Kenjiro Ito

TAS-116, a Highly Selective Inhibitor of Heat Shock Protein 90α and β, Demonstrates Potent Antitumor Activity and Minimal Ocular Toxicity in Preclinical Models

The molecular chaperone HSP90 plays a crucial role in cancer cell growth and survival by stabilizing cancer-related proteins. A number of HSP90 inhibitors have been developed clinically for cancer therapy; however, potential off-target and/or HSP90-related toxicities have proved problematic. The 4-(1H-pyrazolo[3,4-b]pyridine-1-yl)benzamide TAS-116 is a selective inhibitor of cytosolic HSP90α and β that does not inhibit HSP90 paralogs such as endoplasmic reticulum GRP94 or mitochondrial TRAP1. Oral administration of TAS-116 led to tumor shrinkage in human tumor xenograft mouse models accompanied by depletion of multiple HSP90 clients, demonstrating that the inhibition of HSP90α and β alone was sufficient to exert antitumor activity in certain tumor models. One of the most notable HSP90-related adverse events universally observed to differing degrees in the clinical setting is visual disturbance. A two-week administration of the isoxazole resorcinol NVP-AUY922, an HSP90 inhibitor, caused marked degeneration and disarrangement of the outer nuclear layer of the retina and induced photoreceptor cell death in rats. In contrast, TAS-116 did not produce detectable photoreceptor injury in rats, probably due to its lower distribution in retinal tissue. Importantly, in a rat model, the antitumor activity of TAS-116 was accompanied by a higher distribution of the compound in subcutaneously xenografted NCI-H1975 non–small cell lung carcinoma tumors than in retina. Moreover, TAS-116 showed activity against orthotopically transplanted NCI-H1975 lung tumors. Together, these data suggest that TAS-116 has a potential to maximize antitumor activity while minimizing adverse effects such as visual disturbances that are observed with other compounds of this class. Mol Cancer Ther; 14(1); 14–22. ©2014 AACR.

High Potency VEGFRs/MET/FMS Triple Blockade by TAS-115 Concomitantly Suppresses Tumor Progression and Bone Destruction in Tumor-Induced Bone Disease Model with Lung Carcinoma Cells

Approximately 25–40% of patients with lung cancer show bone metastasis. Bone modifying agents reduce skeletal-related events (SREs), but they do not significantly improve overall survival. Therefore, novel therapeutic approaches are urgently required. In this study, we investigated the anti-tumor effect of TAS-115, a VEGFRs and HGF receptor (MET)-targeted kinase inhibitor, in a tumor-induced bone disease model. A549-Luc-BM1 cells, an osteo-tropic clone of luciferase-transfected A549 human lung adenocarcinoma cells (A549-Luc), produced aggressive bone destruction associated with tumor progression after intra-tibial (IT) implantation into mice. TAS-115 significantly reduced IT tumor growth and bone destruction. Histopathological analysis showed a decrease in tumor vessels after TAS-115 treatment, which might be mediated through VEGFRs inhibition. Furthermore, the number of osteoclasts surrounding the tumor was decreased after TAS-115 treatment. In vitro studies demonstrated that TAS-115 inhibited HGF-, VEGF-, and macrophage-colony stimulating factor (M-CSF)-induced signaling pathways in osteoclasts. Moreover, TAS-115 inhibited Feline McDonough Sarcoma oncogene (FMS) kinase, as well as M-CSF and receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. Thus, VEGFRs/MET/FMS-triple inhibition in osteoclasts might contribute to the potent efficacy of TAS-115. The fact that concomitant dosing of sunitinib (VEGFRs/FMS inhibition) with crizotinib (MET inhibition) exerted comparable inhibitory efficacy for bone destruction to TAS-115 also supports this notion. In conclusion, TAS-115 inhibited tumor growth via VEGFR-kinase blockade, and also suppressed bone destruction possibly through VEGFRs/MET/FMS-kinase inhibition, which resulted in potent efficacy of TAS-115 in an A549-Luc-BM1 bone disease model. Thus, TAS-115 shows promise as a novel therapy for lung cancer patients with bone metastasis.

MP21-10 DEVELOPMENT OF AN EFFICIENT BLADDER TUMOR MODEL BY RECOMBINING WITH MESENCHYMAL CELLS

progression remain unclear. Here, we investigated the role of plectin-1 and Fer in bladder cancer cell lines. METHODS: We used the 2 bladder cancer cell lines YTS-1 (muscle invasive) and RT4 (superficial). Expression of plectin-1 and Fer in bladder cancer cells was analyzed by immunofluorescence (IF) staining and immunoblotting (IB). Interaction of plectin-1 and Fer in bladder cancer cells was analyzed by immunoprecipitation (IP). Bladder cancer cells were tested for invasion potential against basal membrane extract (BME) gel or bladder smooth muscle cell (BdSMC) monolayer. We also examined the role of Fer in invasion capacity using Fer knockdown YTS-1 cells. Furthermore, we examined expression of phospho-Fer in bladder cancer tissues. RESULTS: IB and IF staining showed that expression of plectin-1 was much higher in RT4 compared with YTS-1. Although the expression of Fer was decreased in YTS-1, expression of phosphor-Fer was much higher in YTS-1 compared with RT4. Phosphorylation of ERK or AKT which is a downstream signaling molecules of Fer was also upregulated in YTS-1. IP showed that plectin-1 bound to Fer in RT4 but not bound to Fer in YTS-1. In YTS-1, Fer was bound to vimentin. Invasion capacity against BME gel and BdSMC in YTS-1 was much higher than those of RT4. Immunohistochemical analyses of bladder cancer tissues showed that the upregulation of phospho-Fer correlated with poor prognosis of bladder cancer. CONCLUSIONS: These results indicate that plectin-1 depletion in muscle invasive bladder cancer cells promotes phosphorylation of Fer and enhances invasion capacity.

Abstract C128: TAS-116, an orally available HSP90α/β selective inhibitor, has potent antitumor activity in small cell lung cancer as a single agent and in combination with an anthracycline derivative, amrubicin.

Background: The molecular chaperone HSP90 has been considered a promising target for cancer therapy. We have previously reported the discovery of an orally available HSP90α/β selective inhibitor, TAS-116. TAS-116 led to tumor shrinkage in various human tumor xenograft models, while not inducing retinal toxicity in rats including at doses given above the maximum tolerated dose. Extensive small cell lung cancer (SCLC) is one of the most aggressive types of cancer, and has an unmet medical need for more effective treatments. An anthracycline, amrubicin (AMR), has been used to treat platinum resistant SCLC in Japan. Here we report on the possible application of TAS-116 to treat SCLC both as a single agent and in combination with AMR. Materials and Methods: In vitro drug combination effects were evaluated with calculation of the combination index and cell cycle analysis by fluorescence-activated cell sorting. To clarify the mechanism involved in TAS-116’s enhancement of anthracycline-induced apoptosis the extent of apoptosis was measured in combination with several kinase inhibitors including SB 218078 (CHK1) and MK-2206 (AKT). Antitumor activities of TAS-116 alone and in combination with AMR were evaluated in human SCLC xenograft models. Results: TAS-116 induced cell growth arrest and apoptosis in SCLC cell lines by inhibiting both PI3K/AKT and MAPK signaling through downregulation of HSP90 clients such as AKT and RAF1. TAS-116 also showed antitumor activity against human SCLC tumor xenograft models. In in vitro explorative combination studies in SCLC cell lines, structurally-related anthracyclines, AMR and doxorubicin (DOX), both showed synergistic effect upon apoptosis induction in combination with TAS-116. Moreover, TAS-116 enhanced the antitumor activity of AMR in human SCLC tumor xenograft models. Since anthracyclines exert antitumor activity based on their DNA damaging potential through topoisomerase II inhibition, we evaluated whether DNA damage signaling proteins and apoptosis-related proteins were affected by TAS-116 when given in combination with DOX. TAS-116 significantly reduced phosphorylation of CHK1, AKT and ERK1/2. In combination with these kinase inhibitors, SB 218078 significantly and MK-2206 moderately enhanced DOX-mediated apoptosis. Comparatively, TAS-116 enhanced apoptosis to a greater extent than SB 218078. Conclusion: TAS-116 showed antitumor activity against human SCLC tumor xenograft models alone and in combination with AMR. Our data suggest that TAS-116 enhanced the antitumor activity of anthracyclines through the combined actions of abrogation of the DNA damage-induced G2/M checkpoint and the inhibition of a survival signal by CHK1 and AKT downregulation, respectively. These results suggest that TAS-116 could be a promising agent to treat extensive SCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C128. Citation Format: Hiromi Muraoka, Chihoko Yoshimura, Akihiro Hashimoto, Kenjiro Ito, Makoto Kitade, Kazuhiko Yonekura, Shuichi Ohkubo, Teruhiro Utsugi. TAS-116, an orally available HSP90α/β selective inhibitor, has potent antitumor activity in small cell lung cancer as a single agent and in combination with an anthracycline derivative, amrubicin. [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 C128.

Abstract C129: TAS-116, an orally available HSP90α and β selective inhibitor, exhibits synergistic effects in combination with taxanes in EGFR-tyrosine kinase inhibitor-resistant human NSCLC xenograft models through the downregulation of key molecules involved in drug resistance of cancer chemotherapy.

