Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Targeting DHX9 Triggers Tumor-Intrinsic Interferon Response and Replication Stress in Small Cell Lung Cancer.

Activating innate immunity in cancer cells through cytoplasmic nucleic acid sensing pathways, a phenomenon known as "viral mimicry," has emerged as an effective strategy to convert immunologically "cold" tumors into "hot." Through a curated CRISPR-based screen of RNA helicases, we identified DExD/H-box helicase 9 (DHX9) as a potent repressor of double-stranded RNA (dsRNA) in small cell lung cancers (SCLC). Depletion of DHX9 induced accumulation of cytoplasmic dsRNA and triggered tumor-intrinsic innate immunity. Intriguingly, ablating DHX9 also induced aberrant accumulation of R-loops, which resulted in an increase of DNA damage-derived cytoplasmic DNA and replication stress in SCLCs. In vivo, DHX9 deletion promoted a decrease in tumor growth while inducing a more immunogenic tumor microenvironment, invigorating responsiveness to immune-checkpoint blockade. These findings suggest that DHX9 is a crucial repressor of tumor-intrinsic innate immunity and replication stress, representing a promising target for SCLC and other "cold" tumors in which genomic instability contributes to pathology. SIGNIFICANCE: One promising strategy to trigger an immune response within tumors and enhance immunotherapy efficacy is by inducing endogenous "virus-mimetic" nucleic acid accumulation. Here, we identify DHX9 as a viral-mimicry-inducing factor involved in the suppression of double-stranded RNAs and R-loops and propose DHX9 as a novel target to enhance antitumor immunity. See related commentary by Chiappinelli, p. 389. This article is featured in Selected Articles from This Issue, p. 384.

Z-form nucleic acid-binding protein 1 (ZBP1) as a sensor of viral and cellular Z-RNAs: walking the razor's edge.

Z-form nucleic acid-binding protein 1 (ZBP1) detects viral Z-form RNAs (Z-RNAs), activates receptor-interacting protein kinase 3, and triggers cell death during both RNA and DNA virus infections. Such cell death promotes virus clearance by eliminating infected cells and galvanizing antiviral immunity, and is thus often targeted for evasion by virus-encoded suppressors. Recent evidence demonstrates that ZBP1 can also be activated by cellular Z-RNAs transcribed from endogenous retroelements within mammalian genomes. These cellular Z-RNAs, if not edited and neutralized by adenosine deaminase RNA-specific 1, trigger ZBP1-dependent cell death and inflammation, which may drive disease in Aicardi-Goutière's syndrome and related interferonopathies. Thus, while well-controlled activation of ZBP1 by viral Z-RNAs during infections is beneficial, the same pathway can have harmful consequences when inappropriately triggered by cellular Z-RNAs in other disease settings.

Apoptotic cell death in disease-Current understanding of the NCCD 2023.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

Detecting Z-RNA and Z-DNA in Mammalian Cells.

Eukaryotic cells sense and respond to virus infections by detecting conserved virus-generated molecular structures, called pathogen-associated molecular patterns (PAMPs). PAMPs are usually produced by replicating viruses, but not typically seen in uninfected cells. Double-stranded RNA (dsRNA) is a common PAMP produced by most, if not all, RNA viruses, as well as by many DNA viruses. DsRNA can adopt either the right-handed (A-RNA) or the left-handed (Z-RNA) double-helical conformation. A-RNA is sensed by cytosolic pattern recognition receptors (PRRs) such as RIG-1-like receptor MDA-5 and the dsRNA-dependent protein kinase PKR. Z-RNA is detected by Zα domain containing PRRs, including Z-form nucleic acid binding protein 1 (ZBP1) and the p150 subunit of adenosine deaminase RNA specific 1 (ADAR1). We have recently shown that Z-RNA is generated during orthomyxovirus (e.g., influenza A virus) infections and serves as activating ligand for ZBP1. In this chapter, we describe our procedure for detecting Z-RNA in influenza A virus (IAV)-infected cells. We also outline how this procedure can be used to detect Z-RNA produced during vaccinia virus infection, as well as Z-DNA induced by a small-molecule DNA intercalator.

TET1 and TDG Suppress Inflammatory Response in Intestinal Tumorigenesis: Implications for Colorectal Tumors With the CpG Island Methylator Phenotype.

