ZRSR2 Mutations Cause Dysregulated RNA Splicing in MDS

Abstract Recurrent somatic mutations have been uncovered in several components of the spliceosome in Myelodysplastic Syndrome (MDS). Recent high throughput sequencing of large cohorts of MDS has established RNA splicing as the pathway most frequently targeted by somatic mutations. These findings implicate dysregulated RNA splicing in the pathogenesis of MDS. However, the mechanism linking aberrant splicing to the development of MDS is unknown. ZRSR2, a frequently mutated spliceosome gene in MDS is located on the X chromosome. Somatic alterations of ZRSR2 are typically inactivating mutations (frameshift indels, nonsense point mutations or splice site mutations) which are observed predominantly in males. Mutations in ZRSR2are more prevalent in MDS subtypes without ring sideroblasts and chronic myelomonocytic leukemia (CMML) and are associated with elevated bone marrow blasts and higher rate of progression to AML. Although ZRSR2 has been suggested to interact with other splice proteins, U2AF2 and SRSF2, at the 3΄ splice sites during the pre-spliceosome assembly, its precise role in RNA splicing remains unexplored. In this study, we demonstrate that deficiency of ZRSR2 leads to impaired splicing of U12-type introns which are dependent upon the minor spliceosome. RNA-Sequencing of MDS bone marrow harboring inactivating mutations of ZRSR2 revealed aberrant retention of U12-type introns as compared to bone marrow with wild-type ZRSR2. In addition, several U12-type introns displayed mis-splicing associated with recognition of cryptic splice-site. In contrast, the splicing of U2-type introns (dependent upon the major spliceosome) was largely unaffected. The mis-spliced introns were found in several key genes including PTEN, MAPK1, MAPK3, BRAF and E2F2, and the impaired splicing of several introns in ZRSR2 mutant bone marrow was validated experimentally. Further, short hairpin RNA (shRNA) mediated knockdown of ZRSR2 in MDS/AML TF1 cells led to impaired splicing specifically of U12-type introns. We also observe that ectopic expression of ZRSR2 in stable knockdown 293T cells resulted in increase in splicing efficiency of U12-type introns. Further, the downregulation of ZRSR2 in leukemia cells results in reduced growth and clonogenic potential in leukemia cell lines. Moreover, knockdown of ZRSR2 in human CD34+ cells displayed altered differentiation potential towards erythroid and myeloid lineages in vitro. Overall, the dysregulated RNA splicing of U12-type introns in ZRSR2 mutant samples affects several crucial genes involved in cell cycle, signaling, RNA binding and transport. These genes are potential mediators of MDS phenotype. Ours is a first study which demonstrates the functional consequences of ZRSR2 mutations in MDS and identifies a specific role of ZRSR2 in regulating RNA splicing. It underlines aberrant splicing of U12-type introns as a distinctive feature of ZRSR2 mutant MDS. Disclosures Kohlmann: MLL Munich Leukemia Laboratory, Munich, Germany: Employment. Grossmann:MLL Munich Leukemia Laboratory, Munich, Germany: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Aberrant RNA Splicing Contributes to the Pathogenesis of EVI-Rearranged Myeloid Leukemias

Blood ◽

10.1182/blood-2019-124722 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 917-917

Author(s):

Daichi Inoue ◽

Stanley C Lee ◽

Akihide Yoshimi ◽

Justin Taylor ◽

Lillian E Bitner ◽

...

