Pamela M. Holland

Death Receptor Agonists as a Targeted Therapy for Cancer

Apoptosis is integral to normal, physiologic processes that regulate cell number and results in the removal of unnecessary or damaged cells. Apoptosis is frequently dysregulated in human cancers, and recent advancements in our understanding of the regulation of programmed cell death pathways has led to the development of novel agents to reactivate apoptosis in malignant cells. The activation of cell surface death receptors by tumor necrosis factor–related apoptosis-inducing ligand (Apo2L/TRAIL) and death receptor agonists represent an attractive therapeutic strategy to promote apoptosis of tumor cells through the activation of the extrinsic pathway. The observation that Apo2L/TRAIL can eliminate tumor cells preferentially over normal cells has resulted in several potential therapeutics that exploit the extrinsic pathway, in particular, the soluble recombinant human (rh)Apo2L/TRAIL protein and agonist monoclonal antibodies that target death receptors 4 or 5. Many of these agents are currently being evaluated in phase 1 or 2 trials, either as a single agent or in combination with cytotoxic chemotherapy or other targeted agents. The opportunities and challenges associated with the development of death receptor agonists as cancer therapeutics, the status of ongoing clinical evaluations, and the progress toward identifying predictive biomarkers for patient selection and pharmacodynamic markers of response are reviewed. Clin Cancer Res; 16(6); 1701–8

Death receptor agonist therapies for cancer, which is the right TRAIL?

The activation of cell-surface death receptors represents an attractive therapeutic strategy to promote apoptosis of tumor cells. Several investigational therapeutics that target this extrinsic pathway, including recombinant human Apo2L/TRAIL and monoclonal agonist antibodies directed against death receptors-4 (DR4) or -5 (DR5), have been evaluated in the clinic. Although Phase 1/1b studies provided encouraging preliminary results, findings from randomized Phase 2 studies failed to demonstrate significant clinical benefit. This has raised multiple questions as to why pre-clinical data were not predictive of clinical response. Results from clinical studies and insight into why current agents have failed to yield robust responses are discussed. In addition, new strategies for the development of next generation death receptor agonists are reviewed.

Combined therapy with the RANKL inhibitor RANK-Fc and rhApo2L/TRAIL/dulanermin reduces bone lesions and skeletal tumor burden in a model of breast cancer skeletal metastasis

In bone metastases, tumor cells interact with the bone microenvironment to induce osteoclastogenesis, leading to bone destruction and the growth factor release. RANK ligand (RANKL) is essential for osteoclast formation, function, and survival. Tumor cell-mediated osteolysis is thought to occur ultimately via induction of RANKL within the bone stroma, and inhibition of RANKL in models of breast cancer bone metastases blocks tumor-induced osteolysis and reduces skeletal tumor burden. In addition, the skeleton is co-opted by tumor cells and functions as a supportive tumor microenvironment. Inhibition of RANKL, by reducing tumor-induced osteoclastogenesis, may reduce the local release of growth factors and calcium which may potentially enhance the anti-tumor activity of cytotoxic or direct tumor apoptotic agents. Recombinant human Apo2 ligand/ TNF-related apoptosis-inducing ligand (rhApo2L/TRAIL/dulanermin) is a dual pro-apoptotic receptor agonist that preferentially induces apoptosis in cancer cells versus normal cells. We therefore examined RANKL inhibition (using RANK-Fc) in combination with rhApo2L/TRAIL on tumor-induced osteolysis and skeletal tumor burden in a murine intracardiac injection model of MDA-MB-231 breast carcinoma bone metastasis. rhApo2L/TRAIL treatment resulted in a rapid reduction of skeletal tumor burden. Treatment with RANK-Fc prevented osteolytic lesions and reduced skeletal tumor burden. Combining RANK-Fc with rhApo2L/TRAIL was superior to either rhApo2L/TRAIL or RANK-Fc alone at reducing skeletal tumor burden in the bone metastasis model. Our findings show that RANKL inhibition effectively inhibits pathologic osteolysis induced by human breast adenocarcinoma MDA-MB-231 cells in animals with established tumors, and also enhances the ability of rhApo2L/TRAIL to reduce skeletal tumor burden in vivo.

Targeting Apo2L/TRAIL receptors by soluble Apo2L/TRAIL

The early observation that Apo2L/TRAIL preferentially triggers apoptosis in tumor cells over normal cells highlighted its potential as a candidate therapeutic in cancer. Since its identification in the mid-1990s, our increased understanding of Apo2L/TRAIL and Apo2L/TRAIL receptor signaling has led to the development of several agonists designed to promote tumor cell apoptosis through death receptor engagement. Recombinant human Apo2L/TRAIL/dulanermin is unique in that it is the only agonist which binds both Apo2L/TRAIL death receptors. In pre-clinical studies dulanermin demonstrates broad spectrum anti-tumor activity and the ability to cooperate with multiple conventional and targeted therapies. Results from early stage clinical trials indicate that dulanermin is well tolerated and shows some evidence of clinical activity. Not all tumors are likely to be equally sensitive to apoptosis induction by Apo2L/TRAIL. Therefore, an increased understanding of the regulation of Apo2L/TRAIL signaling should aid in the identification of molecular signatures that define a patient population likely to respond. In this review, current knowledge and new insights about Apo2L/TRAIL signaling is discussed with the focus on the development of Apo2L/TRAIL as a cancer therapeutic.

Expression of anti-apoptotic factors modulates Apo2L/TRAIL resistance in colon carcinoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) selectively induces apoptosis in transformed cells. Normal cells and certain tumor cells can evade Apo2L/TRAIL induced cell death, but the determinants of Apo2L/TRAIL sensitivity are poorly understood. To better understand the factors that contribute to Apo2L/TRAIL resistance, we characterized two colon carcinoma lines with pronounced differences in Apo2L/TRAIL sensitivity. Colo205 cells are highly sensitive to Apo2L/TRAIL whereas Colo320 cells are unresponsive. Components of the DISC (death inducing signaling complex) could be immunoprecipitated from both cell lines in response to Apo2L/TRAIL. Sensitizing agents including a proteasome inhibitor conferred Apo2L/TRAIL sensitivity in Colo320 cells, indicating that the apoptotic machinery was intact and functional. We specifically suppressed the expression of Bcl-2, FLIP or XIAP in Colo320 cells. Downregulation of either FLIP or XIAP but not Bcl-2 restored sensitivity of Colo320 cells to Apo2L/TRAIL. Moreover, stable knockdown of XIAP expression in Colo320 subcutaneous tumors resulted in suppression of tumor growth and sensitivity to Apo2L/TRAIL in vivo. Our results indicate that only a specific subset of anti-apoptotic proteins can confer resistance to Apo2L/TRAIL in Colo320 cells. Elucidation of the factors that contribute to Apo2L/TRAIL resistance in tumor cells may provide insight into combination therapies with Apo2L/TRAIL in a clinical setting.

RIP4 Regulates Epidermal Differentiation and Cutaneous Inflammation

The receptor-interacting protein (RIP) family kinase RIP4 interacts with protein kinase C (PKC) isoforms and is implicated in PKC-dependent signaling pathways. RIP4(-/-) mice die at birth with epidermal differentiation defects, causing fusions of all external orifices and loss of the esophageal lumen. To further understand RIP4 function in the skin, we generated transgenic mice with epidermal-specific expression of RIP4 using the human keratin-14 promoter (K14-RIP4). The K14-RIP4 transgene rescued the epidermal phenotype of RIP4(-/-) mice, showing that RIP4 acts autonomously in the epidermis to regulate differentiation. Although RIP4(-/-) mice share many phenotypic similarities with inhibitor kappaB kinase (IKK)alpha(-/-) mice and stratifin repeated epilation (Sfn(Er/Er)) mice, the K14-RIP4 transgene failed to promote epidermal differentiation in these mutant backgrounds. Unexpectedly, topical treatment of K14-RIP4 mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced dramatic, neutrophilic inflammation, an effect that was independent of tumor necrosis factor type 1 receptor (TNFR1/p55) function. Despite their enhanced sensitivity to TPA, K14-RIP4 mice did not have an altered frequency of tumor formation in TPA-promoted skin cancer initiated with 7,12-dimethylbenz[a]anthracene (DMBA). These data suggest that RIP4 functions in the epidermis through PKC-specific signaling pathways to regulate differentiation and inflammation.

Abstract 2275: Characterization of the mechanistic basis of Apo2L/TRAIL-AMG 655-dr5 interactions and cooperative signaling in cancer cells

Clinical studies with multiple death receptor (DR) agonists targeting DR4 and/or DR5 s have failed to advance beyond phase 2 due to limited efficacy. Unexpectedly, treatment with DR5 agonist antibody AMG 655 markedly enhanced Apo2L/TRAIL-induced killing in cancer cells. Strong synergism observed between AMG 655 and Apo2L/TRAIL in multiple cancer cell lines suggests that additional cross-linking provided by AMG 655 can enhance the ability of soluble ligand to initiate an apoptotic signal. Since the Apo2L/TRAIL related TNF family members FasL and TNF are more potent apoptotic agonists in their membrane-bound forms, the synergy observed between soluble Apo2L/TRAIL and AMG 655 suggests that the combination may mimic a membrane bound form of the ligand. In this study, we used biochemical approaches to characterize the Apo2L/TRAIL-AMG 655-DR5 interactions and cooperative signaling. We found membrane-bound Apo2L/TRAIL induced potent apoptotic signaling against cancer cell lines that are resistant to soluble Apo2L/TRAIL. Cooperativity between Apo2L/TRAIL and AMG 655 but not single agent alone promotes caspase 8 activation and DISC complex assembly, suggesting that the combination represents a qualitative change in apoptotic signaling. To better understand the molecular interactions between Apo2L/TRAIL, DR5 and AMG 655, we determined the crystal structure of the ternary complex of Apo2L/TRAIL, the DR5 ectodomain, and the Fab fragment of AMG 655 at 3.3 A resolution. The Apo2L/TRAIL-DR5-AMG 655-Fab complex is composed of an Apo2L/TRAIL homotrimer with three DR5 monomers bound diagonally along the crevices between two neighboring Apo2L/TRAIL molecules, and three AMG 655 Fabs bound to the three DR5 monomers individually. We then modeled the positioning of additional ternary complexes, which results in an array of DR5 molecules on the cell surface in a hexagonal honeycomb pattern, with three DR5 molecules in each node. Using different AMG 655 fragments, we demonstrated that AMG 655-Fab92 but not AMG 655-Fab fragment is sufficient to cooperate with Apo2L/TRAIL suggesting that the two DR5 binding moieties of AMG 655 mediate the effect. These observations illustrate how co-administration of trimeric Apo2L/TRAIL and bivalent AMG 655 may promote super-clustering of DR5, thereby leading to efficient DISC assembly and enhanced apoptotic signaling. The enhanced agonism generated by combining Apo2L/TRAIL and AMG 655 provides insight into the limited efficacy observed in previous clinical trials and suggests testable hypotheses to reconsider death receptor agonism as a therapeutic strategy. Citation Format: Tzu-Hsuan Huang, Wesley Chang, Alexander Long, Xin Huang, Pamela Holland. Characterization of the mechanistic basis of Apo2L/TRAIL-AMG 655-dr5 interactions and cooperative signaling in cancer cells. [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 2275. doi:10.1158/1538-7445.AM2014-2275

Abstract 5429: The combination of Apo2L/Trail and a death receptor 5 agonist antibody leads to enhanced antitumor activity in mouse syngeneic tumor models

Background: Death receptor-5 (DR5, TR2, TRAIL-R2) is a death domain-containing receptor that induces an apoptosis signaling cascade when engaged by Apo2L/TRAIL or a DR5 agonist antibody1. Many tumor cell lines express DR5 and are sensitive to DR5 agonists2. Despite pre-clinical efficacy, several DR5 agonists have failed to advance beyond phase II clinical trials due to poor clinical efficacy. Strong synergystic activity was observed with the combination of Apo2L/TRAIL and AMG655 (DR5 agonist antibody) pre-clinically in vitro and in vivo (manuscript submitted). Although Apo2L/TRAIL and AMG655 were well tolerated in clinical trials when delivered as single agents3, the efficacy and safety profile of combining Apo2L/TRAIL and AMG655 has not been investigated. Objective: To determine a therapeutic index for Apo2L/TRAIL + DR5 agonist antibody combination treatment in mouse syngeneic tumor cell lines and xenograft models. Results: To evaluate both efficacy and tolerability, we utilized a mouse monoclonal antibody, MD5-1, that recognizes the mouse DR5 (mDR5) receptor. In vitro, treatment with Apo2L/TRAIL + MD5-1 enhanced cell killing in multiple mouse tumor lines, which all expressed mDR5 at similar levels. In vivo, we observed marked anti-tumor activity after treatment with Apo2L/TRAIL at 5 mg/kg for 5 days per week and MD5-1 at 1ug/mouse for 2 days per week. In the Renca model, Apo2L/TRAIL + MD5-1 treatment resulted in enhanced efficacy compared to either single agent (> 92% inhibition; p Conclusion: Our preclinical findings provide evidence that a therapeutic index can be achieved for Apo2L/TRAIL in combination with a DR5 agonist antibody and may be an effective strategy for cancer therapy. Citation Format: Timothy Sullivan, Daniel Branstetter, Pamela Holland, Tammy L. Bush. The combination of Apo2L/Trail and a death receptor 5 agonist antibody leads to enhanced antitumor activity in mouse syngeneic tumor models. [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 5429. doi:10.1158/1538-7445.AM2014-5429