Leptomycin Research Articles

Abstract 3515: Combined effects of leptomycin B and doxorubicin on lung cancer

Abstract To develop novel molecularly targeted therapy has become an important focus of research efforts in lung cancer treatment. Our previous study has shown leptomycin B (LMB), by blocking chromosome maintenance region 1 (CRM1), significantly inhibits cell proliferation in lung cancer cells in a p53-dependent manner. These results suggest the potential use of LMB as a new therapeutic regimen for lung cancer treatment. On the other hand, doxorubicin (DOX) is one of the most effective anticancer agents, which induces p53 activation and nuclear accumulation. The objective of this study is to determine the combinative cytotoxic and molecular effects of LMB and DOX on lung adenocarcinoma cell line, A549. Cells were treated with DOX alone, pre-treatment of DOX/LMB and subsequent treatment of LMB/DOX, or DOX and LMB simultaneously. Cell viability was measured by the MTT assay. The inhibitory effects of DOX on A549 were dose and time-dependent (P<0.001). The 50% inhibitory concentrations (IC50s) of DOX at 24, 48, and 72 h were 60.0, 2.2, and 1.7 µM, respectively. The pre-treatment of 0.5 µM DOX for 24 h significantly improved the cytotoxic effects of LMB on A549 cells at 48 h (P<0.05). The resulting IC50 of LMB was decreased more than 2 fold after pre-treatment of DOX (4.4 nM) as compared to that of LMB alone (10.6 nM). However, the pre-treatment of LMB and the simultaneous treatment of DOX and LMB did not change the cytotoxic effects of either treatment of LMB or DOX. p21 and survivin are well-known p53 downstream genes that play important roles in cell cycle arrest and/or apoptosis. The protein expression levels of these two genes were further analyzed by western blots. Our results showed that the protein expression level of p21 was significantly increased while the protein expression level of survivin was significantly decreased in A549 cells treated with either pre-treatment of DOX and LMB or LMB alone in comparison with those of controls. Furthermore, these changes were more pronounced in pre-treatment of DOX and LMB group as compared to those of LMB alone group. Taken together, our data suggest that the combined therapy using an initial DOX treatment (through activating p53) and subsequent LMB treatment (through blocking the CRM1 function to accumulate activated p53 in the cellular nucleus), that lead to the activation of p53 downstream transcripted genes for cell cycle arrest and apoptosis, might be more effective as a new therapeutic strategy for lung cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3515. doi:10.1158/1538-7445.AM2011-3515

Abstract 658: CBS9106 is a novel low toxic reversible oral CRM1 inhibitor with CRM1 degrading activity

Abstract CRM1 plays an important role in the nuclear export of cargo proteins bearing Nuclear Exporting Signal sequences. Leptomycin B (LMB), a well-known irreversible inhibitor of CRM1, possesses strong anti-tumor properties, however, its toxicity prevents it from being clinically useful. In this study, we show that a novel compound, CBS9106, inhibits CRM1-dependent nuclear export and induces G1-arrest and apoptosis in time- and dose-dependent manner in a broad-spectrum of cancer cells including multiple myeloma cells. IC50s of growth inhibition by CBS9106 (48h) are in the range from 46.7 to 366 nM in multiple myeloma cell lines (MM.1R, MM.1S, ARH-77 and RPMI-8226). In contrast with LMB, CBS9106 acts reversibly and reduces CRM1 protein without affecting the mRNA expression level. CBS9106-dependent reduction of CRM1 is abolished by MG132, a proteasome inhibitor. Pretreatment of cells with LMB suppresses CBS9106-induced CRM1 degradation. Moreover, CBS9106-biotin captured CRM1 in a pull down analysis and the amount of CRM1 captured by CBS9106-biotin was reduced by LMB treatment and vice versa. These results suggest that CBS9106 acts on the domain of CRM1 that includes LMB's covalent binding site at Cys-528, leading to proteasome-dependent CRM1 degradation. Oral administration of CBS9106 significantly suppressed tumor growth and prolonged survival duration in xenograft model mice. It caused prolonged reduction of CRM1 protein in tumor xenografts without showing significant body weight loss. Taken together, the results indicate that CBS9106 is a novel CRM1 inhibitor and a promising clinical candidate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 658. doi:10.1158/1538-7445.AM2011-658

