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

The Role of TRAIL Signaling in Cancer: Searching for New Therapeutic Strategies.

Cancer continues to pose a significant threat to global health, with its status as a leading cause of death remaining unchallenged. Within the realm of cancer research, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) stands out as a critical player, having been identified in the 1990s as the tenth member of the TNF family. This review examines the pivotal role of TRAIL in cancer biology, focusing on its ability to induce apoptosis in malignant cells through both endogenous and exogenous pathways. We provide an in-depth analysis of TRAIL's intracellular signaling and intercellular communication, underscoring its potential as a selective anticancer agent. Additionally, the review explores TRAIL's capacity to reshape the tumor microenvironment, thereby influencing cancer progression and response to therapy. With an eye towards future developments, we discuss the prospects of harnessing TRAIL's capabilities for the creation of tailored, precision-based cancer treatments, aiming to enhance efficacy and improve patient survival rates.

Noncanonical TRAIL Signaling Promotes Myeloid-Derived Suppressor Cell Abundance and Tumor Growth in Cholangiocarcinoma

Proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling as a cause of cancer cell death is a well-established mechanism. However, TRAIL-receptor (TRAIL-R) agonists have had very limited anticancer activity in human beings, challenging the concept of TRAIL as a potent anticancer agent. Herein, we aimed to define mechanisms by which TRAIL+ cancer cells can leverage noncanonical TRAIL signaling in myeloid-derived suppressor cells (MDSCs) promoting their abundance in murine cholangiocarcinoma (CCA). Multiple immunocompetent syngeneic, orthotopic models of CCA were used. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of CD45+ cells in murine tumors from the different CCA models was conducted. In multiple immunocompetent murine models of CCA, implantation of TRAIL+ murine cancer cells into Trail-r-/- mice resulted in a significant reduction in tumor volumes compared with wild-type mice. Tumor-bearing Trail-r-/- mice had a significant decrease in the abundance of MDSCs owing to attenuation of MDSC proliferation. Noncanonical TRAIL signaling with consequent nuclear factor-κB activation in MDSCs facilitated enhanced MDSC proliferation. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of immune cells from murine tumors showed enrichment of a nuclear factor-κB activation signature in MDSCs. Moreover, MDSCs were resistant to TRAIL-mediated apoptosis owing to enhanced expression of cellular FLICE inhibitory protein, an inhibitor of proapoptotic TRAIL signaling. Accordingly, cellular FLICE inhibitory protein knockdown sensitized murine MDSCs to TRAIL-mediated apoptosis. Finally, cancer cell-restricted deletion of Trail significantly reduced MDSC abundance and murine tumor burden. Our findings highlight the therapeutic potential of targeting TRAIL+ cancer cells for treatment of a poorly immunogenic cancer.

The Prognostic, Diagnostic, and Therapeutic Potential of TRAIL Signalling in Cardiovascular Diseases

TNF-related apoptosis-inducing ligand (TRAIL) was originally discovered, almost 20 years ago, for its ability to kill cancer cells. More recent evidence has described pleiotropic functions, particularly in the cardiovascular system. There is potential for TRAIL concentrations in the circulation to act as prognostic and/or diagnostic factors for cardiovascular diseases (CVD). Pre-clinical studies also describe the therapeutic capacity for TRAIL signals, particularly in the context of atherosclerotic disease and diseases of the myocardium. Because diabetes mellitus significantly contributes to the progression and pathogenesis of CVDs, in this review we highlight recent evidence for the prognostic, diagnostic, and therapeutic potential of TRAIL signals in CVDs, and where relevant, the impact of diabetes mellitus. A greater understanding of how TRAIL signals regulate cardiovascular protection and pathology may offer new diagnostic and therapeutic avenues for patients suffering from CVDs.

