TRAIL Binding Research Articles

Endogenous TRAIL-R4 critically impacts apoptotic and non-apoptotic TRAIL-induced signaling in cancer cells.

Binding of TRAIL to its death domain-containing receptors TRAIL-R1 and TRAIL-R2 can induce cell death and/or pro-inflammatory signaling. The importance of TRAIL and TRAIL-R1/R2 in tumor immune surveillance and cancer biology has meanwhile been well documented. In addition, TRAIL has been shown to preferentially kill tumor cells, raising hope for the development of targeted anti-cancer therapies. Apart from death-inducing receptors, TRAIL also binds to TRAIL-R3 and TRAIL-R4. Whereas TRAIL-R3 is lacking an intracellular domain entirely, TRAIL-R4 contains a truncated death domain but still a signaling-competent intracellular part. It is assumed that these receptors have anti-apoptotic, yet still not well understood regulatory functions.To analyze the significance of the endogenous levels of TRAIL-R4 for TRAIL-induced signaling in cancer cells, we stably knocked down this receptor in Colo357 and MDA-MB-231 cells and analyzed the activation of apoptotic and non-apoptotic pathways in response to treatment with TRAIL.We found that TRAIL-R4 affects a plethora of signaling pathways, partly in an opposite way. While knockdown of TRAIL-R4 in Colo357 strongly increased apoptosis and reduced clonogenic survival, it inhibited cell death and improved clonogenic survival of MDA-MB-231 cells after TRAIL treatment. Furthermore, TRAIL-R4 turned out to be an important regulator of the expression of a variety of anti-apoptotic proteins in MDA-MB-231 cells since TRAIL-R4-KD reduced the cellular levels of FLIPs, XIAP and cIAP2 but upregulated the levels of Bcl-xL. By inhibiting Bcl-xL with Navitoclax, we could finally show that this protein mainly accounts for the acquired resistance of MDA-MB-231 TRAIL-R4-KD cells to TRAIL-induced apoptosis. Analyses of non-apoptotic signaling pathways revealed that in both cell lines TRAIL-R4-KD resulted in a constitutively increased activity of AKT and ERK, while it reduced AKT activity after TRAIL treatment. Furthermore, TRAIL-R4-KD potentiated TRAIL-induced activation of ERK and p38 in Colo357, and NF-κB in MDA-MB-231 cells. Importantly, in both cell lines the activity of AKT, ERK, p38 and NF-κB after TRAIL treatment was higher in TRAIL-R4-KD cells than in respective control cells.Thus, our data provide evidence for the important regulatory functions of endogenous TRAIL-R4 in cancer cells and improve our understanding of the very complex human TRAIL/TRAIL-R system.

Copy number variation in triple negative breast cancer samples associated with lymph node metastasis

Triple negative breast cancer (TNBC) is a highly metastatic and aggressive subtype of breast cancer and cases presenting with lymph node involvement have worse outcomes. This study aimed to determine the regions of copy number variation (CNV) associated with lymph node metastasis in TNBC patients. CNV analyses were performed in a study cohort of 23 invasive ductal carcinomas (IDCs), 12 lymph node metastases (LNmets), and 7 normal adjacent tissues (NATs); as well as in an independent cohort containing 70 TNBC IDCs and the same 7 NATs. CNV-associated genes were analyzed using GO-enrichment and Pathway analysis. The prognostic role for genes showing CNV-based changes in messenger RNA expression was determined using the Kaplan-Meier plotter database. For the IDCs, there were a number of variations that were common in both the study and independent cohorts in the amplified regions of 1q, 8q, 19 (p and q), 2p, 5p and the deleted regions in 8p followed by 5q, and 19p. The most frequently amplified regions in the LNmets of the study cohort were 4q28.3, 2p, 3q24, 1q21.2, 10p, 12p11.1, 8q, 20p11.22-20p11.21, 21q22.13, 6p22.1 and the most frequently deleted regions were in 1p36.23, 4q21.1 and 5q. A total of 686 (441 amplified and 245 deleted) genes were associated with LNmets. The LNmet-associated genes were highly enriched for “regulation of complement activation,” “regulation of protein activation cascade,” “regulation of humoral immune response,” “oxytocin signalling pathway,” and “TRAIL binding” pathways. Moreover, 6/686 LNmet-associated genes showed CNV-based changes in their mRNA expression of which, high expression of ASPM and KIF14 was significantly associated with worse relapse-free survival. This study has identified several CNV regions in TNBC that could play a major role in metastasis to the lymph node.

