TRAIL Receptor Agonists Research Articles

HvTRA, a novel TRAIL receptor agonist, induces apoptosis and sustained growth retardation in melanoma.

In recent years, new treatment options for malignant melanoma patients have enhanced the overall survival for selected patients. Despite new hope, most melanoma patients still relapse with drug-resistant tumors or experience intrinsic resistance to the therapy. Therefore, novel treatment modalities beneficial for subgroups of patients are needed. TRAIL receptor agonists have been suggested as promising candidates for use in cancer treatment as they preferentially induce apoptosis in cancer cells. Unfortunately, the first generation of TRAIL receptor agonists showed poor clinical efficacy. hvTRA is a second-generation TRAIL receptor agonist with improved composition giving increased potency, and in the present study, we showed hvTRA-induced activation of apoptosis leading to an efficient and sustained reduction in melanoma cell growth in cell lines and xenograft models. Furthermore, the potential of hvTRA in a clinical setting was demonstrated by showing efficacy on tumor cells harvested from melanoma patients with lymph node metastasis in an ex vivo drug sensitivity assay. Inhibition of mutated BRAF has been shown to regulate proteins in the intrinsic apoptotic pathway, making the cells more susceptible for apoptosis induction. In an attempt to increase the efficacy of hvTRA, combination treatment with the mutated BRAF inhibitor vemurafenib was investigated. A synergistic effect by the combination was observed for several cell lines in vitro, and an initial cytotoxic effect was observed in vivo. Unfortunately, the initial increased reduction in tumor growth compared with hvTRA mono treatment was not sustained, and this was related to downregulation of the DR5 level by vemurafenib. Altogether, the presented data imply that hvTRA efficiently induce apoptosis and growth delay in melanoma models and patient material, and the potential of this TRAIL receptor agonist should be further evaluated for treatment of subgroups of melanoma patients.

Abstract 3494: MEDI3039, a novel highly potent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor agonist, induces apoptotic cell death in breast cancer cells

Abstract TRAIL receptor agonists are attractive anti-tumor agents because of their capability to induce apoptosis in cancer cells by activating death receptors 4 and 5 (DR4 and DR5) with little toxicity against normal cells. We previously reported that GST-TRAIL efficiently induced cell death in breast cancer cells, particularly mesenchymal triple negative breast cancer (TNBC) - so called basal B breast cancer cells (Rahman et al., Breast Cancer Res Treat. 2009). Recently, a newly developed multivalent TRAIL receptor agonist designed to activate DR5, has been shown to be a TRAIL super-agonist with significantly enhanced potency in multiple cancer cell lines (Swers et al., Mol Cancer Ther. 2013). We hypothesized that MEDI3039, developed by modification of this TRAIL super-agonist, is a potential new therapeutic agent to be used in human breast cancer treatment. We used 19 human breast cancer cell lines that can be categorized into 4 groups: ER+, HER2 amplified, TNBC basal A and TNBC basal B. MEDI3039- or GST-TRAIL-induced cell death was analyzed by an MTS assay in a 96 well format after 72h of treatment. MEDI3039- or GST-TRAIL-induced caspase activation was measured by Caspase-glo 3/7 assay. Z-VAD-FMK was used as a pan-caspase inhibitor. To verify that MEDI3039 induced apoptosis via DR5, siRNA against DR4 and DR5 were transfected to cells and tested in MTS assay and Western blotting. MEDI3039 induced cell death in MDA-MB231 (TNBC basal B), and the IC50 was 4.71pM. By contrast, GST-TRAIL induced cell death in this cell line with an IC50 of 624 pM (a 132 fold difference). MEDI3039-induced cell death was completely inhibited by Z-VAD-FMK, indicating that cell death was the result of caspase-mediated apoptotic pathway. Knockdown of DR5, but not DR4, inhibited MEDI3039-induced cell death, demonstrating that MEDI3039-mediated apoptosis requires DR5. MEDI3039 induced cell death in multiple breast cancer cell lines, but the sensitivity varied between cell lines from the four different subtypes. The TNBC basal B group was the most sensitive (avg IC50 = 1.4 pM), the TNBC basal A group was next most sensitive (avg IC50 = 203 pM), the HER2 amplified group was less sensitive (avg IC50 = 314 pM), and the ER+ group was the least sensitive to MEDI3039 (avg IC50 = 403 pM). This relative sensitivity of the different subtypes of breast cancer was similar to what was observed with GST-TRAIL. Importantly, MEDI3039 was at least 2 orders of magnitude more potent compared with GST-TRAIL in most cell lines tested. Drug combination experiments indicated that MEDI3039 has synergistic effect with multiple drugs, including cisplatin and the Wee1 inhibitor MK1775. Data from ongoing animal experiments will be presented. In conclusion, MEDI3039 is a potent TRAIL receptor agonist in breast cancer cells and has potential as a cancer drug in breast cancer patients, especially those with TNBC basal B. Citation Format: Yoshimi Greer, Sam Gilbert, David Tice, Stanley Lipkowitz. MEDI3039, a novel highly potent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor agonist, induces apoptotic cell death in breast cancer cells. [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 3494.

