehara: Eisai: Employment. Owa:Eisai: Employment.
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Whisenant, Thomas C., Eigen R. Peralta, Lauren D. Aarreberg, Nina J. Gao, Steven R. Head, Phillip Ordoukhanian, Jamie R. Williamson, and Daniel R. Salomon. "The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells." PLOS ONE 10, no. 12 (December 7, 2015): e0144409. http://dx.doi.org/10.1371/journal.pone.0144409.
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Bogusz, Agata M. "EBV-Negative Monomorphic B-Cell Posttransplant Lymphoproliferative Disorder with Marked Morphologic Pleomorphism and Pathogenic Mutations in ASXL1, BCOR, CDKN2A, NF1, and TP53." Case Reports in Hematology 2017 (2017): 1–8. http://dx.doi.org/10.1155/2017/5083463.
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Posttransplant lymphoproliferative disorders (PTLDs) are a diverse group of lymphoid or plasmacytic proliferations frequently driven by Epstein-Barr virus (EBV). EBV-negative PTLDs appear to represent a distinct entity. This report describes an unusual case of a 33-year-old woman that developed a monomorphic EBV-negative PTLD consistent with diffuse large B-cell lymphoma (DLBCL) 13 years after heart-lung transplant. Histological examination revealed marked pleomorphism of the malignant cells including nodular areas reminiscent of classical Hodgkin lymphoma (cHL) with abundant large, bizarre Hodgkin-like cells. By immunostaining, the malignant cells were immunoreactive for CD45, CD20, CD79a, PAX5, BCL6, MUM1, and p53 and negative for CD15, CD30, latent membrane protein 1 (LMP1), and EBV-encoded RNA (EBER). Flow cytometry demonstrated lambda light chain restricted CD5 and CD10 negative B-cells. Fluorescence in situ hybridization studies (FISH) were negative for cMYC, BCL2, and BCL6 rearrangements but showed deletion of TP53 and monosomy of chromosome 17. Next-generation sequencing studies (NGS) revealed numerous genetic alterations including 6 pathogenic mutations in ASXL1, BCOR, CDKN2A, NF1, and TP53(x2) genes and 30 variants of unknown significance (VOUS) in ABL1, ASXL1, ATM, BCOR, BCORL1, BRNIP3, CDH2, CDKN2A, DNMT3A, ETV6, EZH2, FBXW7, KIT, NF1, RUNX1, SETPB1, SF1, SMC1A, STAG2, TET2, TP53, and U2AF2.
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Adema, Vera, Courtney E. Hershberger, Wencke Walter, Cassandra M. Kerr, Stephan Hutter, Yasunobu Nagata, Hassan Awada, et al. "Hotspot U2AF1 Mutations Determine Missplicing Selectivity: Novel Mechanisms Altering Splicing Factors." Blood 134, Supplement_1 (November 13, 2019): 2985. http://dx.doi.org/10.1182/blood-2019-129367.
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Mutations in splicing factor genes (SF3B1, SRSF2, U2AF1, and ZRSR2) are identified in over 50% of patients diagnosed with myelodysplastic syndrome (MDS). U2AF1 is a U2 auxiliary factor that forms a heterodimer with U2AF2 for the recognition of the 3' splice site (SS) and results in the subsequent recruitment of U2snRNPs during pre mRNA splicing. U2AF1 mutations are present in 11% of MDS and its presence is correlated with an increased risk of progression to AML. Non-canonical mutations are rarely seen in U2AF1 but two highly conserved hotspots (S34, Q157) are frequently seen and result in distinct downstream effects. We performed NGS analysis of a large cohort of patients with myeloid neoplasms (MN; n=3,328) and identified 238 patients with U2AF1 mutations (7%). We analyzed the somatic mutational profile, clonal hierarchy, and splicing profile of patients with U2AF1S34 (n=99), U2AF1Q157 (n=119), and others (n=20; M1/*, A26T/V, R35Q, R118C, E124K, F150L, E152G, C154S, R156H, M172L). The mutational spectrum of U2AF1S34 and U2AF1Q157 was equally dismal but pretty distinct. U2AF1 S34 cases were mostly co-mutated for DNMT3A (5%), TET2 (4%), RUNX1 (2%), ASXL1, CBL, ETV6, KRAS, NRAS (1.3%), STAG2 (1%), CUX1, and TP53 (<1%) while U2AF1Q157 had higher numbers of mutations in ASXL1 (10%), SETBP1, TET2 (3%), NRAS (2%), DNMT3A, PHF6 (2%), JAK2 (2%), CBL, EZH2, TP53 (2%), IDH2, RUNX1, STAG2 (2%), KRAS (1%), and IDH1 (<1%). Mutational rank showed: U2AF1S34 was ancestral in 38% of the cases followed by secondary DNMT3A and NRAS (5%, both). Ancestral U2AF1Q157 was found in 35%, with ASXL1 (19%) being the most common secondary hit. Subclonal U2AF1S34 (44%) was most commonly preceded by DNMT3A (21%), while secondary U2AF1Q157 (40%) had ASXL1 (28%) as the most common first hit. U2AF1S34 and U2AF1Q157 were co-dominant to a miscellanea of mutations. U2AF1S34 cases had a shorter OS than U2AF1Q157 cases (n=82 vs. n=101; 20 vs. 25 mo.; P=.002). Ancestral U2AF1S34 or U2AF1Q157 defined a dismal prognosis compared to secondary U2AF1S34 or U2AF1Q157 (OS: n=63 vs. n=86; 29 vs. 37 mo.; P=.03). To investigate the effects of both mutations on splicing patterns, we analyzed RNA-Seq profiles, followed by the rMATS bioinformatics pipeline to determine alternative splicing (AS) events that were classified as skipping exon (SE), retained intron (RI), and 3' or 5' alternative SS (A3SS, A5SS) (Hershberger, ASH 2019). Overall, 675 AS events in 430 genes were scored significant (pFDR<.05) when the splicing inclusion/exclusion difference was ±10%. U2AF1S34 and U2AF1Q157 caused an equal fraction of SE (79% U2AF1S34; 72% U2AF1Q157). Only 4% of the genes were commonly misspliced by both mutations, while the rest of the genes were uniquely spliced according to each mutation. Some exemplary genes misspliced by both mutations were DDX3X (an RNA helicase) showing a consistent SE at exon 5 and an RI 3-5a and CCNG1 (cell growth regulation) which was enriched for RI 7-7. Among others, U2AF1S34 uniquely affected the splicing of TET2 (SE e3); cell cycle regulators, CDC37L1 (SE), CCNC (SE e8), and HDAC3 (SE e6). We then investigated whether U2AF1 mutations might affect the splicing of other RNA splicing components. This mechanism would lead to the loss of regulation of the spliceosome complex. U2AF1S34 produces selective RIs in SRSF2 (4-4a) and A5SS in SRSF6 (7a and 7). Tumor suppressors and proto-oncogenes were also found to be misspliced by U2AF1S34 including PTEN (RI 3-Ua), CTNNB1 (RI 15-19; 3'SS (19 and 19a), and CCNL1 (RI 4-U). Major regulators of splicing factor activity are phosphatases like PP1R12A and PPP1R12B, which showed an RI 8-7 and an A3SS, respectively. Among genes exclusively misspliced by U2AF1Q157, we identified DEAD-Box helicases [DDX17 (RI), DDX59 (SE e8), DHX29 (RI)], ALAS family members (ALAS1, SE e6; ALAS2, SE e5) and UTX (KDM6A; SE e16). U2AF1Q157 affected the missplicing of DYRK1A (SE e7), a kinase known to phosphorylate SF3B1 at T434 site. In sum, our study suggests that while concurrent mutations in splicing factors lead to lethality, the presence of mutations (as the case of U2AF1S34 and U2AF1Q157) and consequent missplicing of other splicing factors are permissive events in MN and might represent novel mechanisms of disease pathogenesis. Disclosures Walter: MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nazha:Abbvie: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Novartis: Speakers Bureau; Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.
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Correa, Juan Gonzalo, Alberto Alvarez-Larrán, Monica Lopez-Guerra, Juan Carlos Hernandez Boluda, Mar Tormo, María Rozman, Daniel Martínez, Dolors Colomer, Jordi Esteve, and Francisco Cervantes. "Triple Negative Myelofibrosis and Myelodysplastic Syndrome with Fibrosis: Clinico-Biological Characterization and Correlation with Gene Mutations." Blood 132, Supplement 1 (November 29, 2018): 4299. http://dx.doi.org/10.1182/blood-2018-99-115888.
