YM155 Treatment Research Articles

Survivin inhibition ameliorates liver fibrosis by inducing hepatic stellate cell senescence and depleting hepatic macrophage population.

Persistent activation of hepatic stellate cells (HSCs) in the injured liver leads to the progression of liver injury from fibrosis to detrimental cirrhosis. In a previous study, we have shown that survivin protein is upregulated during the early activation of HSCs, which triggers the onset of liver fibrosis. However, the therapeutic potential of targeting survivin in a fully established fibrotic liver needs to be investigated. In this study, we chemically induced hepatic fibrosis in mice using carbon tetrachloride (CCl4) for 6weeks, which was followed by treatment with a survivin suppressant (YM155). We also evaluated survivin expression in fibrotic human liver tissues, primary HSCs, and HSC cell line by histological analysis. αSMA+ HSCs in human and mice fibrotic liver tissues showed enhanced survivin expression, whereas the hepatocytes and quiescent (qHSCs) displayed minimal expression. Alternatively, activated M2 macrophage subtype induced survivin expression in HSCs through the TGF-β-TGF-β receptor-I/II signaling. Inhibition of survivin in HSCs promoted cell cycle arrest and senescence, which eventually suppressed their activation. In vivo, YM155 treatment increased the expression of cell senescence makers in HSCs around fibrotic septa such as p53, p21, and β-galactosidase. YM155 treatment in vivo also reduced the hepatic macrophage population and inflammatory cytokine expression in the liver. In conclusion, downregulation of survivin in the fibrotic liver decreases HSC activation by inducing cellular senescence and modulating macrophage cytokine expression that collectively ameliorates liver fibrosis.

Single-Cell Multi-Ome Analysis Reveals Novel Molecular Mechanisms Underlying Subclonal Response to Survivin Inhibition in Relapsed/Refractory Multiple Myeloma

Multiple myeloma is the second-most common hematological malignancy in the US that remains a challenging disease to cure despite recent advancements in treatment strategies. Drug resistance is a major cause of concern in myeloma chemotherapy. We have created an optimization-regularization-based computational prediction method called secDrug that identified several novel secondary drug (‘secDrug‘) combinations against drug-resistant myeloma (relapsed/refractory multiple myeloma or RRMM). Top among our predicted secDrugs includes the survivin inhibitor, YM155. Survivin is a member of the inhibitor of apoptosis protein (IAP) family that has dual roles in mitosis and apoptosis. YM155 inhibits survivin at the transcriptional level by inhibiting the survivin gene promoter. Several studies have demonstrated the efficacy of YM155 in solid tumors and heme malignancies, including myeloma. However, the precise mechanism of action is not yet known. MM cell lines express high levels of IAPs. We observed high in vitro cytotoxicity of YM155 as a single agent (IC 50 range 0.41 to 12.85nM) as well as in combination with the primary drugs like proteasome inhibitors and Immunomodulatory drugs (IMiDs) against >15 human myeloma cell lines representing innate/refractory and acquired/emerging (relapse) resistance. Next, we explored the molecular mechanisms underlying the efficacy of YM155 using pre-vs-post-treatment tumor whole-transcriptome analysis (RNAseq). The top differentially regulated genes include DDIT3 (CHOP), ATF3, TXNDC5 SEC24D, and DHX15 ( Figure 1A). Ingenuity Pathway Analysis (IPA) predicted that, in addition to regulating apoptosis, survivin inhibition in myeloma significantly upregulated unfolded protein response and downregulated vehicle trafficking and genes associated with immune cell function. Further, we performed single-cell transcriptomics (scRNAseq; 10X Genomics) analysis to explore treatment-induced changes to the subclonal architecture. Our scRNAseq results revealed a distinct shift in clusters representing myeloma single-cell subpopulations following YM155 treatment ( Figure 1B). For example, Cluster 1 (53.11%), characterized by high expression of cell survival markers Ki67 and MYC, was pre-dominant in untreated myeloma cells, while Cluster 4 (52.19%) was the primary subclone in post-treatment tumors. Cluster 3 was shared between the untreated and treated groups. Detailed distinctions between each single-cell cluster will be presented. Furthermore, our scRNAseq data alluded to the benefits of combining survivin-inhibitors and Hsp90-inhibitors based on the expression of drug target genes. Hsp90 inhibitors indirectly target survivin by disrupting the interaction between survivin and Hsp90, resulting in a destabilized survivin protein that ultimately results in survivin degradation. In fact, we have earlier shown the effectiveness of Hsp90 inhibitors (17-AAG, AICAR, and CCT018159) as potent secDrugs against RRMM. Finally, high-dimensional mass cytometry or CyTOF analysis using heavy metal-conjugated proteins in patient bone marrow-derived primary myeloma cells ( ex vivo model system) showed that YM155 induced dose-dependent reduction of several protein markers implicated in cell survival and chemotherapeutic resistance in multiple myeloma, including myc, Ki-67, CD28, CD81, and CD274 (PD-L1). Previous studies have shown that CD28 and CD81 are high-risk immunophenotypes associated with significantly worse prognosis in patients with myeloma. PD-L1 (Programmed death-ligand 1) is a cell-surface glycoprotein expressed in a large number of malignancies that is involved in the termination of immune response and immune evasion. An earlier study has shown that YM155 enhances daratumumab-mediated cellular lysis of multiple myeloma cells. Our RNAseq and CyTOF results thus suggest a potential basis of synergy between YM155 with immunotherapies approved for myeloma, which we will test further. Our approach thus integrates in silico prediction with single-cell multi-ome analysis to identify molecular mechanisms potentially underlying subclonal response to novel combination therapy candidates (secDrugs) for the treatment of RRMM.

Survivin enhances hippocampal neurogenesis and cognitive function in Alzheimer's disease mouse model.

Cognitive impairment is associated with reduced hippocampal neurogenesis; however, the causes of decreased hippocampal neurogenesis remain highly controversial. Here, we investigated the role of survivin in the modulation of hippocampal neurogenesis in AD. To investigate the effect of survivin on neurogenesis in neural stem cells (NSCs), we treated mouse embryonic NSCs with a survivin inhibitor (YM155) and adeno-associated viral survivin (AAV-Survivin). To explore the potential role of survivin expression in AD, AAV9-Survivin or AAV9-GFP were injected into the dentate gyrus (DG) of hippocampus of 7-month-old wild-type and 5XFAD mice. Cognitive function was measured by the Y maze and Morris water maze. Neurogenesis was investigated by BrdU staining, immature, and mature neuron markers. Our results indicate that suppression of survivin expression resulted in decreased neurogenesis. Conversely, overexpression of survivin using AAV-Survivin restored neurogenesis in NSCs that had been suppressed by YM155 treatment. Furthermore, the expression level of survivin decreased in the 9-month-old 5XFAD compared with that in wild-type mice. AAV-Survivin-mediated overexpression of survivin in the DG in 5XFAD mice enhanced neurogenesis and cognitive function. Hippocampal neurogenesis can be enhanced by survivin overexpression, suggesting that survivin could serve as a promising therapeutic target for the treatment of AD.

Exploring MicroRNA and Exosome Involvement in Malignant Pleural Mesothelioma Drug Response.

Simple SummaryMalignant pleural mesothelioma (MPM) is a deadly thoracic malignancy with limited treatment options. Chemotherapy remains the most widely used first-line treatment for unresectable MPM but is hampered by drug resistance issues. Small molecule inhibitors and microRNA mimics have shown promising potential for the treatment of MPM in preclinical studies, but are yet to be successfully implemented in the clinical setting. Our study aims to provide an understanding of the molecular mechanism(s) that mediate drug response in MPM. The inhibitor of apoptosis family member, survivin, has been reported to be over-expressed in MPM and is associated with drug resistance. Therefore, we particularly focused on determining the cellular mechanism(s) that contribute to MPM cell response to a survivin small molecule inhibitor, YM155. Our study provides key information to facilitate a prediction of the potential utility of small molecule inhibitors and microRNA mimics as treatment options for MPM.Malignant pleural mesothelioma (MPM) is a deadly thoracic malignancy and existing treatment options are limited. Chemotherapy remains the most widely used first-line treatment regimen for patients with unresectable MPM, but is hampered by drug resistance issues. The current study demonstrated a modest enhancement of MPM cell sensitivity to chemotherapy drug treatment following microRNA (miRNA) transfection in MPM cell lines, albeit not for all tested miRNAs. This effect was more pronounced for FAK (PND-1186) small molecule inhibitor treatment; consistent with previously published data. We previously established that MPM response to survivin (YM155) small molecule inhibitor treatment is unrelated to basal survivin expression. Here, we showed that MPM response to YM155 treatment is enhanced following miRNA transfection of YM155-resistant MPM cells. We determined that YM155-resistant MPM cells secrete a higher level of exosomes in comparison to YM155-sensitive MPM cells. Despite this, an exosome inhibitor (GW4896) did not enhance MPM cell sensitivity to YM155. Additionally, our study showed no evidence of a correlation between the mRNA expression of inhibitor of apoptosis (IAP) gene family members and MPM cell sensitivity to YM155. However, two drug transporter genes, ABCA6 and ABCA10, were upregulated in the MPM cell lines and correlated with poor sensitivity to YM155.

Effects of YM155 on the proliferation and apoptosis of pulmonary artery smooth muscle cells in a rat model of high pulmonary blood flow-induced pulmonary arterial hypertension

ABSTRACT Introduction Proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) play an important role in the occurrence and development of pulmonary arterial hypertension (PAH). The purpose of this study was to investigate the effects of survivin inhibitor YM155 on the proliferation and apoptosis of PASMCs in rats with PAH induced by high pulmonary blood flow. Methods Thirty male Sprague-Dawley (SD) rats were randomly divided into control, model, and YM155 intervention groups. A rat model of PAH induced by high pulmonary blood flow was established, and it was confirmed by assessments of right-ventricular pressure (RVP) and right ventricular hypertrophy index (RVHI). Immunohistochemical staining and western blot analysis were used to detect the expression of survivin, and the proliferation and apoptosis of PASMCs. Lastly, the effects of in vivo treatment of YM155 were tested. Results The increased expression of survivin mRNA and protein were observed in the model group, accompanied by pulmonary arteriolar wall thickening, lumen stenosis, and perivascular inflammatory cell infiltration. Elevated expression of survivin and pulmonary vascular remodeling were significantly mitigated after YM155 treatment. Specifically, the YM155 intervention group had a significantly lower PASMC proliferation rate and a higher PASMC apoptotic rate. Conclusion YM155 suppressed PASMC proliferation and promoted PASMC apoptosis by inhibiting survivin expression and thereby reducing pulmonary vascular remodeling in high pulmonary blood flow-induced PAH in vivo.

