A phase I study of LCL161, a novel oral pan-inhibitor of apoptosis protein (IAP) antagonist, in Japanese patients with advanced solid tumors.

INTRODUCTION: LCL161 is a novel oral pan-inhibitor of apoptosis protein (IAP) antagonist. LCL161 enhances paclitaxel activity in cell lines and xenograft models. A phase I study of LCL161 combined with paclitaxel for the treatment of Japanese patients with advanced solid tumors was conducted. METHODS: Each patient received oral LCL161 in a single weekly dose on days 1, 8, and 15 of a 21-day treatment cycle. In the second cycle, patients received a combination treatment with weekly paclitaxel (80 mg/m2 ) whenever possible. A Bayesian logistic regression model by escalation with the overdose control principle was used. RESULTS: Nine patients were treated with LCL161 at a dose of 600 mg (five patients) or 1200 mg (four patients). Seven patients were treated with LCL161 plus paclitaxel, and two patients received only LCL161 monotherapy. Because this study was terminated early due to a change in the LCL161 development strategy, the maximum tolerated dose (MTD) was not determined. One patient treated with LCL161 monotherapy at a dose of 1200 mg experienced dose limitind toxicity (grade 3 maculopapular rash). Another patient died on day 86 of bacterial pneumonia, which was suspected to be related to the study treatment. The most common serious adverse events were infections and infestations (n = 3). CONCLUSION: The present study suggests that the risk of infection may increase when LCL161 is combined with paclitaxel, but other conclusions about the MTD, pharmacokinetic profile, and preliminary activity of the combination of LCL161 plus paclitaxel were not drawn.

Exploratory window-of-opportunity trial to investigate the tumor pharmacokinetics/pharmacodynamics of the IAP antagonist Debio 1143 in patients with head and neck cancer.

Inhibitor of apoptosis proteins (IAPs) regulate apoptosis and modulate NF-κB signaling thereby driving expression of genes involved in immune/inflammatory responses. The orally available IAP antagonist Debio 1143 has potential to enhance tumor response to chemoradiotherapy and/or immunotherapy. Patients with pre-operative squamous cell carcinomas of the head and neck (SCCHN) received: Debio 1143 monotherapy (200 mg/day [D]1-15 +/- 2); Debio 1143 (200 mg/day D1-15 +/- 2) plus cisplatin (40 mg/m2 D 1 and 8); cisplatin alone (40 mg/m2 D 1 and 8; EudraCT: 2014-004655-31). Pharmacokinetic/pharmacodynamic effects were assessed in plasma and resected tumors. Primary end point; effect of Debio 1143 on cellular IAP-1 (cIAP-1). Levels of cIAP-1/-2, X-linked inhibitor of apoptosis protein (XIAP), tumor infiltrating lymphocytes (TILs), including CD8+ T cells, programmed cell death protein 1 (PD-1), PD-ligand 1 (PD-L1), and gene expression were also analyzed. Twenty-three of 26 patients completed treatment. In the Debio 1143 monotherapy cohort (n = 13), mean tumor concentrations of Debio 1143 were 18-fold (maximum 55.2-fold) greater than in plasma, exceeding the half-maximal inhibitory concentration for cIAPs and XIAP by 100 to 1000-fold, with significant engagement/degradation of cIAP-1 (p < 0.05). Overall, levels of CD8+ TILs, PD-1, and PD-L1 positive immune cells increased significantly (p < 0.05) following Debio 1143 treatment. Changes were observed in the expression of genes related to NF-κB signaling. Treatments were well-tolerated. Debio 1143 penetrated SCCHN tumors, engaged cIAP-1, and induced immune inflammatory changes in the tumor microenvironment. Based on the mode of action demonstrated here and in previous studies, these data support future combinations of Debio 1143 with immune-checkpoint agents.

Novel targeted mtLivin nanoparticles treatment for disseminated diffuse large B-cell lymphoma.

