Spliceosome Inhibitors Research Articles

The Natural Product N-Palmitoyl-l-leucine Selectively Inhibits Late Assembly of Human Spliceosomes

The spliceosome is a dynamic complex of five structural RNAs and dozens of proteins, which assemble together to remove introns from nascent eukaryotic gene transcripts in a process called splicing. Small molecules that target different components of the spliceosome represent valuable research tools to investigate this complicated macromolecular machine. However, the current collection of spliceosome inhibitors is very limited. To expand the toolkit we used a high-throughput in vitro splicing assay to screen a collection of pre-fractions of natural compounds derived from marine bacteria for splicing inhibition. Further fractionation of initial hits generated individual peaks of splicing inhibitors that interfere with different stages of spliceosome assembly. With additional characterization of individual peaks, we identified N-palmitoyl-l-leucine as a new splicing inhibitor that blocks a late stage of spliceosome assembly. Structure-activity relationship analysis of the compound revealed that length of carbon chain is important for activity in splicing, as well as for effects on the cytological profile of cells in culture. Together these results demonstrate that our combination of in vitro splicing analysis with complex natural product libraries is a powerful strategy for identifying new small molecule tools with which to probe different aspects of spliceosome assembly and function.

The SF3B1 inhibitor spliceostatin A (SSA) elicits apoptosis in chronic lymphocytic leukaemia cells through downregulation of Mcl-1.

The pro-survival Bcl-2 family member Mcl-1 is expressed in chronic lymphocytic leukaemia (CLL), with high expression correlated with progressive disease. The spliceosome inhibitor spliceostatin A (SSA) is known to regulate Mcl-1 and so here we assessed the ability of SSA to elicit apoptosis in CLL. SSA induced apoptosis of CLL cells at low nanomolar concentrations in a dose- and time-dependent manner, but independently of SF3B1 mutational status, IGHV status and CD38 or ZAP70 expression. However, normal B and T cells were less sensitive than CLL cells (P=0.006 and P<0.001, respectively). SSA altered the splicing of anti-apoptotic MCL-1(L) to MCL-1(s) in CLL cells coincident with induction of apoptosis. Overexpression studies in Ramos cells suggested that Mcl-1 was important for SSA-induced killing since its expression inversely correlated with apoptosis (P=0.001). IL4 and CD40L, present in patient lymph nodes, are known to protect tumour cells from apoptosis and significantly inhibited SSA, ABT-263 and ABT-199 induced killing following administration to CLL cells (P=0.008). However, by combining SSA with the Bcl-2/Bcl-x(L) antagonists ABT-263 or ABT-199, we were able to overcome this pro-survival effect. We conclude that SSA combined with Bcl-2/Bcl-x(L) antagonists may have therapeutic utility for CLL.

Molecular identification of the dominant-negative, splicing isoform of the two-pore domain K+ channel K2P5.1 in lymphoid cells and enhancement of its expression by splicing inhibition

The two-pore domain background K+ channel K2P5.1 is expected as a possible therapeutic target for autoimmune and inflammatory disorders and cancers because it plays an important role in maintaining the resting membrane potential and regulation of Ca2+ signaling in T lymphocytes and cancer cells. However, the lack of selective K2P5.1 blockers has led to difficulties conducting experimental studies on this K+ channel. We identified a novel splicing isoform of K2P5.1, K2P5.1B from the mammalian spleen, which lacked the N-terminus of full-length K2P5.1A. A co-immunoprecipitation assay using mice spleen lysates revealed an interaction between K2P5.1A and K2P5.1B in the cytoplasmic C-terminal domain. In a heterologous HEK293 expression system, K2P5.1B inhibited the trafficking of K2P5.1A to the plasma membrane. The alkaline pHe-induced hyperpolarizing response was significantly suppressed in K2P5.1B-transfected human leukemia K562 cells. Enhancement in cell proliferation by the overexpression of K2P5.1A in K562 was significantly prevented by the transfection of K2P5.1B. The spliceosome inhibitor pladienolide B significantly enhanced the relative expression of K2P5.1B in K562, resulting in decreases in the activity of K2P5.1A. K2P5.1B suppresses the function of the K2P5.1 K+ channel in a dominant-negative manner, suggesting that the mRNA splicing mechanisms underlying the transcriptional regulation of K2P5.1B may be a new therapeutic strategy for autoimmune and inflammatory disorders and cancers.

Spliceosome Inhibitor Meayamycin B As a Novel Potential Chemotherapeutic Agent in ALL and AML

IntroductionThe process of pre-mRNA splicing is gaining attention as a contributor to anticancer drug resistance and as a promising novel therapeutic target. Splicing of many genes involved in regulation of apoptosis was found to be altered in tumor cells leading to chemoresistance. Similarly, aberrant splicing of genes engaged in drug metabolism was reported to mediate resistance to several anchor drugs of chemotherapeutic protocols in the treatment of leukemia including daunorubicin, cytarabine and methotrexate. Therefore, targeting the spliceosome holds potential to directly activate apoptosis by inducing pro-apoptotic splicing profiles as well as to sensitize cells displaying drug resistance related to altered splicing of genes involved in drug metabolism. In this respect, meayamycin B (MAMB) is a novel compound, which potently inhibits the SF3B1 subunit, which is one of the core components of the spliceosome complex. MAMB was previously shown to induce shifts in splicing of one of the essential apoptosis regulators Mcl-1 in non-small cell lung cancer cell lines A549 and H1299, hence promoting expression of its pro-apoptotic isoform Mcl-1S. The resulting dominance of Mcl-1S was able to sensitize these tumor cells to Bcl-XL inhibitor leading to induction of cell death. Here we evaluated the in vitro impact of MAMB as a single drug in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).MethodsTo achieve this goal, we first assessed the impact of MAMB in short-term exposure on splicing of selected apoptosis-related genes, in particular Mcl-1, and concomitant induction of apoptosis in 3 human ALL and 3 AML cell lines. In addition, MAMB sensitivity was assessed using a 72h MTT assay in a panel of ALL and AML cell lines including sublines displaying resistance to several conventional chemotherapeutics including methotrexate, bortezomib or imatinib. Finally, we assessed MAMB sensitivity in primary ALL and AML samples, as compared to healthy bone marrow specimens.ResultsAs previously shown in solid tumors, MAMB (0.5-1nM) was able to shift splicing of Mcl-1 but not Bcl-X in leukemic cell lines upon 24h exposure. The observed changes in splicing coincided with enhanced apoptosis as determined by flow cytometry. The fraction of apoptotic cells reached approximately 40% in the parental CCRF-CEM cell line (T-ALL) and 10% in the methotrexate-resistant subline which has lost folylpolyglutamate synthetase (FPGS) activity. Intriguingly, we previously found impaired splicing of FPGS in this methotrexate-resistant cell line as compared to intact splicing in parental cells. It should be emphasized that intact FPGS splicing is a key component of intracellular retention and activity of MTX. Induction of apoptosis in AML cell lines ranged between 17% in OCI-AML3 and 53% in KG1a. When MAMB sensitivity was assessed in the 72h MTT assay, the growth of both ALL and AML cell lines was efficiently inhibited with remarkable IC50 values varying between 0.07 and 0.16 nM. Intriguingly, even tumor cell lines which are resistant to conventional chemotherapeutics with different mechanisms of action, such as methotrexate, imatinib and bortezomib, were highly sensitive to MAMB. In line with our results with leukemic cell lines, both ALL and AML primary samples showed a remarkable sensitivity to MAMB (mean LC50 value 0.42 and 0.43 nM, respectively, Figure 1). The normal bone marrow cells obtained from healthy children showed a slightly higher resistance (mean LC50 value 0.57, p=0.03, Figure).ConclusionsOur results show that MAMB itself may constitute a therapeutic option for patients displaying drug resistance to conventional chemotherapy, especially in AML, which in general tends to be more resistant to current treatment options compared to ALL. In addition, as a splicing modulating agent it may also be used to reverse aberrant splicing profiles of genes involved in drug metabolism, thereby restoring sensitivity to standard chemotherapeutics. This novel paradigm warrants further exploration using drug combination studies. Further investigation in a larger cohort of leukemia patient samples is warranted. Moreover, mice studies will be performed to reveal possible toxicities and in vivo efficacy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

456 SF3B1 mutations induce disease relevant aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition

456 SF3B1 mutations induce disease relevant aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition

Abstract 2932: SF3B1 mutations induce aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition

Abstract Recurrent heterozygous mutations of the spliceosome protein SF3B1 have been identified in myelodysplastic syndromes, chronic lymphocytic leukemia (CLL), breast, pancreatic and skin cancers. SF3B1 is a component of the U2 snRNP complex which binds to the pre-mRNA branch point site and is involved in recognition and stabilization of the spliceosome at the 3′ splice site. To understand the impact of SF3B1 mutations, we compared RNAseq profiles from tumor samples with SF3B1 hotspot mutations (SF3B1-MUT) or wild-type SF3B1 (SF3B1-WT) in breast cancer, melanoma and CLL. This analysis revealed significant increases in the usage of novel alternative splice junctions in SF3B1-MUT samples including selection of alternative 3′ splice sites and less frequently exon skipping. These events induce expression of alternative mRNAs that are translated into novel proteins or aberrant mRNAs that are decayed by cells. A common alternative splicing profile was shared across different hotspot mutations and lineages (e.g. ZDHHC16 and COASY); however, unique alternative splicing profiles were also observed suggesting lineage specific effects. RNAseq analysis of several cell lines with endogenous SF3B1 hotspot mutations confirmed the presence of the same spliced isoforms as observed in tumor samples. To prove that SF3B1-MUT were inducing alternative splicing, transient transfection of several SF3B1 hotspot mutations in 293FT cells induced the expression of the common alternatively spliced genes suggesting functional similarity. Selective shRNA depletion of mutant SF3B1 allele in SF3B1-MUT cells resulted in downregulation of the same splice isoforms. Furthermore, isogenic B-cell lines (NALM-6) expressing the most frequent SF3B1 mutation (K700E) were generated and profiled by RNAseq. As expected, similar alternatively spliced genes were observed in NALM-6 SF3B1-K700E cells exclusively. To investigate the role of nonsense-mediated mRNA decay (NMD) in eliminating aberrant mRNAs induced by SF3B1-MUT, we treated NALM-6 SF3B1-K700E cells with cycloheximide, a translation inhibitor known to inhibit NMD. In the treated samples, expression of several aberrant mRNAs was revealed and some of these transcripts were shown to be downregulated in patient samples. Taken together, these results confirm the association between different SF3B1 hotspot mutations and the presence of novel splice isoforms. We demonstrated that E7107, a potent and selective inhibitor of wild-type SF3B1, also binds and inhibits SF3B1-MUT protein. In addition, E7107 represses the expression of several common aberrant splice mRNA products in SF3B1-MUT cells in vitro and in vivo. When tested in a NALM-6 mouse model, E7107 induced tumor regression and increased the overall survival of animals implanted with NALM-6 SF3B1-K700E cells. These data suggest splicing inhibitors as a promising therapeutic approach for cancer patients carrying SF3B1 mutations. Citation Format: Silvia Buonamici, Kian Huat Lim, Jacob Feala, Eunice Park, Laura Corson, Michelle Aicher, Daniel Aird, Betty Chan, Erik Corcoran, Rachel Darman, Peter Fekkes, Gregg Keaney, Pavan Kumar, Kaiko Kunii, Linda Lee, Xiaoling Puyang, Jose Rodrigues, Anand Selvaraj, Michael Thomas, John Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Peter Smith, Yoshiharu Mizui. SF3B1 mutations induce aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2932. doi:10.1158/1538-7445.AM2014-2932

Spliceostatin hemiketal biosynthesis in Burkholderia spp. is catalyzed by an iron/α-ketoglutarate-dependent dioxygenase.

