Spliceostatin A Research Articles

Spliceostatin C, a component of a microbial bioherbicide, is a potent phytotoxin that inhibits the spliceosome.

Spliceostatin C (SPC) is a component of a bioherbicide isolated from the soil bacterium Burkholderia rinojensis. The chemical structure of SPC closely resembles spliceostatin A (SPA) which was characterized as an anticancer agent and splicing inhibitor. SPC inhibited the growth of Arabidopsis thaliana seedlings with an IC50 value of 2.2 µM. The seedlings exposed to SPC displayed a significant response with decreased root length and number and inhibition of gravitropism. Reverse transcriptase semi-quantitative PCR (RT-sqPCR) analyses of 19 selected genes demonstrated the active impact of SPC on the quality and quantity of transcripts that underwent intron rearrangements as well as up or down expression upon exposure to SPC. Qualitative and quantitative proteomic profiles identified 66 proteins that were significantly affected by SPC treatment. Further proteomics data analysis revealed that spliceostatin C induces hormone-related responses in Arabidopsis seedlings. In silico binding studies showed that SPC binds to a pocket between the SF3B3 and PF5A of the spliceosome.

Spliceostatin A interaction with SF3B limits U1 snRNP availability and causes premature cleavage and polyadenylation

RNA splicing, a highly conserved process in eukaryotic gene expression, is seen as a promising target for anticancer agents. Splicing is associated with other RNA processing steps, such as transcription and nuclear export; however, our understanding of the interaction between splicing and other RNA regulatory mechanisms remains incomplete. Moreover, the impact of chemical splicing inhibition on long non-coding RNAs (lncRNAs) has been poorly understood. Here, we demonstrate that spliceostatin A (SSA), a chemical splicing modulator that binds to the SF3B subcomplex of the U2 small nuclear ribonucleoprotein particle (snRNP), limits U1 snRNP availability in splicing, resulting in premature cleavage and polyadenylation of MALAT1, a nuclear lncRNA, as well as protein-coding mRNAs. Therefore, truncated transcripts are exported into the cytoplasm and translated, resulting in aberrant protein products. Our work demonstrates that active recycling of the splicing machinery maintains homeostasis of RNA processing beyond intron excision.

Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis of mRNA splicing relevant proteins in aging HSPCs.

HSPC (hematopoietic stem/progenitor cell) aging was closely associated with the organism aging, senile diseases and hematopoietic related diseases. Therefore, study on HSPC aging is of great significance to further elucidate the mechanisms of aging and to treat hematopoietic disease resulting from HSPC aging. Little attention had been paid to mRNA splicing as a mechanism underlying HSPC senescence. We used our lab's patented in vitro aging model of HSPCs to analyze mRNA splicing relevant protein alterations with iTRAQ-based proteomic analysis. We found that not only the notable mRNA splicing genes such as SR, hnRNP, WBP11, Sf3b1, Ptbp1 and U2AF1 but also the scarcely reported mRNA splicing relevant genes such as Rbmxl1, Dhx16, Pcbp2, Pabpc1 were significantly down-regulated. We further verified their gene expressions by qRT-PCR. In addition, we reported the effect of Spliceostatin A (SSA), which inhibits mRNA splicing in vivo and in vitro, on HSPC aging. It was concluded that mRNA splicing emerged as an important factor for the vulnerability of HSPC aging. This study improved our understanding of the role of mRNA splicing in the HSPC aging process.

Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression.