Abstract 3849: The antidiabetic drug metformin increases pro-apoptotic effect of TRAIL therapy by reducing X-linked inhibitor of apoptosis protein (XIAP) levels in triple-negative breast cancer cells

Abstract Most metastatic tumors, such as triple negative breast cancer (TNBC) respond poorly to conventional chemotherapy. We have previously demonstrated that targeting TNF-related apoptosis-inducing ligand receptor-2 (TRAIL-R2) might be an effective pathway against metastatic TNBC. However, many TNBCs are resistant to TRAIL receptor agonists. Here we demonstrate that diabetes drug metformin, previously linked to preventing and treating several types of cancer, enhances sensitivity of TNBC cells to recombinant TRAIL and TRAIL-R2 agonistic humanized monoclonal antibody (lexatumumab)-induced apoptosis. We have demonstrated that combination of metformin with lexatumumab or TRAIL augments response to TRAIL therapy and reduce cancer cell viability via induction of apoptosis and inhibiting long-term survival of three different metastatic TNBC cell lines. Intriguingly, metformin treatment also sensitized transformed MCF10A-RasV12 cells, but not untransformed MCF10A-Vector cells, to TRAIL agonist therapy. This effect was associated with reduction of X-linked Inhibitor of Apoptosis Protein (XIAP) levels after metformin treatment. Similar to metformin treatment, inhibition of XIAP by small interfering RNA sensitized breast cancer cells to TRAIL-induced apoptosis. Moreover, metformin treatment in combination with TRAIL more efficiently inhibited primary tumor growth and lung metastases in orthotopic model of TNBC. Overall, our results demonstrate that metformin treatment in combination with targeting TRAIL receptors may be a good strategy to augment the antitumor effects of TRAIL receptors agonistic therapy and provide the foundation for a clinical trial combining dietary metfomin and TRAIL agonists. Citation Format: Elena Strekalova, Dmitry Malin, Vincent Cryns. The antidiabetic drug metformin increases pro-apoptotic effect of TRAIL therapy by reducing X-linked inhibitor of apoptosis protein (XIAP) levels in triple-negative breast cancer cells. [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 3849.

Onto better TRAILs for cancer treatment.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand (Apo2L), is a member of the TNF cytokine superfamily. By cross-linking TRAIL-Receptor (TRAIL-R) 1 or TRAIL-R2, also known as death receptors 4 and 5 (DR4 and DR5), TRAIL has the capability to induce apoptosis in a wide variety of tumor cells while sparing vital normal cells. The discovery of this unique property among TNF superfamily members laid the foundation for testing the clinical potential of TRAIL-R-targeting therapies in the cancer clinic. To date, two of these therapeutic strategies have been tested clinically: (i) recombinant human TRAIL and (ii) antibodies directed against TRAIL-R1 or TRAIL-R2. Unfortunately, however, these TRAIL-R agonists have basically failed as most human tumors are resistant to apoptosis induction by them. It recently emerged that this is largely due to the poor agonistic activity of these agents. Consequently, novel TRAIL-R-targeting agents with increased bioactivity are currently being developed with the aim of rendering TRAIL-based therapies more active. This review summarizes these second-generation novel formulations of TRAIL and other TRAIL-R agonists, which exhibit enhanced cytotoxic capacity toward cancer cells, thereby providing the potential of being more effective when applied clinically than first-generation TRAIL-R agonists.

