1. Volume 43, Issue 3, Pages 432-448 (August 2011)
The Ripoptosome, a Signaling Platform that Assembles in Response to Genotoxic Stress and Loss of IAPs 
Tencho Tenev, Katiuscia Bianchi, Maurice Darding, Meike Broemer, Claudia Langlais, Fredrik Wallberg, Anna Zachariou, Juanita Lopez, Marion MacFarlane, Kelvin Cain, Pascal Meier 
Molecular Cell 
Volume 43, Issue 3, Pages (August 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2011 43, 432-448DOI: (10.1016/j.molcel.2011.06.006)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1
Etoposide Promotes Cell Death via the Formation of a Caspase-8-Activating Platform
(A) Cell lines stably transduced with an inducible FLAG-CrmA transgene were left untreated or treated with etoposide (100 μM) in the presence or absence of CrmA. The inset depicts CrmA expression. Cell death was measured 30 hr after etoposide treatment, using CDE. Similar results were obtained using FACS (Figure S1).
(B) CrmA blocked activation of effector caspases, as measured by hydrolysis of Ac-DEVD-AMC.
(C) Etoposide and doxorubicin induce processing of caspase-8.
(D) Cell death assays in the presence and absence of the indicated siRNA oligos.
(E) Etoposide stimulates the association of caspase-8 to RIP1. Caspase-8 was immunoprecipitated (IP), and the presence of RIP1 and caspase-8 was assayed by immunoblotting. Note, for this and all subsequent caspase-8 pull-down assays, cells were incubated in the presence of z-VAD-fmk, which stabilizes caspase-8-containing complexes (Micheau and Tschopp, 2003).
(F) BE cells were treated with the indicated compounds, and the binding of RIP1 to caspase-8 was assessed.
(G) The indicated cells were treated with the indicated compounds for 6 and 24 hr and analyzed as in (F). The densitometer analysis of these blots is shown in Figure S2.
(H and I) Cells were treated with etoposide (10 μM) in the presence or absence of siRNA oligos (MDA-MB-231) or stable expression of RIP1-shRNA (HT1080). “Mock” indicates cells infected with a targeting vector lacking the shRNA region. Data shown represent mean ± SD of three independent experiments. p values are indicated.
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4. Figure 2
Etoposide-Induced Activation of Caspase-8 and Cell Death Occurs Independently of Death Ligands and Mitochondrial Pathways
(A) Sensitivity chart of five cancer cell lines depicting their response to SM treatment (see Figure S4 for primary data).
(B) Etoposide stimulates depletion of cIAP1, cIAP2, and XIAP proteins independently of RIP1 kinase activity. BE cells were pretreated with Nec-1 (10 μM) for 1 hr before addition of etoposide for 20 hr.
(C) Etoposide and SM stimulated induction of an NF-κB reporter gene. The reporter gene consists of the promoter region of the cIAP2 gene fused to luciferase.
(D) Knockdown of NIK and TAK1 suppressed etoposide-mediated activation of NF-κB signaling.
(E) Expression of IκBSR failed to block etoposide-induced cell death in HT1080 cells. Note, IκBSR blocks canonical and noncanonical NF-κB signaling (Darding et al., 2011).
(F) Cells harboring an inducible IκBSR transgene were left uninduced or induced with doxycycline for 12 hr prior to treatment with etoposide.
(G–I) Treatment with the TNF-blocking antibody Enbrel (10 μg/ml) had no effect on etoposide killing, while it fully suppressed SM-induced caspase activation and cell death in MDA-MB-231 cells (G). Incubation with blocking antibodies against TNF, TRAIL (10 μg/ml), and CD95L (10 μg/ml), either alone or in combination, also failed to suppress etoposide killing (I). Incubation with extremely high concentrations of neutralizing antibodies (α-TNF (50 μg/ml), α-CD95L (50 μg/ml), and/or α-TRAIL (10 μg/ml)) also had no effect (see Figure S5D).
(J) Binding of RIP1 to caspase-8 under various conditions.
(K) Enbrel did not affect the binding of RIP1 to caspase-8 following etoposide treatment.
(L) Exposure to SM or etoposide failed to promote the recruitment of RIP1 to TNF-R1 in the absence of signal (TNF). In contrast, RIP1 was rapidly recruited to complex-I in response to TNF. TNF-R1 was immunopurified using an α-TNF-R1 antibody, and the presence of copurified proteins was analyzed by immunoblotting.
(M) Bcl-2 expression did not affect Ripoptosome formation. Cells stably overexpressing Bcl-2, or corresponding parental cells, were treated with etoposide and analyzed as indicated.
(N) Bcl-2 fully protected HT1080 cells from etoposide-induced cell death, while only partially rescuing MDA-MB-231, which is consistent with the notion that these cells can also die by necroptosis (see Figures 4F–4J). Data represent mean ± SD of three independent experiments. p values are indicated.
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5. Figure 3
The Ripoptosome Is a Large ∼2MDa Complex that Contains RIP1, FADD, Caspase-8, and FLIP
(A) RIP1 binds to FADD and caspase-8 following various death stimuli.
(B) Association of RIP1, FADD, and caspase-8 was confirmed by reciprocal pull-down assays. Flp-In ™T-REx™-HEK293 cells with a single-copy insertion of HA2x/Strep-tagged-RIP1, -caspase-8, or -GFP were incubated with doxycycline to induce expression of the respective transgenes. HA2x/Strep-tagged proteins were purified, and the presence of copurified proteins was assessed by immunoblotting.
(C) The Ripoptosome is a large complex of ∼2MDa. Cells were incubated under the indicated conditions, and lysates were separated on a size-exclusion column. The protein standards are indicated below the panels. Aliquots from each fraction were retained for immunoblot analysis (1st step), and fractions (1–5, 6–10, 11–16, 17–22, and 23–28) were pooled and used for immunoprecipitation assays (2nd step). Shown are the immunoblot analysis of each fraction (1st step) and the corresponding immunoprecipitates of the respective pooled fractions (2nd step).
(D) FLIP is recruited to the Ripoptosome following treatment with SM and etoposide. Notably, significantly more FLIP was present in the complex formed following SM treatment when compared to the one assembled upon treatment with etoposide.
(E and F) RNAi-mediated depletion of FLIP boosts Ripoptosome formation (E) and sensitizes cells to etoposide and SM (F). Data represent mean ± SD of three independent experiments. p values are indicated.
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6. Figure 4
RIP1 Kinase Activity Is Indispensable for Ripoptosome Formation and RIP-Mediated Cell Death
(A) Etoposide-induced activation of caspases requires RIP1 kinase activity. Cells were incubated with etoposide in the presence of Nec-1 or z-VAD-fmk, and caspase activity was assayed.
(B) Nec-1 suppresses Ripoptosome formation.
(C–E) Wild-type RIP1 readily promoted Ripoptosome assembly (C), caspase-8 processing (D), and activation of effector caspases (E). In contrast, RIP1K137A, which carries a mutation of the conserved K137 residue that is required for stabilizing the active form, was ineffective in Ripoptosome formation (C) and activation of caspases (D and E). For this experiment, stable cell lines containing a single-copy insertion of RIP1-WT or RIP1-K137A were generated using Flp-In™T-REx™-HEK293 cells (see Experimental Procedures). Expression was induced with Dox for 6 hr before immunoprecipitation of caspase-8.
(F) Time-lapse video microscopy of the indicated cell lines. While plasma membrane permeabilization is a relatively late event during apoptosis, the time between Annexin V staining and Annexin V/PI-uptake (double positive) can be used as an additional measure, in combination with cellular morphology, to distinguish between apoptosis and necroptosis. Using these criteria, z-VAD-fmk (TNF) solely triggered necroptosis in L929 cells, while etoposide exclusively promoted apoptosis in HT1080 cells. MDA-MB-231 cells, however, died by a mixture of apoptosis and necroptosis.
(G) Bcl-2 expression blocked etoposide-induced cell death in HT1080 cells, but had minimal effect in MDA-MB-231 cells.
(H) z-VAD-fmk merely reduced the Annexin V positive population and did not suppress the appearance of PI-positive cells. FACS analysis of cells treated with the indicated compounds is shown.
(I) Time-lapse microscopy of cells treated with the indicated drugs is shown. Experiments were performed in the presence of 1.5 μM PI, which selectively labels dying cells.
(J) RIP3 is required for caspase activation following etoposide treatment. Shown are the results of two independent RIP3-siRNA oligos.
(K) Gel filtration analysis of the Ripoptosome from Flp-In™T-REx™-HEK293-RIP1-WT cells. Cells were left untreated or induced with Dox in the presence of SM (top right panel) or Nec-1 (bottom right panel). Cell lysates were fractionated on a size-exclusion column and SMART protein purification system, and the fractions were analyzed by immunoblotting. The protein standards are indicated.
(L) SM-mediated depletion of cIAPs sensitizes cells to cell death stimulated by induced expression of RIP1 (6 hr). Data shown represent mean ± SD of three independent experiments. p values are indicated.
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7. Figure 5 cIAP1, cIAP2, and XIAP Regulate the Ripoptosome
(A) Simultaneous knockdown of all three IAPs is required for maximal Ripoptosome formation.
(B) RNAi-mediated depletion of XIAP enhanced SM-mediated Ripoptosome formation (top panel) and cell death (bottom panel) are shown.
(C and D) Matched pairs of wild-type and XIAP-deficient HCT116 cells were treated with the indicated compounds. Ripoptosome formation (C) and cell death (D) was assessed as above.
(E and F) cIAP1−/−/cIAP2−/− double-knockout (DKO) MEFs display elevated levels of active caspases, which occurs independently of NF-κB signaling. Wild-type and DKO MEFs, inducibly expressing IκBSR, were left uninduced or induced with doxycycline for 24 hr. DEVDase (E) and IETDase (F) activity was measured as above.
(G) Induced expression of CrmA (24 hr) inhibited DEVDase activity in DKO MEFs.
(H) DKO MEFs, reconstituted with cIAP1 or cIAP2, were left uninduced or induced with doxycycline for 24 hr. DEVDase activity was measured as above. Expression of cIAP1 and cIAP2 was verified by immunoblotting (inset).
