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A flexible mouse model of liver cancer is used to search for spontaneous mutations arising in tumors initiated by different oncogenic lesions and it is then compared to alterations observed in human cancers. The model has not only identified two oncogenes in the same focal amplification that cooperate during tumerogenesis but has also highlighted the use of integrating mouse models and cancer genomics for the functional annotation of cancer genes.

A novel mammalian protein that may promote apoptosis by binding to and antagonizing IAP proteins has been identified. It is called DIABLO for 'direct IAP-binding protein with low pl.' Coimmunoprecipitation and 2D immobilized pH gradient/SDS PAGE was used, followed by electrospray ionization tandem mass spectrometry.

Inhibitors of apoptosis (IAPs) proteins are critical regulators of innate immune signaling pathways and therefore have potential as drug targets. X-linked IAP (XIAP) and cellular IAP1 and IAP2 (cIAP1 and cIAP2) are E3 ligases that have been shown to be required for signaling downstream of NOD2, an intracellular receptor for bacterial peptidoglycan. We used genetic and biochemical approaches to compare the responses of IAP-deficient mice and cells to NOD2 stimulation. In all cell types tested, XIAP is the only IAP required for signaling immediately downstream of NOD2, while cIAP1 and cIAP2 are dispensable for NOD2-induced nuclear factor kB (NF-kB) and mitogen-activated protein kinase (MAPK) activation. However, mice lacking cIAP1 or TNFR1 have a blunted cytokine response to NOD2 stimulation. We conclude that cIAPs regulate NOD2-dependent autocrine TNF signaling in vivo and highlight the importance of physiological context in the interplay of innate immune signaling pathways. [ABSTRACT FROM AUTHOR]

Summary After exiting the thymus, Foxp3 + regulatory T (Treg) cells undergo further differentiation in the periphery, resulting in the generation of mature, fully suppressive effector (e)Treg cells in a process dependent on TCR signaling and the transcription factor IRF4. Here, we show that tumor necrosis factor receptor superfamily (TNFRSF) signaling plays a crucial role in the development and maintenance of eTreg cells. TNFRSF signaling activated the NF-κB transcription factor RelA, which was required to maintain eTreg cells in lymphoid and non-lymphoid tissues, including RORγt + Treg cells in the small intestine. In response to TNFRSF signaling, RelA regulated basic cellular processes, including cell survival and proliferation, but was dispensable for IRF4 expression or DNA binding, indicating that both pathways operated independently. Importantly, mutations in the RelA binding partner NF-κB1 compromised eTreg cells in humans, suggesting that the TNFRSF-NF-κB axis was required in a non-redundant manner to maintain eTreg cells in mice and humans. [ABSTRACT FROM AUTHOR]

Summary X-linked Inhibitor of Apoptosis (XIAP) deficiency predisposes people to pathogen-associated hyperinflammation. Upon XIAP loss, Toll-like receptor (TLR) ligation triggers RIPK3-caspase-8-mediated IL-1β activation and death in myeloid cells. How XIAP suppresses these events remains unclear. Here, we show that TLR-MyD88 causes the proteasomal degradation of the related IAP, cIAP1, and its adaptor, TRAF2, by inducing TNF and TNF Receptor 2 (TNFR2) signaling. Genetically, we define that myeloid-specific cIAP1 loss promotes TLR-induced RIPK3-caspase-8 and IL-1β activity in the absence of XIAP. Importantly, deletion of TNFR2 in XIAP-deficient cells limited TLR-MyD88-induced cIAP1-TRAF2 degradation, cell death, and IL-1β activation. In contrast to TLR-MyD88, TLR-TRIF-induced interferon (IFN)β inhibited cIAP1 loss and consequent cell death. These data reveal how, upon XIAP deficiency, a TLR-TNF-TNFR2 axis drives cIAP1-TRAF2 degradation to allow TLR or TNFR1 activation of RIPK3-caspase-8 and IL-1β. This mechanism may explain why XIAP-deficient patients can exhibit symptoms reminiscent of patients with activating inflammasome mutations. [ABSTRACT FROM AUTHOR]

