IAPs and RIPK1 mediate LPS-induced cytokine production in healthy subjects and Crohn's disease.

Innate immune activity fuels intestinal inflammation in Crohn's disease (CD), an inflammatory bowel disease. Identification and targeting of new molecular regulators of the innate activity are warranted to control the disease. Inhibitor of apoptosis proteins (IAPs) regulate both cell survival and inflammatory signaling. We investigated the effects of IAP inhibition by second mitochondria-derived activator of caspases (SMAC) mimetics (SMs) on innate responses and cell death to pathogen-associated molecular patterns in peripheral blood mononuclear cells (PBMCs) and monocytes. IAPs inhibited lipopolysaccharide (LPS)-induced expression of proinflammatory interleukin (IL)-1ß, IL-6. Likewise, LPS (but not muramyl dipeptide or Escherichia coli) induced TNF-α was inhibited in CD and control PBMCs. The SM effect was partially reversed by inhibition of receptor-interacting serine/threonine-protein kinase 1 (RIPK1). The effect was mainly cell death independent. Thus, IAP inhibition by SMs leads to reduced production of proinflammatory cytokines and may be considered in the efforts to develop new therapeutic strategies to control CD.

LCL161 enhances expansion and survival of engineered anti-tumor T cells but is restricted by death signaling.

Background: The genesis of SMAC mimetic drugs is founded on the observation that many cancers amplify IAP proteins to facilitate their survival, and therefore removal of these pathways would re-sensitize the cells towards apoptosis. It has become increasingly clear that SMAC mimetics also interface with the immune system in a modulatory manner. Suppression of IAP function by SMAC mimetics activates the non-canonical NF-κB pathway which can augment T cell function, opening the possibility of using SMAC mimetics to enhance immunotherapeutics. Methods: We have investigated the SMAC mimetic LCL161, which promotes degradation of cIAP-1 and cIAP-2, as an agent for delivering transient costimulation to engineered BMCA-specific human TAC T cells. In doing so we also sought to understand the cellular and molecular effects of LCL161 on T cell biology. Results: LCL161 activated the non-canonical NF-κB pathway and enhanced antigen-driven TAC T cell proliferation and survival. Transcriptional profiling from TAC T cells treated with LCL161 revealed differential expression of costimulatory and apoptosis-related proteins, namely CD30 and FAIM3. We hypothesized that regulation of these genes by LCL161 may influence the drug's effects on T cells. We reversed the differential expression through genetic engineering and observed impaired costimulation by LCL161, particularly when CD30 was deleted. While LCL161 can provide a costimulatory signal to TAC T cells following exposure to isolated antigen, we did not observe a similar pattern when TAC T cells were stimulated with myeloma cells expressing the target antigen. We questioned whether FasL expression by myeloma cells may antagonize the costimulatory effects of LCL161. Fas-KO TAC T cells displayed superior expansion following antigen stimulation in the presence of LCL161, suggesting a role for Fas-related T cell death in limiting the magnitude of the T cell response to antigen in the presence of LCL161. Conclusions: Our results demonstrate that LCL161 provides costimulation to TAC T cells exposed to antigen alone, however LCL161 did not enhance TAC T cell anti-tumor function when challenged with myeloma cells and may be limited due to sensitization of T cells towards Fas-mediated apoptosis.

Selective JAK1 inhibitors for the treatment of inflammatory bowel disease.

Janus kinase (JAK) inhibitors, also known as jakinibs, are third-generation oral small molecules that have expanded the therapeutic options for the management of chronic inflammatory diseases, including inflammatory bowel disease (IBD). Tofacitinib, a pan-JAK inhibitor, has spearheaded the new JAK class for IBD treatment. Unfortunately, serious adverse effects, including cardiovascular complications such as pulmonary embolism and venous thromboembolism or even death from any cause, have been reported for tofacitinib. However, it is anticipated that next-generation selective JAK inhibitors may limit the development of serious adverse events, leading to a safer treatment course with these novel targeted therapies. Nevertheless, although this drug class was recently introduced, following the launch of second-generation biologics in the late 1990s, it is breaking new ground and has been shown to efficiently modulate complex cytokine-driven inflammation in both preclinical models and human studies. Herein, we review the clinical opportunities for targeting JAK1 signaling in the pathophysiology of IBD, the biology and chemistry underpinning these target-selective compounds, and their mechanisms of actions. We also discuss the potential for these inhibitors in efforts to balance their benefits and harms.