Abstract B42: Role of TRAIL signaling in tumor killing

Abstract TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is an endogenous ligand, which plays role in immune surveillance and anti-tumor immunity. Binding of TRAIL to its death receptor (DR4 or DR5) leads to receptor oligomerization and recruitment of the death-inducing signaling complex (DISC), activates the caspase-8 and its downstream caspase cascade and ultimately leads to cell death. Although TRAIL induces apoptosis in many tumor cell lines, almost all normal cells are insensitive to TRAIL-induced cell death. The physiological role of TRAIL remains to be elucidated. Recently, scientists have turned their attention to the emerging role of TRAIL in modulating immune responses. TRAIL administration induced anti-inflammatory effects in several autoimmune animal models. All these results suggest an apoptosis-independent role of TRAIL in the immune system. In this study, we studied the role of TRAIL signaling in immune resistance to tumor growth by using the TRAILR-R knockout mice. We demonstrated TRAIL suppressed T cell activation stimulated by anti-CD3/CD28. T cells from TRAIL-R knockout mice resisted to these effects and were with higher proliferation and cytokine production capability to anti-CD3/CD28 stimulation. However, when the melanoma cell lines B16-F10 were inoculated, there was enhanced tumor growth in TRAIL-R knockout mice compared to the wild type control. This finding suggests TRAIL/TRAIL-R interaction may play a role in host against tumor growth. The immunophenotype of the tumor infiltrating lymphocytes and cytokine production are analyzed. Apoptosis and activation of caspases of tumor cells and infiltrating immune cells are also characterized. This study will help to elucidate the role of TRAIL signaling in tumor killing and immune surveillance. Citation Format: Shih-Hong Siao, Ping-Ning Hsu. Role of TRAIL signaling in tumor killing. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B42.

An HPLC method associated with a thermodynamic analysis to compare the binding of TRAIL and its nanovectorized form to death receptors DR4 and DR5 and their relationship to cytotoxicity.

TRAIL is a member of the TNF family of cytokines which induces apoptosis of cancer cells via its binding to its cognate receptors, DR5 a high affinity site and DR4 a site of low affinity. Our working group has recently demonstrated that nanovectorization of TRAIL with single wall carbon nanotubes (abbreviated NPT) enhanced TRAIL affinity to the high affinity site DR5 and increased pro apoptotic potential in different human tumor cell lines. In this paper, the DR4 low affinity site was immobilized on a chromatographic support and the effect of temperature on a wide temperature range 1°C-50°C was studied to calculate the thermodynamic parameters of the binding of TRAIL and NPT to DR4 and DR5 receptors. For the first time the heat capacity changes for the different binding processes were determined. At a physiological pH (7.4) the heat capacity changes for the binding of NPT to DR4 and DR5 were respectively equal to -0.91kJ/molK and -0.28kJ/molK and those obtained for the binding of TRAIL to DR4 and DR5 were respectively equal to -1.54kJ/molK and -1.05kJ/molK. By the use of differential scanning calorimetry (DSC), a phase transition (∼12°C for DR5, ∼4°C for DR4) between a disordered (low temperature) and an ordered (high temperature) solid like state visualized in the receptor structure confirmed the temperature dependence of binding affinity enthalpy ΔH for soluble TRAIL and its nanovectorized form to its cognate receptors. In the low temperature domain, the positive ΔH values contribute non-favourably to the free energy of binding, TRAIL and NPT described similar affinities for DR4 and DR5. For the high temperature domain, negative ΔH values indicated that van der Waals interactions and hydrogen bonding are engaged favourably at the ligand - receptor interface. Above 30°C, their rank-ordered affinities were thus strongly different in the sequence: TRAILDR4<NPTDR4<TRAILDR5< NPTDR5. The nanovectorization of TRAIL enhanced its binding to both DR4 and DR5 receptors at 37°C and could potentially sensitized cancer cells to TRAIL induced apoptosis through simultaneous activation of DR4 and DR5 as described in this paper for the non-small lung carcinoma cell line (H1703), the two hepatocarcinoma cell lines (SK-Hep1, HUH) and the colon carcinoma cell line (HCT116WT). The excellent linear coefficient (r2=1) for the plot comparing NPT cytotoxicity to TRAIL cytotoxicity confirmed a high degree of similarity for the mechanism of cytotoxicity of TRAIL and NPT between these four cell lines expressing DR4 and DR5 receptors. The slope (0.10) and the negative intercept (-1.23) of this plot indicated that NPT was much more efficient in these four cell lines as compared to TRAIL alone. As well, it was shown that the extra cellular acidosis led to the protonation of the TRAIL residue histidine by flipping the His switch to the on position with a concomitant decrease in affinity for DR4 and DR5 receptors. The highest affinity for NPT to DR4 and DR5 receptors observed at low pHs was due to the less accessibility of the His molecular switch to be protonated when TRAIL was immobilized on CNTs.

