Formulation design and evaluation of liposomal sepantronium bromide (YM155), a small-molecule survivin suppressant, based on pharmacokinetic modeling and simulation.

PURPOSE: Sepantronium bromide (YM155) is administered by 168-hour continuous infusions in clinical studies due to its time-dependent pharmacological efficacy and rapid elimination from plasma. To enable more convenient administration, i.e., bolus injections with low frequency, we prepared liposomal formulations of YM155 and evaluated their antitumor activities. METHODS: A kinetic simulation model of liposomal YM155 to predict the free drug concentration in both tumor and plasma was developed. A liposomal formulation with the target drug release rate was prepared based on the simulation. Antitumor activities of the formulation were examined in various tumor xenograft mouse models. In addition, antitumor activities of liposomal formulations with different drug release rates were compared in order to confirm the validity of the simulation-based prediction. RESULTS: Liposomal YM155 with the release half-life of 48 h was prepared as a promising formulation. This formulation showed significantly potent antitumor activities in tumor xenograft models by weekly bolus injections. Further studies demonstrated that this release rate was optimal for YM155 in terms of both efficacy and safety. CONCLUSIONS: We successfully developed a liposomal formulation of YM155 that could substitute for long-term continuous infusion of the drug solution in clinical settings by being given as weekly bolus injections.

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.

Synergistic antitumor activities of sepantronium bromide (YM155), a survivin suppressant, in combination with microtubule-targeting agents in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) has a poor prognosis compared to other subtypes, and effective treatment options are limited to cytotoxic agents, including microtubule-targeting agents, due to the lack of molecular targets. Here, we examined the combined effect of sepantronium bromide (YM155) and microtubule-targeting agents in TNBC models. The combination of YM155 with docetaxel showed synergistic antiproliferative and caspase 3/7-inducing effects in MRK-nu-1 and MDA-MB-453 human TNBC cell lines in vitro. YM155 also synergistically enhanced the efficacies of other microtubule-targeting agents, including paclitaxel and vinorelbine, which induced accumulation of survivin at the G2/M phase, whereas it did not affect the efficacy of doxorubicin. Combination treatment with YM155 and microtubule-targeting agents decreased the accumulation of survivin at the G2/M phase and induced greater apoptosis than either single agent alone. Further, combination treatment with YM155 and docetaxel also had a synergistic antitumor effect, achieving complete regression without exacerbation of body weight loss in all mice, in a MRK-nu-1 human TNBC xenograft model. These results suggest that survivin inhibition synergistically sensitize human TNBC cells to microtubule-targeting agents.

Sepantronium bromide (YM155) induces disruption of the ILF3/p54(nrb) 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.

Sepantronium bromide (YM155) enhances response of human B-cell non-Hodgkin lymphoma to rituximab.

In the treatment of B-cell non-Hodgkin lymphoma (B-NHL) rituximab improves long-term survival in combination with conventional chemotherapy. However, because the majority of patients with B-NHL eventually relapse, the development of more effective therapies is needed. Here, we evaluated the antitumor effects of a combination treatment involving sepantronium bromide (YM155), a first-in-class survivin suppressant, and rituximab in B-NHL xenograft mice models. To determine the efficacy of the combination treatment, YM155- and rituximab-treated B-NHL cell xenografted mice were monitored for tumor size and survival and subjected to 2'-deoxy-2'-(18)F-fluoro-D-glucose ((18)F-FDG) and 3'-(18)F-fluoro-3'-deoxythymidine ((18)F-FLT) positron emission tomography (PET) imaging. In addition, the cell proliferation status of excised tumors was examined by Ki-67 immunohistochemistry. In DB, WSU-DLCL-2, and Mino xenograft-bearing mice, the combination treatment of YM155 and rituximab induced significant tumor growth inhibition and tumor regression compared with either single agent. On day 3 after the initiation of treatment a significant decrease in both (18)F-FDG and (18)F-FLT tumor uptake from pretreatment levels was observed in combination treatment groups. The Ki-67 proliferation index was significantly decreased on day 3 in the xenograft models treated with combination treatment, suggesting that the combination of YM155 and rituximab reduced cell proliferation and glucose metabolism. Furthermore, compared with monotherapy, combination treatment prolonged survival times of severe combined immunodeficient mice with disseminated WSU-FSCCL and Jeko B-NHL tumors. Our findings demonstrate that YM155 and rituximab combination treatment enhances antitumor activity in B-NHL xenografts, and (18)F-FLT and (18)F-FDG PET imaging may allow the early functional evaluation of treatment responses in patients with B-NHL.