Abstract 5537: Ubiquitination of RRM1 by Ring1B (RNF2) promotes its degradation and nuclear export

Abstract RRM1 (ribonucleotide reductase M1) is a key enzyme involved in deoxyribonucleotide (dNTP) synthesis and DNA damage repair. It also plays a critical role in acquired resistance to the chemotherapy drug gemcitabine, which directly inhibits the RR holoenzyme. Despite its significant importance, however, the mechanisms that control RRM1 abundance and subcellular localization are unclear. We have identified a ubiquitin E3 ligase, Ring1B (RNF2), as an RRM1-associated protein in a yeast two-hybrid screen. RRM1 interacts with Ring1B both in endogenous and exogenous situations. Additionally, immunofluorescent staining has shown that Ring1B clearly co-localized with RRM1 in a pattern of diffuse nuclear speckles. We found that the protein level of RRM1 is regulated by a proteasome-mediated degradation pathway. Ring1B induces poly-ubiquitination of RRM1 in vivo. Formation of poly-ubiquitin chains on RRM1 is through both Lys48 (K48) and Lys63 (K63) of the ubiquitin. Our study demonstrates that the cytoplasmic fraction of RRM1 was increased upon Ring1B or ubiquitin overexpression, but significantly decreased after nuclear export inhibition by leptomycin B (LMB). This indicates that RRM1 ubiquitination promotes the translocation of RRM1 from the nucleus to the cytoplasm. We also show that hydrogen peroxide (H2O2) promotes the degradation of RRM1 in dose and time-dependent manners. H2O2 can increase the poly-ubiquitination of RRM1, which suggests that RRM1 ubiquitination may participate in cellular responses to oxidative stress-induced DNA damage repair. In summary, our data propose that Ring1B-induced ubiquitination regulates RRM1 activity through RRM1 degradation and nuclear export. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5537. doi:10.1158/1538-7445.AM2011-5537

Nuclear Localization of Phospholipase D1 Mediates the Activation of Nuclear Protein Kinase Cα and Extracellular Signal-regulated Kinase Signaling Pathways

Recent studies highlight the existence of a nuclear lipid metabolism related to cellular proliferation. However, the importance of nuclear phosphatidylcholine (PC) metabolism is poorly understood. Therefore, we were interested in nuclear PC as a source of second messengers and, particularly, nuclear localization of PC-specific phospholipase D (PLD). In the present study we have identified the nuclear localization sequence (NLS) of PLD1 whose mutation abolished its nuclear import. Recently, we reported that caspase-mediated cleavage of PLD1 generates the N-terminal fragment (NF-PLD1) and C-terminal fragment (CF-PLD1). Here we show that CF-PLD1 but not NF-PLD1, is exclusively imported into the nucleus via its functional NLS, whereas only some portions of intact PLD1 were localized into the nucleus. The NLS of intact PLD1 or CF-PLD1 is required for interaction with importin-β, which is known to mediate nuclear import. The amount of intact PLD1 or CF-PLD1 translocated into nucleus is correlated with its binding affinity with importin-β. Ultimately, nuclear localization of intact PLD1 but not CF-PLD1 mediates the activation of nuclear protein kinase Cα and extracellular signal-regulated kinase signaling pathways. Taken together, we propose that nuclear localization of PLD1 via the NLS and its interaction with importin-β may provide new insights on the functional role of nuclear PLD1 signaling.

