Recombinant Tumor Necrosis Factor-related Apoptosis-inducing Ligand Research Articles

Chemical synthetic approaches to mimic the TRAIL: promising cancer therapeutics.

Apoptosis is programmed cell death that eliminates undesired cells to maintain homeostasis in metazoan. Aberration of this process may lead to cancer genesis. The tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces apoptosis in cancer cells after ligation with death receptors (DR4/DR5) while sparing most normal cells. Therefore, strategies to induce apoptosis in cancer cells by mimicking the TRAIL emerge as a promising therapeutic tool. Hence, approaches are taken to develop TRAIL/DR-based cancer therapeutics. The recombinant soluble TRAIL (rhTRAIL) and death receptor agonistic antibodies were produced and tested pre-clinically and clinically. Pre-clinical and clinical trial data demonstrate that these therapeutics are safe and relatively well tolerated. But some of these therapeutics failed to exert adequate efficacy in clinical settings. Besides these biotechnologically derived therapeutics, a few chemically synthesized therapeutics are reported. Some of these therapeutics exert considerable efficacy in vitro and in vivo. In this review, we will discuss chemically synthesized TRAIL/DR-based therapeutics, their chemical and biological behaviour, design concepts and strategies that may contribute to further improvement of TRAIL/DR-based therapeutics.

Oxaliplatin Enhances the Apoptotic Effect of Mesenchymal Stem Cells, Delivering Soluble TRAIL in Chemoresistant Colorectal Cancer.

A key problem in colorectal cancer (CRC) is the development of resistance to current therapies due to the presence of cancer stem cells (CSC), which leads to poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a protein that activates apoptosis in cancer cells through union with TRAIL death receptors. Cell therapies as delivery systems can produce soluble TRAIL (sTRAIL) and full-length TRAIL (flTRAIL), showing a high capacity to produce apoptosis in vitro and in vivo assays. However, the apoptotic activity of TRAIL as monotherapy had limitations, so it is important to explore other ways to enhance susceptibility to TRAIL. This study evaluated the cytotoxic and proapoptotic activity of soluble TRAIL overexpressed by mesenchymal stem cells (MSC) in an oxaliplatin-resistant CRC cell line. Bone marrow-MSC were lentiviral transduced for soluble TRAIL expression. DR5 death receptor expression was determined in Caco-2 and CMT-93 CRC cell lines. Sensitivity to first-line chemotherapies and recombinant TRAIL was evaluated by half-maximal inhibitory concentrations. Cytotoxic and proapoptotic activity of soluble TRAIL-MSC alone and combined with chemotherapy pre-treatment was evaluated using co-cultures. Caco-2 and CMT-93 cell lines expressed 59.08 ± 5.071 and 51.65 ± 11.99 of DR5 receptor and had IC50 of 534.15 ng/mL and 581.34 ng/mL for recombinant murine TRAIL (rmTRAIL), respectively. This finding was classified as moderate resistance to TRAIL. The Caco-2 cell line showed resistance to oxaliplatin and irinotecan. MSC successfully overexpressed soluble TRAIL and induced cancer cell death at a 1:6 ratio in co-culture. Oxaliplatin pre-treatment in the Caco-2 cell line increased the cell death percentage (50%) and apoptosis by sTRAIL. This finding was statistically different from the negative control (p < 0.05), and activity was even higher with the oxaliplatin-flTRAIL combination. Thus, oxaliplatin increases apoptotic activity induced by soluble TRAIL in a chemoresistant CRC cell line.

Dose-related immunomodulatory effects of recombinant TRAIL in the tumor immune microenvironment

BackgroundIn addition to specifically inducing tumor cell apoptosis, recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has also been reported to influence the cancer immune microenvironment; however, its underlying effects and mechanisms remain unclear. Investigating the immunomodulatory effects and mechanisms of recombinant TRAIL in the tumor microenvironment (TME) may provide an important perspective and facilitate the exploration of novel TRAIL strategies for tumor therapy.MethodsImmunocompetent mice with different tumors were treated with three doses of recombinant TRAIL, and then the tumors were collected for immunological detection and mechanistic investigation. Methodological approaches include flow cytometry analysis and single-cell sequencing.ResultsIn an immunocompetent mouse model, recombinant soluble mouse TRAIL (smTRAIL) had dose-related immunomodulatory effects. The optimal dose of smTRAIL (2 mg/kg) activated innate immune cells and CD8+ T cells, whereas higher doses of smTRAIL (8 mg/kg) promoted the formation of a tumor-promoting immune microenvironment to counteract the apoptotic effects on tumor cells. The higher doses of smTRAIL treatment promoted M2-like macrophage recruitment and polarization and increased the production of protumor inflammatory cytokines, such as IL-10, which deepened the suppression of natural killer (NK) cells and CD8+ T cells in the tumor microenvironment. By constructing an HU-HSC-NPG.GM3 humanized immune system mouse model, we further verified the immunomodulatory effects induced by recombinant soluble human TRAIL (shTRAIL) and found that combinational administration of shTRAIL and trabectedin, a macrophage-targeting drug, could remodel the tumor immune microenvironment, further enhance antitumor immunity, and strikingly improve antitumor effects.ConclusionOur results highlight the immunomodulatory role of recombinant TRAIL and suggest promising therapeutic strategies for clinical application.Graphical