We previously showed that Livin, an inhibitor of apoptosis protein, is specifically cleaved to produce a truncated protein, tLivin, and demonstrated its paradoxical proapoptotic activity. We further demonstrated that mini-tLivin (MTV), a 70 amino acids derivative of tLivin, is a proapoptotic protein as potent as tLivin. Based on these findings, in this study we aimed to develop a venue to target MTV for the treatment of diffuse large B-cell lymphoma (DLBCL). MTV was conjugated to poly (lactide-co-glycolic acid) surface-activated nanoparticles (NPs). In order to target MTV-NPs we also conjugated CD40 ligand (CD40L) to the surface of the NPs and evaluated the efficacy of the bifunctional CD40L-MTV-NPs. In vitro, CD40L-MTV-NPs elicited significant apoptosis of DLBCL cells. In a disseminated mouse model of DLBCL, 37.5% of MTV-NPs treated mice survived at the end of the experiment. Targeting MTV-NPs using CD40L greatly improved survival and 71.4% of these mice survived. CD40L-MTV-NPs also greatly reduced CNS involvement of DLBCL. Only 20% of these mice presented infiltration of lymphoma to the brain in comparison to 77% of the MTV-NPs treated mice. In a subcutaneous mouse model, CD40L-MTV-NPs significantly reduced tumor volume in correlation with significant increased caspase-3 activity. Thus, targeted MTV-NPs suggest a novel approach to overcome apoptosis resistance in cancer.

Bivalent SMAC Mimetics for Treating Cancer by Antagonizing Inhibitor of Apoptosis Proteins.

Inhibitors of apoptosis proteins (IAPs) inhibit caspase activity, allowing various cancers to reduce programmed cell death (apoptosis) and resist drug treatment. The second mitochondrial-derived activator of caspases (SMAC) protein is an endogenous IAP antagonist, which can be considered as a potential anticancer therapy. Small-molecule SMAC mimetics based on the Ala-Val-Pro-Ile motif have been validated as potent IAP antagonists. In particular, most bivalent SMAC mimetics, which target both the baculovirus IAP repeat 2 (BIR2) and BIR3 domains in X-linked IAP (XIAP), antagonize IAPs better than the corresponding monovalent mimetics. Here we focus on strategies for designing bivalent small-molecule SMAC mimetics and progress in using them to antagonize IAPs. We also consider their clinical potential. Our discussion will hopefully help guide further study of these interesting mimetics.

IAP Antagonists Enhance Apoptotic Response to Enzalutamide in Castration-Resistant Prostate Cancer Cells via Autocrine TNF-α Signaling.

BACKGROUND: Castration-resistant prostate cancer (CRPC) remains incurable and identifying effective treatments continues to present a clinical challenge. Although treatment with enzalutamide, a second generation androgen receptor (AR) antagonist, prolongs survival in prostate cancer patients, responses can be limited by intrinsic resistance or acquired resistance. A potential mechanism of resistance to androgen axis inhibition is evasion of apoptosis. Inhibitor of apoptosis proteins (IAPs) are found to be overexpressed in prostate cancer and function to block apoptosis and promote survival signaling. Novel, small-molecule IAP antagonists, such as AEG40995, are emerging as a strategy to induce apoptosis and increase therapeutic response in cancer. METHODS: Human prostate cancer cell lines LNCaP and C4-2 were treated with enzalutamide with or without addition of IAP antagonist AEG40995 and proliferation and survival were determined by MTS and clonogenic assay. Western blot was used to evaluate IAP protein expression changes and PARP-1 cleavage was assessed as indication of apoptosis. Flow cytometry was performed to analyze apoptosis in treated cells. Caspase activity was determined by luminescence assay. Quantitative real-time PCR and immunometric ELISA was used to assess TNF-α (transcript and protein levels, respectively) in response to treatment. RESULTS: In this study, we demonstrate that IAP antagonist AEG40995 exhibits minimal effects on prostate cancer cell proliferation or survival, but rapidly degrades cIAP1 protein. Combination treatment with enzalutamide demonstrates that AEG40995 increases apoptosis and reduces proliferation and clonogenic survival in cell line models of prostate cancer. Mechanistically, we demonstrate that apoptosis in response to enzalutamide and IAP antagonist requires activation of caspase-8, suggesting extrinsic/death receptor apoptosis signaling. Assessment of TNF-α in response to combination treatment with enzalutamide and AEG40995 reveals increased mRNA expression and autocrine protein secretion. Blocking TNF-α signaling abrogates the apoptotic response demonstrating that TNF-α plays a critical role in executing cell death in response to this drug combination. CONCLUSIONS: These findings suggest that IAP antagonists can increase sensitivity and amplify the caspase-mediated apoptotic response to enzalutamide through TNF-α signaling mechanisms. Combination with an IAP antagonist increases enzalutamide sensitivity, lowers the apoptotic threshold and may combat drug resistance in patients with prostate cancer. Prostate 77:866-877, 2017. © 2017 Wiley Periodicals, Inc.