Recent Advances in the Development of Nanodelivery Systems Targeting the TRAIL Death Receptor Pathway

The TRAIL (TNF-related apoptosis-inducing ligand) apoptotic pathway is extensively exploited in the development of targeted antitumor therapy due to TRAIL specificity towards its cognate receptors, namely death receptors DR4 and DR5. Although therapies targeting the TRAIL pathway have encountered many obstacles in attempts at clinical implementation for cancer treatment, the unique features of the TRAIL signaling pathway continue to attract the attention of researchers. Special attention is paid to the design of novel nanoscaled delivery systems, primarily aimed at increasing the valency of the ligand for improved death receptor clustering that enhances apoptotic signaling. Optionally, complex nanoformulations can allow the encapsulation of several therapeutic molecules for a combined synergistic effect, for example, chemotherapeutic agents or photosensitizers. Scaffolds for the developed nanodelivery systems are fabricated by a wide range of conventional clinically approved materials and innovative ones, including metals, carbon, lipids, polymers, nanogels, protein nanocages, virus-based nanoparticles, dendrimers, DNA origami nanostructures, and their complex combinations. Most nanotherapeutics targeting the TRAIL pathway are aimed at tumor therapy and theranostics. However, given the wide spectrum of action of TRAIL due to its natural role in immune system homeostasis, other therapeutic areas are also involved, such as liver fibrosis, rheumatoid arthritis, Alzheimer's disease, and inflammatory diseases caused by bacterial infections. This review summarizes the recent innovative developments in the design of nanodelivery systems modified with TRAIL pathway-targeting ligands.

Post‐translational modification and phenotype

Post‐translational modification and phenotype

Proteome and phosphoproteome profiling of non-small cell lung cancer cell line A549 treated with TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is recognized for its promising therapeutic effects against cancer. However, mechanisms underlying the effect of TRAIL on protein expression, signal transduction, and apoptosis induction remain unclear. We surmised that a systematic analysis of the proteome and phosphoproteome associated with TRAIL signaling may help elucidate the mechanisms involved and facilitate the development of therapeutics. Therefore, we investigated the proteome and phosphoproteome of non-small cell lung cancer cell line A549 treated with TRAIL. Our results indicated that 126 proteins and 1684 phosphosites were markedly differentially expressed between the phosphate-buffered saline- and TRAIL-treated groups. The expression at protein and phosphosite levels were not completely consistent. Gene ontology functional analysis revealed that metal ion (zinc) binding was highly affected by TRAIL treatment. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that almost all pathways that involved differentially expressed phosphosites were associated with apoptosis. We also identified an important kinase, AKT1, and its series of substrates in TRAIL signaling. The results of this study may provide guidance for future research on tumor therapy using TRAIL.

Abstract PO032: Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-TRAIL-R Mediates Cholangiocarcinoma Tumor Immune Evasion by Enhancing Myeloid-Derived Suppressive Cell Population

Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of TNF superfamily, is predominantly expressed on immune cells. Engagement of TRAIL with its cognate receptor, TRAIL-R (TR), leads to cancer cells apoptosis. Although the anti-tumor role of TRAIL-TR has been extensively studied, TRAIL agonists have had very limited anti-cancer activity in human clinical trials. TRAIL signaling can facilitate the myeloid response. However, this potential immunosuppressive function of TRAIL has not been examined extensively in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, has a dense immunosuppressive microenvironment. Accordingly, CCA provides a model to examine the potential immune regulatory function of TRAIL in cancer biology. Methods: Using syngeneic, orthotopic murine models of CCA, (PMID: 29464042), murine CCA cells (SB cells) that express both Trail and Trail receptor (Tr) were implanted into livers of WT Tr− / − and LyzcreTrf/f mice, the latter have a specific deletion of TRAIL-R on myeloid cells. Hence, in this model the host immune cells express Trail but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr− / − mice had a significant reduction in tumor burden compared to WT mice. Myeloid-derived suppressor cells (MDSCs) were significantly decreased in Tr− / − tumors compared to WT tumors. Implantation of SB cells into LyzcreTrf/f mice resulted in a marked reduction in tumor burden and attenuation of MDSC infiltration compared to Lyzcre mice. In vitro functional studies employing MDSCs from mice deficient in Tr (MDSC-Tr −/−) were carried out. Compared to WT MDSCs, coculture of MDSC-Tr− /- with SB cells resulted in a significant reduction in the proliferation and immunosuppressive function of MDSCs. Moreover, following cocultured with SB cells, immunofluorescence analysis demonstrated that MDSC-Tr− / − had a reduction in the nuclear translocation of the NFkB subunit P65 compared to WT MDSCs. These data suggest that TRAIL-TR augments MDSC proliferation via NFkB. In conclusion, we have demonstrated that Tr− / − mice have a significant reduction in CCA tumor burden and MDSC infiltration. These results indicate that TRAIL-TR facilitates tumor immune escape and progression by fostering MDSC immunosuppressive function and infiltration, in an NFkB-dependent manner. Direct targeting of TRAIL on CCA cells is a potential anti-tumor strategy. Citation Format: Emilien Loeuillard, Jingchun Yang, Dong Haidong, Gregory Gores, Sumera Ilyas. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-TRAIL-R Mediates Cholangiocarcinoma Tumor Immune Evasion by Enhancing Myeloid-Derived Suppressive Cell Population [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO032.