BACKGROUND & AIMS: Aberrant DNA methylation is frequent in colorectal cancer (CRC), but underlying mechanisms and pathologic consequences are poorly understood. METHODS: We disrupted active DNA demethylation genes Tet1 and/or Tdg from ApcMin mice and characterized the methylome and transcriptome of colonic adenomas. Data were compared to human colonic adenocarcinomas (COAD) in The Cancer Genome Atlas. RESULTS: There were increased numbers of small intestinal adenomas in ApcMin mice expressing the TdgN151A allele, whereas Tet1-deficient and Tet1/TdgN151A-double heterozygous ApcMin colonic adenomas were larger with features of erosion and invasion. We detected reduction in global DNA hypomethylation in colonic adenomas from Tet1- and Tdg-mutant ApcMin mice and hypermethylation of CpG islands in Tet1-mutant ApcMin adenomas. Up-regulation of inflammatory, immune, and interferon response genes was present in Tet1- and Tdg-mutant colonic adenomas compared to control ApcMin adenomas. This up-regulation was also seen in murine colonic organoids and human CRC lines infected with lentiviruses expressing TET1 or TDG short hairpin RNA. A 127-gene inflammatory signature separated colonic adenocarcinomas into 4 groups, closely aligned with their microsatellite or chromosomal instability and characterized by different levels of DNA methylation and DNMT1 expression that anticorrelated with TET1 expression. Tumors with the CpG island methylator phenotype (CIMP) had concerted high DNMT1/low TET1 expression. TET1 or TDG knockdown in CRC lines enhanced killing by natural killer cells. CONCLUSIONS: Our findings reveal a novel epigenetic regulation, linked to the type of genomic instability, by which TET1/TDG-mediated DNA demethylation decreases methylation levels and inflammatory/interferon/immune responses. CIMP in CRC is triggered by an imbalance of methylating activities over demethylating activities. These mice represent a model of CIMP CRC.

Viral immunity: Basic mechanisms and therapeutic applications-a Keystone Symposia report.

Viruses infect millions of people each year. Both endemic viruses circulating throughout the population as well as novel epidemic and pandemic viruses pose ongoing threats to global public health. Developing more effective tools to address viruses requires not only in-depth knowledge of the virus itself but also of our immune system's response to infection. On June 29 to July 2, 2022, researchers met for the Keystone symposium "Viral Immunity: Basic Mechanisms and Therapeutic Applications." This report presents concise summaries from several of the symposium presenters.

Z-DNA enhances immunotherapy by triggering death of inflammatory cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are a heterogeneous population of cells. At one end of the spectrum are alpha-smooth muscle actin expressing myoCAFs (myofibroblast CAFs) and at the other end are the interferon (IFN) and Janus Kinase/Signal Transducer and Activator of Transcription responsive iCAFs (inflammatory CAFs). Both types of CAFs promote tumor growth. While myoCAFs foster immune exclusion and limit tumor spread, iCAFs create a highly immunosuppressive environment and foster the seeding of distant metastases. However, iCAFs also represent a tumor vulnerability. They are competent to undergo necroptosis, a highly immunogenic form of cell death that is triggered when Z-DNA or Z-RNA (collectively called ZNA) is sensed by the IFN-induced ZNA binding protein 1 (ZBP1). The sequestering of ZNA ligands by the p150 isoform of the double-stranded RNA-specific deaminase ADAR1 protects iCAFs from cell death. ZBP1-dependent necroptosis in iCAFs can be triggered by administering an orally available small molecule that generates sufficient amounts of ZNA to bypass ADAR1 inhibition. The therapeutic approach of targeting Z-prone sequences (called flipons) is agnostic to the mutations driving cancer progression. By exploiting the tumor vulnerability posed by expression of ZBP1-dependent immunogenic cell death pathways in iCAFs, flipon therapeutics offer new hope for improved clinical outcomes.

Caspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis.

