Inhibitor Of Apoptosis Family Research Articles

Comprehensive analysis of prognostic value and immune infiltration of IAPs family in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality rates. The inhibitors of apoptosis (IAP) family act as oncogenes in various tumor types; however, their functions in HCC remain unclear. Here, we used integrated bioinformatics analysis and experimental verification to assess the expression and the prognostic and clinical value of the IAP family in HCC. Using the University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) and the Tumor Immune Estimation Resource (TIMER), we analyzed the expression profiles of IAP family members in HCC tissue, normal tissues, and in patients with different stages and grades of HCC. We further verified the expression level of BIRC2 in 25 HCC samples and matched adjacent normal tissues using quantitative real-time PCR (qRT-PCR), and analyzed its correlation with the marker gene of T-helper type 1 cells (Th1)-STAT1. Meanwhile, the association between BIRC2 and the immunotherapeutic response or immunomodulators was confirmed using the Biomarker Exploration of Solid Tumors (BEST) database. The results showed that NAIP, BIRC2, BIRC3, XIAP, BIRC5, and BIRC6 mRNAs were overexpressed in HCC. The clinical stages, pathological grades, and other clinicopathological features of HCC were closely related to the expression levels of the IAP family members, especially the BIRC2 and BIRC5, which were found to be potential prognostic biomarkers for HCC. Expression of the IAPs was strongly associated with immune cell infiltration. Based on the infiltrative status of various immune cells, HCC patients with high BIRC2 and BIRC5 expression demonstrated poor overall survival (OS) rates. In patients with HCC, BIRC2 expression was noticeably elevated. Concurrently, the expression levels of BIRC2 and STAT1 showed a favorable correlation. BEST database analysis revealed that BIRC2 was a negative predictor of responsiveness to anti-programmed cell death ligand 1 (PD-L1)/cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) inhibitor treatment in HCC, and BIRC2 mRNA expression levels were positively correlated with the expression levels of the immune checkpoint genes programmed cell death protein 1 (PD-1), PD-L1, and CTLA-4 in HCC. Consequently, the IAP family may play a role in carcinogenesis and cancer-immune system interactions in HCC. Our results demonstrate that IAP family members may be viable predictive biomarkers and therapeutic targets for HCC.

BRCA mutations lead to XIAP overexpression and sensitise ovarian cancer to inhibitor of apoptosis (IAP) family inhibitors

BackgroundWe tested the hypothesis that inhibitor of apoptosis family (IAP) proteins may be altered in BRCA1-mutated ovarian cancers and that could affect the sensitivity to IAP inhibitors.MethodsThe levels of IAP proteins were evaluated in human cancers and cell lines. Cell lines were used to determine the effects of IAP inhibitors. The in vivo effects of treatments were evaluated in PDX mouse models.ResultsExpression of X-linked inhibitor of apoptosis (XIAP) is increased in BRCA1-mutated cancers and high levels are associated with improved patient outcomes after platinum chemotherapy. XIAP overexpression is mediated by NF-kB activation and is associated with an optimisation of PARP. BRCA1-mutated cell lines are particularly sensitive to IAP inhibitors due to an inhibitory effect on PARP. Both a BRCA1-mutated cell line with acquired resistance to PARP inhibitors and one with restored BRCA1 remain sensitive to IAP inhibitors. Treatment with IAP inhibitors restores the efficacy of PARP inhibition in these cell lines. The IAP inhibitor LCL161 alone and in combination with a PARP inhibitor, exhibited antitumour effects in PDX mouse models of resistant BRCA2 and 1-mutated ovarian cancer, respectively.ConclusionA clinical trial may be justified to further investigate the utility of IAP inhibitors.

Two-color fluorescent proteins reporting survivin regulation in breast cancer cells for high throughput drug screening.

Reporter gene assay is widely used for high throughput drug screening and drug action mechanism evaluation. In this study, we developed a robust dual-fluorescent reporter assay to detect drugs repressing the transcription of survivin, a cancer biomarker from the inhibitor of apoptosis family, in breast cancer cells cultured in three-dimensional (3D) microbioreactors. Survivin is overexpressed in numerous malignancies but almost silent in normal tissue cells and is considered a lead target for cancer therapy. Breast cancer MCF-7 cells were engineered to express enhanced green fluorescent protein driven by a survivin promoter and red fluorescent protein driven by a cytomegalovirus promoter as internal control to detect changes in survivin expression in cells as affected by drugs. This 3D dual-fluorescent reporter assay was validated with YM155 and doxorubicin, which were known to downregulate survivin in cancer cells, and further evaluated with two widely used anticancer compounds, cisplatin, and epigallocatechin gallate, to evaluate their effects on survivin expression. The results showed that the 3D dual-fluorescent reporter assay was robust for high throughput screening of drugs targeting survivin in breast cancer cells.

Using a Prime-Boost Vaccination Strategy That Proved Effective for High Resolution Epitope Mapping to Characterize the Elusive Immunogenicity of Survivin.