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% (GI(50) ) value was 15 nM. The mean GI(50) values of YM155 were 11 nM for p53 mut/null cell lines and 16 nM 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 (1-10 mg/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.

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.

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.

YM753, a novel histone deacetylase inhibitor, exhibits antitumor activity with selective, sustained accumulation of acetylated histones in tumors in the WiDr xenograft model.

Histone deacetylase (HDAC) inhibitors have been shown to have antitumor activity in vitro and in vivo. Various studies related to their antitumor activity and mechanism of action have been reported for HDAC inhibitors, but the relationship of their antitumor effects to their pharmacodynamic and pharmacokinetic properties in vivo has not ever fully characterized. We report here the discovery of a novel cyclic-peptide-based HDAC inhibitor, YM753. YM753 is a bacteria-derived natural product containing a disulfide bond. It potently inhibited HDAC enzyme with an IC50 of 2.0 nM in the presence of dithiothreitol. YM753 was rapidly converted to a reduced form in tumor cells, and then induced accumulation of acetylated histones, followed by p21WAF1/Cip1 expression, tumor cell growth inhibition and tumor-selective cell death. In an in vitro washout study, YM753 showed prolonged accumulation of acetylated histones in WiDr human colon carcinoma cells. In vivo YM753 dosing of mice harboring WiDr colon tumor xenografts significantly inhibited the tumor growth via sustained accumulation of acetylated histones in the tumor tissue. In a pharmacokinetic study, YM753 rapidly disappeared from the plasma, but its reduced form remained in the tumor tissue. Moreover, the accumulation of acetylated histones induced by YM753 was tumor tissue selective compared to several normal tissues. This study provides evidence that YM753 has antitumor activity that is the result of selective, sustained accumulation of acetylated histones in tumor tissues despite rapid disappearance of the drug from the plasma. These results suggest that the novel HDAC inhibitor, YM753 has attractive pharmacodynamic and pharmacokinetic properties giving it potential as an antitumor agent.

Hes1 and Hes5 control the progenitor pool, intermediate lobe specification, and posterior lobe formation in the pituitary development.

The pituitary gland is composed of two distinct entities: the adenohypophysis, including the anterior and intermediate lobes, and the neurohypophysis, known as the posterior lobe. This critical endocrine organ is essential for homeostasis, metabolism, reproduction, and growth. The pituitary development requires the control of proliferation and differentiation of progenitor cells. Although multiple signaling molecules and transcription factors are required for the proper pituitary development, the mechanisms that regulate the fate of progenitor cells remain to be elucidated. Hes genes, known as Notch effectors, play a crucial role in specifying cellular fates during the development of various tissues and organs. Here, we report that mice deficient for Hes1 and Hes5 display severe pituitary hypoplasia caused by accelerated differentiation of progenitor cells. In addition, this hypoplastic pituitary gland (adenohypophysis) lacks the intermediate lobe and exhibits the features of the anterior lobe only. Hes1 and Hes5 double-mutant mice also lack the neurohypophysis (the posterior lobe), probably due to incomplete evagination of the diencephalon. Thus, Hes genes control not only maintenance of progenitor cells but also intermediate vs. anterior lobe specification during the adenohypophysis development. Hes genes are also essential for the formation of the neurohypophysis.

The proneural bHLH genes Mash1, Math3 and NeuroD are required for pituitary development

Multiple signaling molecules and transcription factors are required for pituitary development. Activator-type bHLH genes Mash1, Math, NeuroD (Neurod) and Neurogenin (Neurog) are well known as key molecules in neural development. Although analyses of targeted mouse mutants have demonstrated involvement of these bHLH genes in pituitary development, studies with single-mutant mice could not elucidate their exact functions, because they cooperatively function and compensate each other. The aim of this study was to elucidate the roles of Mash1, Math3 and NeuroD in pituitary development. Mash1;Math3;NeuroD triple-mutant mice were analyzed by immunohistochemistry and quantitative real-time RT-PCR. Misexpression studies with retroviruses in pituisphere cultures were also performed. The triple-mutant adenohypophysis was morphologically normal, though the lumen of the neurohypophysis remained unclosed. However, in triple-mutant pituitaries, somatotropes, gonadotropes and corticotropes were severely decreased, whereas lactotropes were increased. Misexpression of Mash1 alone with retrovirus could not induce generation of hormonal cells, though Mash1 was involved in differentiation of pituitary progenitor cells. These data suggest that Mash1, Math3 and NeuroD cooperatively control the timing of pituitary progenitor cell differentiation and that they are also required for subtype specification of pituitary hormonal cells. Mash1 is necessary for corticotroph and gonadotroph differentiation, and compensated by Math3 and NeuroD. Math3 is necessary for somatotroph differentiation, and compensated by Mash1 and NeuroD. Neurog2 may compensate Mash1, Math3 and NeuroD during pituitary development. Furthermore, Mash1, Math3 and NeuroD are required for neurohypophysis development. Thus, Mash1, Math3 and NeuroD are required for pituitary development, and compensate each other.