Magnetic albumin immuno-nanospheres as an efficient gene delivery system for a potential use in lung cancer: preparation, in vitro targeting and biological effect analysis.

Magnetic albumin immuno-nanospheres (MAINs), simultaneously loaded with super-paramagnetic iron oxide nanoparticles for targeting application and anticancer gene, plasmid-survivin/shRNA (pshRNA) and modified with anti-EGFR monoclonal antibody Cetuximab for targeting and treatment agents, were prepared for targeting lung cancer. Transmission electron microscopy images and transfection photographs, respectively, showed that magnetic nanoparticles and pshRNA were successfully encased in the albumin nanospheres. The release profiles in vitro indicated that nanospheres had an obvious effect of sustained release of pshRNA. The results of slide agglutination test and immunofluorescence analysis demonstrated that the immuno-nanospheres retained the immuno-reactivity of Cetuximab. The MAINs significantly increased adherence and uptake by GLC-82 lung cancer cells over-expressed epidermal growth factor receptor over a magnetic albumin nanospheres (MANs) control. The pshRNA-loaded MAINs formulation was more effective than equimolar doses of free Cetuximab, single magnetic targeting with pshRNA (pshRNA-loaded MANs) or single monoclonal antibody targeting with pshRNA (pshRNA-loaded AINs) in the treatment of GLC-82 lung cancer cells. Collectively, the study indicates that the novel pshRNA-loaded magnetic immuno-nanospheres represent a promising approach for magnetic and monoclonal antibody-dependent gene targeting in lung cancer therapy.

Caspase-8 activation by TRAIL monotherapy predicts responses to IAPi and TRAIL combination treatment in breast cancer cell lines.

The discovery of cancer cell-selective tumour necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis generated broad excitement and development of TRAIL receptor agonists (TRA) as potential cancer therapy. Studies demonstrating the synergistic combination effect of SMAC mimetics and TRA further suggested potentially effective treatment in multiple tumour settings. However, predictive biomarkers allowing identification of patients that could respond to treatment are lacking. Here, we described a high throughput combination screen conducted across a panel of 31 breast cancer cell lines in which we observed highly synergistic activity between TRAIL and the inhibitors of apoptosis proteins (IAP) inhibitor (IAPi) AZD5582 in ~30% of cell lines. We detected no difference in the expression levels of the IAPi or TRAIL-targeted proteins or common modulators of the apoptotic pathway between the sensitive and resistant cell lines. Synergistic combination effect of AZD5582 and TRAIL correlated with sensitivity to TRAIL, but not to AZD5582 as a single agent. TRAIL treatment led to significantly greater activity of Caspase-8 in sensitive than in resistant cell lines (P=0.002). The majority (12/14) of AZD5582+TRAIL-resistant cell lines retained a functional cell death pathway, as they were sensitive to AZD5582+TNFα combination treatment. This suggested that failure of the TRAIL receptor complex to transduce the death signal to Caspase-8 underlies AZD5582+TRAIL resistance. We developed a 3D spheroid assay and demonstrated its suitability for the ex vivo analysis of the Caspase-8 activity as a predictive biomarker. Altogether, our study demonstrated a link between the functionality of the TRAIL receptor pathway and the synergistic activity of the IAPi+TRA combination treatment. It also provided a rationale for development of the Caspase-8 activity assay as a functional predictive biomarker that could allow better prediction of the response to IAPi+TRA-based therapies than the analysis of expression levels of protein biomarkers.