A nuclear export signal within the structural Gag protein is required for prototype foamy virus replication

BackgroundThe Gag polyproteins play distinct roles during the replication cycle of retroviruses, hijacking many cellular machineries to fulfill them. In the case of the prototype foamy virus (PFV), Gag structural proteins undergo transient nuclear trafficking after their synthesis, returning back to the cytoplasm for capsid assembly and virus egress. The functional role of this nuclear stage as well as the molecular mechanism(s) responsible for Gag nuclear export are not understood.ResultsWe have identified a leptomycin B (LMB)-sensitive nuclear export sequence (NES) within the N-terminus of PFV Gag that is absolutely required for the completion of late stages of virus replication. Point mutations of conserved residues within this motif lead to nuclear redistribution of Gag, preventing subsequent virus egress. We have shown that a NES-defective PFV Gag acts as a dominant negative mutant by sequestrating its wild-type counterpart in the nucleus. Trans-complementation experiments with the heterologous NES of HIV-1 Rev allow the cytoplasmic redistribution of FV Gag, but fail to restore infectivity.ConclusionsPFV Gag-Gag interactions are finely tuned in the cytoplasm to regulate their functions, capsid assembly, and virus release. In the nucleus, we have shown Gag-Gag interactions which could be involved in the nuclear export of Gag and viral RNA. We propose that nuclear export of unspliced and partially spliced PFV RNAs relies on two complementary mechanisms, which take place successively during the replication cycle.

A new cell-based reporter system for sensitive screening of nuclear export inhibitors

Nucleocytoplasmic transport of proteins across the nuclear pore complex (NPC), mediated by the nuclear localization signal (NLS) and the nuclear export signal (NES), is a vital homeostatic process in eukaryotic cells and also in mitogen-activated protein kinase (MEK) signaling molecule in tumor cell proliferation. Some viruses, including the influenza virus and HIV-1, also employ this nuclear export mechanism during their life cycle. Hence, drugs that control nucleocytoplasmic transport of proteins are putative candidate antivirals or anti-cancer agents. Thus, we previously developed a GFP/NES-MDCK reporter cell system for screening novel nuclear export inhibitors. NES signal-conjugated GFP accumulates in the nucleus in the presence of the nuclear export inhibitor leptomycin B (LMB). In this study, a stable GFP/NLS/NES fusion protein-expressing cell line was established, and its potential as a reporter was evaluated. The GFP/NLS/NES-MDCK cell line demonstrates improved nuclear accumulation of GFP in a time-course treatment with LMB. In addition, the dose-response data demonstrated superior sensitivity of GFP/NLS/NES-MDCK over GFP/NES-MDCK cells. As low as 0.01 ng/mL LMB is sufficient to cause accumulation of the GFP fusion protein in the nucleus in GFP/NLS/NES-MDCK cells, while at least 1 ng/mL of LMB is needed for the accumulation of GFP fusion protein in the nucleus of GFP/NES-MDCK cells. These results indicate that the newly established GFP/NLS/NES-MDCK cell line is a potentially powerful tool to screen for novel nuclear export inhibitors.

Anti-influenza viral effects of novel nuclear export inhibitors from Valerianae Radix and <i>Alpinia galanga </i>

Many pathogenic viruses, such as the influenza virus and the Human Immunodeficiency Virus (HIV)-1, are a threat to humans, thus leading to thousands of deaths annually. The development of antiviral drugs is urgent, and it is an essential strategy for the suppression of these infectious diseases. However, regardless of the rapid emergence of many infectious diseases, the development of novel antiviral drugs has been slow, except for the case of the AIDS. In addition, several viruses can easily mutate and escape the inhibitory activity of anti-viral drugs. It was already well-established that HIV escapes from anti-viral drug effects because of the lack of proofreading activity in its reverse transcriptase. It is known that the influenza virus, which is resistant to Tamiflu, is already spread all over the world. Viruses utilize the host cell environment and cellular factors to propagate. Therefore, the development of novel drugs which inhibit viral protein-host protein interactions or cellular functions appear to be good candidates. The influenza virus is unique in replicating in host nuclei, and we therefore focused on the nuclear export processes for the development of anti-influenza viral drugs. We previously reported that leptomycin B (LMB), which inhibited the nuclear export processes via the nuclear export signal (NES) inhibited the nuclear export of influenza viral RNP (vRNP), and resulted in the inhibition of influenza viral propagation. We herein examined novel CRM1 inhibitors, valtrate from Valerianae Radix, and 1'-acetoxychavicol acetate (ACA) from Alpinia galanga as potent inhibitors for the influenza virus replication.