Predicting response to sepantronium bromide (YM155), a survivin suppressant, by PET imaging with [11C]YM155.

INTRODUCTION: Sepantronium bromide (YM155) is a survivin suppressant that induces apoptosis in tumor cells. Although YM155 induces tumor regression in various tumor types in vivo, phase I and II studies demonstrated responding and non-responding patient populations. We investigated 11C-labeled YM155 ([11C]YM155) used as a positron emission tomography (PET) tracer to assess whether tumor uptake of [11C]YM155 correlated with its anti-tumor effect, thereby allowing identification of patients who would respond to YM155 treatment. METHODS: (1) Uptake of YM155 was measured in 39 human cancer cell lines in vitro using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). (2) In vivo tumor uptake was assessed in xenografted mice and total body distribution was evaluated in a cynomolgus monkey using [11C]YM155 with PET/computed tomography (CT) (mice) and PET (monkey) imaging. RESULTS: Intracellular uptake of YM155 in human cancer cell lines correlated well with its in vitro efficacy measured by GI50 (Pearson's r = -0.5709). Similarly, in vivo studies using tumor xenografted mice showed that tumors sensitive to YM155 demonstrated robust uptake of [11C]YM155, whereas insensitive tumors demonstrated low uptake. In the monkey, the biodistribution of [11C]YM155 indicated low accumulation in lung, breast, head, and neck and was only significant in organs involved with drug clearance: i.e. liver, kidneys, and bladder. CONCLUSIONS: Robust uptake of [11C]YM155 by a tumor appears to be a positive predictive marker for a good response to YM155. The findings suggest the potential utility of PET/CT imaging with [11C]YM155 for selection of patients whose tumors are likely to respond to YM155. ADVANCES IN KNOWLEDGE: YM155 efficacy correlated closely with its in vitro intracellular uptake and uptake on [11C]YM155 PET imaging. [11C]YM155 PET may predict tumor sensitivity to YM155. IMPLICATIONS FOR PATIENT CARE: The concept that tumor response can be accurately predicted prior to chemotherapy should be exploited to improve cancer treatment outcomes through judicious patient selection. The small molecule sepantronium bromide (YM155), a survivin suppressant, has been developed for the treatment of several cancers, including non-Hodgkin lymphoma, lung cancer, and breast cancer. The preferentially high in vitro uptake of YM155 by YM155-sensitive cancer cells and the high in vivo uptake of [11C]YM155 in YM155-sensitive tumors demonstrated by PET imaging suggest the potential utility of performing [11C]YM155 PET to allow the identification of patients with YM155-sensitive tumors.

Hes1 and Hes5 are required for differentiation of pituicytes and formation of the neurohypophysis in pituitary development.

The pituitary gland is a critical endocrine organ regulating diverse physiological functions, including homeostasis, metabolism, reproduction, and growth. It is composed of two distinct entities: the adenohypophysis, including the anterior and intermediate lobes, and the neurohypophysis known as the posterior lobe. The neurohypophysis is composed of pituicytes (glial cells) and axons projected from hypothalamic neurons. The adenohypophysis derives from Rathke's pouch, whereas the neurohypophysis derives from the infundibulum, an evagination of the ventral diencephalon. Molecular mechanisms of adenohypophysis development are much better understood, but little is known about mechanisms that regulate neurohypophysis development. Hes genes, known as Notch effectors, play a crucial role in specifying cellular fates during the development of various tissues and organs. Here, we report that the ventral diencephalon fails to evaginate resulting in complete loss of the posterior pituitary lobe in Hes1(-/-); Hes5(+/-) mutant embryos. In these mutant mice, progenitor cells are differentiated into neurons at the expense of pituicytes in the ventral diencephalon. In the developing neurohypophysis, the proliferative zone is located at the base of the infundibulum. Thus, Hes1 and Hes5 modulate not only maintenance of progenitor cells but also pituicyte versus neuron fate specification during neurohypophysis development.

Antitumor efficacy and biodistribution of liposomal sepantronium bromide (YM155), a novel small-molecule survivin suppressant.