Simple SummaryThe generation of tumor-specific T cells remains a pillar of modern cancer immunotherapy. Exogenous vaccines often rely on targeting tumor-associated antigens. The anti-apoptotic protein survivin has been deemed a high priority target due to its overexpression in a wide variety of tumor types. To support the analysis of tumor-associated T cell responses, optimization of epitope mapping would be valuable. A heterologous prime-boost vaccination strategy was designed to target survivin to induce anti-tumor immune responses. However, survivin-specific T cell responses could not be detected in mice. Potential mechanisms to explain this failure were explored. To confirm the robustness of the vaccination platform, enhanced green fluorescent protein (eGFP) was targeted since it has been defined as a protein with relatively low immunogenicity. In this context the vaccination strategy uncovered novel T cell epitopes from eGFP in two strains of mice. This research highlighted the utility of the vaccine platform to triage potential target antigens based on their immunogenicity.Survivin is a member of the inhibitor of apoptosis family of proteins and has been reported to be highly expressed in a variety of cancer types, making it a high priority target for cancer vaccination. We previously described a heterologous prime-boost strategy using a replication-deficient adenovirus, followed by an oncolytic rhabdovirus that generates unprecedented antigen-specific T cell responses. We engineered each vector to express a mutated version of full-length murine survivin. We first sought to uncover the complete epitope map for survivin-specific T cell responses in C57BL/6 and BALB/c mice by flow cytometry. However, no T cell responses were detected by intracellular cytokine staining after re-stimulation of T cells. Survivin has been found to be expressed by activated T cells, which could theoretically cause T cell-mediated killing of activated T cells, known as fratricide. We were unable to recapitulate this phenomenon in experiments. Interestingly, the inactivated survivin construct has been previously shown to directly kill tumor cells in vitro. However, there was no evidence in our models of induction of death in antigen-presenting cells due to treatment with a survivin-expressing vector. Using the same recombinant virus-vectored prime-boost strategy targeting the poorly immunogenic enhanced green fluorescent protein proved to be a highly sensitive method for mapping T cell epitopes, particularly in the context of identifying novel epitopes recognized by CD4+ T cells. Overall, these results suggested there may be unusually robust tolerance to survivin in commonly used mouse strains that cannot be broken, even when using a particularly potent vaccination platform. However, the vaccination method shows great promise as a strategy for identifying novel and subdominant T cell epitopes.

Single Agent and Synergistic Combinatorial Efficacy of First-in-Class Small Molecule Imipridone ONC201 in Hematological Malignancies In Vitro and In Vivo through Apoptosis Via the Integrated Stress Response Pathway

▪ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies.ONC201 was evaluated in 20 human leukemia cell lines (Figure 1): 13 acute myeloid leukemia (AML), 5 acute lymphoblastic leukemia (ALL) and 2 chronic myelogenous leukemia (CML) cell lines. We observed a dose-dependent decrease in cell viability in AML, ALL and CML cells (GI50 1-8 µM). ONC201 also decreased cell viability in 2 primary chronic lymphocytic leukemia (CLL) patient samples with 48-72h treatment. Dose- and time-dependent induction of apoptosis (caspase-3 cleavage), inhibition of Akt and Foxo3a phosphorylation was observed in AML cells treated with ONC201 for 24-48 hr. Sub-G1 analysis confirmed that ONC201 induced apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. ONC201-mediated apoptosis in ALL cells involved PARP cleavage and caspase-9 activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim was upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells, which are known to confer a poor clinical prognosis.Low micromolar concentrations of ONC201 also reduced multiple myeloma (MM) cell viability in a time- and dose-dependent manner. ONC201 reduced cell viability and induced apoptosis in dexamethasone-resistant MM.1R cells with efficacy comparable to dexamethasone-sensitive MM.1S cells (Figure 2). ONC201 caused activation of the integrated stress response (ISR) pathway, as observed with induction of ATF/CHOP in MM cells. XIAP is highly up-regulated in MM cells and sownregulation of IAP family proteins induces apoptosis. ONC201 treatment of MM cells resulted in a dose-dependent decrease in the anti-apoptotic proteins XIAP, cIAP1 and cIAP2.Next, ONC201 was evaluated in 33 human lymphoma cell lines: 3 anaplastic large cell lymphoma (ALCL), 7 Burkitt's lymphoma, 2 cutaneous T-cell lymphoma (CTCL), 8 diffuse large B-cell lymphoma (DLBCL), 1 MCL, 2 nodular sclerosis (NS) and 10 other lymphoma cell lines. ONC201 demonstrated low micromolar range efficacy (GI50 1-6 µM) across all lymphoma subtypes. Low micromolar concentrations of ONC201 reduced cell viability of MCL and DLBCL cell lines and primary cells in a time- and dose-dependent manner (Figure 3). ONC201 caused activation of the ISR pathway, as observed with induction of ATF4 in Pfeiffer DLBCL cells. In patient-derived MCL cells, ONC201 reduced cyclin D1 expression, a diagnostic feature of MCL.We then tested the efficacy of ONC201 in combination with approved therapies in hematological malignancies in vitro and in vivo. ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine confirmed by combination indices in AML cell lines CMK, THP-1, HL60 and ALL Kasumi-2 cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model exhibited superior tumor growth inhibition compared to either agent alone (Figure 4). ONC201 demonstrated synergistic reduction in cell viability in combination with 5-azacytidine that was confirmed by combination indices in AML cell lines HL-60, MOLM14 and MV411. ONC201 synergistically reduced cell viability in combination with dexamethasone or ixazomib in RPMI-8226 MM cells. ONC201 in combination with bortezomib synergistically reduced cell viability in KARPAS 299 ALCL cells and RPMI-8226 MM cells and improved apoptosis induction in UPN2 MCL cells. ONC201 combined with bortezomib was evaluated in a Burkitt's lymphoma xenograft model. Histological evaluation of tumor xenografts revealed a reduced tumor cell density at 3-4 weeks and enhanced CHOP induction at 48 hours with combination treatment compared to either agent alone. The combination treatment did not result in body weight loss.These results serve as a rationale for ongoing ONC201 single agent trials in relapsed/refractory acute leukemia (NCT02392572), non-Hodgkin's lymphoma (NCT02420795), MM (NCT02609230) and combination trial with dexamethasone in MM (NCT02863991). Our findings provide pharmacodynamic biomarkers and identify synergistic combinatorial regimens that are well tolerated and can be explored in the clinic. [Display omitted] DisclosuresPrabhu:Oncoceutics: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Oster:Oncoceutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Allen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc: Equity Ownership, Patents & Royalties.