Abstract P5-03-06: MEDI3039, a novel highly potent tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor agonist, induces apoptotic cell death in breast cancer cells

Abstract TRAIL receptor agonists are attractive anti-tumor agents because of their capability to induce apoptosis in cancer cells by activating death receptors 4 and 5 (DR4 and DR5) with little toxicity against normal cells. We previously reported that GST-TRAIL efficiently induced cell death in breast cancer cells, particularly mesenchymal triple negative breast cancer (TNBC) – so called basal B breast cancer cells (Rahman et al., Adv. Cancer Res. 2009). Recently, a newly developed multivalent TRAIL receptor agonist designed to activate DR5, has been shown to be a TRAIL super-agonist with significantly enhanced potency in multiple cancer cell lines (Swers et al., Mol Cancer Ther. 2013). We hypothesized that MEDI3039, developed from this TRAIL super-agonist, is a potential new therapeutic agent to be used in human breast cancer treatment. As model systems, we used 19 breast cancer cell lines that can be categorized into 4 different groups: ER+, HER2 amplified, TNBC basal A and TNBC basal B. MEDI3039- or GST-TRAIL-induced cell death was analyzed by an MTS assay in 96 well format after 72h of treatment. MEDI3039- or GST-TRAIL-induced caspase activation was measured by Caspase-glo 3/7 assay. Z-VAD-FMK was used as a pan-caspase inhibitor. To verify the receptor for MEDI3039, siRNA against DR4 and DR5 were transfected to cells and tested in MTS assay and Western blotting. MEDI3039 induced cell death in MDA-MB231 (TNBC basal B), and the IC50 was 4.71pM. By contrast, GST-TRAIL induced cell death in this cell line with an IC50 of 624 pM (a 132 fold difference). MEDI3039 and GST-TRAIL induced cell death was completely inhibited by Z-VAD-FMK, indicating that cell death was the result of caspase-mediated apoptotic pathway. Knockdown of DR5, but not DR4, inhibited MEDI3039-induced cell death, demonstrating that MEDI3039-mediated apoptosis requires DR5. MEDI3039 induced cell death in multiple breast cancer cell lines, but the sensitivity varied between cell lines from the four different subtypes. TNBC basal B group was the most sensitive (avg IC50= 1.4 pM), TNBC basal A group was next most sensitive (avg IC50 = 203 pM, HER2 amplified group was less sensitive (avg IC50 = 314 pM), and ER+ group was the least sensitive to MEDI3039 (avg IC50= 403 pM). This was similar to what was observed with GST-TRAIL. Importantly, MEDI3039 was at least 2 orders of magnitude more potent compared with GST-TRAIL in most cell lines tested. Drug combination experiments indicated that MEDI3039 has synergistic effect with multiple drugs, including cisplatin, MK1775. Animal breast cancer xenograft experiments are planned to test the efficacy of MEDI3039 in vivo. Further work to identify biomarker(s) that correlate with MEDI3039 sensitivity, and effective combinations that enhance the toxicity of MEDI3039 especially in the resistant breast cancer subtypes are ongoing. In conclusion, MEDI3039 is a potent TRAIL receptor agonist in breast cancer cells and has potential as a cancer drug in breast cancer patients, especially those with TNBC basal B. Citation Format: Greer YE, Tice D, Lipkowitz S. MEDI3039, a novel highly potent tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor agonist, induces apoptotic cell death in breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-06.

The TRAIL receptor agonist drozitumab targets basal B triple-negative breast cancer cells that express vimentin and Axl.

Previously, we found that GST-tagged tumor necrosis factor-related apoptosis inducing ligand preferentially killed triple-negative breast cancer (TNBC) cells with a mesenchymal phenotype by activating death receptor 5 (DR5). The purpose of this study was to explore the sensitivity of breast cancer cell lines to drozitumab, a clinically tested DR5-specific agonist; identify potential biomarkers of drozitumab-sensitive breast cancer cells; and determine if those biomarkers were present in tumors from patients with TNBC. We evaluated viability, caspase activity, and sub-G1 DNA content in drozitumab-treated breast cancer cell lines and we characterized expression of potential biomarkers by immunoblot. Expression levels of vimentin and Axl were then explored in 177 TNBC samples from a publically available cDNA microarray dataset and by immunohistochemistry (IHC) in tumor tissue samples obtained from 53 African-American women with TNBC. Drozitumab-induced apoptosis in mesenchymal TNBC cell lines but not in cell lines from other breast cancer subtypes. The drozitumab-sensitive TNBC cell lines expressed the mesenchymal markers vimentin and Axl. Vimentin and Axl mRNA and protein were expressed in a subset of human TNBC tumors. By IHC, ~15 % of TNBC tumors had vimentin and Axl expression in the top quartile for both. These findings indicate that drozitumab-sensitive mesenchymal TNBC cells express vimentin and Axl, which can be identified in a subset of human TNBC tumors. Thus, vimentin and Axl may be useful to identify TNBC patients who would be most likely to benefit from a DR5 agonist.