Background and Aims Tumor necrosis factor (TNF) is a cytokine that promotes inflammation and contributes to pathogenesis of inflammatory bowel diseases. Unlike other cells and tissues, intestinal epithelial cells undergo rapid cell death upon exposure to TNF, by unclear mechanisms. We investigated the roles of inhibitor of apoptosis proteins (IAPs) in the regulation of TNF-induced cell death in the intestinal epithelium of mice and intestinal organoids. Methods RNA from cell lines and tissues was analyzed by quantitative polymerase chain reaction, protein levels were analyzed by immunoblot assays. BIRC2 (also called cIAP1) was expressed upon induction from lentiviral vectors in young adult mouse colon (YAMC) cells. YAMC cells, the mouse colon carcinoma cell line MC38, the mouse macrophage cell line RAW 264.7, or mouse and human organoids were incubated with second mitochondrial activator of caspases (Smac)-mimetic compound LCL161 or recombinant TNF-like weak inducer of apoptosis (TNFSF12) along with TNF, and cell death was quantified. C57BL/6 mice with disruption of Xiap, Birc2 (encodes cIAP1), Birc3 (encodes cIAP2), Tnfrsf1a , or Tnfrsf1b ( Tnfrsf1a and b encode TNF receptors) were injected with TNF or saline (control); liver and intestinal tissues were collected and analyzed for apoptosis induction by cleaved caspase 3 immunohistochemistry. We also measured levels of TNF and alanine aminotransferase in serum from mice. Results YAMC cells, and mouse and human intestinal organoids, died rapidly in response to TNF. YAMC and intestinal crypts expressed lower levels of XIAP, cIAP1, cIAP2, and cFLIP than liver tissue. Smac-mimetics reduced levels of cIAP1 and XIAP in MC38 and YAMC cells, and Smac-mimetics and TNF-related weak inducer of apoptosis increased TNF-induced cell death in YAMC cells and organoids—most likely by sequestering and degrading cIAP1. Injection of TNF greatly increased levels of cell death in intestinal tissue of cIAP1-null mice, compared with wild-type C57BL/6 mice, cIAP2-null mice, or XIAP-null mice. Excessive TNF-induced cell death in the intestinal epithelium was mediated TNF receptor 1. Conclusions In a study of mouse and human cell lines, organoids, and tissues, we found cIAP1 to be required for regulation of TNF-induced intestinal epithelial cell death and survival. These findings have important implications for the pathogenesis of TNF-mediated enteropathies and chronic inflammatory diseases of the intestine. [ABSTRACT FROM AUTHOR]

SHARPIN regulates signaling from the tumor necrosis factor (TNF) superfamily and pattern-recognition receptors. An inactivating Sharpin mutation in mice causes TNF-mediated dermatitis. Blocking cell death prevents the phenotype, implicating TNFR1-induced cell death in causing the skin disease. However, the source of TNF that drives dermatitis is unknown. Immune cells are a potent source of TNF in vivo and feature prominently in the skin pathology; however, T cells, B cells, and eosinophils are dispensable for the skin phenotype. We use targeted in vivo cell ablation, immune profiling, and extensive imaging to identify immune populations driving dermatitis. We find that systemic depletion of Langerin+ cells significantly reduces disease severity. This is enhanced in mice that lack Langerhans cells (LCs) from soon after birth. Reconstitution of LC-depleted Sharpin mutant mice with TNF-deficient LCs prevents dermatitis, implicating LCs as a potential cellular source of pathogenic TNF and highlighting a T cell-independent role in driving skin inflammation. [Display omitted] • In vivo depletion of LCs in Sharpin cpdm mice prevents TNF-mediated dermatitis • The Sharpin cpdm dermatitis phenotype is microbiota independent • Sharpin mutation on LCs is necessary for the dermatitis-causing pathological effect • Sharpin mutant LCs need to express TNF for the effect to occur TNF can be a potent cause of skin inflammation, but the origin of disease-causing TNF often remains elusive. Anderton et al. identify Langerhans cells (LCs) as the source of TNF responsible for dermatitis in Sharpin cpdm mice, highlighting a T cell-independent, microbiota-independent role for LCs in driving TNF-mediated skin inflammation. [ABSTRACT FROM AUTHOR]