Abstract 4184: Genetic and pharmacological disruption of survivin suppressed primary and metastatic tumor growth by attenuating TGFB pathway in ovarian cancer

Abstract Introduction: Survivin, a member of the inhibitor of apoptosis family, is upregulated in multiple cancers including ovarian cancer, but is rarely detectable in normal tissues. Therefore, it is a potential drug target. However, targeting survivin is still challenge and small molecule inhibitors failed in clinical trials. It's very important to understand how survivin contributes to tumor development. In this study, we investigated the role of survivin in ovarian cancer using ovarian cancer cell lines in vitro and mouse models in vivo and examined how survivin contributes to ovarian primary ovarian tumor growth and metastasis by examining epithelial to mesenchymal transition (EMT). Methods: To define the role of survivin in EMT in ovarian cancer cells, we established survivin knockout cell lines using lentiviral CRISPR/Cas9 nickase and pharmacologically inhibited survivin with a novel inhibitor MX106. We examined EMT markers, migration and invasion assays in survivin disrupted ovarian cancer cells using Transwell plates. We also assessed primary ovarian tumor growth and metastasis in an orthotopic ovarian cancer mouse models through intrabursal injecting survivin KO cell line and also treatment using MX106. Findings: We found that disruption of survivin expression inhibited EMT by attenuating the TGFβ pathway in ovarian cancer cells. Cell proliferation, migration, and invasion were significantly inhibited in survivin knockout or MX106 treatment in SKOV3 and OVCAR3 cell lines compared to control cells. Inhibition of survivin sensitized cell responses to paclitaxel treatment and overcame chemoresistance in vitro. Lentiviral CRISPR/Cas9 mediated survivin knockout or MX106 treatment efficiently inhibited primary ovarian tumor growth and metastasis in orthotopic ovarian cancer mouse models. Conclusions: Our data demonstrate that inhibition of survivin using either genetic knockout or a novel inhibitor MX106 suppresses primary ovarian tumor growth and metastasis by attenuating TGFβ pathway. MX106, as a novel survivin inhibitor effectively overcame chemoresistance compared to commercial YM155, which provides a novel approach for ovarian cancer therapy. Citation Format: Junming Yue. Genetic and pharmacological disruption of survivin suppressed primary and metastatic tumor growth by attenuating TGFB pathway in ovarian cancer [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 4184.

IAP-Mediated Protein Ubiquitination in Regulating Cell Signaling.

Over the last decade, the E3-ubiquitine ligases from IAP (Inhibitor of Apoptosis) family have emerged as potent regulators of immune response. In immune cells, they control signaling pathways driving differentiation and inflammation in response to stimulation of tumor necrosis factor receptor (TNFR) family, pattern-recognition receptors (PRRs), and some cytokine receptors. They are able to control the activity, the cellular fate, or the stability of actors of signaling pathways, acting at different levels from components of receptor-associated multiprotein complexes to signaling effectors and transcription factors, as well as cytoskeleton regulators. Much less is known about ubiquitination substrates involved in non-immune signaling pathways. This review aimed to present IAP ubiquitination substrates and the role of IAP-mediated ubiquitination in regulating signaling pathways.

Sasa quelpaertensis Nakai extract induces p53-independent apoptosis via the elevation of nitric oxide production in human HCT116 colon cancer cells.

Induction of apoptosis in human cancer cells by Sasa quelpaertensis Nakai has been considered to be a potential therapeutic target for cancer treatment; however, the underlying mechanisms of action are not well understood. The present study investigated the role of nitric oxide (NO•) and inhibitors of apoptosis (IAPs) during apoptosis induced by Sasa quelpaertensis Nakai extracts (SQE) in p53-wild type (WT) and p53-null HCT116 colon carcinoma cells. Trypan blue exclusion and Annexin V/propidium iodide assays were used to test for antiproliferation, and apoptosis and cell cycle. Griess and reverse transcription-polymerase chain reaction and western blotting assays were carried out to assay NO• production, and to detect the mRNA and protein levels of Bcl-2, PARP and IAPs. A colorimetric assay was utilized to measure the time-dependent increase in caspase-3 activity. SQE inhibited cell growth and promoted apoptosis by the elevation of NO• in a dose- and time-dependent manner. In addition, both cell types underwent a reduction in mRNA and protein levels of IAPs (survivin, CIAP-1 and -2, and X-linked inhibitor of apoptosis) as well as anti-apoptotic Bcl-2, whereas an increase in protein expression of poly (ADP-ribose) polymerase 1 and caspase 3 activity was observed; however, an equivalent cytotoxic and apoptotic effect by SQE was observed in p53-WT and p53-null cells. Collectively, the results indicated that SQE-induced apoptosis was independent of p53 status and associated with modulation of endogenous NO• and IAP family gene expression.