Spliceostatins are potent spliceosome inhibitors biosynthesized by a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) system of the trans-acyl transferase (AT) type. Burkholderia sp. FERM BP-3421 produces hemiketal spliceostatins, such as FR901464, as well as analogs containing a terminal carboxylic acid. We provide genetic and biochemical evidence for hemiketal biosynthesis by oxidative decarboxylation rather than the previously hypothesized Baeyer-Villiger oxidative release postulated to be catalyzed by a flavin-dependent monooxygenase (FMO) activity internal to the last module of the PKS. Inactivation of Fe(II)/α-ketoglutarate-dependent dioxygenase gene fr9P led to loss of hemiketal congeners, whereas the mutant was still able to produce all major carboxylic acid-type compounds. FMO mutants, on the other hand, produced both hemiketal and carboxylic acid analogs containing an exocyclic methylene instead of an epoxide, indicating that the FMO is involved in epoxidation rather than Baeyer-Villiger oxidation. Moreover, recombinant Fr9P enzyme was shown to catalyze hydroxylation to form β-hydroxy acids, which upon decarboxylation led to hemiketal FR901464. Finally, a third oxygenase activity encoded in the biosynthetic gene cluster, the cytochrome P450 monooxygenase Fr9R, was assigned as a 4-hydroxylase based on gene inactivation results. Identification and deletion of the gene involved in hemiketal formation allowed us to generate a strain--the dioxygenase fr9P(-) mutant--that accumulates only the carboxylic acid-type spliceostatins, which are as potent as the hemiketal analogs, when derivatized to increase cell permeability, but are chemically more stable.

Genomics-guided discovery of potent anticancer natural products from exotic bacterial species

for 2014 UNTHSC RAD (March 21, 2014; Fort Worth, TX), No. 1144 GENOMICS-GUIDED DISCOVERY OF POTENT ANTICANCER NATURAL PRODUCTS FROM EXOTIC BACTERIAL SPECIES Xiangyang Liu 1,2 , Cheng Wang 2 , Sreya Biswas 2 , Christopher M. Antapli 2 , Hui Zhu 1 , Santosh Thapa 3 and Yi-Qiang Cheng 1,2,4* 1 UNT System College of Pharmacy, 3 Graduate School of Biomedical Sciences, 4 Institute for Cancer Research, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX 76107. 2 Department of Biological Sciences and Department of Chemistry and Biochemistry, University of Wisconsin–Milwaukee, PO Box 413, Milwaukee, WI 53201 Tel: (817) 735-0165; E-mail: yiqiang.cheng@unthsc.edu Abstract. Natural products are small chemical molecules produced by bacteria, fungi or plants. Natural Natural products are small chemical molecules produced by bacteria, fungi or plants. Natural products have made great contributions to medicine, particularly as anticancer drugs or anti-infective drugs. Currently there is a renaissance of natural product discovery due to the development of new discovery technologies and unmet medical needs. We hypothesized that exotic Gram-negative bacterial species can be a good source of diverse natural products. We analyzed the genomes of two bacterial species originally isolated from central Thailand or north Australia, and found that each genome contains multiple natural product biosynthetic gene clusters. Those information facilitated our discovery of two groups of new natural products, named thailandepsins and thailanstatins, that were found to possess potent antiproliferative activities against an array of human cancer cell lines. Additional studies are being conducted in animal models through collaborations. We thus concluded that Gram-negative bacterial species is a good source of diverse natural products, and genomics-guided discovery approach is effective and particularly suitable for small research laboratories with limited resources. Purpose. We hypothesize that exotic Gram-negative bacterial species can be a good source of diverse natural products. The purpose of the research is thus to discover new bioactive natural products from exotic bacterial species. Materials and Methods. Burkholderia thailandensis E264, a Gram-negative beta-proteobacterium strain originally isolated from a rice paddy in central Thailand, was purchased from the American Type Culture Collection (ATCC); Burkholderia thailandensis MSMB43, another Gram-negative beta-proteobacterium strain originally isolated from a water source in north Australia, was obtained from the US Centers for Disease Control (CDC). Bacterial genome analysis and natural product discovery and identification were performed according to standard procedures. Results. Mining the genome of B. thailandensis E264 revealed a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS–PKS) biosynthetic gene cluster that resembles that of FK228 (romidepsin, drug name Istodax) in Chromobacterium violaceum No. 968, which led us to discover thailandepsins A–F, natural analogues of FK228, and potent histone deacetylase inhibitors and antiproliferative agents with GI50 values in the sub-nM range. Mining the genome of B. thailandensis MSMB43 revealed at least 13 biosynthetic gene clusters. Among them one hybrid NRPS–PKS gene cluster is highly homologous to that of FR901464 (a prototype spliceosome inhibitor) in Pseudomonas sp. No. 2663, which led us to discover thailanstatins A–D, natural and more stable analogues of FR901464, and potent pre-mRNA splicing inhibitors and antiproliferative agents with GI50 values in the low nM range. Selected members of those natural products are under intensive collaborative investigations as anticancer drug candidates, and preliminary results are encouraging. Metabolic engineering approach is being undertaken to increase the yield of those potent compounds that are often produced in minute amounts by the wild-type bacteria. Conclusions. Potent new anticancer natural products have been discovered from exotic bacterial species via a genomics-guided discovery approach, which is effective and particularly suitable for small research laboratories with limited resources. We seek additional collaborations for identifying the best possible use of our small collection of potent natural products; we also seek to establish a “Texas Network for Collaborative Natural Product Discovery and Development” for sharing the resources, risks and rewards.

Multiple components of the spliceosome regulate Mcl1 activity in neuroblastoma

Cancer treatments induce cell stress to trigger apoptosis in tumor cells. Many cancers repress these apoptotic signals through alterations in the Bcl2 proteins that regulate this process. Therapeutics that target these specific survival biases are in development, and drugs that inhibit Bcl2 activities have shown clinical activity for some cancers. Mcl1 is a survival factor for which no effective antagonists have been developed, so it remains a principal mediator of therapy resistance, including to Bcl2 inhibitors. We used a synthetic-lethal screening strategy to identify genes that regulate Mcl1 survival activity using the pediatric tumor neuroblastoma (NB) as a model, as a large subset are functionally verified to be Mcl1 dependent and Bcl2 inhibitor resistant. A targeted siRNA screen identified genes whose knockdown restores sensitivity of Mcl1-dependent NBs to ABT-737, a small molecule inhibitor of Bcl2, BclXL and BclW. Three target genes that shifted the ABT-737 IC50 >1 log were identified and validated: PSMD14, UBL5 and PRPF8. The latter two are members of a recently characterized subcomplex of the spliceosome that along with SART1 is responsible for non-canonical 5′-splice sequence recognition in yeast. We showed that SART1 knockdown similarly sensitized Mcl1-dependent NB to ABT-737 and that triple knockdown of UBL5/PRPF8/SART1 phenocopied direct MCL1 knockdown, whereas having no effect on Bcl2-dependent NBs. Both genetic spliceosome knockdown or treatment with SF3b-interacting spliceosome inhibitors like spliceostatin A led to preferential pro-apoptotic Mcl1-S splicing and reduced translation and abundance of Mcl1 protein. In contrast, BN82865, which inhibits the second transesterification step in terminal spliceosome processing, did not have this effect. These findings demonstrate a prominent role for the spliceosome in mediating Mcl1 activity and suggest that drugs that target either the specific UBL5/PRPF8/SART1 subcomplex or SF3b functions may have a role as cancer therapeutics by attenuating the Mcl1 survival bias present in numerous cancers.

Spliceosome Mutations Are Frequent In Patients With Myelodysplastic Syndromes Who Failed Hypomethylating Therapy: Possible Implications Of Spliceosome Inhibitors As Alternative Treatments

Myelodysplastic syndromes (MDS) are a heterogeneous group of blood cancers characterized by bone marrow (BM) failure, peripheral blood cytopenias, dysplasia, chromosomal abnormalities and an increased risk for transformation to acute myeloid leukemia (AML). Patients (pts) with higher risk disease are primarily treated with pharmacologic treatments like hypomethylating therapy (HMT) (5-azacytidine and decitabine). 5-azacytidine (AZA) and decitabine (DAC) can result in overall response rates of 36% with a median duration of response of 15 months and 17-21% with a median duration of response of 10 months, respectively. Pts refractory to HMT have poor outcomes with a median overall survival of ∼4 months. Spliceosome gene mutations are frequently found in certain subtypes of MDS specifically SF3B1 (∼28%), U2AF1 (6-12%) and SRSF2 (6-12%). The prognostic value of spliceosome mutations in different MDS subtypes has been largely investigated while the impact of these mutations on treatment response is still unknown. We aim to investigate the frequency of three commonly mutated spliceosome genes (SF3B1, U2AF1, and SRSF2) in pts who failed HMT in order to define mutational frequency and evaluate the feasibility of targeted therapy with next generation spliceosome inhibitors. We screened a cohort of 120 pts (MDS, 70; MDS/MPN, 33; MDS/sAML, 17; median age: 69; male/female: 85/35) that underwent HMT (AZA: 58; DAC: 21; AZA/DAC: 7; AZA/REV: 25; DAC/REV: 4; AZA/DAC/REV: 5). Forty-eight percent of pts failed HMT therapy as refractory or relapse. We performed Sanger sequencing on BM/peripheral blood DNA for known pathways involved in MDS pathogenesis including methylation (TET2, DNMT3A, IDH1/2), histone (ASXL1, UTX, EZH2), signaling (CBL, N/KRAS), transcription (RUNX1, TP53, JAK2), and RNA splicing (SF3B1, U2AF1, SRSF2). Data analysis was available for 90 pts. We detected a total of 131 mutations in different pathways. In total, spliceosome mutations were observed in 28/90 (31%) of pts. When we analyzed the presence of the mutations in relation to the rate of response, we found that pts who failed HMT have frequent spliceosome mutations: 17/58 (29%). We have reported that molecular mutations in TET2 and DNMT3A can predict response to treatment to HMT (Traina F, Blood (ASH Annual Meeting Abstracts), Nov 2011; 118: 461). Indeed, the frequency of mutations in methylation genes was lower in the group of pts who failed HMT (11/58; 18.9%) compared to pts who achieved hematological response (11/32; 34%). Spliceosome inhibitors have been proposed for targetted therapy in MDS. The presence of spliceosome mutations in pts who failed HMT can open a new era of investigation leading to the possibility of using spliceosome inhibitors in pts who fail conventional therapy. We performed RNA-sequencing analysis on BM cells of pts who failed HMT compared to pts who achieved hematological response (n=2 vs 2) in order to define any specific gene signature explaining the differences in response to HMT. We performed differential gene expression testing on 11,459 expressed genes. In total, 158 genes were differentially expressed at FDR < .2 in responders compared to not responders. We identified several interesting genes involved in tumorigenesis and epigenetic regulation such as YPEL3, and ST14, which were up-regulated in responders vs not responders (FC: 4 and 7.5; P<.00001). In sum, spliceosome mutations are frequent in pts who failed HMT and may affect downstream pathways most probably in the epigenetic network leading to differences in susceptibility to HMT and can open the possibility of treatment with spliceosome inhibitors. Disclosures:No relevant conflicts of interest to declare.