Summary Linear ubiquitination is crucial for innate and adaptive immunity. The linear ubiquitin chain assembly complex (LUBAC), consisting of HOIL-1, HOIP, and SHARPIN, is the only known ubiquitin ligase that generates linear ubiquitin linkages. HOIP is the catalytically active LUBAC component. Here, we show that both constitutive and Tie2-Cre-driven HOIP deletion lead to aberrant endothelial cell death, resulting in defective vascularization and embryonic lethality at midgestation. Ablation of tumor necrosis factor receptor 1 (TNFR1) prevents cell death, vascularization defects, and death at midgestation. HOIP-deficient cells are more sensitive to death induction by both tumor necrosis factor (TNF) and lymphotoxin-α (LT-α), and aberrant complex-II formation is responsible for sensitization to TNFR1-mediated cell death in the absence of HOIP. Finally, we show that HOIP’s catalytic activity is necessary for preventing TNF-induced cell death. Hence, LUBAC and its linear-ubiquitin-forming activity are required for maintaining vascular integrity during embryogenesis by preventing TNFR1-mediated endothelial cell death. [ABSTRACT FROM AUTHOR]

Highlights: [•] TNF and its receptor, TNFR1, help orchestrate an inflammatory response to pathogens. [•] TNFR1 signaling is therefore a frequent target of pathogen manipulation. [•] We describe how the TNFR1 signaling pathway is targeted by pathogen virulence factors and how the different arms of TNFR1 signaling react. [•] We examine recent data showing the importance of other members of the Death Receptor family in the immune response to pathogens. [•] We discuss how understanding pathogen inhibition suggests clinical applications. [ABSTRACT FROM AUTHOR]

In the past 2 years there has been an explosion of information regarding molecules that regulate TNF-R1 signalling, and even reviews published in 2010 are out of date. TNF-R1 activation of NF-κB is a text book example of a signal transduction pathway regulated by ubiquitin and many of the concepts concerning the different roles of ubiquitin chains were first outlined in TNF-R1 signalling. What was once a very simple pathway with clearly defined roles for ubiquitin in regulating TNF-R1 signalling has, however, now become so complicated that we have ‘an embarrassment of riches’ []. The less polite might claim our pathways of TNF-R1 signalling look as complicated as a web constructed by a drug-addled spider []. This review will pick apart only one small strand of the web, and will address the role of ubiquitin in the activation of NF-κB by TNF with a focus on interpreting in vivo results. Nevertheless some of the concepts, for example the role of linear ubiquitin chains in regulating signalling, may be applicable to the family in general. [ABSTRACT FROM AUTHOR]

Smac-mimetics are compounds that target cellular Inhibitor of APoptosis (cIAP) proteins and induce tumor cell death. In this issue of Cancer Cell , Yang and colleagues (2016) identify cIAPs at the CARD11-MALT1-BCL10 complex in the ABC subtype of diffuse large B cell lymphoma (DLBCL), making this malignancy a prime target for these drugs. [ABSTRACT FROM AUTHOR]

Grim is a Drosophila inhibitor of apoptosis (IAP) antagonist that directly interferes with inhibition of caspases by IAPs. Expression of Grim, or removal of DIAP1, is sufficient to activate apoptosis in fly cells. Transient expression of Grim in mammalian cells induces apoptosis, arguing for the conservation of apoptotic pathways, but cytoplasmic expression of the mammalian IAP antagonist Diablo/smac does not. To understand why, we compared Grim and Diablo. Although they have the same IAP binding specificity, only Grim promoted XIAP ubiquitination and degradation. Grim also synergized with XIAP to promote an increase in total cellular ubiquitination, whereas Diablo antagonized this activity. Surprisingly, Grim-induced ubiquitination of XIAP did not require the IAP RING finger. Analysis of a Grim mutant that promoted XIAP degradation, but was not cytotoxic, suggests that Grim killing in transient assays is due to a combination of IAP depletion, blocking of IAP-mediated caspase inhibition, and at least one other unidentified function. Unlike transiently transfected cells, inducible mammalian cell lines can sustain continuous expression of Grim and selective degradation of XIAP without undergoing apoptosis, demonstrating that down-regulation and antagonism of IAPs is not sufficient to cause apoptosis of mammalian cells. [ABSTRACT FROM AUTHOR]

