Small-molecule Survivin Suppressant Research Articles

D-a-Tocopheryl Polyethylene Glycol 1000 Succinate and a small-molecule Survivin suppressant synergistically induce apoptosis in SKBR3 breast cancer cells

Breast cancer is the second in mortality rate malignancy among women. Despite the many advances in breast cancer treatment, there is still a need to improve drug efficacy and reduce non-specific effects. D-alpha-tocopheryl polyethylene glycol succinate (TPGS) is frequently used in the development of drug delivery systems to improve the pharmacokinetics of anti-cancer drugs and reduce multi-drug resistance. We have previously shown that TPGS not only acts as a carrier molecule but also exerts anti-cancer effects. As part of this study, we investigated the effect of TPGS with YM155, a small molecule suppressant of Survivin, in various breast cancer cell lines representing different subtypes of the disease. We aimed to evaluate the presumed synergistic effect of the TPGS-YM155 combination and reveal its mechanism of action. Our results show that the TPGS-YM155 combination acts synergistically to reduce specifically the viability of SKBR3 cells. The combination of these agents reduced activation of the AKT pathway, decreased Survivin and Bcl-2 levels, and induced caspase-dependent and independent apoptosis via the mitochondrial pathway. Importantly, the TPGS-YM155 combination did not significantly affect the viability of MCF-10A normal immortalized cells. In conclusion, the combination of YM155 and TPGS could be a promising approach against SKBR3-type breast cancer.

TAT-IL-24-KDEL-induced apoptosis is inhibited by survivin but restored by the small molecular survivin inhibitor, YM155, in cancer cells.

Interleukin-24 (IL-24) is a cytokine belonging to the IL-10 gene family. This cytokine selectively induces apoptosis in cancer cells, without harming normal cells, through a mechanism involving endoplasmic reticulum (ER) stress response. TAT-IL-24-KDEL is a fusion protein that efficiently enters the tumor cells and locates in the ER. Here we report that TAT-IL-24-KDEL induced apoptosis in human cancer cells, mediated by the ER stress cell death pathway. This process was accompanied by the inhibition of the transcription of an antiapoptotic protein, survivin. The forced expression of survivin partially protected cancer cells from the induction of apoptosis by TAT-IL-24-KDEL, increased their clonogenic survival, and attenuated TAT-IL-24-KDEL-induced activation of caspase-3/7. RNA interference of survivin markedly sensitized the transformed cells to TAT-IL-24-KDEL. Survivin was expressed at higher levels among isolated clones that resistant to TAT-IL-24-KDEL. These observations show the important role of survivin in attenuating cancer-specific apoptosis induced by TAT-IL-24-KDEL. The pharmacological inhibition of survivin expression by a selective small-molecule survivin suppressant YM155 synergistically sensitized cancer cells to TAT-IL-24-KDEL-induced apoptosis in vitro and in vivo. The combined regimen caused significantly higher activation of ER stress and dysfunction of mitochondria than either treatment alone. As survivin is overexpressed in a majority of cancers, the combined TAT-IL-24-KDEL and YM155 treatment provides a promising alternative to the existing therapies.

A multicenter phase II study of sepantronium bromide (YM155) plus rituximab in patients with relapsed aggressive B-cell Non-Hodgkin lymphoma

This phase II study evaluated YM155, a novel small-molecule survivin suppressant, in combination with rituximab in patients with relapsed aggressive B-cell non-Hodgkin lymphoma (NHL) who failed or were not candidates for autologous stem cell transplant (ASCT). During 14-day cycles, 41 patients received YM155 (5mg/m2/d) by continuous intravenous (IV) infusion for 168 hours (day 1–7), and rituximab (375mg/m2) IV on days 1 and 8 during cycles 1–4 and repeated for 4 cycles every 10 cycles. Forty patients (97.6%) had prior rituximab and 15 patients (36.6%) prior ASCT. Most frequent grade 3–4 adverse events were neutropenia (19.5%) and thrombocytopenia (12.2%). In the per-protocol set (n = 34), objective response rate was 50% and median progression-free survival 17.9 months. Median overall survival was not reached at study termination (median follow-up, 23 months). YM155 in combination with rituximab was tolerable with encouraging antitumor activity and durable responses in relapsed aggressive B-cell NHL patients.