Abstract 1315: Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma

Abstract Background and Aims: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable attention as a potential anti-cancer strategy, TRAIL agonists have had very limited anti-cancer activity in humans. TRAIL signaling can attenuate the T lymphocyte response. However, this potential immunosuppressive function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, has a dense immunosuppressive microenvironment. Accordingly, CCA provides a model to examine the potential immune regulatory function of TRAIL in cancer biology. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both Trail and Trail receptor (Tr) were implanted into livers of WT and Tr-/- mice. Hence, in this model the host immune cells express Trail but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr-/- mice had a significant reduction in tumor burden compared to WT mice. Moreover, tumor bearing Tr-/- mice had a significant increase in cytotoxic T lymphocytes (CTLs) and enhanced CTL effector function. Myeloid-derived suppressor cells (MDSCs) were significantly decreased in Tr-/- tumors compared to WT tumors. Furthermore, implantation of SB cells devoid of Trail (SB-Trail-/-) into WT mice resulted in a significant reduction in tumor burden and MDSC infiltration. In vitro functional studies employing SB cells deficient in Tr (SB-Tr-/-) and MDSCs were carried out. There was a significant reduction in proliferation and immunosuppressive function of MDSCs when cocultured with SB-Tr-/- compared to SB cells. These results indicate that TRAIL-TR fosters MDSC growth and immunosuppressive function. In conclusion, we have demonstrated that Tr-/- mice have a significant reduction in CCA tumor burden and MDSC infiltration. Consequently, Tr-/- tumors have enhanced CTL infiltration and function. These data suggest that the TRAIL-TR system mediates tumors immune evasion via MDSCs, and direct targeting of TRAIL on CCA cells is a potential anti-tumor strategy. Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera I. Ilyas. Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma [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 1315.

Abstract P072: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) foster myeloid-derived suppressor cell-mediated tumor immune evasion in cholangiocarcinoma

Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable interest as a potential anti-cancer strategy, TRAIL agonists have had very limited anti-cancer activity in humans. TRAIL signaling in T cells may also potentially provide an immune checkpoint function as it can inhibit T cell activation and proliferation by interfering with T cell receptor signaling. However, this potential immune checkpoint function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, provides a model to examine the potential immune checkpoint function of TRAIL. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both TRAIL and TRAIL receptor (TR) were implanted into livers of WT and Tr−/− mice. Hence, in this model the host immune cells express TRAIL but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr−/− mice had a significant reduction in tumor burden compared to WT mice. Moreover, tumor bearing Tr−/− mice had a significant increase in cytotoxic T lymphocytes (CTLs) and enhanced CTL effector function. However, coculture of T cells with SB cells or SB cells deficient in Tr (SB-Tr−/−) did not result in a significant difference in T cell apoptosis or function, implying that TRAIL-TR is not a direct T cell checkpoint. Myeloid derived suppressor cells (MDSCs) were significantly decreased in Tr−/− tumors compared to WT tumors. Furthermore, implantation of SB cells devoid of Trail (SB-Trail−/−) into WT mice resulted in a significant reduction in tumor burden and MDSC infiltration. Coculture of SB cells with MDSCs from Tr−/− mice attenuated MDSC proliferation and immunosuppression compared to WT MDSCs. Moreover, treatment of MDSCs from Tr−/− mice with TRAIL recombinant protein resulted in a reduction in their proliferation and immunosuppressive function compare to MDSCs from WT mice implying that TRAIL-TR fosters MDSC growth and immunosuppressive function. In conclusion, we have demonstrated that Tr−/− mice have a significant reduction in CCA tumor burden and MDSC infiltration. Consequently, Tr−/− mice bearing tumors have enhanced CTL infiltration and function. These data suggest that the TRAIL-TR system mediates tumor immune evasion via MDSCs. Herein, we suggest that TRAIL-TR appears to function as an indirect T cell checkpoint by augmenting the immunosuppressive effects of MDSCs. Citation Format: Emilien Loeuillard, Juan Wang, Jingchun Yang, Haidong Dong, Gregory Gores, Sumera Ilyas. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) foster myeloid-derived suppressor cell-mediated tumor immune evasion in cholangiocarcinoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P072.