Keyword(s):

Gene Expression ◽

Splice Site ◽

Rna Splicing ◽

Rna Binding ◽

Conflicts Of Interest ◽

Macrocytic Anemia ◽

Ectopic Expression ◽

Mrna Isoforms ◽

Genomic Alterations ◽

Mutant Cells

Inversions between chromosome 3q21 and 3q26 ("inv(3)/t(3;3)") mark an aggressive, poor prognosis form of AML with limited treatment options and also occasionally occur in MDS and CML. Inv(3)/t(3;3) repositions a distal GATA2enhancer from 3q21 to the EVI1locus at 3q26 inducing ectopic EVI1 expression and reducing GATA2 expression. At the same time, additional genomic alterations exist in inv(3)/t(3;3) leukemias but the contributions of these events to inv(3)/t(3;3) leukemia are not well understood. Here we evaluated genomic alterations in 63 patients with inv(3)/t(3;3) leukemia. Mutations in NRAS and the core RNA splicing factor SF3B1 were the most common individual alterations (each occurring in 27% of patients; Fig.A). We next quantified gene expression and splicing in AML samples with and without inv(3)/t(3;3) abnormalities and SF3B1 mutations. Amongst inv(3)/t(3;3) AML, the majority of gene expression changes were driven by the inv(3)/t(3;3) rearrangement while the majority of splicing changes were driven by mutant SF3B1. The most abundant category of splicing change in inv(3)/SF3B1 co-mutant cells was alteration in 3' splice site usage (Fig.B). Intriguingly, one of the most robust changes in splicing in inv3/SF3B1 co-mutant cells was aberrant splicing of EVI1itself such that SF3B1 mutant cells promoted expression of a novel isoform of EVI1using an intron proximal 3' splice site (Fig.C; the official gene name "MECOM" corresponds to the genes MDS1and EVI1at this locus). While several mRNA isoforms of EVI1have been previously described, these all result in loss of EVI1 functional domains. In contrast, the unique EVI1 isoform in SF3B1 mutant/inv(3) AMLs contains in an in-frame insertion of six amino acids in the second zinc finger domain (ZF2) of EVI1 (a region of EVI1 known to be affected by germline mutations in leukemia predisposition syndromes). These data identify that nearly one-third of inv(3) AML patients express a heretofore undescribed isoform of EVI1. Of note, this unannotated EVI1isoform is also present in EVI1expressing/SF3B1K700Emutant leukemias lacking inv(3)). The above findings highlight a novel model where inv(3)/t(3;3) AML is driven by ectopic expression of distinct oncogenic isoforms of EVI1. To test this model and understand the contribution of SF3B1K700Eto inv(3)/t(3;3) AML, we crossed transgenic mice bearing the entire human inv(3)(q21;q26) locus whereby the GATA2enhancer misdirects human EVI1expression ("inv3 mice"; Yamazaki et al. Cancer Cell2014) to Sf3b1K700Econditional knockin mice (Mx1-cre Sf3b1K700E/WT).Given that the human MECOMlocus (coding and noncoding regions) was recapitulated in this mouse model, the concordant novel EVI1isoform was expressed in inv3/Sf3b1K700Emice as in patients. While Mx1-cre Sf3b1K700Emice develop an MDS-like disorder, inv3 mice develop myeloid and lymphoid leukemias with lethality ~300 days after birth. However, expression of the Sf3b1K700E/WTmutation in inv3 hematopoietic cells resulted in a highly penetrant MDS, which transformed to a lethal AML by a median of 241 days (Fig.D;p=0.0021). In the first 6 months following transplant, Mx1-cre inv3 Sf3b1K700Emice had leukopenia, macrocytic anemia, and morphologic dysplasia (Fig.E-F) that eventually transformed to a disease with a high WBC count and large numbers of immature cells around time of death. In competitive reconstitution assays, Mx1-cre inv3 Sf3b1K700Ehematopoietic stem cells (HSCs) failed to differentiate into mature peripheral blood cells despite having a competitive advantage at the level of HSCs (Fig.G-H). RNA-seq of hematopoietic precursors from the above models identified (i) a substantial change in splicing in inv3/Sf3b1K700Emutant leukemias versus those driven by inv3 alone, and (ii) alterations in a host of RNA binding proteins in inv3/Sf3b1K700Emutant leukemias (Fig.I). These data highlight a high occurrence of SF3B1 mutations in inv(3)/t(3;3) leukemias, present a new genetically accurate model for inv(3) AML, and uncover a novel oncogenic isoform of EVI1 expressed in a large proportion of inv(3)/t(3;3) patients. Ongoing work focused on identifying the mechanistic effect of the SF3B1-mutant induced aberrant EVI1isoform may provide novel insight into the role of EVI1 in promoting leukemogenesis and engender development of therapeutic opportunities targeting EVI1splicing. Figure Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2019-123180 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 662-662