Abstract 3493: New, highly potent antibodies to death receptors having Fc mutations to increase antitumor activity

Abstract Death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2 or Apo2) are TNF receptor superfamily members that are receptors for TRAIL (Apo2 ligand), an immunomodulatory cytokine. Binding of TRAIL to DR4 or DR5 can activate the extrinsic apoptotic pathway selectively in tumor cells. Agonist antibodies to death receptors DR4 and DR5 thus have potential for treatment of cancer and may be better than derivatives of TRAIL itself because of their superior pharmacokinetics and receptor selectivity. However, despite dramatic effects in vitro and in preclinical xenograft models, several agonist antibodies to DR4 and especially DR5 have not provided significant therapeutic benefit in clinical trials. While resistance mechanisms in cancer cells are undoubtedly one reason, another factor may be inadequate potency of the antibodies tested. We therefore used intense immunization and screening protocols to develop very potent anti-DR4 and anti-DR5 monoclonal antibodies (mAbs), which were humanized to make the mAbs denoted HuD114 and HuG4.2 respectively. These mAbs were substantially more effective at killing tumor cells in vitro than the mAbs that have been tested in clinical trials. In addition, we introduced one or two mutations into the Fc (constant) regions of these and other potent anti-DR4 and anti-DR5 mAbs in order to increase affinity for the Fc gamma receptor IIb. Cross-linking, as provided by binding of anti-DR4 and anti-DR5 mAbs to Fc gamma receptors on immune cells, is required for effective transmission of an apoptotic signal through the death receptors. Accordingly, introduction of these mutations greatly increased the ability of the mAbs to kill tumor cells in vitro in the presence of human peripheral blood mononuclear cells, and to inhibit the growth of tumor xenografts in mouse models, with two mutations generally more effective than a single mutation. As a complementary approach to increase cross-linking, we also developed bispecific antibodies containing two binding domains from an anti-DR4 mAb and two binding domains from an anti-DR5 mAb. These bispecific mAbs were made in the IgG-like Bs(scFv)4-IgG format consisting of a homodimer of two monomers, each monomer having a single chain Fv from an anti-DR4 mAb linked to a heavy chain constant region, and a single chain Fv from an anti-DR5 mAb linked to a light chain constant region. Such a bispecific mAb was more effective at killing tumor cells than an anti-DR4 or anti-DR5 mAb alone, or even a mixture of anti-DR4 and anti-DR5 mAbs. Based on their potency in vitro and in animal models, we believe that HuD114 and HuG4.2, enhanced by the Fc mutations and/or in bispecific form, have the potential for greater clinical efficacy than previously tested anti-DR4 and anti-DR5 mAbs and thus merit further investigation. Citation Format: Lihong Wang, Yi Ding, Hangil Park, April Zhang, Zhengran Wang, Maximiliano Vasquez, Cary Queen, Jin Kim. New, highly potent antibodies to death receptors having Fc mutations to increase antitumor activity. [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 3493.

Abstract 3513: Promoting TRAIL apoptosis signaling using 17-beta-hydroxywithanolides

Abstract We have previously reported that withanolide E (WE), a steroidal lactone from Physalis peruviana, was highly active in sensitizing various human carcinoma cell lines to TRAIL-mediated apoptosis. Treatment of cancer cells with WE induced a reduction in the levels of the antiapoptotic proteins cFLIPL and cFLIPS, resulting in an increased activation of caspase-8 on subsequent TRAIL binding to its death receptors DR4 or DR5. The reduction in cFLIPL and cFLIPS was due to their destabilization and increased degradation by the proteasome. Interestingly, WE (a 17-beta-hydoxywithanolide, 17-BHW) was a far superior TRAIL sensitizer than more widely studied withaferin A (WFA) and its analogues, which lack the 17-beta-hydroxy group and bear an opposite side chain orientation, exhibit more promiscuous reactivity and are much more directly toxic to cells. Therefore, over 30 natural and semi-synthetic 17-BHWs were evaluated for their ability to promote death ligand-mediated cancer cell death. The 17-BHWs used in this work were obtained by the application of an efficient method of plant biomass production involving our innovative and patented soil-less aeroponic cultivation of P. crassifolia and P. peruviana and by chemical modification of natural withanolides produced by these plants. Our studies identified several 17-BHWs that were 4-8 fold more potent than WE in sensitizing the renal carcinoma cells ACHN to TRAIL-mediated apoptosis. These more active 17-BHWs were also more efficient at reducing cellular levels of cFLIPL and cFLIPS and enhancing caspase-8 activation. Preliminary structure activity relationship (SAR) studies suggested that the enone moiety in ring A was essential for activity. In addition acetoxylation at C-18, an alpha orientation of the lactone group and the double bond at C-24(25) of the lactone ring played important roles in determining the activity of 17-BHWs as TRAIL sensitizers. This suggests that the 17-BHW scaffold is amenable to optimization by a medicinal chemistry approach, which could lead to the identification of highly active natural product-based sensitizers of cancer cells to TRAIL-mediated apoptosis. The cellular molecular target(s) of active 17-BHWs are currently under further investigation. Funded by FNLCR Contract HHSN261200800001E Citation Format: Alan D. Brooks, Ya-ming Xu, E. M. Kithsiri Wijeratne, Poonam Tewary, Curtis J. Henrich, Cheryl L. Thomas, A. A. Leslie Gunatilaka, Thomas J. Sayers. Promoting TRAIL apoptosis signaling using 17-beta-hydroxywithanolides. [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 3513.

