Search

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.

Introduction: Combination anti-CTLA-4 antibody (Ab) + anti-PD-1 Ab is approved in multiple cancer types and has improved clinical outcomes. In addition, atezolizumab + bevacizumab combination therapy is approved for unresectable hepatocellular carcinoma. Given the rapid changes in the therapeutic landscape, it is important to investigate effective treatments for people with prior ICI-based combination therapy. E7386 modulates Wnt/β-catenin signaling, and a phase 1 study in combination with LEN (a receptor tyrosine kinase inhibitor that mainly targets both VEGFR and FGFR), in people with solid tumors (NCT04008797) is ongoing. In this study, we examined the antitumor activity of the combination of E7386 + LEN, with either prior anti-CTLA-4 Ab + anti-PD-1 Ab or anti-PD-1 Ab + aflibercept (a soluble decoy receptor against VEGF), in the mouse lung tumor KLN 205 model. Methods: Antitumor activity of E7386 (25 or 50 mg/kg), LEN (3 mg/kg), and the combination, was evaluated in the KLN 205 model (orally, once daily x 14 days), with or without prior treatments of either anti-CTLA-4 Ab (0.2 mg/head, weekly x 3, intraperitoneally [ip]) + anti-PD-1 Ab (0.2 mg/head, weekly x 3, ip)─ie, CTLA-4 + PD-1; or aflibercept (5 mg/kg) + anti-PD-1 Ab (0.2 mg/head, twice a week x 2, ip)─ie, AFL + PD-1. Mice with relatively rapid-growing tumors and prior treatments were given E7386, LEN, and their combination. RNA-seq analyses were conducted by using tumor tissues collected from mice one day after the prior treatment period or without treatment to analyze gene-expression alterations with prior treatments. Results: In the mouse model without prior treatments, LEN had clear antitumor activity. E7386 had limited antitumor activity as a monotreatment. E7386 + LEN had superior antitumor activity vs LEN in a dose-dependent manner. In addition, E7386 + LEN had superior antitumor activity to each single treatment despite either prior CTLA-4 + PD-1 or AFL + PD-1. E7386 + LEN with prior CTLA-4 + PD-1 had equivalent antitumor activity to LEN without prior treatments. Conversely, E7386 + LEN with prior AFL + PD-1 had strong antitumor activity, including marked tumor regression, compared to both LEN alone and E7386 + LEN without prior treatments. RNA-seq analyses showed that intratumoral Axin2, a target gene in the canonical Wnt-signaling pathway, was upregulated with prior AFL + PD-1—suggesting a potential activation of the Wnt-signaling pathway. Conclusion: E7386 + LEN combination shows antitumor activity in the mouse KLN 205 lung tumor model when there is prior ICI-based (CTLA-4 + PD-1 or AFL + PD-1) combination treatment. Further, E7386 + LEN shows enhanced antitumor activity with prior AFL + PD-1; possibly due to activation of Wnt-signaling pathway during prior treatments. Citation Format: Satoshi Kawano, Yusuke Adachi, Megumi Kuronishi, Kohta Toshimitsu, Kotaro Kodama, Takayuki Kimura, Masahiko Kume, Jialing Shen, Saori Watanabe Miyano, Akira Yokoi, Junji Matsui, Yu Kato, Yasuhiro Funahashi. E7386, a selective inhibitor of the interaction between β-catenin and CREB-binding protein (CBP), in combination with lenvatinib (LEN), exerts antitumor activity in preclinical tumor models with prior immune checkpoint inhibitor (ICI)-based combination treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1830.

Mineral trioxide aggregate (MTA) is an alternative endodontic material that predicts conductive or inductive calcified tissue formation from immature pulp mesenchymal stem cells (IPMSCs). The purpose of this study was to investigate whether MTA could promote reparative odontoblast differentiation via IPMSCs in the early phase of regeneration and compare with calcium hydroxide (CH). Direct pulp capping using calcium hydroxide (CH), MTA, and MTA with platelet-rich plasma (MTA + PRP) was performed on maxillary first molars of 8-week-old male Wistar rats (n = 36). After 3, 7, or 14 days, the teeth were analyzed for mineral density (MD) and volume of MD (VMD) via micro-focusing computed tomography (µCT), nestin, dentin matrix acidic phosphoprotein 1 (DMP1) immunohistochemistry, and real-time PCR for DMP1 mRNA expression. MTA stimulated the early phase differentiation of the IPMSCs into odontoblasts, with positive results for nestin and DMP1 compared with CH. Moreover, MTA + PRP stimulated calcified granule and dentin bridge formation through calcium mineral deposition, following the induction of DMP1 mRNA expression in IPMSCs. Our results suggested that the combination of MTA and PRP is an effective and clinically applicable method for activating endogenous dental pulp stem cells into odontoblasts in the early stages of pulp regeneration.

Despite their outstanding antitumour activity in mice, the limited supply from the natural sources has prevented drug discovery/development based on intact halichondrins. We achieved a total synthesis of C52-halichondrin-B amine (E7130) on a >10 g scale with >99.8% purity under GMP conditions. Interestingly, E7130 not only is a novel microtubule dynamics inhibitor but can also increase intratumoural CD31-positive endothelial cells and reduce α-SMA-positive cancer-associated fibroblasts at pharmacologically relevant compound concentrations. According to these unique effects, E7130 significantly augment the effect of antitumour treatments in mouse models and is currently in a clinical trial. Overall, our work demonstrates that a total synthesis can address the issue of limited material supply in drug discovery/development even for the cases of complex natural products.