Hyaluronic acid-fabricated nanogold delivery of the inhibitor of apoptosis protein-2 siRNAs inhibits benzo[a]pyrene-induced oncogenic properties of lung cancer A549 cells.

Benzo[a]pyrene (BaP), a component of cooking oil fumes (COF), promotes lung cancer cell proliferation and survival via the induction of inhibitor of apoptosis protein-2 (IAP-2) proteins. Thus knockdown of IAP-2 would be a promising way to battle against lung cancer caused by COF. Functionalized gold nanoparticle (AuNP) is an effective delivery system for bio-active materials. Here, biocompatible hyaluronic acid (HA) was fabricated into nanoparticles to increase the target specificity by binding to CD44-over-expressed cancer cells. IAP-2-specific small-interfering RNA (siRNAs) or fluorescein isothiocyanate (FITC) were then incorporated into AuNP-HA. Conjugation of IAP-2 siRNA into AuNPs-HA was verified by the UV-vis spectrometer and Fourier transform infrared spectrometer. Further studies showed that AuNP-HA/FITC were effectively taken up by A549 cells through CD44-mediated endocytosis. Incubation of BaP-challenged cells with AuNP-HA-IAP-2 siRNAs silenced the expression of IAP-2, decreased cell proliferation and triggered pronounced cell apoptosis by the decrease in Bcl-2 protein and the increase in Bax protein as well as the active form of caspases-3. The BaP-elicited cell migration and enzymatic activity of the secreted matrix metalloproteinase-2 were also substantially suppressed by treatment with AuNP-HA-IAP-2 siRNAs. These results indicated that IAP-2 siRNAs can be efficiently delivered into A549 cells by functionalized AuNP-HA to repress the IAP-2 expression and BaP-induced oncogenic events, suggesting the potential therapeutic application of IAP-2 siRNA or other siRNA-conjugated AuNP-HA composites to COF-induced lung cancer and other gene-caused diseases in the future.

Antigen-specific immune responses and clinical outcome after vaccination with glioma-associated antigen peptides and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose in children with newly diagnosed malignant brainstem and nonbrainstem gliomas.

PURPOSE: Diffuse brainstem gliomas (BSGs) and other high-grade gliomas (HGGs) of childhood carry a dismal prognosis despite current treatments, and new therapies are needed. Having identified a series of glioma-associated antigens (GAAs) commonly overexpressed in pediatric gliomas, we initiated a pilot study of subcutaneous vaccinations with GAA epitope peptides in HLA-A2-positive children with newly diagnosed BSG and HGG. PATIENTS AND METHODS: GAAs were EphA2, interleukin-13 receptor alpha 2 (IL-13Rα2), and survivin, and their peptide epitopes were emulsified in Montanide-ISA-51 and given every 3 weeks with intramuscular polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose for eight courses, followed by booster vaccinations every 6 weeks. Primary end points were safety and T-cell responses against vaccine-targeted GAA epitopes. Treatment response was evaluated clinically and by magnetic resonance imaging. RESULTS: Twenty-six children were enrolled, 14 with newly diagnosed BSG treated with irradiation and 12 with newly diagnosed BSG or HGG treated with irradiation and concurrent chemotherapy. No dose-limiting non-CNS toxicity was encountered. Five children had symptomatic pseudoprogression, which responded to dexamethasone and was associated with prolonged survival. Only two patients had progressive disease during the first two vaccine courses; 19 had stable disease, two had partial responses, one had a minor response, and two had prolonged disease-free status after surgery. Enzyme-linked immunosorbent spot analysis in 21 children showed positive anti-GAA immune responses in 13: to IL-13Rα2 in 10, EphA2 in 11, and survivin in three. CONCLUSION: GAA peptide vaccination in children with gliomas is generally well tolerated and has preliminary evidence of immunologic and clinical responses. Careful monitoring and management of pseudoprogression is essential.