Proteomic Analysis of Importin .ALPHA.-interacting Proteins in Adult Mouse Brain

Many transport factors, such as importins and exportins, have been identified, and the molecular mechanisms underlying nucleocytoplasmic transport have been characterized. The specific molecules that are carried by each transport factor and the temporal profiles that characterize the movements of various proteins into or out of the nucleus, however, have yet to be elucidated. Here, we used a proteomic approach to identify molecules that are transported into the nuclei of adult mouse brain cells via importin α5. We identified 48 proteins in total, among which we chose seven to characterize more extensively: acidic (leucine-rich) nuclear phosphoprotein 32 family member A (Anp32a), far upstream element binding protein 1 (FUBP1), thyroid hormone receptor β1 (TRβ1), transaldolase 1, CDC42 effector protein 4 (CDC42-ep4), Coronin 1B, and brain-specific creatine kinase (CK-B). Analyses using green fluorescent protein (GFP)-fused proteins showed that Anp32a, FUBP1, and TRβ1 were localized in the nucleus, whereas transaldolase 1, CDC42-ep4, CK-B, and Coronin 1B were distributed in both the cytoplasm and nucleus. Using a digitonin-permeabilized in vitro transport assay, we demonstrated that, with the exception of CK-B, these proteins were transported into the nucleus by importin α5 together with importin β and Ran. Further, we found that leptomycin B (LMB) treatment increased nuclear CK-B-GFP signals, suggesting that CK-B enters the nucleus and is then exported in a CRM1-dependent manner. Thus, we identified a comprehensive set of candidate proteins that are transported into the nucleus in a manner dependent on importin α5, which enhances our understanding of nucleocytoplasmic signaling in neural cells.

Effect of Nuclear Export Inhibitor Leptomycin B on the Intracellular Localization of HBV Core Protein into Hepatocytes Cell Line Huh-7 and HepG2 Cells

Leptomycin B (LMB) was originally discovered as a potent anti-fungal antibiotic from Streptomyces species. The cellular target of LMB has been identified as the nuclear export receptor CRM-1 or exportin-1, which is involved in nuclear trafficking of cellular RNAs or proteins containing the nuclear export sequence (NES). CRM-1 is the main mediator of nuclear export in many cell types including hepatocyte cell lines. The ability of LMB to inhibit nuclear export has made it a useful tool in the study of the intracellular localization of many regulatory proteins. In this study, we evaluated the effect of nuclear export inhibitor LMB treatment on the intracellular localization of HBV core protein into the hepatocyte cell lines, Huh-7 and HepG2 cells. We also reported the quantification of the distribution of EGFP-Core fusion protein with redundant core NLS as well as SV-40 NLS into cell compartments. Results shown that in Huh-7 cells treatment of LMB caused retention of EGFP-Core fusion protein into the nucleus, so increased the nuclear localization of EGFP-Core and all variants. In HepG2 cells, although not significantly, treatment of LMB increased a number of nuclear localization in all EGFP-Core constructions, even the nuclear localization in HepG2 cells is not so high as in Huh-7 cells. Keywords: Leptomycin B, HBV, core protein, intracellular localization, NLS, Huh-7, HepG2 cell

RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin-dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence-binding protein J (RBP-J)/CBF-1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP-J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP-J/CBF-1-interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP-J/CBF-1 from the nucleus. Functionally, we show that RITA can reverse a Notch-induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch-mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP-J/CBF-1, where its amounts are limiting.

Stepwise Loss of Fluorescent Core Protein V from Human Adenovirus during Entry into Cells

Human adenoviruses (Ads) replicate and assemble particles in the nucleus. They organize a linear double-strand DNA genome into a condensed core with about 180 nucleosomes, by the viral proteins VII (pVII), pX, and pV attaching the DNA to the capsid. Using reverse genetics, we generated a novel, nonconditionally replicating Ad reporter by inserting green fluorescent protein (GFP) at the amino terminus of pV. Purified Ad2-GFP-pV virions had an oversized complete genome and incorporated about 38 GFP-pV molecules per virion, which is about 25% of the pV levels in Ad2. GFP-pV cofractionated with the DNA core, like pV, and newly synthesized GFP-pV had a subcellular localization indistinguishable from that of pV, indicating that GFP-pV is a valid reporter for pV. Ad2-GFP-pV completed the replication cycle, although at lower yields than Ad2. Incoming GFP-pV (or pV) was not imported into the nucleus. Virions lost GFP-pV at two points during the infection process: at entry into the cytosol and at the nuclear pore complex, where capsids disassemble. Disassembled capsids, positive for the conformation-specific antihexon antibody R70, were devoid of GFP-pV. The loss of GFP-pV was reduced by the macrolide antibiotic leptomycin B (LMB), which blocks nuclear export and adenovirus attachment to the nuclear pore complex. LMB inhibited the appearance of R70 epitopes on Ad2 and Ad2-GFP-pV, indicating that the loss of GFP-pV from Ad2-GFP-pV is an authentic step in the adenovirus uncoating program. Ad2-GFP-pV is genetically complete and hence enables detailed analyses of infection and spreading dynamics in cells and model organisms or assessment of oncolytic adenoviral potential.

Association between expression of embryonic lethal abnormal vision‐like protein HuR and cyclooxygenase‐2 in oral squamous cell carcinoma

Increased cytoplasmic HuR expression has been noted in several cancer types, where it may contribute to the increased cyclooxygenase-2 (COX-2) expression observed during tumorigenesis and metastasis. To assess the correlation between COX-2 and HuR in oral squamous cell carcinoma (OSCC), the expression patterns of HuR and COX-2 were assessed via immunohistochemistry analyses of 103 OSCC samples. Cytoplasmic HuR expression was significantly associated with COX-2 expression (p < .025) and lymph node metastasis (p < .050) and distant metastasis (p < .025). In multivariate analysis, cytoplasmic HuR expression was identified as an independent prognostic parameter for reduced overall survival. The inhibition of HuR expression by siRNA or leptomycin B (LMB) caused a reduction in the inducibility of COX-2 in oral cancer cells. Our results indicate that the cytoplasmic expression of HuR is associated with COX-2 expression in OSCCs and HuR can regulate COX-2 expression in oral cancer cell lines.

Novel cis-active structures in the coding region mediate CRM1-dependent nuclear export of IFN-α 1 mRNA

We recently reported the chromosome region maintenance 1 (CRM1)-dependent nuclear export of intron-less human interferon-α1 (IFN-α1) mRNA, which encodes a main effecter of host innate immunity. We show that the coding region of IFN-α1 mRNA forms novel secondary structures that are responsible for the CRM1-dependent export of the transcript. Deletion-mutagenesis, in vivo export assays, and computer analyses of the folding potentials of export-competent fragments revealed the presence of a domain, termed the conserved secondary structure (CSS), comprising two adjacent putative stable stem-loop structures (nt 208-452). Internal deletion-mutagenesis and constitutive export assays of each stem-loop structure demonstrated that subregions 308-322 and 352-434 act as a core element by conferring the export function on the CSS. Leptomycin B (LMB) inhibition of the CRM1 pathway decreased the export of core element RNA, implying that the principal site of CRM1 action for exporting IFN-α1 mRNA resides within the core element. An RNPS1 (RNA-binding protein S1, serine-rich domain) cDNA was isolated by yeast three-hybrid screening, using bait containing two CSS regions. We showed that RNPS1 might recognize IFN-α1 mRNP that includes CRM1. The data demonstrate that interaction between RNA structures in the coding region and CRM1 affects the nucleocytoplasmic translocation of IFN-α1 mRNA.