Targeting abundant survivin expression in liposarcoma: subtype dependent therapy responses to YM155 treatment

PurposeLiposarcoma (LPS) represent the largest group of malignant soft tissue tumours comprising a heterogeneous group of subtypes in which the degrees of chemoresistance and radiosensitivity strongly vary. Consequently, it is of utmost interest to establish novel therapeutic regimens based on molecular targets.MethodsImmunohistochemical staining of survivin was performed in tissue microarrays comprising 49 primary LPS specimens. LPS cell lines were treated with survivin antagonist YM155 and doxorubicin or etoposide alone as well as in combination. Changes in cell viability were investigated and the synergistic effect of a combined therapy analysed.ResultsImmunohistochemistry revealed an abundant expression of survivin in LPS that significantly concurred with less-differentiated tumour subtypes and grading. In vitro, we demonstrated the impact of the survivin inhibitor YM155 on dedifferentiated LPS (DDLPS) and, even more imposing, pleomorphic LPS (PLS) tumour cell viability with a strong induction of apoptosis. A combined treatment of doxorubicin or etoposide with YM155 augmented the cytotoxic effects on DDLPS and PLS cells.ConclusionThese findings support the significant role of survivin in the oncogenesis and progression of LPS subtypes providing a rationale to target survivin in eligible in-vivo models and to pioneer clinical applications of survivin-specific substances unfolding their therapeutic potential in LPS patients prospectively.

Abstract LBA033: YM155 induces DNA damage and cell death in anaplastic thyroid cancer cells by inhibiting DNA topoisomerase IIα at the ATP binding site

Abstract Introduction: Anaplastic thyroid cancer (ATC) is among the most aggressive of all human cancers with a median survival of 4.3 months. Currently, there is no effective treatment for most ATC patients - surgery, radiation, and chemotherapy all fail to significantly prolong ATC patient survival. YM155, first identified as a survivin inhibitor, was highlighted in a high-throughput screen performed by the National Cancer Institute, killing anaplastic thyroid cancer cells in vitro and in vivo. However, there was no association between survivin expression and response to YM155 in clinical trials (not including ATC), and YM155 has been mostly abandoned for development despite favorable pharmacokinetic and toxicity profiles. A number of additional mechanisms have been proposed for YM155. The purpose of this study was to investigate the mechanisms underlying YM155-mediated ATC cell death. Methods: ATC cell line THJ16T was used as the model for this study. AlamarBlue was used to measure cell viability. Immunofluorescent detection of phosphorylated histone H2AX (γ-H2AX, p-Ser139) foci was used as a surrogate marker for double-strand DNA breaks. siRNA was used to knockdown topoisomerase 2α (Top2α), and Lipofectamine-3000 was used to overexpress Top2α plasmids. In-vivo complex of enzyme assay was used to measure the association between Top2α and DNA. Modeling software Molecular Operating Environment and the crystal structure of human Top2α bound to AMP-PNP (Protein Data Bank # 1ZXM) were used to estimate binding affinities for ATP and YM155 in the ATP binding site of Top2α. Site-directed mutagenesis was used to introduce mutation Ser148 → Ala into a recombinant human Top2α. Results: Previously, we presented gene expression data from ATC patients showing significant overexpression of Top2α compared to benign thyroid samples. ATC cell lines that overexpress Top2α are more sensitive to YM155 and exhibit larger increases in DNA damage with YM155 treatment. In this study, ATC cells grown to be resistant to YM155 show decreased expression of Top2α, and overexpression of Top2α re-sensitizes resistant cells to YM155. Molecular modeling shows favorable binding for YM155 in the Top2α ATP binding site and identifies key amino acids for YM155-Top2α interaction. An engineered Top2α mutant abrogates the effect of YM155 as measured by cell viability and DNA damage assays, confirming the contribution of Top2α to YM155 mechanism of action. Conclusions: Our results strongly suggest that Top2α is an important target mediating YM155’s anticancer activity in ATC. We propose that Top2α inhibition by YM155 leads to DNA damage and eventual cell death. Given YM155's potent anti-proliferative effects in ATC cells with sparing of benign thyroid cells, along with a plausible mechanism of action, we suggest further evaluation and optimization of YM155 for future clinical application in ATC. In conclusion, our study lays the foundation for targeting the enzymatic activity of Top2α as a novel therapeutic strategy for the treatment of ATC, with YM155 serving as the predicate drug. Citation Format: Ryan Mackay, Paul Weinberger, Elahe Mahdavian, Qinqin Xu. YM155 induces DNA damage and cell death in anaplastic thyroid cancer cells by inhibiting DNA topoisomerase IIα at the ATP binding site [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA033.

Inhibition of Sp1-mediated survivin and MCL1 expression cooperates with SLC35F2 and myeloperoxidase to modulate YM155 cytotoxicity to human leukemia cells

Although YM155 is reported to suppress survivin (also known as BIRC5) expression in cancer cells, its cytotoxic mechanism in human acute myeloid leukemia (AML) cells has not been clearly resolved. In this study, we analyzed the mechanistic pathways that modulate the sensitivity of human AML U937 and HL-60 cells to YM155. YM155 induced apoptosis in AML cells, which was characterized by p38 MAPK phosphorylation and downregulation of survivin and MCL1 expression. Phosphorylated p38 MAPK causes autophagy-mediated Sp1 degradation, thereby inhibiting the transcription of survivin and MCL1. The reduction of survivin and MCL1 levels further facilitated Sp1 protein degradation through autophagy. The restoration of Sp1, survivin, or MCL1 expression protected U937 and HL-60 cells from YM155-mediated cytotoxicity. U937 and HL-60 cells were continuously exposed to hydroquinone (HQ) to generate U937/HQ and HL-60/HQ cells, which showed increased SLC35F2 expression. The increase in SLC35F2 expression led to an increase in the sensitivity of U937/HQ cells to YM155-mediated cytotoxicity, whereas no such effect was observed in HL-60/HQ cells. Of note, myeloperoxidase (MPO) activity in HL-60 and HL-60/HQ cells enhanced YM155 cytotoxicity in these cells, and the enforced expression of MPO also increased the sensitivity of U937 cells to YM155. Taken together, we conclude that p38 MAPK-modulated autophagy inhibits Sp1-mediated survivin and MCL1 expression, which, in turn, leads to the death of U937 and HL-60 cells following YM155 treatment. In addition, our data indicate that SLC35F2 increases the sensitivity of U937 cells to YM155-mediated cytotoxicity, whereas MPO enhances YM155 cytotoxicity in U937 and HL-60 cells.

YM155 and BIRC5 downregulation induce genomic instability via autophagy-mediated ROS production and inhibition in DNA repair

Activation of autophagy plays a critical role in DNA repair, especially for the process of homologous recombination. Despite upregulation of autophagy promotes both the survival and the death of cells, the pathways that govern the pro-cell death effects of autophagy are still incompletely understood. YM155 is originally developed as an expression suppressant of BIRC5 (an anti-apoptotic molecule) and it has reached Phase I/II clinical trials for the treatment of variety types of cancer. However, the target-specificity of YM155 has recently been challenged as several studies reported that YM155 exhibits direct DNA damaging effects. Recently, we discovered that BIRC5 is an autophagy negative-modulator. Using function-comparative analysis, we found in the current study that YM155 and BIRC5 siRNA both induced early “autophagy-dependent ROS production-mediated” DNA damage/strand breaks and concurrently downregulated the expression of RAD54L, RAD51, and MRE11, which are molecules known for their important roles in homologous recombination, in human cancer (MCF7, MDA-MB-231, and SK-BR-3) and mouse embryonic fibroblast (MEF) cells. Similar to the effects of YM155 and BIRC5 siRNA, downregulation of RAD54L and RAD51 by siRNA induced autophagy and DNA damage/strand breaks in cells, suggesting YM155/BIRC5 siRNA might also induce autophagy partly through RAD54L and RAD51 downregulations. We further observed that prolonged YM155 and BIRC5 siRNA treatment induced autophagic vesicle formation proximal to the nucleus and triggered DNA leakage. In conclusion, our findings reveal a novel mechanism of action of YM155 (i.e. induces autophagy-dependent ROS production-mediated DNA damage) in cancer cells and show the functional complexity of BIRC5 and autophagy involving the modulation of genome stability, highlighting that upregulation of autophagy is not always beneficial to the DNA repair process. Our findings can aid the development of a variety of BIRC5-directly/indirectly targeted anticancer therapies that are currently under pre-clinical and clinical investigations.

Selection of Effective Therapies Using Three-Dimensional in vitro Modeling of Chondrosarcoma.

Purpose: Chondrosarcomas are a group of cartilaginous malignant neoplasms characterized by the deposition of chondrogenic extracellular matrix. Surgical resection is currently the only curative treatment option, due to their high resistance to conventional chemotherapy and radiotherapy. Novel therapeutic treatment options may improve outcome. Predominantly used cell line monolayer in vitro models lack in vivo complexity, such as the presence of extracellular matrix, and differing oxygen access. Hence, we aimed to improve pre-clinical chondrosarcoma research by developing an alginate-based 3D cell culture model.Method: An alginate scaffold was applied to generate spheroids of three chondrosarcoma cell lines (CH2879, JJ012, SW1353). Morphological, histological and immunohistochemical assessment of the spheroids were used to characterize the chondrosarcoma model. Presto blue assay, morphological and immunohistochemical assessment were applied to assess spheroid response to a panel of chemotherapeutics and targeted therapies, which was compared to conventional 2D monolayer models. Synergistic effect of doxorubicin and ABT-737 (Bcl-2 inhibitor) was compared between monolayer and spheroid models using excess over Bliss. A 3D colony formation assay was developed for assessment of radiotherapy response.Results: Chondrosarcoma spheroids produced chondrogenic matrix and remained proliferative after 2 weeks of culture. When treated with chemotherapeutics, the spheroids were more resistant than their monolayer counterparts, in line with animal models and clinical data. Moreover, for sapanisertib (mTOR inhibitor) treatment, a recovery in chondrosarcoma growth, previously observed in mice models, was also observed using long-term treatment. Morphological assessment was useful in the case of YM-155 (survivin inhibitor) treatment where a fraction of the spheroids underwent cell death, however a large fraction remained proliferative and unaffected. Synergy was less pronounced in 3D compared to 2D. A 3D clonogenic assay confirmed increased resistance to radiotherapy in 3D chondrosarcoma spheroids.Conclusion: We demonstrate that the chondrosarcoma alginate spheroid model is more representative of chondrosarcoma in vivo and should be used instead of the monolayer model for therapy testing. Improved selection at in vitro stage of therapeutic testing will increase the amount of information available for experimental design of in vivo animal testing and later, clinical stages. This can potentially lead to increased likelihood of approval and success at clinical trials.

Suppression of Rotenone-Treated Human Breast Cancer Stem Cell Survival Using Survivin Inhibitor YM155 is Associated to Oxidative Stress Modulation.

Background:Despite recent progress in molecular-targeted therapies, breast cancer remains the primary leading cause of cancer related death among women worldwide. Breast cancer stem cells (BCSCs) are believed to be responsible for therapy resistance and cancer recurrence. We recently demonstrated that human BCSCs (CD24-/CD44+) could survive better than their counterpart non-BCSCs (CD24-/CD44-) when treated with rotenone, possibly due to lower levels of reactive oxygen species (ROS) production, high expression of antioxidant manganese superoxide dismutase (MnSOD), and anti-apoptotic survivin. The aim of this study was to verify the role of survivin on human BCSCs survival under oxidative stress modulation by suppressing its expression using YM155, a survivin inhibitor. Methods:Human BCSCs (ALDH+ cells) were treated with YM155 for 24 h prior to treatment with rotenone for a further 6 h. We determined intracellular superoxide levels were determined using dihydroethidium assay, survivin and MnSOD expression using qRT-PCR, survivin protein level using ELISA, as well as cell viability using trypan blue exclusion and acridine orange/ethidium bromide apoptosis assay. Results:Suppression of survivin expression using YM155 could reduce the survival of rotenone-treated BCSCs, which may be associated with oxidative stress modulation, as shown by increased ROS levels and decreased MnSOD expression. We confirm that survivin is responsible for maintaining BCSCs survival under oxidative stress modulation. Furthermore, YM155 could modulate oxidative stress in BCSCs by reducing MnSOD expression and increasing ROS levels. Conclusion:YM155 treatment could be used to overcome BCSCs resistance to oxidative stress-based anticancer therapies.

Safe scarless cassette-free selection of genome-edited human pluripotent stem cells using temporary drug resistance

An efficient gene-editing technique for use in human pluripotent stem cells (hPSCs) has great potential value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However, the extremely low efficiency of gene editing in hPSCs remains as a major technical hurdle. Previously, we demonstrated that YM155, a survivin inhibitor developed as an anti-cancer drug, induces highly selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155 in hPSCs, which is mediated by selective cellular uptake of the drug, is due to the high expression of SLC35F2 in these cells. Knockout of SLC35F2 with CRISPR-Cas9, or depletion with siRNAs, made the hPSCs highly resistant to YM155. Simultaneous editing of a gene of interest and transient knockdown of SLC35F2 following YM155 treatment enabled the survival of genome-edited hPSCs as a result of temporary YM155 resistance, thereby achieving an enriched selection of clonal populations with gene knockout or knock-in. This precise and efficient genome editing approach took as little as 3 weeks and required no cell sorting or the introduction of additional genes, to be a more feasible approach for gene editing in hPSCs due to its simplicity.

Abstract 1838: Accumulation of DNA:RNA hybrids (R-loops) and cell death after YM155 treatment of anaplastic thyroid cancer hints at a novel therapeutic approach

