title: RIP1 protein-dependent assembly of a cytosolic cell death complex is required for inhibitor of apoptosis (IAP) inhibitor-mediated sensitization to lexatumumab-induced apoptosis.
abstract: Searching for new strategies to trigger apoptosis in rhabdomyosarcoma (RMS), we investigated the effect of two novel classes of apoptosis-targeting agents, i.e. monoclonal antibodies against TNF-related apoptosis-inducing ligand (TRAIL) receptor 1 (mapatumumab) and TRAIL receptor 2 (lexatumumab) and small-molecule inhibitors of inhibitor of apoptosis (IAP) proteins. Here, we report that IAP inhibitors synergized with lexatumumab, but not with mapatumumab, to reduce cell viability and to induce apoptosis in several RMS cell lines in a highly synergistic manner (combination index <0.1). Cotreatment-induced apoptosis was accompanied by enhanced activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and caspase-dependent apoptosis. In addition, IAP inhibitor and lexatumumab cooperated to stimulate the assembly of a cytosolic complex containing RIP1, FADD, and caspase-8. Importantly, knockdown of RIP1 by RNA interference prevented the formation of the RIP1·FADD·caspase-8 complex and inhibited subsequent activation of caspase-8, -9, and -3; loss of mitochondrial membrane potential; and apoptosis upon treatment with IAP inhibitor and lexatumumab. In addition, RIP1 silencing rescued clonogenic survival of cells treated with the combination of lexatumumab and IAP inhibitor, thus underscoring the critical role of RIP1 in cotreatment-induced apoptosis. By comparison, the TNFα-blocking antibody Enbrel had no effect on IAP inhibitor/lexatumumab-induced apoptosis, indicating that an autocrine TNFα loop is dispensable. By demonstrating that IAP inhibitors and lexatumumab synergistically trigger apoptosis in a RIP1-dependent but TNFα-independent manner in RMS cells, our findings substantially advance our understanding of IAP inhibitor-mediated regulation of TRAIL-induced cell death.
authors.affiliation: Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, 60528 Frankfurt, Germany.
authors.forename: Farhan
authors.initials: F
authors.lastname: Basit
authors.forename: Robin
authors.initials: R
authors.lastname: Humphreys
authors.forename: Simone
authors.initials: S
authors.lastname: Fulda
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D002469:Cell Separation; D002470:Cell Survival; D003600:Cytosol / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D034622:RNA Interference; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000502:physiology*; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism; D012208:Rhabdomyosarcoma / Q000188:drug therapy* / Q000473:pathology*; D015398:Signal Transduction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D051034:Inhibitor of Apoptosis Proteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; C510234:lexatumumab; C494833:BIRC2 protein, human; D044767:Ubiquitin-Protein Ligases; C506249:RIPK1 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D020169:Caspases; C507357:mapatumumab