CDH1 overexpression sensitizes TRAIL resistant breast cancer cells towards rhTRAIL induced apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is well known for its unique ability to induce apoptosis in cancer cells but not normal cells. However, a subpopulation of cancer cells exist that does not respond to toxic doses of TRAIL. In this study, we aimed to identify key factors regulating TRAIL resistance in breast cancer. rhTRAIL (recombinant human TRAIL) resistant cells (TR) isolated from TRAIL sensitive MDA-MB-231 parental cells (TS) were confirmed using trypan blue assay, cell viability assay and AO/EtBr (acridine orange/ethidium bromide) staining. Microarray was performed followed by analysis using DAVID and Cytoscape bioinformatics software to identify the candidate hub gene. Gene expression of the candidate gene was confirmed using real-time PCR and western blot. Candidate gene was overexpressed via transient transfection to identify its significance in the context of rhTRAIL. Breast cancer patient data was obtained from The Cancer Genome Atlas (TCGA) database. Whole transcriptome analysis identified 4907 differentially expressed genes (DEGs) between TS and TR cells. CDH1 was identified as the candidate hub gene, with 18-degree centrality. We further observed CDH1 protein to be downregulated, overexpression of which increased apoptosis in TR cells after rhTRAIL treatment. TCGA patient data analysis also showed CDH1 mRNA to be low in TRAIL resistant patient group compared to TRAIL sensitive group. CDH1 overexpression sensitizes TR cells towards rhTRAIL induced apoptosis. Therefore, we can hypothesize that CDH1 expression should be taken into account while performing TRAIL therapy in breast cancer.

Electronic Cigarette Exposure Increases the Severity of Influenza a Virus Infection via TRAIL Dysregulation in Human Precision-Cut Lung Slices.

The use of electronic nicotine dispensing systems (ENDS), also known as electronic cigarettes (ECs), is common among adolescents and young adults with limited knowledge about the detrimental effects on lung health such as respiratory viral infections and underlying mechanisms. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a protein of the TNF family involved in cell apoptosis, is upregulated in COPD patients and during influenza A virus (IAV) infections, but its role in viral infection during EC exposures remains unclear. This study was aimed to investigate the effect of ECs on viral infection and TRAIL release in a human lung precision-cut lung slices (PCLS) model, and the role of TRAIL in regulating IAV infection. PCLS prepared from lungs of nonsmoker healthy human donors were exposed to EC juice (E-juice) and IAV for up to 3 days during which viral load, TRAIL, lactate dehydrogenase (LDH), and TNF-α in the tissue and supernatants were determined. TRAIL neutralizing antibody and recombinant TRAIL were utilized to determine the contribution of TRAIL to viral infection during EC exposures. E-juice increased viral load, TRAIL, TNF-α release and cytotoxicity in IAV-infected PCLS. TRAIL neutralizing antibody increased tissue viral load but reduced viral release into supernatants. Conversely, recombinant TRAIL decreased tissue viral load but increased viral release into supernatants. Further, recombinant TRAIL enhanced the expression of interferon-β and interferon-λ induced by E-juice exposure in IAV-infected PCLS. Our results suggest that EC exposure in human distal lungs amplifies viral infection and TRAIL release, and that TRAIL may serve as a mechanism to regulate viral infection. Appropriate levels of TRAIL may be important to control IAV infection in EC users.

Production of Soluble Murine TRAILs in Escherichia coli with Zn2+ Supplementation.