U1 small ribonucleoproteins demonstrate proteopathy in Alzheimer’s disease, and their inhibition modulates the expression of the amyloid precursor protein (APP). We sought to determine whether this effect on the APP expression is a universal result of different kinds of RNA splicing inhibitions. We treated cells with two chemical RNA splicing inhibitors: isoginkgetin (IGK) and spliceostatin A (SSA), in which SSA reduced the APP expression, whereas IGK substantially increased it. The following western blot and reverse transcription polymerase chain reaction analyses showed that the APP expression under the IGK treatment has distinct protein forms, but the total mRNA level was nearly unchanged despite a slight switch within its three major transcripts. Further analysis revealed that the APP-increasing effect of IGK depended on protein translation and might involve inhibition in the degradation system. By immunocytochemistry, the APP likely redistributed from Golgi to endoplasmic reticulum (ER) in cells treated with IGK. When compared to the well-characterized ER-to-Golgi transport inhibitor brefeldin A, IGK showed similar APP expression patterns on the western blot. In summary, we not only determined the diverse effects of RNA splicing inhibition on the APP expression but also found the additional function of IGK on protein subcellular traffic.

Global analysis of pre-mRNA subcellular localization following splicing inhibition by spliceostatin A

Spliceostatin A (SSA) is a methyl ketal derivative of FR901464, a potent antitumor compound isolated from a culture broth of Pseudomonas sp. no. 2663. These compounds selectively bind to the essential spliceosome component SF3b, a subcomplex of the U2 snRNP, to inhibit pre-mRNA splicing. However, the mechanism of SSA's antitumor activity is unknown. It is noteworthy that SSA causes accumulation of a truncated form of the CDK inhibitor protein p27 translated from CDKN1B pre-mRNA, which is involved in SSA-induced cell-cycle arrest. However, it is still unclear whether pre-mRNAs are uniformly exported from the nucleus following SSA treatment. We performed RNA-seq analysis on nuclear and cytoplasmic fractions of SSA-treated cells. Our statistical analyses showed that intron retention is the major consequence of SSA treatment, and a small number of intron-containing pre-mRNAs leak into the cytoplasm. Using a series of reporter plasmids to investigate the roles of intronic sequences in the pre-mRNA leakage, we showed that the strength of the 5′ splice site affects pre-mRNA leakage. Additionally, we found that the level of pre-mRNA leakage is related to transcript length. These results suggest that the strength of the 5′ splice site and the length of the transcripts are determinants of the pre-mRNA leakage induced by SF3b inhibitors.

Abstract 4336: Spliceosome inhibition as a novel therapeutic option in acute leukemia

Abstract Spliceosome targeting is a novel therapeutic strategy, showing promising results in solid tumors and hematological malignancies. Aberrant splicing of genes involved in apoptosis regulation and drug metabolism was shown to confer chemoresistance in various tumor cells, including acute leukemia. Moreover, increased expression of abnormal splice variants cause reduced sensitivity of leukemic cells to crucial components of current treatment protocols, such as glucocorticoids or methotrexate. Therefore, targeting the spliceosome holds potential to modulate drug resistance-related splicing and to eradicate cells, which do not respond to conventional therapy. In this study, we assessed the in vitro sensitivity of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) cells to spliceosome inhibitors, including meayamycin B (MAMB), pladienolide B (PB) and spliceostatin A (SSA). First, the growth inhibitory activity of MAMB was determined using a 72 h MTT assay in a panel of ALL and AML cell lines, including sublines with acquired resistance to conventional chemotherapeutics. Mechanistically, the effect of MAMB, PB and SSA on splicing profiles, cell cycle distribution and apoptosis induction was assessed in time course experiments. Finally, we compared MAMB sensitivity between 10 primary ALL, 10 AML specimens, and 6 healthy bone marrow (BM) specimens. Remarkably, both ALL and the notoriously apoptosis-resistant AML cell lines responded to subnanomolar concentrations of MAMB, with IC50 values (50% growth inhibition in the MTT assay) ranging between 0.07 and 0.16 nM. Moreover, MAMB retained full sensitivity towards leukemic sublines resistant to conventional chemotherapeutics with various modes of action, including methotrexate, dexamethasone, bortezomib and imatinib. MAMB, PB and SSA-induced growth inhibition was associated with time and dose-dependent alterations in splicing profiles of selected apoptosis-related genes (including Mcl-1, Bcl-X, FAS and Casp2), concomitant cell cycle arrest (in G1 and G2/M phases) and apoptosis induction (up to 40% after 24 h exposure to 1nM MAMB). Consistent with cell line observations, both primary ALL and AML specimens showed remarkable response to MAMB (mean LC50 = 0.42 nM, range: 0.26-0.69 nM and 0.43 nM, range: 0.33-0.44 nM, respectively), with a significantly lower MAMB sensitivity of healthy BM samples (mean lethal concentration causing 50% cell kill [LC50] value 0.57, range: 0.39-1.13 nM, p = 0.02). Collectively, this is the first study to demonstrate that spliceosome inhibition constitutes a promising therapeutic option for both ALL and AML patients, including those with acquired resistance to other anti-leukemic drugs. Financial support by KiKa (Children Cancer Free) Citation Format: Anna Wojtuszkiewicz, Rocco Sciarrillo, Gerrit Jansen, Yehuda G. Assaraf, Kazunori Koide, Robert K. Bressin, Upamanyu Basu, Edwin Sonneveld, Godefridus J. Peters, Gertjan J L Kaspers, Jacqueline Cloos. Spliceosome inhibition as a novel therapeutic option in acute leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4336.