SMAC mimetics and RIPK inhibitors as therapeutics for chronic inflammatory diseases.

New therapeutic approaches for chronic inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, and psoriasis are needed because current treatments are often suboptimal in terms of both efficacy and the risks of serious adverse events. Inhibitor of apoptosis proteins (IAPs) are E3 ubiquitin ligases that inhibit cell death pathways and are themselves inhibited by second mitochondria-derived activator of caspases (SMAC). SMAC mimetics (SMs), small-molecule antagonists of IAPs, are being evaluated as cancer therapies in clinical trials. IAPs are also crucial regulators of inflammatory pathways because they influence both the activation of inflammatory genes and the induction of cell death through the receptor-interacting serine-threonine protein kinases (RIPKs), nuclear factor κB (NF-κB)-inducing kinase, and mitogen-activated protein kinases (MAPKs). Furthermore, there is an increasing interest in specifically targeting the substrates of IAP-mediated ubiquitylation, especially RIPK1, RIPK2, and RIPK3, as druggable nodes in inflammation control. Several studies have revealed an anti-inflammatory potential of RIPK inhibitors that either block inflammatory signaling or block the form of inflammatory cell death known as necroptosis. Expanding research on innate immune signaling through pattern recognition receptors that stimulate proinflammatory NF-κB and MAPK signaling may further contribute to uncovering the complex molecular roles used by IAPs and downstream RIPKs in inflammatory signaling. This may benefit and guide the development of SMs or selective RIPK inhibitors as anti-inflammatory therapeutics for various chronic inflammatory conditions.

Sp3-cificity of TNF-α expression promotes the Smac mimetic-mediated killing of cancer cells.

A genome-wide small-interfering RNA-based screen identified the transcription factor Specificity Protein 3 (SP3) as a critical factor for Second mitochondrial-derived activator of caspase (Smac) mimetic-mediated killing of cancer cells. In concert with Nuclear Factor kappa B (NF-κB,) SP3 is required for the expression of the cytokine Tumor Necrosis Factor alpha (TNF-α) under basal and Smac mimetic-stimulated conditions.

Targeted ablation of the cellular inhibitor of apoptosis 1 (cIAP1) attenuates denervation-induced skeletal muscle atrophy.

BACKGROUND: Skeletal muscle atrophy is a pathological condition that contributes to morbidity in a variety of conditions including denervation, cachexia, and aging. Muscle atrophy is characterized as decreased muscle fiber cross-sectional area and protein content due, in part, to the proteolytic activities of two muscle-specific E3 ubiquitin ligases: muscle RING-finger 1 (MuRF1) and muscle atrophy F-box (MAFbx or Atrogin-1). The nuclear factor-kappa B (NF-κB) pathway has emerged as a critical signaling network in skeletal muscle atrophy and has become a prime therapeutic target for the treatment of muscle diseases. Unfortunately, none of the NF-κB targeting drugs are currently being used to treat these diseases, likely because of our limited knowledge and specificity, for muscle biology and disease. The cellular inhibitor of apoptosis 1 (cIAP1) protein is a positive regulator of tumor necrosis factor alpha (TNFα)-mediated classical NF-κB signaling, and cIAP1 loss has been shown to enhance muscle regeneration during acute and chronic injury. METHODS: Sciatic nerve transection in wild-type, cIAP1-null and Smac mimetic compound (SMC)-treated mice was performed to investigate the role of cIAP1 in denervation-induced atrophy. Genetic in vitro models of C2C12 myoblasts and primary myoblasts were also used to examine the role of classical NF-κB activity in cIAP1-induced myotube atrophy. RESULTS: We found that cIAP1 expression was upregulated in denervated muscles compared to non-denervated controls 14 days after denervation. Genetic and pharmacological loss of cIAP1 attenuated denervation-induced muscle atrophy and overexpression of cIAP1 in myotubes was sufficient to induce atrophy. The induction of myotube atrophy by cIAP1 was attenuated when the classical NF-κB signaling pathway was inhibited. CONCLUSIONS: These results demonstrate the cIAP1 is an important mediator of NF-κB/MuRF1 signaling in skeletal muscle atrophy and is a promising therapeutic target for muscle wasting diseases.