Accumulating evidence has demonstrated the immunomodulatory effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in rheumatoid arthritis and the tumor microenvironment, besides its known capacity of specifically inducing the apoptosis of cancer cells. Mice are common available animal models for studying the roles of TRAIL. However, mice express only a single TRAIL receptor (mTRAILR) with an intracellular death domain, in contrast to the two TRAIL receptors (TRAILR1 and TRAILR2) in humans. Moreover, human TRAIL binds weakly to mTRAILR, whereas mouse TRAIL has a high affinity for human TRAIL-Rs. Therefore, we considered that murine TRAIL would be more suitable than human TRAIL for exploring the immunoregulatory effect of TRAIL in immunocompetent mice or when using mouse cells as the target. To our knowledge, the detailed method for the production of recombinant murine TRAIL has not been reported. In this study, we aimed to design and express two soluble forms of murine TRAIL and verify the properties of the protein. Recombinant murine TRAILs were expressed in Escherichia coli BL21 (DE3, and Nichelating affinity chromatography was used for protein purification. SDS-PAGE, GDS-PAGE and HPLC were applied to analyze the protein structure. The cytotoxicity of our purified murine TRAILs was evaluated in the TRAIL-sensitive human breast cancer ZR-75-30 cells and murine breast cancer 4T1 cells. Finally, validation of the tumor-killing ability of the murine protein in vivo. Two soluble forms of murine TRAILs (mT_N99 and mT_N188) were purified and demonstrated with high purity and trimeric structure. In addition, Zn2+ supplement was essential to produce soluble murine TRAILs in E.coli BL21 (DE3). The two purified soluble mTRAILs showed similar cytotoxicity to cancer cells, moreover, mT_N99 also showed a good anti-tumor effect in vivo and is more suitable for the treatment of murine tumor models. A production approach for recombinant murine TRAIL was determined, which covered the design of shortened forms, expression, purification and characterization.

Palmitic Acid Induced a Long-Lasting Lipotoxic Insult in Human Retinal Pigment Epithelial Cells, which Is Partially Counteracted by TRAIL.

Hyperglycaemia and increased circulating saturated fatty acids are key metabolic features of type 2 diabetes mellitus (T2DM) that contribute to diabetic retinopathy pathogenesis. Contrarily, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been shown to improve or prevent T2DM. This study aimed at investigating the effect of TRAIL in an in vitro model of human retinal pigment epithelium: the ARPE-19 cell line, treated with palmitic acid (PA) in the presence of high glucose concentration. PA caused a drop in cellular metabolic activity and cell viability as well as an increase in apoptosis rates, which were paralleled by an upregulation of reactive oxygen species (ROS) generation as well as mitochondrial fragmentation. Despite ARPE-19 cells expressing TRAIL-R2 at the cell surface, TRAIL failed to counteract the cytotoxic effects of PA. However, when TRAIL was used alongside PA and then removed or used alone following PA challenge, it partially attenuated PA-induced lipotoxicity. This effect of TRAIL appeared to rely upon the modulation of inflammation and ROS production. Thus, TRAIL exerted a trophic effect on ARPE-19 cells, which became evident only when the lipotoxic insult was removed. Nevertheless, whether recombinant TRAIL might have a therapeutic potential for the treatment of diabetic retinopathy requires further investigation.

Human Umbilical Cord MSC Delivered-Soluble TRAIL Inhibits the Proliferation and Promotes Apoptosis of B-ALL Cell In Vitro and In Vivo.

The TNF-related apoptosis-inducing ligand (TRAIL) could induce apoptosis of leukemic cells, while showed no cytotoxic effect on normal cells. One of the limitations for application of recombinant TRAIL (rhTRAIL) in leukemia treatment is that the serum half-life of this protein is short. Gene delivery is a good strategy to prolong the half-life of TRAIL. In this study, we genetically engineered umbilical cord-MSCs to continuously express and secrete soluble TRAIL (MSC-sTRAIL), to investigate the effects of MSC-sTRAIL on B-cell acute lymphocytic leukemia (B-ALL) cells. In vitro, MSC-sTRAIL significantly inhibited the proliferation of B-ALL cells by suppressing PI3K/AKT and MEK/ERK signaling pathways, and induced apoptosis of B-ALL cells via the caspase cascade-mediated pathway and mitochondrial-mediated pathway. In vivo, MSC-sTRAIL dramatically inhibited B-ALL cell growth. Meanwhile, B-ALL-induced splenic and renal injuries were significantly alleviated after MSC-sTRAIL treatment. Moreover, the serum levels of MSC-secreted sTRAIL were still high in MSC-sTRAIL treated mice, indicating an extended half-life of sTRAIL. Our study suggests that MSC delivered-TRAIL secretion is a potential therapeutic strategy for B-ALL treatment.

Proscillaridin A Sensitizes Human Colon Cancer Cells to TRAIL-Induced Cell Death.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces cancer cell death by binding to TRAIL receptors. Because of its selective cytotoxicity toward cancer cells, TRAIL therapeutics, such as recombinant TRAIL and agonistic antibodies targeting TRAIL receptors, have garnered attention as promising cancer treatment agents. However, many cancer cells acquire resistance to TRAIL-induced cell death. To overcome this issue, we searched for agents to sensitize cancer cells to TRAIL-induced cell death by screening a small-molecule chemical library consisting of diverse compounds. We identified a cardiac glycoside, proscillaridin A, as the most effective TRAIL sensitizer in colon cancer cells. Proscillaridin A synergistically enhanced TRAIL-induced cell death in TRAIL-sensitive and -resistant colon cancer cells. Additionally, proscillaridin A enhanced cell death in cells treated with TRAIL and TRAIL sensitizer, the second mitochondria-derived activator of caspase mimetic. Proscillaridin A upregulated TRAIL receptor expression, while downregulating the levels of the anti-cell death molecules, cellular FADD-like IL-1β converting enzyme-like inhibitor protein and Mcl1, in a cell type-dependent manner. Furthermore, proscillaridin A enhanced TRAIL-induced cell death partly via O-glycosylation. Taken together, our findings suggest that proscillaridin A is a promising agent that enhances the anti-cancer efficacy of TRAIL therapeutics.