P53-dependent anticancer effects of leptomycin B on lung adenocarcinoma

Leptomycin B (LMB) and/or its derivatives are considered a novel class of cancer therapeutics through blocking chromosome maintenance region 1, which mediates p53 nuclear export. The objectives of the present study were to first evaluate the cytotoxic effects of LMB on a normal human lung epithelial cell line (BEAS-2B) and three human lung adenocarcinoma cell lines with various p53 status (wild type: A549, mutant: NCI-H522, and null: NCI-H358) and then to identify LMB-induced gene expression alterations in human p53 signaling pathway. Cells were treated with 0.01-100 nM LMB or 0.1% ethanol (vehicle control) for 4-72 h. Gene expression analyses using gene array for 84 genes involved in p53-mediated signaling pathways were performed in A549 and NCI-H358 after treatment with 20 nM LMB or vehicle control for 24 h. Cytotoxic results from MTS assays revealed a significant dose- and time-dependent effect of LMB on all cell lines. However, this effect was more pronounced in cancer cells than in normal cells, and cancer cells with p53 wild type tended to be less sensitive than those with p53 mutant or null. A total of 23 genes, predominantly involved in apoptosis and cell cycle/proliferation, were significantly altered in A549 after LMB treatment, while no strong modulating effects were observed in NCI-H358. The protein expression of two selected genes, p21 and survivin, was further confirmed by Western blots. Our results suggest that LMB has anti-cancer potential and provides a new regimen of individualized therapy for lung cancer treatment.

Cloning and functional characterization of the guinea pig apoptosis inhibitor protein Survivin

Background The guinea pig is widely used as a model to study (patho)physiological processes, such as neurodegenerative disorders. Survivin's dual function as an apoptosis inhibitor and a mitotic regulator is crucial not only for ordered development but its modulation seems crucial also under disease conditions. However, data on the expression and function of the guinea pig Survivin protein (Survivin Gp ) are currently lacking. Results Here, we here report the cloning and functional characterization of Survivin Gp . The respective cDNA was cloned from spleen mRNA, containing a 426 bp open reading frame encoding for a protein of 142aa. Survivin Gp displays a high homology to the human and murine orthologue, especially in domains critical for function, such as binding sites for chromosomal passenger complex (CPC) proteins and the nuclear export signal (NES). Notably, phylogenetic analyses revealed that Survivin Gp is more related to humans than to rodents. Ectopic expression studies of a Survivin Gp -GFP fusion confirmed its dynamic intracellular localization, analogous to the human and murine counterparts. In interphase cells, Survivin Gp -GFP was predominantly cytoplasmic and accumulated in the nucleus following export inhibition with leptomycin B (LMB). A typical CPC protein localization during mitosis was observed for Survivin Gp -GFP. Microinjection experiments together with genetic knockout demonstrated that the NES is essential for the anti-apoptotic and regulatory role of Survivin Gp during cell division. In vivo protein interaction assays further demonstrated its dimerization with human Survivin and its interaction with human CPC proteins. Importantly, RNAi-depletion studies show that Survivin Gp can fully substitute for human Survivin as an apoptosis inhibitor and a mitotic effector. Immunohistochemistry, immunofluorescence, and western blotting were employed to detect Survivin expression in guinea pig tissues. Besides its expression in proliferating tissues, such as spleen and liver, we also found Survivin in terminally differentiated cell types. Importantly, Survivin was detectable also in the cochlea, suggesting a potential role for Survivin in the auditory system. Conclusions We provide the first experimental evidence for the expression of Survivin in the guinea pig. As Survivin Gp can substitute for known functions of human Survivin, the guinea pig model will now also allow investigating Survivin's (patho)physiological role and to test Survivin-directed potential therapeutic strategies.

Abstract A19: KPT0127, a novel, potent, and selective inhibitor of Crm1-mediated nuclear export shows selective cytotoxicity for neoplastic over normal cells and is well tolerated in animals