Introduction and objectives: E7130 is a novel anti-cancer agent created from a total synthetic study of the natural compound norhalichondrin B, and is evaluated in a clinical study currently (NCT03444701). Previous pre-clinical study reported that E7130 had both cancer cell proliferation inhibitory activity and tumor microenvironment ameliorating activities featuring vascular remodeling effect and anti-CAF (cancer-associated fibroblasts) effect. Intratumoral hypoxia induces tumor malignancy and causes resistance against anti-cancer drugs including anti-estrogen agents (eg fulvestrant). It is hypothesized that vascular remodeling effect of E7130 mitigates hypoxia-induced resistant mechanism of ER (estrogen receptor) + breast cancer against anti-estrogen agents, resulting in improving anti-tumor activity of fulvestrant in combination with E7130. Experimental procedure: ERα expression levels in human ER+ breast cancer cell lines under hypoxic or normoxic culture condition were examined by FCM (flow cytometry) analysis. The histological changes after treatment with E7130 were examined by immunohistochemical analyses in the MCF-7 subcutaneous xenograft model. Anti-tumor activity of E7130 in combination with fulvestrant were examined in concurrent and in sequential treatment in the MCF-7 model. Summary: In vitro hypoxic culture condition down-regulated ERα expression in MCF-7 and T-47D compared with normoxic condition. E7130 had no effect on the ERα expression level in the cancer cells in vitro. On the contrary, E7130 increased ERα expression level in the cancer cells concomitantly with an increase of CD31-positive endothelial cell number (ie vascular remodeling effect) in MCF-7 xenograft tumor. These results suggested that E7130 up-regulated ERα expression in cancer cells not by cell-autonomous manner but by mitigating intratumoral hypoxia via its vascular remodeling effect. Concurrent combination therapy of E7130 at 90 μg/kg with fulvestrant at 250 mg/kg significantly inhibited the tumor growth compared with each monotherapy. In sequential combination therapy, pretreatment of E7130 followed by fulvestrant showed superior anti-tumor activity to posttreatment of E7130 following fulvestrant. Severe body weight loss was not observed in any treatment groups. Conclusions: The vascular remodeling effect of E7130 increased ERα expression in cancer cells in vivo, and sensitized ER+ breast cancer against fulvestrant to achieve promising combinational anti-tumor activity. Our data provides compelling evidence that E7130 attenuates tumor malignancy by its tumor microenvironment ameliorating effects and improves current anti-cancer therapy in combination with other drugs. Citation Format: Takanori Abe, Satoshi Kawano, Ken Ito, Taro Semba, Kimiyo Tabata, Hiroki Jozawa, Osamu Asano, Yoshito Kishi. Tumor microenvironment ameliorating effect of E7130 sensitized ER+ breast cancer against fulvestrant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1865.

Mineral trioxide aggregate (MTA) cement is widely used in the field of endodontic treatment. We herein synthesized calcium silicates from calcium carbonate and silicon dioxide, with the aim of reducing the cost associated with the MTA. Additionally, we prepared gypsum-containing calcium silicate cement to reduce the setting time while enhancing the mechanical strength. We evaluated the physical properties of this cement and investigated the response of human dental pulp stem cells (hDPSCs) grown in culture media containing cement eluate. Our results revealed that calcium silicates could be easily synthesized in lab-scale. Furthermore, we demonstrate that gypsum addition helps shorten the setting time while increasing the compressive strength of dental cements. The synthesized gypsum-containing calcium silicate cement showed minimal cytotoxicity and did not inhibit the proliferation of hDPSCs. These results suggested that the newly developed calcium silicate material could be a promising pulp capping material.

In our previous study, we demonstrated that hyaluronan induces odontoblastic differentiation of dental pulp stem cells via interactions with CD44. However, it remains unclear whether CD44 expression by dental pulp stem cells is required for odontoblastic differentiation. Therefore, we searched for a compound that induces odontoblastic differentiation of dental pulp stem cells, regardless of the chemical structure and function of hyaluronan, and examined whether CD44 is involved in the induction of odontoblastic differentiation by the compound. Because vitamin K analogues can promote bone formation and tissue calcification, we focused on derivatives of naphthoquinone, the skeleton of vitamin K; we verified whether those compounds could induce odontoblastic differentiation of dental pulp stem cells. We found that dentin sialophosphoprotein, a marker of odontoblasts, was expressed in dental pulp stem cells after treatment with shikonin. The shikonin-induced expression of dentin sialophosphoprotein was inhibited by PI3K, AKT, and mTOR inhibitors. In addition, in dental pulp stem cells transfected with siRNA against CD44, the shikonin-induced expression of dentin sialophosphoprotein was inhibited. Thus, shikonin can stimulate dental pulp stem cells to undergo odontoblastic differentiation through a mechanism involving the AKT–mTOR signaling pathway and CD44. Hyaluronan stimulated dental pulp stem cells to undergo CD44-mediated odontoblastic differentiation in our previous study; the present study indicated that CD44 is necessary for dental pulp stem cells to undergo odontoblastic differentiation. Although expression of CD44 is important for inducing odontoblastic differentiation of dental pulp stem cells, the relationship between the AKT–mTOR signaling pathway and CD44 expression, in the context of shikonin stimulation, has not yet been elucidated. This study suggested that shikonin may be useful for inducing odontoblastic differentiation of dental pulp stem cells, and that it may have clinical applications, including protection of dental pulp.

We previously reported that oral and intestinal absorption of insulin in rats and mice is significantly enhanced in vivo by coadministration with cell-penetrating peptides (CPPs). To evaluate the clinical use of CPPs as absorption enhancers, it is imperative to clarify the mechanisms associated with the permeation-stimulatory effect of CPPs in vitro. The confirmation experiment revealed a discrepancy between in vivo and in vitro effects of CPPs, such as D-octaarginine (D-R8) and L-penetratin, on epithelial permeation of insulin. The present study was designed to determine the factors that work in vivo but are deficient in an in vitro system consisting of Caco-2 cells. The effects of D-R8 and L-penetratin on permeation of insulin through the Caco-2 cell monolayer were partially boosted in fasted-state simulated intestinal fluid (FaSSIF). Contrary to expectation, the effects of CPPs on cellular uptake of insulin and the binding ratio of CPPs to insulin analyzed by surface plasmon resonance in normal buffer and FaSSIF were similar. Also, the effects of CPPs, especially D-R8, on cellular uptake of insulin, were stronger in Caco-2 cell monolayers with microfold cell (M cell)-like properties. These results suggested a key role of intestinal lipids and M cells in the stimulatory effect of CPPs on net epithelial permeation of insulin in vivo.