Plasmodium sporozoites infect the liver and develop into exoerythrocytic merozoites that initiate blood-stage disease. The hepatocyte molecular pathways that permit or abrogate parasite replication and merozoite formation have not been thoroughly explored, and a deeper understanding may identify therapeutic strategies to mitigate malaria. Cellular inhibitor of apoptosis (cIAP) proteins regulate cell survival and are co-opted by intracellular pathogens to support development. Here, we show that cIAP1 levels are upregulated during Plasmodium liver infection and that genetic or pharmacological targeting of cIAPs using clinical-stage antagonists preferentially kills infected hepatocytes and promotes immunity. Using gene-targeted mice, the mechanism was defined as TNF-TNFR1-mediated apoptosis via caspases 3 and 8 to clear parasites. This study reveals the importance of cIAPs to Plasmodium infection and demonstrates that host-directed antimalarial drugs can eliminate liver parasites and induce immunity while likely providing a high barrier to resistance in the parasite. • cIAPs are upregulated during Plasmodium liver infection • Inactivation of cIAPs preferentially kills liver-stage malaria parasites • IAP antagonists induce extrinsic apoptosis of infected hepatocytes and promote immunity • Targeting extrinsic apoptosis signaling could be a way to manage malaria infections Ebert et al. reveal that cellular inhibitor of apoptosis proteins (cIAPs) are upregulated in the liver during Plasmodium infection. Inactivation of cIAPs kills Plasmodium liver stages via TNF-mediated apoptosis of infected hepatocytes, affecting disease and promoting immunity. Targeting extrinsic apoptosis of infected host cells may be an antimalarial avenue. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor (TNF) is a proinflammatory cytokine that is produced and secreted by cytotoxic lymphocytes upon tumor target recognition. Depending on the context, TNF can mediate either pro-survival or pro-death signals. The potential cytotoxicity of T cell-produced TNF, particularly in the context of T cell-directed immunotherapies, has been largely overlooked. However, a spate of recent studies investigating tumor immune evasion through the application of CRISPR-based gene-editing screens have highlighted TNF-mediated killing as an important component of the mammalian T cell antitumor repertoire. In the context of the current understanding of the role of TNF in antitumor immunity, we discuss these studies and touch on their therapeutic implications. Collectively, we provide an enticing prospect to augment immunotherapy responses through TNF cytotoxicity. Cancer immunotherapy aims to enhance antitumor T cell cytotoxicity. Inflammatory cytokines are a part of the cytotoxic T cell arsenal; however, their cytotoxic activity is largely overlooked in the context of immunotherapy. TNF is one such inflammatory cytokine that can signal either pro-survival or pro-death signals depending on the context. Interest in TNF in the immunotherapy field has been reinvigorated by a recent collection of loss-of-function genetic screens in tumor cells, and these have highlighted multiple components of the TNF signaling pathway that can be protective against CD8+ T cell cytotoxicity. Pharmacological targeting of several proteins identified in these preliminary screens might provide a means to reroute tumor TNF signaling towards pro-death, representing a new combinatorial strategy to exploit T cell-derived TNF and boost responses to immunotherapy. [ABSTRACT FROM AUTHOR]

It is becoming clear that “apoptotic” caspases can effect cellular processes other than cell death. A recent paper in Cell points to a novel role of the Drosophila caspase inhibitor DIAP1 as a determinant of cell migration. [Copyright &y& Elsevier]

Malignant cells must circumvent endogenous cell death pathways to survive and develop into cancers. Acquired cell death resistance also sets up malignant cells to survive anticancer therapies. Acute Myeloid Leukemia (AML) is an aggressive blood cancer characterized by high relapse rate and resistance to cytotoxic therapies. Recent collaborative profiling projects have led to a greater understanding of the ‘fearful symmetry’ of the genomic landscape of AML, and point to the development of novel potential therapies that can overcome factors linked to chemoresistance. We review here the most recent research in the genetics of AML and how these discoveries have led, or might lead, to therapies that specifically activate cell death pathways to substantially challenge this ‘fearful’ disease. [ABSTRACT FROM AUTHOR]