Author(s):

Naranie Shanmuganathan ◽

Daniel Thomson ◽

Carol Wadham ◽

Verity A Saunders ◽

Nur Hezrin Shahrin ◽

...

Keyword(s):

Splice Site ◽

Rna Splicing ◽

Research Funding ◽

Cancer Genes ◽

Aberrant Splicing ◽

Cryptic Exon ◽

Large Deletions ◽

Tki Resistance ◽

Splice Junctions ◽

Runx1 Mutation

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

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U2AF1 Driver Mutations in Hematopoietic Disorders Alter but Do Not Abrogate RNA Binding and Enlighten Structural Dependencies of the U2AF-RNA Complex

Blood ◽

10.1182/blood-2019-130759 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1230-1230

Author(s):

Giulia Biancon ◽

Poorval Joshi ◽

Torben Hunck ◽

Yimeng Gao ◽

Valentina Botti ◽

...

Keyword(s):

Cell Cycle ◽

Splice Site ◽

Rna Binding ◽

Meta Analysis ◽

Complex Model ◽

Driver Mutations ◽

Cancer Therapies ◽

Sequence Specificity ◽

Aberrant Splicing ◽

Systematic Analysis

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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Lineage-Specific Aberrant mRNA Splicing By U2AF1 Mutation Alters Erythroid and Granulomonocytic Differentiation

Blood ◽

10.1182/blood.v126.23.142.142 ◽

2015 ◽

Vol 126 (23) ◽

pp. 142-142

Author(s):

Bon Ham Yip ◽

Swagata Roy ◽

Hamid Dolatshad ◽

Jacqueline Shaw ◽

Seishi Ogawa ◽

...

Keyword(s):

