A Bak-dependent mitochondrial amplification step contributes to Smac mimetic/glucocorticoid-induced necroptosis.
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.
Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstraße 3a, 60528 Frankfurt, Germany.
Katharina
K
Rohde
Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstraße 3a, 60528 Frankfurt, Germany.|German Cancer Consortium (DKTK), Heidelberg, Germany.|German Cancer Research Center (DKFZ), Heidelberg, Germany.
Lara
L
Kleinesudeik
Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstraße 3a, 60528 Frankfurt, Germany.
Stefanie
S
Roesler
Institute for Cardiovascular Physiology, Faculty of Medicine, Goethe-University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany.
Oliver
O
Löwe
Functional Proteomics, Centre of Biochemistry, Faculty of Medicine, Goethe-University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany.
Juliana
J
Heidler
Institute for Cardiovascular Physiology, Faculty of Medicine, Goethe-University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany.
Katrin
K
Schröder
Functional Proteomics, Centre of Biochemistry, Faculty of Medicine, Goethe-University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany.
Ilka
I
Wittig
Clinic of Anesthesiology, Intensive-Care Medicine and Pain Therapy, University Hospital, Theodor-Stern Kai 7, 60596 Frankfurt, Germany.|Centre of Biological Chemistry, Molecular Bioenergetics Group, Faculty of Medicine, Goethe-University, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany.
Stefan
S
Dröse
Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Komturstraße 3a, 60528 Frankfurt, Germany.|German Cancer Consortium (DKTK), Heidelberg, Germany.|German Cancer Research Center (DKFZ), Heidelberg, Germany.
Simone
S
Fulda
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D061945:Caspase Inhibitors / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects*; D002460:Cell Line; D003907:Dexamethasone / Q000633:toxicity; D016166:Free Radical Scavengers / Q000494:pharmacology; D005938:Glucocorticoids / Q000633:toxicity*; D006801:Humans; D019169:Jurkat Cells; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism*; D009336:Necrosis / Q000503:physiopathology*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D000906:Antibodies; D061945:Caspase Inhibitors; D016166:Free Radical Scavengers; D005938:Glucocorticoids; D017382:Reactive Oxygen Species; D050998:bcl-2 Homologous Antagonist-Killer Protein; C484771:benzyloxycarbonyl-valyl-aspartic acid fluoromethyl ketone; D003907:Dexamethasone; C570694:MLKL protein, mouse; D011494:Protein Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases