Splicing modulator FR901464 is a potential agent for colorectal cancer in combination therapy.
ABSTRACT: FR901464 (FR) was first described as an anticancer drug and later identified as a modulator of splicing factor 3B subunit 1 (SF3B1). Although the effectiveness of splicing modulators has been investigated in colorectal cancer (CRC) cells, their usefulness in animal experiments has not been confirmed. The association of SF3B1 with CRC progression and the influence of FR on transcriptional activity in CRC has not been fully elucidated. FR showed strong cytotoxicity against CRC cell lines, SF3B1-mutated cancer cell lines, and human fibroblasts with IC50 values less than 1 ng/ml. FR-resistant clones derived from HCT116, DLD1, Lovo, and CT26 cells showed IC50 values greater than 100 ng/ml. SF3B1 sequencing demonstrated low frequencies of SF3B1 mutations in CRC and mutations in codon 1074 of exon 22 in all FR-resistant clones. Unlike hematological malignancies, SF3B1 expression was not associated with CRC progression. Although FR showed significant growth inhibition in a xenograft model of RKO cells, severe toxicity was also induced. These data indicated CRC might be a suitable target of FR unless toxicity occurs. Microarray analysis and real-time quantitative PCR demonstrated downregulation of genes associated with Fanconi anemia (BRCA1 and BRCA2) and 28 driver oncogenes. These data suggested combination treatment of FR with other anticancer drugs whose sensitivity is associated with genes affected by FR treatment. Combination treatment with PARP1 inhibitor olaparib, whose sensitivity was enhanced by BRCA 1/2 deficiency, showed synergistic effects in CRC cells. Our data indicates the potential of FR in combination therapy rather than monotherapy for CRC treatment.
Project description:DLD1, HCT116, DLD1 - derived FR resistant clone DLD FR1, and HCT116 - derived FR resistant clone CT FR1, were assessed for change of gene expression induced by FR901464 Overall design: Cells were incubated with or without 10 ng/ml of FR901464 for 24h.
Project description:FR901464 is a potent anticancer natural product that lowers the mRNA levels of oncogenes and tumor suppressor genes. In this article, we report a convergent enantioselective synthesis of FR901464, which was accomplished in 13 linear steps. Central to the synthetic approach was the diene-ene cross olefin metathesis reaction to generate the C6-C7 olefin without the use of protecting groups as the final step. Additional key reactions include a Zr/Ag-promoted alkynylation to set the C4 stereocenter, a mild and chemoselective Red-Al reduction, a reagent-controlled stereoselective Mislow-Evans-type [2,3]-sigmatropic rearrangement to install the C5 stereocenter, a Carreira asymmetric alkynylation to generate the C4' stereocenter, and a highly efficient ring-closing metathesis-allylic oxidation sequence to form an unsaturated lactone. The decomposition pathways of FR901464's right fragment were studied under physiologically relevant conditions. Facile epoxide opening by beta-elimination gave two enones, one of which could undergo dehydration via its hemiketal to form a furan. To prevent this decomposition pathway, a right fragment was rationally designed and synthesized. This analogue was 12 times more stable than the right fragment of the natural product. Using this more stable right fragment analogue, an FR901464 analogue, meayamycin, was prepared in 13 linear steps. The inhibitions of human breast cancer MCF-7 cell proliferation by synthetic FR901464 and meayamycin were studied, and the GI50 values for these compounds were determined to be 1.1 nM and 10 pM, respectively. Thus, meayamycin is among the most potent anticancer small molecules that do not bind to either DNA or microtubule.
Project description:FR901464 (1) and spliceostatin A (2) are potent inhibitors of spliceosomes. These compounds have shown remarkable anticancer activity against multiple human cancer cell lines. Herein, we describe efficient, enantioselective syntheses of FR901464, spliceostatin A, six corresponding diastereomers and an evaluation of their splicing activity. Syntheses of spliceostatin A and FR901464 were carried out in the longest linear sequence of 9 and 10 steps, respectively. To construct the highly functionalized tetrahydropyran A-ring, we utilized CBS reduction, Achmatowicz rearrangement, Michael addition, and reductive amination as key steps. The remarkable diastereoselectivity of the Michael addition was specifically demonstrated with different substrates under various reaction conditions. The side chain B was prepared from an optically active alcohol, followed by acetylation and hydrogenation over Lindlar's catalyst. The other densely functionalized tetrahydropyran C-ring was derived from readily available (R)-isopropylidene glyceraldehyde through a route featuring 1,2-addition, cyclic ketalization, and regioselective epoxidation. These fragments were coupled together at a late stage through amidation and cross-metathesis in a convergent manner. Six key diastereomers were then synthesized to probe the importance of specific stereochemical features of FR901464 and spliceostatin A, with respect to their in vitro splicing activity.
