Masahiro Takeuchi

YM155, a Novel Small-Molecule Survivin Suppressant, Induces Regression of Established Human Hormone-Refractory Prostate Tumor Xenografts

Various accumulating evidence suggests that survivin, a member of the inhibitor of apoptosis (IAP) family, plays an important role in drug resistance and cancer cell survival in many types of cancer, including hormone-refractory prostate cancer (HRPC). Here, we characterized YM155, a novel small-molecule survivin suppressant, using a survivin gene promoter activity assay. YM155 suppressed expression of survivin and induced apoptosis in PC-3 and PPC-1 human HRPC cell lines at 10 nmol/L. In contrast, YM155 up to 100 nmol/L showed little effect on expression levels of other IAP- or Bcl-2-related proteins. In a s.c. xenografted PC-3 tumor model in mice, 3-day continuous infusions of YM155 at 3 to 10 mg/kg induced massive tumor regression accompanied by suppression of intratumoral survivin. YM155 also completely inhibited the growth of orthotopically xenografted PC-3 tumors. No significant decreases in body weight were observed in mice treated with YM155 during the experimental period. Pharmacokinetic analyses indicated that YM155 is highly distributed to tumors and at concentrations approximately 20-fold higher than those in plasma. Our findings represent the first attempt to show tumor regression and suppression of survivin in p53-deficient human HRPC cells by a single small molecular compound treatment. Further extensive investigation of YM155 in many types of cancer, including HRPC, seems to be worthwhile to develop this novel therapeutic approach.

Broad spectrum and potent antitumor activities of YM155, a novel small‐molecule survivin suppressant, in a wide variety of human cancer cell lines and xenograft models

Antitumor activities of YM155, a novel small‐molecule survivin suppressant, were investigated in a wide variety of human cancer cell lines and xenograft models. YM155 inhibited the growth of 119 human cancer cell lines, with the greatest activity in lines derived from non‐Hodgkin’s lymphoma, hormone‐refractory prostate cancer, ovarian cancer, sarcoma, non‐small‐cell lung cancer, breast cancer, leukemia and melanoma. The mean log growth inhibition of 50% (GI50) value was 15u2003nM. The mean GI50 values of YM155 were 11u2003nM for p53 mut/null cell lines and 16u2003nM for p53 WT cell lines, suggesting that YM155 inhibits the growth of human tumor cell lines regardless of their p53 status. In non‐small‐cell lung cancer (Calu 6, NCI‐H358), melanoma (A375), breast cancer (MDA‐MB‐231) and bladder cancer (UM‐UC‐3) xenograft models, 3‐ or 7‐day continuous infusions of YM155 (1–10u2003mg/kg) demonstrated significant antitumor activity without showing significant bodyweight loss. Tumor regressions induced by YM155 were associated with reduced intratumoral survivin expression levels, increased apoptosis and decreased mitotic indices. The broad and potent antitumor activity presented in the present study is indicative of the therapeutic potential of YM155 in the clinical setting. (Cancer Sci 2011; 102: 614–621)

Antitumor activity of YM155, a selective small-molecule survivin suppressant, alone and in combination with docetaxel in human malignant melanoma models.

Purpose: Aggressive cell growth and chemoresistance are notorious obstacles in melanoma therapy. Accumulating evidence suggests that survivin is preferentially expressed in cancer cells and plays a crucial role in cell division and apoptosis dysfunction. Here, we evaluated the therapeutic potential of YM155, a selective survivin suppressant, alone and in combination with docetaxel using human melanoma models. Experimental Design: A375 and SK-MEL-5 human malignant melanoma cells were treated with siRNA, YM155, and/or docetaxel, and cell viability, mRNA and protein expression levels, cell-cycle distribution, and immunohistochemical staining were then evaluated. Furthermore, the efficacy of YM155 combined with docetaxel was further examined in established xenograft models. Results: Survivin suppression was sufficient to induce spontaneous apoptosis of melanoma cells. YM155 showed nanomolar antiproliferative effects and induced tumor regression in established melanoma xenograft models. Docetaxel showed antitumor activity against melanoma cells, although it also induced survivin upregulation and G2/M mitotic arrest; however, cotreatment with YM155 decreased survivin expression below basal levels. Combination treatment of YM155 and docetaxel induced a greater rate of apoptosis than the sum of the single-treatment rates and promoted tumor regression without enhanced body weight loss in the melanoma xenograft models. Conclusions: Survivin is responsible for the inherent low levels of spontaneous apoptosis in melanoma cells. The concomitant combination of YM155 with docetaxel diminished the accumulation of survivin in G2/M mitotic arrest, and induced more intense apoptosis compared with each single treatment. YM155 in combination with docetaxel is well tolerated and shows greater efficacy than either agent alone in mouse xenograft models. Clin Cancer Res; 17(16); 5423–31. ©2011 AACR.

YM155, a novel survivin suppressant, enhances taxane-induced apoptosis and tumor regression in a human Calu 6 lung cancer xenograft model.

Survivin, an apoptotic inhibitor, is overexpressed in the majority of human tumor types and represents a novel target for anticancer therapy. Taxanes induce a mitotic cell-cycle block through the inhibition of microtubule depolymerization, with subsequent elevated expression/stabilization of survivin. We investigated the administration of survivin suppressant YM155 monobromide (YM155), in combination with docetaxel, in a human non-small-cell lung cancer (NSCLC) xenograft model. Animals received a 7-day continuous infusion of YM155, 2 mg/kg, and/or three bolus doses of docetaxel, 20 mg/kg, according to three dosing schedules: YM155 administered concomitantly with docetaxel, before docetaxel, and after docetaxel. YM155 administered either concomitantly with or before docetaxel showed significant antitumor activity (tumor regression ≥99%), with complete regression of the established human NSCLC-derived tumors in mice (eight of eight and seven of eight animals, respectively). Significantly fewer complete responses (three of eight animals) were achieved when YM155 was administered after docetaxel. No statistically significant decreases in body weight were observed in the combination versus docetaxel groups. YM155 administered concomitantly with docetaxel resulted in significant decreases in mitotic and proliferative indices, and in a significant increase in the apoptosis index. Elevated survivin expression was seen in tumors from mice treated with docetaxel alone; a significant reduction in survivin expression was seen in tumors from mice treated with YM155 alone or in combination with docetaxel, but not in the control group. These results indicate that in a human NSCLC xenograft model YM155 in combination with docetaxel diminished the accumulation of survivin by docetaxel and induced more intense apoptosis and enhanced antitumor activity, compared with single-agent YM155 or docetaxel.

Sepantronium Bromide (YM155) induces disruption of the ILF3/p54nrb complex, which is required for survivin expression

YM155, a small-molecule survivin suppressant, specifically binds to the transcription factor ILF3, which regulates the expression of survivin[1]. In this experiment we have demonstrated that p54(nrb) binds to the survivin promoter and regulates survivin expression. p54(nrb) forms a complex with ILF3, which directly binds to YM155. YM155 induces disruption of the ILF3/p54(nrb) complex, which results in a different subcellular localization between ILF3 and p54(nrb). Thus, identification of molecular targets of YM155 in suppression of the survivin pathway, might lead to development of its use as a novel potential target in cancers.

Interleukin Enhancer-binding Factor 3/NF110 Is a Target of YM155, a Suppressant of Survivin

Survivin is responsible for cancer progression and drug resistance in many types of cancer. YM155 selectively suppresses the expression of survivin and induces apoptosis in cancer cells in vitro and in vivo. However, the mechanism underlying these effects of YM155 is unknown. Here, we show that a transcription factor, interleukin enhancer-binding factor 3 (ILF3)/NF110, is a direct binding target of YM155. The enhanced survivin promoter activity by overexpression of ILF3/NF110 was attenuated by YM155 in a concentration-dependent manner, suggesting that ILF3/NF110 is the physiological target through which YM155 mediates survivin suppression. The results also show that the unique C-terminal region of ILF3/NF110 is important for promoting survivin expression and for high affinity binding to YM155.