Inhibition of NF-kappaB transcriptional activity enhances fucoxanthinol-induced apoptosis in colorectal cancer cells

BackgroundEvidence from epidemiological and experimental studies has shown that the etiology of colorectal cancer (CRC) is related to lifestyle, mainly diet. At the same time, there are many foods and beverages that have been shown to provide protection against CRC. We turned our attention to a traditional Japanese food, brown algae, that contains carotenoids and various functional polyphenols, especially fucoxanthin (FX) and fucoxanthinol (FxOH).ResultsBoth FX and FxOH treatments induced apoptosis in a dose-dependent and time-dependent manner as detected by annexin V / propidium iodide and the presence of a subG1 population in human colon cancer HCT116 cells. This apoptotic effect of FxOH was stronger than that of FX. We also found that nuclear factor-kappa B (NF-κB) transcriptional activity was significantly increased by treatment with ≥5 μM FxOH. Thus, we cotreated the cells with FxOH plus NF-κB inhibitor, and the results demonstrated that this cotreatment strongly enhanced the induction of apoptosis compared with the effects of FxOH or NF-κB inhibitor treatment alone and resulted in X-linked inhibitor of apoptosis (IAP) downregulation.ConclusionsThis study suggested that FxOH is a more potent apoptosis-inducing agent than FX and that its induction of apoptosis is enhanced by inhibiting NF-κB transcriptional activity via suppression of IAP family genes.

Impact of inhibitor of apoptosis proteins on immune modulation and inflammation.

The routes leading to programmed cell death are as tightly regulated as those of cellular growth and proliferation, and a finely synchronized balance between the life and death of cells ensures proper organ size and function. Inhibitors of apoptosis (IAPs) proteins were initially characterized by their ability to directly bind and inhibit apoptotic caspases. However, recent studies have clarified that IAPs are much more functionally versatile, modulating a vast range of signaling pathways that have an impact on antimicrobial responses, tumorigenesis, metastasis and cellular migration. A significant contribution of IAPs in tumorigenesis is their inherent function as E3 ubiquitin ligases to modulate cellular signaling downstream of death receptors or certain pattern recognition receptors. In this review, we focus on modulation of the innate and adaptive immune systems, macrophage plasticity and inflammatory responses by IAP family members. We also explore the rationale to target IAPs pharmacologically for the treatment of a number of inflammatory diseases and cancer.

Characterization of Potent SMAC Mimetics that Sensitize Cancer Cells to TNF Family-Induced Apoptosis.

Members of the Inhibitor of APoptosis (IAP) protein family suppress apoptosis within tumor cells, particularly in the context of immune cell-mediated killing by the tumor necrosis factor (TNF) superfamily cytokines. Most IAPs are opposed endogenously by the second mitochondrial activator of caspases (SMAC), which binds to selected baculovirus IAP repeat (BIR) domains of IAPs to displace interacting proteins. The development of SMAC mimetics as novel anticancer drugs has gained impetus, with several agents now in human clinical trials. To further understand the cellular mechanisms of SMAC mimetics, we focused on IAP family members cIAP1 and cIAP2, which are recruited to TNF receptor complexes where they support cell survival through NF-κB activation while suppressing apoptosis by preventing caspase activation. We established fluorescence polarization (FP) assays for the BIR2 and BIR3 domains of human cIAP1 and cIAP2 using fluorochrome-conjugated SMAC peptides as ligands. A library of SMAC mimetics was profiled using the FP assays to provide a unique structure activity relationship (SAR) analysis compared to previous assessments of binding to XIAP. Potent compounds displayed mean inhibitory binding constants (Ki) of 9 to 27 nM against the BIR3 domains of cIAP1 and cIAP2, respectively. Selected compounds were then characterized using cytotoxicity assays in which a cytokine-resistant human tumor cell line was sensitized to either TNF or lymphotoxin-α (LT-α). Cytotoxicity correlated closely with cIAP1 and cIAP2 BIR3 binding activity with the most potent compounds able to reduce cell viability by 50%. Further testing demonstrated that active compounds also inhibit RIP1 binding to BIR3 of cIAP1 and cIAP2 in vitro and reduce steady-state cIAP1 protein levels in cells. Altogether, these data inform the SAR for our SMAC mimetics with respect to cIAP1 and cIAP2, suggesting that these IAP family members play an important role in tumor cell resistance to cytotoxicity mediated by TNF and LT-α.

Cellular inactivation of nitric oxide induces p53-dependent apoptosis in human melanoma cells

Purpose: To examine the role of endogenous nitric oxide (NO • ) and influence of p53 status during apoptosis induced by a selective iNOS inhibitor, N-[(3-aminomethyl) benzyl] acetamidine (1400W), and/or an NO • scavenger carboxy-PTIO (c-PTIO) in two isogenic human melanoma cell lines, wild-type p53 (A375) and p53 mutant (SK mel-28) cells. Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Annexin V/propidium iodide assay were used to test for antiproliferation and apoptosis, respectively. Griess and reverse transcription-polymerase chain reaction (RT-PCR) reactions were carried out to assay NO • production and the mRNA levels of inhibitors of apoptosis (IAP). Results: c-PTIO and 1400W, alone or in combination, inhibited cell growth and promoted apoptosis via sub-G1 cell cycle arrest mediated by decrease in NO • . Apoptosis was delayed and greatly reduced in magnitude in SK mel-28 cells, underscoring the importance of p53 modulation of the response. In both cell types, apoptosis induced by iNOS inhibition and/or NO • depletion was blocked by an exogenous NO • donor, sodium nitroprusside. It was also found that inhibitors of apoptosis family (survivin, XIAP and cIAP1) were significantly depressed, which appear to play an important role in the regulation of p53- mediated apoptotic response under these conditions. Conclusion: The data obtained provide insight into the mechanism of cell proliferation action of endogenous NO • , based on p53 status, and indicate manipulation of iNOS may offer exciting opportunities to improve the effectiveness of melanoma treatment. Keywords: Apoptosis, Human melanoma cells, Inducible nitric oxide synthase, p53

Abstract 371: The Role of Survivin in Cardiac Regeneration

Heart failure continues to be the major cause of mortality in many countries. How to reduce the loss of cardiac function after injury continues to be a major challenge in cardioprotective treatment. Survivin (SVV) is the smallest member in IAP (Inhibitor of apoptosis) family and has multiple functions such as inhibiting cell apoptosis and enhancing cell proliferation. SVV expresses in nucleus of proliferating cardiomyocyte during heart development but disappear in adult stage. However after injury, SVV is induced in the cytoplasm of cardiomyocyte in peri-infarct region. We established inducible conditional SVV knockout transgenic mouse to investigate the importance of SVV after myocardial infarction. Cardiac-specific SVV knockout mice exhibit poor cardiac function and more apoptotic cardiomyocytes compared to wild-type mice after injury. In order to confirm localization is related to the function of SVV, we have established adenovirus carrying SVV or SVV fused with NLS (nuclear-localization signal). SVV locates in cytoplasm after virus infection but NLS-SVV could be detected in both cytoplasm and nucleus. Both SVV and NLS-SVV inhibited cell apoptosis in vitro but only NLS-SVV enhanced neonatal mouse cardiomyocyte proliferation. Mice received intramyocardial injection of virus shows better ejection fraction after myocardial infarction and less apoptotic cardiomyocytes in peri-infarct region, and more H3P-positive cardiomyocytes are observed in border zone overexpressing NLS-SVV group. We believe this dual functional SVV can be applied in future cardioprotective therapy.

Abstract B13: Differential expressions of stress proteins and miRNAs in circulating exosomes in prostate cancer health disparities

Abstract African American men are about 1.5 times more likely to develop prostate cancer and about 2.5 times more likely to die from prostate cancer. However, there's no known cause of increased rate of morbidity and mortality in these population. Stress is well known factor for the causation of health disparities. Stress is a known inducer of post-transcriptional modification in cellular levels. Survivin, a member of the inhibitor of apoptosis family can get alternatively spliced both transcriptionally and post-transcriptionally. We have shown that survivin and its splice variants are secreted from cells via exosomes, small membrane-bound vesicles used in cell-cell and cell-extracellular communication (1,2). MicroRNAs (miRNA) are small RNA, play regulatory role in tumor development and prognosis. Main objective of the present study is to find out differential expression patterns of stress proteins and miRNA from circulating exosomes deived from prostate cancer in African American (PCa-AA) and Caucasian (PCa-CC) patient populations. This is a novel way of establishing non-invasive early detection intervention that can be tailored to Americans of different ethnicities. In this study, exosomes are collected from sera of PCa-AA and PCa-CC using the commercially available ExoQuick kit. miRNAs are extracted from the exosomes using QIAGEN miRNA kit. Differential expressions of IAPs, wild type survivin and its splice variants are noted in the exosomes of AA-prostate cancer patients' sera compared to CC-prostate cancer sera. Differentially expressed miRNAs are identified by sequencing analysis. We find that this observation will ultimately pave the way for future studies focused on analyzing these biomarkers in larger cohorts of ethnically diverse cancer patients. Citation Format: Salma Khan, TessaRae Stiff, Jennifer Simpson, James C. Lynch, Patrick Leaf, Amber Gonda, saeid mirshahidi, Tino Sanchez, Carlos A. Casiano, Nathan R. Wall. Differential expressions of stress proteins and miRNAs in circulating exosomes in prostate cancer health disparities. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B13.

A Genome-Wide Association Study of Myeloma Survival in Utah Uncovers Germline Variants That May Influence Survival of Multiple Myeloma Patients

Background. Recently, the first genome-wide association study (GWAS) for multiple myeloma (MM) survival was performed. It identified 16p13 as a candidate region for germline risk variants influencing survival1. The Utah study contributed to a successful replication of this region. Here, we perform a GWAS of MM survival in 318 MM cases residing or being treated in Utah to identify additional SNPs associated with MM prognosis.Methods. Utah cases were treated in the Huntsman Cancer Hospital (HCH) and/or Intermountain Healthcare systems, and were ascertained either though the HCH or the Utah Cancer Registry. Cases were genotyped on the Illumina 610Q high-density SNP array containing over 600,000 variants across the genome, imputed to the 1000 Genomes sequence data. Only imputed genotypes with information content >0.7 and certainty >0.9 were considered. For all variants (genotyped or imputed) with minor allele frequency of ≥2%, we estimated genome-wide hazard ratios using Cox models to compare survival between MM carriers of major or minor SNP alleles. Models were adjusted for sex, age at diagnosis, and principal components to adjust for possible population stratification as covariates. Disease stage and treatment were considered in sensitivity analyses to ensure that the hazard ratios were robust to these important factors. All Cox analyses were performed in R and Kaplan-Meier estimates of the survival function were graphed. Suggestive regions (p <1×10-6) were taken forward to replication in the UCSF study (352 patients). Combined analyses using METAL were calculated, including Cochran's Q test of heterogeneity.Results. Of 16 regions with p <1×10-6, we identified four regions on chromosomes 9, 10, 11 and 16 that were replicated in the UCSF data (p =0.007 to 0.06): (1) 9q22 rs12346172, p =5.7×10-7, HR=7.7; (2) 10p12.2 rs67205241, p =8.0×10-7, HR=3.1; (3) 11q22 rs142677811, p =1.3×10-9, HR=8.6 (see Figure 1); and, (4) 16q24 rs87814765, p =8.5×10-7, HR=2.1. These regions maintain genome-wide significance (rs142677811, p =2.1×10-8) or near-significance in a Utah-UCSF Meta analysis. Estimates were not sensitive to differences in staging or treatment.Conclusions. We observed that germline variants near genes PTCH1 (9q22.3; tumor suppressor in the patched gene family), EBLN1 (10p12.31), BIRC2 and BIRC3 (inhibitors of apoptosis; 11q22), YAP1 (oncogene in the Hippo signaling pathway; 11q13) and SLC7A5 (16 q24.3; an L-type amino-acid transporter recently associated with poor prognosis in MM2) were associated with decreased survival among MM patients. Somatic mutation and copy number loss for BIRC2/3, proteins in the inhibitor of apoptosis family, are frequently detected in lymphoid malignancies3. Nuclear ABL1 triggers cell death through its interaction with the Hippo pathway coactivator YAP1 in normal cells; it has been shown that low YAP1 levels prevent nuclear ABL1-induced apoptosis in hematological malignancies4.

Abstract LB-A16: Novel Small Molecule ONC201 Induces Cell Death in Triple Negative and Non-triple Negative Breast Cancer Cells

Abstract Breast cancer is a major cause of cancer related death in United States women, and there is an urgent need for novel targeted therapies with low toxicity and increased efficacy. The TRAIL pathway has been of interest in the field of oncology, but resistance has been identified in cancer cell lines and primary tumors including those of the breast. Only a subset of triple negative breast cancers (TNBC) is sensitive to TRAIL, with other breast cancer molecular subtypes being resistant (Rahman et al., Breast Cancer Res. Treat., 2008). Clinical translation of these findings has been limited, as TRAIL receptor agonistic antibodies have shown minimal effects. We identified a small molecule inducer of the TRAIL pathway, ONC201/TIC10 (Allen et al., Sci. Trans. Med., 2013) that functions through dual inhibition of AKT/ERK, induction of the ATF4/CHOP pathway (Kline et al., Submitted, 2015) and cytotoxic effects dependent on TRAIL/DR5 upregulation. Recently, ONC201 completed its first-in-man phase 1 clinical trial in advanced solid tumors that defined its safety, pharmacokinetics and recommended phase II dose (Stein et al., Abstract C138, 2015 AACR-EORTC meeting). The objective of this work was to investigate ONC201 efficacy in TNBC (TRAIL-sensitive) and non-TNBC (TRAIL-resistant) cells. Using cell viability assays, we demonstrate IC50 values for ONC201 in the low micromolar range for both TNBC (n = 6) and non-TNBC cells (n = 5). These doses are achievable based on human PK. The evaluated non-TNBC cell lines, including those with estrogen receptor positivity and HER2 amplifications, have been shown to be highly resistant to TRAIL therapy. Propidium iodide staining and analysis of SubG1 DNA content indicates that ONC201 induces apoptosis in both TNBC and non-TNBC cells. We also show that a single dose of ONC201 is well tolerated and efficacious in vivo against the MDA-MB-231 triple negative human breast cancer xenograft model. The effects of ONC201 on well-known mechanisms of TRAIL-resistance, such as increases in inhibitor of apoptosis (IAP) family proteins, were investigated to explain sensitivity of TRAIL-resistant breast cancer cells to ONC201. We observed that ONC201 mediates a decrease in expression of IAP family proteins XIAP, c-IAP1, and c-IAP2 across TNBC and non-TNBC cell lines. We have shown that XIAP levels correlate with sensitivity to ONC201-induced apoptosis (Kline et al., Submitted, 2015). We also examined levels of death receptors DR4 and DR5 using flow cytometry, as a known mechanism of TRAIL resistance involves constitutive death receptor endocytosis from the cell surface (Zhang et al., Mol. Cancer Res., 2008). Interestingly, increases in cell surface DR4 and DR5 levels are greater in TNBC cell lines when compared with non-TNBC cell lines. Overall, our findings suggest that ONC201 exerts cytotoxic effects against a broad range of breast cancer cells, including TNBC and non-TNBC subtypes. These effects are observed regardless of the TRAIL sensitivity of the cells, and may be mediated through mechanisms involving downregulation of anti-apoptotic proteins and upregulation of cell surface death receptors. Our work will further understanding of these mechanisms and contribute to development of a preclinical rationale for the use of ONC201 as a treatment for breast cancers. Citation Format: Marie D. Baumeister, Jessica Wagner, Christina L.B. Kline, Joshua E. Allen, David T. Dicker, Wafik S. El-Deiry. Novel Small Molecule ONC201 Induces Cell Death in Triple Negative and Non-triple Negative Breast Cancer Cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A16.

Abstract 5382: Epigenetic targeting of survivin enhances paclitaxel-mediated antitumor activity against non-small-cell lung cancer

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide and has a low 5-year survival rate. The current treatment for lung cancer patients is surgical resection followed by chemotherapy. However, the majority of patients will eventually experience disease progression and require further treatment. Increased evidence indicates that overexpression of survivin, the smallest member of IAP (inhibitor of apoptosis) family, results in poor prognosis, tumor recurrence, and drug resistance in various cancers, including non-small-cell lung cancer (NSCLC), which accounts for approximately 85% of all lung cancer cases. Although the precise mechanism leading to survivin overexpression in cancer cells is not fully understood, approach to effectively downregulate survivin is thought to be required to enhance chemotherapy, reduce relapse, and improve the survival of NSCLC patients. In the present study, we have explored the therapeutic potential of the class I HDAC inhibitor (HDACi) entinostat (or MS-275, SNDX-275, LC Laboratories, Woburn, MA) in combination with paclitaxel in the treatment of NSCLC. We show that entinostat significantly enhances paclitaxel-induced anti-proliferative/anti-survival effects and apoptosis in NSCLC cells. It appears that this HDACi selectively reduces the expression levels of survivin, but not the functionally-related molecules Mcl-1 and Bcl-xL. Further studies reveal that entinostat induces expression of two survivin-targeting miRNAs, miR-203 and miR-542-3p; and that both survivin-specific shRNAs and the mimics of miR-203 and/or miR-542-3p effectively downregulate survivin and dramatically promote NSCLC cells undergoing apoptosis upon paclitaxel treatment. Moreover, in a tumor xenografts model-established from NSCLC cells, entinostat maintains its capability to upregulate miR-203 and downregulate survivin in vivo. Importantly, the combinations of entinostat and paclitaxel exhibit a striking activity, as compared to either agent alone, to inhibit tumor growth. These data indicate that entinostat is efficacious to enhance paclitaxel-mediated anti-NSCLC activity. Our findings suggest that epigenetic targeting of survivin with entinostat or miRNA-replacement therapy may be a novel strategy to re-sensitize NSCLC to chemotherapy. Numerous studies demonstrate that entinostat exerts potent antitumor activity in various human cancers. Its clinic activity in NSCLC is currently being tested in combination with the DNA methyltransferase inhibitor Azacitidine or EGFR inhibitor erlotinib, but not conventional chemotherapy (http://www.clinicaltrial.gov/ct2/results?term = entinostat&amp;Search = Search). Our studies provide a strong rationale to initiate clinical evaluation of the combinatorial effects of entinostat and paclitaxel on NSCLC patients. Citation Format: Shuiliang Wang, Ling Zhu, Zhiyong Zeng, Lianghu Huang, Fengjin Lin, Rong Lin, Jin Wang, Jun Lu, Qinghua Wang, Lingjing Lin, Huiyue Dong, Weizhen Wu, Kai Zheng, Jinquan Cai, Shunliang Yang, Yujie Ma, Shixin Ye, Wei Liu, Yinghao Yu, Bolin Liu, Jianming Tan. Epigenetic targeting of survivin enhances paclitaxel-mediated antitumor activity against non-small-cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5382. doi:10.1158/1538-7445.AM2015-5382

Abstract 17: Xiap over-expression is a poor prognostic marker in breast cancer and can be targeted to induce efficient apoptosis