Project description: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.
Project description:Two unrelated bacterial natural products, FR901464 and pladienolide B, have previously been shown to have significant antitumor activity in vivo. These compounds target the SF3b subunit of the spliceosome, with a derivative of pladienolide (E7107) entering clinical trials for cancer. However, due to the structural complexity of these molecules, their research and development has been significantly constrained. We have generated a set of novel analogues (Sudemycins) that possess the pharmacophore that is common to FR901464 and pladienolide, via a flexible enantioselective route, which allows for the production of gram quantities of drug. These compounds demonstrate cytotoxicity toward human tumor cell lines in culture and exhibit antitumor activity in a xenograft model. Here, we present evidence that Sudemycins are potent modulators of alternative splicing in human cells, both of endogenous genes and from minigene constructs. Furthermore, levels of alternative splicing are increased in tumor cells relative to normal cells, and these modifications can be observed in human tumor xenografts in vivo following exposure of animals to the drug. In addition, the change in the splicing pattern observed with the Sudemycins are similar to that observed with Spliceostatin A, a molecule known to interact with the SF3b subunit of the spliceosome. Hence, we conclude that Sudemycins can regulate the production of alternatively spliced RNA transcripts and these alterations are more prevalent in tumors, as compared to normal cells, following drug exposure. These studies suggest that modulation of alternative splicing may play a role in the antitumor activity of this class of agents.
Project description:Thailanstatin A (TST-A) is a potent antiproliferative natural product discovered by our group from Burkholderia thailandensis MSMB43 through a genome-guided approach. The limited supply of TST-A, due to its low titer in bacterial fermentation, modest stability and very low recovery rate during purification, has hindered the investigations of TST-A as an anticancer drug candidate. Here we report the significant yield improvement of TST-A and its direct precursor, thailanstatin D (TST-D), through metabolic engineering of the thailanstatin biosynthetic pathway in MSMB43. Deletion of tstP, which encodes a dioxygenase involved in converting TST-A to downstream products including FR901464 (FR), resulted in 58% increase of the TST-A titer to 144.7?±?2.3?mg/L and 132% increase of the TST-D titer to 14.6?±?0.5?mg/L in the fermentation broth, respectively. Deletion of tstR, which encodes a cytochrome P450 involved in converting TST-D to TST-A, resulted in more than 7-fold increase of the TST-D titer to 53.2?±?12.1?mg/L in the fermentation broth. An execution of 90?L pilot-scale fed-batch fermentation of the tstP deletion mutant in a 120-L fermentor led to the preparation of 714?mg of TST-A with greater than 98.5% purity. The half-life of TST-D in a phosphate buffer was found to be at least 202?h, significantly longer than that of TST-A or FR, suggesting superior stability. However, the IC50 values of TST-D against representative human cancer cell lines were determined to be greater than those of TST-A, indicating weaker antiproliferative activity. This work enabled us to prepare sufficient quantities of TST-A and TST-D for our ongoing translational research.
Project description:The spliceosome regulates pre-mRNA splicing, which is a critical process in normal mammalian cells. Recently, recurrent mutations in numerous spliceosomal proteins have been associated with a number of cancers. Previously, natural product antitumor agents have been shown to interact with one of the proteins that is subject to recurrent mutations (SF3B1). We report the optimization of a class of tumor-selective spliceosome modulators that demonstrate significant in vivo antitumor activity. This optimization culminated in the discovery of sudemycin D6, which shows potent cytotoxic activity in the melanoma line SK-MEL-2 (IC50 = 39 nM) and other tumor cell lines, including JeKo-1 (IC50 = 22 nM), HeLa (IC50 = 50 nM), and SK-N-AS (IC50 = 81 nM). We also report improved processes for the synthesis of these compounds. Our work supports the idea that sudemycin D6 is worthy of further investigation as a novel preclinical anticancer agent with application in the treatment of numerous human cancers.