Introduction: The fibroblast growth factor receptor (FGFR) signaling has a crucial role in proliferation, survival, migration, and drug resistance of cancer cells, as well as tumor angiogenesis. Moreover, gene alterations of FGF/FGFR were reported after treatment of a CDK4/6 inhibitor and endocrine therapy (ET) in ER(+)/HER2(-) breast cancer (BC) patients, suggesting FGF/FGFR signaling mediates resistance to these therapies. E7090, an orally available FGFR1-3 selective inhibitor, is currently under evaluation in a Phase 2 study in patients with unresectable advanced or metastatic cholangiocarcinoma with FGFR2 gene fusion (NCT04238715), and a Phase 1 study as a monotherapy or in combination with fulvestrant (FUL) or exemestane for ER(+)/HER2(-) BC patients (NCT04572295). In this study, we present the roles of FGF/FGFR signaling in resistance to a CDK4/6 inhibitor and/or ET, and activities of E7090 on drug resistance in preclinical ER(+)/HER2(-) BC cell lines and PDx models. Results: Antitumor activities of E7090 (25 or 50mg/kg, Q1Dx14 or x21, p.o.) were evaluated using five ER(+)/HER2(-) BC PDx models, OD-BRE-0438, -0450, -0188, -0704, and IM-BRE-556 with or without prior treatment of FUL (5 mg/mouse, Q7Dx2, s.c.)+palbociclib (PAL, 100mg/kg, Q1Dx14, p.o.). Among them, OD-BRE-0438 and -0704 showed higher sensitivities to E7090 with prior FUL+PAL than without it, and E7090 showed tumor regression in the OD-BRE-0438 model only with prior FUL+PAL. In these two models, FUL+PAL treatment upregulated expressions of several FGF ligands mRNA (qPCR) and FGFRs protein (IHC) in tumors. To evaluate the activities of FGF/FGFR signaling on sensitivities to FUL or FUL+PAL, in vitro cell proliferation assay using ER(+)/HER2(-) BC cell lines, MCF-7, ZR-75-1, and HCC1428 were performed in the presence of FGF2 and FGF10, and decreased sensitivities to FUL or FUL+PAL were observed. Furthermore, co-treatment with E7090 restored the sensitivity to those drugs in cultured cell lines. In consistent with in vitro results, FUL in combination with E7090 showed a combination antitumor activity in the OD-BRE-0438 model, in which expressions of several FGF ligands mRNA and FGFR2 protein in tumors showed a tendency of upregulation by FUL. Conclusion: In ER(+)/HER2(-) BC PDx models, FGFs/FGFRs expression in tumors were induced by treatment of FUL and/or PAL, and antitumor activity of E7090 was enhanced with prior FUL+PAL. In vitro study using cultured ER(+)/HER2(-) BC cell lines indicated that sensitivity against FUL or FUL+PAL was decreased by FGFR activation. Furthermore, a combination antitumor activity of E7090 and FUL was observed in the OD-BRE-0438 model. These data suggest that activation of FGF/FGFR signaling confer the resistance to a CDK4/6 inhibitor and/or ET, and E7090 shows antitumor activity against ER(+)/HER2(-) BC with prior a CDK4/6 inhibitor and/or ET. Citation Format: Satoshi Kawano, Sayo Fukushima, Kyoko Nishibata, Ryu Gejima, Yuki Niwa, Yasuhiro Funahashi, Saori Watanabe Miyano. Effect of E7090, an FGFR1-3 selective inhibitor, on resistance to a CDK4/6 inhibitor and endocrine therapy in ER(+)/HER2(-) breast cancer preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1414.

We previously reported that eribulin mesylate (eribulin), a tubulin‐binding drug (TBD), could remodel tumor vasculature (i.e. increase tumor vessels and perfusion) in human breast cancer xenograft models. However, the role of this vascular remodeling in antitumor effects is not fully understood. Here, we investigated the effects of eribulin‐induced vascular remodeling on antitumor activities in multiple human cancer xenograft models. Microvessel densities (MVD) were evaluated by immunohistochemistry (CD31 staining), and antitumor effects were examined in 10 human cancer xenograft models. Eribulin significantly increased MVD compared to the controls in six out of 10 models with a correlation between enhanced MVD levels and antitumor effects (R 2 = 0.54). Because of increased MVD, we next used radiolabeled liposomes to examine whether eribulin treatment would result in increased tumoral accumulation levels of these macromolecules and, indeed, we found that eribulin, unlike vinorelbine (another TBD) enhanced them. As eribulin increased accumulation of radiolabeled liposomes, we postulated that this treatment might enhance the antitumor effect of Doxil (a liposomal anticancer agent) and facilitate recruitment of immune cells into the tumor. As expected, eribulin enhanced antitumor activity of Doxil in a post‐erlotinib treatment H1650 (PE‐H1650) xenograft model. Furthermore, infiltrating CD11b‐positive immune cells were significantly increased in multiple eribulin‐treated xenografted tumors, and natural killer (NK) cell depletion reduced the antitumor effects of eribulin. These findings suggest a contribution of the immune cells for antitumor activities of eribulin. Taken together, our results suggest that vascular remodeling induced by eribulin acts as a microenvironment modulator and, consequently, this alteration enhanced the antitumor effects of eribulin.

Background and objectives: Natural products have been a rich source of inspiration for drug discovery as demonstrated by the fact that over one-third of current therapeutic agents are natural products or compounds derived from them. Particularly, when the supply of a natural product from the natural source is limited, organic synthesis can offer a solution. However, with increasing structural complexity, this approach becomes exponentially more challenging, as a synthesis must meet various requirements, including high overall efficiency, scalability, cost-effectiveness, and product purity as well as conforming to good manufacturing practice (GMP) regulations. Despite the outstanding in vivo antitumor activity in mice, the limited supply from the natural sources has prevented drug development based on intact halichondrins. Methods: We successfully synthesized 19.5 g of C52-halichondrin-B alcohol with 99.84% purity via a total synthesis. From 15.0 g of this material, we obtained 11.5 g of C52-halichondrin-B amine (E7130) with 99.81% purity under GMP conditions. With totally synthetic E7130, a novel microtubule dynamics inhibitor, we studied the activities in both of in vitro and in vivo. The inhibitory activity of E7130 towards tubulin polymerization was analyzed in the cell-free system in which tubulin polymerization could be monitored by fluorescence enhancement due to the incorporation of a fluorescence reporter into the microtubules during polymerization. We analyzed in vivo antitumor activities using both a human cancer cell line orthotopic transplantation mouse model and subcutaneous xenograft models. The immunohistochemical analyses were performed with tumor tissues collected from mouse models to analyze histological changes after treatment with E7130. Results: E7130 is a novel microtubule dynamics inhibitor with exceedingly potent in vitro and in vivo anticancer activities. Significantly, E7130 not only is cytotoxic, but can also increase CD31-positive endothelial cells in tumors and reduce α-SMA-positive cancer-associated fibroblasts at pharmacologically relevant compound concentrations. Notably, this dual activity has been recognized for the first time through this study. According to these unique tumor microenvironment ameliorative effects, E7130 can augment the effect of other antitumor treatments in mouse models. In particular, a dose of 90 µg/kg, one-half of the maximum tolerated dose in mice, showed a prominent combinational effect with cetuximab, which suggests that E7130 has a different mechanism than other microtubule-targeted drugs. Conclusions: E7130 ameliorates the tumor microenvironment to improve cancer treatment when used in combination with other compounds. The data provide compelling evidence that E7130 is a promising molecular-targeting anticancer agent. Citation Format: Satoshi Kawano, Takanori Abe, Ken Ito, Kenzo Yahata, Kazunobu Kira, Tsuyoshi Akagi, Makoto Asano, Kentaro Iso, Yuki Sato, Fumiyoshi Matsuura, Isao Ohashi, Yasunobu Matsumoto, Minetaka Isomura, Takeo Sasaki, Takashi Fukuyama, Yusuke Miyashita, Yosuke Kaburagi, Akira Yokoi, Osamu Asano, Takashi Owa, Yoshito Kishi. E7130 derived from total synthesis of halichondrin as a novel tumor-microenvironment ameliorator [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4179.