Cytoplasmic levels of cFLIP determine a broad susceptibility of breast cancer stem/progenitor-like cells to TRAIL.

BackgroundThe clinical application of TRAIL receptor agonists as a novel cancer therapy has been tempered by heterogeneity in tumour responses. This is illustrated in breast cancer, where TRAIL is cytotoxic in cell lines of mesenchymal origin but refractory in lines with an epithelial-like phenotype. However, it is now evident that intra-tumour heterogeneity includes a minority subpopulation of tumour-initiating stem/progenitor-like cells (CSCs) that possess mesenchymal characteristics. We hypothesised therefore that TRAIL may target these phenotypically distinct CSC-like cells that are common to most - if not all - breast cancers, thus impacting on the source of malignancy in a much broader range of breast tumour subtypes than previously envisaged.MethodsWe used colony formation, tumoursphere, flow cytometry and xenograft tumour initiation assays to observe the TRAIL sensitivity of CSC-like cells in a panel of two mesenchymal-like (TRAIL-sensitive) and four epithelial-like (TRAIL-resistant) breast cancer cell lines. Subcellular levels of the endogenous TRAIL inhibitor, cFLIP, were determined by western blot and immunofluorescence microscopy. The effect of the subcellular redistribution of cFLIP on TRAIL sensitivity and Wnt signalling was determined using cFLIP localisation mutants and the TOPFlash reporter assay respectively.ResultsTRAIL universally suppressed the clonal expansion of stem/progenitors in all six of the breast cancer cell lines tested, irrespective of their phenotype or overall sensitivity to TRAIL. A concomitant reduction in tumour initiation was confirmed in the TRAIL-resistant epithelial cell line, MCF-7, following serial dilution xenotransplantation. Furthermore TRAIL sensitivity of breast CSCs was inversely proportional to the relative cytoplasmic levels of cFLIP while overexpression of cFLIP in the cytosol using subcellular localization mutants of cFLIP protected these cells from cytotoxicity. The accumulation of nuclear cFLIP on the other hand did not influence TRAIL cytotoxicity but instead promoted Wnt-dependent signalling.ConclusionThese data propose a novel role for TRAIL as a selective CSC agent with a broad specificity for both epithelial and mesenchymal breast tumour subtypes. Furthermore we identify a dual role for cFLIP in the maintenance of breast CSC viability, dependent upon its subcellular distribution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0478-y) contains supplementary material, which is available to authorized users.

Triptolide enhances the tumoricidal activity of TRAIL against renal cell carcinoma.

Renal cell carcinoma (RCC) is resistant to traditional cancer therapies, and metastatic RCC (mRCC) is incurable. The shortcomings in current therapeutic options for patients with mRCC provide the rationale for the development of novel treatment protocols. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has proven to be a potent inducer of tumor cell death in vitro and in vivo, and a number of TRAIL death receptor agonists (recombinant TRAIL or TRAIL death receptor-specific mAb) have been developed and tested clinically. Unfortunately the clinical efficacy of TRAIL has been underwhelming and is likely due to a number of possible mechanisms that render tumors resistant to TRAIL, prompting the search for drugs that increase tumor cell susceptibility to TRAIL. The objective of this study was to determine the effectiveness of combining the diterpene triepoxide triptolide, or its water-soluble prodrug, Minnelide, with TRAIL receptor agonists against RCC in vitro or in vivo, respectively. TRAIL-induced apoptotic death of human RCC cells was increased in the presence of triptolide. The triptolide-induced sensitization was accompanied by increased TRAIL-R2 (DR5) and decreased heat shock protein 70 expression. In vivo treatment of mice bearing orthotopic RCC (Renca) tumors showed the combination of Minnelide and agonistic anti-DR5 mAb significantly decreased tumor burden and increased animal survival compared to either therapy alone. Our data suggest triptolide/Minnelide sensitizes RCC cells to TRAIL-induced apoptosis through altered TRAIL death receptor and heat shock protein expression.

Therapeutic applications of TRAIL receptor agonists in cancer and beyond.

