Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes

• Published in: Cell death and differentiation 2013-09, Vol.20 (9), p.1161-1173
• Main Authors: Basit, F, Cristofanon, S, Fulda, S
• Format: Article
• Language: English
• Subjects: 631/154/436/2388; 631/67/1798; 631/80/82/2344; Apoptosis; Autophagy; Autophagy - drug effects; Autophagy - genetics; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Biochemistry; Biomedical and Life Sciences; Cancer research; Cell Biology; Cell Cycle Analysis; Cell death; Cell Death - drug effects; Cell Death - genetics; Cell Line; Chromosomes; Cytotoxicity; Fas-Associated Death Domain Protein - metabolism; GTPase-Activating Proteins - antagonists & inhibitors; GTPase-Activating Proteins - genetics; GTPase-Activating Proteins - metabolism; HEK293 Cells; Humans; Imidazoles - pharmacology; Indoles - pharmacology; Kinases; Life Sciences; Medical prognosis; Medical research; Membranes; Microtubule-Associated Proteins - genetics; Microtubule-Associated Proteins - metabolism; Necrosis - genetics; Original Paper; Pediatrics; Phagosomes - drug effects; Proteins; Pyrroles - pharmacology; Receptor-Interacting Protein Serine-Threonine Kinases - genetics; Receptor-Interacting Protein Serine-Threonine Kinases - metabolism; RNA Interference; RNA, Small Interfering; Stem Cells; Tumor necrosis factor-TNF
• ISSN: 1350-9047; 1476-5403
• DOI: 10.1038/cdd.2013.45

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.