Abstract PO-064: ONC212 stimulates cytotoxic T-cell killing, increases tumor-immune cell interactions, and promotes tumor regression in combination with TLY012 in a PDAC murine model

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer with a 4.2% 5-year survival rate. There is an urgent need for innovative treatments for patients suffering from PDAC. Patients with PDAC often have immunosuppressed tumors that are apoptosis-resistant and have limited immune cell infiltration and/or activation. Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces cancer cell apoptosis during innate immunity, while ONC212 is a potent fluorinated second-generation imipridone currently in IND-enabling studies that induces TRAIL signaling and the integrated stress response (ISR). TLY012 is a novel PEG’ylated trimeric TRAIL receptor agonist being clinically developed to overcome the short half-life of TRAIL. We hypothesized that combining a TRAIL pathway inducer and a TRAIL receptor agonist may be efficacious in PDAC and may activate immune cell killing based on prior work with imipridone ONC201. Immune cell co-culture experiments were conducted using PANC1 PDAC cells and TALL-104 CD8+ cytotoxic T-cells at a 1:1 effector-to-target cell ratio with or without ONC212. We observed a significant increase in T-cell killing of PDAC cells in the co-culture assay following treatment with ONC212 at several doses as compared to controls. We noted an increase in tumor-immune cell interactions as measured by the number of T-cells that were directly touching tumor cells at each timepoint. The BxPC-3 immunodeficient murine PDAC model revealed an increase in natural killer (NK) cell tumor infiltration by immunohistochemistry (IHC) staining for NK1.1 after 30 days of 50 mg/kg ONC212 treatment three times a week. The combination of ONC212 and TLY012 was evaluated using subcutaneously-implanted KPC-Luc PDAC cells in a syngeneic immunocompetent C57BL/6 mouse model. Mice treated with 25 mg/kg ONC212 alone twice a week, 10 mg/kg TLY012 alone twice a week, or combination therapy with ONC212 and TLY012 showed statistically significant decreases in tumor growth compared to controls after 5 weeks of treatment (n=6 mice per treatment condition) by tumor volume and in vivo bioluminescent imaging. We are analyzing murine plasma cytokine profiles, changes in tumor-infiltrating NK- and T-cells by flow cytometric and IHC analysis of murine spleen and KPC-Luc tumor samples. We report a novel immune stimulation of the TRAIL pathway and T-cell activation by ONC212 plus TLY012 leading to cytotoxic anti-PDAC responses in vivo. Citation Format: Kelsey E. Huntington, Anna Louie, Young Lee, Jared Mompoint, Isacco Ferrarini, Aakash Jhaveri, Varun V. Prabhu, Allen Melemed, Seulki Lee, Wafik S. El-Deiry. ONC212 stimulates cytotoxic T-cell killing, increases tumor-immune cell interactions, and promotes tumor regression in combination with TLY012 in a PDAC murine model [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-064.

Abstract 2738: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates tumor immune evasion in cholangiocarcinoma

Abstract Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor superfamily, is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable interest as a potential anti-cancer strategy, TR agonists have had very limited anti-cancer activity in humans. TRAIL signaling in T cells may also potentially provide an immune checkpoint function as it can inhibit T cell activation and proliferation by interfering with T cell receptor signaling. However, this potential immune checkpoint function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a malignancy of the bile ducts, provides a model to examine the potential immune checkpoint function of TRAIL. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both TRAIL and TRAIL receptor (TR) were implanted into livers of WT C57BL/6J and Tr−/− mice. Hence, in this model the host immune cells express TRAIL but not the receptor; they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to potential TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed and tumor and immune characterization (via flow cytometry) was conducted. Results: Implantation of SB cells into Tr−/− mice result in a significant reduction in tumor volumes compared to WT mice. Tumor-bearing Tr−/− mice had a significant infiltration of cytotoxic T lymphocytes (CTLs) (CD45+CD3+CD8+CD11a+) and enhanced CTL effector function. Moreover, Tr−/− mice tumors had a significant decrease in granulocytic and monocytic myeloid-derived suppressor cell (MDSCs) as well as tumor-associated macrophages (TAMs) (CD45+F4/80+CD11b+CD206+) compared to WT mice tumors. Conclusion: Using a unique syngeneic orthotopic implantation model of murine CCA, we demonstrate that CCA cells expressing TRAIL in a mouse genetically deficient for TR exhibit reduced tumor volumes enhanced CTL infiltration and function, and reduced MDSC and TAM infiltration into the tumors. These data suggest that TRAIL has a potential immune checkpoint function, and targeting TRAIL signaling with consequent augmentation of CTL function maybe a promising therapeutic approach in human cancers. Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera Ilyas. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates tumor immune evasion in cholangiocarcinoma [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 2738.

Abstract 940: The combinatorial activity of Eftozanermin ABBV621, a novel and potent TRAIL receptor agonist fusion protein, in pre-clinical models of hematologic malignancies

Abstract The Combinatorial Activity of Eftozanermin (ABBV-621), a Novel and Potent TRAIL Receptor Agonist Fusion Protein, in Pre-clinical Models of Hematologic Malignancies Cell death can be initiated through activation of the extrinsic and intrinsic apoptotic signaling pathways. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, preferentially triggers the extrinsic apoptotic pathway by binding as a trimer to two closely related cell surface death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Receptor trimerization leads to the formation of the death-inducing signaling complex (DISC) to recruit and activate downstream caspases that ultimately leads to apoptotic cell death. Because TRAIL signaling induces apoptosis, several TRAIL receptor agonists have been developed for the treatment of cancer. ABBV-621 is a novel, second generation TRAIL receptor agonist that is an engineered fusion protein consisting of an IgG1-Fc linked to a single chain trimer of TRAIL subunits resulting in a total of six death receptor binding sites per molecule to maximize receptor clustering that is currently being tested in Phase I clinical trials (NCT03082209).To expand upon the potential therapeutic utility of ABBV-621, we tested the combinatorial activity of ABBV-621 with numerous standard-of-care (SoC) therapeutics and targeted agents in diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines. The in vitro results led to selection of agents to combine with ABBV-621 for in vivo studies. In DLBCL cell line-derived xenograft (CDX) preclinical models, we observed combination activity of ABBV-621 with pevonedistat (PEV) a selective NEDD8 inhibitor. Additionally, synergistic activity was observed with ABBV-621 with either bendamustine (BED) or rituximab (RTX) alone, or BED/RTX together. In AML, we observed compelling combination activity of ABBV-621 with PEV in cell line-derived xenograft (CDX) models. In MM, combination of ABBV-621 plus bortezomib (BTZ) resulted in deeper anti-tumorigenic activity than either agent alone in several CDX models. The pre-clinical data presented here support expanding the indications and settings where ABBV-621 may have utility. A clinical trial assessing the activity of ABBV-621 in combination with bortezomib and dexamethasone in R/R MM patients is planned. Disclosures:All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Morey Smith, Sha Jin, Dong Cheng, Haichao Zhang, Jason Huska, Deborah Widomski, Vicky Bontcheva, Greg Buchanan, Susan Morgan-Lappe, Darren Phillips, Stephen Tahir. The combinatorial activity of Eftozanermin ABBV621, a novel and potent TRAIL receptor agonist fusion protein, in pre-clinical models of hematologic malignancies [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 940.

Weighted miRNA co-expression network reveals potential roles of apoptosis related pathways and crucial genes in thoracic aortic aneurysm

BackgroundThoracic aortic aneurysm (TAA) is a potentially life-threatening disease for which few medical therapies are available. Thus, it is critically important to investigate the underlying molecular mechanisms of TAA, and identify potential targets for TAA treatment.MethodsDifferentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) were screened, and a weighted correlation network analysis (WGCNA) was employed to construct a weighted miRNA co-expression network using GSE110527. The DEMs were then mapped into the whole co-expression network of all samples, and a DEM coexpression network was created. Molecular Complex Detection (MCODE) was used to identify crucial miRNAs. Target genes were predicted using the miRTarbase database, and further screened by identifying genes that overlapped with the DEGs of GSE26155. The screened target genes were validated using GSE9106, and the successfully validated genes were considered as crucial genes. Finally, a miRNA risk score for diagnosing TAA was calculated by undertaking a least absolute shrinkage and selection operator (LASSO) regression.ResultsThe tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) signaling pathway was found in DEM functional enrichment. Crucial miRNAs were identified and target genes were predicted and associated with the regulation of the TRAIL signaling pathway. Next, 113 important target genes were identified as overlapping with the DEGs of GSE26155. These genes were further validated, and 5 successfully validated genes were considered as crucial genes. Finally, the miRNA risk score calculated by the LASSO regression was shown to have potential diagnostic value.ConclusionsWe performed a WGCNA analysis to construct a weighted miRNA co-expression network, predicted target genes of crucial miRNAs, identified crucial genes, and finally calculated a miRNA risk score. The results showed that pathways and genes associated with apoptosis appear to play an important role in TAA pathogenesis, and that medications targeting apoptosis might slow TAA progression. Future in vitro and in vivo experimental studies need to be undertaken to further validate our findings and investigate the mechanistic details of these crucial miRNAs and crucial genes.

The multifaceted role of TRAIL signaling in cancer and immunity.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can lead to the induction of apoptosis in tumor or infected cells. However, activation of TRAIL signaling may also trigger nonapoptotic pathways in cancer and in nontransformed cells, that is, immune cells. Here, we review the current knowledge on noncanonical TRAIL signaling. The biological outcomes of TRAIL signaling in immune and malignant cells are presented and explained, with a focus on the role of TRAIL for natural killer (NK) cell function. Furthermore, we highlight the technical difficulties in dissecting the precise molecular mechanisms involved in the switch between apoptotic and nonapoptotic TRAIL signaling. Finally, we discuss the consequences thereof for a therapeutic manipulation of TRAIL in cancer and possible approaches to bypass these difficulties.

Targeting eIF4A using rocaglate CR‑1‑31B sensitizes gallbladder cancer cells to TRAIL‑mediated apoptosis through the translational downregulation of c‑FLIP.

Induction of the apoptosis of tumor cells is a promising therapeutic approach for the treatment of cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a novel type of anticancer drug. However, gallbladder cancer cells (GBC) exhibit strong resistance to TRAIL. The aim of the present study was to assess the effect of rocaglate CR-1-31B (CR-31), an inhibitor of eukaryotic translation initiation factor 4A (eIF4A), on the sensitization of cells to TRAIL-induced apoptosis in TRAIL-resistant GBC. eIF4A was highly abundant in GBC tissues and cell lines (GBC-SD and SGC-996). GBC cells were treated using TRAIL and/or CR-31 and then apoptosis and TRAIL signaling were detected in vitro. CR-31 enhanced the sensitivity of TRAIL-resistant GBC cells, due to the CR-31-mediated eIF4A translational downregulation of c-FLIP and the subsequent activation of the caspase cascade. Furthermore, GBC-SD tumor xenografts models were established and the effects of CR-31 in vivo were assessed. CR-31 significantly reduced the growth and initiated the apoptosis of tumor cells, suggesting that CR-31 also increased sensitivity in vivo. Taken together, the results of the present study show that CR-31 treatment countered the resistance to TRAIL in GBC cells in vitro and in vivo. Therefore, eIF4A may serve as a novel therapeutic target and its combination with TRAIL-CR-31 as a therapy may serve as a novel strategy for GBC treatment.

Zika Virus Induces Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL)-Mediated Apoptosis in Human Neural Progenitor Cells.

Zika virus (ZIKV) remains as a public health threat due to the congenital birth defects the virus causes following infection of pregnant women. Congenital microcephaly is among the neurodevelopmental disorders the virus can cause in newborns, and this defect has been associated with ZIKV-mediated cytopathic effects in human neural progenitor cells (hNPCs). In this study, we investigated the cellular changes that occur in hNPCs in response to ZIKV (African and Asian lineages)-induced cytopathic effects. Transmission electron microscopy showed the progress of cell death as well as the formation of numerous vacuoles in the cytoplasm of ZIKV-infected hNPCs. Infection with both African and Asian lineages of ZIKV induced apoptosis, as demonstrated by the increased activation of caspase 3/7, 8, and 9. Increased levels of proinflammatory cytokines and chemokines (IL-6, IL-8, IL-1β) were also detected in ZIKV-infected hNPCs, while z-VAD-fmk-induced inhibition of cell death suppressed ZIKV-mediated cytokine production in a dose-dependent manner. ZIKV-infected hNPCs also displayed significantly elevated gene expression levels of the pro-apoptotic Bcl2-mediated family, in particular, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Furthermore, TRAIL signaling led to augmented ZIKV-mediated cell death and the knockdown of TRAIL-mediated signaling adaptor, FADD, resulted in enhanced ZIKV replication. In conclusion, our findings provide cellular insights into the cytopathic effects induced by ZIKV infection of hNPCs.

The Combinatorial Activity of Eftozanermin (ABBV-621), a Novel and Potent TRAIL Receptor Agonist Fusion Protein, in Pre-Clinical Models of Hematologic Malignancies

Cell death can be initiated through activation of the extrinsic and intrinsic apoptotic signaling pathways. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, preferentially triggers the extrinsic apoptotic pathway by binding as a trimer to two closely related cell surface death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Receptor trimerization leads to the formation of the death-inducing signaling complex (DISC) to recruit and activate downstream caspases that ultimately leads to apoptotic cell death. Because TRAIL signaling induces apoptosis, several TRAIL receptor agonists have been developed for the treatment of cancer. ABBV-621 is a novel, second generation TRAIL receptor agonist that is an engineered fusion protein consisting of an IgG1-Fc linked to a single chain trimer of TRAIL subunits resulting in a total of six death receptor binding sites per molecule to maximize receptor clustering that is currently being tested in Phase I clinical trials (NCT03082209). To expand upon the potential therapeutic utility of ABBV-621, we tested the combinatorial activity of ABBV-621 with numerous standard-of-care (SoC) therapeutics and targeted agents in diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines. Thein vitroresults led to selection of agents to combine with ABBV-621 forin vivostudies. In DLBCL cell line-derived xenograft (CDX) preclinical models, we observed combination activity of ABBV-621 with pevonedistat (PEV) a selective NEDD8 inhibitor. Additionally, synergistic activity was observed with ABBV-621 with either bendamustine (BED) or rituximab (RTX) alone, or BED/RTX together. In AML, we observed compelling combination activity of ABBV-621 with PEV in cell line-derived xenograft (CDX) models. In MM, combination of ABBV-621 plus bortezomib (BTZ) resulted in deeper anti-tumorigenic activity than either agent alone in several CDX models. The pre-clinical data presented here support expanding the indications and settings where ABBV-621 may have utility. A clinical trial assessing the activity of ABBV-621 in combination with bortezomib and dexamethasone in R/R MM patients is planned. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Disclosures Smith: AbbVie:Current Employment, Current equity holder in publicly-traded company.Jin:AbbVie:Current Employment, Current equity holder in publicly-traded company.Chen:AbbVie:Current Employment, Current equity holder in publicly-traded company.Zhang:AbbVie:Current Employment, Current equity holder in publicly-traded company.Huska:AbbVie:Current Employment, Current equity holder in publicly-traded company.Widomski:AbbVie:Current Employment, Current equity holder in publicly-traded company.Bontcheva:AbbVie:Current Employment, Current equity holder in publicly-traded company.Buchanan:AbbVie:Current Employment, Current equity holder in publicly-traded company.Morgan-Lappe:AbbVie:Current Employment, Current equity holder in publicly-traded company.Phillips:AbbVie:Current Employment, Current equity holder in publicly-traded company.Tahir:AbbVie:Current Employment, Current equity holder in publicly-traded company.

Mcl-1 targeting strategies unlock the proapoptotic potential of TRAIL in melanoma cells.

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis selectively in cancer cells. For melanoma, the targeting of TRAIL signaling appears highly attractive, due to pronounced TRAIL receptor expression in tumor tissue. However, mechanisms of TRAIL resistance observed in melanoma cells may limit its clinical use. The Bcl-2 family members are critical regulators of cell-intrinsic apoptotic pathways. Thus, the antiapoptotic Bcl-2 protein myeloid cell leukemia 1 (Mcl-1) is overexpressed in many tumor types and was linked to chemotherapy resistance in melanoma. In this study, we evaluated the involvement of antiapoptotic Bcl-2 proteins (Bcl-2, Bcl-xL , Bcl-w, Mcl-1, Bcl-A1, and Bcl-B) in TRAIL resistance. They were targeted by small interfering RNA-mediated silencing in TRAIL-sensitive (A-375, Mel-HO) and in TRAIL-resistant melanoma cell lines (Mel-2a, MeWo). This highlighted Mcl-1 as the most efficient target to overcome TRAIL resistance. In this context, we investigated the effects of Mcl-1-targeting microRNAs as well as the Mcl-1-selective inhibitor S63845. Both miR-193b and S63845 resulted in significant enhancement of TRAIL-induced apoptosis, associated with decreased cell viability. Apoptosis induction was mediated by caspase-3 processing as well as by Bax and Bak activation, indicating the critical involvement of intrinsic apoptosis pathways. These data may indicate a high relevance of Mcl-1 targeting also in melanoma therapy. Furthermore, the data may suggest to consider the use of the tumor suppressor miR-193b as a strategy for countering TRAIL resistance in melanoma.

Insight into the divergent role of TRAIL in non-neoplastic neurological diseases.

Tumour necrosis factor–related apoptosis‐inducing ligand (TRAIL) is a member of the tumour necrosis factor (TNF) superfamily which mainly induces apoptosis of tumour cells and transformed cell lines with no systemic toxicity, whereas they share high sequence homology with TNF and CD95L. These unique effects of TRAIL have made it an important molecule in oncology research. However, the research on TRAIL‐related antineoplastic agents has lagged behind and has been limited by the extensive drug resistance in cancer cells. Given the several findings showing that TRAIL is involved in immune regulation and other pleiotropic biological effects in non‐malignant cells, TRAIL and its receptors have attracted widespread attention from researchers. In the central nervous system (CNS), TRAIL is highly correlated with malignant tumours such as glioma and other non‐neoplastic disorders such as acute brain injury, CNS infection and neurodegenerative disease. Many clinical and animal studies have revealed the dual roles of TRAIL in which it causes damage by inducing cell apoptosis, and confers protection by enhancing both pro‐ and non‐apoptosis effects in different neurological disorders and at different sites or stages. Its pro‐apoptotic effect produces a pro‐survival effect that cannot be underestimated. This review extensively covers in vitro and in vivo experiments and clinical studies investigating TRAIL. It also provides a summary of the current knowledge on the TRAIL signalling pathway and its involvement in pathogenesis, diagnosis and therapeutics of CNS disorders as a basis for future research.

Andrographolide sensitizes human renal carcinoma cells to TRAIL‑induced apoptosis through upregulation of death receptor 4

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in cancer cells, with minimal toxicity to normal tissues. However, accumulating evidence suggests that certain cancer types are insensitive to TRAIL signaling. The aim of this study was to identify an effective combination regimen, which can overcome TRAIL resistance in renal cancer cell. Herein, we found that human renal carcinoma cells (RCCs) are widely resistant to TRAIL-mediated growth inhibition and subsequently identified that andrographolide (Andro), a major constituent of Andrographis paniculate, an annual herbaceous plant in the family Acanthaceae, counteracts TRAIL resistance in RCCs. Combined treatment with TRAIL and Andro suppressed cell viability as determined by MTS and proliferation as determined by EdU in a dose-dependent manner and inactivated the clonogenic and migration ability of RCCs. Andro significantly enhances TRAIL-mediated cell cycle arrest at the G2/M phase as determined by flow cytometry and senescence. Moreover, Andro restored TRAIL signaling, which in turns activated pro-apoptosis caspases as determined by immunoblot assay. The TRAIL receptor, death receptor (DR)4, but not DR5, was found to be significantly upregulated in Andro-treated RCC cells, which contributed to the role of Andro as a TRAIL sensitizer. The present study demonstrated that the combined treatment of Andro and TRAIL has potential therapeutic value against renal cancer.