The transcription factor SP3 drives TNF-α expression in response to Smac mimetics.

The controlled production and downstream signaling of the inflammatory cytokine tumor necrosis factor-α (TNF-α) are important for immunity and its anticancer effects. Although chronic stimulation with TNF-α is detrimental to the health of the host in several autoimmune and inflammatory disorders, TNF-α-contrary to what its name implies-leads to cancer formation by promoting cell proliferation and survival. Smac mimetic compounds (SMCs), small-molecule antagonists of inhibitor of apoptosis proteins (IAPs), switch the TNF-α signal from promoting survival to promoting death in cancer cells. Using a genome-wide siRNA screen to identify factors required for SMC-to-TNF-α-mediated cancer cell death, we identified the transcription factor SP3 as a critical molecule in both basal and SMC-induced production of TNF-α by engaging the nuclear factor κB (NF-κB) transcriptional pathway. Moreover, the promotion of TNF-α expression by SP3 activity confers differential sensitivity of cancer versus normal cells to SMC treatment. The key role of SP3 in TNF-α production and signaling will help us further understand TNF-α biology and provide insight into mechanisms relevant to cancer and inflammatory disease.

Combination of IAP Antagonists and TNF-α-Armed Oncolytic Viruses Induce Tumor Vascular Shutdown and Tumor Regression.

Smac mimetic compounds (SMCs) are anti-cancer drugs that antagonize Inhibitor of Apoptosis proteins, which consequently sensitize cancer cells to death in the presence of proinflammatory ligands such as tumor necrosis factor alpha (TNF-α). SMCs synergize with the attenuated oncolytic vesicular stomatitis virus (VSVΔ51) by eliciting an innate immune response, which is dependent on the endogenous production of TNF-α and type I interferon. To improve on this SMC-mediated synergistic response, we generated TNF-α-armed VSVΔ51 to produce elevated levels of this death ligand. Due to ectopic expression of TNF-α from infected cells, a lower viral dose of TNF-α-armed VSVΔ51 combined with treatment of the SMC LCL161 was sufficient to improve the survival rate compared to LCL161 and unarmed VSVΔ51 co-therapy. This improved response is attributed to a bystander effect whereby the spread of TNF-α from infected cells leads to the death of uninfected cells in the presence of LCL161. In addition, the treatments induced vascular collapse in solid tumors with a concomitant increase of tumor cell death, revealing another mechanism by which cytokine-armed VSVΔ51 in combination with LCL161 can kill tumor cells. Our studies demonstrate the potential for cytokine-engineered oncolytic virus and SMCs as a new combination immunotherapy for cancer treatment.

Publisher Correction: Smac mimetics synergize with immune checkpoint inhibitors to promote tumour immunity against glioblastoma.

This corrects the article DOI: 10.1038/ncomms14278.

Inhibitor of apoptosis proteins, NAIP, cIAP1 and cIAP2 expression during macrophage differentiation and M1/M2 polarization.

Monocytes and macrophages constitute the first line of defense of the immune system against external pathogens. Macrophages have a highly plastic phenotype depending on environmental conditions; the extremes of this phenotypic spectrum are a pro-inflammatory defensive role (M1 phenotype) and an anti-inflammatory tissue-repair one (M2 phenotype). The Inhibitor of Apoptosis (IAP) proteins have important roles in the regulation of several cellular processes, including innate and adaptive immunity. In this study we have analyzed the differential expression of the IAPs, NAIP, cIAP1 and cIAP2, during macrophage differentiation and polarization into M1 or M2. In polarized THP-1 cells and primary human macrophages, NAIP is abundantly expressed in M2 macrophages, while cIAP1 and cIAP2 show an inverse pattern of expression in polarized macrophages, with elevated expression levels of cIAP1 in M2 and cIAP2 preferentially expressed in M1. Interestingly, treatment with the IAP antagonist SMC-LCL161, induced the upregulation of NAIP in M2, the downregulation of cIAP1 in M1 and M2 and an induction of cIAP2 in M1 macrophages.

Smac mimetics synergize with immune checkpoint inhibitors to promote tumour immunity against glioblastoma.

Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells.

How genetic testing can lead to targeted management of XIAP deficiency-related inflammatory bowel disease.