Splicing Factor 3b Subunit 1 (SF3B1) Heterozygous Mice Manifest a Hematologic Phenotype Similar To Low Risk Myelodysplastic Syndromes With Ring Sideroblasts

The link between SF3B1 mutation and the ring sideroblast (RS) phenotype in myelodysplastic syndromes (MDS) was solidified by the identification of RS in Sf3b1 heterozygous (Sf3b1+/-) mice. The identification of SF3B1 mutations in refractory anemia with RS (RARS) and RARS with thrombocytosis (RARS-T) showed the importance of RNA splicing in MDS biology. Furthermore, it opened the possibility of targeted therapy using spliceosome inhibitors in RARS/-T. However, many questions remain unanswered in linking SF3B1 dysfunction to MDS biology like the downstream targets of this gene. The identification of a robust murine model is essential to study a specific molecularly defined disease-phenotype and develop targeted therapies. We identified occasional RS in the bone marrow (BM) of Sf3b1+/- which are rarely found in current mouse models of MDS (Beachy SH, Hematol Oncol Clin North Am, 2010). However, aside from RS in the BM no other MDS features were found. Sf3b1+/- mice were originally engineered as a means to study the interaction between polycomb (PcG) genes and other protein-complexes (Isono K, Genes Dev, 2005). Homozygous Sf3b1-/- mice died at the stage of pre-implantation of the embryos while Sf3b1+/- appear healthy. Several tools have been tested to model MDS in genetically engineered mice targeting key genes in MDS. However, the creation of an ideal mouse model resembling distinct morphologic MDS subtypes is still lacking. To define a mouse model useful for preclinical therapeutic studies, we evaluated the hematologic features of Sf3b1+/- and Sf3b1+/+ mice during a long term follow-up. Five Sf3b1+/- and 5 Sf3b1+/+ mice were followed over time until 12 months of age. Blood was drawn from the retro-orbital vein every month starting from 6 months of age. Using two-sample Wilcoxon test we compared standard hematologic parameters finding a significant difference over the time between Sf3b1+/- and Sf3b1+/+: hemoglobin (g/dL) 6.9 ±0.73 vs 10.0 ±1.6 (P=0.008), red blood cells (M/uL) 8.3±0.5 vs 5.9±1.0 (P=0.008), platelets (K/uL) 731±105 vs 579±93 (P=0.008), and mean corpuscular volume (fL) 47.8±1.5 vs 45.1±1.1 (P=0.032). We did not detect any significant difference in other parameters although lymphocytes were more represented vs neutrophils, eosinophils and monocytes in Sf3b1+/- vs Sf3b1+/+ (6.3K/uL ±3.1 vs 5.8 ±1.8; P=1). Analysis of the BM, showed no difference in cell number between Sf3b1+/- (n=7) and Sf3b1+/+ (n=7) (44.1±9.1 vs 43.2 ±11; P=0.62). However, distinct dyserythropoiesis such as nuclear budding or irregular nuclei in Wright-Giemsa and occasional RS in Prussian blue stains were noted in Sf3b1+/- which were not present in Sf3b1+/+. In support of the iron overload seen in SF3B1 mutant patients (pts), a similar observation was made in Sf3b1+/- by light microscopy and rhodamine based- flow cytometry to quantify mitochondrial iron (Visconte, Abstract #64897). We also characterized the transcriptome of Sf3b1+/- and Sf3b1+/+. Total RNA was isolated from BM of age/gender matched mice, polyA cDNA was prepared from 3ug of RNA and Mouse RNA-sequencing was run on Illumina HiSeq2000. 200 exons were found differentially used in Sf3b1+/- vs Sf3b1+/+. Chromosome 1 contains the highest number of genes with at least 1 exon alternatively used similar to what we observed in SF3B1 mutant pts. In total 22 genes showed stronger differential expression in Sf3b1+/- vs Sf3b1+/+. Sf3b1 was down-regulated as expected (MFC: 0.74) in Sf3b1+/-. Studies in Sf3b1+/- mice show that Sf3b1 protein physically interacts with Class II PcG proteins (PRC1) which are relevant in MDS. When we interrogated PcG genes and others, we found lower mRNA levels of ezh2 (MFC: 0.06) and npm1 and tpr53 (MFC: 0.01 and 0.28) and no difference in asxl1 and runx1 (MFC:1.22 and 1.1) in Sf3b1+/- vs Sf3b1+/+. Jak2, dock8, and uhrf2 showed significant (P=.0003) higher expression in Sf3b1+/-. MDS is a heterogeneous disease characterized by genetic and non-genetic causes. Introduction of secondary events implicated in MDS pathogenesis can modify the phenotype of Sf3b1+/- mice. In sum, Sf3b1+/- mice after 6 months of follow-up developed macrocytic anemia, thrombocytosis, RS and dyserythropoiesis akin to human RARS/-T. Furthermore, transcriptome analysis shows exon usage/ gene expression changes similar to human SF3B1 mutants lending support that Sf3b1+/- can serve as a mouse model for studying the biology of human low risk MDS specifically that of RARS/-T. Disclosures:No relevant conflicts of interest to declare.

BCL-2 Family Of Genes Is a Key Regulator In The Pathogenesis Of SF3B1 Mutant and Wild Type MDS With Ring Sideroblasts and Represents a Novel Drug Target In This Disease

