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Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly–ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146, resulting in proteasomal degradation of complex 2, thereby limiting cell death. Expression of the ADP-ribose–binding/hydrolyzing severe acute respiratory syndrome coronavirus 2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix ‘brace’ that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO)., Necroptosis is a regulated form of inflammatory cell death driven by activated MLKL. Here, the authors identify a mutation in the brace region that confers constitutive activation, leading to lethal inflammation in homozygous mutant mice and providing insight into human mutations in this region.

Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER+) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER+ breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER+ cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.

Receptor Interacting Protein Kinase 1 (RIPK1) is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is post-translationally regulated by well characterised ubiquitylation and phosphorylation events, as well as caspase-8 mediated cleavage1–7. The physiological relevance of this cleavage remains unclear, though it is believed to inhibit activation of RIPK3 and necroptosis8. Here we show that heterozygous missense mutations p.D324N, p.D324H and p.D324Y prevent caspase cleavage of RIPK1 in humans and result in early-onset periodic fever episodes and severe intermittent lymphadenopathy, a condition we designate ‘Cleavage-resistant RIPK1-Induced Autoinflammatory’ (CRIA) syndrome. To define the mechanism for this disease we generated a cleavage-resistant Ripk1D325A mutant mouse strain. While Ripk1-/- mice die postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by combined loss of Casp8 and Ripk3 but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, however the mice died before weaning from multi organ inflammation in a RIPK3 dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3 dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but maintains inflammatory homeostasis throughout life.

Tumor necrosis factor (TNF) is an inflammatory cytokine that, upon binding to its receptor TNFR1, can drive cytokine production, cell survival, or cell death and is a major component of an organism’s anti-pathogen repetoire1,2. TNF stimulation leads to the formation of two distinct signalling complexes, a well-defined membrane bound complex (complex 1), and a less well characterised cytosolic death inducing complex (complex 2). Using mass spectrometry, we identified the ADP-ribosyltransferase, tankyrase-1 (TNKS1/TNKS/ARTD5/PARP5a) as a novel native complex 2 component. Following a TNF-induced death stimulus TNKS1 is recruited to complex 2, resulting in complex 2 poly(ADP-ribosyl)ation (PARylation). Tankyrase inhibitors sensitise cells to TNF-induced death, which is correlated with increased complex 2 assembly. Tankyrase-mediated PARylation promotes recruitment of the E3 ligase RNF146 and RNF146 deficiency or proteasome inhibition results in increased levels of complex 2, suggesting that RNF146 causes proteasomal degradation of complex 2. Several viruses express ADP-ribose binding macrodomain proteins, and expression of the SARS-CoV-2 or VEEV macrodomain markedly sensitises cells to TNF-induced death. This suggests that ADP-ribosylation serves as yet another mechanism to detect pathogenic interference of TNF signalling and retaliate with an inflammatory cell death.

MAPK-activated protein kinase 2 (MK2) has diverse roles in cancer. In response to chemotherapy, MK2 inhibition is synthetically lethal to p53-deficiency. While TP53 deletion is rare in glioblastomas, these tumors often carry TP53 mutations. Here, we show that MK2 inhibition strongly attenuated glioblastoma cell proliferation through p53wt stabilization and senescence. The senescence-inducing efficacy of MK2 inhibition was particularly strong when cells were co-treated with the standard-of-care temozolomide. However, MK2 inhibition also increased the stability of p53 mutants and enhanced the proliferation of p53-mutant stem cells. These observations reveal that in response to DNA damaging chemotherapy, targeting MK2 in p53-mutated cells produces a phenotype that is distinct from the p53-deficient phenotype. Thus, MK2 represents a novel drug target in 70% glioblastomas harboring intact TP53 gene. However, targeting MK2 in tumors with TP53 mutations may accelerate disease progression. These findings are highly relevant since TP53 mutations occur in over 50% of all cancers.

SUMMARYWe have isolated a mouse strain with a single missense mutation in the gene encoding MLKL, the essential effector of necroptotic cell death. The resulting substitution lies within the two-helix ‘brace’ and confers constitutive, RIPK3 independent, killing activity to MLKL. Mice homozygous forMlklD139Vdevelop lethal inflammation within days of birth, implicating the salivary glands and pericardium as hotspots for necroptosis and inflammatory infiltration. The normal development ofMlklD139Vhomozygotes until birth, and the absence of any overt phenotype in heterozygotes provides importantin vivoprecedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common humanMLKLpolymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, CRMO.