Abstract Introduction: Anaplastic thyroid cancer (ATC) is a rapidly fatal disease with a median survival of less than 6 months. Although rare, ATC is responsible for a disproportionate number of thyroid cancer deaths and is highly resistant to common cancer therapies. YM155 is a small molecule drug identified in a screen by the National Cancer Institute that potently kills ATC cells in vitro and in vivo. We have previously shown the ability of YM155 to inhibit ATC cell proliferation, however, the mechanism is still unclear. This study investigates the DNA damage and accumulation of R-loops that is seen with YM155 treatment of ATC cells. R-loops are structures characterized by a DNA:RNA hybrid with a displaced single strand of DNA and can be formed by transcription-replication collision, nucleotide depletion, and other situations where transcription is stalled. Materials & Methods: Clonogenic assays were performed in four ATC cell lines (ACT1, THJ11T, THJ16T, and THJ29T) to study the effect of YM155 on cell survival. DNA damage was measured by COMET assay and by counting foci of phosphorylated histone H2AX (γH2AX). R-loops were measured via immunofluorescent microscopy with S9.6 anti-R-loop antibody and anti-nucleolin antibody in conjunction with an AI algorithm using inFormTM cell segmentation software to quantify non-nucleolar nuclear R-loop signal. Results: We show in multiple ATC cell lines that YM155 potently kills ATC cells at low nanomolar concentrations. COMET assay found significant increases in DNA damage after treatment with 10 nM YM155, with large increases in tail moment seen by 24 hours in three ATC cell lines (p &lt 0.001 for all) with no change in primary benign thyroid (BT) cells. Similar trends were seen when measuring formation of γH2AX foci, a marker for double strand DNA breaks. R-loop immunofluorescence more than doubled with 10 nM YM155 treatment (p &lt 0.001) similar to hydroxyurea, a positive control. This finding was confirmed when addition of RNAseH, which is specific for RNA in DNA:RNA hybrids, reduced R-loop signal down to baseline levels. Conclusion: YM155's ability to kill ATC cells at low concentrations while sparing BT cells coincides with increases in DNA damage and accumulation of R-loops. A variety of factors aid in the resolution of R-loops, and we have previously shown patient data exhibiting overexpression of DNA topoisomerase genes TOP1 and TOP2A. Combined with published information describing the ability of YM155 to inhibit Topoisomerase Iiα (Top2α) activity, these results suggest a newly revealed, targetable aspect of ATC pathophysiology and a potential novel role for the involvement of Top2α in the resolution of R-loops. Citation Format: Ryan Mackay, Qinqin Xu, Paul Weinberger. Accumulation of DNA:RNA hybrids (R-loops) and cell death after YM155 treatment of anaplastic thyroid cancer hints at a novel therapeutic approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1838.

MP51-16 DECREASING SURVIVIN BY YM155 REVERSES CABAZITAXEL RESISTANCE IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (MP51)1 Apr 2020MP51-16 DECREASING SURVIVIN BY YM155 REVERSES CABAZITAXEL RESISTANCE IN PROSTATE CANCER Takeshi Miyao*, Hidekazu Koike, Akira Otsu, Daisuke Oka, Masanori Aoki, Hiroshi Nakayama, Yoshiyuki Miyazawa, Seiji Arai, Yoshitaka Sekine, Hiroshi Matsui, Yasuhiro Shibata, and Kazuhiro Suzuki Takeshi Miyao*Takeshi Miyao* More articles by this author , Hidekazu KoikeHidekazu Koike More articles by this author , Akira OtsuAkira Otsu More articles by this author , Daisuke OkaDaisuke Oka More articles by this author , Masanori AokiMasanori Aoki More articles by this author , Hiroshi NakayamaHiroshi Nakayama More articles by this author , Yoshiyuki MiyazawaYoshiyuki Miyazawa More articles by this author , Seiji AraiSeiji Arai More articles by this author , Yoshitaka SekineYoshitaka Sekine More articles by this author , Hiroshi MatsuiHiroshi Matsui More articles by this author , Yasuhiro ShibataYasuhiro Shibata More articles by this author , and Kazuhiro SuzukiKazuhiro Suzuki More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000913.016AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cabazitaxel has exhibited promising anticancer activity for the treatment of prostate cancer. However, many patients acquire resistance to therapeutic agents leading to treatment failure. The objective of this study was to determine whether treatment with YM155, a novel small molecule inhibitor of survivin, could reverse Cabazitaxel resistance in a Cabazitaxel-resistant prostate cancer. METHODS: We induced a Cabazitaxel-resistant prostate cancer cell line (22Rv1-CR). We showed that survivin gene expression was significantly up-regulated in 22Rv1-CR compared with that in its parent cells (22Rv1). Therefore, we hypothesized that targeting of survivin in 22Rv1-CR could reverse the resistant phenotype in tumor cells, thereby enhancing the therapeutic efficacy of Cabazitaxel. We used both in vitro and in vivo models to test the efficacy of YM155 either as a single agent or in combination with Cabazitaxel. RESULTS: In 22Rv1-CR cells, YM155 significantly decreased survivin gene expression levels and cell proliferation in a dose-dependent manner in vitro. In addition, YM155 treatment significantly reversed Cabazitaxel resistance in 22Rv1-CR cells. In a nude mouse tumor xenograft model, YM155 significantly enhanced the antitumor effects of Cabazitaxel in 22Rv1-CR tumor. CONCLUSIONS: Our results suggest a potentially novel strategy to use YM155 to overcome the resistance in tumor cells, thereby enhancing the effectiveness of Cabazitaxel-therapy in prostate cancer. Source of Funding: none © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e770-e770 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takeshi Miyao* More articles by this author Hidekazu Koike More articles by this author Akira Otsu More articles by this author Daisuke Oka More articles by this author Masanori Aoki More articles by this author Hiroshi Nakayama More articles by this author Yoshiyuki Miyazawa More articles by this author Seiji Arai More articles by this author Yoshitaka Sekine More articles by this author Hiroshi Matsui More articles by this author Yasuhiro Shibata More articles by this author Kazuhiro Suzuki More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract 722: Epigenetic regulation of the Birc5 promoter explains mechanism of action of YM-155 in synovial sarcoma