The inhibitor of apoptosis (IAP) proteins are important ubiquitin E3 ligases that regulate cell survival and oncogenesis. The cIAP1 and cIAP2 paralogs bear three N-terminal baculoviral IAP repeat (BIR) domains and a C-terminal E3 ligase RING domain. LAP antagonist compounds, also known as Smac mimetics, bind the BIR domains of IAPs and trigger rapid RING-dependent autoubiquitylation, but the mechanism is unknown. We show that RING dimerization is essential for the E3 ligase activity of cIAP1 and cIAP2 because monomeric RING mutants could not interact with the ubiquitin-charged E2 enzyme and were resistant to Smac mimetic-induced autoubiquitylation. Unexpectedly, the BIR domains inhibited cIAP1 RING dimerization, and cIAP1 existed predominantly as an inactive monomer, However, addition of either mono-or bivalent Smac mimetics relieved this inhibition, thereby allowing dimer formation and promoting E3 ligase activation. In contrast, the cIAP2 dimer was more stable, had higher intrinsic E3 ligase activity, and was not highly activated by Smac mimetics. These results explain how Smac mimetics promote rapid destruction of cIAP1 and suggest mechanisms for activating cIAP1 in other pathways. [ABSTRACT FROM AUTHOR]

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-κB, but also regulates caspase-8 activation and cell death. Although overexpressioñ of RIPK1 can cause caspase-8-dependent cell death, when RIPK1-/- cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities (1, 2). To determine how RIPK1 promotes cell survival, we compared wild-type and RIPIKF-/- cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1-/- cells caused TRAF2 and clAP 1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-κB2 to p52, a decrease in levels of cFLIPL, and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1. [ABSTRACT FROM AUTHOR]

Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamlly (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials, IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-κB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that R1NG dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-κB, however, delayed activation of NF-κB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-κB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required. [ABSTRACT FROM AUTHOR]

Abstract: Background/aims: Dysregulation of the cell cycle is frequently associated with tumor development. Hepatitis B virus (HBV) is associated with a significant risk of developing hepatocellular carcinoma but the effects of HBV on cell cycle regulation are not completely understood. Methods: We have used a recombinant adeno-HBV model system to investigate the effect of infection with HBV and the replication defective lamivudine resistant mutant rtM204I mutant on hepatocyte cell cycle and cell viability. Results: Huh7 cells synchronised at the G1/S phase of the cell cycle were arrested at the G2/M following infection with rAdHBV-wt and rAdHBV-M204I. This was accompanied by increased levels of p21cip1, p-cdc2, cyclins D, A and B. Cell viability was reduced and cleaved caspase 3 levels were increased in HBV- and rtM204I-infected cells. rAdHBV-M204I-infected Huh7 cells also demonstrated significant up-regulation of phospho-ERK, phospho-Akt, p53 and phospho-Mdm2 compared to mock-infected cells. These changes were comparable to those following infection of Huh7 cells with rAdHBV-wt. Conclusion: Our results suggest that HBV, regardless of phenotype, produces cell cycle arrest and reduced hepatocyte viability. Perturbations in these cellular processes are likely to underlie HBV-associated liver oncogenic transformation and may help explain the ongoing risk of developing hepatocellular carcinoma in individuals in whom the lamivudine resistant rtM204I mutant emerges. [Copyright &y& Elsevier]

Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to clAP 1 and cIAP2 (clAP 1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cLAP 1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-κB-inducing kinase stability and suppress constitutive noncanonical NF-κB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-κB activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-κB signaling and that efficient activation of NF-κB by TNFR1 requires the recruitment of cIAP1/2 by TRAF2. [ABSTRACT FROM AUTHOR]

