scholarly journals MDS-Associated Splicing Factor Mutations Promote Alternative Splicing of a Differentiation-Impaired CSF3R Isoform

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3128-3128
Author(s):  
Borwyn Wang ◽  
Hrishikesh M Mehta ◽  
Seth J. Corey
Keyword(s):  
Alternative Splicing ◽  
K562 Cells ◽  
Chromosome 7 ◽  
Splicing Factor ◽  
Class I ◽  
Expression Vectors ◽  
Aberrant Splicing ◽  
Cd34 Cells ◽  
Neutrophil Differentiation ◽  
Class Iv

Abstract Background. Myelodysplastic syndromes (MDS) constitute the most common group of bone marrow failure disorders, characterized by ineffective hematopoiesis and a significant risk of transformation to AML. Efforts to understand the molecular basis of MDS led to the identification of acquired somatic mutations in the RNA splicing factors SF3B1, U2AF1, SRSF2, ZRSR2, and LUC7L2 in 45-85% of adult MDS patients. How they perturb hematopoiesis and promote expansion of abnormal clones remain unknown. Elevated levels of alternate isoforms of granulocyte colony stimulating factor receptor (CSF3R) patients with MDS and other myeloid neoplasias have been reported with a strong correlation reported with loss of chromosome 7. Constitutive splicing produces the canonical Class I CSF3R isoform that supports proliferation and differentiation. Alternative splicing promoting intron excision in CSF3R exon 17 results in a differentiation-defective Class IV CSF3R isoform. We hypothesized that aberrant splicing activity of MDS-associated splicing factor mutations promote Class IV CSF3R expression, resulting in dysgranulopoiesis. Methods. We constructed a CSF3R minigene that was transiently expressed in K562 cells along with eukaryotic expression vectors containing cDNAs for wild-type or mutant forms of SF3B1, U2AF1, or SRSF2. To determine role of LUC7L2, a splicing factor residing on chromosome 7, the CSF3R minigene was transiently expressed in K562 cells with knockout LUC7L2. Class IV or Class I was measure by qPCR using specific primers. Putative exonic splicing enhancer motifs (ESEs) within exon 17 were deleted in the minigene and transfected into K562 cells expressing either wildtype or mutant SRSF2. To determine impact of mutant SRSF2 on granulopoiesis, wildtype or mutant SRSF2 were overexpressed in GCSF-treated CD34+ cells and morphology was assessed on day 14. To determine impact of Class IV isoform on granulopoiesis, Lin-, Sca-1+, and c-Kit+ (LSK) cells from Csf3r null mice were transduced with Class I or Class IV and colony forming unit (CFU) assay for granulopoiesis was performed with Methocult 3534. Colony scores and cell morphology were assessed on day 8. Results. K562 cells with transient expression of mutant SRSF2 P95H and LUC7L2 knockout resulted in statistically significant increase in Class IV:I CSF3R mRNA ratios normalized to a minigene-only control. Interestingly, expression of mutant U2AF1 S34F significantly decreased Class IV:I while U2AF1 Q157P increased Class IV:I ratios. K562 cells expressing mutant SF3B1 K700E did not show significant differences in Class IV splicing. To further investigate how SRSF2 regulates CSF3R splicing, we deleted two putative SRSF2-binding ESE motifs (ESE1 and ESE2) within exon 17 of the CSF3R minigene and transfected them into K562 cells stably expressing either wildtype SRSF2 or mutant P95H, P95L, or P95R. Deletion of either ESE1 or ESE2 resulted in decreased Class IV:I in all SRSF2 P95 mutant cells compared to minigene-only and wildtype controls. Next, we assessed the effect of SRSF2 P95 mutations on neutrophil differentiation in CD34+ cells. Overexpression of SRSF2 P95H, P95L, or P95R led to increased neutrophilic precursors compared to untransduced CD34+ cells. To determine the effect of a single CSF3R isoform on granulopoiesis, we cultured LSK cells from Csf3r null mice transduced with either Class I or Class IV on MethoCult 3534 with addition of GCSF. On day 8, cells expressing Class I had more total colony numbers with significantly higher CFU-GM colonies than cells expressing Class IV compared to empty vector control. Conclusions. We demonstrated that mutated SRSF2, U2AF1, and LUC7L2 deficiency alters CSF3R splicing in its terminal exon. Interestingly, mutations on different residues of the same gene (U2AF1) had opposing effects on CSF3R splicing. Mutant SRSF2 resulted in increased intron excision to promote increased levels of Class IV isoform. Mutation of two putative SRSF2 binding sites within CSF3R exon 17 reversed the increased splicing promoted by mutant SRSF2, further support our observation that CSF3R is a target for mutant SRSF2. Our observation with overexpression of mutant SRSF2 P95 in CD34+ cells suggests that defective neutrophil differentiation is related to increased Class IV. Our findings shed insights into how aberrant splicing of CSF3R drives MDS progression and provides a new model of dysgranulopoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals The SF3B1 K700E Mutation Induces R-Loop Accumulation and Associated DNA Damage

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4219-4219 ◽  
Author(s):  
Shalini Singh ◽  
Doaa Ahmed ◽  
Hamid Dolatshad ◽  
Dharamveer Tatwavedi ◽  
Ulrike Schulze ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
K562 Cells ◽  
Splicing Factor ◽  
Leukemia Cells ◽  
Cd34 Cells ◽  
Damage Response ◽  
Healthy Control ◽  
Atr Activation ◽  
Mutant Cells

The myelodysplastic syndromes (MDS) are common myeloid malignancies. Mutations in genes involved in pre-mRNA splicing (SF3B1, SRSF2, U2AF1 and ZRSR2) are the most common mutations found in MDS. There is evidence that some spliceosomal components play a role in the maintenance of genomic stability. Splicing is a transcription coupled process; splicing factor mutations affect transcription and may lead to the accumulation of R-loops (RNA-DNA hybrids with a displaced single stranded DNA). Mutations in the splicing factors SRSF2 and U2AF1 have been recently shown to increase R-loops formation in leukemia cell lines, resulting in increased DNA damage, replication stress and activation of the ATR-Chk1 pathway. SF3B1 is the most frequently mutated splicing factor gene in MDS, but a role for mutated SF3B1 in R-loop accumulation and DNA damage has not yet been reported in hematopoietic cells. We have investigated the effects of the common SF3B1 K700E mutation on R-loop formation and DNA damage response in MDS and leukemia cells. R-loop signals and the DNA damage response were measured by immunofluorescence staining using S9.6 and anti-γ-H2AX antibodies respectively. Firstly, we studied K562 (myeloid leukemia) cells with the SF3B1 K700E mutation and isogenic SF3B1 K700K wildtype (WT) K562 cells. K562 cells with SF3B1 mutation showed a significant increase in the number of S9.6 foci [Fold change (FC) 2.01, p<0.001] and in the number of γ-H2AX foci (FC 2.32, p<0.001), indicating increased R-loops and DNA damage, compared to SF3B1 WT K562 cells. Moreover, we observed increased Chk1 phosphorylation at Ser345, a hallmark of activation of the ATR pathway, in SF3B1 mutant K562 cells. Next, we analyzed induced pluripotent stem cells (iPSCs) that we generated from the bone marrow cells of one SF3B1 mutant MDS patient and of one healthy control. A significant increase in R-loops and DNA damage response was observed in an iPSC clone harboring SF3B1 mutation compared to another iPSC clone without SF3B1 mutation obtained from same MDS patient (S9.6 mean fluorescence intensity - FC 1.72, p<0.001; γ-H2AX foci - FC 1.34, p=0.052) and to iPSCs from the healthy control (S9.6 mean fluorescence intensity - FC 1.53, p<0.001; γ-H2AX foci - FC 1.61, p=0.006). In addition, bone marrow CD34+ cells from a SF3B1 mutant MDS patient showed increased R-loops (as measured by number of S9.6 foci) compared to CD34+ cells from a MDS patient without splicing factor mutations (FC 1.9) and from a healthy control (FC 2.6). To investigate whether the observed DNA damage and ATR activation in SF3B1 mutant K562 cells result from induced R-loops, we overexpressed RNASEH1 (encoding an enzyme that degrades the RNA in RNA:DNA hybrids) to resolve R-loops in these cells. RNASEH1 overexpression significantly reduced the number of S9.6 (FC 0.51, p<0.001) and γ-H2AX foci (FC 0.63, p=0.035) in SF3B1 mutant K562 cells compared to SF3B1 WT K562 cells. RNASEH1 overexpression also resulted in decreased Chk1 phosphorylation, indicating suppression of ATR pathway activation in SF3B1 mutant K562 cells. To determine the functional importance of ATR activation associated with SF3B1 mutation, we evaluated the sensitivity of SF3B1 mutant cells towards the ATR inhibitor VE-821. SF3B1 mutant K562 cells showed preferential sensitivity towards VE-821 compared to SF3B1 WT K562 cells. Chk1 is a critical substrate of ATR, and we next investigated the effects of Chk1 inhibition in SF3B1 mutant cells. Interestingly, SF3B1 mutant K562 cells demonstrated preferential sensitivity towards the Chk1 inhibitor UCN-1 (IC50 61.8 nM) compared to SF3B1 WT K562 cells (IC50 267 nM), suggesting that ATR activation is important for the survival of SF3B1 mutant cells. SF3B1 mutant K562 cells were preferentially sensitive to the splicing modulator Sudemycin D6 (IC50 53.2 nM) compared to SF3B1 WT K562 cells (IC50 130.7 nM). The effects of VE-821 and UCN-1 on SF3B1 mutant K562 cells were enhanced by Sudemycin D6 (Combination index <1), indicating synergy. In summary, our results show that the SF3B1 mutation leads to accumulation of R-loops and associated DNA damage resulting in activation of the ATR pathway in MDS and leukemia cells. Thus different mutated splicing factors have convergent effects on R-loop elevation leading to DNA damage. Moreover, our data suggest that Chk1 inhibition, alone or in combination with splicing modulators, may represent a novel therapeutic strategy to target SF3B1 mutant cells. Disclosures Schuh: Janssen: Speakers Bureau; Verastem: Speakers Bureau; Kite: Speakers Bureau; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau. Wiseman:Novartis, Celgene: Consultancy, Honoraria.

Profound Signaling Abnormalities Due to Cytoplasmic Variants of CSF3R in Patients with Myelodysplastic/Myeloproliferative Syndromes Associated with CSF3R Mutations or Mutations of Splicing Factor U2AF1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3792-3792
Author(s):  
Hrishikesh Mehta ◽  
Hideki Maskishima ◽  
Muneyoshji Futami ◽  
Wei-Ming Kao ◽  
Bartlomiej P Przychodzen ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Research Funding ◽  
Exon Skipping ◽  
Hematopoietic Cell ◽  
Class I ◽  
Leukemia Cell Line ◽  
Genetic Mutations ◽  
Nonsense Mutations ◽  
Alternatively Spliced ◽  
Class Iv

Abstract Abstract 3792 Myelodysplastic Syndromes (MDS) are a group of bone marrow disorders closely related to acute myeloid leukemia (AML). Even though a number of genetic mutations have been recently identified in patients with MDS, their contributions to MDS pathogenesis remains poorly understood. Some of these genetic mutations involve transcription factors, but they are also found in AML: TET2, EZH2, and ASXL1. One group of mutations distinct to MDS are those encoding proteins involved in RNA splicing (e.g. U2AF1/U2AF35, ZRSR2, SRSF2, SF3B1). Based on RNA-Seq of MDS/AML patients, we report exon skipping in the 3′ end of the CSF3R transcript, which encodes the granulocyte colony-stimulating factor receptor (GCSFR), in a patient carrying the S34F mutation in the U2AF1 gene. U2AF1 is one of the more recurrent genes affected by mutation in MDS, and it is associated with progression to secondary AML. The S34F mutation in U2AF1 is a gain of function mutation that promotes excess splicing and exon skipping. Alternative splicing of CSF3R results in 7 transcripts, of which the two most common are Class I and Class IV. There are putative splicing sequences within exon 17 of the CSF3R locus; GT (GU) at the 5′ site and AG at the 3′ site - a recognition sequence for U2AF1. In addition, we also identified mutations affecting CSF3R in two patients with chronic myelomonocytic leukemia (GCSFR T595I or Q726X), one patient with Refractory Cytopenias with Multilineage Dysplasia and Ring Sideroblasts (GCSFR W650L), and one patient with primary AML (GCSFR G659fs). In the last case, this mutation affects the Class III transcript of CSF3R, an alternatively spliced form expressed highly in the placenta. The Class IV isoform lacks much of the C-terminal domain, similar to the protein produced by nonsense mutations found in patients with severe congenital neutropenia who develop MDS/AML or the patient we identified. Little is known about the signaling-phenotype relationship of a mutant or alternatively-spliced GCSFR. To address these questions, we first studied the expression patterns of Class I and Class IV GCSFR in the human NB4 promyelocytic leukemia cell line and primary human hematopoietic stem (CD34+) cells induced to differentiate into neutrophils. Quantitative PCR of Class I and Class IV transcripts showed a positive feedback loop for Class I. Expression of the Class IV transcript was downregulated during hematopoietic cell differentiation. Scatchard analysis showed no differences between the two receptors in high-affinity Kd (∼ 500 nM) for the GCSF-GCSFR. Because Class IV lacks the C-terminal di-leucyl motif that facilitates internalization, we measured internalization rates and found that indeed the Class IV internalized more slowly and less completely. Using an MTT assay to measure proliferation we observed Class IV isoform had lower proliferative capacity at lower GCSF concentrations (0.1 – 2 nM GCSF); however at higher GCSF dose (>100 nM) its proliferative response was greater than Class I. Using western blotting we observed that the Class IV isoform showed weaker signaling via the JAK/STAT and ERK1/2 pathways, but had higher Lyn activity when treated with 100 ng/ml GCSF. To determine effects on differentiation, we made chimeric human growth hormone receptor-GCSFR for transfection into murine 32D cells. 32D cells express low levels of murine GCSFR, thus we made the chimeric receptor. Expression of Class IV Receptor impaired their differentiation (as demonstrated by morphology and Gr-1 expression). We are now developing a mouse model of perturbed hematopoiesis due to dysregulated expression of Class IV GCSFR. Altogether, our studies show that S34F mutation of U2AF1 splicing gene is associated with exon skipping of CSF3R. This would result in expression of a C-terminal truncated GCSFR, similar to that observed in patients with nonsense mutations or alternative splicing. A C-terminal truncated GCSFR causes aberrant hematopoietic cell proliferation, altered post-receptor signaling events, and impaired myeloid differentiation. Our findings and those involving GCSFR E785K in high-risk MDS (Wolfler et al, Blood 105:3731, 2005) strongly suggest that aberrant signaling by alterations in the C-terminus of the GCSFR contributes to the pathogenesis of MDS. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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

Blood ◽  
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.

scholarly journals Identification of T-Cell Receptors Specific to Antigens Presented By HLA-B4002 and B5401 in Acquired Aplastic Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3751-3751
Author(s):  
Kohei Hosokawa ◽  
Eiji Kobayashi ◽  
Yoshiki Akatsuka ◽  
Luis Espinoza ◽  
Noriharu Nakagawa ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hla Class I ◽  
K562 Cells ◽  
Cell Receptor ◽  
Class I ◽  
Company Limited ◽  
Cd34 Cells ◽  
Ifn Γ ◽  
A Current

[Background] Leukocytes that lack HLA class I alleles derived from hematopoietic stem progenitor cells (HSPCs) that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations are often detected in acquired aplastic anemia (AA) patients. The presence of HLA class I allele-lacking leukocytes provides compelling evidence that cytotoxic T lymphocytes (CTLs) are involved in the development of AA. Our recent study showed that, among several HLA-class I alleles that are likely to be lost as a result of 6pLOH, HLA-B*40:02 is the most frequently lost allele in AA. Therefore, HLA-B*4002 is thought to play a critical role in the autoantigen presentation by HSPCs to CTLs. We previously identified the T-cell receptor (TCR) sequences from bone marrow (BM) CD8+ T cells in two CsA-dependent AA patients possessing B4002-lacking leukocytes (Case 1, Espinoza et al, Blood Adv, 2018) and B5401-lacking leukocytes (Case 2, Elbadry et al, Haematologica, 2019) by single-cell T-cell receptor (TCR) sequencing. Identifying the TCRs specific to antigens presented by these HLA class I alleles should allow us to screen autoantigens in AA. [Method] We established B4002+ or B5401+ K562 cell lines expressing CD80 and CD137L for the screening of antigen-specific T cell responses. To identify ligands of the TCR, we transfected peripheral blood (PB) T cells with a retrovirus vector containing different TCR cDNA derived from BM T cells and examined their responses to B4002+CD80+CD137L+ or B5401+CD80+CD137L+ K562 cells. Specific responses of each TCR transfectant to K562 cells or iPSC-derived CD34+ cells were determined using an enzyme-linked immunosorbent assay for detecting IFN-γ. Deep TCR sequencing of a current PB sample taken from the same patients was performed to determine whether or not T cells with specific TCRs persisted after successful immunosuppressive therapy (IST). [Results] In Case 1, two TCR transfectants (TCR-K1 and TCR-K2 which were the third- and second-most frequent TCRs in the BM T cells, respectively) secreted greater IFN-γ levels (1730 pg/mL and 2157 pg/mL, respectively) in response to B4002+CD80+CD137L+ K562 cells than those secreted by the other six transfectants (710 to 1184 pg/mL, respectively). TCR-K1 and TCR-K2 did not respond to an A2402+ counterpart (Figure). Notably, deep TCR sequencing of a current PB sample taken from Case 1 nine years after BM sampling revealed the persistence of the TCR-K1 sequence, suggesting that TCR-K1 may be responsible for CsA dependency of this patient. Deep TCR sequencing of other three AA patients with B4002-lacking leukocytes revealed decreased diversity of the T cell repertoire in CD8+ T cells but failed to reveal the same TCR motifs as Case 1. In Case 2, two TCR transfectants (TCR-K3 and TCR-K4) showed a specific response to B5401+CD80+CD137L+ K562 cells. Furthermore, these 2 TCR transfectants secreted higher amounts of IFN-γ (1.7 and 2.0 folds for TCR-K3 and TCR-K4, respectively) in response to wild-type iPSC-derived CD34+ cells than to B5401(-) CD34+ cells. [Conclusions] Our results suggest that these TCR transfectants recognized some intrinsic antigens derived from K562 cells in a B4002 or B5401-restricted manner. These TCR transfectants are the ideal tools for screening libraries of cDNA expressed by B4002+ COS/293T cells to identify autoantigens in AA. Figure Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Chugai Pharmaceutical Co.,Ltd: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Kyowa Kirin: Honoraria; Novartis Pharma K.K: Honoraria.

scholarly journals The Splicing Factor RBM17 Supports Leukemia Stem Cell Self-Renewal

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1238-1238
Author(s):  
Lina Liu ◽  
Ana Vujovic ◽  
Joshua Xu ◽  
Kristin Hope ◽  
Yu Lu
Keyword(s):  
Stem Cell ◽  
Alternative Splicing ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Splicing Factor ◽  
Binding Motif ◽  
Aberrant Splicing ◽  
Self Renewal ◽  
Hematopoietic Stem

Background: Acute myeloid leukemia (AML) is thought to be sustained by sub-populations of leukemia stem cells (LSCs), which possess the capacity for self-renewal and differentiation and are believed to be responsible for disease initiation, relapse and chemoresistance. There is therefore an urgent need to develop therapies that target the LSC population to achieve effective AML treatment. To date there has been limited success in this endeavor, highlighting the importance of gaining a more comprehensive understanding of the mechanistic elements that underpin LSC function. Aberrant alternative splicing is recognized as a key driver of cancer. In the context of AML, genome-wide sequencing studies have shown that approximately one third of genes are differentially spliced in primitive CD34+ cells in AML patients compared to those obtained from normal controls. In particular, LSCs also have a unique splicing profile when compared to normal aging HSCs, underscoring the importance of understanding the mechanistic controls of aberrant splicing in LSC function. Results: Previous studies examining the link between aberrant splicing and AML focused on spliceosome genes with somatic mutations in AML patients. To examine mechanisms that mediate aberrant alternative splicing in LSC beyond splicing factor mutations, we performed a data-mining survey of 203 known mRNA splicing factors. Strikingly, RNA-binding motif protein 17 (RBM17) is the only splicing factor that is both strongly linked to poor AML prognosis and significantly elevated in LSC-enriched subsets of primary AML samples based on a recent study of 78 AML samples with normal karyotype. RBM17 has been implicated in regulating alternative splicing and cancer chemotherapy resistance. However, its function in AML or LSCs is not known. From our studies, we found that the level of RBM17 protein is elevated in the phenotypically primitive subsets of primary AML samples (n=8, RBM17+%: 67.42% in CD34+ versus 34.66% in CD34-fractions). Depletion of RBM17 with shRNAs in 3 human primary AML samples resulted in reduced colony formation compared to shScramble controls. More importantly, knockdown of RBM17 in a primary AML sample greatly impeded AML engraftment in immune-deficient mice (mean of 0.89 control versus mean of 0.1671 and 0.3171 shRNAs), suggesting that RBM17 is required for the stem and progenitor potential of AML and maintenance of LSC populations. Intriguingly, in contrast to the situation for the malignant hierarchy, the level of RBM17 in normal HSCs is lower than that in more committed cell populations in the normal hematopoietic system. To determine if RBM17 plays different roles in malignant LSC and normal hematopoietic stem and progenitor cells (HSPCs), we depleted RBM17 with shRNAs in human cord blood (CB) derived CD34+ HSPCs, and found RBM17 knockdown had negligible adverse impact on both CB total colony and primitive GEMM colony outputs and yielded no increase in apoptosis, indicating no defects were apparent to primitive cells of CB as read out in vitro. To uncover the molecular mechanisms underlying the role of RBM17 in LSC functions, we preformed RBM17 eCLIP-seq in the K562 and HL60 human leukemic cell lines. We then cross-analyzed the CLIP-seq datasets with a published ENCODE RNA-seq dataset (RBM17 knockdown in K562 cells), where we found RBM17 directly binds to transcripts of stem cell program-related genes and regulates the splicing of these genes, including MADD (MAP kinase activating death domain) and MRPS18C (mitochondrial ribosomal protein S18C). We further demonstrated that the splicing patterns of MADD and MRPS18C are mediated by RBM17 in primary AML samples. In our ongoing functional validation experiments, isoform-specific knockdown of MADD or MRPS18C splice variants downstream of RBM17 impeded colony forming capacity and induced myeloid differentiation in the MOLM13 AML cell line. These results suggest that RBM17-mediated splicing events impact primitive cell function in AML. Conclusion: We have identified RBM17 as a novel LSC-regulating factor, plays an important role in maintaining AML LSC function through regulating the alternative splicing of stem cell program-related genes. The potential LSC-selective role for RBM17, along with its downstream splicing events, represent promising putative therapeutic targets whose modulation could offer attractive therapeutic windows in AML treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Splicing Genomics Events in Cervical Cancer: Insights for Phenotypic Stratification and Biomarker Potency

