Synthetic ligands of death receptor 5 display a cell-selective agonistic effect at different oligomerization levels

Abstract

DR4 (Death Receptor 4) and DR5 (Death Receptor 5) are two potential targets for cancer therapy due to their ability to trigger apoptosis of cancer cells, but not normal ones, when activated by their cognate ligand TRAIL (TNF related apoptosis-inducing ligand). Therapies based on soluble recombinant TRAIL or agonist antibodies directed against one of the receptors are currently under clinical trials. However, TRAIL-R positive tumor cells are frequently resistant to TRAIL induced apoptosis. The precise mechanisms of this resistance are still not entirely understood. We have previously reported on synthetic peptides that bind to DR5 (TRAILmim/DR5) and induce tumor cell apoptosis in vitro and in vivo. Here, we showed that while hexameric soluble TRAIL is able to efficiently kill the DR5 positive lymphoma Jurkat or the carcinoma HCT116, these cells are resistant to apoptosis induced by the divalent form of TRAILmim/DR5 and are poorly sensitive to apoptosis induced by an anti-DR5 agonist monoclonal antibody. This resistance can be restored by the cross-linking of anti-DR5 agonist antibody but not by the cross-linking of the divalent form of TRAILmim/DR5. Interestingly, the divalent form of TRAILmim/DR5 that induced apoptosis of DR5 positive BJAB cells, acts as an inhibitor of TRAIL-induced apoptosis on Jurkat and HCT116 cells. The rapid internalization of DR5 observed when treated with divalent form of TRAILmim/DR5 could explain the antagonist activity of the ligand on Jurkat and HCT116 cells but also highlights the independence of the mechanisms responsible for internalization and activation when triggering the DR5 apoptotic cascade.

Keywords: DR5; agonist; apoptosis; oligomerization; peptides.

Figure 1. Differential apoptogenic activity of SPK and TRAIL^mim/DR5

BJAB Jurkat and HCT116 cells A. were stained with monoclonal antibody directed against DR5 or a control isotype coupled to phycoerythrin (PE) and analyzed by flow cytometry. Results in B. showed fold increase of the mean of fluorescence intensity between control isotype and TRAIL-R2 specific labeling that was calculated to compare level of TRAIL-R2 expression on the three cells lines. BJAB, Jurkat and HCT116 cells were treated with stepwise 2-fold increasing concentrations of SPK C. or divalent TRAIL^mim/DR5 peptide (2d) D. After 16 hours of incubation, apoptosis was determined as the percentage of Annexin V–positive cells by flow cytometry analysis. Results are expressed as mean % apoptosis of 3 independent experiments +/− SD.

Figure 2. Different requirement of DR5 oligomerization for apoptosis induction

BJAB A., Jurkat B. and HCT116 C. cells were treated with an agonist antibody directed against DR5 processed for different levels of valencies: monovalent (anti-DR5-Fab), divalent (anti-DR5), and tetravalent (anti-DR5-CL). After 16 hours of incubation cell viability was determined using the MTS assay. Results are expressed as % of cell viability inhibition according to the following formula viable cell % =(OD (treatment) / OD (100% viability) * 100) were cells incubated with medium alone were considered as 100% of viability. Results are expressed as the OD mean of 3 independent experiments ± SEM.

Figure 3. Tetravalent version of TRAIL^mim/DR5 peptide is not sufficient for triggering Jurkat cell death

Jurkat A. or BJAB B. cells were treated with the divalent TRAIL^mim/DR5 peptide (2d) and the tetravalent version (2ta). After 16 hours of incubation, cell viability was determined using the MTS assay as described in Figure 2. Kinetic curves of divalent and tetravalent TRAIL^mim/DR5 peptides binding to immobilized human TRAIL-R2 (density of TRAIL-R2 was 65 fmol/mm²) were obtained by SPR experiments (C, D). Peptides 2d and 2ta were tested at concentrations of 5000, 1666, 555, 185, 61 nM. The response is expressed in fmol/mm² (1000RU= 1mg/mm²). The apparent k_off were evaluated by fitting (black curves) the post-injection phase of the curves and values are the mean of three experiments. E. Responses obtained at 5μM were normalized to the stoichiometry of interaction.

Figure 4. Divalent TRAIL^mim/DR5 inhibits SPK mediated Jurkat and HCT116 cells apoptosis

Jurkat A. or HCT116 B. cells were treated with a solution consisting of a constant concentration of SPK (5ng/mL) or an agonist antibody (5ng/mL) directed against Fas and stepwise 2-fold increasing concentration of divalent (2d) or monovalent (2m) TRAIL^mim/DR5 peptides. After 16h of incubation, apoptosis was determined as the percentage of Annexin V–positive cells by flow cytometry. Data are expressed as the percentage of inhibition of apoptosis.

Figure 5. SPK and divalent TRAIL^mim/DR5 induce DR5 internalization on BJAB Jurkat and HCT116 cell lines

BJAB A., Jurkat B. and HCT116 C. cells were treated with SPK or divalent (2d) TRAIL^mim/DR5 peptide for different period of times. Cells were subsequently processed for surface expression of DR5 analysis by flow cytometry. The panels show histograms for selected times during a kinetic analysis of DR5 internalization after treatment with divalent (2d) TRAIL^mim/DR5. The right panels represent the percentage of DR5 internalization upon treatments calculated using the mean fluorescence intensity values at each time point. D. BJAB, Jurkat or HCT116 cells were treated for 30 minutes with vehicle, monovalent (2m) or divalent (2d) TRAIL^mim/DR5 peptides. Cells were subsequently processed for either surface expression of DR5 analysis by localization of DR5 by immunocytochemistry. Results showed representative images of DR5 localization in the three cell lines.