Abstract CRM1 (Xpo1) is the major export factor for proteins from the nucleus to the cytoplasm, including tumor suppressors (TSPs) and other modulators of proliferative responses such as p53, FOXO, c-Abl, pRB and IκB. Leptomycin B (LMB), a non-drug like natural product is a potent inhibitor of CRM1-mediated nucleocytoplasmic transport. LMB and related analogs trap TSPs and other proteins in the nucleus, forcing neoplastic cells into apoptosis while normal cells undergo reversible cell cycle arrest. However, LMB has limited efficacy in vivo due to its severe gastrointestinal toxicity. Here, we describe our lead compound KPT-0127 a novel small molecule, water soluble, drug-like, selective, irreversible CRM1 antagonist. Like LMB, KPT-0127 forms a covalent bond with Cys528 in the CRM1 cargo-binding pocket, abrogating most, but not all, export functions of CRM1. KPT-0127 exerts a potent (EC50 300-400nM) and prolonged inhibition of CRM1- mediated HIV-1 Rev, forkhead (FOXO), and p53 nuclear export in a variety of normal and transformed cell lines. In cytotoxicity assays, KPT-0127 showed high potency in most hematologic cancer cell lines (EC50 &amp;lt;500nM, with leukemia and lymphoma lines often &amp;lt;100nM) and variable activity in solid tumor cell lines (EC50 90-2000nM), with colon and melanoma cells being the most sensitive (EC50&amp;lt; 100 nM). By contrast, normal cells were largely unaffected by treatment with KPT-0127 (EC50 &amp;gt;5-10µM). Studies in the HCT-116 colon cancer cell line suggested that KPT-0127 dose dependently increases the nuclear levels of p53 and appears to induce cell cycle arrest at both the G1/S and G2/M checkpoints, prior to inducing apoptosis. In normal peripheral blood mononuclear cells (PBMCs) and in Hut78 leukemia cells, KPT-0127 potently increased the nuclear levels of IκB. However, KPT-0127 induced cell death of Hut78 cells with no effect on normal PBMCs. In drug combination studies, KPT-0127 showed additive or synergistic cytotoxicity activity with either 5-FU, carboplatin, or doxorubicin. Mechanism of action studies demonstrated that the Cys528 residue in the cargo-binding pocket of CRM1 is essential for the inhibitory effect of KPT-0127. Mutagenesis of Cys528 to a Ser completely abrogated KPT-0127 inhibition. KPT-0128, the transisomer of KPT-0127, shows little effect on both HIV-Rev nuclear export and in cytotoxicity assays (EC50s &amp;gt; 10μM), supporting the specificity of KPT-0127 for CRM1. Moreover, the selectivity of KTP-0127 was demonstrated across a panel of 37 proteins including several cysteine proteases. In single dose mouse toxicology studies, KPT-0127 was generally well tolerated (oral or SC) up to 560 mg/kg, and no deaths were observed. SC dosing daily for 5 days up to 100mg/kg (the highest dose tested) showed no behavioral, clinical chemistry, or hematogical effects in mice; Pharmacokinetics of KPT-0127 is adequate and in vivo efficacy studies are currently being performed; results will be presented. All together, these data demonstrate that KPT-0127 represents a novel, tumor selective, and well-tolerated irreversible Crm1 inhibitor which may be suitable for clinical development both as a single agent and in combination with standard therapies. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A19.

Pericentrin contains five NESs and an NLS essential for its nucleocytoplasmic trafficking during the cell cycle

Pericentrin, a conserved centrosomal component, provides the structural scaffold to anchor numerous centrosomal proteins, and thus plays an essential role in the organization and function of the centrosome and the mitotic spindle. Although pericentrin was shown to localize in the cytoplasm and reported to be sensitive to leptomycin B (LMB), a specific inhibitor of Crm1, the regions within pericentrin that serve as signals for transporting in and out of the nucleus have not yet been identified. In this study, we identified five novel nuclear export signals (NESs) in pericentrin with diverse export activities. All of the five NESs could bind to Crm1 in a LMB-sensitive way when mediating the nuclear export of pericentrin. We also demonstrated that the region of amino acids 8-42 in pericentrin contains a tripartite nuclear localization signal (NLS) consisting of three clusters of basic amino acids. The NLS of pericentrin binds to importin beta directly or via the adaptor importin alpha to form the import complex, which could be disrupted by RanQ69L, a dominant-negative Ran GTPase possessing high affinity for importin beta. Furthermore, we found that mutation of the NESs in full-length pericentrin results in both nuclear and cytoplasmic localization, and mutation of the NLS abolishes the nuclear import of pericentrin. On the basis of these results, we suggest that the NESs and NLS of pericentrin are essential for its subcellular localization and nucleocytoplasmic trafficking during the cell cycle.