Abstract 2586: ASP8273 selectively inhibits mutant EGFR signal pathway and induces tumor shrinkage in EGFR mutated tumor models

Activating mutations of epidermal growth factor receptor (EGFR) are associated with the high sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) like erlotinib. However, acquired resistance limits the clinical efficacy of EGFR-TKIs, which is the most commonly caused by T790M mutation in EGFR. Second generation EGFR-TKIs such as afatinib are able to inhibit T790M mutation but the clinical efficacy in T790M positive patients is limited due to severe side effects associated with wild type (WT) EGFR inhibition. ASP8273 is a mutant-selective irreversible EGFR inhibitor currently in clinical trials (ClinicalTrials.gov Identifier: NCT02113813, NCT02192697). We have previously reported that ASP8273 covalently binds to mutant EGFR via C797 and inhibits kinase activity of mutant EGFR, which results in anti-tumor activity in xenograft models. To further explore the selectivity and the activity of ASP8273 on mutant EGFR, we evaluated effects of ASP8273 and other EGFR-TKIs on EGFR signal pathway, cell growth and anti-tumor activity. Phosphorylation of EGFR, ERK and Akt was determined by Western blot after treatment with EGFR-TKIs at 10, 100 and 1000nM. Apoptosis induction was analyzed by detecting caspase activity after 24h treatment with EGFR-TKIs. Anti-tumor activity of ASP8273 was evaluated in mice xenografted with HCC827 (deletion in exon 19 [del ex19]), NCI-H1975 (T790M/L858R), A431 (WT), and a patient derived LU1868 (T790M/L858R). ASP8273 selectively inhibited phosphorylation of EGFR and its down-stream signal pathway, ERK and Akt from 10nM in HCC827 and NCI-H1975 while inhibitory effects were only detected at 1000nM in A431.In NCI-H1650 (del ex19), ASP8273 inhibited cell growth with an IC50 value of 70nM while other EGFR-TKIs were only partially effective. ERK and Akt phosphorylation were diminished after ASP8273 treatment at 1000nM, however, other EGFR-TKIs only partially reduced the phosphorylation levels of these proteins. ASP8273 potently enhanced the caspase activity in NCI-H1650 after 24h treatment, which is concordant with signal and cell growth inhibitory effect. In HCC827 and NCI-H1975 xenograft models, ASP8273 induced tumor regression at 10, 30 and 100mg/kg without affecting body weight. ASP8273 also produced tumor growth inhibition from 10mg/kg in the NSCLC patient derived tumor xenograft, LU1868 which express T790M/L858R. On the other hand, ASP8273 did not produce significant tumor growth inhibition at 10 and 30mg/kg in A431 xenograft model. ASP8273 selectively inhibited mutant EGFR compared to WT EGFR in preclinical models, showing activity in mutant EGFR cell line which is resistant to other EGFR-TKIs including AZD9291 and CO-1686. These results indicate the potential of ASP8273 to induce tumor shrinkage in patients with mutant EGFR positive tumors including those that do not respond to other EGFR-TKIs despite EGFR mutation. Citation Format: Satoshi Konagai, Hideki Sakagami, Hiroko Yamamoto, Hiroaki Tanaka, Takahiro Matsuya, Shinya Mimasu, Yusuke Tomimoto, Masamichi Mori, Hiroyuki Koshio, Masaaki Hirano, Sadao Kuromitsu, Masahiro Takeuchi. ASP8273 selectively inhibits mutant EGFR signal pathway and induces tumor shrinkage in EGFR mutated tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2586. doi:10.1158/1538-7445.AM2015-2586

Identification of a novel 300-kDa factor termed IκBαE3-F1 that is required for ubiquitinylation of IκBα

Destruction of IκB by ubiquitinylation is required for signal‐dependent activation of NF‐κB. The IκBα ubiquitin‐ligase activity associated with phosphorylated IκBα (pIκBα) in HeLa cells was almost completely lost by washing under stringent conditions including 1 M NaCl; nevertheless, an SCFβTrCP complex containing Skp1, Cullin‐1, and F‐box/WD40 protein βTrCP was still bound to pIκBα, suggesting the existence of a putative factor that is loosely associated with pIκBα and may collaborate with SCFβTrCP. The factor was named IκBαE3‐F1 and was partially purified from HeLa cells. Gel filtration analysis revealed that IκBαE3‐F1 has an apparent molecular mass of approximately 300 kDa.