Bone Marrow ◽

Cell Growth ◽

Mouse Model ◽

Progenitor Cells ◽

Splice Site ◽

Myeloid Cells ◽

Mrna Splicing ◽

Splicing Factor ◽

Aberrant Splicing ◽

Cd34 Cells

Abstract Splicing factor genes are the most common targets of somatic mutations in myelodysplastic syndromes (MDS). The splicing factor U2AF1 is an auxiliary factor that forms a heterodimer for the recognition of the 3′ splice site during pre-mRNA splicing. Heterozygous mutations of U2AF1 occur in ~10% of MDS patients and are predominantly located at S34 and Q157 within the zinc fingers domains. Recently an inducible transgenic mouse model expressing mutant U2AF1 S34F demonstrated altered hematopoiesis and aberrant pre-mRNA splicing in hematopoietic progenitor cells. MDS are clonal stem-cell disorders characterized by ineffective hematopoiesis in one or more myeloid lineages of the bone marrow. To investigate the effects of U2AF1 S34F mutation on hematopoiesis, U2AF1 S34F mutant (S34F) and U2AF1 wild type (WT) were overexpressed in human bone marrow CD34+ progenitor cells by retroviral transduction and the cells were differentiated along erythroid and granulomonocytic lineages. S34F erythroblasts exhibited impaired erythroid differentiation compared to WT and empty vector (EV) controls. A significant increase in CD71-CD235a- non-erythroid cells (p≤0.02, n=7) followed by a significant decrease in CD71+CD235a+ (p≤0.002, n=7) and CD71-CD235a+ (p=0.005, n=7) erythroid cells was observed in S34F erythroblasts from day 11 to 14 using flow cytometry, when compared to WT and EV controls. Moreover, S34F inhibited formation and hemoglobinization of BFU-E colonies from bone marrow CD34+ cells in colony forming cell (CFC) assays compared to WT (p=0.002, n=7) and EV (p=0.0006, n=7) controls. S34F erythroblasts also exhibited impaired cell growth and increased apoptosis (Annexin V+) compared to WT (p<0.05, n=6-8) and EV (p≤0.01, n=6-8) controls. Thus, the S34F mutation results in impaired erythropoiesis. S34F perturbed the granulomonocytic lineage by skewing differentiation of myeloid cells towards granulocytes. A reduction in the CD11b+ population was observed in S34F myeloid cells compared to WT (p≤0.001, n=9) and EV (p≤0.001, n=9) controls from day 11 to 14. An increase in granulocytes (CD15+, p≤0.001, n=5) followed by a concomitant decrease in monocytes (CD14+,p=0.026, n=5) was also observed in S34F myeloid cells on day 20 compared to WT and EV controls. Morphological analysis of myeloid cells confirmed a reduction in monocytes caused by an expansion of granulocyte eosinophils. Moreover, S34F bone marrow CD34+ cells produced a significantly higher number of CFU-G (p=0.035, n=5) with a decrease in the number of CFU-M (p≤0.03, n=5) in myeloid CFC assays compared to WT (p≤0.01, n=7) and EV (p≤0.01, n=7) controls. S34F myeloid cells exhibited impaired cell growth associated with G2/M cell cycle arrest compared to WT (p=0.0003, n=6) and EV (p=0.0002, n=6) controls. To investigate aberrant splicing events, we performed RNA sequencing on individual erythroid (BFU-E) and granulomonocytic (CFU-G and CFU-M) colonies formed by S34F, WT and EV transduced bone marrow CD34+ cells (n=3 each). By comparison with WT and EV colonies of the same lineage, we observed that S34F differentially alters the splicing pattern in different lineages. We have observed aberrant splicing of many genes, including BCOR and H2AFY, two genes previously shown to be aberrantly spliced in common myeloid progenitors from a U2AF1 S34F mouse model. The transcriptional co-repressor BCOR is commonly mutated in MDS/AML. Alternative 3' splice site usage in BCOR, resulting in reduced expression of its long isoform, was observed in S34F granulomonocytic colonies, but not in S34F erythroid colonies. In contrast, reduced expression of isoform 1.1 of H2AFY (a member of H2A histone family), due to mutually exclusive exons, was observed in both S34F erythroid and granulomonocytic colonies. Deregulation in isoform expression levels in BCOR and H2AFY was validated by isoform-specific qRT-PCR in S34F transduced cells compared to WT (p≤0.015, n=5) and EV (p≤0.045, n=5) controls. We are currently introducing these isoform imbalances into bone marrow CD34+ cells as they differentiate towards the erythroid and granulomonocytic lineages to elucidate the lineage-specific effect of S34F. Our results indicate that U2AF1 S34F mutant alters erythroid and granulomonocytic differentiation by inducing lineage-specific aberrant splicing patterns, providing new insights into the molecular pathogenesis of U2AF1 mutant MDS. Disclosures No relevant conflicts of interest to declare.

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IKZF2, a Novel Target of MSI2 RNA-Binding Protein Plays an Oncogenic Role in Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.440.440 ◽

2015 ◽

Vol 126 (23) ◽

pp. 440-440

Author(s):

Sun Mi Park ◽

Angela Thornton ◽

Ly P. Vu ◽

Sagar Chhangawala ◽

Gerard Minuesa ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Myeloid Leukemia ◽

