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Mitochondrial permeabilization engages NF-κB-dependent anti-tumour activity under caspase deficiency

Abstract

Apoptosis represents a key anti-cancer therapeutic effector mechanism. During apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically kills cells even in the absence of caspase activity. Caspase activity can also have a variety of unwanted consequences that include DNA damage. We therefore investigated whether MOMP-induced caspase-independent cell death (CICD) might be a better way to kill cancer cells. We find that cells undergoing CICD display potent pro-inflammatory effects relative to apoptosis. Underlying this, MOMP was found to stimulate NF-κB activity through the downregulation of inhibitor of apoptosis proteins. Strikingly, engagement of CICD displays potent anti-tumorigenic effects, often promoting complete tumour regression in a manner dependent on intact immunity. Our data demonstrate that by activating NF-κB, MOMP can exert additional signalling functions besides triggering cell death. Moreover, they support a rationale for engaging caspase-independent cell death in cell-killing anti-cancer therapies.

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Figure 1: Mitochondrial permeabilization can engage necroptosis as a form of CICD.
Figure 2: MOMP induces TNF synthesis under caspase-deficient conditions.
Figure 3: Mitochondrial permeabilization activates NF-κB.
Figure 4: MOMP activates NF-κB through IAP downregulation and NIK activation.
Figure 5: Mitochondrial permeabilization initiates an NF-κB-dependent pro-inflammatory response.
Figure 6: MOMP-dependent NF-κB activity promotes macrophage activation.
Figure 7: CICD displays enhanced anti-tumorigenic effects versus apoptosis.
Figure 8: CICD anti-tumorigenic effects requires NF-κB and intact immunity.

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Acknowledgements

This work was supported by a Cancer Research UK Programme Foundation award (C40872/A20145) (S.W.G.T.) and a Cancer Research UK studentship. J.L. was supported, in part, by the University of Lyon and by fellowships from Fondation ARC pour la Recherche sur le Cancer and Hospices Civils de Lyon. G.I. was supported by an EMBO long-term postdoctoral fellowship (ALTF 55–2013).C.R.-P. was supported by the Fondation ARC pour la Recherche sur le Cancer and the Agence Nationale de la Recherche (LABEX SIGNALIFE ANR-11-LABX-0028-01). We thank J. Silke (Walter and Eliza Hall Institute), M. Martins (MRC Toxicology Unit), F. Zhang (Massachusetts Institute of Technology), D. Murphy (Beatson Institute) and D. Graczyk (Polish Academy of Sciences) for reagents, A. Keith, B. Clark, T. Gilbey, M. O’Prey (Beatson Institute, UK), D. Vaughan, L. Lapienyte, V. Imbert (Université de Nice-Sophia-Antipolis), M. Hou and C. Thompson (University of Glasgow) for technical assistance. We acknowledge support from Cancer Research UK Glasgow Centre (C596/A18076), the Histology and BSU facilities at the Cancer Research UK Beatson Institute (C596/A17196) and Centre Méditerranéen de Médecine Moléculaire animal facility. Finally, we thank C. Winchester (Beatson Institute) and members of the Tait laboratory for reviewing the manuscript.

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Authors

Contributions

E.G. and S.W.G.T. conceived the study, and designed the work plan together with K.B., J.-E.R. and S.M. Experimental work: E.G., B.Z., S.D., F.B., C.C., K.C., A.R., J.L., G.I., E.P. and N.Y. Development and contribution of reagents: C.R.-P., L.F., E.W., D.L., S.O., L.T., N.P., D.L., L.M., H.W. and A.O. Data analysis: E.G., B.Z., S.D., F.B., K.C., A.R., J.L., G.I., E.P., K.M.R., K.B., J.-E.R. and S.W.G.T. Intellectual input: E.G., N.Y., M.L.A., A.O., J.-E.R., K.B. and S.W.G.T. Manuscript writing: E.G. and S.W.G.T.

Corresponding author

Correspondence to Stephen W. G. Tait.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 MOMP can engage necroptosis under caspase-deficient conditions by the induction of TNF-synthesis.

(A) Control or MCL-1 expressing SVEC cells were immunoblotted for MCL-1 and β-Actin; this experiment was performed once. (B) Control or MCL-1 expressing SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (10 μM) for 48 h, immunostained for cytochrome c and analysed by confocal microscopy. Representative images from three independent experiments. Scale bar represents 20 μM. (C) Quantification of cells that had undergone MOMP in (B), minimum 300 cells counted per condition. Vector-ctrl: n = 17 individual fields, vector-ABT-737/QVD: n = 18 individual fields, MCL-1-ctrl: n = 16 individual fields, MCL-1-ABT-737/QVD: n = 17 individual fields ± SEM. (D) Control or RIPK3SH SVEC cells were immunoblotted for RIPK3 and β –actin; this experiment was performed once. (E) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) +/- Q-VD-OPh (30 μM) then immunoblotted for indicated proteins. Representative images from three independent experiments. (F) Cell lysates from Control and RIPK3SH BCL-xL dependent SVEC cells were immunoblotted for RIPK3 and β-Actin; this experiment was performed once. (G) Vector or RIPK3SH BCL-xL dependent SVEC cells were treated with TNF (20 ng/ml) and zVAD-FMK (50 μM) +/- necrostatin-1 (30 μM). Cell death was measured by SYTOX Green uptake, a representative time-point is shown (16 h). n = 4 independent experiments; mean values ± S.E.M. (H) Lysates from control or MLKL-deleted BCL-xL dependent SVEC cells were immunoblotted for MLKL and β-Actin; this experiment was performed once. (I) Cell lysates from BCL-xL dependent SVEC cells expressing CRISPR control (vector) or BAK/BAK targeting CRISPR constructs were immunoblotted for BAX, BAK and β-Actin; this experiment was performed once. (J) Cell lysates from BCL-xL dependent SVEC cells expressing CRISPR control (vector) or a Caspase-9 targeting CRISPR construct were immunoblotted for Caspase-9 and β-Actin; this experiment was performed once. (K) BCL-xL dependent SVEC cells deleted for MLKL (MLKLCRISPR) or cells expressing empty vector (vectorCRISPR) were treated with ABT-737 (10 μM) and Tnf expression was measured by qRT-PCR. Data represent the mean of triplicate samples and are representative of three independent experiments. p < 0.05, p < 0.01, P < 0.001 determined by Tukey-corrected two way ANOVA (C), two-tailed unpaired t-test (G). Unprocessed original scans of blots are shown in Supplementary Figure 9. Statistical source data can be found in Supplementary Table 5.

Supplementary Figure 2 Mitochondrial permeabilisation activates NF-κB.

(A) Quantification of cells that have undergone MOMP [. Minimum 300 cells counted per condition. VectorCRISPR-ctrl, BAX/BALCRISPR-ctrl and ABT-737/QVD: n = 15 individual fields, vectorCRISPR-ABT-737/QVD: n = 16 individual fields ± SEM. (B) Control or APAF-1 knockdown BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM)(1h) and quantitatively immunostained for p65. Minimum 300 cells counted per condition. Vector- and APAF-1SH-ctrl: n = 15 individual fields, vector- and APAF-1SH- ABT-737: n = 16 individual fields ± SEM. (C) Vector or Caspase-9 deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) for 3 h then quantitatively immunostained for nuclear p65. Minimum 300 cells counted per condition. Both groups, n = 15 individual fields ± SEM. (D) Cell lysates from vector, IKKβ or NEMO deleted BCL-xL dependent SVEC cells were immunoblotted for IKKβ, NEMO and β-Actin expression; this experiment was performed once. (E) Vector, IKKβ or NEMO deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) or TNF (20 ng/ml) for 4 h, immunostained and quantified for nuclear p65. Minimum 580 cells counted per condition. All groups, n = 18 individual fields ± SEM. (F) BCL-xL dependent MEF and HeLa cells were treated with ABT-737 (10 μM), Q-VD-OPh (30 μM) or TNF (20ng/ml) (3 h and 8h respectively), immunostained and quantified for nuclear p65. Minimum 300 cells counted per condition. MEF-ctrl and TNF: n = 15 individual fields, MEF-ABT-737, ABT-737/QVD: n = 16 individual fields, HeLa-ctrl, ABT-737, ABT-737/QVD: n = 16 individual fields, HeLa-TNF: n = 15 fields ± SEM. (G) BCL-xL dependent SVEC cells treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) or TNF (20 ng/ml) for 1 hour +/- Enbrel (50 μg/ml) were immunostained and quantified for nuclear p65. Minimum 450 cells counted per condition. n = 15 individual fields to all groups besides untreated-ABT-737/QVD group: n = 18 individual fields ± SEM. (H) Control or MLKL-deleted, BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) for 6 h, immunostained and quantified for nuclear p65. Minimum 400 cells counted per condition. VectorCRISPR-ctrl and ABT-737/QVD: n = 15 individual fields, MLKLCRISPR-ctrl: n = 14 individual fields, MLKLCRISPR-ABT-737/QVD: n = 15 individual fields, vectorCRISPR- and MLKLCRISPR- TNF: n = 9 individual fields ± SEM of three independent experiments. (I) Control or IκBSR BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) or TNF (20 ng/ml) for 1 h and immunostained for p65. Representative images from three independent experiments. Scale bar represents 30 μM. (J) BCL-xL dependent SVEC cells stably expressing IκBSR were treated with TNF (20 ng/ml). Cell death was measured by IncuCyte imager with a representative time-point is shown (13 h). n = 3 independent experiments; mean values ± S.E.M (K) Vector or IκBSR BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM). Cell death was measured using an IncuCyte imager with a representative time-point shown 16 h. n = 3 independent experiments; mean values ± S.E.M. p < 0.05, p < 0.01, P < 0.001; Tukey-corrected two way ANOVA (AC,E,G,H); Tukey-corrected one way ANOVA (F); two-tailed unpaired t-test (J,K). Unprocessed original scans of blots are shown in Supplementary Figure 9.