Myelodysplastic syndromes (MDS) are a group of neoplastic diseases characterized by impairment in hematopoietic cell differentiation which leads to disease related consequences like cytopenias and cellular dysplasia. Mutations in splicing factor 3b subunit 1 (SF3B1) are the most frequently identified genetic abnormalities in refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T). However, the mechanisms whereby cells acquire clonal dominance and evolution, cytopenias and impairment in differentiation remain unknown. To understand the role of SF3B1 mutations in the pathogenesis of RARS/-T, we analyzed the transcriptome of SF3B1 mutant (MUT) and wild type (WT) RARS/-T patients (pts). RNA was isolated from bone marrow of healthy subjects (n=3), SF3B1 MUT (n=3) and WT (n=3) RARS/-T, and other MDS (n=5). cDNA was made from RNA (1.5-3ug) and fragmented for library preparation. RNA-sequencing was performed on 20 million sequence reads on an Illumina HiSeq2000 and aligned to the human genome 19. RNA-splicing patterns were analyzed by a published bioinformatics algorithm at three levels (exon usage, gene expression, and pathway analysis) (Visconte V; Blood. 2012). Differential exon usage identified 271 and 71 genes with at least 1 exon alternatively spliced in SF3B1 MUT, WT, and healthy subjects (P<.05). SF3B1 preferentially affected the 3'-end regions. The chromosomes (chrs) with the highest number of genes displaying differential exon usage were chr1:30, chr17:25, chr7:22, and chrs19: 20, chr11:17, chrs:6 and 2:15 following by chrs:8, 5, and 16: 11. No significant differences in mRNA levels of genes relevant to MDS (U2AF1, TET2, DNMT3A, ASXL1, EZH2, TP53, RUNX1, ETV6, CBL, IDH1/2, and N/KRAS) were found leading us to think that SF3B1 affects alternative targets. Using GSEA/ MiSigDB v3.0 analysis we found a specific molecular signature: out of 27 mitochondrial gene sets, one gene set differed between SF3B1 MUT and WT RARS pts (P=.01). This gene set contains 11 genes involved in mitochondrial membrane/organization/biogenesis network. BCL-2 family genes were highly expressed: BCL-2 (mean: 22.8 vs 68.1 vs 15.2), BNIP3 (mean: 50.1 vs 55.3 vs 31.9) and BCL2-L1 (mean: 3221 vs 2905 vs 595) in SF3B1 MUT vs WT pts and vs healthy subjects. Changes in BCL2 mRNA levels were also confirmed by western blotting in MUT (n=2) vs WT (n=2). In myeloid cell lineage, the relationship between BCL-2 protein levels is inversely related to the maturation stage with myeloblasts and promyelocytes having higher BCL-2 while more differentiated cells like metamyelocytes and polymorphonuclear cells have less to no BCL-2 expression. Contrary to prevailing knowledge and consistent with what would be expected in explaining MDS persistence by development of clonal advantage, in our hands RARS/-T pts were not characterized by increased cleaved PARP and cellular stress as evidenced by no difference in reactive oxygen species between SF3B1 MUT and WT (93.83% ± 7.94 vs 83.28% ± 13.82; n=3 vs 4) further supported by the overexpression of an antiapoptotic gene BCL-2. We previously presented the biological rationale of the different clinical outcomes between SF3B1 MUT and WT (Visconte V; Blood (ASH Annual Meeting Abstracts), Nov 2012;120: 922). SF3B1 WT carries more high risk chromosomal defects and higher g-H2AX levels indicative of greater DNA damage. Higher g-H2AX and genomic instability are associated with BCL-2 over-expressing lymphoid malignancies (Laulier C; Cancer Res. 2011) which may explain the higher number of chromosomal defects and propensity for leukemia transformation in WT pts. SF3B1 WT tends to respond less to hypomethylating therapy (HMT) vs MUT (n=2 vs 2). Cells presenting an immature hematopoietic phenotype have lower response rate to HMT in-vitro. Indeed, differences in erythroid maturation were found in SF3B1 MUT vs WT RARS. SF3B1 MUT showed higher mRNA levels of GATA-1, a marker of hematopoietic maturation (FC: 4.0), and a down-regulation (FC: 0.48) of GATA-2, an early hematopoietic marker vs WT. In conclusion, BCL-2 is a key regulator in the pathogenesis of RARS/-T and may provide clonal advantage. Differences in BCL-2 expression can explain different survival outcomes in SF3B1 MUT vs WT. BCL-2 may be a novel pathway in the pathogenesis of RARS/-T opening the possibility that a BCL2- antagonist either with/without a spliceosome inhibitor will be an ideal therapeutic approach in RARS/-T. Disclosures:No relevant conflicts of interest to declare.

Spliceosome Gene Mutations Are Frequently Found In JAK2 Negative Myelofibrosis and Associated With Worse Clinical Outcomes

Myelofibrosis (MF), a Philadelphia chromosome negative myeloproliferative neoplasm (MPN) requires clonal markers for accurate diagnosis according to the 2008 World Health Organization classification scheme. However, molecular mutations in Janus Kinase 2 (JAK2) are found in only 50% of MF patients (pts). Various somatic mutations identified in other myeloid cancers have also been found in MF with pre-existing JAK2 V617F mutations. We previously reported in 57 pts diagnosed with Triple Negative MF (TN-MF: JAK2 exon12/14 and wild type (WT) MPL) the absence of mutations in genes commonly identified in other myeloid malignancies, specifically TET2, DNMT3A, CBL, IDH1/2, SH3B2 (LNK), N/KRAS and EZH2. Only somatic mutations in ASXL1 were identified in a small cohort of TN-MF (16%). Mutations in components of the RNA splicing machinery have been recently identified in varying frequencies in different myeloid malignancies. We performed RNA-sequencing on 2 samples (JAK2 mutant (MUT) and 1 JAK2 WT) and found that 2 spliceosome genes (U2AF1 and SF3B1) had higher mRNA expression levels in the JAK2 WT (MFC=0.76 and 0.82, respectively) compared to JAK2 MUT patient. We hypothesized that somatic mutations in RNA splicing factor genes occur in TN-MF pts and may be helpful in the biological characterization of this group of pts. Therefore, we isolated DNA from bone marrow (BM) or peripheral blood mononuclear cells from a cohort of MF pts (N=132; JAK2 MUT:75 and JAK2 WT:57) and performed Sanger sequencing for SF3B1 (exons 13-16), U2AF1 (exons 2, 6 and 7), and SRSF2 (exons 1 and 2). Baseline characteristics of pts and clinical data including hematologic parameters, BM results, and presence of splenomegaly by palpation were collected. Pts were stratified based on the Dynamic International Prognostic Scoring System-Plus risk as high=40, Int-2=51, Int-1=11 and low-risk=5 pts. The mean duration of follow-up was 16.5 months in JAK2 MUT and 12.8 months in JAK2 WT. Spliceosome mutations were found in 35/132 (27%) (SRSF2=17%, U2AF1=8% and SF3B1=2%) of pts. Interestingly, the frequency of spliceosome mutations in JAK2 WT was higher (31%; SRSF2=26%, U2AF1= 5%, SF3B1=0) compared to JAK2 MUT (18%; SRSF2=10%, U2AF1=6%, SF3B1=2%) pts. Of note, the 2 pts with SF3B1 mutations (K700E) had ring sideroblast in the BM (occasional and 50%). A large number of WT JAK2 patients (50%) had concomitant SRSF2 and ASXL1 mutations. Both SRSF2 and ASXL1 have been associated with poor prognosis in MF. Indeed, in our JAK2 WT cohort, pts who harbored SRSF2 mutations had a higher mean percentage of BM blasts compared to the ones that were SRSF2 WT (5.6% vs 1.8%; P=.006). Moreover, JAK2 WT pts carrying spliceosome mutations had more severe anemia as shown by lower hemoglobin (8.98 g/dL vs 10.5) and higher leukocyte counts (27.5 x109/L vs 20.5) compared to WT cases. Furthermore, we noticed that they also had higher frequency of RBC (Red Blood Cells) transfusions compared to WT (60% vs 40%) cases. Spleen examination by palpation below the left sub-costal margin showed that JAK2 WT cases carrying spliceosome mutations had larger splenomegaly compared to WT cases [10.1 cm (range 4-26), vs 8.7cm (range 2-20); P=0.05]. In conclusion, molecular alterations in the spliceosome machinery are frequently found in MF pts who are WT for JAK2/MPL and are associated with higher BM blast percentage, more severe anemia, higher leukocyte counts, transfusion dependence and more prominent splenomegaly. The observation of appreciable frequency of spliceosome mutations in JAK2 WT MF pts opens the possibility of using spliceosome inhibitors in the management of this disease group. Disclosures:No relevant conflicts of interest to declare.