Abstract 5607: Combining PD-1 inhibition with the TRAIL receptor agonist TLY012 promotes pancreatic ductal adenocarcinoma tumor regression in an immunocompetent mouse model

Abstract Introduction: An immunosuppressed, apoptosis-resistant phenotype characterizes pancreatic ductal adenocarcinoma (PDAC). Both inflammation and tumorigenesis are influenced by the Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) pathway. TLY012 is pegylated recombinant TRAIL protein and is an orphan drug for systemic sclerosis and chronic pancreatitis. The potential synergy between TLY012 and anti-mouse PD-1 antibody was investigated in a syngeneic immunocompetent heterotopic mouse model. Methods: KPC-luc PDAC cells were subcutaneously implanted into C57Bl/6 mice. Treatment combined 10 mg/kg TLY012 three times per week with 10 mg/kg anti-mouse PD-1 antibody twice weekly and were compared to individual treatment and control groups. Cytokine profiling via 32-plex Luminex immunoassay was completed after 7 days and after three months of treatment. Flow cytometry at three months evaluated NK and T cell profiles in the spleen and tumors. Results: PDAC tumor-bearing mice treated with the combination of anti-mouse PD-1 and TLY012 had slower tumor growth and reduced tumor volume at 70 days compared to either drug alone (all p&amp;lt;0.005). B-cell activating factor, which can be a marker of PDAC tumor progression, was decreased in single and dual treatment groups at three months of treatment. This factor showed the earliest decrease in the dual treatment group, with a cytokine level 44% of that of control mice at 7 days. Long-term dual treatment mice also had the highest levels of proinflammatory cytokines interferon gamma (average 5.6 times control level, p=0.046), CCL5 (average 14.1 times control level, p=0.048), and interleukin-3 (IL-3, average 71.1 times control level, p=0.0053). Flow cytometry showed trends toward decreased circulating regulatory T cells, increased NK cells, and a higher proportion of CD8+ T cells within tumors in the dual treatment group. Conclusions: The combination of anti-mouse PD-1 and TLY012 prevents growth of PDAC in an immunocompetent mouse model. The combination also increases tumor-infiltrating CD8+ T cells, decreases circulating Treg cells and alters cytokine expression. CCL5 is a T-cell chemokine, and both interferon gamma and IL-3 are produced by activated T cells and macrophages, and have proinflammatory, antitumor effects. Combining TLY012 and anti-mouse PD-1 creates these changes in immune cell and cytokine levels, suggesting induction of a more proinflammatory immune environment. The proinflammatory state may contribute to decreased tumor growth. Citation Format: Anna D. Louie, Kelsey E. Huntington, Young Lee, Jared Mompoint, Jinxuan Wu, Seulki Lee, Wafik S. El-Deiry. Combining PD-1 inhibition with the TRAIL receptor agonist TLY012 promotes pancreatic ductal adenocarcinoma tumor regression in an immunocompetent mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5607.

Lentiviral interferon: A novel method for gene therapy in bladder cancer

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Repositioning the Role of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) on the TRAIL to the Development of Diabetes Mellitus: An Update of Experimental and Clinical Evidence.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF protein superfamily, represents a multifaceted cytokine with unique biological features including both proapoptotic and pro-survival effects in different cell types depending on receptor interactions and local stimuli. Beyond its extensively studied anti-tumor and immunomodulatory properties, a growing body of experimental and clinical evidence over the past two decades suggests a protective role of TRAIL in the development of type 1 (T1DM) and type 2 (T2DM) diabetes mellitus. This evidence can be briefly summarized by the following observations: (i) acceleration and exacerbation of T1DM and T2DM by TRAIL blockade or genetic deficiency in animal models, (ii) prevention and amelioration of T1DM and T2DM with recombinant TRAIL treatment or systemic TRAIL gene delivery in animal models, (iii) significantly reduced circulating soluble TRAIL levels in patients with T1DM and T2DM both at disease onset and in more advanced stages of diabetes-related complications such as cardiovascular disease and diabetic nephropathy, (iv) increase of serum TRAIL levels in diabetic patients after initiation of antidiabetic treatment and metabolic improvement. To explore the underlying mechanisms and provide mechanistic links between TRAIL and diabetes, a number of animal and in vitro studies have reported direct effects of TRAIL on several tissues involved in diabetes pathophysiology such as pancreatic islets, skeletal muscle, adipose tissue, liver, kidney, and immune and vascular cells. Residual controversy remains regarding the effects of TRAIL on adipose tissue homeostasis. Although the existing evidence is encouraging and paves the way for investigating TRAIL-related interventions in diabetic patients with cardiometabolic abnormalities, caution is warranted in the extrapolation of animal and in vitro data to the clinical setting, and further research in humans is imperative in order to uncover all aspects of the TRAIL-diabetes relationship and delineate its therapeutic implications in metabolic disease.

EV-T synergizes with AZD5582 to overcome TRAIL resistance through concomitant suppression of cFLIP, MCL-1, and IAPs in hepatocarcinoma.

Hepatocellular carcinoma (HCC) is an aggressive malignancy, and its effective treatment has been hampered by drug resistance. Extracellular vesicle (EV) delivery of TNF-related apoptosis-inducing ligand (TRAIL) (EV-T) was demonstrated to be superior to recombinant TRAIL (rTRAIL) for cancer treatment previously. And AZD5582, a potent antagonist of inhibitors of apoptosis proteins (IAPs) can potentiate apoptosis-based cancer therapies. However, the combination of EV-T and AZD5582 has never been examined for their possible apoptosis inducing synergism in cancers. In this study, we proposed and tested the combination of EV-T and AZD5582 as a potential novel therapy for effective treatment of HCC. Two HCC lines Huh7 and HepG2 that are both resistant to rTRAIL were examined. The results confirmed that AZD5582 and EV-T are synergistic for apoptosis induction in some cancer lines including Huh7 and HepG2 while sparing normal cells. More importantly, this study revealed that TRAIL sensitization by AZD5582 is mediated through the concomitant suppression of anti-apoptotic factors including cFLIP, MCL-1, and IAPs (XIAP, Survivin and cIAP-1). Particularly the downregulation of cFLIP and IAP's appeared to be essential and necessary for the synergism between AZD5582 and TRAIL. In vivo, we first time demonstrated that the combined therapy with low doses of AZD5582 and EV-Ts triggered drastically enhanced apoptosis leading to the complete eradication of Huh7 tumor development without any apparent adverse side effects examined. We thus have unraveled the important molecular mechanism underlying TRAIL sensitization by AZD5582, rationalizing the next development of a combination therapy with AZD5582 and EV-T for HCC treatment. KEY MESSAGES: It confirmed the TRAIL sensitization by AZD5582, a potent antagonist of IAPs in hepatocarcinoma. It revealed that the sensitization is via the concomitant suppression of antiapoptotic factors including cFLIP, MCL-1, and IAPs. The downregulation of cFLIP and IAPs like Survivin appeared to be essential and necessary for the synergism between AZD5582 and nanosomal TRAIL. In vivo the combined therapy with AZD5582 and nanosomal TRAIL led to complete eradication of hepatocarcinoma tumors. This study has rationalized the next development of a combination therapy with AZD5582 and nanosomal TRAIL for cancer treatment.

Enhanced extrinsic apoptosis of therapy-induced senescent cancer cells using a death receptor 5 (DR5) selective agonist.

Genotoxic agents are widely used anti-cancer therapies because of their ability to interfere with highly proliferative cells. An important outcome of these interventions is the induction of a state of permanent arrest also known as cellular senescence. However, senescent cancer cells are characterized by genomic instability and are at risk of escaping the growth arrest to eventually facilitate cancer relapse. The tumor necrosis factor related apoptosis inducing ligand (TRAIL) signals extrinsic apoptosis via Death Receptors (DR) 4 and 5, while Decoy Receptors (DcR) 1 and 2, and Osteoprotegerin (OPG) are homologous to death receptors but incapable of transducing an apoptotic signal. The use of recombinant TRAIL as an anti-cancer strategy in combination with chemotherapy is currently in development, and a major question remains whether senescent cancer cells respond to TRAIL. Here, we show variable sensitivity of cancer cells to TRAIL after senescence induction, and upregulation of both pro-apoptotic and anti-apoptotic receptors in therapy-induced senescent cancer cells. A DR5-selective TRAIL variant (DHER), unable to bind to DcR1 or OPG, was more effective in inducing apoptosis of senescent cancer cells compared to wild-type TRAIL. Importantly, no apoptosis induction was observed in non-cancerous cells, even at the highest concentrations tested. Our results suggest that targeting DR5 can serve as a novel therapeutic strategy for the elimination of therapy-induced senescent cancer cells.

Tumor necrosis factor‑related apoptosis‑inducing ligand is a novel transcriptional target of runt‑related transcription factor1.

Runt-related transcription factor 1 (RUNX1), which is also known as acute myeloid leukemia 1 (AML1), has been frequently found with genomic aberrations in human leukemia. RUNX1 encodes a transcription factor that can regulate the expression of hematopoietic genes. In addition, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) performs an important function for malignant tumors in immune surveillance. However, the regulatory mechanism of TRAIL expression remain to be fully elucidated. In the present study, tetradecanoylphorbol 13-acetate-treated megakaryocytic differentiated K562 cells was used to examine the effect of RUNX1 on TRAIL expression. Luciferase assay series of TRAIL promoters for the cells co-transfected with RUNX1 and core-binding factor β (CBFβ) expression vectors were performed to evaluate the nature of TRAIL transcriptional regulation. Electrophoresis mobility shift assay of the RUNX1 consensus sequence of the TRAIL promoter with recombinant RUNX1 and CBFβ proteins was also performed. BloodSpot database analysis for TRAIL expression in patients with acute myeloid leukemia were performed. The expression of TRAIL, its receptor Death receptor 4 and 5 and RUNX1 in K562 cells transfected with the RUNX1 expression vector and RUNX1 siRNA were evaluated by reverse transcription-quantitative PCR (RT-qPCR). TRAIL and RUNX1-ETO expression was also measured in Kasumi-1 cells transfected with RUNX1-ETO siRNA and in KG-1 cells transfected with RUNX1-ETO expression plasmid, both by RT-qPCR. Cell counting, lactate dehydrogenase assay and cell cycle analysis by flow cytometry were performed on Kasumi-1, KG-1, SKNO-1 and K562 cells treated with TRAIL and HDAC inhibitors sodium butyrate or valproic acid. The present study demonstrated that RUNX1 is a transcriptional regulator of TRAIL. It was initially found that the induction of TRAIL expression following the megakaryocytic differentiation of human leukemia cells was RUNX1-dependent. Subsequently, overexpression of RUNX1 was found to increase TRAIL mRNA expression by activating its promoter activity. Additional analyses revealed that RUNX1 regulated the expression of TRAIL in an indirect manner, because RUNX1 retained its ability to activate this promoter following the mutation of all possible RUNX1 consensus sites. Furthermore, TRAIL expression was reduced in leukemia cells carrying the t(8;21) translocation, where the RUNX1-ETO chimeric protein interfere with normal RUNX1 function. Exogenous treatment of recombinant TRAIL proteins was found to induce leukemia cell death. To conclude, the present study provided a novel mechanism, whereby TRAIL is a target gene of RUNX1 and TRAIL expression was inhibited by RUNX1-ETO. These results suggest that TRAIL is a promising agent for the clinical treatment of t(8;21) AML.

Abstract 52: Mechanistic evaluation of anti-DR5 IgM antibody IGM-8444 with potent tumor cytotoxicity, without in vitro hepatotoxicity

Abstract Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF) receptor superfamily that activates the extrinsic apoptotic pathway when bound and multimerized by its ligand, TNF-related apoptosis inducing ligand (TRAIL). DR5 is broadly expressed on solid and hematologic cancers and has been targeted with both recombinant TRAIL and agonistic antibodies in the clinic. However, these therapeutics have been unsuccessful due to lack of efficacy or due to hepatotoxicity. We have developed IGM-8444, an engineered pentameric IgM with 10 binding sites specific for DR5, which is designed to multimerize DR5 to selectively and potently induce tumor cell apoptosis while sparing hepatocytes. Here, we describe the rationale behind the selection of IGM-8444 as our clinical candidate. A panel of agonistic DR5 antibodies were evaluated for DR5 binding affinity, epitope, and in vitro potency versus hepatotoxicity. Antibodies formatted as an IgM showed enhanced potency when compared to an IgG with the same binding domain. IGM-8444 binds an epitope on DR5 within cysteine-rich domain 1 (CRD1) that competes with TRAIL binding. While the binding affinities of the panel of anti-DR5 antibodies were comparable, IGM-8444 was selected from a subset of anti-DR5 IgM antibodies capable of potently killing tumor cells without exhibiting cytotoxicity of primary human hepatocytes in vitro. Further mechanistic studies examined the kinetics of apoptotic induction by IGM-8444 and other DR5 agonists. Interestingly, we noted that DR5 agonists with the fastest kinetics of tumor cell apoptotic induction also displayed the most hepatotoxicity in vitro. In spite of the kinetic differences, IGM-8444 has similar maximal cytotoxicity in vitro and comparable anti-tumor efficacy in xenograft mouse tumor models when compared with an IgM antibody targeting a different DR5 epitope. In cynomolgus monkeys, IGM-8444 showed no evidence of hepatotoxicity or other adverse events when dosed repeatedly up to 30 mg/kg, the highest dose tested. These preclinical properties of IGM-8444 provide an opportunity for enhanced tumor cytotoxicity without additional hepatotoxicity when combined with standard of care agents. Indeed, we have demonstrated enhanced anti-tumor efficacy by combining IGM-8444 with chemotherapies such as 5-FU and irinotecan in colorectal cancer models, as well as combining with Bcl-2 inhibitor ABT-199 in hematological malignancy models. In summary, we have evaluated the mechanism by which IGM-8444 agonizes DR5, which potently kills tumor cells without accompanying hepatotoxicity. IGM-8444 is currently being evaluated in a Phase 1 study as a single agent and in combination with chemotherapy-based regimens in patients with solid cancers and NHL (NCT04553692). Citation Format: Beatrice T. Wang, Tasnim Kothambawala, Kevin C. Hart, Xingjie Chen, Melanie Desbois, Susan E. Calhoun, Poonam Yakkundi, Rodnie A. Rosete, Yuan Cao, Katie Cha, Thomas J. Matthew, Ling Wang, Paul R. Hinton, Maya K. Leabman, Genevive Hernandez, Maya F. Kotturi, Eric W. Humke, Angus M. Sinclair, Bruce A. Keyt. Mechanistic evaluation of anti-DR5 IgM antibody IGM-8444 with potent tumor cytotoxicity, without in vitro hepatotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 52.

Abstract 1044: ONC201 as a novel anti-cancer therapeutic via modulation of inhibitors of apoptosis and up-regulation of DR5 in gastric adenocarcinoma

Abstract Gastric adenocarcinoma is commonly asymptomatic early in its course, leading to late-stage diagnosis at a time when patients are inoperable. Currently available chemotherapy options are non-targeted and highly toxic, leading to a poor 5-year survival of only 31.5%. ONC201 is a small molecule imipridone anti-cancer therapy discovered by our lab that induces cell death in a variety of malignant tumors via a multitude of mechanisms including activation of the integrated stress response (ISR) leading to up-regulation of TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis after binding to the death receptor 5 (DR5). We investigated the ability of ONC201 to induce cell death in gastric adenocarcinoma cells when used in combination with recombinant human TRAIL (rhTRAIL), and its affect on DR5 cell surface expression and expression of inhibitors of apoptosis (IAP) in order to further establish mechanism of action. AGS (caspase 8, KRAS, PIK3CA mutant, HER2 amplified), SNU-1 (KRAS and MLH1 mutant, microsatellite instable), SNU-5 (p53 mutant) and SNU-16 (p53 mutant) gastric adenocarcinoma cell lines were treated with ONC201 and rhTRAIL and viability assays were performed to determine cell line sensitivities. All lines were then treated with combination therapy based on sensitivities and viability assays were performed in order to determine combination indices. Cell death was verified with sub-G1 analysis via flow cytometry and protein expression was established via western blotting. All cell lines exhibited strong synergy in response to dual therapy with ONC201 and rhTRAIL, with combination indices &amp;lt;0.6 at doses that did not induce cell death in normal fibroblast cells. Synergy was confirmed via flow cytometry analysis with increased cells in the sub-G1 phase of the cell cycle with dual therapy in excess of what would be expected from an additive effect. On western blot analysis, apoptosis was confirmed with increased cleavage of PARP, caspase 8 and caspase 3 after dual treatment with ONC201 and rhTRAIL. Flow cytometry revealed increased cell surface expression of DR5 with ONC201 therapy, and western blot analysis showed that ONC201 lead to up-regulation of ATF-4 and CHOP, indicating activation of the ISR, and down-regulation of anti-apoptotic CIAP-2 and XIAP in all cell lines except AGS, as well as FLIP in all cell lines except SNU-16. These in vitro results suggest that ONC201 in combination with rhTRAIL may be an effective and non-toxic option for the treatment of gastric adenocarcinoma by inducing apoptosis via activation of the ISR, increased cell surface expression of DR5 and down-regulation of inhibitors of apoptosis. We are currently applying this regimen to an organoid model, as well as a murine sub-cutaneous xenograft model using AGS and SNU-1 cells. Citation Format: Cassandra Susan Parker, Lanlan Zhou, Varun Prabhu, Josh Allen, Wafik El-Deiry. ONC201 as a novel anti-cancer therapeutic via modulation of inhibitors of apoptosis and up-regulation of DR5 in gastric adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1044.

TRAIL-R3 Expression on Myeloid Leukemic Blasts Is Related to a Shortened Overall Survival.

Since chemotherapy and transplantation can cure only around 35% of patients with acute myeloid leukemia (AML), there is still need for complementary and targeted treatment modalities. One of them could be the use of TNF-related apoptosis-inducing ligand (TRAIL). TRAIL is expressed by effector T cells and induces apoptosis via the death receptor intrinsic pathway. Activation of this pathway in addition to the mitochondrial pathway (by chemotherapy) has synergistic effects in vitro. In human, 4 membrane bound receptors have been identified: two of them (R1 and R2) contain a functional death domain and are capable of starting the apoptotic cascade, and two others (R3 and R4) lack a functional death domain and function as decoy receptors. Most normal cells express R3 and R4, where many tumor cells express R1 and R2. This makes soluble recombinant TRAIL an attractive candidate for targeted therapy; phase 1 clinical studies for solid tumors are launched. Until now, sparse data on TRAIL sensitivity of myeloid leukemic cells have demonstrated low TRAIL sensitivity. We investigated blood and bone-marrow samples of 113 patients with AML for TRAIL receptor expression by flow-cytometry. In contrast with published data, we found presumably (pro-apoptotic) R1 and R2 expression (mean percentage positive cells 16% and 34%, range 0–79% and 0–97% respectively) versus R3 and R4 expression (mean 9% and 10%, range 0–71% an 0–45%) indicating a TRAIL sensitive profile for myeloid blasts. Surprisingly, the expression of the anti-apoptotic R3 strongly correlated to survival. Expression of > 25% blasts positive for R3 resulted in shortened overall survival (p=0.0051), see figure 1. In multivariate analysis R3 expression remained a significant prognostic factor next to cytogenetics (p=0.03 and p= 0.015 respectively). In vitro studies on 4 myeloid leukemic cell lines confirmed TRAIL sensitivity in cell lines that expressed R1 and R2. Furthermore, simultaneous expression of R3 clearly reduced the amount of apoptosis, suggesting that TRAIL effects are inhibited by binding to R3. TRAIL induction studies on fresh leukemic samples are currently underway in our laboratory.In conclusion, our data suggest that, in contrast to earlier reports, there might be a role for TRAIL in apoptosis induction of AML blasts. R3 expression is a strong predictor for overall survival and modulation of R3 might yield additional new therapeutic options for AML patients. [Display omitted]

TRAIL-Armed ER Nanosomes Induce Drastically Enhanced Apoptosis in Resistant Tumor in Combination with the Antagonist of IAPs (AZD5582).

Although mesenchymal stem cells (MSCs) can be engineered to deliver the TNF-related apoptosis-inducing ligand (TRAIL) as an effective anticancer therapy, the clinical application is hampered by the costly manufacturing of therapeutic MSCs. Therefore, it is needed to find an alternative cell-free therapy. In this study, TRAIL-armed endoplasmic reticulum (ER)-derived nanosomes (ERN-T) are successfully prepared with an average size of 70.6nm in diameter from TRAIL transduced MSCs. It is demonstrated that the ERN-T is significantly more efficient for cancer cell killing than the soluble recombinant TRAIL (rTRAIL). AZD5582 is an antagonist of the inhibitors of apoptosis proteins (IAPs), and its combination with ERN-T induces strikingly enhanced apoptosis in cancerous but not normal cells. AZD5582 sensitizes resistant cancer cells to TRAIL through concomitant downregulation of IAP members like XIAP and the Bcl2 family member Mcl-1. Intravenously infused ERN-Ts accumulate in tumors for over 48 h indicating good tumor tropism and retention. The combination of ERN-T and AZD5582 drastically promotes therapeutic efficacy comparing with the cotreatment by rTRAIL and AZD5582 in a subcutaneous MDA-MB-231 xenograft tumor model. The data thus demonstrate that ERN-T can be a novel cell-free alternative to TRAIL-expressing MSC-based anticancer therapy and its efficacy can be drastically enhanced through combination with AZD5582.

Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows promising antitumor activity in preclinical studies. However, the efficacy of recombinant TRAIL in clinical trials is compromised by its short serum half-life and low in vivo stability. Induction of endogenous TRAIL may overcome the limitations and become a new strategy for cancer treatment. Here, we discovered that metformin increased TRAIL expression and induced apoptosis in triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) cells. Metformin did not alter the expression of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Metformin-upregulated TRAIL was secreted into conditioned medium (CM) and found to be functional, since the CM promoted TNBC cells undergoing apoptosis, which was abrogated by a recombinant TRAIL-R2-Fc chimera. Moreover, blockade of TRAIL binding to DR4/DR5 or specific knockdown of TRAIL expression significantly attenuated metformin-induced apoptosis. Studies with a tumor xenograft model revealed that metformin not only significantly inhibited tumor growth but also elicited apoptosis and enhanced TRAIL expression in vivo. Collectively, we have demonstrated that upregulation of TRAIL and activation of death receptor signaling are pivotal for metformin-induced apoptosis in TNBC and NSCLC cells. Our studies identify a novel mechanism of action of metformin exhibiting potent antitumor activity via induction of endogenous TRAIL.