Background and objectives: Despite the outstanding antitumor activity of halichondrins in mice, the limited supply from the natural sources has prevented drug development using intact halichondrins. We achieved a total synthesis of C52-halichondrin-B amine (E7130) under good manufacturing practice (GMP) conditions. E7130 is not only a novel microtubule dynamics inhibitor, but also a novel tumor-microenvironment ameliorator. E7130 can increase intratumoral CD31-positive endothelial cells and reduce α-SMA-positive cancer-associated fibroblasts (CAFs) at pharmacologically relevant compound concentrations. Here, we analyzed the molecular mechanisms of the α-SMA-positive CAF reduction with E7130. Methods: The expression levels of α-SMA, ECM proteins, ER-TR7 (pan-fibroblast marker), and Ki67 were examined using E7130-treated tumors. In vitro co-culture experiments, normal human fibroblasts were co-cultured with human cancer cells. To examine the effects of E7130 on the TGF-β-induced CAF activation, normal human fibroblasts were treated with TGF-β (final concentration of 1 ng/mL) and E7130 at the several concentrations, and gene expression analysis and immunocytochemical analysis were conducted. Results: E7130 reduced α-SMA-positive CAF and malignant ECM proteins without changing total fibroblast number in tumors. The in vitro co-culture system revealed that TGF-β from cancer cells activate normal human fibroblast and increased α-SMA expression. In addition, treatment with E7130 interfered with α-SMA induction by TGF-β in fibroblasts without growth inhibitory activity. We further found that E7130 inhibited TGF-β-induced PI3K/AKT/mTOR pathway, which resulted in the reduction of α-SMA expression in fibroblasts. Moreover, TGF-β treatment enhanced β-tubulin expression and focal adhesions formation, which were diminished by co-treatment with E7130 in fibroblasts. Many signaling complexes are assembled in focal adhesion sites, and those complexes dispatch several downstream signals, including those involved in the PI3K/AKT/mTOR pathway. Conclusions: Drug discovery using halichondrins was based on their outstanding in vivo antitumor (inhibitory) activity in mice and bone metastasis in mouse melanoma model first reported in 1996. From these results, we hypothesized that halichondrins are not simple microtubule-targeted compounds in tumor cell, and have developed E7130, which harbors unique tumor microenvironment ameliorating effects. Here we proved that E7130 impedes the TGF-β-induced myofibroblast transdifferentiation process without killing the fibroblast by disrupting microtubule network formation, which is important for focal adhesion assembly and thereby the downstream activation of the PI3K/AKT/mTOR pathway. Citation Format: Takanori Abe, Satoshi Kawano, Ken Ito, Kenzo Yahata, Kazunobu Kira, Tsuyoshi Akagi, Makoto Asano, Kentaro Iso, Yuki Sato, Fumiyoshi Matsuura, Isao Ohashi, Yasunobu Matsumoto, Minetaka Isomura, Takeo Sasaki, Takashi Fukuyama, Yusuke Miyashita, Yosuke Kaburagi, Akira Yokoi, Osamu Asano, Takashi Owa, Yoshito Kishi. Mechanism of action analysis of anti-CAF activity of E7130, a novel tumor-microenvironment ameliorator [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4183.

Inhibitors of the protein methyltransferase Enhancer of Zeste Homolog 2 (EZH2) may have significant therapeutic potential for the treatment of B cell lymphomas and other cancer indications. The ability of the scientific community to explore fully the spectrum of EZH2-associated pathobiology has been hampered by the lack of in vivo-active tool compounds for this enzyme. Here we report the discovery and characterization of EPZ011989, a potent, selective, orally bioavailable inhibitor of EZH2 with useful pharmacokinetic properties. EPZ011989 demonstrates significant tumor growth inhibition in a mouse xenograft model of human B cell lymphoma. Hence, this compound represents a powerful tool for the expanded exploration of EZH2 activity in biology.

Almost all cancers show intrinsic and/or evasive resistance to vascular endothelial growth factor (VEGF) inhibitors by multiple mechanisms. Serum angiopoietin-2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. From these clinical observations, the Ang2 and Tie2 (its receptor) axis has been focused on as a promising target. Here, we show a novel strategy to circumvent the resistance by combining multi-tyrosine kinase inhibitors lenvatinib (VEGF receptor, fibroblast growth factor receptor, and RET inhibitor) and golvatinib (E7050; c-Met, Tie2, and EphB4 inhibitor). Tie2 identifies a highly pro-angiogenic macrophage subset, Tie2-expressing macrophages (TEM). Angi-Tie2 and EphB4-EphrinB2 signaling plays critical roles in pericyte-mediated vessel stabilization. In vitro analyses suggested that golvatinib combined with lenvatinib inhibited pericyte-mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib and lenvatinib inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable, and no macroscopic change was observed. These preclinical studies suggest that modulation of the tumor microenvironment by a strategic and well-tolerated combination of multi-targeting tyrosine kinase inhibitors may sensitize cancer to VEGF inhibitors.

Introduction Pulp tissue regeneration is becoming a reality after discovery of mesenchymal stem cells (MSCs) residing in the pulp tissues through various clinical innovations, although MSC transplantation into the pulp space has met with challenges of in vitro cell expansion and cultures. As a way to circumvent the regulatory and technical complexities of in vitro MSC culture, we investigated the use of minced pulp tissues as a source of pulpal MSCs for tissue regeneration. Methods We characterized the phenotype of cells explanted from minced pulp (MP), namely MP-derived MSCs (MP-MSCs), compared with dental pulp stem cells (DPSCs) established from pulp tissues by enzyme digestion. Phenotypic characterization included replication kinetics, immunophenotyping, and multilineage differentiation. Using the tooth slice model, we assessed odonto/osteogenic differentiation of DPSCs, MP-MSCs, and minced pulp tissues in situ. Results In vitro replication of MP-MSCs occurred more rapidly during the initial phase of subcultures compared with DPSCs; however, MP-MSCs arrived at senescence at population doubling 47, whereas DPSCs replicated until population doubling 64, indicating shorter replicative lifespan. MP-MSCs also demonstrated stronger odonto/osteogenic differentiation than DPSCs by alkaline phosphatase activity and the protein expression. Both MP-MSCs and DPSCs demonstrated odonto/osteogenic and adipogenic differentiation capacities. Both cell types also showed mineralized tissue formation in the tooth slice model. Seeding minced pulp tissue on poly-L-lactic acid scaffold allowed for migration of MP-MSCs from the tissues and odontogenic differentiation with dentin sialophosphoprotein expression in the tooth slice model. Conclusions These data indicated that MP may be an alternative source of pulpal MSCs that may allow de novo pulp-dentin regeneration without the need for in vitro culture and expansion.

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.

Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor (HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF-induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted.

Eribulin mesilate (eribulin), a first-in-class halichondrin B-based microtubule dynamics inhibitor, has been shown to promote vascular remodeling and reversal of epithelial-mesenchymal transition (EMT) apart from its antimitotic activity in breast cancer models.Anti-proliferative activity of eribulin was examined in vitro and in vivo in several human soft tissue sarcoma (STS) cell lines. To assess tumor blood perfusion and phenotypic changes, eribulin was investigated in a leiomyosarcoma xenograft and in vitro in liposarcoma and leiomyosarcoma cell lines.Eribulin showed anti-proliferative activity in vitro against all six cell lines investigated, with 50% inhibitory concentration values of around 1 nmol/l, as well as significant antitumor activity against four xenografts in vivo. In addition, eribulin significantly enhanced tumor blood perfusion in xenografts and induced morphological changes and up-regulation of differentiation marker genes.In pre-clinical models, eribulin showed anti-proliferative activity against a variety of histopathological subtypes of STS. Eribulin might also cause tumor vasculature remodeling to enhance tumor blood perfusion and induce tumor cell differentiation.

The catalytic activities of covalent and ATP-dependent chromatin remodeling are central to regulating the conformational state of chromatin and the resultant transcriptional output. The enzymes that catalyze these activities are often contained within multiprotein complexes in nature. Two such multiprotein complexes, the polycomb repressive complex 2 (PRC2) methyltransferase and the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler have been reported to act in opposition to each other during development and homeostasis. An imbalance in their activities induced by mutations/deletions in complex members (e.g. SMARCB1) has been suggested to be a pathogenic mechanism in certain human cancers. Here we show that preclinical models of synovial sarcoma—a cancer characterized by functional SMARCB1 loss via its displacement from the SWI/SNF complex through the pathognomonic SS18-SSX fusion protein—display sensitivity to pharmacologic inhibition of EZH2, the catalytic subunit of PRC2. Treatment with tazemetostat, a clinical-stage, selective and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity reverses a subset of synovial sarcoma gene expression and results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX fusion-positive cells in vitro. Treatment of mice bearing either a cell line or two patient-derived xenograft models of synovial sarcoma leads to dose-dependent tumor growth inhibition with correlative inhibition of trimethylation levels of the EZH2-specific substrate, lysine 27 on histone H3. These data demonstrate a dependency of SS18-SSX-positive, SMARCB1-deficient synovial sarcomas on EZH2 enzymatic activity and suggests the potential utility of EZH2-targeted drugs in these genetically defined cancers.

Integrin α6β4 is abundantly expressed in normal epithelial cells and forms hemidesmosomes, one of cell–extracellular matrix junctions. In many types of cancer cells, integrin α6β4 is up-regulated, laminin, an integrin α6β4-binding extracellular matrix protein, is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and a facilitation of their invasion. It was previously shown that integrin α6β4 interacts with ErbB1 and ErbB2 and enhances cell proliferation and motility. Here we show that integrin α6β4 interacts with ErbB3 but not with ErbB1, ErbB2 or ErbB4, and enhances the heregulin-induced, ErbB3/ErbB2 heterodimer-mediated DNA synthesis, but not cell motility, in A549 cells.

Aiming at creating an apical barrier material that promotes recovery of defects in the root canal, a composite of apatite and collagen was developed. To achieve this, reconstitution and cross-link of atelocollagen, and calcification of reconstituted collagen fibers, were conducted under controlled stirring at 37°C. Deposits were identified as CO3-containing apatite and its crystallinity was comparable to that of bone apatite. After 20 hours of calcification, the composite contained apatite of about 60 wt%. The composite and saline mixture had a pH of about 9. The mixture was easily set into a narrow space, such as the root canal, and was liquid permeable after being set into a root canal. The biological property of the composite was examined in vivo using the molar teeth of dogs. After 8 weeks, a thin layer of new bone-like tissue was formed along the inner wall of root canal. Without the composite paste, such a new bone-like tissue was not observed. Thus, the apatite/collagen composite showed potential as an apical barrier material with osteoconductivity.

The purpose of this study was to evaluate the cleaning effect of root canal walls using strong acid electrolytic water (SAEW) as a root canal irrigant, and to investigate the influence of SAEW on the root canal dentin by micro-hardness test. Forty-three single-rooted, single-canaled teeth were instrumented using standard step-back technique with K-files. Irrigation was performed using distilled water, 5.25% NaOCl and 3% H(2)O(2), SAEW, or 15% EDTA solution in five groups. Samples were prepared to be examined under a scanning electron microscope (SEM) and micro Vickers hardness (H(V)) test machine. Our results showed that the root cleaning effects of the combined use of SAEW and NaOCl solution as root canal irrigants were equivalent to those in the group with NaOCl and 15% EDTA. When SAEW was used for 1 min under ultrasonic vibration, no decreases in the hardness of dentin inside the root canal were detected.

Eribulin mesilate (Eribulin), a first-in-class halichondrin B-based microtubule dynamics inhibitor, has been reported to remodel tumor vasculature (i.e., improvement of tumor vessels and perfusion) in human breast cancer xenograft models; however, the role of this vascular remodeling in anti-tumor effects is not fully understood. Here, we investigated the effects of eribulin-induced vascular remodeling on anti-tumor activities in multiple human cancer xenograft models. Microvessel densities (MVDs) were evaluated by immunohistochemistry (CD31 staining), and anti-tumor effects were examined in 10 human cancer xenograft models treated with eribulin. Eribulin treatment significantly increased MVDs compared to the corresponding control groups in 7 out of 10 models with a correlation between enhanced MVD levels and anti-tumor effects (R2=0.55). Because of observed increases in MVDs, we next utilized 111In-labeled PEGylated liposomes to examine if eribulin treatment would result in increased tumoral accumulation levels of such liposomes, and indeed, we found that eribulin, unlike vinorelbine another tubulin inhibitor, enhanced them. Since eribulin treatment increased accumulation of 111In-labeled PEGylated liposomes, we postulated that this treatment might enhance anti-tumor effect of Doxil (a liposomal anti-cancer agent). As expected, eribulin enhanced anti-tumor activity of Doxil in the PE-H1650 xenograft model. Then, we evaluated whether eribulin facilitated the recruitment of immune cells into the tumor. Infiltrating CD11b-positive immune cells were significantly increased in the multiple eribulin-treated xenografted tumors, and natural killer (NK) cell depletion reduced anti-tumor effects of eribulin. Collectively, these findings suggested a contribution of the immune cells for anti-tumor activities of eribulin. Taken together, our results obtained from multiple human cancer xenograft models suggested that vascular remodeling induced by eribulin therapy would act as a microenvironment modulator, and contributed to show anti-tumor effects of eribulin. Citation Format: Ken Ito, Shusei Hamamichi, Takanori Abe, Tsuyoshi Akagi, Hiroshi Shirota, Satoshi Kawano, Makoto Asano, Osamu Asano, Akira Yokoi, Junji Matsui, Izumi O. Umeda, Hirofumi Fujii. Eribulin modulates tumor microenvironment through vascular remodeling for antitumor effect in multiple mouse xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5927. doi:10.1158/1538-7445.AM2017-5927

The catalytic activities of covalent and ATP-dependent chromatin remodeling are central to regulating the conformational state of chromatin and the resultant transcriptional output. The enzymes that catalyze these activities are often contained within multiprotein complexes in nature. Two such multiprotein complexes, the polycomb repressive complex 2 (PRC2) methyltransferase and the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler have been reported to act in opposition to each other during development and homeostasis. An imbalance in their activities induced by mutations/deletions in complex members (e.g. SMARCB1) has been suggested to be a pathogenic mechanism in certain human cancers. Here we show that preclinical models of synovial sarcoma-a cancer characterized by functional SMARCB1 loss via its displacement from the SWI/SNF complex through the pathognomonic SS18-SSX fusion protein-display sensitivity to pharmacologic inhibition of EZH2, the catalytic subunit of PRC2. Treatment with tazemetostat, a clinical-stage, selective and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity reverses a subset of synovial sarcoma gene expression and results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX fusion-positive cells in vitro. Treatment of mice bearing either a cell line or two patient-derived xenograft models of synovial sarcoma leads to dose-dependent tumor growth inhibition with correlative inhibition of trimethylation levels of the EZH2-specific substrate, lysine 27 on histone H3. These data demonstrate a dependency of SS18-SSX-positive, SMARCB1-deficient synovial sarcomas on EZH2 enzymatic activity and suggests the potential utility of EZH2-targeted drugs in these genetically defined cancers.

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.

Cubic InN films have been grown on MgO (1 0 0) substrates with cubic GaN buffer layers by pulsed laser deposition (PLD). It has been found that cubic InN (1 0 0) films grow on the GaN (1 0 0)/MgO (1 0 0) structure with an in-plane epitaxial relationship of [0 0 1] InN ∥[0 0 1] GaN ∥[0 0 1]MgO. The phase purity of a cubic InN film grown at 440 °C was as high as 99% that can probably be attributed to the enhanced surface migration of film precursors in case of PLD. These results indicate that PLD is a suitable technique for the growth of high-quality cubic InN films, and will makes it possible to fabricate optical and electron devices based on cubic InN films.

In normal epithelial cells, integrin α(6)β(4) is abundantly expressed and forms hemidesmosomes, which is a cellular structure that mediates cell-extracellular matrix binding. In many types of cancer cells, integrin α(6)β(4) is up-regulated, laminin is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and facilitation of their invasion. We previously showed that the immunoglobulin-like cell adhesion molecule Necl-2 (Nectin-like molecule 2), known as a tumor suppressor, inhibits cancer cell movement by suppressing the ErbB3/ErbB2 signaling. We show here that Necl-2 interacts in cis with integrin α(6)β(4). The binding of Necl-2 with integrin β(4) was mediated by its extracellular region. In human colorectal adenocarcinoma Caco-2 cells, integrin α(6)β(4) was localized at hemidesmosomes. Small interfering RNA-mediated suppression of Necl-2 expression enhanced the phorbol ester-induced disruption of the integrin α(6)β(4) complex at hemidesmosomes, whereas expression of Necl-2 suppressed the disruption of this structure. These results indicate that tumor-suppressive functions of Necl-2 are mediated by the stabilization of the hemidesmosome structure in addition to the inhibition of the ErbB3/ErbB2 signaling.

Integrin alpha(6)beta(4) is abundantly expressed in normal epithelial cells and forms hemidesmosomes, one of cell-extracellular matrix junctions. In many types of cancer cells, integrin alpha(6)beta(4) is up-regulated, laminin, an integrin alpha(6)beta(4)-binding extracellular matrix protein, is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and a facilitation of their invasion. It was previously shown that integrin alpha(6)beta(4) interacts with ErbB1 and ErbB2 and enhances cell proliferation and motility. Here we show that integrin alpha(6)beta(4) interacts with ErbB3 but not with ErbB1, ErbB2 or ErbB4, and enhances the heregulin-induced, ErbB3/ErbB2 heterodimer-mediated DNA synthesis, but not cell motility, in A549 cells.

