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.
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Abbreviations
- ERMS:
-
embryonal rhabdomyosarcoma
- ARMS:
-
alveolar rhabdomyosarcoma
- BafA1:
-
Bafilomycin A1
- Nec-1:
-
necrostatin-1
- CAM:
-
chorioallantoic membrane
- TNFα:
-
tumor necrosis factor α
- RIP:
-
receptor-interacting protein
- RMS:
-
rhabdomyosarcoma
- zVAD.fmk:
-
N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone
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Acknowledgements
We thank D. Brücher for expert technical assistance, C. Hugenberg for expert secretarial assistance and Dr. J. Cinatl for help with microscopy. This work has been partially supported by grants from the Bundesministerium für Forschung und Technologie (01GM1104C) and the Deutsche Krebshilfe (to SF).
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Basit, F., Cristofanon, S. & Fulda, S. Obatoclax (GX15-070) triggers necroptosis by promoting the assembly of the necrosome on autophagosomal membranes. Cell Death Differ 20, 1161–1173 (2013). https://doi.org/10.1038/cdd.2013.45
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DOI: https://doi.org/10.1038/cdd.2013.45
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