Inhibitors of apoptosis proteins in experimental benign prostatic hyperplasia: effects of serenoa repens, selenium and lycopene.

BACKGROUND: The apoptosis machinery is a promising target against benign prostatic hyperplasia (BPH). Inhibitors of apoptosis proteins (IAPs) modulate apoptosis by direct inhibition of caspases. Serenoa Repens (SeR) may be combined with other natural compounds such as Lycopene (Ly) and Selenium (Se) to maximize its therapeutic activity in BPH. We investigated the effects of SeR, Se and Ly, alone or in association, on the expression of four IAPs, cIAP-1, cIAP-2, NAIP and survivin in rats with experimental testosterone-dependent BPH. Moreover, caspase-3, interleukin-6 (IL-6) and prostate specific membrane antigen (PSMA) have been evaluated.Rats were administered, daily, with testosterone propionate (3 mg/kg/sc) or its vehicle for 14 days. Testosterone injected animals (BPH) were randomized to receive vehicle, SeR (25 mg/kg/sc), Se (3 mg/kg/sc), Ly (1 mg/kg/sc) or the SeR-Se-Ly association for 14 days. Animals were sacrificed and prostate removed for analysis. RESULTS: BPH animals treated with vehicle showed unchanged expression of cIAP-1 and cIAP-2 and increased expression of NAIP, survivin, caspase-3, IL-6 and PSMA levels when compared with sham animals. Immunofluorescence studies confirmed the enhanced expression of NAIP and survivin with a characteristic pattern of cellular localization. SeR-Se-Ly association showed the highest efficacy in reawakening apoptosis; additionally, this therapeutic cocktail significantly reduced IL-6 and PSMA levels. The administration of SeR, Se and Ly significantly blunted prostate overweight and growth; moreover, the SeR-Se-Ly association was most effective in reducing prostate enlargement and growth by 43.3% in treated animals. CONCLUSIONS: The results indicate that IAPs may represent interesting targets for drug therapy of BPH.

Recombinant attenuated Salmonella typhimurium carrying a plasmid co-expressing ENDO-VEGI151 and survivin siRNA inhibits the growth of breast cancer in vivo.

The aim of this study was to investigate the antitumor effect of a plasmid co-expressing ENDO-VEGI151 and survivin siRNA on breast cancer in nude mice, and to explore the feasibility of attenuated Salmonella typhimurium (S. typhimurium) as a delivery vector for cancer gene therapy in vivo. Three recombinant expression plasmids pENDO­VEGI151 (pEV), pSurvivin-siRNA (psi-survivin) and co-expressing plasmid pENDO-VEGI151/survivin­siRNA (pEV/si-survivin), were transferred into the attenuated S. typhimurium strain SL7207, respectively. MDA-MB-231 cells were infected with these recombinants in vitro, and the expression of ENDO-VEGI151 and survivin was detected. In order to detect S. typhimurium distribution and gene delivery efficiency in vivo, the plasmid pEGFP-N1 which encodes green fluorescent protein was transferred into SL7207, and the recombinant known as SL-pEGFP was orally administered to tumor-bearing nude mice. The gene transfer efficiency, distribution and survival time of the SL-pEGFP in vivo were evaluated by detection of GFP fluorescence. SL-pEGFP not only infected the cancer cells effectively, but also allowed the survival and expression of specific genes mainly in the xenografts of nude mice. To further identify the anticancer effects of these recombinants in vivo, mice burdened with xenografts were randomly divided into 6 groups, which were subjected to intragastric administration of vehicle, SL7207, SL-pcDNA3.1, SL-pEV, SL-psi-survivin and SL-pEV/si-survivin, respectively. Eight weeks after implantation, tumor size, weight, inhibition rate, intratumoral microvessel density (MVD), apoptotic index (AI), ENDO­VEGI151 and survivin expression were evaluated. Compared with the SL-pEV or SL-psi-survivin-treated groups, the growth of tumors was significantly reduced in the SL-pEV/si-survivin group with an inhibition rate of 90.28 vs. 69.12 and 65.61%, respectively. MVD and the expression of survivin were decreased significantly in the SL-pEV/si-survivin-treated group, while AI increased significantly in the SL-pEV/si-survivin-treated group. These results indicated that attenuated S. typhimurium carrying the dual function plasmid pEV/si-survivin cannot only be specifically enriched in the tumor tissue, but also showed a synergistic antitumor effect in vivo.