(I) Schematic diagram of the Flp-In™T-REx™-HEK293shcIAP1 cell system (Lopez et al., 2011). “TRE,” tetracycline response element; “UBC,” ubiquitin promoter; “FRT,” flippase recognition target; “Tet Op,” tetracycline operon; “Tet-R,” tetracycline repressor protein; “rtTA3,” reverse tetracycline transactivator (rtTA3).
(J–L) Doxycycline treatment induces simultaneous knockdown of endogenous cIAP1 and expression of wild-type cIAP1, cIAP1-ER/AA, and cIAP2. Ripoptosome formation (J) and DEVDase assays (K) were conducted as above. The functionality of the system was validated by monitoring cIAP1 knockdown (lane 2), cIAP reconstitution (lanes 4, 6, and 8), and p100 processing (L). Data shown represent mean ± SD of three independent experiments. p values are indicated.
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8. Figure 6
IAPs Regulate the Ripoptosome by Targeting Components of This Complex for Ubiquitylation and Inactivation
(A) cIAP1 targets endogenous RIP1 for ubiquitylation in a TNF-independent manner. Cells were cotransfected with the indicated constructs, and ubiquitylated proteins were purified under denaturing conditions.
(B) cIAP1 and XIAP target components of the Ripoptosome for ubiquitylation in vitro. HA-Strep-RIP1 and RIP1-associated proteins were purified from Flp-In™T-REx™-HEK293RIP1-HA2x/Strep cells and incubated in an in vitro ubiquitylation mixture with the indicated IAPs. Autoubiquitylation of cIAP1 and XIAP is shown in the panels to the right. ∗ and ∗∗ mark cross-reactive bands.
(C) cIAP1, cIAP2 and XIAP coimmunoprecipitate RIP1 from cellular extracts. The indicated constructs were expressed in HEK293T cells, and HA-tagged proteins were immunoprecipitated with α-HA antibodies and analyzed by immunoblotting.
(D) In vitro ubiquitylation reaction with either wild-type Ub or K48R- and K63R-Ub mutants is shown.
(E) Proteasome inhibition causes Ripoptosome accumulation. Cells were treated with lactacystin (10 μM) for the indicated time points, and Ripoptosome formation was assessed.
(F) RIP1-K377A mutant is significantly more potent than RIP1-WT in stimulating Ripoptosome assembly and activating caspases. Stable Flp-In™T-REx™-HEK293 containing RIP1-WT or RIP1-K377R was assayed for Ripoptosome formation and caspase activation.
(G and H) cIAP1 target FLIP for ubiquitylation in a z-VAD-dependent manner. XIAP also ubiquitylates FLIP under these conditions. HEK293T cells were cotransfected with the indicated constructs, and ubiquitylated proteins were purified under denaturing conditions. Data shown represent mean ± SD of three independent experiments. p values are indicated.
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9. Figure 7
Etoposide and Ripoptosome Formation Convert Proinflammatory Cytokines into Prodeath Signals
(A and B) The indicated cell lines were treated with etoposide in the presence and absence of TNF (10 ng/ml) for 16 hr. Cell death (A) and DEVDase activity (B) were measured as above.
(C) TWEAK enhances etoposide-induced caspase activation.
(D) Cell death induced by etoposide/TNF treatment was blocked by CrmA. Note, CrmA completely blocked cell death under these conditions, presumably because it suppresses caspase-8 homodimers but not caspase-8-FLIP heterodimers, which in turn can block RIP-mediated necroptosis (Oberst et al., 2011). Data shown represent mean ± SD of three independent experiments. p values are indicated.
(E and F) Exogenously added TNF (100 ng/ml) (E) or TWEAK (100 ng/ml) and LIGHT (100 ng/ml) (F) enhance the association of RIP1 with caspase-8 in etoposide-treated cells.
(G) Time-lapse video microscopy of cells in the presence of Annexin V-FITC and PI.
(H) Model. Genotoxic damage and depletion of IAPs result in Ripoptosome formation. Under baseline conditions, the majority of RIP1 is in a closed configuration. A small fraction of RIP1 (referred to as “open” configuration) is targeted for Ub-mediated inactivation by IAPs. FLIP-caspase-8 heterodimers also negatively regulate RIP1. Upon genotoxic stress, or treatment with SM, RIP1 is derepressed, which allows it to bind to partner proteins such as FADD and caspase-8 to form the Ripoptosome in a TNF-independent manner. Unmodified RIP1 is also more abundantly recruited to death receptors and TNF-R1 following their stimulation with the respective ligands. In the situation of TNF-R1, this leads to detachment of RIP1 from the receptor to form complex-IIB, which is reminiscent of the Ripoptosome, but originates from complex-I at TNF-R1. Importantly, the Ripoptosome can form independently of death ligands or mitochondrial pathways. Ripoptosome assembly stimulates caspase-8 activation and/or necroptosis, depending on cellular context. The kinase activity of RIP1 is thereby required for Ripoptosome assembly and RIP-mediated induction of apoptosis and necroptosis.
Molecular Cell  , DOI: ( /j.molcel )
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