Upregulation of p27 cyclin-dependent kinase inhibitor and a C-terminus truncated form of p27 contributes to G1 phase arrest

Potent anti-cancer compounds FR901464 and its methyl-ketal derivative spliceostatin A (SSA) inhibit cell cycle progression at G1 and G2/M phases. These compounds bind to the spliceosome and inhibit the splicing reaction. However, the molecular mechanism underlying G1 arrest after SSA treatment remains unknown. In this study, we found that ~90% of SSA-treated cells arrested at G1 phase after cell cycle synchronization. SSA treatment caused upregulation of the p27 cyclin-dependent kinase inhibitor both at mRNA and protein levels. In addition to p27, we observed expression of p27*, a C-terminal truncated form of p27 that is translated from CDKN1B (p27) pre-mRNA accumulated after splicing inhibition. Overexpression of p27 or p27* inhibited the exit from G1 phase after a double thymidine block. Conversely, knocking down of p27 by siRNA partially suppressed the G1 phase arrest caused by SSA treatment. There results suggest that G1 arrest in SSA-treated cells is caused, at least in part, by upregulation of p27 and p27*.

Splicing Inhibition By Spliceostatin_A Is a Promising Therapeutic Strategy in Chronic Lymphocytic Leukaemia

SF3B1 is a critical component of the RNA splicing machinery that achieves successful transcription and provides the functional diversity of protein species through alternative splicing. Recent studies have identified SF3B1 mutations in several tumours. In chronic lymphocytic leukemia (CLL) these mutations are associated with altered splicing, reduced survival and resistance to treatment. We therefore hypothesised that inhibition of the spliceosome may be an effective therapeutic strategy in SF3B1 mutated individuals.Initially, we utilized Spliceostatin A (SSA), an agent known to inhibit SF3B1 and alter gene splicing in various cell types. We showed that in CLL cells (SF3B1 WT and mutated samples), SSA treatment resulted in a dose-dependent increase in the un-spliced mRNA of DNAJB1 and RIOK3, two genes known to be regulated by SF3B1. We then assessed the ability of SSA to kill CLL cells with an SF3B1 mutant (n=12) or wild-type (n=28) genotype over a 24 hour period. CLL cells, regardless of SF3B1 mutational status, underwent apoptosis in a dose- (Figure A) and time-dependent manner (data not shown), with a mean IC50 of 5.1 nM. Interestingly, SSA induced more apoptosis in IGHV unmutated-CLL (U-CLL) compared to mutated-CLL samples (M-CLL) (p=0.037) and was active in 17p-deleted CLL (n=7), an aggressive disease sub-type that exhibits pronounced resistance to standard treatments. Normal B and T cells were significantly more resistant to SSA treatment than CLL samples purified from age-matched normal donors (B-cells p=0.006, T-cells p=0.001) and CLL derived T cells (p=0.003).SSA-induced apoptosis proceeded via a caspase-dependent mechanism (inhibited by ZVAD.FMK) coincident with elevations in Noxa. No changes in Bcl-2 or Bcl-xL were observed. Given the known ability of Noxa to interact with, and destabilise, Mcl-1, we next assessed the ability of SSA to modulate Mcl-1, and observed a substantial decrease in protein expression (Figure B, p=0.028). SSA treatment also altered the splicing of MCL-1; reducing the proportion of full length MCL-1L and increasing the amount of MCL-1s transcripts produced. The key role of Mcl-1 in regulating SSA-induced apoptosis was also observed in Ramos cells where over-expression of Mcl-1L, significantly protected them from SSA-induced apoptosis (p=0.001). Furthermore, in CLL cells exposed to CD40L and IL-4, factors known