Abstract YM-155 is an anti-cancer therapy that has advanced into 11 different human clinical trials to treat various cancers. This apoptosis-inducing therapy indirectly affects the protein levels of Survivin (Gene: Birc5), but the molecular underpinnings of the mechanism remain largely unknown. Synovial sarcoma is another cancer with high protein expression of Survivin. We investigated whether YM-155 would be a viable therapeutic option to treat synovial sarcoma. We, therefore, applied YM-155 therapy to human synovial sarcoma cell lines and a genetically engineered mouse model of synovial sarcoma. We discovered that YM-155 exhibited nanomolar potency against human synovial sarcoma cell lines and the treated mice with synovial sarcoma demonstrated a 50% reduction in tumor volume compared to control treated mice. We further investigated the mechanism of action of YM-155 by looking at the change of modifications of the histone tails that were near the Birc5 promoter. Using chomatin immunoprecipitation (ChIP) we discovered that the histone epigenetic marks of H3K27 for the Birc5 promoter changed upon YM-155 treatment. H3K27me3 increased whereas the H3K27ac decreased, highlighting the decrease of the protein Survivin occurs through epigenetic silencing of the gene’s promoter. The treatment of YM-155 was accompanied by an increase in NFkB protein expression, which indicates an attempt of the cell to initiate a positive feedback due to the decrease in Survivin expression. This combination of molecular events eventually resulted in Caspace 3/7/8 upregulation and death of the sarcoma cells. Citation Format: Aleksander Mika, Sarah Luelling, Adriene Pavek, Neil Parkinson, Alexandra Heyneman, Jared J. Barrott. Epigenetic regulation of the Birc5 promoter explains mechanism of action of YM-155 in synovial sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 722.

Epigenetic Changes at the Birc5 Promoter Induced by YM155 in Synovial Sarcoma.

YM155 is an anti-cancer therapy that has advanced into 11 different human clinical trials to treat various cancers. This apoptosis-inducing therapy indirectly affects the protein levels of survivin (gene: Birc5), but the molecular underpinnings of the mechanism remain largely unknown. Synovial sarcoma is a rare soft-tissue malignancy with high protein expression of survivin. We investigated whether YM155 would be a viable therapeutic option to treat synovial sarcoma. YM155 therapy was applied to human synovial sarcoma cell lines and to a genetically engineered mouse model of synovial sarcoma. We discovered that YM155 exhibited nanomolar potency against human synovial sarcoma cell lines and the treated mice with synovial sarcoma demonstrated a 50% reduction in tumor volume compared to control treated mice. We further investigated the mechanism of action of YM155 by looking at the change of lysine modifications of the histone tails that were within 250 base pairs of the Birc5 promoter. Using chromatin immunoprecipitation (ChIP)-qPCR, we discovered that the histone epigenetic marks of H3K27 for the Birc5 promoter changed upon YM155 treatment. H3K27me3 and H3K27ac increased, but the net result was decreased Birc5/survivin expression. Furthermore, the combination of molecular events resulted in caspase 3/7/8 upregulation and death of the sarcoma cells.

Abstract B06: Overcoming hypoxia-induced resistance is essential for effective survivin targeting in acute myeloid leukemia