Abstract B21: Use of polymeric nanoparticles for the delivery of YM155 to glioma cells in vitro and in vivo

Abstract YM155 (Sepantronium Bromide) is a potent survivin inhibitor that is the subject of clinical trials for multiple types of cancer including melanoma, lypmphoma, lung cancer, prostate and breast cancer. YM155 is a therapeutic candidate for the treatment of glioblastoma (GBM), however, its rapid plasma clearance and minimal blood-brain barrier penetration are obstacles to clinical translation (1). The encapsulation of drugs in nanoparticles can help increase drug circulation time by reducing clearance, and nanoparticles have the ability to increase drug concentration in the tumor through the enhanced permeability and retention effect (2). Here, we encapsulated YM155 in poly(lactic acid)-b-poly(ethylene glycol) nanoparticles (NPs) and evaluated the potency of YM155 in 4 glioma cell lines in vitro. Additionally, we investigated the ability of systemically administered NPs to inhibit tumor growth in vivo in mice bearing orthotopic gliomas. In vitro potency of YM155 was measured by CellTiter Glo in 3 immortalized GBM lines (GL261, U118, U87), as well as in patient derived tumor cells (GB7). Tumor growth inhibition was evaluated in C57 albino mice bearing orthotopic GL261-luc2 tumors. Mice were treated for 3 consecutive days with saline, free YM155 or NPs by tail vein injection, and tumor growth was measured by bioluminescence using an IVIS imaging system. All cells tested were sensitive to both free and encapsulated YM155. GL261 was the least sensitive of the cells tested, with an IC50 of 196 nM, while U118 and U87 had IC50s of 61 and 32 nM, respectively. The patient derived GB7 cells were the most sensitive to YM155, with an IC50 of 22 nM. Tumors grew exponentially for mice treated with free YM155 or saline, whereas tumor growth was halted in 2/5 subjects after one cycle of treatment with NPs. This work shows the potency of YM155 against multiple glioma cell lines in vitro. In vivo, free YM155 does not affect the growth of an orthotopic glioma; however, our initial treatment study provides evidence systemically administered YM155-loaded NPs allow for the delivery of a therapeutically relevant dose to orthotopic GBM. Success of this work shows the ability to encapsulate a water soluble drug in polymeric nanoparticles, which can be used to generate new options in the treatment of GBM. Further work will evaluate drug efficacy in additional patient derived cells and investigate the ability of NP encapsulated YM155 alone or in conjunction with radiation to treat mice bearing patient derived xenograft tumors.

Antitumor effect of YM155, a novel small-molecule survivin suppressant, via mitochondrial apoptosis in human MFH/UPS.

Survivin is a member of the inhibitor of apoptosis family, which is known to inhibit mitochondrial apoptosis. Survivin is highly expressed in cancers and plays an important role in cancer cell survival, and increased survivin expression is an unfavorable prognostic marker in cancer patients. YM155, a novel small-molecule survivin suppressant, selectively suppresses survivin expression, resulting in the induction of apoptosis in various malignancies. However, the roles of survivin in human malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma (MFH/UPS) have not been studied. In the present study, we examined survivin expression in human musculoskeletal tumor tissues, and the effect of survivin inhibition by siRNA or YM155 on apoptotic activity in human MFH/UPS cell lines. In tumor tissues, mRNA expression of survivin was significantly higher in MFH/UPS samples than in benign schwannomas. Moreover, in vitro studies revealed that both survivin siRNA and YM155 suppressed survivin expression and inhibited MFH/UPS cell proliferation in a dose- and a time-dependent manner. Further, the numbers of apoptotic cells significantly increased with YM155 treatment. In vivo, tumor volume in YM155-treated groups was significantly reduced without significant bodyweight loss. Increased apoptotic activity along with decreased survivin expression was also observed in YM155-treated tumors. The findings in this study strongly suggest that survivin suppressants, including YM155, contribute to the suppression of human MFH/UPS cell growth via promoting mitochondrial apoptosis, and that survivin may be a potent therapeutic target for the novel treatment of human MFH/UPS.

The over-expression of survivin enhances the chemotherapeutic efficacy of YM155 in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. The inability of chemotherapeutic drugs to selectively target HCC tumor cells because of their predominant resistant phenotype to most conventional anticancer agents bestows a major obstacle for the clinical management of HCC. In this report, we have examined and demonstrated the remarkable heterogeneity of expression of survivin and its phosphorylated active form (p-survivin) in HCC patients' tissues and cell lines. Furthermore, the expression of survivin and p-survivin in HCC cell lines was found to be associated with response to the small-molecule survivin suppressant YM155. Therefore, in the HCC cell lines that express elevated level of survivin and p-survivin, YM155 efficiently inhibited their proliferation, induced cell cycle arrest and apoptosis resulting in DNA damage through the dysregulation of cell-cycle checkpoint-related regulatory genes. Importantly, YM155 yielded significantly better therapeutic effect than sorafenib when tested in an orthotopic mouse model using patient-derived HCC xenografts with elevated survivin and p-survivin expression. Our results clearly demonstrated that the level of survivin and p-survivin expression could serve as molecular predictive biomarkers to select potential YM155-responsive patients, in a move towards delivering precision medicine for HCC patients.

Abstract 4369: The microRNA-218-survivin axis regulates cervical cancer cell migration and invasion

Abstract MicroRNA (miR)-218 down-regulation has been reported in numerous human malignancies. In cervical cancer, we identified that lower miR-218 expression was significantly associated with poorer overall survival, disease-free survival, and pelvic/para-aortic lymph node recurrence. Further analyses of cervical cancer data from The Cancer Genome Atlas (TCGA) identified that this down-regulation was associated with a genomic locus loss (hsa-mir-218-1:4p15.31, hsa-mir-218-2:5q34, n=105). The objective of the current study was to elucidate the cellular and molecular functions of miR-218. MiR-218 transfection into cervical cancer cells (SiHa and ME-180) significantly reduced cell migration (by 66% and 89%, respectively), invasion (by 49% and 67%, respectively), and clonogenic capacity (by 42% and 53%, respectively), relative to control-transfected cells (P<0.05). In order to identify clinically relevant miR-218 target genes, we used an integrated trimodal approach, incorporating DNA microarray (Affymetrix Human Genome U133 Plus 2.0) analyses of 79 clinical samples, miR-218 transfection, and miRDB target prediction. The most significant target was survivin (BIRC5); miR-218 transfection confirmed a reduction in survivin mRNA and protein expression in both SiHa and ME-180 cells. Furthermore, a direct interaction between the survivin-3′UTR and miR-218 was validated using a luciferase reporter assay. siRNA knockdown of survivin in SiHa and ME-180 significantly reduced cell migration (by 76% and 83%, respectively), invasion (by 79% and 88%, respectively), and clonogenic capacity (by 98% and 97%, respectively), relative to control cells (P<0.05). YM155, a small-molecule survivin suppressant, effectively reduced survivin mRNA and protein levels in a concentration- and time-dependent manner. This compound correspondingly decreased cervical cancer cell proliferation and clonogenic survival. Our results suggest that the miR-218-survivin axis plays an important role in cervical cancer progression. Citation Format: Ryunosuke Kogo, Christine How, Jeff Bruce, Willa Shi, Kenneth W. Yip, Laurie Ailles, Fei-Fei Liu. The microRNA-218-survivin axis regulates cervical cancer cell migration and invasion. [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 4369. doi:10.1158/1538-7445.AM2014-4369

Survivin family proteins as novel molecular determinants of doxorubicin resistance in organotypic human breast tumors.

IntroductionThe molecular determinants of breast cancer resistance to first-line anthracycline-containing chemotherapy are unknown.MethodsWe examined the response to doxorubicin of organotypic cultures of primary human breast tumors ex vivo with respect to cell proliferation, DNA damage and modulation of apoptosis. Samples were analyzed for genome-wide modulation of cell death pathways, differential activation of p53, and the role of survivin family molecules in drug resistance. Rational drug combination regimens were explored by high-throughput screening, and validated in model breast cancer cell types.ResultsDoxorubicin treatment segregated organotypic human breast tumors into distinct Responder or Non Responder groups, characterized by differential proliferative index, stabilization of p53, and induction of apoptosis. Conversely, tumor histotype, hormone receptor or human epidermal growth factor receptor-2 (HER2) status did not influence chemotherapy sensitivity. Global analysis of cell death pathways identified survivin and its alternatively spliced form, survivin-ΔEx3 as uniquely overexpressed in Non Responder breast tumors. Forced expression of survivin-ΔEx3 preserved cell viability and prevented doxorubicin-induced apoptosis in breast cancer cell types. High-throughput pharmacologic targeting of survivin family proteins with a small-molecule survivin suppressant currently in the clinic (YM155) selectively potentiated the effect of doxorubicin, but not other chemotherapeutics in breast cancer cell types, and induced tumor cell apoptosis.ConclusionsSurvivin family proteins are novel effectors of doxorubicin resistance in chemotherapy-naive breast cancer. The incorporation of survivin antagonist(s) in anthracycline-containing regimens may have improved clinical activity in these patients.

