Efficacy of a triple treatment with irradiation, agonistic TRAIL receptor antibodies and EGFR blockade.

BACKGROUND AND PURPOSE: : Since the efficacy of a single targeted agent in combination with ionizing radiation is limited by putative treatment resistances, a rationally designed triple treatment consisting of an agonistic antibody targeting either TRAIL-R1 (mapatumumab) or TRAIL-R2 (lexatumumab), radiation and an epidermal growth factor receptor-(EGFR-)inhibiting antibody (cetuximab) was tested. MATERIAL AND METHODS: : Induction of apoptosis after triple treatment was determined in Colo205, HCT116 and FaDu cells by Hoechst 33342 stain. The degree of interaction was determined by isobologram analysis. A knockout variant of HCT116 was used to examine Bax dependence of the triple treatment. The role of Akt/PKB signaling was analyzed using the phosphatidylinositol 3-kinase inhibitor LY294002. Clonogenic assays were performed to examine the effect on clonogenic survival of tumor cells. RESULTS: : A synergistic effect of radiation, cetuximab and agonistic TRAIL-R antibodies was demonstrated in cell lines derived from colorectal tumors or head-and-neck cancers. The efficacy of this multimodal approach was dependent on Bax and inhibition of Akt/PKB in the cell systems used. The results also show a positive impact on clonogenic cell death in several cell lines. CONCLUSION: : These data suggest that rationally designed multimodal therapy approaches integrating radiation with more than one targeted agent will open new perspectives in radiation oncology.

Efficacy of triple therapies including ionising radiation, agonistic TRAIL antibodies and cisplatin.

The detection of molecular targeted agents is a milestone in cancer treatment. Despite the achievements, the efficacy of single targeted agents in combination with radiotherapy is limited by putative treatment resistance. We therefore tested a rationally designed triple therapy consisting of an agonistic antibody against either TRAIL-R1 (mapatumumab/HGS-ETR1) or TRAIL-R2 (lexatumumab/HGS-ETR2) in combination with the established chemotherapeutic drug cisplatin in a panel of solid tumour cell lines derived from head and neck as well as colorectal carcinomas. Induction of apoptosis after monotherapy, double or triple treatment was determined in FaDu (squamous cancer cell line of the head and neck), Colo205 and HCT116 cells (colorectal adenocarcinoma cell lines) by Hoechst 33342 stain. Double and triple therapies were compared using analysis of variance followed by post hoc tests. The degree of interaction was determined by 3D-isobologram analysis. A knockout variant of HCT116 was used to examine Bax-dependence of the triple therapy to gain insight into the underlying molecular signaling pathways possibly responsible for the observed effects. Dose-response relationships revealed different baseline activities of the modalities dependent on cell type. Triple therapy was more effective than double therapy in most cases according to the induction of apoptosis. Furthermore, a synergistic efficacy of the triple therapy was demonstrated in a subset of tumour cell lines. The efficacy of this multimodal approach was highly dependent on the presence of Bax. Our data suggest that targeted agents can be effectively added to existing multimodal therapy approaches which might open new perspectives in radiation oncology.

Drug evaluation: lexatumumab, an intravenous human agonistic mAb targeting TRAIL receptor 2.

Human Genome Sciences Inc, under license from Cambridge Antibody Technology Ltd, is developing lexatumumab, an intravenous human agonistic mAb to TNF-related apoptosis-inducing ligand (TRAIL) receptor 2, for the potential treatment of cancer. Phase II clinical trials of lexatumumab are underway.

Irradiation specifically sensitises solid tumour cell lines to TRAIL mediated apoptosis.

BACKGROUND: TRAIL (tumor necrosis factor related apoptosis inducing ligand) is an apoptosis inducing ligand with high specificity for malignant cell systems. Combined treatment modalities using TRAIL and cytotoxic drugs revealed highly additive effects in different tumour cell lines. Little is known about the efficacy and underlying mechanistic effects of a combined therapy using TRAIL and ionising radiation in solid tumour cell systems. Additionally, little is known about the effect of TRAIL combined with radiation on normal tissues. METHODS: Tumour cell systems derived from breast- (MDA MB231), lung--(NCI H460) colorectal--(Colo 205, HCT-15) and head and neck cancer (FaDu, SCC-4) were treated with a combination of TRAIL and irradiation using two different time schedules. Normal tissue cultures from breast, prostate, renal and bronchial epithelia, small muscle cells, endothelial cells, hepatocytes and fibroblasts were tested accordingly. Apoptosis was determined by fluorescence microscopy and western blot determination of PARP processing. Upregulation of death receptors was quantified by flow cytometry. RESULTS: The combined treatment of TRAIL with irradiation strongly increased apoptosis induction in all treated tumour cell lines compared to treatment with TRAIL or irradiation alone. The synergistic effect was most prominent after sequential application of TRAIL after irradiation. Upregulation of TRAIL receptor DR5 after irradiation was observed in four of six tumour cell lines but did not correlate to tumour cell sensitisation to TRAIL. TRAIL did not show toxicity in normal tissue cell systems. In addition, pre-irradiation did not sensitise all nine tested human normal tissue cell cultures to TRAIL. CONCLUSIONS: Based on the in vitro data, TRAIL represents a very promising candidate for combination with radiotherapy. Sequential application of ionising radiation followed by TRAIL is associated with an synergistic induction of cell death in a large panel of solid tumour cell lines. However, TRAIL receptor upregulation may not be the sole mechanism by which sensitation to TRAIL after irradiation is induced.

Death receptor ligands: new strategies for combined treatment with ionizing radiation.

The major goal of modern radiation oncology is the achievement of a maximal tumor control with minimal normal tissue damage. However, normal tissue tolerance may preclude the application of tumoricidal radiation doses. In order to overcome this limitation, strategies either to increase normal tissue tolerance or to reduce the radiation dose required may prove beneficial. In this regard, attempts to minimize the required radiation dose by reducing the number of malignant clonogenic cells are promising. Therefore, therapies which induce programmed cell death (apoptosis) in tumor cells, may prove to be suitable approaches. TRAIL (TNFalpha-related apoptosis inducing ligand)/Apo2L is a very promising member of the family of death ligands. The ligand preferentially induces apoptotic cell death in a wide range of tumor cells but not in normal cells. TRAIL/Apo2L triggers apoptosis even in cells not undergoing apoptosis in response to radiation, since ionizing radiation induce apoptosis by a different pathway as death ligands although an overlapping set of molecules is involved. Combination of both modalities has been shown to induce additive or synergistic apoptotic effects and eradication of clonogenic tumor cells thereby increasing the therapeutic efficacy. The present article reviews this novel biological strategy for optimized radiotherapy based on the combination of ionizing irradiation and death receptor triggered cell death.

Influence of hypoxia on TRAIL-induced apoptosis in tumor cells.

PURPOSE: Tumor hypoxia reduces the efficacy of radiotherapy, many types of chemotherapy, and tumor necrosis factor-alpha (TNF-alpha). TRAIL (TNF-alpha-related apoptosis-inducing ligand) is a ligand for death receptors of the TNF superfamily shown to be selectively toxic for tumor cells and thereby a promising antineoplastic tool. The impact of hypoxia on TRAIL-induced apoptosis was examined in this study. METHODS AND MATERIALS: Apoptosis induction and growth rates of various tumor cell lines under hypoxia were evaluated in vitro. Biologically effective induction of hypoxia was verified by determination of hypoxia-inducible factor-1 (HIF-1) activation. The efficacy of TRAIL- and radiation-induced apoptosis under different oxygen conditions was quantified in vitro. The impact of Bcl-2 on TRAIL-induced apoptosis under hypoxia or normoxia was evaluated by comparing cells expressing Bcl-2 with a vector control. RESULTS: Moderate hypoxia caused no growth retardation or apoptosis, but led to activation of HIF-1 as a prerequisite of hypoxic gene induction. Cellular responses to TRAIL differed considerably among the cell lines tested. Hypoxia reduced radiation-induced, but not TRAIL-induced, apoptosis in the tested cell lines. Hypoxia did not induce Bcl-2 expression. Bcl-2 had a minor impact on the efficacy of TRAIL-induced apoptosis. CONCLUSION: Taken together, the data indicate that TRAIL is clearly effective under conditions of proven hypoxia.

Molecular requirements for the combined effects of TRAIL and ionising radiation.

BACKGROUND AND PURPOSE: Previously it was shown that combination of death ligand TRAIL and irradiation strongly increases cell kill in several human tumour cell lines. Since Bcl-2 overexpression did not strongly interfere with the efficacy, components of the mitochondrial death pathway are not required for an effective combined treatment. In the present study the minimal molecular prerequisites for the efficacy of a combined treatment were determined. MATERIALS AND METHODS: Apoptosis induction in control, caspase-8 and FADD negative Jurkat cells, BJAB control and FADD-DN cells was analysed by FACS. Activation of caspase-8, -10 and -3 and cleavage of PARP was determined by immunoblotting. TRAIL receptors were activated using recombinant human TRAIL. Surface expression of TRAIL receptors DR4 and DR5 was analysed by FACS. RESULTS: Jurkat T-cells express the agonistic DR5 receptor but not DR4. Presence of FADD was found to be essential for TRAIL induced apoptosis. Caspase-8 negative cells show very low rates of apoptosis after prolonged stimulation with TRAIL. No combined effects of TRAIL with irradiation could be found in FADD-DN overexpressing and FADD deficient cells. However, the combination of TRAIL and irradiation clearly lead to a combined effect in caspase-8 negative Jurkat cells, albeit with reduced death rates. In these cells activation of the alternative initiator caspase-10 could be detected after combined treatment. CONCLUSION: Our data show that a combined therapy with TRAIL and irradiation will only be effective in cells expressing at least one agonistic TRAIL receptor, FADD and caspase-8 or caspase-10.

Glioma tropism of lentivirally transduced hematopoietic progenitor cells.

Experimental gliomas attract hematopoietic progenitor cells (HPC) in vivo. HPC are therefore promising candidates for a cell-based delivery of therapeutic molecules to experimental gliomas. A therapeutic application requires efficient genetic manipulation of the cellular vector and a lack of tumorigenicity. Here, we studied the impact of lenti-viral transduction on the glioma tropism of human or murine HPC. Transduction of human or murine HPC with a GFP lentivirus (lenti-GFP) did not interfere with the glioma-mediated attraction of HPC. Bone marrow reconstitution of C57Bl/6 mice with syngeneic GFP-transgenic lineage-depleted bone marrow cells (lin- BM) was as efficient as reconstitution with syngeneic lin- BM transduced ex vivo with lenti-GFP. SMA-560 gliomas growing orthotopically in lenti-GFP-reconstituted VM/Dk mice recruited GFP-positive bone marrow-derived cells. Thus, lentiviral transduction did not interfere with the attraction of exogenously injected HPC or endogenous bone marrow-derived cells by experimental gliomas. Lenti-GFP-HPC implanted directly into tumor-free brains were not tumorigenic. The intravenous injection of lenti-GFP-HPC in glioma-bearing mice did not alter the survival of otherwise untreated animals and had no impact on the survival benefit conferred by cerebral irradiation. Taken together, genetic manipulation of HPC with lenti-GFP neither made these cells tumorigenic nor interfered with their glioma tropism.

Aurora kinase inhibitor ZM447439 induces apoptosis via mitochondrial pathways.

ZM447439 (ZM) is a potent and selective inhibitor of aurora-A and -B kinase with putative anti-tumoral activity. Inhibitors of aurora kinases were shown to induce apoptosis in vitro and in vivo. To investigate the underlying mechanisms, cell death pathways triggered by ZM was analysed in HCT-116 colorectal cancer cells. Through correlation of polyploidization and apoptosis in different knockout cells, the interrelation of these cellular responses to ZM was investigated. ZM induced apoptosis in a concentration- and time-dependent manner. ZM-induced apoptosis was associated with an upregulation of p53, breakdown of the mitochondrial membrane potential (DeltaPsim) and activation of caspase-3. To precisely define key components for ZM-induced apoptosis, knockout cells lacking p53, Bak, Bax or both Bak and Bax were used. Lack of p53 reduced ZM-induced apoptosis and breakdown of DeltaPsim, while lack of Bak, Bax or both almost completely inhibited apoptosis and breakdown of DeltaPsim. Since no difference in apoptosis induction was detectable between HCT-116 cells lacking Bak, Bax or both, apoptosis induction depended non-redundantly on both Bak and Bax. Phenomenally, ZM induced notable polyploidization in all examined cells, especially in p53-/- cells. A correlation between polyploidization and apoptosis was observed in wild-type, and also in p53-/- cells, albeit with a modest extent of apoptosis. Moreover, in Bak-/-, Bax-/- and Bak/Bax-/- cells apoptosis was totally inhibited in spite of the strongest polyploidization, suggesting apoptosis may be a secondary event following polyploidization in HCT-116 cells. Thus ZM-induced apoptosis depends not only on polyploidization, but also on the intracellular apoptotic signaling.

Combined treatment with lexatumumab and irradiation leads to strongly increased long term tumour control under normoxic and hypoxic conditions.

PURPOSE: The combination of ionizing radiation with the pro-apoptotic TRAIL receptor antibody lexatumumab has been shown to exert considerable synergistic apoptotic effects in vitro and in short term growth delay assays. To clarify the relevance of these effects on local tumour control long-term experiments using a colorectal xenograft model were conducted. MATERIALS AND METHODS: Colo205-xenograft bearing NMRI (nu/nu) nude mice were treated with fractionated irradiation (5x 3 Gy, d1-5) and lexatumumab (0.75 mg/kg, d1, 4 and 8). The tumour bearing hind limbs were irradiated with graded single top up doses at d8 under normoxic (ambient) and acute hypoxic (clamped) conditions. Experimental animals were observed for 270 days. Growth delay and local tumour control were end points of the study. Statistical analysis of the experiments included evaluation of tumour regrowth and local tumour control. RESULTS: Combined treatment with irradiation and lexatumumab led to a pronounced tumour regrowth-delay when compared to irradiation alone. The here presented long-term experiments revealed a highly significant rise of local tumour control for normoxic (ambient) (p = 0. 000006) and hypoxic treatment (p = 0. 000030). CONCLUSION: Our data show that a combination of the pro-apoptotic antibody lexatumumab with irradiation reduces tumour regrowth and leads to a highly increased local tumour control in a nude mouse model. This substantial effect was observed under ambient and more pronounced under hypoxic conditions.

Combination of the pro-apoptotic TRAIL-receptor antibody mapatumumab with ionizing radiation strongly increases long-term tumor control under ambient and hypoxic conditions.

PURPOSE: Mapatumumab, an agonistic tumor necrosis factor-related apoptosis inducing ligand-receptor antibody, exerts highly synergistic apoptotic effects in vitro and in short-term growth delay assays when combined with irradiation. Because it remained unclear in how far these effects influence local tumor control, long-term experiments using a colorectal xenograft model were undertaken. MATERIAL AND METHODS: Experiments were performed with irradiation (5 x 3 Gy, d1-5) and mapatumumab (10 mg/kg) in Colo205-xenograft-bearing NMRI (nu/nu) nude mice. Graded top up doses were delivered on the tumor-bearing hind leg under ambient and hypoxic conditions; follow-up was 270 days. Growth delay and local tumor control were end points of the study. Statistical analysis of the experiments included calculation of tumor regrowth and local tumor control. RESULTS: After combined treatment, a pronounced tumor regrowth-delay was observed when compared with irradiation alone. Long-term experiments revealed a highly significant increase in local tumor control for ambient (p = 0.00076) and hypoxic treatment (p = 0.000069). CONCLUSIONS: The present data demonstrate for the first time that combination of a pro-apoptotic antibody with irradiation results in evidently reduced tumor regrowth times and subsequently highly increased local tumor control under normoxic and hypoxic conditions in a xenograft mouse model.

[Death inducing ligands in combination with ionizing radiation: objective and current knowledge]. / Zelltodliganden in Kombination mit ionisierender Strahlung: Rationale und Kenntnisstand.

BACKGROUND: Apart from optimization of the radiation technology, future new strategies in radiation oncology will focus on the biological optimisation leading to improved adaptation of the tumor therapy on each tumor entity. In this regard, different areas of biological research may be distinguished: prediction, development of new cytotoxic agents, improvement of the tolerance of normal tissue and the optimisation of radiochemotherapy. MATERIAL AND METHOD: For the development of new strategies the knowledge of molecular mechanisms of radiation induced cell death is essential. In the present article a novel biological strategy for optimisation of radiotherapy is introduced. RESULTS: Distinct pathways mediate apoptosis in response to death receptor stimulation and ionizing radiation although an overlapping set of molecules is involved. TRAIL (TNF alpha-related apoptosis inducing ligand) is a type-II membrane protein belonging to the TNF family, which preferentially induces apoptotic cell death in a wide variety of tumor cells but not in normal cells. Based on the assumption of distinct signalling pathways, combination of TRAIL and ionizing radiation seems ideal to increase the therapeutic efficacy. Our study presents the rationale, own data, data of other groups as well as the current status of the latest findings regarding the function and physiological role of the TRAIL death ligand. CONCLUSIONS: Apoptosis resistance towards ionising radiation is not associated with a general apoptosis resistance. Death ligands, for example TRAIL trigger apoptosis even in cells not undergoing apoptosis in response to radiation. A combination of both modalities induces additive or synergistic effects in regard to apoptosis induction and eradication of clonogenic tumor cells. Thus, the combination of TRAIL with radiation proofs that novel treatment strategies may be developed on the basis of molecular or cell biological research.

Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal.

Efficient engulfment of the intact cell corpse is a critical end point of apoptosis, required to prevent secondary necrosis and inflammation. The presentation of "eat-me" signals on the dying cell is an important part of this process of recognition and engulfment by professional phagocytes. Here, we present evidence that apoptotic cells secrete chemotactic factor(s) that stimulate the attraction of monocytic cells and primary macrophages. The activation of caspase-3 in the apoptotic cell was found to be required for the release of this chemotactic factor(s). The putative chemoattractant was identified as the phospholipid, lysophosphatidylcholine. Further analysis showed that lysophosphatidylcholine was released from apoptotic cells due to the caspase-3 mediated activation of the calcium-independent phospholipase A(2). These data suggest that in addition to eat-me signals, apoptotic cells display attraction signals to ensure the efficient removal of apoptotic cells and prevent postapoptotic necrosis.