TRAIL/Apo-2L is a member of the TNF superfamily first described as an apoptosis-inducing cytokine in 1995. Similar to TNF and Fas ligand, TRAIL induces apoptosis in caspase-dependent manner following TRAIL death receptor trimerization. Because tumor cells were shown to be particularly sensitive to this cytokine while normal cells/tissues proved to be resistant along with being able to synthesize and release TRAIL, it was rapidly appreciated that TRAIL likely served as one of our major physiologic weapons against cancer. In line with this, a number of research laboratories and pharmaceutical companies have attempted to exploit the ability of TRAIL to kill cancer cells by developing recombinant forms of TRAIL or TRAIL receptor agonists (e.g., receptor-specific mAb) for therapeutic purposes. In this review article we will describe the biochemical pathways used by TRAIL to induce different cell death programs. We will also summarize the clinical trials related to this pathway and discuss possible novel uses of TRAIL-related therapies. In recent years, the physiological importance of TRAIL has expanded beyond being a tumoricidal molecule to one critical for a number of clinical settings - ranging from infectious disease and autoimmunity to cardiovascular anomalies. We will also highlight some of these conditions where modulation of the TRAIL/TRAIL receptor system may be targeted in the future.

Methionine Deprivation Induces a Targetable Vulnerability in Triple-Negative Breast Cancer Cells by Enhancing TRAIL Receptor-2 Expression.

Many neoplasms are vulnerable to methionine deficiency by mechanisms that are poorly understood. Because gene profiling studies have revealed that methionine depletion increases TNF-related apoptosis-inducing ligand receptor-2 (TRAIL-R2) mRNA, we postulated that methionine stress sensitizes breast cancer cells to proapoptotic TRAIL-R2 agonists. Human triple (ER/PR/HER2)-negative breast carcinoma cell lines were cultured in control or methionine-free media. The effects of methionine depletion on TRAIL receptor expression and sensitivity to chemotherapy or a humanized agonistic TRAIL-R2 monoclonal antibody (lexatumumab) were determined. The melanoma-associated antigen MAGED2 was silenced to delineate its functional role in sensitizing TNBC cells to methionine stress. An orthotopic TNBC model was utilized to evaluate the effects of dietary methionine deficiency, lexatumumab, or the combination. Methionine depletion sensitized TNBC cells to lexatumumab-induced caspase activation and apoptosis by increasing TRAIL-R2 mRNA and cell surface expression. MCF-10A cells transformed by oncogenic H-Ras, but not untransformed cells, and matrix-detached TNBC cells were highly sensitive to the combination of lexatumumab and methionine depletion. Proteomics analyses revealed that MAGED2, which has been reported to reduce TRAIL-R2 expression, was suppressed by methionine stress. Silencing MAGED2 recapitulated features of methionine deprivation, including enhanced mRNA and cell surface expression of TRAIL receptors and increased sensitivity to TRAIL receptor agonists. Dietary methionine deprivation enhanced the antitumor effects of lexatumumab in an orthotopic metastatic TNBC model. Methionine depletion exposes a targetable defect in TNBC cells by increasing TRAIL-R2 expression. Our findings provide the foundation for a clinical trial combining dietary methionine restriction and TRAIL-R2 agonists. Clin Cancer Res; 21(12); 2780-91. ©2015 AACR.

Distinct effects of TRAIL on the mitochondrial network in human cancer cells and normal cells: role of plasma membrane depolarization.

Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a promising anticancer drug due to its tumor-selective cytotoxicity. Here we report that TRAIL exhibits distinct effects on the mitochondrial networks in malignant cells and normal cells. Live-cell imaging revealed that multiple human cancer cell lines and normal cells exhibited two different modes of mitochondrial responses in response to TRAIL and death receptor agonists. Mitochondria within tumor cells became fragmented into punctate and clustered in response to toxic stimuli. The mitochondrial fragmentation was observed at 4 h, then became more pronounced over time, and associated with apoptotic cell death. In contrast, mitochondria within normal cells such as melanocytes and fibroblasts became only modestly truncated, even when they were treated with toxic stimuli. Although TRAIL activated dynamin-related protein 1 (Drp1)-dependent mitochondrial fission, inhibition of this process by Drp1 knockdown or with the Drp1 inhibitor mdivi-1, potentiated TRAIL-induced apoptosis, mitochondrial fragmentation, and clustering. Moreover, mitochondrial reactive oxygen species (ROS)-mediated depolarization accelerated mitochondrial network abnormalities in tumor cells, but not in normal cells, and TRAIL caused higher levels of mitochondrial ROS accumulation and depolarization in malignant cells than in normal cells. Our findings suggest that tumor cells are more prone than normal cells to oxidative stress and depolarization, thereby being more vulnerable to mitochondrial network abnormalities and that this vulnerability may be relevant to the tumor-targeting killing by TRAIL.

Fractional killing arises from cell-to-cell variability in overcoming a caspase activity threshold.