Sepantronium bromide (YM155) exhibits time-dependent antitumor activity, although the plasma half-life of YM155 after a bolus intravenous (i.v.) administration is very short. Therefore, greater antitumor efficacy is obtained by continuous infusion than by bolus i.v. administration. In the present study, we attempted to liposomalize YM155 to obtain a longer circulation time than that achieved by bolus i.v. administration and yet retain sufficient antitumor activity. Encapsulation of YM155 in polyethylene glycol-coated liposomes extended the half-life of the drug, and high tumor accumulation of the drug was observed. Bolus i.v. administration of liposomal YM155 by a weekly administration regimen showed antitumor activity comparable to that obtained by the continuous infusion without severe toxicity in a murine xenograft model. Therefore, this liposomal formulation can be a new dosage form of YM155 that achieves sufficient efficacy and safety and is a more convenient administration regimen for users. It should be noted that liposomal YM155 showed unexpectedly high accumulation in the kidneys. This is a specific finding for liposomal YM155, offering important information for the consideration of the potential toxicity of liposomal YM155.

Combination of YM155, a survivin suppressant, with bendamustine and rituximab: a new combination therapy to treat relapsed/refractory diffuse large B-cell lymphoma.

PURPOSE: There remains an unmet therapeutic need for patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). The purpose of this study was to evaluate the therapeutic potential of sepantronium bromide (YM155), a survivin suppressant, in combination with either bendamustine or both bendamustine and rituximab using DLBCL models. EXPERIMENTAL DESIGN: Human DLBCL cell lines, DB, SU-DHL-8, and WSU-DLCL2, were treated with YM155 in combination with bendamustine. Cell viability, apoptosis induction, protein expression, and cell-cycle distribution were evaluated. Furthermore, antitumor activities of YM155, in combination with bendamustine or both bendamustine and rituximab, were evaluated in mice bearing human DLBCL xenografts. RESULTS: The combination of YM155 with bendamustine showed greater cell growth inhibition and sub-G1 population than either agent alone. YM155 inhibited bendamustine-induced activation of the ATM pathway and accumulation of survivin at G2-M phase, with greater DNA damage and apoptosis than either single agent alone. In a DLBCL DB murine xenograft model, YM155 enhanced the antitumor activity of bendamustine, resulting in complete tumor regression without affecting body weight. Furthermore, YM155 combined with bendamustine and rituximab, decreased FLT-PET signals in lymph nodes and prolonged overall survival of mice bearing disseminated SU-DHL-8, an activated B-cell-like (ABC)-DLBCL xenografts when compared with the combination of either rituximab and bendamustine or YM155 with rituximab. CONCLUSIONS: These results support a clinical trial of the combination of YM155 with bendamustine and rituximab in relapsed/refractory DLBCL.

Combination of YM155, a survivin suppressant with a STAT3 inhibitor: a new strategy to treat diffuse large B-cell lymphoma.

Survivin and STAT3 pathway have been reported to be important for the growth of diffuse large B-cell lymphoma (DLBCL) cells. Here we investigated the antitumor activity of sepantronium bromide (YM155), a survivin suppressant, in combination with STAT3 inhibitors in DLBCL cell lines in vitro. YM155 synergistically enhanced STAT3 inhibitors (AG490 and STA-21)-induced apoptosis in DLBCL cell lines. Moreover, rituximab, which shows inhibitory activity against STAT3, also sensitized DLBCL cell lines to YM155 regardless of sensitivity to rituximab. These results suggest that combining the inhibition of survivin with STAT3 pathway is an attractive and potentially effective way for the treatment of DLBCL.

Radiosynthesis, biodistribution and imaging of [11C]YM155, a novel survivin suppressant, in a human prostate tumor-xenograft mouse model.

INTRODUCTION: Sepantronium bromide (YM155) is an antitumor drug in development and is a first-in-class chemical entity, which is a survivin suppressant. We developed a radiosynthesis of [(11)C]YM155 to non-invasively evaluate its tissue and tumor distribution in mice bearing human prostate tumor xenografts. METHODS: Methods utilizing [(11)C]acetyl chloride and [(11)C]methyl triflate, both accessible with automated radiosynthesis boxes, were evaluated. The O-methylation of ethanolamine-alkolate with [(11)C]methyl triflate proved to be the key development toward a rapid and efficient process. The whole-body distribution of [(11)C]YM155 in PC-3 xenografted mice was examined using a planar positron imaging system (PPIS). RESULTS: Sufficient quantities of radiopharmaceutical grade [(11)C]YM155 were produced for our PET imaging and distribution studies. The decay corrected (EOB) radiochemical yield was 16-22%, within a synthesis time of 47 min. The radiochemical purity was higher than 99%, and the specific activity was 29-60 GBq/µmol (EOS). High uptake levels of radioactivity (%ID/g, mean±SE) were observed in tumor (0.0613±0.0056), kidneys (0.0513±0.0092), liver (0.0368±0.0043) and cecum (0.0623±0.0070). The highest tumor uptake was observed at an early time point (from 10 min after) following injection. Tumor-to-blood and tumor-to-muscle uptake ratios of [(11)C]YM155, at 40 min after injection, were 26.5 (±2.9) and 25.6 (±3.6), respectively. CONCLUSION: A rapid method for producing a radiopharmaceutical grade [(11)C]YM155 was developed. An in vivo distribution study using PPIS showed high uptake of [(11)C]YM155 in tumor tissue. Our methodology may facilitate the evaluation and prediction of response to YM155, when given as an anti-cancer agent.