Abstract Breast cancer is the most common cancer in females and is ranked only second to lung cancer in cancer-related deaths all over the world in women. Despite improvement in diagnosis and management of breast cancer, the survival rate of this disease for stage 4 disease is still only 22%. X-linked Inhibitor of Apoptosis (XIAP) is a member of the inhibitor of apoptosis family of proteins that has been shown to be over-expressed in a variety of cancers including breast cancer leading to an overall poor survival. However, the role of XIAP in breast cancer from Middle Eastern region has not been fully explored. We therefore examined the expression of XIAP in a cohort of 964 breast cancer cases by immunohistochemistry and found that XIAP was over-expressed in 29.5% (284/964) of the cases and was found to be directly associated with clinical parameters such as tumor size (p = 0.0044), extra nodal extension (p = 0.0041) and poorly differentiated breast cancer (p&amp;lt;0.0001). In addition, XIAP over-expression was also significantly associated with activated AKT (p&amp;lt;0.0001), Ki-67 (p&amp;lt;0.0001) and PARP (p&amp;lt;0.0001). Finally, XIAP over-expression in our cohort of breast cancer was an independent poor prognostic marker in multivariate analysis. Next, we investigated inhibition of XIAP using a specific inhibitor; Embelin on a panel of breast cancer cell lines and found that Embelin treatment led to inhibition of cell viability and induction of apoptosis in breast cancer cells. In order to identify the exact pathway involved in XIAP inhibition, we treated breast cancer cells with different doses of Embelin and found that there was down-regulation of XIAP leading to activation of caspases-9, -3 and PARP. We also found that Embelin treatment caused cleavage of caspase-8 and truncation of Bid leading to activation of the mitochondrial apoptotic pathway and efficient apoptosis. Finally, as our clinical data demonstrated a significant association between XIAP over-expression and activation of AKT in breast cancer samples, we synergistically induced apoptosis in breast cancer cells using sub-optimal doses of Embelin and PI3-kinase inhibitor; LY294002. These data suggest that XIAP may be playing an important role in the pathogenesis of breast cancer and XIAP can be therapeutically targeted either alone or in combination with PI3-kinase inhibition to induce efficient apoptosis in breast cancer cells. Citation Format: Azhar R. Hussain, Maqbool Ahmed, Rong Bu, Shaham Beg, Alanood M. Alrashed, Roxanne Melosantos, Dahish S. Ajarim, Shahab Uddin, Khawla S. AlKuraya. Xiap over-expression is a poor prognostic marker in breast cancer and can be targeted to induce efficient apoptosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 17. doi:10.1158/1538-7445.AM2015-17

Expression of IAP family proteins and its clinical importance in breast cancer patients.

Inhibitor of apoptosis (IAP) family proteins is involved in mechanisms of resistance to apoptosis in various cancer cells. The aim of this study was to assess the expression of selected IAP proteins such as XIAP, cIAP-1, cIAP-2 and survivin in breast cancer patients and evaluates their relationship with the prognostic and predictive factors and their impact to overall survival (OS) and progression free survival (PFS). The study was conducted with the use of tissue samples prospectively collected from 92 previously untreated female breast cancer patients. The control encompassed 10 fibroadenoma patients. The expression of XIAP, cIAP-1, cIAP-2 and survivin was assessed using flow multicolor cytometry. XIAP expression was present in 99 % of the breast cancer patients (91/92) with the median expression 13.65% (range 1-66.8%). Expression of XIAP in breast cancer was significantly higher compared to the control group (p=0.006). Median expression of cIAP-1, cIAP-2 and survivin in the study group was 25.95% (range 0.8-83.7%), 16.7% (range 1-53.2%) and 4.6% (range 0-43%) respectively. In the rank Spearman test, strong correlations (p<0.001) were seen among the expressions of XIAP, cIAP-2 and survivin, in all combination. Additionally, week correlation between XIAP and cIAP-1 was observed (p=0.02). The median expression of XIAP and survivin was significantly higher in more advanced tumors (stages pT2/pT3 vs. pT1). The median PFS and OS in breast cancer group were 46.15 and 47.1 months respectively. No significant correlations were observed among expressions of IAP family proteins and survival. However, low expression of XIAP in breast cancer showed trend to longer PFS (p=0.08). XIAP, cIAP-1 cIAP-2 and survivin participate in antiapoptotic mechanisms in breast cancer and XIAP and survivin seem to have the most significant prognostic importance. Further studies are needed to establish more complete prognostic and predictive values of IAP family proteins in breast cancer patients.

Tumor necrosis factor-α-induced apoptosis of gastric cancer MKN28 cells: Accelerated degradation of the inhibitor of apoptosis family members

The role of the inhibitor of apoptosis (IAP) family members in tumor necrosis factor-α (TNF-α)-induced apoptosis of human gastric cancer MKN28 cells was explored. TNF-α induced up-regulation of cIAP2, whereas cycloheximide (CHX) induced down-regulation of XIAP and survivin. Degradation of cIAP1 and XIAP, but not survivin, was accelerated by co-treatment of cells with TNF-α and CHX, and TNF-α-induced up-regulation of cIAP2 was inhibited by BMS-345541 (NF-κB inhibitor). Treatment of MKN28 cells with TNF-α plus CHX induced degradation of survivin and activation of caspase-8 and -3, followed by degradation of cIAP1 and XIAP and apoptosis. Proteasome inhibitors (MG132 and epoxomicin) suppressed TNF-α plus CHX-induced degradation of survivin, cIAP1, and XIAP as well as apoptosis. A caspase inhibitor (z-VAD-fmk) suppressed TNF-α plus CHX-induced apoptosis, but allowed degradation of survivin, cIAP1 and XIAP. TNF-α receptor 1 and 2 were expressed on MKN28 cells. The magnitude of apoptosis induced by TNF-α plus BMS-345541 was much less than that induced by TNF-α plus CHX. These findings suggest that TNF-α plus CHX-induced apoptosis of gastric cancer MKN28 cells may be caused by accelerated degradation of the IAP family members (survivin, cIAP1, and XIAP), in addition to inhibition of NF-κB-dependent synthesis of anti-apoptotic molecules.