X-linked lymphoproliferative disease type 2 (XLP-2, OMIM 300635) is a primary immunodeficiency caused by the loss of X chromosome-linked inhibitor of apoptosis (XIAP), the X-linked inhibitor of apoptosis gene at Xq25. XLP-2 individuals are susceptible to several specific and potentially fatal infections, such as Epstein-Barr virus (EBV). Children with XIAP-related XLP-2 may present with either familial hemophagocytic lymphohistiocytosis, often triggered in response to EBV infection, or with a treatment-refractory severe pediatric form of inflammatory bowel disease (IBD) that might be diagnosed as Crohn disease. However, this monogenic cause of IBD is distinct from adult Crohn disease (a polygenic and multifactorial disease) in its etiology and responsiveness to therapy. XLP-2 and the associated IBD symptoms are managed by a reduced-intensity conditioning regimen with an allogeneic hematopoietic stem cell transplantation that causes resolution of gastrointestinal symptoms. Exome sequencing has enabled identification of XIAP-deficient diseased individuals and has altered their morbidity by providing potentially lifesaving strategies in a timely and effective manner. Here, we summarize XLP-2 IBD treatment history and patient morbidity/mortality since its original identification in 2006. Since XLP-2 is rare, cases are probably undergiagnosed or misdiagnosed. Consideration of XLP-2 in children with severe symptoms of IBD can prevent serious morbidities and mortality, avoid unnecessary procedures, and expedite specific targeted therapy.Genet Med 19 2, 133-143.

Oncolytic virus synergizes with Smac mimetic compounds to induce rhabdomyosarcoma cell death in a syngeneic murine model.

Rhabdomyosarcoma (RMS), a neoplasm characterized by undifferentiated myoblasts, is the most common soft tissue tumour in children. Therapeutic resistance is common in RMS and is often caused by acquired defects in the cellular apoptotic program. Smac mimetic compounds (SMCs) are a novel class of inhibitor of apoptosis (IAP) antagonists that are currently under clinical development as cancer therapeutics. We previously reported that cIAP1 is overexpressed in human primary RMS tumours and in patient-derived RMS cell lines where it drives resistance to apoptosis. In this study, we investigated whether inflammatory cytokine production triggered by activators of innate immunity synergizes with LCL161 to induce bystander killing of RMS cells in vitro and in vivo. Indeed, we show that innate immune stimuli (oncolytic virus (VSVΔ51-GFP), interferon γ (IFNγ), and tumour necrosis factor-like weak inducer of apoptosis (TWEAK)) combine with SMCs in vitro to reduce cell viability in the Kym-1 RMS cancer cell line. Other human RMS cell lines (RH36, RH41, RD, RH18, RH28, and RH30) and the murine RMS cell line 76-9 are resistant to treatment with LCL161 alone or in combination with immune stimulants in in vitro cell viability assays. In contrast, we report that the combination of LCL161 and VSVΔ51-GFP reduces tumour volume and prolongs survival in a 76-9 syngeneic murine model. Our results support further exploration of the combined use of IAP antagonists and innate immune stimuli as a therapeutic approach for RMS cancers.

Combinatorial cancer immunotherapy strategies with proapoptotic small-molecule IAP antagonists.

Members of the inhibitor of apoptosis (IAP) family control several critical aspects of innate immunity, cell death, and tumorigenesis. Small molecule antagonists that target specific IAP oncoproteins, primarily cIAP1 and cIAP2, but potentially also XIAP and Livin, modulate distinct immune signal transduction pathways that can lead to an increased sensitivity of tumors cells to cytokine-mediated apoptosis. These antagonists are based on the structure of an endogenous cellular IAP inhibitor called Smac. Smac is normally sequestered within the mitochondria and is released into the cytoplasm upon cell death stimuli, thereby overcoming the anti-apoptotic action of the IAPs. The therapeutic usefulness of recombinant tumoricidal cytokines to treat cancer patients is principally limited due to their unacceptable adverse side effects. Therefore, investigators have sought to develop alternative regimens that do not rely on exogenously delivered death ligands. These approaches include the stimulation of the immune system with oncolytic virus-based agents or Toll-like receptor agonists in combination with Smac mimetics. Similarly, preclinical combination immunotherapy studies reveal that recombinant interferon synergizes with Smac mimetics to kill cancer. This strategy opens up new therapeutic avenues for anti-cancer therapy by modulating specific immune-mediated death pathways employing unique dual-pronged combinatorial approaches.