Rna Binding ◽

Leukemia Cell ◽

Viable Cell Number ◽

Human Leukemia ◽

Self Renewal ◽

Hematopoietic Stem ◽

Renewal Program

Abstract Deregulated epigenetic program is found in many cancers, and genetic aberrations of histone methyltransferases contribute to transformation in myeloid leukemias. Post-transcriptional regulation in leukemia has recently been highlighted as a novel way for maintaining the leukemia stem cell (LSC) program. We have recently demonstrated that Msi2 is required for LSC function in a murine MLL-AF9 leukemia model. We determined that MSI2 maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting and retaining efficient translation of critical MLL regulated transcription factors including Hoxa9, Myc and Ikzf2. Despite extensive studies implicating Myc and Hoxa9 in leukemia, the role for Ikzf2 in myeloid leukemia is not known. Ikzf2 is a member of the Ikaros transcription factor family and regulates lymphocyte development by controlling regulatory T-cell function. Ikzf2 is highly expressed in Hematopoietic Stem Cells (HSC) and to investigate if Ikzf2 is involved in HSC function, we utilized mice that have a specific deletion of Ikzf2 in the hematopoietic system through the Vav-cre system. We found similar frequencies for different populations in the stem, progenitor and mature cells in the bone marrow of Ikzf2f/f and Ikzf2D/D mice. Colony assays of isolated Lin- Sca1+ c-Kit+ (LSK) cells from Ikzf2f/f and Ikzf2D/D mice resulted in a comparable number of myeloid progenitor colonies. Furthermore, noncompetitive transplant of Ikzf2f/f and Ikzf2D/D bone marrow cells showed similar chimerism after 34 months indicating that I kzf2D/D mice have normal HSC function and hematopoiesis. To interrogate the role of Ikzf2 in acute myeloid leukemia we utilized the MLL-AF9 retroviral transduction model. Intracellular flow cytometry showed that IKZF2 is highly expressed in the LSC population compared to the non-LSCs. We then transduced Ikzf2f/f and Ikzf2D/D LSK cells with MLL-AF9 and found that Ikzf2 deletion results in a ten-fold reduction in colony formation compared to Ikzf2f/f cells. Transplantation of transduced cells results in delayed leukemia progression with reduced disease burden. Secondary transplantation of the initiation experiment exhibited a significant delay in leukemogenesis in the Ikzf2D/D compared to the Ikzf2f/f mice (median survival of 32 and 19.5 days, respectively). The role for Ikzf2 in maintenance was assessed with an inducible puro-creER system, which resulted in 80% decrease in viable cell number within 24hrs of 4-hydroxytamoxifen (4-OHT) treatment. Flow cytometric analysis showed that the Ikzf2-deficient cells had increased apoptosis and differentiation, shown by AnnexinV/7-AAD and Mac1 expression respectively. Furthermore, inducible deletion of Ikzf2 using puro-creER system in vivo revealed that Ikzf2 deletion leads to a delay in leukemia after tamoxifen administration in mice. These results indicate that Ikzf2 is required for both leukemia initiation and maintenance. To determine a role for IKZF2 in human leukemia cells, we performed Ikzf2 knockdown experiments with shRNAs in Kasumi-1, KG1, KCL22 and MOLM13 cells. Ikzf2 depletion resulted in decreased cell growth and increased apoptosis compared to cells infected with scramble shRNA. To determine the mechanism for how IKZF2 controls leukemia cell survival and self-renewal, we performed gene expression profiling of the Ikzf2-deficient Vav-cre LSCs and demonstrated enrichment in signatures for self-renewal loss, increased differentiation, loss of Myc-regulated genes and loss for targets of Hoxa9 and Meis1. Further analysis overlapping our MSI2 HITS-CLIP data and our differentially regulated genes revealed a strong enrichment suggesting that the MSI2 bound targets are transcriptionally regulated by IKZF2. Lastly, ATAC-sequencing of Ikzf2f/f and Ikzf2D/D LSCs revealed alterations in chromatin accessibility that correlated closely with differentially expressed genes. Utilizing the ATAC-seq data we predicted that HOXA9 and MYC sites were significantly altered. We validated that MYC RNA and protein levels were reduced in both murine and human AML cell lines. In contrast to its known tumor suppressor role in hypodiploid B-ALL and T-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell initiation and maintenance. Thus, we provide evidence that Ikzf2 can regulate c-MYC expression helping in maintaining the stem cell self-renewal program in LSCs. Disclosures No relevant conflicts of interest to declare.

