Abstract
Necroptosis is a form of programmed cell death that critically depends on RIP3 and MLKL. However, the contribution of mitochondria to necroptosis is still poorly understood. In the present study, we discovered that mitochondrial perturbations play a critical role in Smac mimetic/Dexamethasone (Dexa)-induced necroptosis independently of death receptor ligands. We demonstrate that the Smac mimetic BV6 and Dexa cooperate to trigger necroptotic cell death in acute lymphoblastic leukemia (ALL) cells that are deficient in caspase activation due to absent caspase-8 expression or pharmacological inhibition by the caspase inhibitor zVAD.fmk, since genetic silencing or pharmacological inhibition of RIP3 or MLKL significantly rescue BV6/Dexa-induced necroptosis. In addition, RIP3 or MLKL knockout mouse embryonic fibroblasts (MEFs) are protected from BV6/Dexa/zVAD.fmk-induced cell death. In contrast, antagonistic antibodies against the death receptor ligands TNFα, TRAIL or CD95 ligand fail to rescue BV6/Dexa-triggered cell death. Kinetic studies revealed that prior to cell death BV6/Dexa treatment causes hyperpolarization of the mitochondrial membrane potential (MMP) followed by loss of MMP, reactive oxygen species (ROS) production, Bak activation and disruption of mitochondrial respiration. Importantly, knockdown of Bak significantly reduces BV6/Dexa-induced loss of MMP and delays cell death, but not ROS production, whereas ROS scavengers attenuate Bak activation, indicating that ROS production occurs upstream of BV6/Dexa-mediated Bak activation. Consistently, BV6/Dexa treatment causes oxidative thiol modifications of Bak protein. Intriguingly, knockdown or knockout of RIP3 or MLKL protect ALL cells or MEFs from BV6/Dexa-induced ROS production, Bak activation, drop of MMP and disruption of mitochondrial respiration, demonstrating that these mitochondrial events depend on RIP3 and MLKL. Thus, mitochondria might serve as an amplification step in BV6/Dexa-induced necroptosis. These findings provide new insights into the role of mitochondrial dysfunctions during necroptosis and have important implications for the development of novel treatment approaches to overcome apoptosis resistance in ALL.
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Abbreviations
- ALL:
-
acute lymphoblastic leukemia
- cIAP:
-
cellular IAP
- Dexa:
-
Dexamethasone
- FSC/SSC:
-
forward/side scatter
- HDAC:
-
histone deacetylase
- IAP:
-
Inhibitor of Apoptosis
- MEFs:
-
mouse embryonic fibroblasts
- MLKL:
-
mixed lineage kinase domain-like
- MMP:
-
mitochondrial membrane potential
- MPTP:
-
mitochondrial permeability transition pore
- NEM:
-
N-ethylmaleimide
- NF-κB:
-
Nuclear Factor kappaB
- NSA:
-
necrosulfonamide
- PI:
-
propidium iodide
- RING:
-
really interesting new gene
- RIP:
-
Receptor-Interacting Protein
- ROS:
-
reactive oxygen species
- siRNA:
-
small interfering RNA
- TNF:
-
tumor necrosis factor
- TRAIL:
-
tumor-necrosis-factor-related apoptosis-inducing ligand
- XIAP:
-
X-linked IAP
References
Taylor RC, Cullen SP, Martin SJ . Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol 2008; 9: 231–241.
He S, Wang L, Miao L, Wang T, Du F, Zhao L et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 2009; 137: 1100–1111.
Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M et al. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 2009; 137: 1112–1123.
Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 2009; 325: 332–336.
Sun L, Wang H, Wang Z, He S, Chen S, Liao D et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 2012; 148: 213–227.
Zhao J, Jitkaew S, Cai Z, Choksi S, Li Q, Luo J et al. Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci USA 2012; 109: 5322–5327.
Hitomi J, Christofferson DE, Ng A, Yao J, Degterev A, Xavier RJ et al. Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 2008; 135: 1311–1323.
Vanden Berghe T, Linkermann A, Jouan-Lanhouet S, Walczak H, Vandenabeele P . Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol 2014; 15: 135–147.
Wang H, Sun L, Su L, Rizo J, Liu L, Wang LF et al. Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell 2014; 54: 133–146.
Dondelinger Y, Declercq W, Montessuit S, Roelandt R, Goncalves A, Bruggeman I et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep 2014; 7: 971–981.
Oberst A, Dillon CP, Weinlich R, McCormick LL, Fitzgerald P, Pop C et al. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature 2011; 471: 363–367.
Marshall KD, Baines CP . Necroptosis: is there a role for mitochondria? Front Physiol 2014; 5: 323.
Fulda S . Regulation of necroptosis signaling and cell death by reactive oxygen species. Biol Chem 2016; 397: 657–660.
Fulda S, Vucic D . Targeting IAP proteins for therapeutic intervention in cancer. Nat Rev Drug Discov 2012; 11: 109–124.
Pui CH, Relling MV, Downing JR . Acute lymphoblastic leukemia. N Engl J Med 2004; 350: 1535–1548.
Stanulla M, Schrappe M . Treatment of childhood acute lymphoblastic leukemia. Semin Hematol 2009; 46: 52–63.
Fulda S . Tumor resistance to apoptosis. Int J Cancer 2009; 124: 511–515.
Fulda S . Therapeutic opportunities for counteracting apoptosis resistance in childhood leukaemia. Br J Haematol 2009; 145: 441–454.
Fulda S . Promises and challenges of smac mimetics as cancer therapeutics. Clin Cancer Res 2015; 21: 5030–5036.
Varfolomeev E, Blankenship JW, Wayson SM, Fedorova AV, Kayagaki N, Garg P et al. IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell 2007; 131: 669–681.
Vince JE, Wong WW, Khan N, Feltham R, Chau D, Ahmed AU et al. IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis. Cell 2007; 131: 682–693.
Wang L, Du F, Wang X . TNF-alpha induces two distinct caspase-8 activation pathways. Cell 2008; 133: 693–703.
Belz K, Schoeneberger H, Wehner S, Weigert A, Bonig H, Klingebiel T et al. Smac mimetic and glucocorticoids synergize to induce apoptosis in childhood ALL by promoting ripoptosome assembly. Blood 2014; 124: 240–250.
Roesler S, Eckhardt I, Wolf S, Fulda S . Cooperative TRAIL production mediates IFNalpha/Smac mimetic-induced cell death in TNFalpha-resistant solid cancer cells. Oncotarget 2016; 7: 3709–3725.
Mukhopadhyay P, Rajesh M, Haskó G, Hawkins BJ, Madesh M, Pacher P . Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. Nat Protoc 2007; 2: 2295–2301.
Fulda S, Galluzzi L, Kroemer G . Targeting mitochondria for cancer therapy. Nat Rev Drug Discov 2010; 9: 447–464.
Batinic-Haberle I, Cuzzocrea S, Reboucas JS, Ferrer-Sueta G, Mazzon E, Di Paola R et al. Pure MnTBAP selectively scavenges peroxynitrite over superoxide: comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury, an SOD-specific Escherichia coli model and carrageenan-induced pleurisy. Free Radic Biol Med 2009; 46: 192–201.
Irrinki KM, Mallilankaraman K, Thapa RJ, Chandramoorthy HC, Smith FJ, Jog NR et al. Requirement of FADD, NEMO, and BAX/BAK for aberrant mitochondrial function in tumor necrosis factor alpha-induced necrosis. Mol Cell Biol 2011; 31: 3745–3758.
Tischner D, Manzl C, Soratroi C, Villunger A, Krumschnabel G . Necrosis-like death can engage multiple pro-apoptotic Bcl-2 protein family members. Apoptosis 2012; 17: 1197–1209.
Karch J, Kanisicak O, Brody MJ, Sargent MA, Michael DM, Molkentin JD . Necroptosis interfaces with MOMP and the MPTP in mediating cell death. PLoS One 2015; 10: e0130520.
Karch J, Kwong JQ, Burr AR, Sargent MA, Elrod JW, Peixoto PM et al. Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice. Elife 2013; 2: e00772.
Baines CP . The mitochondrial permeability transition pore and the cardiac necrotic program. Pediatr Cardiol 2011; 32: 258–262.
D'Alessio M, De Nicola M, Coppola S, Gualandi G, Pugliese L, Cerella C et al. Oxidative Bax dimerization promotes its translocation to mitochondria independently of apoptosis. FASEB J 2005; 19: 1504–1506.
Schenk B, Fulda S . Reactive oxygen species regulate Smac mimetic/TNFalpha-induced necroptotic signaling and cell death. Oncogene 2015; 34: 5796–5806.
Vanlangenakker N, Vanden Berghe T, Bogaert P, Laukens B, Zobel K, Deshayes K et al. cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production. Cell Death Differ 2011; 18: 656–665.
Ardestani S, Deskins DL, Young PP . Membrane TNF-alpha-activated programmed necrosis is mediated by Ceramide-induced reactive oxygen species. J Mol Signal 2013; 8: 12.
Tait SW, Oberst A, Quarato G, Milasta S, Haller M, Wang R et al. Widespread mitochondrial depletion via mitophagy does not compromise necroptosis. Cell Rep 2013; 5: 878–885.
Temkin V, Huang Q, Liu H, Osada H, Pope RM . Inhibition of ADP/ATP exchange in receptor-interacting protein-mediated necrosis. Mol Cell Biol 2006; 26: 2215–2225.
Kasof GM, Prosser JC, Liu D, Lorenzi MV, Gomes BC . The RIP-like kinase, RIP3, induces apoptosis and NF-kappaB nuclear translocation and localizes to mitochondria. FEBS Lett 2000; 473: 285–291.
Chen W, Zhou Z, Li L, Zhong CQ, Zheng X, Wu X et al. Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. J Biol Chem 2013; 288: 16247–16261.
Morgan MJ, Kim YS . The serine threonine kinase RIP3: lost and found. BMB Rep 2015; 48: 303–312.
Fakler M, Loeder S, Vogler M, Schneider K, Jeremias I, Debatin KM et al. Small molecule XIAP inhibitors cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells and overcome Bcl-2-mediated resistance. Blood 2009; 113: 1710–1722.
Hacker S, Dittrich A, Mohr A, Schweitzer T, Rutkowski S, Krauss J et al. Histone deacetylase inhibitors cooperate with IFN-gamma to restore caspase-8 expression and overcome TRAIL resistance in cancers with silencing of caspase-8. Oncogene 2009; 28: 3097–3110.
Birkenmeier K, Drose S, Wittig I, Winkelmann R, Kafer V, Doring C et al. Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma are highly dependent on oxidative phosphorylation. Int J Cancer 2016; 138: 2231–2246.
Fulda S, Strauss G, Meyer E, Debatin KM . Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells. Blood 2000; 95: 301–308.
Leichert LI, Gehrke F, Gudiseva HV, Blackwell T, Ilbert M, Walker AK et al. Quantifying changes in the thiol redox proteome upon oxidative stress in vivo . Proc Natl Acad Sci USA 2008; 105: 8197–8202.
Acknowledgements
We thank D. Vucic for kindly providing BV6 and C. Hugenberg for expert secretarial assistance. This work has been partially supported by grants from Edith von Heyden-Vermächtnis (to KR), Nachlässe Maria Christine Held und Erika Hecker (to KR), Nachlass Martha Schmelz (to KR), DFG SFB815 (to SF, IW, JH, OL, KS), BMBF (to SF), Wilhelm-Sander-Stiftung (to SF) and IUAP VII (to SF).
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Rohde, K., Kleinesudeik, L., Roesler, S. et al. A Bak-dependent mitochondrial amplification step contributes to Smac mimetic/glucocorticoid-induced necroptosis. Cell Death Differ 24, 83–97 (2017). https://doi.org/10.1038/cdd.2016.102
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DOI: https://doi.org/10.1038/cdd.2016.102
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