BRD9 is a druggable component of interferon-stimulated gene expression and antiviral activity.

Interferon (IFN) induction of IFN-stimulated genes (ISGs) creates a formidable protective antiviral state. However, loss of appropriate control mechanisms can result in constitutive pathogenic ISG upregulation. Here, we used genome-scale loss-of-function screening to establish genes critical for IFN-induced transcription, identifying all expected members of the JAK-STAT signaling pathway and a previously unappreciated epigenetic reader, bromodomain-containing protein 9 (BRD9), the defining subunit of non-canonical BAF (ncBAF) chromatin-remodeling complexes. Genetic knockout or small-molecule-mediated degradation of BRD9 limits IFN-induced expression of a subset of ISGs in multiple cell types and prevents IFN from exerting full antiviral activity against several RNA and DNA viruses, including influenza virus, human immunodeficiency virus (HIV1), and herpes simplex virus (HSV1). Mechanistically, BRD9 acts at the level of transcription, and its IFN-triggered proximal association with the ISG transcriptional activator, STAT2, suggests a functional localization at selected ISG promoters. Furthermore, BRD9 relies on its intact acetyl-binding bromodomain and unique ncBAF scaffolding interaction with GLTSCR1/1L to promote IFN action. Given its druggability, BRD9 is an attractive target for dampening ISG expression under certain autoinflammatory conditions.

Loss of cIAP1 in Endothelial Cells Limits Metastatic Extravasation through Tumor-Derived Lymphotoxin Alpha.

In this study, we determined whether Smac mimetics play a role in metastasis, specifically in circulation, tumor extravasation and growth in a metastatic site. Reports suggest inducing the degradation of IAPs through use of Smac mimetics, alters the ability of the tumor cell to metastasize. However, a role for the immune or stromal compartment in affecting the ability of tumor cells to metastasize upon loss of IAPs has not been defined. To address this open question, we utilized syngeneic tumor models in a late-stage model of metastasis. Loss of cIAP1 in the endothelial compartment, rather than depletion of cIAP2 or absence of cIAP1 in the hematopoietic compartment, caused reduction of tumor load in the lung. Our results underline the involvement of the endothelium in hindering tumor cell extravasation upon loss of cIAP1, in contrast to the immune compartment. Endothelial specific depletion of cIAP1 did not lead to cell death but resulted in an unresponsive endothelium barrier to permeability factors causing a decrease in tumor cell extravasation. Surprisingly, lymphotoxin alpha (LTA), and not TNF, secreted by the tumor cells, was critical for the extravasation. Using TCGA, we found high LTA mRNA expression correlated with decreased survival in kidney carcinoma and associated with advanced disease stage. Our data suggest that Smac mimetics, targeting cIAP1/2, reduce metastasis to the lung by inhibiting tumor cell extravasation.

Correction: TNFR2 induced priming of the inflammasome leads to a RIPK1-dependent cell death in the absence of XIAP.

The original version of this article contained an error in the name of one of the co-authors (Erika Owsley). This has been corrected in the PDF and HTML versions.

TNFR2 induced priming of the inflammasome leads to a RIPK1-dependent cell death in the absence of XIAP.

The pediatric immune deficiency X-linked proliferative disease-2 (XLP-2) is a unique disease, with patients presenting with either hemophagocytic lymphohistiocytosis (HLH) or intestinal bowel disease (IBD). Interestingly, XLP-2 patients display high levels of IL-18 in the serum even while in stable condition, presumably through spontaneous inflammasome activation. Recent data suggests that LPS stimulation can trigger inflammasome activation through a TNFR2/TNF/TNFR1 mediated loop in xiap-/- macrophages. Yet, the direct role TNFR2-specific activation plays in the absence of XIAP is unknown. We found TNFR2-specific activation leads to cell death in xiap-/- myeloid cells, particularly in the absence of the RING domain. RIPK1 kinase activity downstream of TNFR2 resulted in a TNF/TNFR1 cell death, independent of necroptosis. TNFR2-specific activation leads to a similar inflammatory NF-kB driven transcriptional profile as TNFR1 activation with the exception of upregulation of NLRP3 and caspase-11. Activation and upregulation of the canonical inflammasome upon loss of XIAP was mediated by RIPK1 kinase activity and ROS production. While both the inhibition of RIPK1 kinase activity and ROS production reduced cell death, as well as release of IL-1ß, the release of IL-18 was not reduced to basal levels. This study supports targeting TNFR2 specifically to reduce IL-18 release in XLP-2 patients and to reduce priming of the inflammasome components.