Afadin is an actin-filament-binding protein that binds to nectin, an immunoglobulin-like cell-cell adhesion molecule, and plays an important role in the formation of adherens junctions. Here, we show that afadin, which did not bind to nectin and was localized at the leading edge of moving cells, has another role: enhancement of the directional, but not random, cell movement. When NIH3T3 cells were stimulated with platelet-derived growth factor (PDGF), afadin colocalized with PDGF receptor, αvβ3 integrin and nectin-like molecule-5 at the leading edge and facilitated the formation of leading-edge structures and directional cell movement in the direction of PDGF stimulation. However, these phenotypes were markedly perturbed by knockdown of afadin, and were dependent on the binding of afadin to active Rap1. Binding of Rap1 to afadin was necessary for the recruitment of afadin and the tyrosine phosphatase SHP-2 to the leading edge. SHP-2 was previously reported to tightly regulate the activation of PDGF receptor and its downstream signaling pathway for the formation of the leading edge. These results indicate that afadin has a novel role in PDGF-induced directional cell movement, presumably in cooperation with active Rap1 and SHP-2.

ErbB2 and ErbB3, members of the EGF receptor/ErbB family, form a heterodimer upon binding of a ligand, inducing the activation of Rac small G protein and Akt protein kinase for cell movement and survival, respectively. The enhanced ErbB3/ErbB2 signaling causes tumorigenesis, invasion, and metastasis. We found here that the ErbB3/ErbB2 signaling is regulated by immunoglobulin-like Necl-2, which is down-regulated in various cancer cells and serves as a tumor suppressor. The extracellular region of ErbB3, but not ErbB2, interacted in cis with that of Necl-2. This interaction reduced the ligand-induced, ErbB2-catalyzed tyrosine phosphorylation of ErbB3 and inhibited the consequent ErbB3-mediated activation of Rac and Akt, resulting in the inhibition of cancer cell movement and survival. These inhibitory effects of Necl-2 were mediated by the protein-tyrosine phosphatase PTPN13 which interacted with the cytoplasmic tail of Necl-2. We describe here this novel mechanism for silencing of the ErbB3/ErbB2 signaling by Necl-2.

Fruiting-inducing activity and antifungal properties of lipid components in the phylum Annelida were examined. Some amphoteric cerebrosides carrying a phosphocholine group showed fruiting-inducing activity on Schizophyllum commune, and one of them possessed activity comparable to that of Sch II, the most potent substance known. Furthermore, alkyl lysophosphatidylcholines were found to have an inhibitory effect on the growth of phytopathogenic fungi, Alternaria kikuchiana and Phomopsis mali. The relationship between structure and biological activities is discussed.

Although vascular endothelial growth factor (VEGF) inhibitors provide significant clinical benefit, they often require dose reductions or even withdrawals due to their severe toxicities. Furthermore, almost all cancers show intrinsic and/or evasive resistance to VEGF inhibitors by multiple mechanisms. Serum angiopoietin-2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. Response to lenvatinib (E7080; VEGFR1-3 inhibitor) is also reported to correlate with low Ang2 level in differentiated thyroid cancer and endometrial cancer. From these clinical observations, Ang2 and its receptor Tie2 has been focused as promising targets. Here, we demonstrated mechanisms of resistance induced by Ang2 and a novel strategy to circumvent the resistance by combination of multi-tyrosine kinase inhibitors (TKIs), lenvatinib and golvatinib (E7050; c-Met, Tie2, EphB4 inhibitor). Tie2 defines a highly pro-angiogenic macrophage subset, Tie2-expressing macrophage (TEM). Ang-Tie2 and EphB4-EphrinB2 signaling play critical roles in pericyte-mediated vessel stabilization. Ectopic expression of Ang2 in thyroid cancer conferred resistance to lenvatinib and enhanced pericyte-associated endothelial network development and TEM infiltration. In vitro analyses suggested that golvatinib/lenvatinib combination inhibited pericyte-mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib/lenvatinib combination inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable in mice, and no macroscopic change was observed. These results suggest that modulation of tumor microenvironment by strategic and well-tolerated combination of multi-targeting TKIs sensitizes cancer to VEGF inhibitors, which warrants further clinical investigation to determine the clinical benefit of anti-angiogenesis cancer therapy. Citation Format: Youya Nakazawa, Satoshi Kawano, Junji Matsui, Yasuhiro Funahashi, Osamu Tohyama, Hiroki Muto, Takayuki Nakagawa, Tomohiro Matsushima. Maximizing the efficacy of anti-angiogenesis cancer therapy: A multi-targeting strategy by tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2980. doi:10.1158/1538-7445.AM2014-2980

Background Eribulin mesylate is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B. Eribulin is a tubulin-binding drug and approved in many countries worldwide for treatment of certain patients with advanced breast cancer. Here we investigated antiproliferative and antiangiogenic effects of eribulin on vascular cells, human umbilical vein endothelial cells (HUVECs) and human brain vascular pericytes (HBVPs), in vitro in comparison with another tubulin-binding drug, paclitaxel. Methods HUVECs and HBVPs were treated with either eribulin or paclitaxel and their antiproliferative effects were evaluated. Global gene expression profiling changes caused by drug treatments were studied using Affymetrix microarray platform and custom TaqMan Low Density Cards. To examine effects of the drugs on pericyte-driven in vitro angiogenesis, we compared lengths of capillary networks in co-cultures of HUVECs with HBVPs. Results Both eribulin and paclitaxel showed potent activities in in vitro proliferation of HUVECs and HBVPs, with the half-maximal inhibitory concentrations (IC50) in low- to sub-nmol/L concentrations. When gene expression changes were assessed in HUVECs, the majority of affected genes overlapped for both treatments (59%), while in HBVPs, altered gene signatures were drug-dependent and the overlap was limited to just 12%. In HBVPs, eribulin selectively affected 11 pathways (p