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 130
Author(s):  
Flavia Zita Francies ◽  
Sheynaz Bassa ◽  
Aristotelis Chatziioannou ◽  
Andreas Martin Kaufmann ◽  
Zodwa Dlamini
Keyword(s):  
Cervical Cancer ◽  
Alternative Splicing ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Therapy Resistance ◽  
Splicing Factors ◽  
Aberrant Splicing ◽  
Molecular Alteration ◽  
Common Cancer ◽  
Potential Biomarkers

Gynaecological cancers are attributed to the second most diagnosed cancers in women after breast cancer. On a global scale, cervical cancer is the fourth most common cancer and the most common cancer in developing countries with rapidly increasing mortality rates. Human papillomavirus (HPV) infection is a major contributor to the disease. HPV infections cause prominent cellular changes including alternative splicing to drive malignant transformation. A fundamental characteristic attributed to cancer is the dysregulation of cellular transcription. Alternative splicing is regulated by several splicing factors and molecular changes in these factors lead to cancer mechanisms such as tumour development and progression and drug resistance. The serine/arginine-rich (SR) proteins and heterogeneous ribonucleoproteins (hnRNPs) have prominent roles in modulating alternative splicing. Evidence shows molecular alteration and expression levels in these splicing factors in cervical cancer. Furthermore, aberrant splicing events in cancer-related genes lead to chemo- and radioresistance. Identifying clinically relevant modifications in alternative splicing events and splicing variants, in cervical cancer, as potential biomarkers for their role in cancer progression and therapy resistance is scrutinised. This review will focus on the molecular mechanisms underlying the aberrant splicing events in cervical cancer that may serve as potential biomarkers for diagnosis, prognosis, and novel drug targets.

scholarly journals Practice parameter: Temporal lobe and localized neocortical resections for epilepsy

Neurology ◽  
2003 ◽  
Vol 60 (4) ◽  
pp. 538-547 ◽  
Author(s):  
J. Engel ◽  
S. Wiebe ◽  
J. French ◽  
M. Sperling ◽  
P. Williamson ◽  
...  
Keyword(s):  
Temporal Lobe ◽  
Controlled Trial ◽  
Mesial Temporal Lobe Epilepsy ◽  
Class I ◽  
Partial Seizures ◽  
Complex Partial Seizures ◽  
Single Class ◽  
Intention To Treat ◽  
Epileptic Syndromes ◽  
Class Iv

Objectives/Methods: To examine evidence for effectiveness of anteromesial temporal lobe and localized neocortical resections for disabling complex partial seizures by systematic review and analysis of the literature since 1990.Results: One intention-to-treat Class I randomized, controlled trial of surgery for mesial temporal lobe epilepsy found that 58% of patients randomized to be evaluated for surgical therapy (64% of those who received surgery) were free of disabling seizures and 10 to 15% were unimproved at the end of 1 year, compared with 8% free of disabling seizures in the group randomized to continued medical therapy. There was a significant improvement in quantitative quality-of-life scores and a trend toward better social function at the end of 1 year for patients in the surgical group, no surgical mortality, and infrequent morbidity. Twenty-four Class IV series of temporal lobe resections yielded essentially identical results. There are similar Class IV results for localized neocortical resections; no Class I or II studies are available.Conclusions: A single Class I study and 24 Class IV studies indicate that the benefits of anteromesial temporal lobe resection for disabling complex partial seizures is greater than continued treatment with antiepileptic drugs, and the risks are at least comparable. For patients who are compromised by such seizures, referral to an epilepsy surgery center should be strongly considered. Further studies are needed to determine if neocortical seizures benefit from surgery, and whether early surgical intervention should be the treatment of choice for certain surgically remediable epileptic syndromes.

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