Abstract 2464: DART® molecules with enhanced DR5 agonistic activity for improved cancer cell cytotoxicity

Abstract Introduction: Death Receptor 5 (DR5) belongs to the tumor necrosis factor receptor superfamily. The target and its associated signaling pathway are preferentially active on cancer cells. DR5 elicits pro-apoptotic signal through receptor oligomerization upon binding of TRAIL or an agonistic mAb. Chemotherapy and radiation also can synergize with the DR5 pathway. Various therapeutics targeting DR5 have been generated but clinical outcomes have been generally disappointing. Here we describe optimization of the agonistic activity of anti-DR5 mAbs by enabling multivalent target engagement via engineering into DARTs; covalently-linked Fv-based diabodies optimized for manufacturability and stability (1) whose modular nature facilitates tailoring avidity and PK properties to the specific needs of the application. Methods: Anti-DR5 mAbs were selected from a panel of mAbs generated from cancer cell immunizations and characterized for binding properties. DARTs with varying anti-DR5 specificities, valency and incorporation of either a wild type IgG1 Fc or an Fc mutated to eliminate FcγR binding, were expressed in CHO cells and purified to homogeneity. DR5 DARTs and mAbs were characterized across a panel of cell lines including those derived from colorectal, lung, pancreatic, breast and prostate cancer and by IHC on normal and tumor tissue specimens. Results: mAbs from whole cancer cell immunizations displaying differential expression on both normal and cancer tissues were subjected to antigen identification and identified a subset with reactivity to DR5. Binding analyses revealed DR5 mAbs recognizing non-overlapping epitopes that do not block the TRAIL-DR5 interaction. Upon secondary cross-linking or when combined, the DR5 mAbs induced apoptosis across multiple cancer cell lines. Further potency enhancement independent of secondary cross-linking was obtained by the engineered multivalent DR5-targeting DARTs. Dose-dependent growth inhibition assay demonstrated that the DR5-targeting DARTs delivered significantly greater potency than TRAIL (≥100-fold) across a broad range of cancer cell lines. Furthermore, the DR5-targeting DARTs maintained the exquisite tumor/normal differential reactivity displayed by the parental DR5 mAbs from which they were derived. Combination of an HDAC inhibitor with DR5-targeting DARTs revealed maintenance of synergistic activity and potential to overcome pathway resistance. Conclusion: Whole cancer cell immunizations yielded DR5 mAb candidates with desirable binding and functional properties. Through incorporation of the anti-DR5 mAb specificities into multivalent DART molecules, we have generated a new class of therapeutics that may overcome the limitations of existing DR5-based therapeutics. The data support the use of DR5-targeting DARTs to target this apoptotic pathway in multiple cancer cells. Reference: (1) Johnson et al. 2010. J Mol Bio. 399:436-49 Citation Format: Jonathan C. Li, Kalpana Shah, Jill Rillema, Francine Chen, Doug Smith, Steve Burke, Valentina Ciccarone, Sergey Gorlatov, Ralph Alderson, James Tamura, Ezio Bonvini, Syd Johnson, Paul A. Moore. DART® molecules with enhanced DR5 agonistic activity for improved cancer cell cytotoxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2464. doi:10.1158/1538-7445.AM2015-2464

Variants of Osteoprotegerin Lacking TRAIL Binding for Therapeutic Bone Remodeling in Osteolytic Malignancies.

Osteolytic bone damage is a major cause of morbidity in several metastatic pathologies. Current therapies using bisphosphonates provide modest improvement, but cytotoxic side effects still occur prompting the need to develop more effective therapies to target aggressive osteoclastogenesis. Increased levels of receptor activator of NF-κB ligand (TNFSF11/RANKL), leading to RANKL-RANK signaling, remain the key axis for osteoclast activation and bone resorption. Osteoprotegerin (TNFRSF11B/OPG), a decoy receptor for RANKL, is significantly decreased in patients who present with bone lesions. Despite its potential in inhibiting osteoclast activation, OPG also binds to TNF-related apoptosis-inducing ligand (TNFSF10/TRAIL), making tumor cells resistant to apoptosis. Toward uncoupling the events of TRAIL binding of OPG and to improve its utility for bone remodeling without inducing tumor resistance to apoptosis, OPG mutants were developed by structural homology modeling based on interactive domain identification and by superimposing models of OPG, TRAIL, and its receptor DR5 (TNFRSF10B) to identify regions of OPG for rational design. The OPG mutants were purified and extensively characterized for their ability to decrease osteoclast damage without affecting tumor apoptosis pathway both in vitro and in vivo, confirming their potential in bone remodeling following cancer-induced osteolytic damage. OPG variants were developed that lack TRAIL binding, yet retain RANKL binding and suggest new possibilities for therapeutic targeting in osteolytic malignancies.

