Diep Chau

IAPs limit activation of RIP kinases by TNF receptor 1 during development

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.

IAPs must limit activation of RIP kinases by TNF Receptor 1 to prevent embryonic lethality

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.

cIAPs and XIAP regulate myelopoiesis through cytokine production in an RIPK1- and RIPK3-dependent manner

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.

Targeting p38 or MK2 Enhances the Anti-Leukemic Activity of Smac-Mimetics.

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.

A RIPK2 inhibitor delays NOD signalling events yet prevents inflammatory cytokine production

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.

MK2 Phosphorylates RIPK1 to Prevent TNF-Induced Cell Death

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.

Lymphotoxin α induces apoptosis, necroptosis and inflammatory signals with the same potency as tumour necrosis factor.

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.

TAK1 is required for survival of mouse fibroblasts treated with TRAIL, and does so by NF-κB dependent induction of cFLIPL

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-κB and JNK signalling pathways. To determine the role of TGF-β-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-κB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-κB, protected TAK1−/− MEFs against TRAIL killing, suggesting that TAK1 activation of NF-κB 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-κB 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-κB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.

Combination of IAP antagonist and IFNγ activates novel caspase-10- and RIPK1-dependent cell death pathways.

Peptido-mimetic inhibitor of apoptosis protein (IAP) antagonists (Smac mimetics (SMs)) can kill tumour cells by depleting endogenous IAPs and thereby inducing tumour necrosis factor (TNF) production. We found that interferon-γ (IFNγ) synergises with SMs to kill cancer cells independently of TNF− and other cell death receptor signalling pathways. Surprisingly, CRISPR/Cas9 HT29 cells doubly deficient for caspase-8 and the necroptotic pathway mediators RIPK3 or MLKL were still sensitive to IFNγ/SM-induced killing. Triple CRISPR/Cas9-knockout HT29 cells lacking caspase-10 in addition to caspase-8 and RIPK3 or MLKL were resistant to IFNγ/SM killing. Caspase-8 and RIPK1 deficiency was, however, sufficient to protect cells from IFNγ/SM-induced cell death, implying a role for RIPK1 in the activation of caspase-10. These data show that RIPK1 and caspase-10 mediate cell death in HT29 cells when caspase-8-mediated apoptosis and necroptosis are blocked and help to clarify how SMs operate as chemotherapeutic agents.

Correction: Targeting p38 or MK2 Enhances the Anti-Leukemic Activity of Smac-Mimetics (Cancer Cell (2016) 30(3) (499–500) (S1535610816000350) (10.1016/j.ccell.2016.01.006))

Corrections Targeting p38 or MK2 Enhances the Anti-Leukemic Activity of Smac-Mimetics Najoua Lalaoui, Kay Hänggi, Gabriela Brumatti, Diep Chau, Nhu-Y.N. Nguyen, Lazaros Vasilikos, Lisanne M. Spilgies, Denise A. Heckmann, Chunyan Ma, Margherita Ghisi, Jessica M. Salmon, Geoffrey M. Matthews, Elisha de Valle, Donia M. Moujalled, Manoj B. Menon, Sukhdeep Kaur Spall, Stefan P. Glaser, Jennifer Richmond, Richard B. Lock, StephenM. Condon, Raffi Gugasyan, Matthias Gaestel, Mark Guthridge, RickyW. Johnstone, LenkaMunoz, AndrewWei, Paul G. Ekert, David L. Vaux, W. Wei-Lynn Wong, and John Silke* *Correspondence: silke@wehi.edu.au http://dx.doi.org/10.1016/j.ccell.2016.08.009 (Cancer Cell 29, 145–158; February 8, 2016) After the publication of this paper, the authors found four small errors in Figure 3. In Figure 3A, the label p38 on the right side of the second blot from the top should be p38, and the label MK2 on the right side of the second blot from the bottom should be MK2. In addition, the pTAK1 and pMK2 blots in Figure 3C were inadvertently stretched out during figure preparation. These errors have now been corrected here and in the article online. The authors apologize for these errors and any inconvenience that may have resulted.