to induce Mcl-1 expression, SSA-induced apoptosis was significantly reduced (p=0.008). Taken together, these data implicate aberrant Mcl-1 splicing and Noxa upregulation in the apoptosis induced after SSA exposure. CD40L and IL-4 are proposed to be key factors preventing the apoptosis of CLL cells in the proliferative niche. Therefore, to explore possible strategies for overcoming this resistance, we investigated combining SSA with the Bcl-2/Bcl-xL inhibitor ABT-263 and showed that dual exposure to both inhibitors significantly increased apoptosis overcoming the protective effects of IL-4/CD40L (p=0.004; Figure C).In conclusion, our data shows that SSA induces apoptosis at nanomolar concentrations in CLL cells, independently of SF3B1 mutational status, through effects on Mcl-1. In combination with ABT-263, we show that SSA is particular active and can overcome the protective effects of CD40L and IL-4, suggesting promising clinical utility. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

U2 snRNP is required for expression of the 3' end of genes.

Pre-mRNA in eukaryotes is subjected to mRNA processing, which includes capping, polyadenylation, and splicing. Transcription and mRNA processing are coupled, and this coupling stimulates mRNA processing; however, the effects of mRNA processing on transcription are not fully understood. In this study, we found that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA), or by an antisense oligonucleotide to U2 snRNA, caused gene-specific 3′-end down-regulation. Removal of SSA from the culture media restored expression of the 3′ ends of genes, suggesting that U2 snRNP is required for expression of the 3′ end of genes. Finally, we found that SSA treatment caused accumulation of Pol II near the 5′ end of 3′-end down regulated genes, such as CDK6, SMEK2 and EGFR, indicating that SSA treatment led to transcription elongation arrest on these genes. These findings suggest that U2 snRNP is important for production of full length mRNA probably through regulation of transcription elongation, and that a novel checkpoint mechanism prevents pre-mRNA from accumulating as a result of splicing deficiencies, and thereby prevents production of aberrant proteins that might be translated from pre-mRNAs through the arrest of transcription elongation.

U2 snRNP is required for the phosphorylation of Pol II CTD Ser2 and efficient transcription elongation (560.4)

Pre‐mRNA in eukaryotes is subjected to post‐transcriptional modifications, such as capping, polyadenylation and splicing. Transcription and post‐transcriptional modifications are coupled and this coupling stimulates post‐transcriptional modifications, however, the effects of post‐transcriptional modifications on transcription are not fully understood. Here, we report that inhibition of U2 snRNP by a splicing inhibitor, spliceostatin A (SSA), and an antisense morpholino oligonucleotide to U2 snRNA caused 3’ end down regulation in a gene‐specific manner. We also show that U2 snRNP inhibition resulted in the dephosphorylation of second serine residues (Ser2) within heptad repeats of the C‐terminal domain (CTD) of Pol II, which is important for transcription elongation; this dephosphorylation was correlated with splicing inactivation. Interestingly, SSA treatment caused accumulation of Pol II near the 5’ ends of genes, indicating that SSA treatment led to transcription elongation arrest. These findings suggest that a novel checkpoint mechanism prevent pre‐mRNA accumulation and the production of aberrant proteins translated from pre‐mRNA upon splicing deficiencies through the dephosphorylation of CTD Ser2.

