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XLSX file - 132K, Table S2: Effects of EPZ-6438 on Gene Expression in EZH2 Mutant Lymphoma Cells.

PDF file - 811K, Fig. S1. Effects of EPZ-6438 on Gene Promoter H3K27 Methylation, Recruitment of PRC2 Components to Gene Promoters, and Cell Cycle, in WSU-DLCL2 Cells. Fig. S2: Effects of EPZ-6438 on Gene Expression in EZH2 Mutant Lymphoma Cell Lines. Fig. S3: Pharmacokinetic Profiles of EPZ-6438 in Rats and Mice. Fig. S4: EPZ-6438 Compound Levels in Plasma and WSU-DLCL2 Xenograft Tumor Homogenates from Mice Dosed for 7 or 28 Days. Fig. S5: In vivo Effects of EPZ-6438 in Lymphoma Xenograft Models. Fig. S6: Body Weights of Mice during Xenograft Efficacy Studies.

PDF file - 25K, Table S1: LCC Values for EPZ-6438 for WSU-DLCL2 Human Lymphoma Cells Dosed Either Continuously or After Compound Washout.

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers. Mol Cancer Ther; 13(4); 842–54. ©2014 AACR.

The list of Topologically significant genes by RNA-Seq analysis

The Wnt/β-catenin signaling pathway plays crucial roles in embryonic development and the development of multiple types of cancer, and its aberrant activation provides cancer cells with escape mechanisms from immune checkpoint inhibitors. E7386, an orally active selective inhibitor of the interaction between β-catenin and CREB binding protein, which is part of the Wnt/β-catenin signaling pathway, disrupts the Wnt/β-catenin signaling pathway in HEK293 and adenomatous polyposis coli (APC)-mutated human gastric cancer ECC10 cells. It also inhibited tumor growth in an ECC10 xenograft model and suppressed polyp formation in the intestinal tract of ApcMin/+ mice, in which mutation of Apc activates the Wnt/β-catenin signaling pathway. E7386 demonstrated antitumor activity against mouse mammary tumors developed in mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice. Gene expression profiling using RNA sequencing data of MMTV-Wnt1 tumor tissue from mice treated with E7386 showed that E7386 downregulated genes in the hypoxia signaling pathway and immune responses related to the CCL2, and IHC analysis showed that E7386 induced infiltration of CD8+ cells into tumor tissues. Furthermore, E7386 showed synergistic antitumor activity against MMTV-Wnt1 tumor in combination with anti-PD-1 antibody. In conclusion, E7386 demonstrates clear antitumor activity via modulation of the Wnt/β-catenin signaling pathway and alteration of the tumor and immune microenvironments, and its antitumor activity can be enhanced in combination with anti-PD-1 antibody.Significance:These findings demonstrate that the novel anticancer agent, E7386, modulates Wnt/β-catenin signaling, altering the tumor immune microenvironment and exhibiting synergistic antitumor activity in combination with anti-PD-1 antibody.

Supplementary Table S1, Table S4, Table S5, Table S6, Table S7. Supplementary Table S1: The list of DEGs by nCounter analysis (|Fold change| > 1.25, P < 0.1). Supplementary Table S4: Top 10 enriched pathways identified by RNA-Seq analysis in MMTV-Wnt1 model. Supplementary Table S5: The result of CIBERSORT analysis. Supplementary Table S6: The lists of antibody for flow cytometry analysis. Supplementary Table S7: Comparison of Physicochemical property between E7386 and C-82.