The Survivin Suppressant YM155 Overcomes Microenvironment-Mediated Immune Resistance In a Humanized Multiple Myeloma Mouse Model

BackgroundDurable tumor regression can be achieved in Multiple Myeloma (MM) patients by immunotherapeutic approaches, such as allogeneic stem cell transplantation and donor lymphocyte infusion. However, the low rate of patients, who reach a sustained remission, impedes the success of this therapeutic approach. Currently, much emphasis is placed on the role of the tumor microenvironment in tumor cell immune escape. We previously described that different accessory cells of the microenvironment significantly inhibited the lysis of MM cell lines by T cells in a cell-cell contact dependent manner. Further analysis revealed that this immune escape could be attributed to the induction of a cell adhesion-mediated immune resistance (CAM-IR) of MM against T cell lysis in vitro. Further in vitro studies identified up-regulation of survivin in the MM cells as one of the potential mechanisms of immune resistance. Moreover, co-culture with YM155, a small molecule survivin suppressant, could abrogate accessory cell induced resistance in vitro. Methods and ResultsWe investigated the influence of a human bone marrow microenvironment on the anti tumor activity of T cell therapy in a human-mouse hybrid model (Groen et al. Blood 2012). Immune-deficient RAG2-/- γc-/- -mice were implanted subcutaneously either with uncoated scaffolds or scaffolds coated with human mesenchymal stromal cells (MSC), which generates a human bone marrow environment. Luciferase transduced MM cell line UM9 injected directly in both types of scaffolds grew into MM tumors and were treated with Myeloma reactive minor Histocompatibility antigen specific T cell clones. The T cells induced effective anti-myeloma responses against tumors developed in the uncoated scaffolds. In contrast, and consistent with the in vitro results, no anti-tumor effect was observed in the MSC coated scaffolds. Thus confirming in vivo a microenvironment induced resistance of MM cells to T cell kill. We next investigated if the resistance could be abrogated by combination therapy with T cells and YM155.To this end, mice with tumors in human MSC coated scaffolds were treated with T cells alone, YM155 alone, or the combination of both. YM155- nor T cell-monotherapy induced tumor regression on evaluation of tumor growth based on bioluminescent signal. Moreover, the combination of YM155 with T cells demonstrated a substantial anti-tumor effect. ConclusionYM155 treatment can sensitize myeloma cells to T cell mediated anti-tumor effects and more importantly can overcome microenvironment-mediated resistance of MM cells to T cell treatment. These findings support further development of CAM-IR modulating agents in combination with immune therapy. Disclosures:Minnema:Janssen Cilag: Consultancy, Honoraria. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

The Survivin Suppressant YM155 (Sepantronium Bromide) Has Potent Pre-Clinical Activity Against Pediatric Acute Myeloid Leukemia

