title: Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes.
abstract: Obatoclax (GX15-070), a small-molecule inhibitor of antiapoptotic Bcl-2 proteins, has been reported to trigger cell death via autophagy. However, the underlying molecular mechanisms have remained elusive. Here, we identify GX15-070-stimulated assembly of the necrosome on autophagosomal membranes as a key event that connects GX15-070-stimulated autophagy to necroptosis. GX15-070 predominately induces a non-apoptotic form of cell death in rhabdomyosarcoma cells, as evident by lack of typical apoptotic features such as DNA fragmentation or caspase activation and by insensitivity to the broad-range caspase inhibitor zVAD.fmk. Instead, GX15-070 triggers massive accumulation of autophagosomes, which are required for GX15-070-induced cell death, as blockade of autophagosome formation by silencing of Atg5 or Atg7 abolishes GX15-070-mediated cell death. Co-immunoprecipitation studies reveal that GX15-070 stimulates the interaction of Atg5, a constituent of autophagosomal membranes, with components of the necrosome such as FADD, RIP1 and RIP3. This GX15-070-induced assembly of the necrosome on autophagosomes occurs in a Atg5-dependent manner, as knockdown of Atg5 abrogates formation of this complex. RIP1 is necessary for GX15-070-induced cell death, as both genetic and pharmacological inhibition of RIP1 by shRNA-mediated knockdown or by the RIP1 inhibitor necrostatin-1 blocks GX15-070-induced cell death. Similarly, RIP3 knockdown rescues GX15-070-mediated cell death and suppression of clonogenic survival. Interestingly, RIP1 or RIP3 silencing has no effect on GX15-070-stimulated autophagosome formation, underlining that RIP1 and RIP3 mediate cell death downstream of autophagy induction. Of note, GX15-070 significantly suppresses tumor growth in a RIP1-dependent manner in the chorioallantoic membrane model in vivo. In conclusion, GX15-070 triggers necroptosis by promoting the assembly of the necrosome on autophagosomes. These findings provide novel insights into the molecular mechanisms of GX15-070-induced non-apoptotic cell death.
authors.affiliation: Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstr. 3a, Frankfurt 60528, Germany.
authors.forename: F
authors.initials: F
authors.lastname: Basit
authors.forename: S
authors.initials: S
authors.lastname: Cristofanon
authors.forename: S
authors.initials: S
authors.lastname: Fulda
mesh_terms: D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D000071187:Autophagy-Related Protein 5; D000071193:Autophagy-Related Protein 7; D016923:Cell Death / Q000187:drug effects* / Q000235:genetics; D002460:Cell Line; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009336:Necrosis / Q000235:genetics; D010588:Phagosomes / Q000187:drug effects*; D011758:Pyrroles / Q000494:pharmacology*; D034622:RNA Interference; D034741:RNA, Small Interfering; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: C495773:Atg5 protein, mouse; C502661:Atg7 protein, mouse; D000071187:Autophagy-Related Protein 5; C505534:Fadd protein, mouse; D053200:Fas-Associated Death Domain Protein; D020690:GTPase-Activating Proteins; D007093:Imidazoles; D007211:Indoles; D008869:Microtubule-Associated Proteins; D011758:Pyrroles; D034741:RNA, Small Interfering; C490620:Ralbp1 protein, mouse; C507699:necrostatin-1; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C485615:Ripk3 protein, mouse; D000071193:Autophagy-Related Protein 7; C520962:obatoclax