Immunogenic enhancement and clinical effect by type-I interferon of anti-apoptotic protein, survivin-derived peptide vaccine, in advanced colorectal cancer patients.

We previously identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80-88, recognized by CD8+ cytotoxic T lymphocytes (CTL). Subsequently, we attempted clinical trials with this epitope peptide alone for some malignancies, resulting in clinical and immunological responses, although their potential was not strong enough for routine clinical use as a cancer vaccine. In the current study, to assess whether immunogenicity of the survivin-2B80-88 peptide could be enhanced with other vaccination protocols, we performed clinical trials in advanced colon cancer patients with two vaccination protocols: (i) survivin-2B80-88 plus incomplete Freund's adjuvant (IFA); and (ii) survivin-2B80-88 plus IFA and a type-I interferon (IFN), IFNα. Our data clearly indicated that, although the effect of survivin-2B80-88 plus IFA was not significantly different from that with survivin-2B80-88 alone, treatment with the vaccination protocol of survivin-2B80-88 plus IFA and IFNα resulted in clinical improvement and enhanced immunological responses of patients. Tetramer analysis of survivin-2B80-88 peptide-specific CTL demonstrated that such CTL were increased at least twofold after vaccination with this protocol in four of eight patients. In these patients, enzyme-linked immunosorbent spot (ELISPOT) results were also enhanced. Subsequent study of single-cell clone separation by cell sorting of peptide-specific CTL showed that each CTL clone was indeed not only peptide-specific but also cytotoxic against human cancer cells in the context of the expression of both HLA-A24 and survivin molecules. Taken together, these results indicate that vaccination of colon cancer patients with survivin-2B80-88 plus IFA and IFNα can be considered to be a very potent immunotherapeutic regimen, and that this protocol might work for other cancers.

Protection of CHO cells by transfer of survivin driven by ovarian-specific promoter OSP-2.

Chemotherapy is the major therapy for cancer in clinic. However, chemotherapeutic agents can harm the other tissues/organs besides cancer. Thus, there are great interests in protecting the innocents by the transfer of protective genes. There are two problems to be solved, one is the selection of protective genes and the other is the orientation of the exotic genes. Recent researches demonstrated that the principal mechanism of chemotherapeutics was through apoptosis. Hereby, introduction of anti-apoptosis genes might interrupt the processes of apoptosis to avoid side effect from chemotherapeutics. On the other hand, tissue-specific promoters, which control gene expression in a tissue-specific manner, might be an alternative tool to guarantee the location of target genes. In this research, we applied gene therapy to chemoprotection using anti-apoptosis gene survivin and ovarian-specific promoter OSP-2. The results showed that OSP-2 could specifically drive the expression of survivin in ovarian cells and survivin could protect cells via inhibiting apoptosis. This might put a light on the future of chemoprotective gene therapy.