Despite aggressive chemotherapy and a high remission induction rate, approximately one third of children with acute myeloid leukemia (AML) relapse. Therefore, novel and more effective treatments are urgently needed. Survivin is an inhibitor-of-apoptosis protein that plays a key role in regulating cell division, proliferation and apoptosis (Altieri et al, Nat Rev Cancer, 2008). Furthermore, high expression of survivin and its splice variants have been shown to be associated with poor clinical outcome in AML (Moore et al, ASH Abstract 3555, 2011; Carter et al, Blood, 2012). The small-molecule survivin suppressant YM155 (sepantronium bromide) has been demonstrated to have pre-clinical activity against a range of solid cancers and leukemias, although data in AML is limited, particularly in pediatric models. Therefore, we undertook a comprehensive pre-clinical evaluation of YM155 in AML, with a focus on pediatric disease.When tested in vitro against a diverse panel of 9 AML cell lines, YM155 potently inhibited cell viability with a median IC50 of 0.03 µM (range 0.001 - 0.680 µM; Table 1). All 4 pediatric cell lines tested (Kasumi-1, MV4-11, THP-1 and CMK) were particularly sensitive to YM155, with IC50 values in the range of 0.010 - 0.053 µM. Of note, YM155 had generally similar in vitro efficacy to daunorubicin, but was more potent than cytarabine in 7 of the 9 cell lines.Table 1In vitro sensitivity of AML cell lines to YM155, cytarabine and daunorubicin, as measured by 72 hour cell viability (resazurin reduction) assays. Values indicate median IC50(µM) from at least 3 independent experiments. *Pediatric AML cell lines. NS, not significant.AML Cell LineCharacteristic FeatureYM155CytarabineDaunorubicinP value (YM155 vs. Cytarabine)HL-60Complex karyotype0.0010.4810.019NSKasumi-1*t(8;21)(q22;q22)0.0100.1120.0220.011RUNX1-CBFA2T1c-KITmut(N822K)ML-2t(6;11)(q27;q23)0.0100.0620.0050.027OCI/AML3NPM1mut0.011>100.020<0.001MV4-11*t(4;11)(q21;q23)0.0301.2630.004<0.001MLL-AF4FLT3-ITDTHP-1*t(9;11)(p21;q23)0.0367.0440.027<0.001CMK*AML assoc. with Down Syndrome0.0530.4280.078<0.001HELAML M60.5310.0780.1310.011ME-1inv(16)(p13q22)0.6800.3900.298NSCBFB-MYH11Cell cycle analyses demonstrated concentration-dependent increases in the sub-G1 fraction of cell lines treated with YM155, suggestive of apoptosis. An apoptotic mechanism of cell death was confirmed by an increase in annexin V positivity, measured by flow cytometry. Consistent with the proposed mechanism of action, YM155 treatment also caused down-regulation of total survivin protein expression in AML cell lines.Furthermore, in vitro assays demonstrated that YM155 was cytotoxic against a panel of 7 primary, diagnostic bone marrow samples from children with AML (median age 4.8 years, range 1.3-15.7). Patient samples were obtained from the Queensland Children's Tumour Bank and had diverse cytogenetic and molecular characteristics, including CBF-AML, monosomy 7, MLL gene rearrangements and FLT3-ITD. Similar to experiments in established cell lines, YM155 treatment caused an apoptotic response, as evidenced by induction of annexin V positivity.In conclusion, YM155 has potent pre-clinical activity against a broad panel of AML cell lines and patient samples. Pediatric AML appears to be particularly sensitive to YM155. These data suggest that YM155-mediated inhibition of survivin is a potentially beneficial therapeutic strategy for AML, particularly childhood disease, and warrants further pre-clinical and clinical evaluation. Disclosures:No relevant conflicts of interest to declare.

Phase II randomized, open-label study of YM155 (sepantronium bromide) plus docetaxel versus docetaxel alone as first-line treatment for HER2 negative metastatic breast cancer.

548 Background: YM155 (YM) is a small molecule survivin suppressant. In a phase I/II study of YM plus docetaxel (D) in solid tumors evidence of anti-tumor activity was observed in women with human epidermal growth factor 2 non-overexpressing (HER2 negative) metastatic breast cancer (mBC). Methods: This was a randomized study of YM plus D versus D as 1st line treatment in subjects with HER2 negative mBC. Eligibility criteria were: ECOG &lt; 1, no prior chemotherapy for mBC, and at least one measurable lesion. Primary endpoint was progression free survival (PFS); secondary endpoints were: objective response rate (ORR), overall survival (OS), duration of response (DOR), clinical benefit rate (CBR), time to response (TTR) and safety. YM was administered at 5 mg/m2/day as a 168 hr continuous infusion followed by 14 Day (d) observation and D was administered at 75 mg/m2over 1 hr on d1 every 21d. In the control arm, D was dosed per investigator choice q 21d. Results: 101 subjects were randomized (50 YM + D; 51 D). Median (m) age 55 (range: 25 – 79), 25% had triple negative disease, &gt; 60% had bone and lymph mets, 86% had prior therapy for BC. mPFS (days) was 251 (95%CI: 176 – 333) YM + D vs 252 (95%CI: 202-433) D (p=0.34). ORR, CBR and TTR (YM+D; D): 26% vs. 25.5%; 82% vs. 84.3% and 45 vs 59 d. OS data are immature but showed no difference (p=0.911). Adverse events [AEs (&gt; 25%)] [YM + D% vs D %]: neutropenia 83 vs 84, alopecia 62.5 vs 53, fatigue 50 vs 41.2, nausea 35.4 vs 41.2, leucopenia 27 vs 33 and dyspnoea 33 vs 14. Common (&gt;10%) serious AEs [YM + D% vs D%]: febrile neutropenia 21 vs 8 and neutropenia 10 vs 8. Conclusions: Preclinical and clinical evidence suggested the combination of YM + D may offer additional benefit to D alone in subjects with mBC. This study showed no difference in efficacy, but the combination appeared to be well tolerated. Clinical trial information: NCT01038804.