Use of decoy receptor 3 to block gemcitabine-induced apoptosis via binding with TRAIL in pancreatic cancer.

238 Background: Most of human pancreatic cancer cell lines are resistant to chemotherapeutics-induced apoptosis including gemcitabine. This study is to address the role of decoy receptor3 (DcR3) in pancreatic cancer resistant to chemotherapeutics. Methods: siRNA targeted for DcR3 was used to down-regulate DcR3 expression in pancreatic cancer cells. On exploring for the pathway of DcR3 blocking Gemcitabine-induced apoptosis, up or down-regulated DcR3 level of pancreatic cancer cells were treated with Gemcitabine and apoptosis susceptibility was evaluated with apoptosis analysis. To investigate whether DcR3 is involved in TRAIL, FasL or LIGHT-mediated apoptosis, PARP and apoptotic cells staining were determined under different DcR3 level. Through ELISA-like binding analysis, immunoprecipitation and FACS, TRAIL binding with DcR3 was tested. In vivo experiment, it is to investigate whether down-regulation of DcR3 affects gemcitabine-induced apoptosis and tumor growth delay. Results: It demonstrates that DcR3 down-regulation sensitizes cells to gemcitabine-induced apoptosis and additional DcR3 level can impair gemcitabine-induced apoptosis in both of cells. It is proved that DcR3 blocks gemcitabine-induced apoptosis via binding with TRAIL which was thought not to bind with DcR3 previously. Furthermore, Regulation of DcR3 changes the amount of membrane-bound TRAIL which is correlated with gemcitabine-stimulation. In vivo experiment of AsPC-1 xenografts, stable down-regulation of DcR3 significantly enhances gemcitabine-induced apoptosis and tumor growth delay. Conclusions: DcR3 may play important role resistant to gemcitabine-induced apoptosis via binding with TRAIL in some of pancreatic cancer cells lines. Targeting DcR3 represents a promising strategy to enhance the anti-tumor activity of chemo-agent in pancreatic cancer, which has important clinical implications.

Candidate Gene Study of TRAIL and TRAIL Receptors: Association with Response to Interferon Beta Therapy in Multiple Sclerosis Patients

TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10−4, pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.

Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes

Bioluminescence imaging (BLI) has shown to be crucial for monitoring in vivo biological processes. So far, only dual bioluminescence imaging using firefly (Fluc) and Renilla or Gaussia (Gluc) luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates. Here, we characterized a codon-optimized luciferase from Vargula hilgendorfii (Vluc) as a reporter for mammalian gene expression. We showed that Vluc can be multiplexed with Gluc and Fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin, coelenterazine, and D-luciferin substrates, respectively. We applied this triple imaging system to monitor the effect of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) delivered using an adeno-associated viral vector (AAV) on brain tumors in mice. Vluc imaging showed efficient sTRAIL gene delivery to the brain, while Fluc imaging revealed a robust antiglioma therapy. Further, nuclear factor-κB (NF-κB) activation in response to sTRAIL binding to glioma cells death receptors was monitored by Gluc imaging. This work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology, oncology, virology, and neuroscience.

Differential roles of CD95L and TRAIL in activation-induced cell death of cytotoxic T lymphocytes (110.22)

Abstract Cytotoxic T lymphocytes can be differentiated into type 1 (Tc1) and type 2 (Tc2) subsets as their T helper counterparts. Interestingly, our investigation in activation-induced cell death mechanisms reveals that CD95L mediates cell death in Tc1 lymphocytes and that Tc2 lymphocytes require TRAIL. Cell death in both subsets is also dependent on caspases. Inhibiting CD95L binding to its receptors in Tc1 lymphocytes from wild type mice displays altered Tc1 lymphocyte death, while Inhibiting TRAIL binding to its receptors in Tc2 lymphocytes shows altered Tc2 lymphocyte death. Activation-induced cell death in both Tc1 and Tc2 lymphocyte subsets can be blocked by pan caspase inhibitors. Moreover, Tc2 lymphocytes differentiated from TRAIL knockout mice show alleviated cell death. Rescued Tc2 lymphocytes secrete significantly higher amounts of IL-4, IL-5 and IL-10 while Tc1 lymphocytes produce higher IFN-γ and TNF-α. Our results indicate that CD95L and TRAIL play differential seminal roles in activation-induced cell death of these two cytotoxic T lymphocyte subsets, and that their different sensitivity to activation-induced cell death can alter CD8+ phenotype. With our study we conclude that Tc1 lymphocyte activation-induced cell death is mediated by CD95L while Tc2 lymphocytes undergo TRAIL-dependent activation-induced cell death.