When cells are exposed to death ligands such as TRAIL, a fraction undergoes apoptosis and a fraction survives; if surviving cells are re-exposed to TRAIL, fractional killing is once again observed. Therapeutic antibodies directed against TRAIL receptors also cause fractional killing, even at saturating concentrations, limiting their effectiveness. Fractional killing arises from cell-to-cell fluctuations in protein levels (extrinsic noise), but how this results in a clean bifurcation between life and death remains unclear. In this paper, we identify a threshold in the rate and timing of initiator caspase activation that distinguishes cells that live from those that die; by mapping this threshold, we can predict fractional killing of cells exposed to natural and synthetic agonists alone or in combination with sensitizing drugs such as bortezomib. A phenomenological model of the threshold also quantifies the contributions of two resistance genes (c-FLIP and Bcl-2), providing new insight into the control of cell fate by opposing pro-death and pro-survival proteins and suggesting new criteria for evaluating the efficacy of therapeutic TRAIL receptor agonists.

Abstract P5-10-05: TRAIL receptor agonists target basal B triple negative breast Cancer (TNBC) that expresses vimentin and axl

Abstract Background: Tumor Necrosis Factor Related Ligand (TRAIL) triggers apoptosis by binding to cell surface receptors. We previously showed that TRAIL receptor agonists preferentially kill TNBC cells with mesenchymal features (Basal B cell lines) through activation of TRAIL Receptor 2 (TRAIL-R2). We used preclinical models to identify predictive biomarkers of TRAIL sensitivity and tested expression of these markers in TNBC patient (pt) samples. Methods: We tested sensitivity to the TRAIL-R2 specific agonist drozitumab in vitro using cell viability and caspase assays. We examined expression of the mesenchymal proteins Axl and vimentin(vim) in breast cancer cell lines including TNBC by immunoblotting and using commercially available cDNA microarray data sets. Next, we performed an exploratory analysis on IHC tumor expression of vim and Axl in 53 African American pts with TNBC diagnosed between February 2003 and February 2009. In a retrospective cohort study, overall survival (OS) was calculated from date of surgery until date of death or last follow up. Disease-free survival (DFS) was calculated from the date that the pt was identified as being disease free until date of recurrence or date of last followed without recurrence. The significance of the difference among Kaplan-Meier curves was determined by a log-rank test. Axl, vim, and age at diagnosis values were divided approximately into quartiles based on data from all available pts before being used in actuarial analyses. A Cox proportional hazards analysis was also performed to determine if Axl or vim retained prognostic value after adjusting for other factors jointly associated with outcome. All p-values were two-tailed. Results: As previously demonstrated with TRAIL, drozitumab selectively killed Basal B TNBC cell lines. Gene analysis and protein expression demonstrated that vim and Axl were selectively expressed in drozitumab sensitive Basal B cells. Analysis of cDNA microarray data sets showed that approximately 40% of TNBC express high levels of both Axl and vim. IHC confirmed that expression of Axl and vim seen on cDNA microarray was in TNBC tumor cells. In an exploratory analysis of the relationship of vim and Axl expression to OS and DFS, Axl, vim, stage, and response to neoadjuvant chemotherapy were factors found to be potentially associated with OS in univariate analyses while Axl, vim, age and stage were associated with at least trends towards significance with respect to DFS in univariate analyses. Cox models showed that higher vim levels (p=0.08) and stage I and II disease (p=0.024) were potentially associated jointly with OS, while higher Axl levels (p=0.05), age (p=0.016) and stage I and II disease (p=0.0007) were jointly associated with DFS. Conclusions: Preclinical data suggest that expression of vim and Axl can identify TRAIL Receptor agonist sensitive TNBC cells. Based on microarray and IHC, a subset of TNBC tumors express these markers in tumor cells. In our exploratory analysis with limited pts, greater vim and Axl expression were associated with a trend towards better DFS and OS. Vim and Axl may be useful predictive biomarkers for identifying TNBC pts in whom to test TRAIL receptor agonists. Research funding: Safeway Foundation and National Cancer Institute. Citation Format: Ciara C O'Sullivan, Laleh Amiri-Kordestani, Sara Sinclair, Kathryn J Chavez, Jennifer L Dine, Brandon Stone, Stephen M Hewitt, Seth M Steinberg, Sandra M Swain, Stanley Lipkowitz. TRAIL receptor agonists target basal B triple negative breast Cancer (TNBC) that expresses vimentin and axl [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-10-05.

Safety and tolerability of TRAIL receptor agonists in cancer treatment.

Targeting the death receptor pathway of apoptosis represents a promising approach for the development of novel cancer therapeutics, since death receptors on the cell surface are directly linked to the apoptotic machinery. The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor/ligand system is of particular interest among the death receptor superfamily for therapeutic targeting in cancer, since TRAIL has been reported to preferentially induce apoptosis in cancer cells, while sparing non-malignant cells. Evaluation of TRAIL receptor agonists in clinical trials has revealed that they are, in principle, well-tolerated but exert limited efficacy in unselective patient populations. Currently, the challenge resides in the development of rational TRAIL-based combination therapies with potent TRAIL receptor agonists in order to exploit the potential of death receptor targeting for cancer therapy.

TIC10/ONC201: a bend in the road to clinical development.

Tumor necrosis factor (TNF) –related apoptosis-inducing ligand (TRAIL) is an immuno-surveillance cytokine that is expressed in various tissues and cells [1, 2]. The expression of TRAIL on immune cells plays a role in immune surveillance in the prevention of tumors and metastasis [3]. TRAIL is attractive as an anti-tumor agent because of its capability to induce apoptosis in cancer cells by activating death receptors 4 and 5 (DR4 and DR5) with little toxicity against normal cells [2]. Despite its robust cell killing of tumor cells in vitro, efficacy of TRAIL receptor agonists has been limited in clinical trials, and this limited activity is thought to be due to lack of reliable predictive biomarkers of sensitivity and drug properties that limit efficacy such as short serum half-life, stability, and bio-distribution [3, 4].

Role of mitochondria in apoptotic and necroptotic cell death in the developing brain

Hypoxic–ischemic encephalopathy induces secondary brain injury characterized by delayed energy failure. Currently, therapeutic hypothermia is the sole treatment available after severe intrapartum asphyxia in babies and acts to attenuate secondary loss of high energy phosphates improving both short- and long-term outcome. In order to develop the next generation of neuroprotective therapies, we urgently need to understand the underlying molecular mechanisms leading to cell death. Hypoxia–ischemia creates a toxic intracellular environment including accumulation of reactive oxygen/nitrosative species and intracellular calcium after the insult, inducing mitochondrial impairment. More specifically mitochondrial respiration is suppressed and calcium signaling is dysregulated. At a certain threshold, Bax-dependent mitochondrial permeabilization will occur leading to activation of caspase-dependent and apoptosis-inducing factor-dependent apoptotic cell death. In addition, hypoxia–ischemia induces inflammation, which leads to the release of TNF-α, TRAIL, TWEAK, FasL and Toll-like receptor agonists that will activate death receptors on neurons and oligodendroglia. Death receptors trigger apoptotic death via caspase-8 and necroptotic cell death through formation of the necrosome (composed of RIP1, RIP3 and MLKL), both of which converge at the mitochondria.

Abstract 2272: Understanding resistance to Trail and Trail agonists

Abstract Trail and Trail agonists induce apoptotic cell death in cancer cells while sparing normal cells making them promising drug candidates. However, Trail and Trail agonists tested so far in clinical trials have had disappointing results. We hypothesize that one reason for the lack of success could be resistance to Trail receptor agonism. In order to understand the underling mechanisms, we set out to generate a panel of Trail resistant models. Following weeks of continuous treatment with Trail, resistant cells began to emerge. Interestingly, no cross resistance to other cytotoxic agents such as doxorubicin, taxol and camptothecin was observed in the Trail receptor agonist resistant cells suggesting that the resistance mechanisms were specific to the extrinsic cell death pathway. We examined a series of factors such as ligand binding, surface receptor levels and caspase cascade activation. Here we highlight two specific mechanisms observed in a colorectal cell line DLD1 and a pancreatic cancer cell line PSN1. In both resistant models, the activation of the intrinsic and extrinsic caspase cascades were severely diminished starting at caspase-8. In the PSN1 model, TRAILR2 surface expression is significantly suppressed. Reduction of TRAILR2 by siRNA in the parental PSN1 cells also resulted in significant reduction of Trail receptor agonist activity in contrast to what was observed in the parental DLD1 cells. On the other hand, expression of caspase-8 proenzyme was severely attenuated in the DLD1 resistant cells. There was no significant change of caspase-8 mRNA expression found in resistant DLD1 suggesting post-translational control. Together, we conclude that Trail resistance is mainly due to early extrinsic pathway blockage such as down regulation of caspase-8 and TRAILR2. Citation Format: Hong Chen, Rosa A. Carrasco, Hui Feng, Robert E. Hollingsworth, Zhan Xiao, David A. Tice. Understanding resistance to Trail and Trail agonists. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2272. doi:10.1158/1538-7445.AM2014-2272

Getting TRAIL back on track for cancer therapy.

Unlike other members of the TNF superfamily, the TNF-related apoptosis-inducing ligand (TRAIL, also known as Apo2L) possesses the unique capacity to induce apoptosis selectively in cancer cells in vitro and in vivo. This exciting discovery provided the basis for the development of TRAIL-receptor agonists (TRAs), which have demonstrated robust anticancer activity in a number of preclinical studies. Subsequently initiated clinical trials testing TRAs demonstrated, on the one hand, broad tolerability but revealed, on the other, that therapeutic benefit was rather limited. Several factors that are likely to account for TRAs' sobering clinical performance have since been identified. First, because of initial concerns over potential hepatotoxicity, TRAs with relatively weak agonistic activity were selected to enter clinical trials. Second, although TRAIL can induce apoptosis in several cancer cell lines, it has now emerged that many others, and importantly, most primary cancer cells are resistant to TRAIL monotherapy. Third, so far patients enrolled in TRA-employing clinical trials were not selected for likelihood of benefitting from a TRA-comprising therapy on the basis of a valid(ated) biomarker. This review summarizes and discusses the results achieved so far in TRA-employing clinical trials in the light of these three shortcomings. By integrating recent insight on apoptotic and non-apoptotic TRAIL signaling in cancer cells, we propose approaches to introduce novel, revised TRAIL-based therapeutic concepts into the cancer clinic. These include (i) the use of recently developed highly active TRAs, (ii) the addition of efficient, but cancer-cell-selective TRAIL-sensitizing agents to overcome TRAIL resistance and (iii) employing proteomic profiling to uncover resistance mechanisms. We envisage that this shall enable the design of effective TRA-comprising therapeutic concepts for individual cancer patients in the future.

Tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL).

The concept to exploit death receptors for cancer therapy is very attractive, since these cell surface receptors have a direct connection to the intracellular cell death machinery. Among the death receptor superfamily, the tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) receptor/ligand system is of special interest. TRAIL receptor agonists have recently entered the stage of clinical evaluation for the treatment of human cancers. Further insights into the regulatory mechanisms of TRAIL signaling will help to better understand the determinants of TRAIL sensitivity versus resistance of human cancers.

Selective CDK9 inhibition overcomes TRAIL resistance by concomitant suppression of cFlip and Mcl-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many cancer cells without causing toxicity in vivo. However, to date, TRAIL-receptor agonists have only shown limited therapeutic benefit in clinical trials. This can, most likely, be attributed to the fact that 50% of all cancer cell lines and most primary human cancers are TRAIL resistant. Consequently, future TRAIL-based therapies will require the addition of sensitizing agents that remove crucial blocks in the TRAIL apoptosis pathway. Here, we identify PIK-75, a small molecule inhibitor of the p110α isoform of phosphoinositide-3 kinase (PI3K), as an exceptionally potent TRAIL apoptosis sensitizer. Surprisingly, PI3K inhibition was not responsible for this activity. A kinome-wide in vitro screen revealed that PIK-75 strongly inhibits a panel of 27 kinases in addition to p110α. Within this panel, we identified cyclin-dependent kinase 9 (CDK9) as responsible for TRAIL resistance of cancer cells. Combination of CDK9 inhibition with TRAIL effectively induced apoptosis even in highly TRAIL-resistant cancer cells. Mechanistically, CDK9 inhibition resulted in downregulation of cellular FLICE-like inhibitory protein (cFlip) and Mcl-1 at both the mRNA and protein levels. Concomitant cFlip and Mcl-1 downregulation was required and sufficient for TRAIL sensitization by CDK9 inhibition. When evaluating cancer selectivity of TRAIL combined with SNS-032, the most selective and clinically used inhibitor of CDK9, we found that a panel of mostly TRAIL-resistant non-small cell lung cancer cell lines was readily killed, even at low concentrations of TRAIL. Primary human hepatocytes did not succumb to the same treatment regime, defining a therapeutic window. Importantly, TRAIL in combination with SNS-032 eradicated established, orthotopic lung cancer xenografts in vivo. Based on the high potency of CDK9 inhibition as a cancer cell-selective TRAIL-sensitizing strategy, we envisage the development of new, highly effective cancer therapies.