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Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing

eLife ◽

10.7554/elife.07938 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 40

Author(s):

Li Zhang ◽

Ngoc-Tung Tran ◽

Hairui Su ◽

Rui Wang ◽

Yuheng Lu ◽

...

Keyword(s):

Cell Fate ◽

Protein Level ◽

Rna Splicing ◽

Messenger Rna ◽

Rna Binding ◽

Leukemia Cell ◽

Terminal Differentiation ◽

Curative Therapy ◽

Protein Arginine Methyltransferase 1 ◽

Acute Megakaryocytic Leukemia

RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.

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Insights of Non-canonical Splice-site Variants on RNA Splicing in Patients with Congenital Hypothyroidism

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgab737 ◽

2021 ◽

Author(s):

Najla Albader ◽

Minjing Zou ◽

Huda A BinEssa ◽

Saba Abdi ◽

Anwar F Al-Enezi ◽

...

Keyword(s):

Congenital Hypothyroidism ◽

Splice Site ◽

Rna Splicing ◽

Mrna Splicing ◽

Next Generation Sequencing Data ◽

Splice Sites ◽

Sequencing Data ◽

Aberrant Splicing ◽

Hormone Synthesis ◽

Generation Sequencing

Abstract Context Congenital hypothyroidism (CH) is caused by mutations in the genes for thyroid hormone synthesis. In our previous investigation of CH patients, ~53% of patients had mutations in either coding exons or canonical splice-sites of causative genes. Non-canonical splice-sites variants in the intron were detected but their pathogenic significance was not known. Objective To evaluate non-canonical splice-site variants on pre-mRNA splicing of CH-causing genes. Methods Next-generation sequencing data of 55 CH cases in 47 families were analyzed to identify rare intron variants. The effects of variants on pre-mRNA splicing were investigated by minigene RNA-splicing assays. Results Four intron variants were found in 3 patients: SLC26A4 c.1544 + 9C>T and c.1707 + 94C>T in one patient, and SLC5A5 c.970-48G>C and c.1652-97A>C in two other patients. The c.1707 + 94C>T and c.970-48G>C caused exons 15 and 16 skipping, and exon 8 skipping, respectively. The remaining variants had no effect on RNA splicing. Furthermore, we analyzed 28 previously reported non-canonical splice-site variants (4 in TG and 24 in SLC26A4). Among them, 15 variants (~54%) resulted in aberrant splicing and 13 variants had no effect on RNA splicing. These data were compared with three variant-prediction programs (FATHMM-XF, FATHMM-MKL, and CADD). Among 32 variants, FATHMM-XF, FATHMM-MKL, and CADD correctly predicted 20 (63%), 17 (53%), and 26 (81%) variants, respectively. Conclusions Two novel deep intron mutations have been identified in SLC26A4 and SLC5A5, bringing the total number of solved families with disease-causing mutations to ~45% in our cohort. Approximately 46% (13/28) reported non-canonical splice-site mutations do not disrupt pre-mRNA splicing. CADD provides highest prediction accuracy of non-canonical splice-site variants.

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Aberrant Upregulation Of Alternatively Spliced Isoforms Of Son In Myeloid Leukemia: Implications In Hematopoiesis and Leukemogenesis

Blood ◽

10.1182/blood.v122.21.3731.3731 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3731-3731

Author(s):

Jung-Hyun Kim ◽

Christopher J Hickey ◽

E.Y. Erin Ahn

Keyword(s):