Background: In advanced NSCLC, EGFR-tyrosine kinase inhibitors (EGFR-TKI) have shown limited effect on lung squamous cell carcinoma (LSCC) or NSCLC harboring either a mutation in the K-RAS oncogene or lacking an activated mutation in EGFR. Though several cytotoxic drugs have been used in these EGFR-TKI-resistant settings, clinical outcomes have been generally poor. It has been shown that several antiapoptotic molecules regulated by PI3K/AKT and MAPK signaling, are involved in drug resistance to cancer chemotherapy. One of the possible approaches to overcome such drug resistance is via simultaneous inhibition of multiple signaling pathways. We have previously reported on an orally available highly selective HSP90α and β inhibitor, TAS-116. Because TAS-116 leads to downregulation of multiple molecules involved in various signaling pathways through HSP90 inhibition, we investigated the potential utility of TAS-116 on LSCC and K-RAS mutated NSCLC in combination with taxanes. Materials and Methods: In vitro drug combination effects were evaluated with calculation of combination index. The amount and the phosphorylation status of cellular proteins were determined by western blot with appropriate antibodies. Antitumor activities of TAS-116 alone and in combination with taxanes were evaluated in human NSCLC xenograft models. The inhibition and induction study of cytochrome P450 (CYP) by TAS-116 were evaluated using human liver microsomes and human cryopreserved hepatocytes, respectively. Results: TAS-116 showed synergistic effects in combination with docetaxel (DTX) and paclitaxel (PTX) against LSCC cell lines. TAS-116 enhanced taxane-mediated apoptosis. To clarify the mode of action in these combinations, we measured the amount and the phosphorylation status of apoptosis-related proteins. TAS-116 decreased phosphorylation of BAD and YBX1, through the downregulation of upstream kinases, AKT and RAF1. TAS-116 synergistically inhibited tumor growth in both human LSCC and K-RAS mutated NSCLC xenograft models in combination with PTX or DTX. With regard to drug-drug interaction potential at effective concentrations, TAS-116 did not inhibit or induce CYP3A4 which is important in taxane metabolism. Conclusion: TAS-116 in combination with taxanes enhanced apoptosis induction and exhibited synergistic antitumor activity against LSCC and K-RAS mutated NSCLC xenograft models through downregulation of key factors involved in drug resistance in cancer chemotherapy. The combination of TAS-116 and taxanes could be a promising approach to treat EGFR-TKI-resistant NSCLC including LSCC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C129. Citation Format: Hiromi Muraoka, Akira Kanoh, Akihiro Hashimoto, Kenjiro Ito, Takamasa Suzuki, Kouichi Takahashi, Chihoko Yoshimura, Makoto Kitade, Kazuhiko Yonekura, Shuichi Ohkubo, Teruhiro Utsugi. TAS-116, an orally available HSP90α and β selective inhibitor, exhibits synergistic effects in combination with taxanes in EGFR-tyrosine kinase inhibitor-resistant human NSCLC xenograft models through the downregulation of key molecules involved in drug resistance of cancer chemotherapy. [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 C129.

Abstract 1785: MET/VEGFR dual inhibition and prominent safety profile of TAS-115 are favorable for the combination with chemotherapeutic drugs

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL MET is known as a proto-oncogene, which involves in growth, migration, metastasis and angiogenesis in tumor progression. Recently, it is reported that MET also functions as chemo and EGFR-TKI resistant factor in cancer cells. Particularly, HGF/MET signal seemed to reduce the potency of conventional chemotherapeutic drugs in clinical. Meanwhile, VEGF/VEGFR signal plays an important role in angiogenesis in tumor growth, and is essential for the survival signal of endothelial cells(ECs) to protect from the damage by chemotherapeutic drugs. Some VEGFR inhibitors have already been approved for the treatment of several carcinomas with monotherapy. Many clinical combination trials with VEGFR inhibitor and chemotherapeutic agents have been tried, but its therapeutic benefit are restricted. It is highly desire for improving therapeutic benefit by combination therapy with VEGFR inhibitor and standard chemotherapy. TAS-115, a novel oral MET/VEGFR dual inhibitor, demonstrated not only potent efficacy but also prominent safety profiles achieved by improvement of PK profile and cellular selectivity. Potent MET/VEGFR dual inhibition provides significant anti-tumor effect from lower dose for TAS-115. On the one hand, the improvement of PK profile and cell selectivity led to better tolerability. As a result, therapeutic window (MTD/ED50) of TAS-115 became wider than that of the pre-existing VEGFR or MET/VEGFR inhibitors. These profiles give TAS-115 the long-term dosing at full effective dose without severe toxicity. Moreover, in vitro study, HGF completely suppressed the induction of apoptosis by 5-FU or Paclitaxel in cancer cells expressed MET. Meanwhile, VEGF abolished the cytotoxic effect by these chemotherapeutic drugs against ECs. It was suggested that harmonization of both HGF and VEGF in tumor greatly contributed to the resistance against conventional chemotherapeutic drugs. The MET/VEGFR dual inhibition by TAS-115 improves the sensitivity of chemotherapeutic drugs for both cancer cells and ECs via suppression of function as chemo-resistance factor by MET and VEGFR. In fact, the combination therapies with TAS-115 and conventional chemotherapeutic drugs caused tumor regression or potent growth inhibition without sever body weight loss in several xenograft models. Notably, TAS-115 markedly enhanced anti-tumor effect of Paclitaxel, and brought tumor shrinkage of approximately 80% in the combination. More important feature is that TAS-115 has disturbed rapid tumor re-growth following Paclitaxel treatment and the higher safety profiles of TAS-115 permits combination therapy with chemotherapeutic drugs in chronic treatment. It is expected TAS-115 demonstrates prominent therapeutic benefit in combination with chemotherapy based on the synergistic action of the potent MET/VEGFR dual inhibition and higher safety profiles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1785. doi:1538-7445.AM2012-1785