Supplementary Materials and Methods

The Wnt/β-catenin signaling pathway plays crucial roles in embryonic development and the development of multiple types of cancer, and its aberrant activation provides cancer cells with escape mechanisms from immune checkpoint inhibitors. E7386, an orally active selective inhibitor of the interaction between β-catenin and CREB binding protein, which is part of the Wnt/β-catenin signaling pathway, disrupts the Wnt/β-catenin signaling pathway in HEK293 and adenomatous polyposis coli (APC)-mutated human gastric cancer ECC10 cells. It also inhibited tumor growth in an ECC10 xenograft model and suppressed polyp formation in the intestinal tract of ApcMin/+ mice, in which mutation of Apc activates the Wnt/β-catenin signaling pathway. E7386 demonstrated antitumor activity against mouse mammary tumors developed in mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice. Gene expression profiling using RNA sequencing data of MMTV-Wnt1 tumor tissue from mice treated with E7386 showed that E7386 downregulated genes in the hypoxia signaling pathway and immune responses related to the CCL2, and IHC analysis showed that E7386 induced infiltration of CD8+ cells into tumor tissues. Furthermore, E7386 showed synergistic antitumor activity against MMTV-Wnt1 tumor in combination with anti-PD-1 antibody. In conclusion, E7386 demonstrates clear antitumor activity via modulation of the Wnt/β-catenin signaling pathway and alteration of the tumor and immune microenvironments, and its antitumor activity can be enhanced in combination with anti-PD-1 antibody. Significance: These findings demonstrate that the novel anticancer agent, E7386, modulates Wnt/β-catenin signaling, altering the tumor immune microenvironment and exhibiting synergistic antitumor activity in combination with anti-PD-1 antibody.

Background Type 2 diabetes mellitus is characterized by insulin resistance and pancreatic β-cell dysfunction. A decrease in β-cell mass, which occurs during the progression of Type 2 diabetes mellitus, contributes to impaired insulin secretion. Mulberry leaves contain various nutritional components that exert anti-diabetic and anti-atherogenic effects. The present study analyzed the effects of mulberry leaf intake on pancreatic β-cells to clarify the mechanisms underlying its anti-diabetic function. Methods Mulberry leaves (Morus alba L.) were dried at 180 °C for 8 s in a hot-air mill and fed to obesity/Type 2 diabetes mellitus db/db mouse models at 5% (w/w) as part of a normal diet from 7 to 10, 15, or 20 weeks of age. An intraperitoneal glucose tolerance test was then performed on the mice. To evaluate the β-cell mass, the pancreas was subjected to immunohistological analysis with an anti-insulin antibody. A TUNEL assay and immunohistological analysis with a proliferation marker was also performed. Expression levels of endoplasmic reticulum stress-responsible genes and proliferation markers were assessed by quantitative RT-PCR. Results Intake of mulberry leaves maintained the β-cell function of db/db mice. Moreover, oral administration of mulberry leaves significantly decreased cell death by reducing endoplasmic reticulum stress in the pancreas. Mulberry leaves significantly increased proliferation of β-cells and the expression of pancreatic duodenal homeobox1 mRNA in the pancreas. Conclusion Considered together, these results indicate that dietary mulberry leaf administration can maintain insulin levels and pancreatic β-cell mass, at least in part, by suppressing endoplasmic reticulum stress in Type 2 diabetes mellitus mouse models.

Sunitinib malate (sunitinib) is an orally available, multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activities. Although sunitinib is effective for the treatment of patients with gastrointestinal stromal tumor, advanced renal cell carcinoma, or pancreatic neuroendocrine tumor, adverse cardiac events associated with sunitinib administration have been reported. Here, we examined the effect of geldanamycin, an inhibitor of heat shock protein (Hsp) 90, on sunitinib-induced cytotoxicity in cardiomyocytes. First, we found that treatment with geldanamycin or other Hsp90 inhibitors (tanespimycin, ganetespib, or BIIB021) significantly attenuated sunitinib-induced cytotoxicity in rat H9c2 cardiomyocytes, suggesting a drug-class effect of Hsp90 inhibitors. We then examined the mechanisms underlying sunitinib-induced cytotoxicity and found that sunitinib induced autophagy in H9c2 cells and that pretreatment with geldanamycin inhibited the induction of autophagy by promoting degradation of the autophagy-related proteins Atg7, Beclin-1, and ULK1. Pharmacological assessment with autophagy inhibitors confirmed that geldanamycin attenuated the cytotoxicity of sunitinib by interfering with autophagy. In addition, we found that the molecular chaperone Hsp70, which is induced by geldanamycin, was not involved in the attenuation of sunitinib-induced cytotoxicity. Finally, to provide more clinically relevant data, we confirmed that geldanamycin attenuated sunitinib-induced cytotoxicity in human induced pluripotent stem cell-derived cardiomyocytes. Together, these data suggest that geldanamycin attenuates sunitinib-induced cytotoxicity in cardiomyocytes by inhibiting the autophagy pathway. Thus, the further investigation of combination or sequential treatment with an Hsp90 inhibitor and sunitinib is warranted as a potential strategy of attenuating the cardiotoxicity associated with sunitinib administration in the clinical setting.

Eribulin mesylate is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. Some tubulin-binding drugs are known to have antivascular (antiangiogenesis or vascular-disrupting) activities that can target abnormal tumor vessels. Using dynamic contrast-enhanced MRI analyses, here we show that eribulin induces remodeling of tumor vasculature through a novel antivascular activity in MX-1 and MDA-MB-231 human breast cancer xenograft models. Vascular remodeling associated with improved perfusion was shown by Hoechst 33342 staining and by increased microvessel density together with decreased mean vascular areas and fewer branched vessels in tumor tissues, as determined by immunohistochemical staining for endothelial marker CD31. Quantitative RT-PCR analysis of normal host cells in the stroma of xenograft tumors showed that eribulin altered the expression of mouse (host) genes in angiogenesis signaling pathways controlling endothelial cell-pericyte interactions, and in the epithelial-mesenchymal transition pathway in the context of the tumor microenvironment. Eribulin also decreased hypoxia-associated protein expression of mouse (host) vascular endothelial growth factor by ELISA and human CA9 by immunohistochemical analysis. Prior treatment with eribulin enhanced the anti-tumor activity of capecitabine in the MDA-MB-231 xenograft model. These findings suggest that eribulin-induced remodeling of abnormal tumor vasculature leads to a more functional microenvironment that may reduce the aggressiveness of tumors due to elimination of inner tumor hypoxia. Because abnormal tumor microenvironments enhance both drug resistance and metastasis, the apparent ability of eribulin to reverse these aggressive characteristics may contribute to its clinical benefits.

Introduction Cardiac hypertrophy is a leading cause of many cardiovascular diseases, including heart failure, but its pathological mechanism is not fully understood. This study used highly purified human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes to produce an in vitro hypertrophy model and characterize its gene expression and electrophysiological properties. Methods For 7 days we cultured hiPSC-derived cardiomyocytes plated at high (2800–4800 cells/mm 2 ) or low (500–1200 cells/mm 2 ) cell density and assessed their cell size with confocal and fluorescence microscopy, their electrophysiological and pharmacological responses with multi-electrode array systems, and their gene expression patterns by using DNA microarray technology and quantitative PCR. We used quantitative PCR and Western blotting to compare the expression of potassium-channel genes between the hiPSC-derived cardiomyocytes and human fetal and adult hearts. Results The hiPSC-derived cardiomyocytes showed spontaneous beating and similar pattern of α-actinin molecules regardless of plating density. However, cells plated at low density had the following characteristics compared with those at high density: 1) significant enlargement in size; 2) significant increase or decrease in expression of the cardiac hypertrophy-characteristic genes NPPA , ATP2A2 , ANKRD1 and MYL2 in accordance with the progression of hypertrophy; 3) significant reduction in responses to the inhibitors of cardiac slow delayed-rectifier K + current ( I Ks ), chromanol 293B and HMR1556, in a cell-density-dependent manner; and 4) significant reduction in the expression of the KCNQ1 and KCNJ2 genes coding the K + ion channels conducting each I Ks and cardiac inward rectifier outward K + current ( I K1 ). Discussion The enlargement, hypertrophy-characteristic and potassium ion channels gene expression of hiPSC-derived cardiomyocytes suggest that low-density plating was sufficient to induce cardiac hypertrophy. This model may be useful in elucidating mechanisms underlying the onset and progress of cardiac hypertrophy, because these cells can be cultured for several weeks.

Target-protein degradation is an emerging field in drug discovery and development. In particular, the substrate-receptor proteins of the cullin-ubiquitin ligase system play a key role in selective protein degradation, which is an essential component of the anti-myeloma activity of immunomodulatory drugs (IMiDs), such as lenalidomide. Here, we demonstrate that a series of anticancer sulfonamides NSC 719239 (E7820), indisulam, and NSC 339004 (chloroquinoxaline sulfonamide, CQS) induce proteasomal degradation of the U2AF-related splicing factor coactivator of activating protein-1 and estrogen receptors (CAPERα) via CRL4DCAF15 mediated ubiquitination in human cancer cell lines. Both CRISPR-Cas9-based knockout of DCAF15 and a single amino acid substitution of CAPERα conferred resistance against sulfonamide-induced CAPERα degradation and cell-growth inhibition. Thus, these sulfonamides represent selective chemical probes for disrupting CAPERα function and designate DCAFs as promising drug targets for promoting selective protein degradation in cancer therapy.

Indirect effects of drugs on ion channel expression levels on plasma membrane are focused as the cause of QT prolongation, and we explored the chronic effects of QT-prolonging drugs on the slow component of the delayed-rectifier potassium current (IKs). Chinese Hamster Ovary cells expressing IKs channels were constructed by transfecting KCNQ1/KCNE1 genes, and the IKs values were measured using IonWorks Quattro in the population patch-clamp mode. After 24 hours of treatment with IKs blockers (HMR1556, L-768673, or chromanol 293B) or human Ether-à-go-go related gene channel trafficking inhibitors (amiodarone,17-AAG, brefeldin A, pentamidine, thioridazine, or probucol), brefeldin A, pentamidine, and probucol decreased IKs. Probucol, which is a cholesterol-lowering drug and clinically reported to cause QT prolongation, potently inhibited the IKs in a concentration-dependent manner, with a half maximal inhibitory concentration of 149.1 nM. A reduction in the IKs by 1 μM of probucol was observed beginning 2 hours after treatment, and the current was reduced by about 80% at 24 hours. The activation and deactivation time constants of residual IKs currents became faster compared with that in the vehicle-treatment group. Acute application of probucol did not directly inhibit IKs channels at concentrations of up to 10 μM. Western blotting analysis indicated the reduction of multimeric complex of KCNQ1 proteins by probucol treatment but not monomeric form. These results suggest that chronic probucol treatment may contribute to QT prolongation in humans by decreasing the functional IKs channel complexes.

Using gene knockout mice of particular genes is one of the most effective methods in conducting successful study on the mode of action of target gene products in targeted organs. So called the knockout technology is now a powerful tool that can lead us to find clear understanding on difficult questions such as the effects of full antagonist against target molecules. Cacna1b ( 1B) gene knockout mouse was generated to study mechanisms of N-type calcium (Ca2+) channel. The model was able to overcome physiological obstacles in studies of N-type Ca2+ channel selective blockers, such as unspecific binding to structurally similar molecules, and failed distribution to targeted organs. In the case of N-type Ca2+ channel studies, knockout technology was successfully applied to various cardiovascular, sympathetic, nociceptive, sleepawake cycles, metabolic and neurodegenerative experiments using homozygous mutants of the 1B gene that turned out to be viable. These studies were able to confirm not only the predicted phenotypes, but were able to present completely unexpected phenotypes that are great interest for future study. Thus the outputs from the knockout mouse studies lead to gain the proof of concept as a drug for specific inhibitors of the gene products and enabled us to make further prediction of side-effects of these inhibitors in the drug discovery and development process.

Pladienolide is a naturally occurring antitumor macrolide that was discovered by using a cell-based reporter gene expression assay controlled by the human vascular endothelial growth factor promoter. Despite the unique mechanisms of action and prominent antitumor activities of pladienolides B and D in diverse in vitro and in vivo systems, their target protein has remained unclear. We used 3H-labeled, fluorescence-tagged and photoaffinity/biotin (PB)-tagged 'chemical probes' to identify a 140-kDa protein in splicing factor SF3b as the binding target of pladienolide. Immunoblotting of an enhanced green fluorescent protein fusion protein of SF3b subunit 3 (SAP130) revealed direct interaction between the PB probe and SAP130. The binding affinities of pladienolide derivatives to the SF3b complex were highly correlated with their inhibitory activities against reporter gene expression and cell proliferation. Furthermore, pladienolide B impaired in vivo splicing in a dose-dependent manner. Our results demonstrate that the SF3b complex is a pharmacologically relevant protein target of pladienolide and suggest that this splicing factor is a potential antitumor drug target.

Stresses that impair the function of the endoplasmic reticulum (ER) lead to an accumulation of unfolded protein in the ER. Under these conditions, the expression of a variety of genes involved in preventing the accumulation of the unfolded proteins is induced. Yeast Hrd1p is an ER stress-inducible ER membrane protein that acts as a ubiquitin ligase (E3) with a RING finger motif and plays a role in the ubiquitination of proteins in the ER. We report here the identification and characterization of a human homolog to yeast Hrd1p. The predicted structures are highly conserved from yeast to humans. Indeed, human HRD1 was localized to the ER and ubiquitinated its substrates. Furthermore, it was found that human HRD1 was up-regulated by ER stress via IRE1 and ATF6, which are ER stress transducers. Interestingly, 293 cells stably expressing wild-type HRD1, but not the C329S mutant, afforded resistance to ER stress-induced apoptosis. These results suggest that the production of HRD1 is up-regulated to protect against ER stress-induced apoptosis by degrading unfolded proteins accumulated in the ER.

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers. Mol Cancer Ther; 13(4); 842–54. ©2014 AACR.

Background: Eribulin mesilate (eribulin), a non-taxane microtubule dynamics inhibitor, has shown trends towards greater overall survival (OS) compared with progression-free survival in late-stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional, previously unrecognised antitumour mechanisms beyond its established antimitotic activity. To investigate this possibility, eribulin's effects on the balance between epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) in human breast cancer cells were investigated. Methods: Triple negative breast cancer (TNBC) cells, which are oestrogen receptor (ER−)/progesterone receptor (PR−)/human epithelial growth receptor 2 (HER2−) and have a mesenchymal phenotype, were treated with eribulin for 7 days, followed by measurement of EMT-related gene and protein expression changes in the surviving cells by quantitative real-time PCR (qPCR) and immunoblot, respectively. In addition, proliferation, migration, and invasion assays were also conducted in eribulin-treated cells. To investigate the effects of eribulin on TGF-β/Smad signalling, the phosphorylation status of Smad proteins was analysed. In vivo, the EMT/MET status of TNBC xenografts in mice treated with eribulin was examined by qPCR, immunoblot, and immunohistochemical analysis. Finally, an experimental lung metastasis model was utilised to gauge the metastatic activity of eribulin-treated TNBC in the in vivo setting. Results: Treatment of TNBC cells with eribulin in vitro led to morphological changes consistent with transition from a mesenchymal to an epithelial phenotype. Expression analyses of EMT markers showed that eribulin treatment led to decreased expression of several mesenchymal marker genes, together with increased expression of several epithelial markers. In the TGF-β induced EMT model, eribulin treatment reversed EMT, coincident with inhibition of Smad2 and Smad3 phosphorylation. Consistent with these changes, TNBC cells treated with eribulin for 7 days showed decreased capacity for in vitro migration and invasiveness. In in vivo xenograft models, eribulin treatment reversed EMT and induced MET as assessed by qPCR, immunoblot, and immunohistochemical analyses of epithelial and mesenchymal marker proteins. Finally, surviving TNBC cells pretreated in vitro with eribulin for 7 days led to decreased numbers of lung metastasis when assessed in an in vivo experimental metastasis model. Conclusions: Eribulin exerted significant effects on EMT/MET-related pathway components in human breast cancer cells in vitro and in vivo, consistent with a phenotypic switch from mesenchymal to epithelial states, and corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo. These preclinical findings may provide a plausible scientific basis for clinical observations of prolonged OS by suppression of further spread of metastasis in breast cancer patients treated with eribulin.

The effect of the intracerebroventricular (i.c.v.) injection of relaxin-3 (RLX3) was evaluated using anxiety-related behavioral tests in rats. RLX3-injected animals showed normal locomotion activity in a habituated environment and declined anxiety cognition in the elevated plus maze test and the shock probe-burying test. The measurement of spontaneous locomotor activity in a novel environment also suggested that RLX3 reduced the stress response. To elucidate the regulatory mechanisms of the downstream signaling pathways underlying RLX3 activity and its relation to anxiolytic and hyperphagic behavior phenotypes, RLX3-i.c.v.-injected rat hypothalamic responses were examined using a microarray analysis. Ingenuity Pathway Analysis software listed the phenotype-relating genes and they showed characteristic expression patterns in the rat hypothalamus. When peptidome data sets for the same listed genes was analyzed using a semi-quantitative approach, the expressions of two neuropeptides were found to have increased. One of these neuropeptides, oxytocin (Oxt), exhibited increased expression in both the microarray and the peptidomic analysis, and a Western blot analysis validated the mass spectrometry results. A cross-omics data analysis is useful for predicting downstream signaling pathways, and the anxiolytic-like behavior of RLX3 may be mediated by an oxytocin signaling pathway in rats. These results suggest that RLX3 acts as an anxiolytic peptide and that the downstream pathways mediated by its receptors may be potential candidates for the treatment of anxieties in the future.

DNA methyltransferase inhibitors (DNMTIs), including decitabine (DAC) and azacitidine (AZA), have recently been highlighted for the treatment of high-risk myelodysplastic syndrome (MDS); however, their action mechanisms have not been clearly defined. Therefore, we investigated the effects of DNMTIs on MDS-derived cell lines in vitro. An MDS-derived cell line MDS92 and its blastic subline MDS-L and HL-60 were used. All three cell lines were sensitive to DNMTIs, but MDS-L was the most susceptible. DAC-induced cell death in MDS-L was preceded by DNA damage–induced G2 arrest via a p53-independent pathway. AZA did not influence the pattern of cell cycle, although it induced DNA damage response. The IC 50 of DAC or AZA on MDS-L cells was associated with the dose inducing the maximal hypomethylation in long interspersed nuclear elements-1 (LINE-1) methylation assay. AZA suppressed the level of methylation in a time-dependent manner (days 4, 7, and 10), whereas DAC maintained the level of methylation from day 4 to 11. The protein expression of DNMT1 and DNMT3a decreased with the suppression of growth and methylation. We conclude that this study provides in vitro models for understanding the effects of DNMTIs on cell growth and gene regulation, including differences in the possible action mechanism of DAC and AZA.

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca(2+) channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca(2+) channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca(2+) channel α(1B)-deficient (α(1B)(-/-)) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35-55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α(1B)(-/-) mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α(1B)(-/-) mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α(1B)(-/-) mice and was significantly inhibited by a selective N-type Ca(2+) channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca(2+). These results suggest that the N-type Ca(2+) channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.

Using gene knockout mice of particular genes is one of the most effective methods in conducting successful study on the mode of action of target gene products in targeted organs. So called the knockout technology is now a powerful tool that can lead us to find clear understanding on difficult questions such as the effects of full antagonist against target molecules. Cacna1b (alpha(1B)) gene knockout mouse was generated to study mechanisms of N-type calcium (Ca(2+)) channel. The model was able to overcome physiological obstacles in studies of N-type Ca(2+) channel selective blockers, such as unspecific binding to structurally similar molecules, and failed distribution to targeted organs. In the case of N-type Ca(2+) channel studies, knockout technology was successfully applied to various cardiovascular, sympathetic, nociceptive, sleep-awake cycles, metabolic and neurodegenerative experiments using homozygous mutants of the alpha(1B) gene that turned out to be viable. These studies were able to confirm not only the predicted phenotypes, but were able to present completely unexpected phenotypes that are great interest for future study. Thus the outputs from the knockout mouse studies lead to gain the proof of concept as a drug for specific inhibitors of the gene products and enabled us to make further prediction of side-effects of these inhibitors in the drug discovery and development process.

Endoplasmic reticulum (ER) stress is defined as an accumulation of unfolded proteins in the endoplasmic reticulum. 4-phenylbutyrate (4-PBA) has been demonstrated to promote the normal trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutant from the ER to the plasma membrane and to restore activity. We have reported that 4-PBA protected against cerebral ischemic injury and ER stress-induced neuronal cell death. In this study, we revealed that 4-PBA possesses chemical chaperone activity in vitro, which prevents the aggregation of denatured alpha-lactalbumin and bovine serum albumin (BSA). Furthermore, we investigated the effects of 4-PBA on the accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R) pathologically relevant to the loss of dopaminergic neurons in autosomal recessive juvenile parkinsonism (AR-JP). Interestingly, 4-PBA restored the normal expression of Pael-R protein and suppressed ER stress induced by the overexpression of Pael-R. In addition, we showed that 4-PBA attenuated the activation of ER stress-induced signal transduction pathways and subsequent neuronal cell death. Moreover, 4-PBA restored the viability of yeasts that fail to induce an ER stress response under ER stress conditions. These results suggest that 4-PBA suppresses ER stress by directly reducing the amount of misfolded protein, including Pael-R accumulated in the ER.

Background: Eribulin mesylate (eribulin), a non-taxane microtubule dynamics inhibitor, has unexpectedly shown greater effects on overall survival compared to progression-free survival in late stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional, previously-unrecognized anti-tumor mechanisms beyond its established antimitotic activity. To investigate this possibility, eribulin's effects on the balance of EMT/ MET in MX-1 human breast cancer cells were investigated. Methods: MX-1 human breast cancer cells, which are ER-/PR-/HER2- and have a mesenchymal phenotype, were treated with eribulin for 7 days, followed by measurement of EMT-related gene and protein expression changes by qPCR and immunoblot, respectively. In addition, the EMT/MET status of MX-1 xenografts in mice treated with eribulin were also examined by qPCR, immunoblot, and immunohistochemical analysis. Proliferation, migration and invasion assays were also conducted. Finally, an experimental lung metastasis model was utilized to gauge the metastatic activity of eribulin treated MX-1 cells. Results: Treatment of MX-1 cells with eribulin in vitro led to morphological changes consistent with transition from a mesenchymal to an epithelial phenotype. Gene expression analyses showed that eribulin treatment led to decreased expression of several mesenchymal marker genes, together with increased expression of several epithelial marker genes. Furthermore, protein levels of epithelial marker E-cadherin were increased following eribulin treatment, while protein levels of mesenchymal markers N-cadherin and vimentin were decreased. Consistent with these changes, MX-1 cells treated with low levels of eribulin for one week showed decreased capacity for in vitro migration and invasiveness. In the MX-1 xenograft model in vivo, eribulin treatment induced MET as assessed by qPCR, immunoblot, and immunohistochemical analyses of epithelial and mesenchymal marker genes and proteins. Finally, MX-1 cells treated in vitro with low levels of eribulin for one week led to decreased numbers of lung metastases when assessed in an in vivo experimental metastasis model. Conclusions: Eribulin exerted significant effects on EMT/MET-related pathway components in human breast cancer cells in vitro and in vivo, consistent with a phenotypic switch from mesenchymal to epithelial states, and corresponding to observed decreases in migration and invasiveness in vitro as well as experimental metastasis in vivo. These preclinical findings provide a scientific basis for clinical observations of prolonged overall survival without corresponding effects on progression-free survival in metastatic breast cancer patients treated with eribulin. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A296. Citation Format: Taku Yoshida, Galina Kuznetsov, Takayuki KImura, Naoko H. Sugi, Hajime Shimizu, Yoichi Ozawa, Yoshiaki Sato, Taisuke Uehara, Hideki Watanabe, Kentaro Takahashi, Kentaro Matsuura, Mai Uesugi, Osamu Toyama, Mamoru Yanagimachi, Yasuhiro Funahashi, Junji Matsui. Eribulin mesylate suppresses experimental metastasis in the MX-1 human breast cancer model by reversing phenotypic states between epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A296.

Objective: Eribulin mesylate (ERI) is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. The objective of this study was to examine the effect of ERI on tumor vasculature with immunohistchemical (IHC) analysis and gene expression profiling (GEP) in normal host cells, such as endothelial cells and vascular mural cells within tumor microenvironments in human BCC xenograft models Methods: Anti-tumor activity of ERI was examined at doses of 1.5 and 3.0 mg/kg, i.v. at day 1, in human BCC MX-1, MDA-MB-231 and MDA-MB-453 sc xenografts in nude mice. For IHC and GEP analysis, tumor tissues were collected at day 4 and day 8. IHC analysis was performed using mouse CD31 antibody to stain endothelial cells. Microvessel density (MVD) and vessel perimeter were determined by using Aperio Image Scope. GEP analysis for mouse host and human tumor cells within tumor tissues was done by using mouse and human TaqMan Low Density Arrays (TLDAs) consisting of a set of 92 genes related to angiogenesis, metastasis/EMT and cell differentiation signal pathways. Results shows % of non-treatment group (NT). Results: ERI showed significant anti-tumor activity against all three human BCC xenografts in a dose dependent manner. IHC analysis showed that ERI altered morphology of tumor vasculature day 8 after treatments and increased number of vessels with small size of perimeter (300um) in both MX-1 and MDA-MB-231 xenograft models (p Conclusions: ERI induced re-modeling of tumor vasculature in human BCC xenograft models. GEP related to angiogenesis and EMT/metastasis pathway was significantly affected with ERI treatment in host cells under tumor microenvironments. ERI might cause remodeling of tumor vasculature by regulating GEP in host cells. Further investigation may be warranted to examine if the activity of ERI against host cells in tumor tissues contributed to anti-tumor activity of ERI. Citation Format: Junji Matsui, Osamu Toyama, Mitsuhiro Ino, Taro Semba, Mai Uesugi, Hiroki Muto, Judith L. Oestreicher, Kentaro Takahashi, Kentaro Matsuura, Yoshiaki Sato, Taisuke Uehara, Takayuki Kimura, Hideki Watanabe, Yoichi Ozawa, Makoto Asano, Yusuke Adachi, Ken Aoshima, Yasuhiro Funahashi. Eribulin caused re-modeling of tumor vasculature altering gene expression profiling in angiogenesis and Epithelial Mesenchymal Transition (EMT) signaling pathway of host cells within human breast cancer cell (BCC) xenografts in nude mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1413. doi:10.1158/1538-7445.AM2013-1413