The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)-dependent necroptosis in vivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8- or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL (MlklFLAG/FLAG), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. Casp8-/-MlklFLAG/FLAG mice were viable and displayed phosphorylated MLKL in a variety of tissues, together with dramatically increased expression of ZBP1 compared to Casp8+/+ mice. Studies in vitro revealed an increased expression of ZBP1 in cells lacking FADD or Caspase-8, which was suppressed by reconstitution of Caspase-8 or FADD. Ablation of ZBP1 in Casp8-/-MlklFLAG/FLAG mice suppressed spontaneous MLKL phosphorylation in vivo. ZBP1 expression and downstream activation of RIPK3 and MLKL in cells lacking Caspase-8 or FADD relied on a positive feedback mechanism requiring the nucleic acid sensors cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and TBK1 signaling pathways. Our study identifies a molecular mechanism whereby Caspase-8 and FADD suppress spontaneous necroptotic cell death.

Reovirus Activated Cell Death Pathways.

Mammalian orthoreoviruses (ReoV) are non-enveloped viruses with segmented double-stranded RNA genomes. In humans, ReoV are generally considered non-pathogenic, although members of this family have been proven to cause mild gastroenteritis in young children and may contribute to the development of inflammatory conditions, including Celiac disease. Because of its low pathogenic potential and its ability to efficiently infect and kill transformed cells, the ReoV strain Type 3 Dearing (T3D) is clinical trials as an oncolytic agent. ReoV manifests its oncolytic effects in large part by infecting tumor cells and activating programmed cell death pathways (PCDs). It was previously believed that apoptosis was the dominant PCD pathway triggered by ReoV infection. However, new studies suggest that ReoV also activates other PCD pathways, such as autophagy, pyroptosis, and necroptosis. Necroptosis is a caspase-independent form of PCD reliant on receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and its substrate, the pseudokinase mixed-lineage kinase domain-like protein (MLKL). As necroptosis is highly inflammatory, ReoV-induced necroptosis may contribute to the oncolytic potential of this virus, not only by promoting necrotic lysis of the infected cell, but also by inflaming the surrounding tumor microenvironment and provoking beneficial anti-tumor immune responses. In this review, we summarize our current understanding of the ReoV replication cycle, the known and potential mechanisms by which ReoV induces PCD, and discuss the consequences of non-apoptotic cell death-particularly necroptosis-to ReoV pathogenesis and oncolysis.

ZBP1 promotes inflammatory responses downstream of TLR3/TLR4 via timely delivery of RIPK1 to TRIF.

ZBP1 is widely recognized as a mediator of cell death for its role in initiating necroptotic, apoptotic, and pyroptotic cell death pathways in response to diverse pathogenic infection. Herein, we characterize an unanticipated role for ZBP1 in promoting inflammatory responses to bacterial lipopolysaccharide (LPS) or double-stranded RNA (dsRNA). In response to both stimuli, ZBP1 promotes the timely delivery of RIPK1 to the Toll-like receptor (TLR)3/4 adaptor TRIF and M1-ubiquitination of RIPK1, which sustains activation of inflammatory signaling cascades downstream of RIPK1. Strikingly, ZBP1-mediated regulation of these pathways is important in vivo, as Zbp1−/− mice exhibited resistance to LPS-induced septic shock, revealed by prolonged survival and delayed onset of hypothermia due to decreased inflammatory responses and subsequent cell death. Further findings revealed that ZBP1 promotes sustained inflammatory responses by mediating the kinetics of proinflammatory "TRIFosome" complex formation, thus having a profound impact downstream of TLR activation. Given the well-characterized role of ZBP1 as a viral sensor, our results exemplify previously unappreciated crosstalk between the pathways that regulate host responses to bacteria and viruses, with ZBP1 acting as a crucial bridge between the two.

ADAR1 masks the cancer immunotherapeutic promise of ZBP1-driven necroptosis.