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Abstract Introduction: Triple negative primary myelofibrosis (TN-PMF) and myelodysplastic syndromes with fibrosis (F-MDS) are rare entities, often difficult to distinguish each other. Currently, no specific molecular markers allowing a precise differential diagnosis are available. In this sense, next generation techniques (NGS) might be useful to distinguish between both entities and to refine prognosis. Methods: Thirty-nine patients with TN-PMF (n=16) or F-MDS (n=23) were analyzed, Targeted NGS was performed in 28 cases (10 TN-PMF and 18 F-MDS) using the Sophia Genetics Myeloid Tumor Solution Panel including the following genes: ABL1, ASXL1, BRAF, CALR, CBL,CEBPA, CSF3R,CSNK1A1,DNMT3A, ETV6, EZH2, FLT3,HRAS, IDH1, IDH2, JAK2, KIT, KMT2A, KRAS, MPL, NPM1, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2, WT1 and ZRSR2. High molecular risk (HMR) was defined by the presence of any mutation in ASXL1, SRSF2, EZH2 or IDH1/2 genes. Clinical and biological data at diagnosis were compared among both groups of patients, including bone marrow cytogenetics, bone marrow histology, and the results from NGS analysis. The probabilities of survival and progression to acute leukemia were estimated by the Kaplan Meier method using the log rank test for comparisons. Results: Median age at diagnosis for the whole series was 63 years (range 42-87). Cytogenetic abnormalities were detected in 16 (57%) out of 28 assessable patients, being classified as low risk (n=17), intermediate risk (n=5), and high risk (n=6) There were no significant differences among TN-PMF and F-MDS regarding age, sex, hematological values, spleen size or cytogenetic risk or the number of blasts in peripheral blood or bone marrow. Mutations were detected by NGS in 26 out of 28 (93%) patients. Median number of mutations was 3 (Range: 1-7). Most frequent mutated genes were: ASXL1 n=10 (36%), TET2 n=7 (25%), SRSF2 n=6 (21%), DNMT3A n=6 (21%), U2AF1 n=5 (18%), ETV6 n=4 (14%), SETBP1 n=3 (11%), RUNX1 n=3 (11%), EZH2 n=2 (7%), TP53 n=2 (7%), ZRSR2 n=2 (7%) and KMT2A n=2 (7%). Mutations in either TET2, U2AF1, SETBP1, TP53 or RUNX1 genes were observed in only 1 patient with TN-PMF in contrast with 12 out of 18 (67%) F-MDS cases (p=0.006). HMR mutations were detected in 12 cases (43%) with 7 of them (21%) carrying two or more HMR mutations. There were no significant differences among TN-PMF and F-MDS regarding the total number of mutations per case or in the presence of HMR mutations. Median survival was 1.6 and 3.7 years for patients with TN-PMF and F-MDS, respectively (p=0.4). Leukocyte count > 25x109/L and presence of blasts ≥ 1% in peripheral blood were associated with a significantly lower survival. High risk cytogenetics and ≥ 2 HMR mutations were associated with a tendency towards lower survival. Median survival was 1.6 years in patients with ≥ 3 mutations in comparison with 8.3 years in those with < 3 mutations, respectively (p=0.03). Progression to acute myeloid leukemia was observed in 11 patients, resulting in a probability of 32% at 3 years. There were no significant differences in the probability of progression to acute leukemia among TN-PMF and F-MFDS. Time to acute leukemia was significantly shorter in patients with ≥ 3 mutations (3-year probability 45% versus 16%, p=0.039). Conclusions: TN-PMF and F-MDS showed a high rate of mutations in myeloid genes with TET2, U2AF1, SETBP1, TP53 or RUNX1 being more frequently mutated in F-MDS. Despite survival being short in both entities, NGS allowed the identification of a subgroup of patients with especially poor prognosis characterized by the presence of ≥ 3 mutations. Disclosures Hernandez Boluda: Novartis: Consultancy; Incyte: Consultancy. Cervantes:Hospital Clinic Barcelona: Employment; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees.