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.