Only a small proportion of patients with cancer show lasting responses to immune checkpoint blockade (ICB)-based monotherapies. The RNA-editing enzyme ADAR1 is an emerging determinant of resistance to ICB therapy and prevents ICB responsiveness by repressing immunogenic double-stranded RNAs (dsRNAs), such as those arising from the dysregulated expression of endogenous retroviral elements (EREs)1-4. These dsRNAs trigger an interferon-dependent antitumour response by activating A-form dsRNA (A-RNA)-sensing proteins such as MDA-5 and PKR5. Here we show that ADAR1 also prevents the accrual of endogenous Z-form dsRNA elements (Z-RNAs), which were enriched in the 3' untranslated regions of interferon-stimulated mRNAs. Depletion or mutation of ADAR1 resulted in Z-RNA accumulation and activation of the Z-RNA sensor ZBP1, which culminated in RIPK3-mediated necroptosis. As no clinically viable ADAR1 inhibitors currently exist, we searched for a compound that can override the requirement for ADAR1 inhibition and directly activate ZBP1. We identified a small molecule, the curaxin CBL0137, which potently activates ZBP1 by triggering Z-DNA formation in cells. CBL0137 induced ZBP1-dependent necroptosis in cancer-associated fibroblasts and reversed ICB unresponsiveness in mouse models of melanoma. Collectively, these results demonstrate that ADAR1 represses endogenous Z-RNAs and identifies ZBP1-mediated necroptosis as a new determinant of tumour immunogenicity masked by ADAR1. Therapeutic activation of ZBP1-induced necroptosis provides a readily translatable avenue for rekindling the immune responsiveness of ICB-resistant human cancers.

Loss of Ribosomal Protein Paralog Rpl22-like1 Blocks Lymphoid Development without Affecting Protein Synthesis.

Ribosomal proteins are thought to primarily facilitate biogenesis of the ribosome and its ability to synthesize protein. However, in this study, we show that Rpl22-like1 (Rpl22l1) regulates hematopoiesis without affecting ribosome biogenesis or bulk protein synthesis. Conditional loss of murine Rpl22l1 using stage or lineage-restricted Cre drivers impairs development of several hematopoietic lineages. Specifically, Tie2-Cre-mediated ablation of Rpl22l1 in hemogenic endothelium impairs the emergence of embryonic hematopoietic stem cells. Ablation of Rpl22l1 in late fetal liver progenitors impairs the development of B lineage progenitors at the pre-B stage and development of T cells at the CD44-CD25+ double-negative stage. In vivo labeling with O-propargyl-puromycin revealed that protein synthesis at the stages of arrest was not altered, indicating that the ribosome biogenesis and function were not generally compromised. The developmental arrest was associated with p53 activation, suggesting that the arrest may be p53-dependent. Indeed, development of both B and T lymphocytes was rescued by p53 deficiency. p53 induction was not accompanied by DNA damage as indicated by phospho-γH2AX induction or endoplasmic reticulum stress, as measured by phosphorylation of EIF2α, thereby excluding the known likely p53 inducers as causal. Finally, the developmental arrest of T cells was not rescued by elimination of the Rpl22l1 paralog, Rpl22, as we had previously found for the emergence of hematopoietic stem cells. This indicates that Rpl22 and Rpl22l1 play distinct and essential roles in supporting B and T cell development.

Viral Z-RNA triggers ZBP1-dependent cell death.

Z-DNA Binding protein 1 (ZBP1) activates Receptor Interacting Protein Kinase 3 (RIPK3) -dependent cell death during lytic infection by members of the orthomyxovirus, herpesvirus and poxvirus families. ZBP1 possesses two Zα domains capable of selective binding to Z-DNA, as well as to Z-RNA. We have now unveiled Z-RNA as the ligand that activates ZBP1 in cells infected with orthomyxoviruses (influenza A and B viruses) and the poxvirus vaccinia virus (VACV). Orthomyxovirus Z-RNA is sensed by ZBP1 in the nucleus of infected cells, resulting in nuclear activation of RIPK3, consequent rupture of the nucleus, and hyper-inflammatory 'nuclear necroptosis'. VACV-generated Z-RNA accumulates in the cytoplasm, where it is sequestered from ZBP1 by E3, the viral E3L gene product. In viruses where the E3 Zα domain has been mutated, ZBP1 senses Z-RNA and triggers RIPK3-dependent necroptosis in the cytoplasm. Z-RNA is thus a new viral pathogen-associated molecular pattern (PAMP).

Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis.

Necroptosis mediated by Z-nucleic-acid-binding protein (ZBP)1 (also called DAI or DLM1) contributes to innate host defense against viruses by triggering cell death to eliminate infected cells. During infection, vaccinia virus (VACV) protein E3 prevents death signaling by competing for Z-form RNA through an N-terminal Zα domain. In the absence of this E3 domain, Z-form RNA accumulates during the early phase of VACV infection, triggering ZBP1 to recruit receptor interacting protein kinase (RIPK)3 and execute necroptosis. The C-terminal E3 double-strand RNA-binding domain must be retained to observe accumulation of Z-form RNA and induction of necroptosis. Substitutions of Zα from either ZBP1 or the RNA-editing enzyme double-stranded RNA adenosine deaminase (ADAR)1 yields fully functional E3 capable of suppressing virus-induced necroptosis. Overall, our evidence reveals the importance of Z-form RNA generated during VACV infection as a pathogen-associated molecular pattern (PAMP) unleashing ZBP1/RIPK3/MLKL-dependent necroptosis unless suppressed by viral E3.