Summary TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-κB, p38α, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production., Graphical Abstract, Highlights • Phosphorylation of RIPK1 by MK2 acts as survival checkpoint in TNF signaling • TNF-induced activation of MK2 results in global RIPK1 phosphorylation • MK2-mediated phosphorylation suppresses RIPK1 kinase activation and cell death • Complex-II originates from RIPK1 in complex-I as well as cytosolic RIPK1, Jaco et al. show that MK2 directly phosphorylates RIPK1 at residue S321, suppressing the cytotoxic potential of RIPK1 and acting as a checkpoint within the TNF signaling pathway.

Loss of inhibitor of apoptosis proteins (IAPs), particularly cIAP1, can promote production of tumor necrosis factor (TNF) and sensitize cancer cell lines to TNF-induced necroptosis by promoting formation of a death-inducing signaling complex containing receptor-interacting serine/threonine-protein kinase (RIPK) 1 and 3. To define the role of IAPs in myelopoiesis, we generated a mouse with cIAP1, cIAP2, and XIAP deleted in the myeloid lineage. Loss of cIAPs and XIAP in the myeloid lineage caused overproduction of many proinflammatory cytokines, resulting in granulocytosis and severe sterile inflammation. In vitro differentiation of macrophages from bone marrow in the absence of cIAPs and XIAP led to detectable levels of TNF and resulted in reduced numbers of mature macrophages. The cytokine production and consequent cell death caused by IAP depletion was attenuated by loss or inhibition of TNF or TNF receptor 1. The loss of RIPK1 or RIPK3, but not the RIPK3 substrate mixed lineage kinase domain-like protein, attenuated TNF secretion and thereby prevented apoptotic cell death and not necrosis. Our results demonstrate that cIAPs and XIAP together restrain RIPK1- and RIPK3-dependent cytokine production in myeloid cells to critically regulate myeloid homeostasis.

Inhibitor of apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP (X-linked IAP) regulate apoptosis and cytokine receptor signalling, but their overlapping functions make it difficult to distinguish their individual roles. To do so, we deleted the genes for IAPs separately and in combination. While lack of any one of the IAPs produced no overt phenotype in mice, deletion of cIap1 with cIap2 or Xiap resulted in mid-embryonic lethality. In contrast, Xiap−/−cIap2−/− mice were viable. The death of cIap2−/−cIap1−/− double mutants was rescued to birth by deletion of tumour necrosis factor (TNF) receptor 1, but not TNFR2 genes. Remarkably, hemizygosity for receptor-interacting protein kinase 1 (Ripk1) allowed Xiap−/−cIap1−/− double mutants to survive past birth, and prolonged cIap2−/−cIap1−/− embryonic survival. Similarly, deletion of Ripk3 was able to rescue the mid-gestation defect of cIap2−/−cIap1−/− embryos, as these embryos survived to E15.5. cIAPs are therefore required during development to limit activity of RIP kinases in the TNF receptor 1 signalling pathway.

Birinapant is a smac-mimetic (SM) in clinical trials for treating cancer. SM antagonize inhibitor of apoptosis (IAP) proteins and simultaneously induce tumor necrosis factor (TNF) secretion to render cancers sensitive to TNF-induced killing. To enhance SM efficacy, we screened kinase inhibitors for their ability to increase TNF production of SM-treated cells. We showed that p38 inhibitors increased TNF induced by SM. Unexpectedly, even though p38 is required for Toll-like receptors to induce TNF, loss of p38 or its downstream kinase MK2 increased induction of TNF by SM. Hence, we show that the p38/MK2 axis can inhibit or promote TNF production, depending on the stimulus. Importantly, clinical p38 inhibitors overcame resistance of primary acute myeloid leukemia to birinapant.

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.

Using combined chromatographic methods, two asterosaponins (compounds 1 and 2), including a new compound novaeguinoside E (compound 1), and six glycosylated polyhydroxysteroids (compounds 3–8) were isolated from a methanol extract of the starfish Culcita novaeguineae. Their structures were determined on the basis of spectroscopic data (1H and 13C NMR, HSQC, HMBC, 1H–1H COSY, ROESY, and HRESI-MS) and by comparison with the literature values. The new compound 1 represents the third example of asterosaponins containing the 5α-cholesta-9(1l)-en-3β,6α,20,22-tetraol aglycone. Among isolated compounds, 4–7 exhibited moderate to weak cytotoxic activities against five human cancer cell lines such as Hep-G2 (hepatoma), KB (epidermoid carcinoma), LNCaP (prostate cancer), MCF7 (breast cancer), and SK-Mel2 (melanoma).