Bone Marrow ◽

Rna Splicing ◽

Myeloid Leukemia ◽

Rna Binding ◽

Bone Marrow Cells ◽

Splice Variants ◽

Full Length ◽

Hematopoietic Stem ◽

Short Isoforms ◽

Marrow Cells

Abstract SON is a large, poorly characterized nuclear speckle protein. The key structural domains comprising the SON protein are the serine/arginine-rich (RS) domain, single stranded RNA-, and double stranded RNA-binding domains, in addition to several long repeating amino acid sequences likely to facilitate its scaffolding function. Previously, we identified SON as a key factor for efficient RNA splicing of diverse genes related to cell cycle, DNA repair, and survival signaling pathways (Molecular Cell, 2011, 42:185). We also demonstrated that SON inhibits transcriptional activation of the promoter associated to the miR-23a-27a-24 cluster, which harbors a regulator for the expression of GATA-2, a known hematopoietic stem cell regulator (J. Biol. Chem. 2013, 288:5381). Moreover, SON is highly expressed in hematopoietic tissues/organs, and particularly upregulated in hematopoietic stem cells and leukemic blasts. These results suggest potential roles of SON in both normal hematopoiesis and hematological malignancies. Based on the nucleotide sequence of the SON gene, several databases predict that the primary transcript of SON contains alternative exons, and inclusion of these alternative exons during RNA splicing may generate C-terminally truncated short isoforms (splice variants) which lack RNA-binding motifs. However, expression of these short SON isoforms has not been confirmed in any types of cells and functions of the isoforms remain unexplored. Using 3’ rapid amplification of cDNA end (3’ RACE), we confirmed that short SON isoforms are indeed expressed in hematopoietic cells, and the included alternative exon provides the 3’ UTR and polyadenylation signal different from those of full-length SON transcript. To further examine SON isoform expression, we designed primers targeting specific alternative exons of SON and screened the level of full-length SON and the short isoforms. Our quantitative PCR data showed that lineage marker negative (Lin-) bone marrow cells highly express full-length SON, but not short isoforms, when compared to total bone marrow cells. Interestingly, we found that the levels of short SON isoforms are noticeably high in acute myeloid leukemia (AML) patient bone marrow cells, while full-length SON is downregulated when compared to normal bone marrow cells. These expression patterns were consistent with data analyses in a cancer microarray database (Oncomine). Furthermore, we found that short isoforms, but not full-length SON, were downregulated during PMA-induced differentiation of HL-60 and K562 cells. These results suggest that while full-length SON is the major form in normal hematopoietic stem cells/progenitors, the production of short SON isoforms through alternative exon inclusion is aberrantly activated in leukemic cells, likely due to a factor associated with impaired differentiation. To address whether the short SON isoforms affect the function of full-length SON in RNA splicing, we examined RNA splicing efficiency using a minigene containing SON-dependent exon-intron junction. As we expected, the short isoform alone, which lacks RNA-binding motifs, was not able to process minigene RNA splicing. However, to our surprise, co-expression of a short isoform together with full-length SON caused an increase in spliced RNA product from a minigene. These results suggest that short isoform expression potentiates the full-length SON function during exon recognition. Taken together, our results reveal that inclusion/skipping of the alternative exons within the primary SON transcript is abnormally regulated in myeloid leukemia, resulting in upregulation of short splice variants of SON. Furthermore, fine-tuning of RNA splicing efficiency by short SON isoforms implicates potential roles of these isoforms in RNA processing and global gene expression during hematopoiesis and leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Elucidation of the aberrant 3′ splice site selection by cancer-associated mutations on the U2AF1

Nature Communications ◽

10.1038/s41467-020-18559-6 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Hisashi Yoshida ◽

Sam-Yong Park ◽

Gyosuke Sakashita ◽

Yuko Nariai ◽

Kanako Kuwasako ◽

...

Keyword(s):

Splice Site ◽

Site Selection ◽

Rna Splicing ◽

Single Amino Acid ◽

Splice Site Selection ◽

Aberrant Splicing ◽

Serious Disease ◽

New Treatments ◽

Target Rna ◽

Single Amino Acid Mutation

Abstract The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.

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