Abstract Background: Although the clinical outcome of pediatric acute myeloid leukemia (AML) has improved over the past few decades, approximately 30% of patients relapse. The overall survival (OS) rate of pediatric patients with primary refractory or relapsed AML is about 30%. Resistance to therapy presents a great challenge for AML patients. Therefore, there is a critical need to understand mechanisms of resistance, as well as to identify novel therapies that will improve OS. The leukemia-initiating cells reside in special hypoxic niches in the bone marrow. Thus, studying drug response of AML cells within a hypoxic environment is essential for more accurate prediction of patients’ response to therapy. We performed functional drug screening using AML cell lines, which demonstrated a differential response of AML cell lines to the survivin-targeting agent YM155 in normoxia versus hypoxia. Survivin (BIRC5), an inhibitor of apoptosis, is overexpressed in a variety of pediatric blood cancers compared to normal cells. We hypothesize that survivin may be an attractive target for therapy in pediatric AML. The overall objective of this study was to study mechanisms of AML response to YM155 in normoxia and hypoxia and survivin’s potential as a therapeutic target in pediatric AML. Purpose: The aims of the present work were (1) to study BIRC5 expression in publicly available databases of pediatric AML including the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative, and (2) to determine the potential mechanisms of hypoxia-altered response to YM155 in AML cells. Methods and Results: In the present study, the TARGET AML gene-expression profiling data available on Affymetrix human gene 1.1 ST arrays (HuGene 1.1) were used to study BIRC5 expression in comparison to HuGene 1.1 datasets from Gene Expression Omnibus (GEO) containing profiling data from adult AML and normal tissues. BIRC5 expression was significantly higher in pediatric AML in comparison to cytogenetically normal (CN) adult AML, and in relapsed pediatric AML in comparison to matched samples at diagnosis. In order to identify novel targeted therapies in AML we used a panel of AML cell lines to screen a library of 50 compounds in normoxia and hypoxia. The survivin-targeting agent YM155 was found to be one of the most cytotoxic compounds in the majority of AML cell lines in normoxia. However, hypoxia conferred resistance to this drug. The IC50 of YM155 in AML cell lines in normoxia ranged from 1.5 nM-8 µM, with an increase of 2-100 fold in hypoxia (IC50 from 22 nM-14 µM). The Mv4-11 cell line that harbors a FLT3-ITD mutation was selected for further mechanistic studies. Hypoxia conferred resistance to YM155 in Mv4-11 cells through inhibition of apoptosis. In order to identify the underlying molecular mechanisms, we performed RNA-seq of YM155-treated Mv4-11 cells in normoxia and hypoxia, and identified a set of over-represented genes that were validated by quantitative RT-PCR. The mRNA expression of HIF2alpha, p21WAF1, and STAT3 was overexpressed after 9h of YM155 treatment, while PIM2 was downregulated in normoxia but not in hypoxia, in comparison to vehicle-treated control. Preliminary data suggest a role of stem cell genes in hypoxia-induced resistance to YM155. Ongoing studies are directed towards determining the functions of the identified genes in resistance. Conclusion: Our data demonstrate that survivin (BIRC5) is overexpressed in pediatric AML in comparison to adult CN-AML, and may be an attractive target for pediatric AML therapy. Additionally, YM155 is highly cytotoxic to AML cell lines in normoxia; however, for its effective use in the clinic, cotargeting hypoxia-induced resistance factors is essential. Citation Format: David W. Lee, Justin Montoya, Kieran Finch, Jack Wilkinson-Dix, Robert J. Arceci, Daniel H. Wai, Eiman Aleem. Overcoming hypoxia-induced resistance is essential for effective survivin targeting in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B06.

Adaptation to chronic exposure to sepantronium bromide (YM155), a prototypical survivin suppressant is due to persistent DNA damage-response in breast cancer cells.

Sepantronium bromide (YM155), originally developed against the anti-apoptotic protein survivin, performed exceptionally well in pre-clinical and phase I clinical trials. However, in phase II trials of several cancer types including breast cancer it performed poorly. Additionally, no definitive correlation between survivin level and response to therapy was found. In an attempt to understand the true reason of the late-stage failure of this promising drug, we developed YM155-resistant MCF-7 breast cancer cell line and characterized side-by-side with the drug-naïve parental cell line. Chronic YM155 treatment resulted in downregulation of survivin expression yet triggered cellular responses typical of adaptation to persistent DNA damage. Lowering endogenous antioxidant glutathione level and activity of cell cycle check-point kinase restored YM155 activity. Thus, contrary to its development as a survivin suppressant, YM155 primarily acts as a chemotherapeutic drug causing oxidative stress-mediated DNA damage. Adaptation to long-term exposure to YM155 can be prevented and/or overcome by interfering with detoxification and DNA damage-response pathways. Finally, proteins associated with DNA damage-response pathway will be more appropriate as predictive biomarkers of YM155 in breast tumor cells.

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

IntroductionSepantronium 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. ResultsIntracellular 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. ConclusionsRobust 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 knowledgeYM155 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 careThe 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.

YM155 decreases radiation-induced invasion and reverses epithelial\u2013mesenchymal transition by targeting STAT3 in glioblastoma

BackgroundRadiotherapy constitutes a standard arm of therapy in the multimodal treatment of patients with glioblastoma (GBM). Ironically, studies have recently revealed that radiation can augment malignant progression, by promoting migration and invasion, which make the disease especially difficult to cure. Here, we investigated the anticancer effects of YM155, a purported radiosensitizer, in GBM cell lines.MethodsGBM cell lines U251 and U87 were treated with YM155 to assess cytotoxicity and activity of the molecule in vitro. Nude mice were implanted with cells to generate orthotopic xenografts for in vivo studies. Response of cells to treatment was examined using cell viability, immunofluorescence, wound healing, and the Transwell invasion assay. Molecules potentially mediating response were examined through western blot analysis, phospho-kinase arrays, and qPCR. Cells were transfected with siRNA knockdown and gene expression constructs to identify molecular mediators of response.ResultsYM155 reduced viability of U251 and U87 cells and enhanced radiosensitivity through inhibition of homologous recombination. Besides, YM155 decreased invasion caused by radiation and led to expression changes in molecular markers associated with EMT. STAT3 was one of 10 molecules identified on a phosphokinase array exhibiting significant change in phosphorylation under YM155 treatment. Transfection with STAT3 siRNAs or expression constructs demonstrated that EMT changes were achieved by inhibiting the phosphorylation of STAT3 and were survivin-independent. Finally, combining YM155 and radiation in orthotopic xenografts reduced growth and prolonged overall survival of animals.ConclusionsYM155 decreased radiation-induced invasion in GBM cell lines in vitro and in vivo through inhibition of STAT3.