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

Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several types of tumors, however, some tumor shows intrinsic resistance to VEGFR inhibitors. In addition, patients initially responding develop acquired resistance to VEGFR inhibitors after varying periods of time. Thus it is urgent to develop strategy to overcome anti-VEGF therapy resistance. Hepatocyte growth factor (HGF)/Met signaling is activated in various types of tumors. Recently, it is reported that HGF/Met signaling contributes to resistance to tyrosine kinase inhibitors such as EGFR, BRAF, ALK inhibitors. This suggests that Met inhibitors may be promising in treating HGF/Met activated anti-VEGF therapy-resistant tumors. We have identified lenvatinib (E7080) as a highly-potent VEGFR inhibitor and phase III trial is ongoing in thyroid cancer. Lenvatinib inhibited the growth of HUVEC induced by VEGF, however, addition of HGF to HUVEC conferred resistance to lenvatinib. This resistance was cancelled by the addition of golvatinib (E7050) which is identified as a potent Met/EphB4 inhibitor and phase I trial of the lenvatinib/golvatinib combination therapy is ongoing. In mouse models bearing various tumor cell lines expressing VEGF and HGF, treatment with lenvatinib alone demonstrated only moderate antitumor activity, however, combination with golvatinib demonstrated synergistic antitumor activity with decrease of tumor vessel density. Furthermore, it is reported that EphB4 has critical roles in tumor vessel development and maturation. Our unique in vitro assay systems, endothelial cells/pericyte 2D co-culture assay and endothelial cells/pericyte 3D co-culture sprouting assay, suggested that EphB4 inhibitory activity of golvatinib disrupted anti-VEGF therapy-resistant tumor vascular in the combination with lenvatinib. Together, these data demonstrated that combination therapy with golvatinib may be promising to overcome anti-VEGF therapy resistance. Citation Format: Satoshi Kawano, Takayuki Nakagawa, Yuya Nakazawa, Yoshiaki Sato, Tomohiro Matsushima, Yasuhiro Funahashi. Combination of VEGFR inhibitor lenvatinib (E7080) and Met/EphB4 inhibitor golvatinib (E7050) overcomes VEGFR inhibitor-resistant tumor vascular. [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 1619. doi:10.1158/1538-7445.AM2013-1619

Abstract 3712 The coupled enzymatic activity of wild-type and mutant EZH2 results in hyper-trimethylation of histone H3 lysine 27 (H3K27), which drives lymphomagenesis in heterozygous patients bearing the EZH2 mutations. Our group has previously reported that selective inhibition of EZH2 in cell culture results in selective killing of lymphoma cells bearing EZH2 mutations, with minimal effect on non-mutant lymphoma cells, suggesting that EZH2 enzymatic activity is a required driver of proliferation in the mutant-bearing cells [Knutson et al. (2012) Nature Chemical Biology, in press]. Through iterative medicinal chemistry we have developed a selective inhibitor of EZH2 with good pharmacological properties, E7438. E7438 binds to the enzyme in a manner competitive with S-adenosyl methionine (SAM) and a Ki for wild-type EZH2 of 2.5 ± 0.5 nM. The compound potently inhibits all known mutants of EZH2 that have been identified in non-Hodgkin lymphoma (NHL) patient samples. E7438 displays about 35-fold less activity against the closely related enzyme EZH1, and is >4500-fold selective with respect to all other protein methyltransferases tested. Lymphoma cells treated with E7438 display concentration- and time-dependent loss of H3K27 methylation with no effect on the methylation status of any other histone sites. The loss of H3K27 methylation results in selective killing of EZH2 mutant-bearing lymphoma cell lines. E7438 displays good oral bioavailability and pharmacokinetic properties. Various EZH2 mutant-bearing human lymphoma tumors were subcutaneously implanted in nude, SCID or NSG mice. Oral administration of E7438 to tumor bearing mice resulted in significant anti-tumor activity. The responses ranged from dose-dependent tumor growth inhibition to complete and sustained regressions. For example, KARPAS422 tumors in nude mice showed complete tumor elimination after 28 days of dosing, with mice remaining tumor free for up to 90 days after treatment cessation. Figure 1. E7438 causes complete and sustained tumor regression in a KARPAS422 nude mouse xenograft model of EZH2-mutated NHL. Dosing was on a BID schedule. * P< 0.05, Repeated measures ANOVA, Dunnett's post test vs. vehicle. Figure 1. E7438 causes complete and sustained tumor regression in a KARPAS422 nude mouse xenograft model of EZH2-mutated NHL. Dosing was on a BID schedule. * P< 0.05, Repeated measures ANOVA, Dunnett's post test vs. vehicle. Mice and rats tolerated E7438 administration well at doses representing high multiples of doses that show antitumor activity in mice. Activity against the EZH2 target in both species was demonstrated by dose-dependent diminution of H3K27me3 levels, assessed by ELISA, in samples of tumor, bone marrow, skin and peripheral blood mononuclear cells (PBMCs). Highly sensitive detection of existing H3K27me3 signal could also be observed with this ELISA assay in drug-naïve samples of human PBMCs. The ability to measure dose-dependent changes in H3K27me3 levels in skin and PBMCs portends the use of signal from these surrogate tissues as a non-invasive pharmacodynamics biomarker in human clinical trials. Disclosures: Keilhack: Epizyme Inc.: Employment, Equity Ownership. Yokoi:Eisai Co., Ltd.: Employment. Knutson:Epizyme Inc.: Employment, Equity Ownership. Wigle:Epizyme Inc.: Employment, Equity Ownership. Warholic:Epizyme Inc.: Employment, Equity Ownership. Kawano:Eisai Co., Ltd.: Employment. Minoshima:Eisai Co., Ltd.: Employment. Huang:Eisai Inc.: Employment. Kuznetsov:Eisai Inc.: Employment. Kumar:Eisai Inc.: Employment. Klaus:Epizyme, Inc.: Employment, Equity Ownership. Allain:Epizyme Inc.: Employment, Equity Ownership. Raimondi:Epizyme Inc.: Employment, Equity Ownership. Porter Scott:Epizyme: Employment, Equity Ownership. Chesworth:Epizyme: Employment, Equity Ownership. Moyer:Epizyme: Employment, Equity Ownership. Uenaka:Eisai Co., Ltd.: Employment. Copeland:Epizyme Inc.: Employment, Equity Ownership. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Kuntz:Epizyme Inc.: Employment, Equity Ownership.

We have investigated the structural and optical properties of semipolar r-plane GaN{1102} films grown on nearly-lattice-matched ZnO substrates with room-temperature (RT) epitaxial GaN buffer layers, putting special emphasis on the effect of surface treatment of the ZnO substrates. The full-width at half-maximum values of X-ray rocking curves for 1-µm-thick r-plane GaN layers grown at 700 °C on these RT-buffer layers, as measured using various X-ray incidence geometries, are in a range from 313 to 598 arcsec. Photoluminescence peaks attributable to structural defects in the r-plane GaN films have been shown to be reduced, and the near-band-edge emission has been enhanced by approximately 5 times by the use of atomically-flat r-plane ZnO substrates prepared by high-temperature annealing in air inside a box made of ZnO.