Inhibitors of apoptosis (IAPs) regulate intestinal immunity and inflammatory bowel disease (IBD) inflammation.

The inhibitor of apoptosis (IAP) family members, notably cIAP1, cIAP2, and XIAP, are critical and universal regulators of tumor necrosis factor (TNF) mediated survival, inflammatory, and death signaling pathways. Furthermore, IAPs mediate the signaling of nucleotide-binding oligomerization domain (NOD)1/NOD2 and other intracellular NOD-like receptors in response to bacterial pathogens. These pathways are important to the pathogenesis and treatment of inflammatory bowel disease (IBD). Inactivating mutations in the X-chromosome-linked IAP (XIAP) gene causes an immunodeficiency syndrome, X-linked lymphoproliferative disease type 2 (XLP2), in which 20% of patients develop severe intestinal inflammation. In addition, 4% of males with early-onset IBD also have inactivating mutations in XIAP. Therefore, the IAPs play a greater role in gut homeostasis, immunity and IBD development than previously suspected, and may have therapeutic potential.

Smac mimetics combined with innate immune stimuli create the perfect cytokine storm to kill tumor cells.

A dual immunotherapy approach employing small-molecule inhibitors of apoptosis (IAP) protein antagonists in combination with innate immune stimuli has proven to be highly synergistic and effective in animal tumor models. This strategy overcomes many of the limitations of either single agent therapy and our results suggest that the combination could be easily and effectively translated to the clinic.

Role of the TWEAK-Fn14-cIAP1-NF-κB Signaling Axis in the Regulation of Myogenesis and Muscle Homeostasis.

Mammalian skeletal muscle maintains a robust regenerative capacity throughout life, largely due to the presence of a stem cell population known as "satellite cells" in the muscle milieu. In normal conditions, these cells remain quiescent; they are activated upon injury to become myoblasts, which proliferate extensively and eventually differentiate and fuse to form new multinucleated muscle fibers. Recent findings have identified some of the factors, including the cytokine TNFα-like weak inducer of apoptosis (TWEAK), which govern these cells' decisions to proliferate, differentiate, or fuse. In this review, we will address the functions of TWEAK, its receptor Fn14, and the associated signal transduction molecule, the cellular inhibitor of apoptosis 1 (cIAP1), in the regulation of myogenesis. TWEAK signaling can activate the canonical NF-κB signaling pathway, which promotes myoblast proliferation and inhibits myogenesis. In addition, TWEAK activates the non-canonical NF-κB pathway, which, in contrast, promotes myogenesis by increasing myoblast fusion. Both pathways are regulated by cIAP1, which is an essential component of downstream signaling mediated by TWEAK and similar cytokines. This review will focus on the seemingly contradictory roles played by TWEAK during muscle regeneration, by highlighting the interplay between the two NF-κB pathways under physiological and pathological conditions. We will also discuss how myogenesis is negatively affected by chronic conditions, which affect homeostasis of the skeletal muscle environment.

Smac mimetics and innate immune stimuli synergize to promote tumor death.

Smac mimetic compounds (SMC), a class of drugs that sensitize cells to apoptosis by counteracting the activity of inhibitor of apoptosis (IAP) proteins, have proven safe in phase 1 clinical trials in cancer patients. However, because SMCs act by enabling transduction of pro-apoptotic signals, SMC monotherapy may be efficacious only in the subset of patients whose tumors produce large quantities of death-inducing proteins such as inflammatory cytokines. Therefore, we reasoned that SMCs would synergize with agents that stimulate a potent yet safe "cytokine storm." Here we show that oncolytic viruses and adjuvants such as poly(I:C) and CpG induce bystander death of cancer cells treated with SMCs that is mediated by interferon beta (IFN-ß), tumor necrosis factor alpha (TNF-α) and/or TNF-related apoptosis-inducing ligand (TRAIL). This combinatorial treatment resulted in tumor regression and extended survival in two mouse models of cancer. As these and other adjuvants have been proven safe in clinical trials, it may be worthwhile to explore their clinical efficacy in combination with SMCs.