Abstract B1: Antitumor activity of TSU-68 in PDGF-BB overexpressing U-87MG xenograft model: contribution of PDGFR blocking action.

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. TSU-68, a new drug under clinical study that inhibits an angiogenic receptor tyrosine kinase KDR, which play a crucial role in tumor-induced vascularization, inhibits angiogenesis and the growth of human tumor xenografts in vivo. This compound is also a potent inhibitor of PDGFRα and PDGFRβ kinases. In this study, PDGF-BB overexpressing human tumor model was used to characterize the in vivo activity of TSU-68 as a PDGFR blocking agent. In vivo xenograft mice study, TSU-68 treatment (once daily p.o. dosing for 28 days) exhibited potent antitumor activity in PDGF-BB overexpressing U-87MG (U-87MG/PDGF-9) xenograft (T/C 28.2%) in comparison to parental U-87MG xenograft (T/C 69.7%). Expression level of VEGF of U-87MG/PDGF-9 cells was similar to that of parental U-87MG cells. PDGF-BB was only detected in plasma of mice bearing U-87MG/PDGF-9 xenograft. To identify the action of TSU-68 on U-87MG/PDGF-9 tumor, in vitro cell proliferation assay, ex vivo study, and immunohistological examination of xenografts were carried out. TSU-68 inhibited cell proliferation of U-87MG cells induced by PDGF-BB but not by FBS. In U-87MG/PDGF-9 xenograft tumors, PDGFR was highly phosphorylated and a single oral dose of TSU-68 (200 mg/kg) inhibited phosphorylation of PDGFR in the tumors. Immunohistochemical analyses revealed reduced Ki-67 staining in the tumors from TSU-68-treated mice relative to the vehicle control. The Ki-67 labeling index, calculated as the percentage of positive staining cells of total nucleated cells in a field was 47.0 ± 8.5 vs. 31.8 ± 4.5 (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B1.

Abstract 3315: TAC-101 induces the differentiation of cancer stem-like cells

TAC-101 (4-[3,5-bis(trimethylsilyl)benzamido] benzoic acid), is an orally bioavailable synthetic retinoid, binding to nuclear retinoic acid receptor (RAR), activates selectively RAR alpha transcriptional. And TAC-101 has demonstrated anti-tumor activity and proplongation of survival in primary and metastatic liver cancer pre-clinical models. In Phase I study, the lymph nodes of a non-small cell lung cancer patient with nodal metastasis returned to normal size, constituting a complete radiologic response. In a Phase I/II trial of TAC-101 in patients with advanced hepatocellular carcinoma, TAC-101 showed significant disease stabilization, 2 PRs after discontinuing TAC-101 treatment, and prolonged median survival, suggesting that TAC-101 provides meaningful patient9ss benefit. Recently, many researchers have reported correlation between cancer stem-like cells and tumor malignancy, such as therapy-resistance. In addition, retinoids were reported to reduce the number of cells with the cancer stem cell marker. So, we hypothesize that TAC-101 induces the differentiation of cancer stem-like cells, and contributes life span prolongation of patients of solid tumor. We cultured A549 cancer cells in mediummedium containing 0.1 and 1 μM of TAC-101 for 8 weeks and analyzed the expression of several stem cell markers using flow cytomoetry technique. Detection method of side population was validated by the result that fumitremorgin C, inhibitor of ABCG2, could eliminate the side population. We found that TAC-101 reduced side-population, CD326 (EpCAM) positive population and standard deviation of side scatter. From these results, it is estimated that treatment of TAC-101 for a certain period of time will be enough to induce of differentiation and to reduce cancer stem-like cells, may result in the tumor shrinkage and prolongation of patient9s survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3315.