Overcoming erlotinib resistance in EGFR mutation-positive non-small cell lung cancer cells by targeting survivin.

Loss of PTEN was recently shown to contribute to resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) in EGFR mutation-positive non-small cell lung cancer (NSCLC) through activation of the protein kinase AKT. We previously showed that downregulation of the expression of the antiapoptotic protein survivin by EGFR-TKIs contributes to EGFR-TKI-induced apoptosis in EGFR mutation-positive NSCLC cells. We have now investigated the role of survivin expression in EGFR-TKI resistance induced by PTEN loss. The EGFR-TKI erlotinib did not affect survivin expression or induce apoptosis in EGFR mutation-positive NSCLC cells with PTEN loss. Downregulation of survivin either by transfection with a specific short interfering RNA or by exposure to the small-molecule survivin suppressor YM155 reversed erlotinib resistance in such cells in vitro. Furthermore, combination therapy with YM155 and erlotinib inhibited the growth of tumors formed by EGFR mutation-positive, PTEN-deficient NSCLC cells in nude mice to a greater extent than did treatment with either drug alone. These results thus indicate that persistent activation of signaling by the AKT-survivin pathway induced by PTEN loss underlies a mechanism of resistance to erlotinib-induced apoptosis in EGFR mutation-positive NSCLC. They further suggest that the targeting of survivin has the potential to overcome EGFR-TKI resistance in EGFR mutation-positive NSCLC.

Suppression of survivin promoter activity by YM155 involves disruption of Sp1-DNA interaction in the survivin core promoter.

YM155, a novel survivin suppressant, shows potent antitumor activity against various human cancers and is currently in phase II clinical trials. In this study, we investigated whether YM155 selectively inhibits survivin transcription. We hypothesize that inhibition of survivin transcription plays a role in YM155-mediated survivin inhibition. We found that YM155 inhibited survivin promoter activity, while it showed minimal inhibitory effect on four control gene promoters in transfection and luciferase activity assay experiments, indicating its selectivity. Transfection of various survivin promoter-luciferase constructs followed by luciferase assays revealed that the survivin core promoter (269 bp) plays a major role in YM155-mediated inhibitory effects. However, flow cytometry analysis indicated that inhibition of survivin promoter activity by YM155 is cell cycle-independent without G1 cell arrests. Electrophoretic mobility shift assays (EMSA) identified that YM155 abrogates nuclear proteins binding to the region of -149 to -71, in which Sp1 is a major candidate, and that YM155 treatment induces Sp1 re-subcellular localization without inhibiting its expression. Forced expression of Sp1 neutralized YM155-mediated downregulation of survivin promoter activity. Consistently, mutation of the identified Sp1 sites in the oligonucleotide probe diminished DNA-protein interactions in EMSA experiments, and mutation of the Sp1 sites in the survivin promoter-luciferase construct diminished survivin promoter activity. These findings indicate that YM155 inhibition of survivin expression is at least in part through its inhibition of survivin transcription by disruption of Sp1 interaction with the region of -149 to -71 in the survivin core promoter.

Antitumor effects of YM155, a novel survivin suppressant, against human aggressive non-Hodgkin lymphoma.

YM155, a novel small-molecule that down-regulates survivin, exhibits broad, potent antitumor activity against a range of human tumors. We evaluated the activity of YM155 in aggressive non-Hodgkin lymphoma. In a number of diffuse large B-cell lymphoma lines, YM155 exhibited 50% growth inhibition with values between 0.23 and 3.9 nM. Within in vivo xenograft models, continuous infusion of YM155 eradicated large, established subcutaneous WSU-DLCL-2 and Ramos tumors, with sustained efficacy observed through 4 cycles of YM155 therapy. YM155 increased survival significantly versus rituximab in disseminated Ramos models. This study suggests that YM155 may represent an effective treatment for aggressive lymphomas.