Netrin G1 Promotes Pancreatic Tumorigenesis through Cancer-Associated Fibroblast-Driven Nutritional Support and Immunosuppression.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell-mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SIGNIFICANCE: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.See related commentary by Sherman, p. 230.This article is highlighted in the In This Issue feature, p. 211.

Cell-specific activation of RIPK1 and MLKL after intracerebral hemorrhage in mice.

Receptor-interacting protein kinase-1 (RIPK1) is a master regulator of cell death and inflammation, and mediates programmed necrosis (necroptosis) via mixed-lineage kinase like (MLKL) protein. Prior studies in experimental intracerebral hemorrhage (ICH) implicated RIPK1 in the pathogenesis of neuronal death and cognitive outcome, but the relevant cell types involved and potential role of necroptosis remain unexplored. In mice subjected to autologous blood ICH, early RIPK1 activation was observed in neurons, endothelium and pericytes, but not in astrocytes. MLKL activation was detected in astrocytes and neurons but not endothelium or pericytes. Compared with WT controls, RIPK1 kinase-dead (RIPK1D138N/D138N) mice had reduced brain edema (24 h) and blood-brain barrier (BBB) permeability (24 h, 30 d), and improved postinjury rotarod performance. Mice deficient in MLKL (Mlkl-/-) had reduced neuronal death (24 h) and BBB permeability at 24 h but not 30d, and improved post-injury rotarod performance vs. WT. The data support a central role for RIPK1 in the pathogenesis of ICH, including cell death, edema, BBB permeability, and motor deficits. These effects may be mediated in part through the activation of MLKL-dependent necroptosis in neurons. The data support development of RIPK1 kinase inhibitors as therapeutic agents for human ICH.

Somatic Epigenetic Silencing of RIPK3 Inactivates Necroptosis and Contributes to Chemoresistance in Malignant Mesothelioma.

PURPOSE: Receptor-interacting protein kinase 3 (RIPK3) phosphorylates effector molecule MLKL to trigger necroptosis. Although RIPK3 loss is seen in several human cancers, its role in malignant mesothelioma is unknown. This study aimed to determine whether RIPK3 functions as a potential tumor suppressor to limit development of malignant mesothelioma. EXPERIMENTAL DESIGN: RIPK3 expression was examined in 66 malignant mesothelioma tumors and cell lines. Promoter methylation and DNMT1 siRNA studies were performed to assess the mode of RIPK3 silencing in RIPK3-deficient malignant mesothelioma cells. Restoration of RIPK3 expression in RIPK3-negative malignant mesothelioma cells, either by treatment with 5-aza-2'-deoxycytidine or lentiviral expression of cDNA, was performed to assess effects on cell viability, necrosis, and chemosensitization. RESULTS: Loss of RIPK3 expression was observed in 42/66 (63%) primary malignant mesotheliomas and malignant mesothelioma cell lines, and RT-PCR analysis demonstrated that downregulation occurs at the transcriptional level, consistent with epigenetic silencing. RIPK3-negative malignant mesothelioma cells treated with 5-aza-2'-deoxycytidine resulted in reexpression of RIPK3 and chemosensitization. Ectopic expression of RIPK3 also resulted in chemosensitization and led to necroptosis, the latter demonstrated by phosphorylation of downstream target MLKL and confirmed by rescue experiments. Mining of RIPK3 expression and survival outcomes among patients with malignant mesothelioma available from The Cancer Genome Atlas repository revealed that promoter methylation of RIPK3 is associated with reduced RIPK3 expression and poor prognosis. CONCLUSIONS: These data suggest that RIPK3 acts as a tumor suppressor in malignant mesothelioma by triggering necroptosis and that epigenetic silencing of RIPK3 by DNA methylation impairs necroptosis and contributes to chemoresistance and poor survival in this incurable disease.