Abstract 5007: Nuclear targeted Deleted in liver cancer 1 exhibited reduced tumor suppressive function both in vitro and in vivo

Abstract The tumor suppressor Deleted in liver cancer 1 (DLC1) protects cell from transformation by catalyzing the GTP hydrolysis of active RhoA. Besides the RhoGAP activity, accumulating evidences have supported that localization at focal adhesions and interaction with tensin protein are key factors guiding the tumor suppressive activity of DLC1. In this study, we provided new evidence that DLC1 was a dynamic protein which shuttles between cytoplasm and nucleus instead of statically staying in the cytoplasm. Subcellular fractionation assay showed that DLC1 protein can be detected in cytoplasmic and nuclear fractions. Treatment of Leptomycin B (LMB), a nuclear export blocker, could lead to the nuclear retention of exogenous and endogenous DLC1 in different human cancer cell lines. Detail examination of relevant DLC1 mutants had shown that the center region of DLC1 was necessary and sufficient for its nuclear localization. We transiently expressed a NLS fusion DLC1 (NLS-DLC1) with preferential nuclear localization in SMMC HCC cells and found that NLS-DLC1 was less potent in suppressing colony formation and actin stress fiber formation. To study the tumor suppressive function of nuclear DLC1 both in vitro and in vivo, we employed retroviral transduction to stably express NLS-DLC1 in a p53−/− RasV12 hepatoblast model. We found that NLS-DLC1 expressing cells proliferated faster in vitro and exhibited increased tumorigenicity upon nude mice injection when compared with the non-nuclear targeted DLC1. Although the function of nuclear DLC1 remains to be answered, our results clearly demonstrated that nuclear localization of DLC1 may serve as a potential mechanism in negatively regulating its tumor suppressive activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5007.

Anguinomycins and Derivatives: Total Syntheses, Modeling, and Biological Evaluation of the Inhibition of Nucleocytoplasmic Transport

The preparation of the polyketide natural products anguinomycin C and D is reported based on key steps such as Negishi stereoinversion cross coupling, Jacobsen Cr(III)-catalyzed Hetero Diels-Alder reaction, Evans B-mediated syn-aldol chemistry, and B-alkyl Suzuki-Miyaura cross coupling. The configuration of both natural products was established as (5R,10R,16R,18S,19R,20S). Biological evaluation demonstrated that these natural products are inhibitors of the nuclear export receptor CRM1, leading to shutdown of CRM1-mediated nuclear protein export at concentrations above 10 nM. Analogues of anguinomycin and leptomycin B (LMB) have been prepared, and the simple alpha,beta-unsaturated lactone analogue 4 with a truncated polyketide chain retains most of the biological activity (inhibition above 25 nM). The structural basis for this inhibition has been demonstrated by modeling the transport inhibitors into X-ray crystal structures, thus highlighting key points for successful and strong biological action of anguinomycin and LMB.

The Final Step in 5.8S rRNA Processing Is Cytoplasmic in Saccharomyces cerevisiae

The 18S rRNA component of yeast (Saccharomyces cerevisiae) 40S ribosomes undergoes cytoplasmic 3' cleavage following nuclear export, whereas exported pre-60S subunits were believed to contain only mature 5.8S and 25S rRNAs. However, in situ hybridization detected 3'-extended forms of 5.8S rRNA in the cytoplasm, which were lost when Crm1-dependent preribosome export was blocked by treatment with leptomycin B (LMB). LMB treatment rapidly blocked processing of 6S pre-rRNA to 5.8S rRNA, leading to TRAMP-dependent pre-rRNA degradation. The 6S pre-rRNA was coprecipitated with the 60S export adapter Nmd3 and cytoplasmic 60S synthesis factor Lsg1. The longer 5.8S+30 pre-rRNA (a form of 5.8S rRNA 3' extended by approximately 30 nucleotides) is processed to 6S by the nuclear exonuclease Rrp6, and nuclear pre-rRNA accumulated in the absence of Rrp6. In contrast, 6S to 5.8S processing requires the cytoplasmic exonuclease Ngl2, and cytoplasmic pre-rRNA accumulated in strains lacking Ngl2. We conclude that nuclear pre-60S particles containing the 6S pre-rRNA bind Nmd3 and Crm1 and are exported to the cytoplasm prior to final maturation by Ngl2.