Markedly additive antitumor activity with the combination of a selective survivin suppressant YM155 and alemtuzumab in adult T-cell leukemia

AbstractAdult T-cell leukemia (ATL) is an aggressive malignancy of CD4+CD25+ lymphocytes caused by human T-cell lymphotropic virus type 1. Currently, there is no accepted curative therapy for ATL. In gene expression profiling, the antiapoptotic protein survivin (BIRC5) demonstrated a striking increase in ATL, and its expression was increased in patient ATL cells resistant to the anti-CD52 monoclonal antibody alemtuzumab (Campath-1H). In this study, we investigated the antitumor activity of a small-molecule survivin suppressant YM155 alone and in combination with alemtuzumab in a murine model of human ATL (MET-1). Both YM155 alone and its combination with alemtuzumab demonstrated therapeutic efficacy by lowering serum soluble IL-2Rα (sIL-2Rα) levels (P < .001) and prolonged the survival of tumor-bearing mice (P < .0001). Moreover, the combination of YM155 with alemtuzumab demonstrated markedly additive antitumor activity by significantly lowering serum sIL-2Rα levels and improving the survival of leukemia-bearing mice compared with monotherapy with either YM155 (P < .001) or alemtuzumab (P < .05). More significantly, all mice that received the combination therapy survived and were tumor free >6 months after treatment. Our data support a clinical trial of the combination of YM155 with alemtuzumab in ATL. This trial was registered at www.clinicaltrials.gov as #NCT00061048.

YM155, a Small-Molecule Survivin Suppressant, Mainly Targets Primary CLL Cells Actively Proliferating and Overcomes Microenvironment-Mediated CLL Cell Protection

Abstract 3868Chronic lymphocytic leukemia (CLL) has been historically considered an accumulative disease. However, recent studies demonstrate the relevance of proliferation centers found in lymphoid organs (lymph nodes [LN] and bone marrow [BM]),which provide survival and proliferation signals to CLL cells. In CLL, immunohistochemical analyses show that survivin is mainly expressed in CLL cells that are actively proliferating in proliferation centers. Interestingly, increased expression of survivin in peripheral blood CLL cells correlates with worse response to treatment. YM155 (Astellas®), a survivin suppressant, has demonstrated promising antitumoral activity in a wide variety of human cancer cell lines and xenograft models, and nowadays it is being evaluated in several phase II clinical trials. As survivin is usually expressed in proliferating cells, herein we tested the effects of YM155 on CLL cells co-cultured in a system mimicking the conditions that they are encountering in the proliferation centers. Primary CLL cells from 20 patients were co-cultured with BM stromal cells along with CD40 ligand and oligodeoxynucleotides-CpG. Optimal proliferative response was observed after 48 hours of co-culture (mean % Ki-67 expression, 1.52±0.06 in cells in suspension vs. 11.78±6.73 in co-culture, p<0.001). Therefore, CLL cells were subsequently treated with YM155 and/or fludarabine at this time point. By Western Blot, increasing survivin expression in co-culture, was completely abrogated by treatment with YM155 50nM for 24 hours. Induction of apoptosis was assessed by flow cytometry (FC) measurement of annexin V and propidium iodide (PI). Mean LD50 of YM155 was 430.4 nM (95%CI 227.3–815.3 nM) for CLL cells cultured in suspension, whereas it was 7.7 times lower (56.25nM, 95%CI 39.71–79.7 nM) in proliferative conditions, revealing that apoptotic cell death induced by inhibition of survivin is mainly affecting proliferating CLL cells. Unlike treatment with YM155, CLL cells treated with 5μg/ml fludarabine showed significantly higher viability in co-culture than in suspension (normalized mean of viability: 60.2%±16.9% vs. 23.4%±7.1%, p<0.001), thus confirming the microenvironment-induced chemoresistance. Of note, the addition of 50nM YM155 overcame this chemoresistance by significantly increasing fludarabine-induced cytotoxicity (normalized mean of viability: 42.1%±7.6% for fludarabine vs. 5.2%±1.9% for the combination; p<0.05). Prior to any treatment, 98.54%±0.12% of cells in suspension were arrested at G0/G1, whereas the co-culture increased the number of CLL cells in S/G2/M phase from 0.89%±0.31% on day 0 to 44.96%±23% at 72 hours (p=0.05). The addition of fludarabine, YM155, or the combination of both drugs after 48 hours of co-culture induced a significant G1 arrest at 72 hours (% cells in G1 phase: control, 55.04%±13.28%; fludarabine, 88.26%±7.94%; YM155, 81.61%±8.47%; combination, 90.75±4.68; p<0.01). Finally, YM155 treatment caused a drastic reduction of 23.31%±9.52% in Ki67 expression, whereas the combination of YM155 with fludarabine caused a significant decrease of 49.64%±4.12% (p<0.001), an effect that fludarabine alone could not achieve. Following this, we also analyzed the effect of YM155 on the proliferative compartment defined by Calissano, C et al (Mol Med, 2011; CD5high/CXCR4dim). Firstly, we observed that the co-culture induced an increase in this fraction (from 3.28%±1.12% to 9.66%±2.83%, p<0.05). Treatment with YM155 alone or combined with fludarabine reduced this proliferative fraction (control: 13.29%±4.21%; YM155: 5.82%±1.24%[p<0.05]; combination: 4.12%±1.14%[p<0.01]), whereas the proportion of the non-proliferative compartments where not affected by these treatments. Altogether, these data show that reproducing signals from the microenvironment in ex vivo co-cultures can promote proliferation and protect CLL cells from apoptosis induced by cytotoxic agents such as fludarabine. In this setting, YM155 treatment is able to efficiently overcome microenvironment-mediated cell protection and proliferation and has specific effect on actively proliferating CLL cells. Combination experiments revealed that YM155 strongly potentiates fludarabine cytotoxicity, especially in proliferating CLL cells chemoresistants to fludarabine alone. Finally, these results open the door to future clinical trials inhibiting survivin in CLL. Disclosures:No relevant conflicts of interest to declare.

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 p54nrb binds to the survivin promoter and regulates survivin expression. p54nrb forms a complex with ILF3, which directly binds to YM155. YM155 induces disruption of the ILF3/p54nrb complex, which results in a different subcellular localization between ILF3 and p54nrb. 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.

Abstract 2749: YM155, a small molecule survivin suppressant, sensitizes human B-cell non-Hodgkin lymphoma to rituximab-induced apoptosis

Abstract Survivin is a member of the inhibitor of apoptosis (IAP) family of proteins, and is highly expressed in various tumor types. Given its preferential expression in tumor cells, and its ability to block apoptosis and regulate cancer cell proliferation, survivin appears to be an attractive target for cancer therapy. YM155 is a small molecule survivin suppressant and shows in vitro antiproliferative activity against the broad spectrum of human cancer cell lines, including B-cell non-Hodgkin lymphoma (B-NHL). The continuous infusion of YM155 induced the tumor regression in B-NHL xenograft mice models. Furthermore, our recent study indicated that the combination treatment of YM155 and rituximab induced significant tumor growth inhibition and tumor regression without weight loss. In the present study, we examined the in vitro combination efficacy of YM155 and rituximab, and its underlying signaling pathways involved in the combination effect of YM155 and rituximab. The combination of YM155 at 1 nmol/L and rituximab at 2 ug/mL induced higher sub G1 compared with each single treatment in WSU-DLCL-2 and SU-DHL-4. Rituximab at 2ug/mL decreased phospho-STAT3 level, meanwhile, did not decreased phospho-p38 MAPK and phospho-AKT levels in WSU-DLCL-2 and SU-DHL4. To further characterize the growth inhibitory effect of inactivation of STAT3 pathway, we investigated the cell growth inhibition by AG490, a JAK2 inhibitor and STA-21, a STAT3 dimerization inhibitor in WSU-DLCL-2 and SU-DHL4. The treatment of AG-490 or STA-21 decreased cell viability in dose-dependent manner. Furthermore, the combination of YM155 at 1 nmol/L and AG490 at 10μmol/L or STA-21 at 6μmol/L induced higher apoptosis rate that was evident by the sub G1 fraction and annexin-V induction compared with each single treatment. These results suggested that the growth of human B-NHL is STAT3 dependent manner and the inhibition of STAT3 pathway sensitizes human B-NHL to YM155 induced apoptosis. The present study suggested that survivin suppression by YM155 and the convergence of disabling inhibition of STAT-dependent survival pathway by ritximab would contribute the synergistic combination efficacy. A phase 2 study with YM155 plus rituximab is ongoing in relapsed patients with diffuse large B cell lymphoma. 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 2749. doi:1538-7445.AM2012-2749

Pharmacokinetics, distribution and excretion of YM155 monobromide, a novel small‐molecule survivin suppressant, in male and pregnant or lactating female rats

YM155 monobromide is a novel small-molecule survivin suppressant. The pharmacokinetics, distribution and excretion of YM155/[14C]YM155 were investigated using males and pregnant or lactating female rats after a single intravenous bolus administration. For the 0.1, 0.3 and 1 mg/kg YM155 doses given to male rats, increases in area under the plasma concentration-time curves were approximately proportional to the increase in the dose level. After administering [14C]YM155, radioactivity concentrations in the kidney and liver were highest among the tissues in both male and pregnant rats: e.g. 14.8- and 5.24-fold, respectively, and higher than in plasma at 0.1 h after dosing to male rats. The YM155 concentrations in the brain were lowest: 25-fold lower than in plasma. The transfer of radioactivity into fetuses was low (about 2-fold lower than in plasma). In lactating rats, the radioactivity was transferred into milk at a level 8- to 21-fold higher than for plasma. Radioactivity was primarily excreted in feces (64.0%) and urine (35.2%). The fecal excretion was considered to have occurred mainly by biliary excretion and partly by secretion across the gastrointestinal membrane from the blood to the lumen.

Utility of P-Glycoprotein and Organic Cation Transporter 1 Double-Transfected LLC-PK1 Cells for Studying the Interaction of YM155 Monobromide, Novel Small-Molecule Survivin Suppressant, with P-Glycoprotein

1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide (YM155 monobromide), a novel small molecule that downregulates survivin and exhibits potent antitumor activity, is hydrophilic and cationic. Although previous studies have shown that influx transporters play important roles in the uptake of YM155 into hepatocytes and possibly into cancer cells, efflux transporters have yet to be investigated. In this study, we assessed the interaction of YM155 with P-glycoprotein [multidrug resistance 1 (MDR1)/ATP-binding cassette B1] using two kinds of transcellular transport systems: Caco-2 and MDR1-expressing LLC-PK1 cells (LLC-MDR1). We also used a newly established LLC-OCT1/MDR1 cell line, which expresses basal YM155 uptake transporter organic cation transporter1 (OCT1) and apical MDR1. Direct interaction between YM155 and MDR1 and other efflux transporters was evaluated using transporter-expressing membrane vesicles. A bidirectional transporter assay using Caco-2 and LLC-MDR1 cells showed low permeability and no vectorial transport of YM155, suggesting that YM155 is not a substrate of MDR1. However, vectorial transport across LLC-OCT1/MDR1 cells was identified, which was inhibited by the MDR1 inhibitor cyclosporine A, clearly indicating that YM155 is in fact a substrate of MDR1. Insufficient expression of basal uptake transporter of YM155 in Caco-2 and LLC-MDR1 might have confounded conclusions regarding YM155 and MDR1. Using the transporter-expressing vesicles, MDR1-mediated transport was most significantly involved in YM155 transport among the efflux transporters examined. In conclusion, these findings suggest that YM155 is a substrate of MDR1, and that MDR1 may play an important role in the pharmacokinetics of YM155. Transcellular assays lacking basal uptake transporters may be inaccurate in the assessment of hydrophilic compounds that have poor membrane permeability by passive diffusion.

Mcl-1 downregulation by YM155 contributes to its synergistic anti-tumor activities with ABT-263

YM155, a small-molecule survivin suppressant, exhibits anti-tumor activities in vitro, in vivo and in clinical trials. However, the mechanism of YM155 action remains unclear. In this study, YM155 was administered to a panel of cell lines and the effects of YM155 on Bcl-2 family members were analyzed. Our results show that YM155 strikingly downregulates Mcl-1 in a broad spectrum of cancer cell lines and that the Mcl-1 modulation occurs at the transcriptional level, independently of survivin modulation or caspase activity. Furthermore, analysis of the contribution of Mcl-1 or survivin downregulation to YM155-induced cell death in vitro showed that knockdown of Mcl-1 sensitizes cells to YM155-induced cytotoxicity. Finally, our data demonstrate that downregulation of Mcl-1 by YM155 synergistically lowers the threshold of Bcl-2 family member inhibitor ABT-263-induced cell death. Our findings reveal a novel mechanism by which survivin-independent Mcl-1 suppression plays a critical role in YM155-mediated anti-tumor activities. YM155 treatment in combination with ABT-263 thus affords a new strategy for cancer treatment.

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.

Effect of YM155, a potent survivin suppressant, on the antitumor effect of rituximab in diffuse large B-cell lymphoma (DLBCL) xenograft models: FLT-/FDG-PET imaging studies.

8087 Background: YM155 is a novel small-molecule survivin suppressant, and is currently under development for the treatment of non- Hodgkin's lymphoma in a phase II clinical trial. Therapy monitori...