Abstract 239: A novel multimeric protein scaffold stimulates apoptotic signaling through TRAILR2

Abstract TRAILR2 (DR5, TNFRSF10B) is a member of the TNF-receptor superfamily that mediates programmed cell death following stimulation with its ligand. Binding of homotrimeric ligand TRAIL to TRAILR2 induces oligomerization of the receptors and triggers the apoptotic pathway through induction of caspases. Although TRAILR2 is found to be expressed in a number of normal cell types it is routinely found at high levels in many cancer types. Since TRAIL has been shown to preferentially induce apoptosis in cancer cells versus normal cells, several TRAIL receptor agonists including monoclonal antibodies as well as the ligand itself, are currently in clinical development. Here, we describe a novel protein scaffold (Tn3) based on a fibronectin type-III domain from the protein Tenascin that was engineered to specifically agonize TRAILR2. Connecting TRAILR2-binding Tn3 domains together significantly improved the in vitro potency of the molecule in several cancer cell models. For example, a hexameric TRAILR2-binding Tn3 construct has an EC50 of 0.5 pM in a 72 hour cell viability assay with Colo-205 cells. This is approximately 2 logs better than TRAIL. Furthermore, a subset of TRAIL-resistant cancer cell lines derived from multiple tissues of origin is sensitive to the multimeric Tn3. The improved potency over TRAIL was demonstrated in colorectal cancer xenograft models. In addition, tumor regression was also observed at doses as low as 0.3 mg/kg. These results suggest that a multimeric Tn3 protein scaffold targeting TRAILR2 has more potent anti-tumor activity in vitro and in vivo compared than the natural ligand. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 239. doi:1538-7445.AM2012-239

Abstract 5382: Etoposide enhances TRAIL cytotoxicity by increase of TRAIL-R2 expression via a NF-κB pathway in neuroblastoma cells

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which induces apoptosis in tumor cells while not in normal cells. TRAIL binds with two types of receptors which are apoptosis-inducing death receptors (TRAIL-R1 and TRAIL-R2) and non-apoptosis-inducing decoy receptors (TRAIL-R3 and TRAIL-R4). TRAIL has been considered as a promising agent for selective cancer therapy, although some cancer cells show resistance to apoptosis induction by this ligand. In this study, we examined whether combination treatment of chemotherapeutic drugs with TRAIL induces synergistic cell death in neuroblastoma cells. We showed that pre-treatment with sublethal concentrations of etoposide followed by exposure to TRAIL synergistically enhanced apoptotic cell death compared to treatment with etoposide or TRAIL alone in SK-N-MC cells (28 ± 2% versus 12 ± 3% or 2 ± 2% apoptotic cell death in 48 h), while not in IMR-32 cells (26 ± 4% versus 26 ± 7% or 0.5 ± 0.6% apoptotic cell death in 48 h). These synergistic cell death was inhibited by addition of fusion chimera protein (recombinant human TRAIL-R2: Fc), which can block TRAIL binding to TRAIL-R1 or TRAIL-R2. In addition, pre-treatment with sublethal concentrations of etoposide in SK-N-MC cells induced degradation of IκBα, resulting in NF-κB activation and up-regulation of TRAIL-R2 expression. The increase of TRAIL-R2 expression resulted in TRAIL-induced synergistic cell deaths, which were effectively inhibited by exposure to NF-κB inhibitor. Moreover, we showed that combination treatment significantly decreased the Mcl-1 expression and increased caspase-3 activation compared to treatment with etoposide or TRAIL alone. Our data suggest that pre-treated etoposide activates NF-κB by down-regulation of IκBα, and then NF-κB up-regulates TRAIL-R2 expression enhancing TRAIL-induced apoptosis in neuroblastoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5382.

Human monomeric antibody fragments to TRAIL-R1 and TRAIL-R2 that display potent in vitro agonism

Apoptosis through the TRAIL receptor pathway can be induced via agonistic IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer.

Generation of new TRAIL mutants DR5-A and DR5-B with improved selectivity to death receptor 5

TRAIL (tumor necrosis factor (TNF) related apoptosis-inducing ligand) has been introduced as an extrinsic pathway inducer of apoptosis that does not have the toxicities of Fas and TNF. However, the therapeutic potential of TRAIL is limited because of many primary tumor cells are resistant to TRAIL. Despite intensive investigations, little is known in regards to the mechanisms underlying TRAIL selectivity and efficiency. A major reason likely lies in the complexity of the interaction of TRAIL with its five receptors, of which only two DR4 and DR5 are death receptors. Binding of TRAIL with decoy receptors DcR1 and DcR2 or soluble receptor osteoprotegerin (OPG) fail to induce apoptosis. Here we describe design and expression in Escherichia coli of DR5-selective TRAIL variants DR5-A and DR5-B. The measurements of dissociation constants of these mutants with all five receptors show that they practically do not interact with DR4 and DcR1 and have highly reduced affinity to DcR2 and OPG receptors. These mutants are more effective than wild type TRAIL in induction of apoptosis in different cancer cell lines. In combination with the drugs targeted to cytoskeleton (taxol, cytochalasin D) the mutants of TRAIL induced apoptosis in resistant Hela cells overexpressing Bcl-2. The novel highly selective and effective DR5-A and DR5-B TRAIL variants will be useful in studies on the role of different receptors in TRAIL-induced apoptosis in sensitive and resistant cell lines.

A cell-based high-throughput screen to identify synergistic TRAIL sensitizers

We have developed a high-throughput screen (HTS) to search for novel molecules that can synergize with TRAIL, thus promoting apoptosis of ACHN renal tumor cells in a combinatorial fashion. The HTS detects synthetic compounds and pure natural products that can pre-sensitize the cancer cells to TRAIL-mediated apoptosis, yet have limited toxicity on their own. We have taken into account the individual effects of the single agents, versus the combination, and have identified hits that are synergistic, synergistic-toxic, or additive when combined with TRAIL in promoting tumor cell death. Preliminary mechanistic studies indicate that a subset of the synergistic TRAIL sensitizers act very rapidly to promote cleavage and activation of caspase-8 following TRAIL binding. Caspase-8 is an apical enzyme that initiates programmed cell death via the extrinsic apoptotic pathway. Thus, these TRAIL sensitizers may potentially reduce resistance of tumor cells to TRAIL-mediated apoptosis. Two representative sensitizers were found to increase levels of p53 but did not inhibit the proteasome, suggesting that early DNA damage-sensing pathways may be involved in their mechanisms of action.

Acquired Resistance to Tumor Necrosis Factor-Related Apoptosis- Inducing Ligand (TRAIL): Histone Deacetylase Inhibitors Resensitize TRAIL-Resistant Jurkat Acute Lymphocytic Leukemia Cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a death-ligand from the TNF family. TRAIL induces programmed cell death by the cell-extrinsic p53-independent apoptotic pathway. A potential of TRAIL as cancer-specific therapeutic agent has been proposed and is preclinically and clinically tested. Development of TRAIL-resistant clones in the TRAIL-sensitive tumor cells may be a serious complication of TRAIL based cancer therapy. Jurkat acute lymphocytic leukemia cells are sensitive to TRAIL-induced apoptosis, as well as other apoptosis inducing ligands from TNF family, Fas and TNF-alpha. Jurkat cells express only one of the four receptors for TRAIL, death receptor 5 (DR5). Prolonged exposure of TRAIL-sensitive Jurkat cells to recombinant soluble TRAIL (1000 ng/mL) resulted in the establishment of three TRAIL-resistant (TR) Jurkat cell subclones, Jurkat TR1, TR2, and TR3. The Jurkat TR subclones were also resistant to TNF-alpha and Fas ligand, suggesting disruption of the extrinsic apoptotic pathway. TRAIL-resistant subclone TR1, but not TR2 and TR3, demonstrated decreased susceptibility to undergo apoptosis in response to histone-deacetylase inhibitors, valproic acid (VA), sodium butyrate (SB) and suberoylanilide hydroxamic acid (SAHA) and was resistant to fludarabine.Flow cytometry analysis showed Jurkat TR subclones had unchanged expression of cell surface death receptor DR5, Fas, and receptors for TNF-alpha, TNF-R1 and TNF-R2, compared to TRAIL-sensitive Jurkat cells. Analysis of death-inducing signaling complex (DISC) formation by immunoprecipitation (anti-TRAIL, anti-DR5) and subsequent western blotting (anti-caspase 8, anti-FADD) clearly demonstrated that the DISC formation in response to TRAIL binding to DR5 was significantly decreased in subclones TR2 and TR3, but remained unchanged in subclone TR1 compared to TRAIL-sensitive Jurkat cells.To gain further insight into potential molecular aletarations associated with acquired TRAIL resistance of Jurkat subclones, we measured gene expression of several key apoptotic regulators, including receptors for TRAIL, cFLIP, BCL2 family, IAP family, HSP family members in TRAIL-resistant and TRAIL-sensitive Jurkat cells and did not detect any significant (>2-fold) change. These results suggest acquired TRAIL resistance of Jurkat cells might be mediated by changes on the protein rather than mRNA level. We analyzed whether the TRAIL-resistant Jurkat cells could be resensitized to TRAIL-induced apoptosis by pretreatment with diverse inhibitors of important prosurvival pathways, including inhibitors of proteosynthesis (cycloheximid), inhibitors of transcription (actinomycin D), NFkB inhibitors (bortezomib, SN-50), PI3K-Akt-mTOR inhibitors (rapamycine, LY294002, Hsp90 inhibitor (17-AAG), cyclin-dependent kinase inhibitors (roscovitine), casein kinase II inhibitors (DRB), or histone deacetylase inhibitors (HDACi: SAHA, VA, SB). Pretreatment with HDAC inhibitors for 12 hour was able to resensitize all three TRAIL-resistant Jurkat subclones to TRAIL-induced apoptosis. The percentage of apoptotic cells of HDACi-pretreated subclones was 70–95% 24 h after the exposure to TRAIL compared to 5–15% apoptosis for HDACi-untreated TRAIL-exposed controls, and to 10–15% apoptosis for HDACi-treated TRAIL unexposed controls.We established TRAIL-resistant subclones from the original TRAIL-sensitive Jurkat cells. Acquired resistance to TRAIL was not mediated by downregulation of TRAIL death receptor DR5 and was associated with (cross)resistance to TNFa and Fas ligand, suggesting disruption of cell-extrinsic apoptotic pathway. We assume diverse molecular mechanisms were involved in the development of TRAIL-resistant subclones upon exposure to TRAIL, as exemplified by disrupted formation of DISC in case of subclones TR2 and TR3 and normal DISC formation and fludarabine resistance in subclone TR1, suggesting deregulated apoptotic pathway downstream of DISC. Finally, we observed that HDACi resensitized the TRAIL-resistant subclones to TRAIL. The results provide substantiation for combinatorial approaches in the potential TRAIL-based therapies of hematological malignancies.

Role of IG20 Splice Variants in TRAIL Resistance

Tumor necrosis factor receptor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis primarily in cancer cells with little or no effect on normal cells; therefore, it has the potential for use in cancer therapy. TRAIL binding to death receptors DR4 and DR5 triggers the death-inducing signal complex formation and activation of procaspase-8, which in turn activates caspase-3, leading to cell death. Like FasL, TRAIL can trigger type 1 (caspase-8 --> caspase-3) or type 2 (caspase-8 --> Bid cleavage --> capsase-9 --> caspase-3) apoptotic pathways depending on the cell type. Some cancers are resistant to TRAIL treatment because most molecules in the TRAIL signaling pathway, including FLIPs and IAPs, can contribute to resistance. In addition, we have identified an essential role for splice variants of the IG20 gene in TRAIL resistance.

P-glycoprotein enhances TRAIL-triggered apoptosis in multidrug resistant cancer cells by interacting with the death receptor DR5

The death-inducing cytokine TRAIL is a promising agent for anticancer therapy since it preferentially kills cancer versus normal cells; however, some cancer cells are TRAIL-resistant. We initially explored whether overexpression of the MDR1 gene product P-glycoprotein (P-gp), which causes multidrug resistance (MDR) in cancer cells, also contributes to TRAIL-resistance. Surprisingly, our results revealed that P-gp-overexpression enhances TRAIL-induced apoptosis not only in neoplastic cells transfected with the MDR1 gene but also in MDR variants selected with cytotoxic anticancer agents. Mechanistic analysis of TRAIL-induced apoptosis in the MDR1-transfected MCF-7 breast cancer cell line BC-19 revealed that TRAIL-triggered significantly more apoptosis in these cells compared with parental MCF-7 cells by binding to the TRAIL receptor DR5. DR5 but not DR4 engagement by TRAIL attenuated cellular ATP levels by robustly stimulating P-gp ATPase activity, and thus triggered P-gp-dependent apoptosis by depletion of the cellular ATP pool. In addition to hyperactive P-gp-mediated ATP hydrolysis, TRAIL-induced, P-gp-potentiated apoptosis was associated with activation of caspases-6, -7, -8, and -9; Bid cleavage; and mitochondrial depolarization. P-gp interacted with the TRAIL receptors DR4, DR5, and DcR1 in plasma membranes and enhanced TRAIL binding to DR5. Interestingly, the decreased level of the decoy TRAIL receptor, DcR1, in BC-19 cells further sensitized these cells to TRAIL. Therefore, both extrinsic and intrinsic apoptosis pathways are involved in this process. These findings for the first time reveal that TRAIL treatment preferentially causes apoptosis in P-gp-overexpressing MDR cells, and suggests significant clinical implications for the use of TRAIL in treating neoplasms that have failed chemotherapy.