Objectives: Eribulin mesylate (ERI) is a simplified synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B. We examined effects of ERI and Paclitaxel (PTX) on blood vessel cells by gene expression profiling (GEP) in mono- and co-culture assays of pericytes and endothelial cells. Activity of ERI and PTX against cell growth inhibition and pericyte-driven in vitro angiogenesis was also studied. Methods: We first assessed IC50s of ERI and PTX in 4-day growth inhibition assay in isolated primary human brain vascular pericytes (HBVP) and human umbilical vein endothelial cells (HUVEC). Based on identified IC50s, cells were treated with 10X IC50s of ERI or PTX and GEP was analyzed at 24 h using either Affimetrix Human Genome U133 Plus 2.0 arrays or custom TaqMan Low Density Cards (TLDA) designed with 92 genes related to angiogenesis, metastasis/ Epithelial Mesenchymal Transition (EMT) and cell differentiation signal pathways. Inhibitory activity of drugs on the length of capillary-like networks was analyzed in co-culture of HUVEC with HBVP. Results: ERI and PTX inhibited cell growth of HBVP at IC50s of 1.2 and 3.1nM and HUVEC was more sensitive than HBVP by 1.9 and 3.3 fold in cell growth assay, respectively. In HUVEC, most genes were down-regulated by both ERI and PTX treatments, while in HBVP about equal number of genes were up- or down-regulated with microarray analysis. Interestingly, 63% affected genes in HUVECs overlapped for both treatments. In HBVPs, altered gene signatures were drug-dependent and an overlap was limited by 16%. ERI specifically affected genes in HIF1 and caveolar-mediated signaling pathways while PTX regulated genes in HER2, PI3K/AKT and HGF signaling pathways among others. We confirmed obtained altered GEP using TLDA and identified 42 significant genes differentially regulated by ERI and PTX in HBVP. To examine effects on pericyte-driven in vitro angiogenesis, we compared length of capillary-like networks in co-culture of HUVEC with HBVP. ERI disrupted capillary-like networks starting at about 2 nM, while PTX showed limited inhibitory activity by less than 50% even at 100-1000 nM for 4 days treatments. In co-culture assay, TLDA data showed decreased expression levels of angiogenesis-related genes DLL4 (14% compare to control), PDGFRB (70%) and metastasis/EMT-related genes ZEB1 (53%), TGFB3 (54 %), VIM (62 %) after treatment with 10X IC50s of ERI (p Conclusions: Endothelial cells and pericytes responded differently to ERI and PTX treatments and effects of ERI on GEP of pericytes were distinct from PTX. ERI inhibited pericyte-driven in vitro angiogenesis at sub nM using co-culture assay of HUVEC with HBVP. Further analysis of the role of ERI on GEP of pericytes and pericyte-driven angiogenesis in anti-tumor activity will be warranted. Citation Format: Sergei I. Agoulnik, Judith L. Oestreicher, Noel H. Taylor, Mai Uesugi, Hiroki Muto, Satoshi Kawano, Kentaro Takahashi, Kentaro Matsuura, Ken Aoshima, Junji Matsui, Yasuhiro Funahashi. Eribulin and Paclitaxel differentially affect gene expression profiling of blood vessel cells and in vitro angiogenesis in co-cultures of human endothelial cells with pericytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3830. doi:10.1158/1538-7445.AM2013-3830