Project description:Herceptin is considered an essential treatment option for double negative breast cancer. Resveratrol and didox are known chemopreventive agents with potential anticancer properties. The aim of the current study is to investigate the influence of resveratrol and didox on the cytotoxicity profile of herceptin in HER-2 receptor positive and HER-2 receptor negative breast cancer cell lines (T47D and MCF-7 cell lines, respectively). The IC50's of herceptin in T47D and MCF-7 were 0.133?±?0.005?ng/ml and 23.3795?±?1.99?ng/ml respectively. Equitoxic combination of herceptin with resveratrol or didox in T47D significantly reduced the IC50 to 0.052?±?0.001 and 0.0365?±?0.001?ng/ml, respectively and similar results were obtained in MCF-7. The gene expression of BCL-xl was markedly decreased in T47D cells following treatment with herceptin/resveratrol compared to herceptin alone. Immunocytochemical staining of HER-2 receptor in T47D cells showed a significant reduction after treatment with herceptin/resveratrol combination compared to herceptin alone. On the contrary, herceptin/didox combination had no significant effect on HER-2 receptor expression. Cell cycle analysis showed an arrest at G2/M phase for both cell lines following all treatments. In conclusion, herceptin/resveratrol and herceptin/didox combinations improved the cytotoxic profile of herceptin in both T47D and MCF-7 breast cancer cell lines.
Project description:Fludarabine refractoriness (FR) represents an unsolved clinical problem of chronic lymphocytic leukemia (CLL) management. Although next-generation sequencing studies have led to the identification of a number of genes frequently mutated in FR-CLL, a comprehensive evaluation of the FR-CLL genome has not been reported. Toward this end, we studied 10 FR-CLLs by combining whole-exome sequencing and copy number aberration (CNA) analysis, which showed an average of 16.3 somatic mutations and 4 CNAs per sample. Screening of recurrently mutated genes in 48 additional FR-CLLs revealed that ~70% of FR-CLLs carry ?1 mutation in genes previously associated with CLL clinical course, including TP53 (27.5%), NOTCH1 (24.1%), SF3B1 (18.9%), and BIRC3 (15.5%). In addition, this analysis showed that 10.3% of FR-CLL cases display mutations of the FAT1 gene, which encodes for a cadherin-like protein that negatively regulates Wnt signaling, consistent with a tumor suppressor role. The frequency of FAT1-mutated cases was significantly higher in FR-CLL than in unselected CLLs at diagnosis (10.3% vs 1.1%, P = .004), suggesting a role in the development of a high-risk phenotype. These findings have general implications for the mechanisms leading to FR and point to Wnt signaling as a potential therapeutic target in FR-CLL.
Project description:Our previous studies indicated that tumor invasion and 5-flurouracil (5-FU) resistance in colorectal cancer (CRC) was more affected by cytoplasmic localization of expressed Nrf2 (cNrf2) than by nuclear localization (nNrf2), indicating a need for novel antitumor agents to overcome 5-FU resistance and improve outcomes in patients with CRC. In the present study, 20 nitrogen-substituted anthra[1,2-c][1,2,5] thiadiazole-6,11-dione derivatives were collected to verify the compound most able to suppress cell growth in nuclear location sequence (NLS)-mutated Nrf2-transfected shNrf2-HCT116 stable clones that have high cNrf2 expression. The MTT assay indicated that these high-cNrf2-expressing shNrf2-HCT116 stable clones exhibited the lowest percentage survival when treated with RV-59 than with the other 19 compounds. As expected, the high-cNrf2-expressing cells also showed a higher value for the inhibitory concentration of 50% cell survival (IC50) for 5-FU when compared with Nrf2-knockdown HCT116 stable clones (17.74 ?M vs. 5.34 ?M). Interestingly, a lower RV-59 IC50 value was seen in the high-cNrf2-expressing stable clones than in the Nrf2-knockdown stable clones (3.55 ?M vs. 16.81 ?M). A similar low RV-59 IC50 value was observed in high-cNrf2-expressing NLS-mutated Nrf2-transfected shNrf2-HCT116 stable clones and p53 null (-/-) HCT116 cells (4.2 ?M vs. 4.4 ?M), whereas the IC50 value was 17.6 ?M in normal colon FHC epithelial cells. Colony-forming assays confirmed that RV-59 treatment inhibited colony formation in NLS-mutated Nrf2-transfected shNrf2-HCT116 stable clones and in p53-/- HCT116 cells. Annexin-V/PI staining showed an involvement of apoptosis in the inhibitory effect of RV-59 on cell viability. A nude mouse xenograft tumor model showed that RV-59 efficiently suppressed tumor growth induced by transplanted NLS-mutated Nrf2-transfected shNrf2-HCT116 stable clones without affecting the body weight of the nude mice over the 37 day experimental period. These results strongly suggest that RV-59 may be a novel antitumor agent for suppression of 5-FU resistance and may have therapeutic potential for improving outcomes in patients with cNrf2-expressing tumors.