SMAC mimetics promote NIK-dependent inhibition of CD4+ TH17 cell differentiation.

Second mitochondria-derived activator of caspase (SMAC) mimetics (SMs) are selective antagonists of the inhibitor of apoptosis proteins (IAPs), which activate noncanonical NF-κB signaling and promote tumor cell death. Through gene expression analysis, we found that treatment of CD4+ T cells with SMs during T helper 17 (TH17) cell differentiation disrupted the balance between two antagonistic transcription factor modules. Moreover, proteomics analysis revealed that SMs altered the abundance of proteins associated with cell cycle, mitochondrial activity, and the balance between canonical and noncanonical NF-κB signaling. Whereas SMs inhibited interleukin-17 (IL-17) production and ameliorated TH17 cell-driven inflammation, they stimulated IL-22 secretion. Mechanistically, SM-mediated activation of NF-κB-inducing kinase (NIK) and the transcription factors RelB and p52 directly suppressed Il17a expression and IL-17A protein production, as well as the expression of a number of other immune genes. Induction of IL-22 production correlated with the NIK-dependent reduction in cMAF protein abundance and the enhanced activity of the aryl hydrocarbon receptor. Last, SMs also increased IL-9 and IL-13 production and, under competing conditions, favored the differentiation of naïve CD4+ T cells into TH2 cells rather than TH17 cells. These results demonstrate that SMs shape the gene expression and protein profiles of TH17 cells and inhibit TH17 cell-driven autoimmunity.

Necroptosis Execution Is Mediated by Plasma Membrane Nanopores Independent of Calcium.

Necroptosis is a form of regulated necrosis that results in cell death and content release after plasma membrane permeabilization. However, little is known about the molecular events responsible for the disruption of the plasma membrane. Here, we find that early increase in cytosolic calcium in TNF-induced necroptosis is mediated by treatment with a Smac mimetic via the TNF/RIP1/TAK1 survival pathway. This does not require the activation of the necrosome and is dispensable for necroptosis. Necroptosis induced by the activation of TLR3/4 pathways does not trigger early calcium flux. We also demonstrate that necroptotic plasma membrane rupture is mediated by osmotic forces and membrane pores around 4 nm in diameter. This late permeabilization step represents a hallmark in necroptosis execution that is cell and treatment independent and requires the RIP1/RIP3/MLKL core. In support of this, treatment with osmoprotectants reduces cell damage in an in vivo necroptosis model of ischemia-reperfusion injury.

RIPK1/RIPK3 promotes vascular permeability to allow tumor cell extravasation independent of its necroptotic function.

Necroptosis is an inflammatory form of programmed cell death requiring receptor-interacting protein kinase 1, 3 (RIPK1, RIPK3) and mixed lineage kinase domain-like protein (MLKL). The kinase of RIPK3 phosphorylates MLKL causing MLKL to form a pore-like structure, allowing intracellular contents to release and cell death to occur. Alternatively, RIPK1 and RIPK3 have been shown to regulate cytokine production directly influencing inflammatory immune infiltrates. Recent data suggest that necroptosis may contribute to the malignant transformation of tumor cells in vivo and we asked whether necroptosis may have a role in the tumor microenvironment altering the ability of the tumor to grow or metastasize. To determine if necroptosis in the tumor microenvironment could promote inflammation alone or by initiating necroptosis and thereby influencing growth or metastasis of tumors, we utilized a syngeneic tumor model of metastasis. Loss of RIPK3 in the tumor microenvironment reduced the number of tumor nodules in the lung by 46%. Loss of the kinase activity in RIPK1, a member of the necrosome also reduced tumor nodules in the lung by 38%. However, the loss of kinase activity in RIPK3 or the loss of MLKL only marginally altered the ability of tumor cells to form in the lung. Using bone marrow chimeras, the decrease in tumor nodules in the Ripk3-/- appeared to be due to the stromal compartment rather than the hematopoietic compartment. Transmigration assays showed decreased ability of tumor cells to transmigrate through the vascular endothelial layer, which correlated with decreased permeability in the Ripk3-/- mice after tumor injection. In response to permeability factors, such as vascular endothelial growth factor, RIPK3 null endothelial cells showed decreased p38/HSP27 activation. Taken together, our results suggest an alternative function for RIPK1/RIPK3 in vascular permeability leading to decreased number of metastasis.

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.

TNF induced inhibition of Cirbp expression depends on RelB NF-κB signalling pathway.

The circadian clock is required for the rhythmic expression of a plethora of genes that orchestrate metabolism, sleep-wake behaviour and the immune response to pathogens. The cold-inducible RNA binding protein (CIRBP) is required for high amplitude expression of clock genes. Moreover, CIRBP protects the expression of clock genes from the inhibitory effects of tumour necrosis factor (TNF). However, since TNF represses Cirbp expression, the protective effect of CIRBP is lost. Here, we show that the TNF effect on Cirbp requires the non-canonical NF-κB signalling pathway. While a knock down of RelA does not alter the effects of TNF on Cirbp, a knock down of RelB represses this effect. In addition, the data indicate that p50 and p52 are required in the TNF induced inhibition of Cirbp. These results show that Cirbp expression in TNF treated cells is regulated via the non-canonical NF-κB pathway.

Targeting of Fn14 Prevents Cancer-Induced Cachexia and Prolongs Survival.

The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14, when expressed in tumors, causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor, rather than host, is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia, thereby extending lifespan and improving quality of life for cancer patients.

RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL.

RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3-caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1ß inflammatory responses independent of MLKL and necroptotic cell death.

Autoreactive T cells induce necrosis and not BCL-2-regulated or death receptor-mediated apoptosis or RIPK3-dependent necroptosis of transplanted islets in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: Type 1 diabetes results from T cell-mediated destruction of pancreatic beta cells. The mechanisms of beta cell destruction in vivo, however, remain unclear. We aimed to test the relative roles of the main cell death pathways: apoptosis, necrosis and necroptosis, in beta cell death in the development of CD4(+) T cell-mediated autoimmune diabetes. METHODS: We altered expression levels of critical cell death proteins in mouse islets and tested their ability to survive CD4(+) T cell-mediated attack using an in vivo graft model. RESULTS: Loss of the B cell leukaemia/lymphoma 2 (BCL-2) homology domain 3-only proteins BIM, PUMA or BID did not protect beta cells from this death. Overexpression of the anti-apoptotic protein BCL-2 or combined deficiency of the pro-apoptotic multi-BCL2 homology domain proteins BAX and BAK also failed to prevent beta cell destruction. Furthermore, loss of function of the death receptor Fas or its essential downstream signalling molecule Fas-associated death domain (FADD) in islets was also not protective. Using electron microscopy we observed that dying beta cells showed features of necrosis. However, islets deficient in receptor-interacting serine/threonine protein kinase 3 (RIPK3), a critical initiator of necroptosis, were still normally susceptible to CD4(+) T cell-mediated destruction. Remarkably, simultaneous inhibition of apoptosis and necroptosis by combining loss of RIPK3 and overexpression of BCL-2 in islets did not protect them against immune attack either. CONCLUSIONS/INTERPRETATION: Collectively, our data indicate that beta cells die by necrosis in autoimmune diabetes and that the programmed cell death pathways apoptosis and necroptosis are both dispensable for this process.

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.

Inhibitor of apoptosis proteins limit RIP3 kinase-dependent interleukin-1 activation.

Interleukin-1ß (IL-1ß) is a potent inflammatory cytokine that is usually cleaved and activated by inflammasome-associated caspase-1. To determine whether IL-1ß activation is regulated by inhibitor of apoptosis (IAP) proteins, we treated macrophages with an IAP-antagonist "Smac mimetic" compound or genetically deleted the genes that encode the three IAP family members cIAP1, cIAP2, and XIAP. After Toll-like receptor priming, IAP inhibition triggered cleavage of IL-1ß that was mediated not only by the NLRP3-caspase-1 inflammasome, but also by caspase-8 in a caspase-1-independent manner. In the absence of IAPs, rapid and full generation of active IL-1ß by the NLRP3-caspase-1 inflammasome, or by caspase-8, required the kinase RIP3 and reactive oxygen species production. These results demonstrate that activation of the cell death-inducing ripoptosome platform and RIP3 can generate bioactive IL-1ß and implicate them as additional targets for the treatment of pathological IL-1-driven inflammatory responses.

In TNF-stimulated cells, RIPK1 promotes cell survival by stabilizing TRAF2 and cIAP1, which limits induction of non-canonical NF-kappaB and activation of caspase-8.

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 overexpression 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. To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1(-/-) cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1(-/-) cells caused TRAF2 and cIAP1 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 cFLIP(L), and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.

Characterization of the apoptotic response of human leukemia cells to organosulfur compounds.

BACKGROUND: Novel therapeutic agents that selectively induce tumor cell death are urgently needed in the clinical management of cancers. Such agents would constitute effective adjuvant approaches to traditional chemotherapy regimens. Organosulfur compounds (OSCs), such as diallyl disulfide, have demonstrated anti-proliferative effects on cancer cells. We have previously shown that synthesized relatives of dysoxysulfone, a natural OSC derived from the Fijian medicinal plant, Dysoxylum richi, possess tumor-specific antiproliferative effects and are thus promising lead candidates. METHODS: Because our structure-activity analyses showed that regions flanking the disulfide bond mediated specificity, we synthesized 18 novel OSCs by structural modification of the most promising dysoxysulfone derivatives. These compounds were tested for anti-proliferative and apoptotic activity in both normal and leukemic cells. RESULTS: Six OSCs exhibited tumor-specific killing, having no effect on normal bone marrow, and are thus candidates for future toxicity studies. We then employed mRNA expression profiling to characterize the mechanisms by which different OSCs induce apoptosis. Using Gene Ontology analysis we show that each OSC altered a unique set of pathways, and that these differences could be partially rationalized from a transcription factor binding site analysis. For example, five compounds altered genes with a large enrichment of p53 binding sites in their promoter regions (p < 0.0001). CONCLUSIONS: Taken together, these data establish OSCs derivatized from dysoxysulfone as a novel group of compounds for development as anti-cancer agents.

Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment.

A role for cellular inhibitors of apoptosis (IAPs [cIAPs]) in preventing CD95 death has been suspected but not previously explained mechanistically. In this study, we find that the loss of cIAPs leads to a dramatic sensitization to CD95 ligand (CD95L) killing. Surprisingly, this form of cell death can only be blocked by a combination of RIP1 (receptor-interacting protein 1) kinase and caspase inhibitors. Consistently, we detect a large increase in RIP1 levels in the CD95 death-inducing signaling complex (DISC) and in a secondary cytoplasmic complex (complex II) in the presence of IAP antagonists and loss of RIP1-protected cells from CD95L/IAP antagonist-induced death. Cells resistant to CD95L/IAP antagonist treatment could be sensitized by short hairpin RNA-mediated knockdown of cellular FLICE-inhibitory protein (cFLIP). However, only cFLIP(L) and not cFLIP(S) interfered with RIP1 recruitment to the DISC and complex II and protected cells from death. These results demonstrate a fundamental role for RIP1 in CD95 signaling and provide support for a physiological role of caspase-independent death receptor-mediated cell death.

Another facet of ubiquitylation: death.

Ubiquitylation of caspase-8 by the Cullin3 E3 ligase allows its translocation to cytosolic aggregates in the cell by p62/sequestosome-1, increasing caspase-8 activation and thus leading to TRAIL-induced cell death.