Abstract B188: Preclinical antitumor activity of ASP8273, a mutant-selective irreversible EGFR inhibitor in an AXL-overexpressing NSCLC model

Background: Activating mutations of epidermal growth factor receptor (EGFR) are associated with the high sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) like erlotinib. However, acquired resistance limits the clinical efficacy of EGFR-TKIs. Several mechanisms of the acquired resistance have been described, including gatekeeper T790M mutation and MET amplification. Overexpression of AXL, a receptor type tyrosine kinase and/or its ligand, GAS-6 have been identified in a subset of NSCLC patients with acquired resistance, and inhibition of AXL has been shown to restore sensitivity to erlotinib in nonclinical resistant models, suggesting that AXL is also involved in EGFR-TKI resistance of NSCLC. ASP8273 is a mutant-selective irreversible EGFR inhibitor currently in clinical trials (ClinicalTrials.gov Identifier: NCT02113813, NCT02192697, NCT02500927). We have previously reported that ASP8273 selectively inhibited kinase activities of EGFR activating and T790M mutations compared to wild type EGFR, and induced tumor regression in HCC827 and NCI-H1975 xenografted mice. In this study, we evaluated the antitumor activity of ASP8273 against AXL overexpressing PC-9 cells. Method: PC-9 vec. cells and PC-9 AXL cells were constructed by the infection of pMXs-puro vector and AXL coding vector, respectively into PC-9, a NSCLC cell line with EGFR exon 19 del activating mutation. Inhibitory effects of ASP8273 and other EGFR-TKIs, including erlotinib on EGFR and AXL signaling and cell proliferation were investigated in PC-9 vec. and PC-9 AXL cells. In vivo antitumor effect was also examined in subcutaneously implanted with PC-9 AXL. Result: ASP8273 at 1 μmol/L inhibited cell growth associated with the inhibition of EGFR, AXL, AKT, and ERK phosphorylation in PC-9 AXL. On the other hand, EGFR-TKIs at 1 μmol/L did not show cell growth inhibition in PC-9 AXL cells, whereas those showed cell growth inhibition in PC-9 vec. cells. EGFR-TKIs inhibited the phosphorylation of EGFR but not AXL, AKT, and ERK in PC-9 AXL cells. In an in vivo xenograft model using PC-9 AXL, once-daily oral administration of ASP8273 at 50 mg/kg induced statistically significant tumor growth inhibition although the other EGFR-TKIs did not. Conclusion: These results suggest that overexpression of AXL confers resistance to EGFR-TKIs in NSCLC cells and ASP8273 may show antitumor activity against EGFR-TKIs-resistant NSCLC patients with AXL expression. Citation Format: Naoki Kaneko, Hiroaki Tanaka, Satoshi Konagai, Hiroko Yamamoto, Hideki Sakagami, Tomohiro Eguchi, Takahiro Matsuya, Masamichi Mori, Hiroyuki Koshio, Tadashi Terasaka, Masaaki Hirano, Sadao Kuromitsu, Masahiro Takeuchi. Preclinical antitumor activity of ASP8273, a mutant-selective irreversible EGFR inhibitor in an AXL-overexpressing NSCLC model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B188.

Abstract 4756: YM155 suppresses survivin expression by disrupting the ILF3/p54nrb transcription factor complex

Survivin, a member of the anti-apoptosis proteins family, is highly expressed in all primary tumor types, and is responsible for cancer progression and drug resistance in many types of cancer. YM155, a small chemical compound, selectively suppressed the expression of survivin and induced apoptosis in cancer cells both in vitro and in vivo. However, the mechanisms underlying the suppression of survivin expression by YM155 are unknown. In order to identify the molecular targets and analyze the drug mechanism of action, affinity purification was performed using an active analogue of YM155. We identified interleukin enhancer-binding factor 3 (ILF3) as the binding target of YM155. From the complex analysis of ILF3 and survivin promoter sequence, we also found that ILF3 forms a complex with p54nrb transcription factor, and then binds to the survivin promoter. Overexpression of ILF3 enhanced survivin promoter activity, which was attenuated by YM155 in a concentration-dependent manner. Furthermore, the ILF3/p54nrb complex was disrupted by YM155, thus dispersing the components in the nucleus.In conclusion, our study suggests that binding to ILF3 by YM155 causes the dissociation of the ILF3/p54nrb complex, thus inhibits ILF3 dependent survivin expression. 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 4756. doi:1538-7445.AM2012-4756