Splicing inhibition induces gene expression through canonical NF‐κB pathway and extracellular signal‐related kinase activation

Splicing, a process for mRNA maturation, is essential for correct gene expression after transcription. However, recent studies also suggest that splicing affects transcription, but its mechanism remains elusive. We previously reported that treatment with spliceostatin A (SSA), a specific splicing inhibitor targeting the splicing factor SF3b, leads to transcriptional activation of a small subset of genes. To investigate the underlying mechanism we utilized luciferase reporters driven by the Interleukin 8 (IL-8) and cytomegalovirus (CMV) promoters, as both recruit a similar set of transcription factors. We also found that SSA treatment led to increased extracellular signal-regulated protein kinase (ERK) activity and that chemical inhibition of ERK also led to decreased promoter activation. Systematic deletion studies suggested that NF-κB activation is mainly responsible for SSA-induced promoters activation.

Non-transmissible Sendai virus vector encoding c-myc suppressor FBP-interacting repressor for cancer therapy.

To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element (FUSE)-binding protein-interacting repressor (FIR). Endogenous c-Myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR (HA-FIR) expression vector. A fusion gene-deficient, non-transmissible, Sendai virus (SeV) vector encoding FIR cDNA, SeV/dF/FIR, was prepared. SeV/dF/FIR was examined for its gene transduction efficiency, viral dose dependency of antitumor effect and apoptosis induction in HeLa (cervical squamous cell carcinoma) cells and SW480 (colon adenocarcinoma) cells. Antitumor efficacy in a mouse xenograft model was also examined. The molecular mechanism of the anti-tumor effect and c-Myc suppression by SeV/dF/FIR was examined using Spliceostatin A (SSA), a SAP155 inhibitor, or SAP155 siRNA which induce c-Myc by increasing FIR∆exon2 in HeLa cells. FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression. Thus, FIR expressing vectors are potentially applicable for cancer therapy. FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2 (FIR∆exon2), counteracting FIR for c-Myc protein expression. Furthermore, FIR forms a complex with SAP155 and inhibits mutual well-established functions. Thus, both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application. SeV/dF/FIR, a cytoplasmic RNA virus, was successfully prepared and showed highly efficient gene transduction in in vivo experiments. Furthermore, in nude mouse tumor xenograft models, SeV/dF/FIR displayed high antitumor efficiency against human cancer cells. SeV/dF/FIR suppressed SSA-activated c-Myc. SAP155 siRNA, potentially produces FIR∆exon2, and led to c-Myc overexpression with phosphorylation at Ser62. HA-FIR suppressed endogenous c-Myc expression and induced apoptosis in HeLa and SW480 cells. A c-myc transcriptional suppressor FIR expressing SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells. SeV/dF/FIR showed strong tumor growth suppression with no significant side effects in an animal xenograft model, thus SeV/dF/FIR is potentially applicable for future clinical cancer treatment.

Establishment of a Monitoring System to Detect Inhibition of mRNA Processing

A number of proteins complete mRNA processing in the nucleus, thus, inhibitor of mRNA processing is worth finding to analyze the mechanism of mRNA maturation in detail. Here, we established a monitoring system for mRNA processing using a test compound, spliceostatin A (SSA), which inhibits mRNA splicing. This system should serve to facilitate the discovery of novel compounds from natural resources that inhibit mRNA processing.

Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A

Spliceostatin A (SSA) is a stabilized derivative of a Pseudomonas bacterial fermentation product that displays potent anti-proliferative and anti-tumor activities in cancer cells and animal models. The drug inhibits pre-mRNA splicing in vitro and in vivo and binds SF3b, a protein subcomplex of U2 small nuclear ribonucleoprotein (snRNP), which is essential for recognition of the pre-mRNA branch point. We report that SSA prevents interaction of an SF3b 155-kDa subunit with the pre-mRNA, concomitant with nonproductive recruitment of U2 snRNP to sequences 5' of the branch point. Differences in base-pairing potential with U2 snRNA in this region lead to different sensitivity of 3' splice sites to SSA, and to SSA-induced changes in alternative splicing. Indeed, rather than general splicing inhibition, splicing-sensitive microarray analyses reveal specific alternative splicing changes induced by the drug that significantly overlap with those induced by knockdown of SF3b 155. These changes lead to down-regulation of genes important for cell division, including cyclin A2 and Aurora A kinase, thus providing an explanation for the anti-proliferative effects of SSA. Our results reveal a mechanism that prevents nonproductive base-pairing interactions in the spliceosome, and highlight the regulatory and cancer therapeutic potential of perturbing the fidelity of splice site recognition.

Spliceostatin A blocks angiogenesis by inhibiting global gene expression including VEGF

Spliceostatin A (SSA) is a methylated derivative of an antitumor natural product FR901464, which specifically binds and inhibits the SF3b spliceosome sub-complex. To investigate the selective antitumor activity of SSA, we focused on the regulation of vascular endothelial growth factor (VEGF) mRNA, since VEGF is a key regulatory component in tumor angiogenesis and known for the intricate regulation of mRNA processing, such as alternative splicing. We found that in HeLa cells SSA reduced the amount of both mRNA and protein of VEGF. Spliceostatin A not only inhibited the splicing reaction of VEGF pre-mRNA but also reduced the total amount of VEGF's transcripts, while SSA affected GAPDH mRNA to a lesser extent. Given a significant reduction in VEGF gene expression, SSA was expected to possess anti-angiogenic activity in vivo. Indeed, SSA inhibited cancer cell-derived angiogenesis in vivo in a chicken chorioallantoic membrane (CAM) assay. The inhibition of angiogenesis with SSA was abolished by addition of exogenous VEGF. We also performed global gene expression analyses of HeLa cells and found that the expression levels of 38% of total genes including VEGF decreased to <50% of the basal levels following 16 h of SSA treatment. These results suggest that the global interference of gene expression including VEGF in tumor cells is at least one of the mechanisms by which SSA (or FR901464) exhibits its strong antitumor activity.

Spliceostatin A inhibits spliceosome assembly subsequent to prespliceosome formation

Pre-mRNA splicing is catalyzed by the large ribonucleoprotein spliceosome. Spliceosome assembly is a highly dynamic process in which the complex transitions through a number of intermediates. Recently, the potent anti-tumor compound Spliceostatin A (SSA) was shown to inhibit splicing and to interact with an essential component of the spliceosome, SF3b. However, it was unclear whether SSA directly impacts the spliceosome and, if so, by what mechanism, which limits interpretation of the drugs influence on splicing. Here, we report that SSA inhibits pre-mRNA splicing by interfering with the spliceosome subsequent to U2 snRNP addition. We demonstrate that SSA inhibition of spliceosome assembly requires ATP, key pre-mRNA splicing sequences and intact U1 and U2 snRNAs. Furthermore all five U snRNAs in addition to the SSA molecule associate with pre-mRNA during SSA inhibition. Kinetic analyses reveal that SSA impedes the A to B complex transition. Remarkably, our data imply that, in addition to its established function in early U2 snRNP recruitment, SF3b plays a role in later maturation of spliceosomes. This work establishes SSA as a powerful tool for dissecting the dynamics of spliceosomes in cells. In addition our data will inform the design of synthetic splicing modulator compounds for targeted anti-tumor treatment.