Supplementary Figure 3 MOMP activates NF-κB through IAP down-regulation and NIK activation.

(A) Model of IAP downregulation and NIK activation following SMAC-mimetic treatment (left) and hypothesis following MOMP (right). (B) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) +/- MG132 (10 μM) for 4 h. Cell lysates were probed by immunoblot for indicated proteins. Representative images from three independent experiments. (C) BCL-xL dependent MEF or HeLa cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for indicated times. Cell lysates were probed by immunoblot for indicated proteins. Representative images from one (MEF) and three (HeLa) independent experiments. (D) Cell lysates from control, NIK-deleted or cIAP2 overexpressing BCL-xL dependent SVEC cells were immunoblotted for indicated proteins. Where noted cells were incubated for 4 h with MG132 (10 μM); this experiment was performed once. (E) Control or NIK-deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 6 h, immunostained for p65 and analysed by confocal microscopy. Scale bar represents 30 μM. Representative images from three independent experiments. (F) Control or cIAP2 overexpressing BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 6 h, immunostained for p65 and analysed by confocal microscopy. Scale bar represents 30 μM. : Representative images from three independent experiments. (G) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for the indicated time-points. Cell lysates were probed by Western blot for p100/p52 and β-Actin. Representative images from three independent experiments. (H) Cell lysates from control, p100/p52-deleted or RelB-deleted SVEC eGFP-tBID 2A BCL-xL were immunoblotted for indicated proteins; this experiment was performed once. (I) Control or p100/p52-deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) or TNF (20 ng/ml) for 6 h and quantitatively immunostained for nuclear p65. Minimum 590 cells counted per condition. VectorCRISPR- p100/52CRISPR- ctrl: n = 16 individual fields, vectorCRISPR- p100/52CRISPR- ABT-737: n = 17 individual fields, vectorCRISPR- p100/52CRISPR- TNF: n = 15 individual fields ± SEM. (J) Control or RelB-deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) or TNF (20 ng/ml) for 6 h, immunostained and quantified for nuclear p65 Minimum 470 cells counted per condition. n = 16 individual fields for all groups besides vectorCRISPR- RelBCRISPR-TNF groups: n = 15 ± SEM. (K) SVEC cells transfected with siRNA targeting SMAC, OMI or both were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 4 h. Cell lysates were immunoblotted for indicated proteins. Representative images from three independent experiments. p < 0.05, p < 0.01, P < 0.001; Tukey-corrected two way ANOVA (I,J). Unprocessed original scans of blots are shown in Supplementary Figure 9.

Supplementary Figure 4 Mitochondrial permeabilisation initiates an NF-κB dependent pro-inflammatory response.

(A) BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 8h. Conditioned media was Luminex assayed for cytokine levels. (B) Control or IκBSR stably expressing BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 8h. Conditioned media was Luminex assayed for cytokine levels. A,B data represent the mean of duplicate samples and are representative of two independent experiments. (C) Control or MLKL-deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM). qRT-PCR was performed for indicated cytokines. (D) Control, IKKβ or NEMO-deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM). qRT-PCR was performed for indicated cytokines. (E) Cell lysates from control and p65-deleted BCL-xL dependent SVEC cells were immunoblotted for p65 and actin; this experiment was performed once. (F) Control or p65-deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 6 h and immunostained for nuclear p65; this experiment was performed once. Scale bar represents 30 μM. (G) Control or p65-deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM). qRT-PCR was performed for indicated cytokines. (H) Control or p100/p52-deleted BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM). Cytokine qRT-PCR was performed. (I) Lysates from control or STING-deleted BCL-xL dependent SVEC cells were immunoblotted for STING and Actin; this experiment was performed once. (J) BCL-xL dependent SVEC cells were treated with EtBr (0.5 μg/ml, 7 days) qPCR was performed to assess mtDNA levels. (K) BCL-xL dependent SVEC cells expressing a non-targeting CRISPR construct (control) or deleted for STING were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 8 h. Cytokine qRT-PCR was performed. (L) Control or ρ0BCL-xL dependent SVEC cells were treated with ABT-737 (10 μM) and Q-VD-OPh (30 μM) for 8 h and cytokine qRT-PCR was performed. For C,D,G,H,JL data represent the mean of triplicate samples and is a representative experiment of three independent experiments. Statistical source data can be found in Supplementary Table 5. Unprocessed original scans of blots are shown in Supplementary Figure 9.

