Endocytosis is crucial for various aspects of cell homeostasis. Here, we show that proapoptotic death receptors (DRs) trigger selective destruction of the clathrin-dependent endocytosis machinery. DR ...stimulation induced rapid, caspase-mediated cleavage of key clathrin-pathway components, halting cellular uptake of the classic cargo protein transferrin. DR-proximal initiator caspases cleaved the clathrin adaptor subunit AP2α between functionally distinct domains, whereas effector caspases processed clathrin's heavy chain. DR5 underwent ligand-induced, clathrin-mediated endocytosis, suggesting that internalization of DR signaling complexes facilitates clathrin-pathway targeting by caspases. An endocytosisblocking, temperature-sensitive dynamin-1 mutant attenuated DR internalization, enhanced caspase stimulation downstream of DRs, and increased apoptosis. Thus, DR-triggered caspase activity disrupts clathrin-dependent endocytosis, leading to amplification of programmed cell death.
Apomab is a fully human monoclonal antibody that induces programmed cell death through the proapoptotic receptor DR5 in various cancer cells but not in normal cells. Several lung cancer cell lines ...express DR5 and exhibit apoptosis in response to apomab in vitro.
We investigated the efficacy of apomab and its interaction with chemotherapy in xenograft models based on human NCI-H460 non-small-cell lung carcinoma cells. In an established model of s.c. tumor xenografts, apomab or Taxol plus carboplatin chemotherapy delayed tumor progression, whereas combined treatment caused tumor regression and a substantially longer growth delay. To test apomab activity in a setting that may more closely mimic lung cancer pathology in patients, we developed a lung orthotopic model.
In this model, microcomputed tomography imaging showed that apomab, chemotherapy, or combination treatment significantly inhibited tumor growth compared with vehicle, whereas the combination caused greater inhibition in tumor growth relative to chemotherapy or apomab. Similarly, histologic analysis revealed that apomab, chemotherapy, or the combination significantly reduced tumor size compared with vehicle, whereas the combination induced significantly greater reduction in tumor size than did chemotherapy or apomab. Furthermore, combined treatment improved 105-day survival relative to vehicle (P = 0.0023) as well as to apomab (P = 0.0445) or chemotherapy (P = 0.0415).
These results show a positive interaction of apomab with chemotherapy, evidenced by significant inhibition of tumor growth as well as improved survival, thus supporting further investigation of this therapeutic approach in lung cancer patients.
Apoptosis-inducing ligand 2 (Apo2L, also called TRAIL), a member of the tumor necrosis factor (TNF) family, induces apoptosis in a variety of human tumor cell lines but not in normal cells Wiley, S. ...R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C.-P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995) Immunity 3, 673−682; Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Ashkenazi, A. (1996) J. Biol. Chem. 271, 12687−12690. Here we describe the structure of Apo2L at 1.3 Å resolution and use alanine-scanning mutagenesis to map the receptor contact regions. The structure reveals a homotrimeric protein that resembles TNF with receptor-binding epitopes at the interface between monomers. A zinc ion is buried at the trimer interface, coordinated by the single cysteine residue of each monomer. The zinc ion is required for maintaining the native structure and stability and, hence, the biological activity of Apo2L. This is the first example of metal-dependent oligomerization and function of a cytokine.
The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with ...activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemotherapeutic agent docetaxel or an anti-vascular endothelial growth factor antibody. Antitumor activity of h10H5 is associated with decreased AKT activation and glucose uptake and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors and furthermore illustrate a new method of monitoring its activity noninvasively in vivo via 2-fluoro-2-deoxy-d-glucose-positron emission tomography imaging.
The anti-FcRH5/CD3 T cell-dependent bispecific antibody (TDB) targets the B cell lineage marker FcRH5 expressed in multiple myeloma (MM) tumor cells. We demonstrate that TDBs trigger T cell receptor ...activation by inducing target clustering and exclusion of CD45 phosphatase from the synapse. The dimensions of the target molecule play a key role in the efficiency of the synapse formation. The anti-FcRH5/CD3 TDB kills human plasma cells and patient-derived myeloma cells at picomolar concentrations and results in complete depletion of B cells and bone marrow plasma cells in cynomolgus monkeys. These data demonstrate the potential for the anti-FcRH5/CD3 TDB, alone or in combination with inhibition of PD-1/PD-L1 signaling, in the treatment of MM and other B cell malignancies.
•Prevalence of FcRH5 expression in multiple myeloma is 100%•Anti-FcRH5/CD3 TDB redirects T cells to kill myeloma cells•Target clustering and CD45 exclusion activate T cells•Anti-FcRH5/CD3 TDB is a highly efficacious immunotherapy for myeloma
Li et al. report that the size and epitope location of the target play a key role in the efficiency of T cell activation induced by T cell-dependent bispecific antibodies (TDBs). They develop a TDB targeting FcRH5 expressed in all multiple myeloma tumor cells and show its potential in treating this disease.
Inhibiting the programmed death-1 (PD-1) pathway is one of the most effective approaches to cancer immunotherapy, but its mechanistic basis remains incompletely understood. Binding of PD-1 to its ...ligand PD-L1 suppresses T-cell function in part by inhibiting CD28 signaling. Tumor cells and infiltrating myeloid cells can express PD-L1, with myeloid cells being of particular interest as they also express B7-1, a ligand for CD28 and PD-L1. Here we demonstrate that dendritic cells (DCs) represent a critical source of PD-L1, despite being vastly outnumbered by PD-L1
macrophages. Deletion of PD-L1 in DCs, but not macrophages, greatly restricted tumor growth and led to enhanced antitumor CD8
T-cell responses. Our data identify a unique role for DCs in the PD-L1-PD-1 regulatory axis and have implications for understanding the therapeutic mechanism of checkpoint blockade, which has long been assumed to reflect the reversal of T-cell exhaustion induced by PD-L1
tumor cells.
Apo2L/TRAIL exhibits enhanced apoptotic activity in tumor xenograft models when used in combination with the topoisomerase 1 inhibitor CPT-11. To investigate the cellular mechanisms involved in this ...increased tumor-killing activity, a series of in vitro experiments were conducted using the human colon carcinoma cell line (HCT116). Apo2L/TRAIL induced a transient upregulation of DR5 mRNA, while CPT-11 increased both death and decoy receptor expression. Upregulation of decoy receptors by CPT-11 was partially inhibited by co-administration of Apo2L/TRAIL. CPT-11 treatment resulted in accumulation of cells at G(2)M-phase and correlated with a substantial increase in the protein levels of the cyclin-dependent kinase inhibitor p21. However, cells co-treated with CPT-11 and Apo2L/TRAIL, or pretreated with CPT-11 for up to 24 h followed by 2 h Apo2L/TRAIL, resulted in a caspase-dependent degradation of p21, reversal of G(2)-M phase arrest with a concomitant increase in apoptosis. The sequential treatment produced the greatest induction of DR5 and DR4, caspase-3-like cleavage/activation and p21 degradation, as well as increased apoptosis. These data indicate that the up-regulation of Apo2L/TRAIL ligand and its death receptors as well as cleavage of p21 protein in the Apo2L/TRAIL plus CPT-11 treatment contributes to the positive cooperation between these agents in enhancing tumor cell apoptosis.
Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR ...sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth
. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to
stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.
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