The ability to remove unwanted proteins is an important cellular feature. Classically, this involves the enzymatic addition of ubiquitin moieties followed by degradation in the proteasome. Nedd4 proteins are ubiquitin ligases important not only for protein degradation, but also for protein trafficking. Nedd4 proteins can bind to target proteins either by themselves or through adaptor protein Ndfip1 (Nedd4 family-interacting protein 1). An alternative mechanism for protein removal and trafficking is provided by exosomes, which are small vesicles (50-90-nm diameter) originating from late endosomes and multivesicular bodies (MVBs). Exosomes provide a rapid means of shedding obsolete proteins and also for cell to cell communication. In the present work, we show that Ndfip1 is detectable in exosomes secreted from transfected cells and also from primary neurons. Compared with control, Ndfip1 increases exosome secretion from transfected cells. Furthermore, while Nedd4, Nedd4-2, and Itch are normally absent from exosomes, expression of Ndfip1 results in recruitment of all three Nedd4 proteins into exosomes. Together, these results suggest that Ndfip1 is important for protein trafficking via exosomes, and provides a mechanism for cargoing passenger proteins such as Nedd4 family proteins. Given the positive roles of Ndfip1/Nedd4 in improving neuronal survival during brain injury, it is possible that exosome secretion provides a novel route for rapid sequestration and removal of proteins during stress. [ABSTRACT FROM AUTHOR]

Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death that are highly expressed in many cancers. Cell death caused by antagonists that bind to IAP proteins is associated with their ubiquitylation and degradation. The RING domain at the C terminus of IAP proteins is pivotal. Here we report the crystal structures of the cIAP2 RING domain homodimer alone, and bound to the ubiquitin-conjugating (E2) enzyme UbcH5b. These structures show that small changes in the RING domain accompany E2 binding. By mutating residues at the E2-binding surface, we show that autoubiquitylation is required for regulation of IAP abundance. Dimer formation is also critical, and mutation of a single C-terminal residue abrogated dimer formation and E3 ligase activity was diminished. We further demonstrate that disruption of E2 binding, or dimerization, stabilizes IAP proteins against IAP antagonists in vivo. [ABSTRACT FROM AUTHOR]

Death ligands not only activate a death program but also regulate inflammatory signalling pathways, for example, through NF-κB induction. Although tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and TNF both activate NF-κB in human keratinocytes, only TRAIL potently induces apoptosis. However, when induction of NF-κB was inhibited with a kinase dead IKK2 mutant (IKK2-KD), TNF- but not TRAIL-induced apoptosis was dramatically enhanced. Acquired susceptibility to TNF-induced apoptosis was due to increased caspase-8 activation. To investigate the mechanism of resistance of HaCaT keratinocytes to TNF-induced apoptosis, we analyzed a panel of NF-κB-regulated effector molecules. Interestingly, the inhibitor of apoptosis protein (IAP) family member cIAP2, but not cIAP1, X-linked inhibitor of apoptosis, TNF receptor-associated factor (TRAF)-1, or TRAF2, was downregulated in sensitive but not in resistant HaCaT keratinocytes. Surprisingly, however, stable inducible expression of cIAP2 was not sufficient to render IKK2-KD-sensitized keratinocytes resistant to TNF, and reduction of cIAP2 alone did not increase the sensitivity of HaCaT keratinocytes to TNF. In conclusion, we demonstrate that inhibition of NF-κB dramatically sensitizes human keratinocytes to TNF- but not to TRAIL-induced apoptosis and that this sensitization for TNF was largely independent of cIAP2. Our data thus clearly exclude the candidates proposed to date to confer TNF apoptosis resistance and suggest the function of an unanticipated effector of NF-κB critical for the survival of HaCaT keratinocytes upstream or at the level of caspase-8 activation.Journal of Investigative Dermatology (2008) 128, 1134–1147; doi:10.1038/sj.jid.5701141; published online 8 November 2007 [ABSTRACT FROM AUTHOR]

The suppressors of cytokine signaling (SOCS) proteins inhibit cytokine action by direct interaction with Janus kinases or activated cytokine receptors. In addition to the N-terminal and Src homology 2 domains that mediate these interactions, SOCS proteins contain a C-terminal SOCS box. DNA data base searches have identified a number of other protein families that possess a SOCS box, of which the ankyrin repeat and SOCS box-containing (Asb) proteins constitute the largest. Although it is known that the SOCS proteins are involved in the negative regulation of cytokine signaling, the biological and biochemical functions of the Asbs are largely undefined. Using a proteomics approach, we demonstrate that creatine kinase B (CKB) interacts with Asb-9 in a specific, SOCS box-independent manner. This interaction increases the polyubiquitylation of CKB and decreases total CKB levels within the cell. The targeting of CKB for degradation by Asb-9 was primarily SOCS box-dependent and suggests that Asb-9 acts as a specific ubiquitin ligase regulating levels of this evolutionarily conserved enzyme. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is an aggressive disease with a current 5-year survival rate of only ∼25%, therefore development of effective treatments towards resistant AML is urgently needed. Potential mechanisms of therapy resistance include overexpression of members of the inhibitor of apoptosis protein (IAP) family. Natural IAP antagonists such as second-mitochondria-derived activator of caspases (Smac) exist, leading to the pharmaceutical development of Smac-mimetics (SMs). The clinical SM birinapant has been trialed in AML and solid cancers, as well as in hepatitis B virus (HBV), with variable and limited success. Using a high-throughput strategy, we screened 5,700 bioactive compounds and identified clinical drugs that overcome birinapant resistance through inhibition of multidrug resistance protein 1 (MDR1). We show that 3rd generation MDR1 inhibitors synergise with birinapant and other SMs to kill AMLs. We use mass spectrometry to show inhibition of MDR1 in SM resistant leukemias increased intracellular levels of birinapant, indicating birinapant is a novel substrate of MDR1. Genetic deletion of MDR1 confirmed combination synergy is mediated specifically by MDR1. Furthermore, we observed a strong correlation between MDR1 expression/activity and sensitivity to birinapant-based therapies in AML cells. Strikingly, combination treatment sensitised leukemic stem cells (LSCs) to birinapant killing and was well-tolerated and effective in treating leukemia in vivo. Moreover, MDR1 inhibitors potentiated birinapant-mediated killing in patient leukemia and ovarian cancer cells, and in HBV infected liver cells in vivo. This study identifies MDR1 as a biomarker of SM therapy and gives clear rationale for MDR1 screening, leading to informed SM clinical trials and a personalised therapy for AML patients. [ABSTRACT FROM AUTHOR]

Resistance to cell death is one of the main problems in Acute Myeloid Leukemia (AML) contributing to treatment failure, disease relapse and a low 5-year survival rate (<30%). Thus, understanding cell death related genes function during the progression and treatment of the disease, is important for the development of more efficient therapies. Necroptosis is an alternative form of programmed cell death that occurs independent of caspase activity and could be exploited as an anti-cancer therapy to overcome apoptosis resistance. The receptor-interacting protein kinase 1 (RIPK1) is key effector of necroptosis, a regulator of apoptosis and inflammatory processes, and is essential for normal haematopoiesis. Mutations in RIPK1 have a significant impact in human health. Patients with RIPK1 deficiency present severe inflammation and immunodeficiency. In cancers, RIPK1 has been indicated as both tumor suppressor and promoter. In AML, loss of RIPK1 is correlated with unfavourable prognosis. Using murine models of AML that recapitulate human disease, we investigated the relevance of RIPK1 in AML development. Interestingly, loss of Ripk1 accelerated leukemogenesis driven by MLL translocations. RIPKI deficient leukaemias were characterised by rapid infiltration of blasts in the bone marrow, spleen and liver accompanied by high levels of granulocyte colony stimulating factor (G-CSF). Concurrently, intravital imaging revealed increased bone marrow infiltration of Ripk1-/- AMLs 10 days after transplant. Moreover, loss of G-CSF partially rescues the aggressive phenotype, suggesting a role for this cytokine in the progression of MLL driven AML. Together our results indicate tumor suppressor role for RIPK1 signalling in AML, providing new therapeutic opportunities for these leukaemias. [ABSTRACT FROM AUTHOR]