A Cyclopropane-Derived Stable Analog Of Fd-895 Induces Apoptosis and Inhibition Of mRNA Splicing In Lymphoma and Chronic Lymphocytic Leukemia: A Novel Therapeutic Approach

Abnormalities in RNA splicing have been implicated in tumorigenesis and cancer progression. Recent studies have defined RNA splicing as in attractive target for development of effective anticancer therapy. However, significant hurdles need to be solved before spliceosome inhibitors are ready for clinical trial applications. The work presented here addresses a major obstacle, specifically the design of active and metabolically stable spliceosome / RNA splicing inhibitors.We previously reported that FD-895 and Pladienolide B (PLAD-B) induce apoptosis viathe inhibition of RNA splicing in chronic lymphocytic leukemia (Kashyap et al, Blood 2012; 120: 3890). However, FD-895 and PLAD-B hydrolyze relatively rapidly in aqueous solution. To address this issue, we applied semi-synthesis and total synthesis to reveal key structure activity relationships (SARs) regulating the RNA splicing inhibition of FD-895. This information was applied to synthesize a panel of cyclopropane analogs (CypB) that showed improved biochemical stability and potent pro-apoptotic activity in a panel of lymphoma cell lines and primary lymphoma - leukemia cells from patients with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL).A series of cyclopropane analogs and FD-895 were tested for hydrolysis and stability using a combination of NMR and mass spectral methods. Based on stability, we selected a first generation cyclopropane analog, CypB, to conduct our experiments. Primary leukemia - lymphoma cells as well as cell lines were incubated with increasing concentrations of FD-895, and CypB over a gradient of 10-1,000 nM. We observed rapid development of intron retention within 15 min after incubation at concentrations ranging from 10-100 nM using a series of spliceosome related genes (DNAJB1, RIOK3, and BRD2). Contrary to that, intron retention was not observed after treatment with chemotherapy controls (fludarabine and etoposide). By using qRT-PCR, we were able to quantify unspliced mRNA forms induced by FD-895, and CypB but not by chemotherapy agents.In addition, we observed a close correlation between the level of intron retention and development of apoptosis induced by FD-895 and CypB in all cell lines and primary leukemia - lymphoma samples (IC50 range: 3-1,000 nM). Apoptosis induced by these compounds was also observed in chemotherapy resistant patient samples independently of TP53 function, deletion or mutational status.To understand the molecular mechanism of FD-895 and CypB induced apoptosis, we analyzed the expression profile of alternative spliced forms of genes related to apoptosis (mcl-1 and Bcl-X) by RT-PCR. Interestingly, we observed that FD-895, and CypB induced the expression of small alternative spliced forms that are known to be pro-apoptotic. This was not observed after incubation with control chemotherapy agents.In conclusion, using a SAR strategy we were able to optimize novel FD-895 based cyclopropane analogs. These compounds induce early mRNA intron retention in different lymphoma cell lines and primary lymphoma - leukemia samples derived from patients. Furthermore, these analogs induced apoptosis in a TP53 independent manner and induce the expression of pro-apoptotic alternatively spliced RNA genes. Our studies showed that combining medicinal chemistry and cancer biology, we were able to discover labile functional epoxide groups present in FD-895 that can be removed enhancing its stability and preserving the pro-apoptotic activity of this compound. This synthesis and optimization strategy provides new avenues for analog design in an effort to develop spliceosome inhibitors that can be used for cancer treatment. Current efforts are underway to complete these translational studies. Disclosures:Kipps:Bristol-Myers Squibb: Research Funding.

Test-Firing Ammunition for Spliceosome Inhibition in Cancer

E7107 is a derivative of the pladienolide family of natural product spliceosome inhibitors, which targets the U2 small nuclear ribonucleoprotein (snRNP) subunit SF3b. The results of a first-in-human trial with E7107 have been reported, representing an important translational step toward the goal of modulating RNA splicing for cancer therapy. Clin Cancer Res; 19(22); 6064-6. ©2013 AACR.

Phase I Pharmacokinetic and Pharmacodynamic Study of the First-in-Class Spliceosome Inhibitor E7107 in Patients with Advanced Solid Tumors

To assess the safety, tolerability, pharmacokinetics, pharmacodynamics, and clinical activity of E7107 administered as 5-minute bolus infusions on days 1, 8, and 15 in a 28-day schedule. Patients with solid tumors refractory to standard therapies or with no standard treatment available were enrolled. Dose levels of 0.6 to 4.5 mg/m(2) were explored. Forty patients [24M/16F, median age 61 years (45-79)] were enrolled. At 4.5 mg/m(2), dose-limiting toxicity (DLT) consisted of grade 3 diarrhea, nausea, and vomiting and grade 4 diarrhea, respectively, in two patients. At 4.0 mg/m(2), DLT (grade 3 nausea, vomiting, and abdominal cramps) was observed in one patient. Frequently occurring side effects were mainly gastrointestinal. After drug discontinuation at 4.0 mg/m(2), one patient experienced reversible grade 4 blurred vision. The maximum tolerated dose (MTD) is 4.0 mg/m(2). No complete or partial responses during treatment were observed; one patient at 4.0 mg/m(2) had a confirmed partial response after drug discontinuation. Pharmacokinetic analysis revealed a large volume of distribution, high systemic clearance, and a plasma elimination half-life of 5.3 to 15.1 hours. Overall drug exposure increased in a dose-dependent manner. At the MTD, mRNA levels of selected target genes monitored in peripheral blood mononuclear cells showed a reversible 15- to 25-fold decrease, whereas unspliced pre-mRNA levels of DNAJB1 and EIF4A1 showed a reversible 10- to 25-fold increase. The MTD for E7107 using this schedule is 4.0 mg/m(2). Pharmacokinetics is dose-dependent and reproducible within patients. Pharmacodynamic analysis revealed dose-dependent reversible inhibition of pre-mRNA processing of target genes, confirming proof-of-principle activity of E7107.

Enantioselective syntheses of FR901464 and spliceostatin A: potent inhibitors of spliceosome.

Enantioselective syntheses of FR901464 and spliceostatin A, potent spliceosome inhibitors, are described. The synthesis of FR901464 has been accomplished in a convergent manner in 10 linear steps (20 total steps). The A-tetrahydropyran ring was constructed from (R)-isopropylidene glyceraldehyde. The functionalized tetrahydropyran B-ring was synthesized utilizing a Corey-Bakshi-Shibata reduction, an Achmatowicz reaction, and a stereoselective Michael addition as the key steps. Coupling of A- and B-ring fragments was accomplished via cross-metathesis.

Stabilized Cyclopropane Analogs of the Splicing Inhibitor FD-895

Targeting the spliceosome with small molecule inhibitors provides a new avenue to target cancer by intercepting alternate splicing pathways. Although our understanding of alternate mRNA splicing remains poorly understood, it provides an escape pathway for many cancers resistant to current therapeutics. These findings have encouraged recent academic and industrial efforts to develop natural product spliceosome inhibitors, including FD-895 (1a), pladienolide B (1b), and pladienolide D (1c), into next-generation anticancer drugs. The present study describes the application of semisynthesis and total synthesis to reveal key structure-activity relationships for the spliceosome inhibition by 1a. This information is applied to deliver new analogs with improved stability and potent activity at inhibiting splicing in patient derived cell lines.

Genomics-Guided Discovery of Thailanstatins A, B, and C As Pre-mRNA Splicing Inhibitors and Antiproliferative Agents from Burkholderia thailandensis MSMB43

Mining the genome sequence of Burkholderia thailandensis MSMB43 revealed a cryptic biosynthetic gene cluster resembling that of FR901464 (4), a prototype spliceosome inhibitor produced by Pseudomonas sp. No. 2663. Transcriptional analysis revealed a cultivation condition in which a regulatory gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, named thailanstatins A (1), B (2), and C (3), were isolated from the fermentation broth of B. thailandensis MSMB43. Thailanstatins are proposed to be biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase pathway. They differ from 4 by lacking an unstable hydroxyl group and by having an extra carboxyl moiety; those differences endow thailanstatins with a significantly greater stability than 4 as tested in phosphate buffer at pH 7.4. In vitro assays showed that thailanstatins inhibit pre-mRNA splicing as potently as 4, with half-maximal inhibitory concentrations in the single to sub-μM range. Cell culture assays indicated that thailanstatins also possess potent antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the single nM range. This work provides new chemical entities for research and development and new structure-activity information for chemical optimization of related spliceosome inhibitors.

Defects in spliceosomal machinery: a new pathway of leukaemogenesis.

Proper splicing of pre-mRNA is required for protein synthesis and therefore is a fundamental cellular function. The discovery of a variety of somatic spliceosomal mutations in haematological malignancies, including myeloid neoplasms and chronic lymphocytic leukaemia has pointed to a new leukaemogenic pathway involving spliceosomal dysfunction. Theoretically, spliceosomal mutations can lead to activation of incorrect splice sites, intron retention or aberrant alternative splicing occurring in patterns generated by mutations of individual spliceosomal proteins. Such events can produce a defective balance between protein isoforms leading to functional consequences including defective regulation of proliferation and differentiation. The observed pattern of occurrence of highly specific missense mutations, coupled with the lack of nonsense mutations and deletions, implies a gain-of-function or better gain-of-dysfunction mechanism. Incorrect splicing of downstream genes, such as tumour suppressor genes, may result in haploinsufficient expression through nonsense-mediated mRNA decay. Thus, spliceosomal mutations may, depending on the pattern of affected proteins, lead to similar functional effects on tumour suppressor genes as chromosomal deletions, epigenetic silencing or inactivating/hypomorphic mutations. The prognostic value of the most common mutations and their phenotypic association in the clinical setting is currently under investigation. It is likely that spliceosomal mutations may indicate sensitivity to spliceosome inhibitors applied in the form of a synthetic lethal approach. This review discusses the most current aspects of spliceosomal research in the context of haematological malignancies.