Intracellular nucleotide binding and oligomerization domain (NOD) receptors recognize antigens including bacterial peptidoglycans and initiate immune responses by triggering the production of pro-inflammatory cytokines through activating NF-κB and MAP kinases. Receptor interacting protein kinase 2 (RIPK2) is critical for NOD-mediated NF-κB activation and cytokine production. Here we develop and characterize a selective RIPK2 kinase inhibitor, WEHI-345, which delays RIPK2 ubiquitylation and NF-κB activation downstream of NOD engagement. Despite only delaying NF-κB activation on NOD stimulation, WEHI-345 prevents cytokine production in vitro and in vivo and ameliorates experimental autoimmune encephalomyelitis in mice. Our study highlights the importance of the kinase activity of RIPK2 for proper immune responses and demonstrates the therapeutic potential of inhibiting RIPK2 in NOD-driven inflammatory diseases.

Both of the TNF superfamily ligands, TNF and LTα, can bind and signal through TNFR1 and TNFR2, yet mice mutant for each have different phenotypes. Part of this difference is because LTα but not TNF can activate Herpes Virus Entry Mediator and also heterotrimerise with LTβ to activate LTβR, which is consistent with the similar phenotypes of the LTα and LTβR deficient mice. However, it has also been reported that the LTα3 homotrimer signals differently than TNF through TNFR1, and has unique roles in initiation and exacerbation of some inflammatory diseases. Our modeling of the TNF/TNFR1 interface compared to the LTα3/TNFR1 structure revealed some differences that could affect signalling by the two ligands. To determine whether there were any functional differences in the ability of TNF and LTα3 to induce TNFR1-dependent apoptosis or necroptosis, and if there were different requirements for cIAPs and Sharpin to transmit the TNFR1 signal, we compared the ability of cells to respond to TNF and LTα3. Contrary to our hypothesis, we were unable to discover differences in signalling by TNFR1 in response to TNF and LTα3. Our results imply that the reasons for the conservation of LTα are most likely due either to differential regulation, the ability to signal through Herpes Virus Entry Mediator or the ability of LTα to form heterotrimers with LTβ. Structured digital abstract LT alpha physically interacts with RIPK1 and cIAP1 by tandem affinity purification (View interaction) TNF and TNF bind by blue native page (View interaction) LT alpha and LT alpha bind by blue native page (View interaction) TNF physically interacts with cIAP1 and RIPK1 by tandem affinity purification (View interaction)

Inhibitor of apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP (X-linked IAP) regulate apoptosis and cytokine receptor signalling, but their overlapping functions make it difficult to distinguish their individual roles. To do so, we deleted the genes for IAPs separately and in combination. While lack of any one of the IAPs produced no overt phenotype in mice, deletion of cIap1 with cIap2 or Xiap resulted in mid-embryonic lethality. In contrast, Xiap(-/-)cIap2(-/-) mice were viable. The death of cIap2(-/-)cIap1(-/-) double mutants was rescued to birth by deletion of tumour necrosis factor (TNF) receptor 1, but not TNFR2 genes. Remarkably, hemizygosity for receptor-interacting protein kinase 1 (Ripk1) allowed Xiap(-/-)cIap1(-/-) double mutants to survive past birth, and prolonged cIap2(-/-)cIap1(-/-) embryonic survival. Similarly, deletion of Ripk3 was able to rescue the mid-gestation defect of cIap2(-/-)cIap1(-/-) embryos, as these embryos survived to E15.5. cIAPs are therefore required during development to limit activity of RIP kinases in the TNF receptor 1 signalling pathway.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a "death ligand"-a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-kappaB and JNK signalling pathways. To determine the role of TGF-beta-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1-/- MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-kappaB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-kappaB, protected TAK1-/- MEFs against TRAIL killing, suggesting that TAK1 activation of NF-kappaB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-kappaB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1-/- MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1-NF-kappaB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor kappaB (NF-kappaB) signaling, and sensitize cells to tumor necrosis factor alpha (TNFalpha). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1-Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1-TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-kappaB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFalpha-induced death occurs. TWEAK-induced loss of the cIAP1-TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFalpha-induced death, whereas primary cells remain resistant. Conversely, cIAP1-TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFalpha sensitization. Lysosomal degradation of cIAP1-TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells.