Recombinant Human TRAIL Research Articles

Nano-TRAIL: a promising path to cancer therapy.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand, also called apo-2 ligand (TRAIL/Apo-2L), is a cytokine that triggers apoptosis by binding to TRAIL-R1 (DR4) and TRAIL-R2 (DR5) death receptors. Apoptosis occurs through either the extrinsic or intrinsic pathway. The administration of recombinant human TRAIL (rhTRAIL) or TRAIL-receptor (TRAIL-R) agonists promotes apoptosis preferentially in cancerous cells over normal cells in vitro; this phenomenon has also been observed in clinical studies. The limited efficacy of rhTRAIL in clinical trials could be attributed to drug resistance, short half-life, targeted delivery issues, and off-target toxicities. Nanoparticles are excellent drug and gene delivery systems characterized by improved permeability and retention, increased stability and biocompatibility, and precision targeting. In this review, we discuss resistance mechanisms to TRAIL and methods to overcome TRAIL resistance by using nanoparticle-based formulations developed for the delivery of TRAIL peptides, TRAIL-R agonists, and TRAIL genes to cancer cells. We also discuss combinatorial approaches of chemotherapeutic drugs with TRAIL. These studies demonstrate TRAIL's potential as an anticancer agent.

Enhanced expression of death receptor 5 is responsible for increased cytotoxicity of theophylline in combination with recombinant human TRAIL in MDA-MB-231 breast cancer cells.

Theophylline has been reported to induce cytotoxicity and cell cycle arrest in cancer cells. On the other hand, TRAIL, a secretory ligand, is known for its unique ability to induce cell death only in tumor cells. In the present study, we elucidated the mechanism behind the cytotoxic effect of theophylline in combination with recombinant human TRAIL (rhTRAIL) on cancer cell line MDA-MB-231. Cytotoxicity of theophylline in combination with TRAIL was measured via trypan blue assay and MTT assay. Protein levels were assessed using Western hybridization. Reactive oxygen species (ROS) levels were measured using 2',7'-dichlorofluorescin diacetate and mitochondrial membrane potential (MMP) assay was conducted using tetramethylrhodamine, ethyl ester. We observed theophylline in combination with rhTRAIL to be significantly cytotoxic to the cancer cells in comparison to theophylline and rhTRAIL alone. Next, western hybridization showed combination treatment to upregulate cleaved form of caspase-8, 9 and 3, in comparison to the cells treated with rhTRAIL and theophylline alone. Theophylline in combination also increased the levels of ROS and reduced MMP. Interestingly, combination treatment increased the protein level of death receptor 5 (DR5), sensitizing the cells towards TRAIL-induced apoptosis. Theophylline in combination with TRAIL significantly increases cytotoxicity in the MDA-MB-231 breast cancer cell line when compared to theophylline and rhTRAIL alone via upregulation of DR5 levels.

TRAIL promotes epithelial-to-mesenchymal transition by inducing PD-L1 expression in esophageal squamous cell carcinomas

BackgroundTumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) was initially considered an immunity guard; however, its function remains controversial. Besides immune cells, lung and colon cancer cells have also been reported to express TRAIL, which can promote tumor invasion and metastasis. However, the biological function and underlying mechanism of action of TRAIL in esophageal squamous cell carcinoma (ESCC) remain poorly elucidated.MethodsThe ESCC cells stemness, migration, and proliferation ability was assessed by sphere formation, Transwell, and CCK8 assay. The stemness- and epithelial-mesenchymal transition (EMT)- related genes expression levels were analyzed by Western blot and RT-qPCR. The signal activation was conducted by Western blot. The xenograft mouse experiments and lung metastasis model were performed to confirm our findings in vitro.ResultsHerein, we found that TRAIL is a negative predictor in patients with ESCC. To further investigate the biological function of TRAIL, we established TRAIL knockdown and overexpression ESCC cell lines and found that TRAIL induced EMT and promoted tumor aggressiveness. Furthermore, we demonstrated that TRAIL- overexpressing cells upregulated PD-L1 expression, which was dependent on the p-ERK/STAT3 signaling pathway. We obtained similar results when using recombinant human TRAIL. Finally, we validated the biological role and mechanism of action of TRAIL in vivo.ConclusionsThese findings demonstrate that TRAIL promotes ESCC progression by enhancing PD-L1 expression, which induces EMT. This may explain the failure of TRAIL preclinical trials.

Designed ferritin nanocages displaying trimeric TRAIL and tumor-targeting peptides confer superior anti-tumor efficacy

TRAIL is considered a promising target for cancer therapy because it mediates activation of the extrinsic apoptosis pathway in a tumor-specific manner by binding to and trimerizing its functional receptors, DR4 or DR5. Although recombinant human TRAIL has shown high potency and specificity for killing cancer cells in preclinical studies, it has failed in multiple clinical trials for several reasons, including a very short half-life mainly caused by instability of the monomeric form of TRAIL and rapid renal clearance of the off-targeted TRAIL. To overcome such obstacles, we developed a TRAIL-active trimer nanocage (TRAIL-ATNC) that presents the TRAIL ligand in its trimer-like conformation by connecting it to a triple helix sequence that links to the threefold axis of the ferritin nanocage. We also ligated the tumor-targeting peptide, IL4rP, to TRAIL-ATNC to enhance tumor targeting. The developed TRAIL-ATNCIL4rP showed enhanced agonistic activity compared with monomeric TRAIL. The in vivo serum half-life of TRAIL-ATNCIL4rP was ~ 16-times longer than that of native TRAIL. As a consequence of these properties, TRAIL-ATNCIL4rP exhibited efficacy as an anti-tumor agent in vivo against xenograft breast cancer as well as orthotopic pancreatic cancer models, highlighting the promise of this system for development as novel therapeutics against cancer.

IBCL-282: Genetic Polymorphisms of Death Receptor 4 (TRAIL-R1) and the Susceptibility to B-Non-Hodgkin Lymphoma among Egyptians

Context: Genetic polymorphisms of death receptor 4 (DR4), called tumor necrosis factor-related apoptosis-inducing ligand-death receptor 1 (TRAIL-R1), were involved with susceptibility to many cancers; however, there is a dearth of reports about B-non-Hodgkin lymphomas (B-NHLs), particularly from Arab-African nations. Objective: To assess the possible association between TRAIL-R1-A1322G, -C626G, and -A683C polymorphisms and B-NHL risk among Egyptians. Design: This case-control study involved 100 adult Egyptian B-NHL patients recruited from The National Cancer Institute, Cairo University. The control group consisted of 150 unrelated, age- and gender-matched healthy, Egyptian volunteers. Setting: The study is applicable in oncology clinics. Patients or other participants: B-NHL patients were diagnosed according to the WHO classification (2016). The extent of the disease was categorized according to theAnn Arbor classification, the performance status of the patients was assessed by Eastern Cooperative Oncology Group criteria, and their response to therapy was evaluated. Interventions: Genotyping of TRAIL-R1-C626G, -A683C, and -A683C was performed using PCR-based molecular assays. Main outcome measures: The highly polymorphic TRAIL-R1 gene was allied with cancer susceptibility; however, these findings are still debatable. Results: The frequency of the polymorphic alleles of -C626G and -A1322G were significantly higher in B-NHL patients compared to controls with almost two-fold increased risk of B-NHL (OR=1.76; 95% CI=1.01–3.08 and OR=2.1; 95% CI=1.61–3.74 respectively), while there was no statistical difference in the distribution of -A683C polymorphic allele between B-NHL patients and controls (OR=1; 95% CI=0.5–2). Combined genotypes analysis revealed that co-inheritance of the polymorphic genotypes of TRAIL-R1-C626G and -A1322G SNPs was associated with almost three-fold increased risk of B-NHL, while co-inheritance of the variant genotypes of -A683C and -A1322G SNPs or those of the three studied SNPs was not. Conclusions: TRAIL-R1-C626G and -A1322G polymorphisms could be considered molecular risk factors for B-NHL in Egyptian populations. Furthermore, the growing interest in TRAIL-based interventions has led to the development of recombinant human TRAIL (rhTRAIL) as a promising target therapy. Accordingly, screening for TRAIL-R1 gene polymorphisms is mandatory for selecting patients who will gain benefit from this novel therapeutic modality.

Apoptosis-related gene expression can predict the response of ovarian cancer cell lines to treatment with recombinant human TRAIL alone or combined with cisplatin

OBJECTIVES:The objectives of this study were to determine the sensitivity of ovarian cancer (OC) cell lines (TOV-21G and SKOV-3) to cisplatin and to the recombinant human TRAIL (rhTRAIL), and to evaluate the expression profile of TNFRSF10B, TNFRSF10C, TP53TG5, MDM2, BAX, BCL-2 and CASPASE-8 genes and their participation in the resistance/susceptibility mechanism of these tumor cell lines.METHODS:To determine the IC50 values associated with Cisplatin and rhTRAIL, inhibition of cell growth was observed using MTT assays in two human OC cell lines (SKOV-3 and TOV-21G). The analysis of gene expression was performed using quantitative real-time polymerase chain reaction (qRT-PCR).RESULTS:Both cell lines had different susceptibility profiles to the tested drugs. In the SKOV-3 cell line, the IC50 values for cisplatin and for rhTRAIL were 270.83 ug/mL and 196.5 ng/mL, respectively. The same concentrations were used for TOV-21G. Different gene expression profiles were observed in each tested cell line. CASPASE-8 and TNFRSF10B expression levels could predict the response of both the cell lines to rhTRAIL alone or the response to a combination of rhTRAIL and cisplatin. In addition, we observed a relationship between BCL-2 and BAX expression that may be helpful in estimating the proliferation rate of the OC cell lines.CONCLUSION:SKOV-3 and TOV-21G respond differently to cisplatin and rhTRAIL exposure, and expression of CASPASE-8 and TNFRSF10B are good predictors of responses to these treatments.

PB2071 GENETIC VARIATIONS IN TUMOR NECROSIS FACTOR‐RELATED APOPTOSIS‐INDUCING LIGAND RECEPTOR‐1 (TRAIL‐R1) GENE AND THE SUSCEPTIBILITY TO B CELL NON‐HODGKIN LYMPHOMA IN EGYPT

Background:Apoptosis is essential for normal tissue homeostasis, cellular differentiation and development. Inhibition or blockade of Tumor Necrosis Factor‐Related Apoptosis‐Inducing Ligand‐Receptor 1 (TRAIL‐R1) may alter its apoptotic function, and subsequently provide growth advantage to neoplastic cells. Genetic polymorphisms in TRAIL‐R1 gene were involved with susceptibility to many cancers, however dearth of reports about B‐non‐Hodgkin lymphomas (B‐NHLs) particularly from Arab African nations.Aims:To assess the possible association between TRAIL‐R1 ‐A1322G, ‐C626G and ‐A683 C and polymorphisms and B‐NHL risk among Egyptians.MethodsGenotyping of TRAIL‐R1 ‐C626G, ‐A683 C and ‐A683 C was performed to 100 B‐NHL patients and 150 controls.Results:The frequency of polymorphic alleles of ‐A1322G and ‐C626G was higher in B‐NHL compared to controls with almost twofold increased risk of B‐NHL (OR = 2.04; 95%CI = 1.02‐4.07 and OR = 1.76; 95%CI = 1.01‐3.07 respectively), while there was no statistical difference in the distribution of ‐A683 C polymorphic allele (OR = 1; 95%CI = 0.5‐2). Combined genotypes analysis revealed that co‐existence of the three variants conferred fourfold increased risk (OR = 4.1; 95% CI = 1.01‐17.63), while co‐inheritance of ‐C626G and ‐A1322G, and co‐inheritance of ‐A683 C and ‐A1322G conferred fivefold and threefold increased the risk respectively.Summary/Conclusion:TRAIL‐R1–C626G and ‐A1322G polymorphisms could be considered as molecular risk factors for B‐NHL in Egyptian population. Furthermore, the growing interest in TRAIL‐based interventions has led to the development of recombinant human TRAIL (rhTRAIL) as a promising target therapy. Accordingly, screening for TRAIL‐R1 gene polymorphisms is mandatory for selecting patients who will gain benefit from this novel therapeutic modality

The regulation of combined treatment-induced cell death with recombinant TRAIL and bortezomib through TRAIL signaling in TRAIL-resistant cells

BackgroundMultiple trials have attempted to demonstrate the effective induction of cell death in TRAIL-resistant cancer cells, including using a combined treatment of recombinant TRAIL and various proteasome inhibitors. These studies have yielded limited success, as the mechanism of cell death is currently unidentified. Understanding this mechanism’s driving forces may facilitate the induction of cell death in TRAIL-resistant cancer cells.MethodsThree kinds of recombinant soluble TRAIL proteins were treated into TRAIL-resistant cells and TRAIL-susceptible cells, with or without bortezomib, to compare their respective abilities to induce cell death. Recombinant TRAIL was treated with bortezomib to investigate whether this combination treatment could induce tumor regression in a mouse syngeneic tumor model. To understand the mechanism of combined treatment-induced cell death, cells were analyzed by flow cytometry and the effects of various cell death inhibitors on cell death rates were examined.ResultsILz:rhTRAIL, a recombinant human TRAIL containing isoleucine zipper hexamerization domain, showed the highest cell death inducing ability both in single treatment and in combination treatment with bortezomib. In both TRAIL-resistant and TRAIL-susceptible cells treated with the combination treatment, an increase in cell death rates was dependent upon both the dose of TRAIL and its intrinsic properties. When a syngeneic mouse tumor model was treated with the combination of ILz:rhTRAIL and bortezomib, significant tumor regression was seen as a result of the effective induction of cancer cell death. The combination treatment-induced cell death was both inhibited by TRAIL blocking antibody and caspase-dependent. However, it was not inhibited by various ER stress inhibitors and autophagy inhibitors.ConclusionsThe combination treatment with ILz:rhTRAIL and bortezomib was able to induce cell death in both TRAIL-susceptible and TRAIL-resistant cancer cells through the intracellular TRAIL signaling pathway. The efficiency of cell death was dependent on the properties of TRAIL under the environment provided by bortezomib. The combination treatment-induced cell death was not regulated by bortezomib-induced ER stress response or by autophagy.

IAPs cause resistance to TRAIL-dependent apoptosis in follicular thyroid cancer.

Follicular thyroid cancer's (FTC) excellent long-term prognosis is mainly dependent on postoperative radioactive iodine (RAI) treatment. However, once the tumour becomes refractory, the 10-year disease-specific survival rate drops below 10%. The aim of our study was to evaluate the prognostic and biological role of the TRAIL system in FTC and to elucidate the influence of small-molecule-mediated antagonisation of inhibitor of apoptosis proteins (IAPs) on TRAIL sensitivity in vitro Tissue microarrays were constructed from forty-four patients with histologically confirmed FTC. Expression levels of TRAIL and its receptors were correlated with clinicopathological data and overall as well as recurrence-free survival. Non-iodine-retaining FTC cell lines TT2609-bib2 and FTC133 were treated with recombinant human TRAIL alone and in combination with Smac mimetics GDC-0152 or Birinapant. TRAIL-R2/DR5 as well as TRAIL-R3/DcR1 and TRAIL-R4/DcR2 were significantly higher expressed in advanced tumour stages. Both decoy receptors were negatively associated with recurrence-free and overall survival. TRAIL-R4/DcR2 additionally proved to be an independent negative prognostic marker in FTC (HR = 1.446, 95% CI: 1.144-1.826; P < 0.001). In vitro, the co-incubation of Birinapant or GDC-0152 with rh-TRAIL-sensitised FTC cell lines for TRAIL-induced apoptosis, through degradation of cIAP1/2. The TRAIL system plays an important role in FTC tumour biology. Its decoy receptors are associated with poor prognosis as well as earlier recurrence. The specific degradation of cIAP1/2 sensitises FTC cells to TRAIL-induced apoptosis and might highlight a new point of attack in patients with RAI refractory disease.

Improvement of Pharmacokinetic Profile of TRAIL via Trimer-Tag Enhances its Antitumor Activity in vivo

TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) has long been considered a tantalizing target for cancer therapy because it mediates activation of the extrinsic apoptosis pathway in a tumor-specific manner by binding to and trimerizing its functional receptors DR4 or DR5. Despite initial promise, both recombinant human TRAIL (native TRAIL) and dimeric DR4/DR5 agonist monoclonal antibodies (mAbs) failed in multiple human clinical trials. Here we show that in-frame fusion of human C-propeptide of α1(I) collagen (Trimer-Tag) to the C-terminus of mature human TRAIL leads to a disulfide bond-linked homotrimer which can be expressed at high levels as a secreted protein from CHO cells. The resulting TRAIL-Trimer not only retains similar bioactivity and receptor binding kinetics as native TRAIL in vitro which are 4–5 orders of magnitude superior to that of dimeric TRAIL-Fc, but also manifests more favorable pharmacokinetic and antitumor pharmacodynamic profiles in vivo than that of native TRAIL. Taken together, this work provides direct evidence for the in vivo antitumor efficacy of TRAIL being proportional to systemic drug exposure and suggests that the previous clinical failures may have been due to rapid systemic clearance of native TRAIL and poor apoptosis-inducing potency of dimeric agonist mAbs despite their long serum half-lives.

Abstract 2316: Elevated H-Ras suppresses death receptor-mediated apoptosis in cancer cells

Abstract TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis through its death receptors (DRs) 4 and/or 5 expressed on the surface of target cells. Despite its selectivity in killing cancer cells over most normal cells, recombinant human TRAIL or its receptor agonists (monoclonal antibodies against DR4 or DR5) encountered resistance in many tumor cells while the underlying mechanisms remain partially understood. Here, we show that wild-type H-Ras GTPase is systemically upregulated in TRAIL-resistant cells compared to TRAIL-sensitive cells. The elevated H-Ras expression correlated with a deficiency of DR4 and DR5 on plasma membrane in TRAIL-resistant cell lines. Notably, knockdown of H-Ras in TRAIL-resistant cells successfully restored the surface expression of DR4 and DR5, thereby sensitizing the cells to TRAIL-induced apoptosis. Consistently, ectopic expression of H-Ras in TRAIL-sensitive cells reduced surface DR4 and DR5 which was associated with a loss of TRAIL sensitivity. By contrast, the status of K-Ras or its mutations was not casually linked to TRAIL receptor expression or TRAIL sensitivity across the panel of cancer cell lines tested. These data suggest that H-Ras may play a distinct role to negatively regulate TRAIL receptors and apoptosis. The upregulated H-Ras could be a predictor of tumor resistance to DR-targeted agents and a potential therapeutic target for combinational therapy to achieve better treatment outcomes. Citation Format: Su-Ryun Kim. Elevated H-Ras suppresses death receptor-mediated apoptosis in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2316. doi:10.1158/1538-7445.AM2017-2316

Abstract 3842: Design and engineering of TRAIL fusion proteins for cancer therapy

Abstract Protein-based agonists of apoptotic death receptors have shown remarkable preclinical efficacy but limited clinical response. The short circulating half-life of recombinant human TRAIL and the necessity of Fc-mediated clustering for potentiating agonistic antibodies against DR4 and DR5 have been proposed to be major impediments to the clinical success of this class. To address these limitations we have created Fc-scTRAIL, a single fusion polypeptide consisting of an IgG1 Fc region followed by three successive TRAIL monomers connected by two fifteen-amino acid linkers. While Fc-scTRAIL showed potent activity in vitro, we observed a low TM (48 °C) and rapid inactivation in serum indicating protein instability. Subsequently, we applied a directed evolution approach using yeast surface display to identify mutations that would stabilize the TRAIL trimer. When individual mutations were transferred to the Fc-scTRAIL format, we observed a dramatic increase in the TM (66-70 °C) while the combination of three mutations improved serum stability by ten-fold. Stabilized Fc-scTRAIL shows greater pro-apoptotic activity across a panel of cancer cell lines when compared to mapatumumab (anti-DR4) and drozitumab (anti-DR5), or the combination of antibodies even in the presence of anti-Fc cross-linking. Moreover, anti-Fc did not improve Fc-scTRAIL activity suggesting that the hexavalent design of the molecule maximizes death receptor activation. Currently, in vivo evaluation of Fc-scTRAIL for pharmacokinetic properties and activity is underway. We believe this format, when combined with an appropriate patient selection strategy, will result in improved clinical outcomes. Citation Format: Eric M. Tam, Hannah Hudson, Tamara Dake, Sara Ghassemifar, Andreas Raue, Yasmin Hashambhoy-Ramsay, Stephen L. Sazinsky, Anahita Daruwalla, Neeraj Kohli, Lihui Xu, Charlotte F. Mc Donagh, Birgit Schoeberl, Diana H. Chai. Design and engineering of TRAIL fusion proteins for cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3842.

NF-kB Regulation in T-cells in Pregnancy is Mediated via Fas/FasL Interactions: The Signal for which is Derived from Exosomes Present in Maternal Plasma

Background: Regulated suppression of maternal Th1 immunity is necessary for normal pregnancy since inappropriate Th1 responses results in increased pregnancy loss and complications including intrauterine growth restriction (IUGR). We have shown that suppression of the p65 subunit of NF-κB in maternal T-cells underlies this change in T-cell responses. This study aimed to determine mechanism(s) that control p65 suppression. Methods and findings: Maternal plasma contained particulate factor’s that suppress p65 and induce T-cell apoptosis in Jurkat T-cells and the factor’s was positive for FasL and TRAIL. Both the Fas activating antibody CH11 and recombinant human TRAIL induced Jurkat apoptosis. Specific Fas activation resulted in p65 suppression which was reversed in the presence of the Fas inactivating antibody ZB4. Despite inducing apoptosis, recombinant TRAIL did not suppress p65 expression. Maternal T-cells expressed increased Fas relative to non-pregnant controls. Fas activation in Jurkats resulted in p65 suppression thus limited IL-2 and IFNA¯ÂÂ§ transcription in response to PMA/ionomycin stimulation. In contrast, partial knockdown of Fas in Jurkats prevented suppression of p65 in response to CH11, leading to increased IL-2 and IFNA¯ÂÂ§ production when stimulated with PMA/ionomycin. FasL+ Exosomes isolated from the particulate fraction of maternal plasma specifically induced p65 suppression in Jurkats since suppression was completely reversed with ZB4. In pregnancies complicated with IUGR where Th1 cytokine production is increased, placental expression of FasL was reduced compared to normal controls. Conclusion: Taken together these data suggest that pregnancy derived FasL+ exosomes in maternal plasma regulate p65 levels in circulating T-cells through Fas activation. The expression of Fas on T-cells and FasL on exosomes both dictate the level of p65 suppression and the level of cytokine production in response to stimulation throughout pregnancy. Inappropriate expression of FasL in placental derived exosomes may underlie one mechanism that is abnormal in complications of pregnancy including IUGR.

TRAIL in Combination with Subtoxic 5-FU Effectively Inhibit Cell Proliferation and Induce Apoptosis in Cholangiocarcinoma Cells.

In the past decade, the incidence and mortality rates of cholangiocarcinoma (CCA) have been increasing worldwide. The relatively low responsiveness of CCA to conventional chemotherapy leads to poor overall survival. Recently, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has emerged as the most promising anti-cancer therapeutic agent since it is able to selectively induce apoptosis of tumor cells but not normal cells. In this study, we aimed to investigate the therapeutic effect of TRAIL in CCA cell lines (M213, M214 and KKU100) compared with the immortal biliary cell line, MMNK1, either alone or in combination with a subtoxic dose of 5-fluorouracil (5-FU). We found that recombinant human TRAIL (rhTRAIL) was a potential agent which significantly inhibited cell proliferation and mediated caspase activities (caspases 8, 9 and 3/7) and apoptosis of CCA cells. The combined treatment of rhTRAIL and 5-FU effectively enhanced inhibition of CCA cell growth with a smaller effect on MMNK1. Our finding suggests TRAIL to be a novel anti-cancer therapeutic agent and advantage of its combination with a conventional chemotherapeutic drug for effective treatment of CCA.

Effects and mechanism of arsenic trioxide in combination with rmhTRAIL in multiple myeloma

The anti-tumor potential of arsenic trioxide (ATO) and recombinant mutant human TRAIL (rmhTRAIL) has been confirmed in various kinds of tumors. However, the effects and mechanism of the two drugs in combination in multiple myeloma (MM) have not been established. In this study, we evaluated the proliferation inhibition and apoptosis induction effects of ATO and rmhTRAIL as single agents and in combination on the MM cell lines RPMI8226 and U266. Then, we used high-performance liquid chromatography and mass spectrometry to find differentially expressed proteins before and after drug treatment and to analyze the mechanism underlying the effect of ATO and rmhTRAIL on MM cells. Results indicated that ATO and rmhTRAIL had synergistic or additive effects on proliferation inhibition and apoptosis induction in MM cells, and the mechanism underlying the two-drug combination might involve regulation of the expression of several proteins affected by ATO and rmhTRAIL. Our study provides evidence of a potential new combination treatment strategy for MM.

Abstract LB-113: A novel role of Withaferin A as an effective pro-apoptotic receptor agonist (PARA) and insights into the underlying molecular mechanisms

Abstract Introduction: Despite significant progress towards screening efforts and targeted therapies, breast cancer is still a leading cause of cancer-related mortality among women. Novel therapeutic strategies are clearly needed for effective targeting of breast cancer. Natural products continue to generate interest for identification of potential chemopreventive and therapeutic agents. Withaferin A (WFA) is a bioactive small molecule that has been proposed as a new-generation molecule capable of eliciting growth inhibitory effect on cancer cells. The molecular mechanisms by which WFA mediates inhibition of breast cancer remain elusive. The present study was designed to systematically elucidate the underlying mechanisms by which WFA inhibits growth and metastatic potential of breast cancer cells. Results: Because many cellular signaling events involve induced-phosphorylation of key targets, in the present study we utilized phosphokinase arrays to gain insight into the intricacies of WFA-mediated signaling networks and discovered that WFA induces RSK phosphorylation in breast cancer cells. Further analysis revealed an integral role of ERK in WFA-mediated RSK activation and WFA-induced ERK/RSK signaling resulted in concomitant upregulation and activation of Elk1(ETS-like transcription factor-1) and CHOP (C-EBP homology protein) leading to transcriptional upregulation of Death Receptor protein-5 (DR5). It is interesting to note that in recent years, many phase 1 and 2 single agent and combination clinical trials have been conducted to examine the efficacy and safety of recombinant human TRAIL (dulanermin, (Amgen/Genentech)), and the agonistic monoclonal antibodies to DR5 (lexatumumab (Human Genome Sciences), conatumumab (Amgen), drozitumab (Genentech), tigatuzumab (Daiichi-Sankyo) and LBY135 (Novartis)). Studies with these pro-apoptotic receptor agonists (PARAs) have been encouraging but no full phase 3 studies have been performed suggesting the need for novel PARAs with improved properties. Here, we show that WFA is more effective than three other known DR5 activators, TRAIL, etoposide and celecoxib, in activation of DR5 in breast cancer cells hence, appears to be a good candidate for further development as an effective DR5 inducer. WFA treatment effectively inhibited breast tumorigenesis in vivo and analysis of WFA-treated xenografts and MMTV-neu mice tumors showed increased activation of ERK/RSK axis and DR5 along with elevated nuclear accumulation of Elk1 and CHOP. Conclusion: These data provided first in vitro and in vivo evidence of the integral role of a previously unrecognized crosstalk between WFA and ERK/RSK and CHOP/Elk1 axes in breast tumor growth inhibition. Also, our findings may potentially open new avenues of research on the role of withaferin A as a novel pro-apoptotic receptor agonist (PARA). Citation Format: Arumugam Nagalingam, Panjamurthy Kuppusamy, Neeraj Saxena, Dipali Sharma. A novel role of Withaferin A as an effective pro-apoptotic receptor agonist (PARA) and insights into the underlying molecular mechanisms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-113. doi:10.1158/1538-7445.AM2014-LB-113

Histone Deacetylase Inhibitor Sensitizes Apoptosis-Resistant Melanomas to Cytotoxic Human T Lymphocytes through Regulation of TRAIL/DR5 Pathway

Modern immune therapies (PD-1/PD-L1 and CTLA-4 checkpoints blockade and adoptive cell transfer) have remarkably improved the response rates of metastatic melanoma. These modalities rely on the killing potential of CTL as proximal mediator of antimelanoma responses. Mechanisms of tumor resistance to and the predominant cytotoxic pathway(s) used by melanoma-reactive CTL are important outcome determinants. We hypothesized that downmodulation of death receptors (DRs) in addition to aberrant apoptotic signaling might confer resistance to death signals delivered by CTL. To test these two hypotheses, we used an in vitro model of MART CTL-resistant melanoma sublines. TCR-transgenic and patient-derived CTLs used the TRAIL cytotoxic pathway through DR5. Furthermore, recombinant human TRAIL and drozitumab (anti-DR5 agonistic mAb) were used to explicitly verify the contribution of the DR5/TRAIL pathway in killing melanomas. CTL resistance was due to DR5 downregulation and an inverted ratio of pro- to antiapoptotic molecules, both of which were reversed by the histone deacetylase inhibitor suberoylanilide hydroxanic acid. Apoptosis negative (c-IAP-2 and Bcl-xL) and positive (DR5) regulators were potential incriminators partly regulating CTL sensitivity. These preclinical findings suggest that exposure to this chromatin remodeling drug of immune-resistant melanomas can skew toward an intracellular proapoptotic milieu, increase DR expression, and overcome acquired immune resistance.

Immunoglobulin Fc domain fusion to TRAIL significantly prolongs its plasma half-life and enhances its antitumor activity.

TRAIL (Apo2L) is a potent inducer of cell death. Interest in TRAIL has increased, following the observation that TRAIL can selectively kill a wide variety of human cancer cells without killing normal cells both in vitro and when grown as xenografts. Therefore, TRAIL has been proposed as a promising anticancer agent and currently is being tested in clinical trials. However, recombinant TRAIL has a very short plasma half-life, which limits its therapeutic potential. To overcome this limitation, we investigated the ability of the human IgG1 fragment crystallizable region (Fc) to enhance TRAIL stability. In this report, we show that Fc-TRAIL chimeric protein displays higher specific activity in vitro and a significantly longer half-life in mice than recombinant human TRAIL (rh-TRAIL). No short-term toxicity, especially liver toxicity, was observed. More importantly, Fc-TRAIL was much more effective in inhibiting tumor growth in a xenograft tumor model compared with rh-TRAIL. Our data suggest that fusion of Fc to TRAIL is able to improve the bioavailability and activity of TRAIL both in vitro and in vivo, and Fc-TRAIL may be explored for future clinical applications in cancer treatment and prevention.

TRAIL/DR5 Signaling Promotes Macrophage Foam Cell Formation by Modulating Scavenger Receptor Expression

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been shown to have protective effects against atherosclerosis. However, whether TRAIL has any effects on expression of macrophage scavenger receptors and lipid uptake has not yet been studied. Macrophage lines RAW264.7 and THP-1, and mouse primary peritoneal macrophages, were cultured in vitro and treated with recombinant human TRAIL. Real-time PCR and western blot were performed to measure mRNA and protein expressions. Foam cell formation was assessed by internalization of acetylated and oxidized low-density lipoproteins (LDL). Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. We found that TRAIL treatment increased expression of scavenger receptor (SR)-AI and SR-BI in a time- and dose-dependent manner, and this effect was accompanied by increased foam cell formation. These effects of TRAIL were abolished by a TRAIL neutralizing antibody or in DR5 receptor-deficient macrophages. The increased LDL uptake by TRAIL was blocked by SR-AI gene silencing or the SR-AI inhibitor poly(I:C), while SR-BI blockade with BLT-1 had no effect. TRAIL-induced SR-AI expression was blocked by the inhibitor of p38 mitogen-activated protein kinase, but not by inhibitors of ERK1/2 or JNK. TRAIL also induced apoptosis in macrophages. In contrast to macrophages, TRAIL showed little effects on SR expression or apoptosis in vascular smooth muscle cells. In conclusion, our results demonstrate that TRAIL promotes macrophage lipid uptake via SR-AI upregulation through activation of the p38 pathway.

Abstract 2059: Kinase library siRNA screen identifies KSR1 as a synergistic therapeutic target in combination with TIC10.

Abstract Apo2L/TRAIL is an endogenous antitumor protein that possesses efficacy-limiting properties such as short serum half-life, stability, cost, and limitations in delivery when used as a recombinant protein therapeutic. TIC10 is a first-in-class small molecule inducer of the human TRAIL gene that improves these unfavorable drug properties to ultimately increase the efficacy and utility of TRAIL. TIC10 upregulates TRAIL gene transcription by activating the pro-apoptotic transcription factor Foxo3a, which is achieved through dual inhibition of Akt and MEK/ERK. We conducted a siRNA screen using a kinase library to identify kinases that affect TIC10 response in cancer cells assayed by cell viability assays to gain mechanistic insight into the molecular mechanisms of TIC10 and potentially identify a combinatorial target to improve the activity of TIC10. Execution and validation of the siRNA screen revealed 4 kinases (DGKD, SGKL, STK23, KSR) that improved the therapeutic response to TIC10 upon siRNA-mediated knockdown at 36 hours, but not 12 hours, post-treatment in HCT116 human colon cancer cells. We next assayed the effect of knocking down these 4 kinases on cancer cell cytotoxic response to recombinant human TRAIL. These studies revealed no effect with kinase knockdowns, suggesting that the mechanisms of sensitization lie upstream of TIC10-mediated TRAIL production. Network analysis of the “hit” kinases and the previously described mechanism of TIC10 revealed rational overlap of signaling pathways and kinetics. Among the 4 identified kinases, KSR1 possessed the most direct connection to the putative mechanism of TIC10 that involves the dual inhibition of Akt and the MAPK pathway. KSR1 is a MAPK scaffold protein that positively regulates the MAPK signaling pathway. Based on these observations, we are testing the hypothesis that inhibiting KSR1 in combination with TIC10 cooperatively inhibits MAPK signaling to increase TRAIL production. Together, these results suggest that KSR1 is a key player in the cytotoxic response to TIC10 and that combining TIC10 and an inhibitor of KSR1 may result in improved therapeutic response. Citation Format: Joshua E. Allen, Wafik S. El-Deiry. Kinase library siRNA screen identifies KSR1 as a synergistic therapeutic target in combination with TIC10. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2059. doi:10.1158/1538-7445.AM2013-2059