Supplementary Figure 5 MOMP dependent NF-κB activity promotes macrophage activation.

(AC) Media from Control or (A) APAF-1SH. (B) Caspase-9 deleted (C) BAX/BAK deleted, BCL-xL dependent SVEC cells that had been treated with ABT-737 (10 μM, 8h) was added to BMDMs (16h). Macrophage activation profile was measured by staining for M1 surface marker (CD86+) or M2 surface marker (CD206+) and flow-cytometry. n = 3 independent experiments; mean values ± S.E.M. (DH) Media from Control or (D) MLKL-deleted (E) p65-deleted (F) NEMO-deleted (G) NIK-deleted or (H) cIAP2 overexpressing BCL-xL dependent SVEC cells that had been treated with ABT-737 (10 μM)/Q-VD-OPh (30 μM, 8h) was added to BMDMs (for 16h). qRT-PCR was performed on BMDMs for M1 marker expression. (I) Murine primary pancreatic tumour cells (Myc-PDAC) were treated with ABT-737 (10 μM)/Q-VD-OPh (10 μM). Tnf and Ifnβ mRNA levels were measured by qRT-PCR. Data represent the mean of triplicate samples and are representative of two independent experiments. (J) Media from BCL-xL dependent SVEC cells treated with ABT-737 (10 μM)/Q-VD-OPh (30 μM, 8h) was added to BMDMs in the presence or absence of Enbrel (50 μg/ml, 16h). For JL qRT-PCR was performed on BMDMs to assess M1 marker expression. (K) Media from BCL-xL dependent SVEC cells treated with ABT-737 (10 μM). Q-VD-OPh (30 μM, 8h) was added to wild type or TNFR-I−/− BMDMs. (L) Media from BCL-xL dependent SVEC cells treated with ABT-737 (10 μM)/Q-VD-OPh (30 μM, 8h) was added to BMDMs +/- neutralizing antibody against IFNβ (10 μg/ml) +/- Enbrel (50 μg/ml). For DH and JL data represent the mean of triplicate samples and are representative of three independent experiments with the exception of Il-1β and Il-18 in J and Il-1β and Il-18 and Il-6 in L, which are representative of two independent experiments. p < 0.05, p < 0.01, P < 0.001; Tukey-corrected one way ANOVA (AC). Statistical source data can be found in Supplementary Table 5.

Supplementary Figure 6 CICD displays enhanced anti-tumorigenic effects versus apoptosis.

(A) Left: Immunoblot confirming APAF-1 knock-down in BCL-2 dependent CT26 cells; n = 1. Right panel: Control or APAF-1sh CT26 cells were treated with ABT-263 (10 μM) and Q-VD-OPh (30 μM) for 8 h, immunostained for cytochrome c and quantitatively analysed for MOMP, minimum 700 cells counted per condition. For all groups n = 16 individual fields ± SEM. (B) Experimental setup. (C) Representative IHC images (n = 3 mice) of cleaved-Caspase-3, taken from vector or APAFsh eGFP-tBID 2A BCL-2 CT26 tumour sections. Mice were treated with vehicle or ABT-263 (twice in 7 days) and sacrificed one day post-final treatment. Scale bar represents 100 μM. (D) Control or APAFsh BCL-2 dependent CT26 cells were injected into Balb/C mice. Tumour bearing mice were treated with vehicle or ABT-263 (twice in 7 days) and harvested one day post-final treatment. Serum levels of indicated cytokines were measured by Luminex assay. n = 3 independent experiments; mean values ± S.E.M. (E) Control or APAF-1SH BCL-2 dependent CT26 cells were injected subcutaneously in Balb/C mice (5x105 cells/mouse). Tumour bearing mice were treated with either vehicle or ABT-263 (twice in 7 days) and harvested one-day post-final treatment. Macrophage tumour infiltration was measured by quantifying F4/80+ DAPI population. Vector-vehicle and ABT-263, APAF-1SH-vehicle: n = 9 mice, APAF-1SH-ABT-263: n = 8 mice ± SD. (F) Representative immunohistochemistry images (n = 3 mice) of F4/80 staining (macrophages), from control or APAF-1 knockdown CT26 tumour sections. Mice had been treated with vehicle or ABT-263 (twice in 7 days) and harvested one-day post-final treatment. Scale bar represents 100 μM. (G) Representative FACS plots (n = 3 mice) of macrophage tumour infiltration (F4/80+ DAPI population) and activation status (MHC-II+ or/and CD206+) from mice that were treated with vehicle/ABT-263 (twice in 7 days) and culled one-day post-final treatment. (H) Control or APAFSH BCL-2 dependent CT26 cells were injected into Balb/C mice. Tumour-bearing mice were treated with vehicle or ABT-263 (twice in 7 days) and harvested one day post-final treatment. Highly granulated macrophage activation status was measured. Vector-vehicle and ABT-263, APAF-1SH-vehicle: n = 9 mice, APAF-1SH-ABT-263: n = 8 mice ± SD. p < 0.05, p < 0.01, P < 0.001; two-tailed unpaired t-test (H). Unprocessed original scans of blots are shown in Supplementary Figure 9.

Supplementary Figure 7 CICD anti-tumourigenic effects requires NF-κB and intact immunity.

(A) Mice bearing control or APAF-1SH, BCL-2 dependent CT26 tumours were treated +/- ABT-263 then divided into responders (R), non-responders (NR) and ulcerated (U) groups. Post-treatment, individual tumours were normalized (%) to starting volume (mm3). Arrows indicate treatment points. (B,C) Samples generated in A were measured for: (B) tumour infiltrating M2-like macrophages (Arg1+, CD206+) and (C) tumour infiltrating total T cell (CD3+) CD4+ and CD8+ T cell subsets by flow cytometry. (B,C) Data were normalized to total live tumour cells and tumour weight and represents the mean ± SD of tumours. Individual n number per sub-group is provided in the figure. (D) BCL-2 dependent CT26 cells (control vs APAF-1SH) were injected into NSG mice and tumour volume (mm3) measured; Data represents the mean ± SD of tumours.; n = 6 mice per group. (E) Mice bearing APAF-1 knockdown, BCL-2 dependent CT26 tumours were treated with anti-Thy.1 and T cell levels were measured in splenic samples. Representative FACS plots were shown; n = 3 mice per group. (F) Mice bearing control or APAF-1 knockdown, BCL-2 dependent CT26 tumours were treated with anti-Thy.1. Splenic samples were measured for T cell (CD3+), macrophage (F4/80+) and B cell (CD19+) levels; n = 3 mice per group. (G) Mice bearing APAF-1 knockdown, BCL-2 dependent CT26 tumours were pre-treated with isotype control or anti-Thy.1. Mice were treated with vehicle or ABT-263 (treatments indicated by black arrows) together with isotype control or anti-Thy.1 (treatments indicated by red arrows). Tumour growth was monitored every 2 days (mm3). APAF-1SH + Isotype ctrl + ABT-263: n = 10 mice, APAF-1SH + anti-Thy.1 + ABT-263: n = 9 mice ± SEM of tumours. (H) Cell lysates from control or NEMOSH BCL-2 dependent CT26 cells were immunoblotted for NEMO and α-Tubulin; this experiment was performed once. (I) Vector or NEMOSH APAF-1SH BCL-2 dependent CT26 cells were injected into mice. Tumour bearing mice were treated +/- ABT-263 (2 times over two weeks). Chi-square test was applied at the earliest time point (day 12 post-treatment), where complete regression was observed after final treatment of ABT-263; APAF-1SH-vector + vehicle:n = 9, APAF-1SH-vector + ABT-263: n = 10, APAF-1SH-NEMOSH + vehicle: n = 8, APAF-1SH-NEMOSH + ABT-263: n = 8. p < 0.05, p < 0.01, P < 0.001; Dunn’s-corrected Kruskal-Wallis (B,C), two-tailed unpaired t-test (F), two-tailed Chi-Square (I). Unprocessed original scans of blots are shown in Supplementary Figure 9.

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Giampazolias, E., Zunino, B., Dhayade, S. et al. Mitochondrial permeabilization engages NF-κB-dependent anti-tumour activity under caspase deficiency. Nat Cell Biol 19, 1116–1129 (2017). https://doi.org/10.1038/ncb3596

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