Zα Domain Research Articles

Thermodynamic analysis of Zα domain-nucleic acid interactions.

DNA/RNA molecules adopting the left-handed conformation (Z-form) have been attributed with immunogenic properties. However, their biological role and importance have been a topic of debate for many years. The discovery of Z-DNA/RNA binding domains (Zα domains) in varied proteins that are involved in the innate immune response, such as the interferon inducible form of the RNA editing enzyme ADAR1 (p150), Z-DNA binding protein 1 (ZBP1), the fish kinase PKZ and the poxvirus inhibitor of interferon response E3L, indicates important roles of Z-DNA/RNA in immunity and self/non-self-discrimination. Such Zα domain-containing proteins recognize left-handed Z-DNA/RNA in a conformation-specific manner. Recent studies have implicated these domains in virus recognition. Given these important emerging roles for the Zα domains, it is pivotal to understand the mechanism of recognition of the Z-DNA/Z-RNA by these domains. To this end, we assessed the binding thermodynamics of Zα domain from ORF112 and ADAR1 on T(CG)3 and T(CG)6 oligonucleotides which have high propensity to adopt the Z-conformation. Our study highlights important differences in the mode of oligonucleotide binding by the two Zα domains originating from different proteins. Site-directed mutagenesis was employed together with isothermal titration calorimetry to tease apart finer details of the binding thermodynamics. Our work advances the understanding on binding thermodynamics of Zα domains to their cognate nucleic acid substrates and paves the ground for future efforts to gain a complete appreciation of this process.

ADAR1 averts fatal type I interferon induction by ZBP1

Mutations of the ADAR1 gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi–Goutières syndrome and bilateral striatal necrosis1–3. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA4,5. Hemizygous ADAR1 mutations in the Zα domain cause type I IFN-mediated pathologies in humans2,3 and mice6–8; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains9, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1mZα/– mice). Adar1mZα/– mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology in Adar1mZα/– mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused by ADAR1 mutations.

ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3-6, and mutations similar to those found in patients with AGS cause autoinflammation7-12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7-10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient inthe expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.

ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), continues to cause significant morbidity and mortality in the ongoing global pandemic. Understanding the fundamental mechanisms that govern innate immune and inflammatory responses during SARS-CoV-2 infection is critical for developing effective therapeutic strategies. While IFN-based therapies are generally expected to be beneficial during viral infection, clinical trials in COVID-19 have shown limited efficacy and potential detrimental effects of IFN treatment during SARS-CoV-2 infection. However, the underlying mechanisms responsible for this failure remain unknown. In this study, we found that IFN induced ZBP1-mediated inflammatory cell death, PANoptosis, in human and murine macrophages and in the lungs of mice infected with β-coronaviruses, including SARS-CoV-2 and mouse hepatitis virus (MHV). In patients with COVID-19, expression of the innate immune sensor ZBP1 was increased in immune cells from those who succumbed to the disease compared with those who recovered, further suggesting a link between ZBP1 and pathology. In mice, IFN-β treatment following β-coronavirus infection increased lethality, and genetic deletion of Zbp1 or its Zα domain suppressed cell death and protected the mice from IFN-mediated lethality during β-coronavirus infection. Overall, our results identify that ZBP1 induced during coronavirus infection limits the efficacy of IFN therapy by driving inflammatory cell death and lethality. Therefore, inhibiting ZBP1 activity may improve the efficacy of IFN therapy, paving the way for the development of new and critically needed therapeutics for COVID-19 as well as other infections and inflammatory conditions where IFN-mediated cell death and pathology occur.

Searching for New Z-DNA/Z-RNA Binding Proteins Based on Structural Similarity to Experimentally Validated Zα Domain.

Z-DNA and Z-RNA are functionally important left-handed structures of nucleic acids, which play a significant role in several molecular and biological processes including DNA replication, gene expression regulation and viral nucleic acid sensing. Most proteins that have been proven to interact with Z-DNA/Z-RNA contain the so-called Zα domain, which is structurally well conserved. To date, only eight proteins with Zα domain have been described within a few organisms (including human, mouse, Danio rerio, Trypanosoma brucei and some viruses). Therefore, this paper aimed to search for new Z-DNA/Z-RNA binding proteins in the complete PDB structures database and from the AlphaFold2 protein models. A structure-based similarity search found 14 proteins with highly similar Zα domain structure in experimentally-defined proteins and 185 proteins with a putative Zα domain using the AlphaFold2 models. Structure-based alignment and molecular docking confirmed high functional conservation of amino acids involved in Z-DNA/Z-RNA, suggesting that Z-DNA/Z-RNA recognition may play an important role in a variety of cellular processes.

Deciphering the Biological Significance of ADAR1-Z-RNA Interactions.

Adenosine deaminase acting on RNA 1 (ADAR1) is an enzyme responsible for double-stranded RNA (dsRNA)-specific adenosine-to-inosine RNA editing, which is estimated to occur at over 100 million sites in humans. ADAR1 is composed of two isoforms transcribed from different promoters: p150 and N-terminal truncated p110. Deletion of ADAR1 p150 in mice activates melanoma differentiation-associated protein 5 (MDA5)-sensing pathway, which recognizes endogenous unedited RNA as non-self. In contrast, we have recently demonstrated that ADAR1 p110-mediated RNA editing does not contribute to this function, implying that a unique Z-DNA/RNA-binding domain α (Zα) in the N terminus of ADAR1 p150 provides specific RNA editing, which is critical for preventing MDA5 activation. In addition, a mutation in the Zα domain is identified in patients with Aicardi–Goutières syndrome (AGS), an inherited encephalopathy characterized by overproduction of type I interferon. Accordingly, we and other groups have recently demonstrated that Adar1 Zα-mutated mice show MDA5-dependent type I interferon responses. Furthermore, one such mutant mouse carrying a W197A point mutation in the Zα domain, which inhibits Z-RNA binding, manifests AGS-like encephalopathy. These findings collectively suggest that Z-RNA binding by ADAR1 p150 is essential for proper RNA editing at certain sites, preventing aberrant MDA5 activation.

Conformational exchange of the Zα domain of human RNA editing enzyme ADAR1 studied by NMR spectroscopy

Z-DNA binding proteins (ZBPs) play important roles in RNA editing, innate immune responses, and viral infections. Numerous studies have implicated a role for conformational motions during ZBPs binding upon DNA, but the quantitative intrinsic conformational exchanges of ZBP have not been elucidated. To understand the correlation between the biological function and dynamic feature of the Zα domains of human ADAR1 (hZαADAR1), we have performed the 15N backbone amide Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments on the free hZαADAR1 at two different magnetic fields at 35 °C. The robust inter-dependence of parameters in the global fitting process using multi-magnetic field CPMG profiles allows us characterizing the dynamic properties of conformational changes in hZαADAR1. This study found that free hZαADAR1 exhibited the conformational exchange with a kex of 5784 s−1 between the states “A” (89% population) and “B” (11% population). The different hydrophobic interactions among helices α1, α2, and α3 between these two states might correlate with efficient Z-DNA binding achieved by the hydrogen bonding interactions between its side-chains and the phosphate backbone of Z-DNA.

Adenosine-to-inosine editing of endogenous Z-form RNA by the deaminase ADAR1 prevents spontaneous MAVS-dependent type I interferon responses

SummaryNucleic acids are powerful triggers of innate immunity and can adopt the Z-conformation, an unusual left-handed double helix. Here, we studied the biological function(s) of Z-RNA recognition by the adenosine deaminase ADAR1, mutations in which cause Aicardi-Goutières syndrome. Adar1mZα/mZα mice, bearing two point mutations in the Z-nucleic acid binding (Zα) domain that abolish Z-RNA binding, displayed spontaneous induction of type I interferons (IFNs) in multiple organs, including in the lung, where both stromal and hematopoietic cells showed IFN-stimulated gene (ISG) induction. Lung neutrophils expressed ISGs induced by the transcription factor IRF3, indicating an initiating role for neutrophils in this IFN response. The IFN response in Adar1mZα/mZα mice required the adaptor MAVS, implicating cytosolic RNA sensing. Adenosine-to-inosine changes were enriched in transposable elements and revealed a specific requirement of ADAR1’s Zα domain in editing of a subset of RNAs. Thus, endogenous RNAs in Z-conformation have immunostimulatory potential curtailed by ADAR1, with relevance to autoinflammatory disease in humans.

ADAR1 interaction with Z-RNA promotes editing of endogenous double-stranded RNA and prevents MDA5-dependent immune activation.

Loss of function of adenosine deaminase acting on double-stranded RNA (dsRNA)-1 (ADAR1) causes the severe autoinflammatory disease Aicardi-Goutières syndrome (AGS). ADAR1 converts adenosines into inosines within dsRNA. This process called A-to-I editing masks self-dsRNA from detection by the antiviral dsRNA sensor MDA5. ADAR1 binds to dsRNA in both the canonical A-form and the poorly defined Z conformation (Z-RNA). Mutations in the Z-RNA-binding Zα domain of ADAR1 are common in patients with AGS. How loss of ADAR1/Z-RNA interaction contributes to disease development is unknown. We demonstrate that abrogated binding of ADAR1 to Z-RNA leads to reduced A-to-I editing of dsRNA structures formed by base pairing of inversely oriented short interspersed nuclear elements. Preventing ADAR1 binding to Z-RNA triggers an MDA5/MAVS-mediated type I interferon response and leads to the development of lethal autoinflammation in mice. This shows that the interaction between ADAR1 and Z-RNA restricts sensing of self-dsRNA and prevents AGS development.

High-throughput screening to identify potential inhibitors of the Zα domain of the adenosine deaminase 1 (ADAR1).

Adenosine deaminases acting on RNA 1 (ADAR1) are enzymes involved in editing adenosine to inosine in the dsRNAs of cells associated with cancer development. The p150 isoform of ADAR1 is the only isoform containing the Zα domain that binds to both Z-DNA and Z-RNA. The Zα domain is suggested to modulate the immune response and could be a suitable target for antiviral treatment and cancer immunotherapy. In this study, we aimed to identify potential inhibitors for ADAR1 protein that bind the Zα domain using molecular docking and simulation tools. Virtual docking and molecular dynamics simulation approaches were used to screen the potential activity of 2115 FDA-approved compounds on the Zα domain of ADAR1 and filtered for to obtain the top-scoring hits. The top three compounds with the best XP Gscore—namely alendronate (−7.045), etidronate (−6.923), and zoledronate (−6.77)—were subjected to 50 ns simulations to characterize complex stability and identify the fundamental interactions that contribute to inhibition of the ADAR1 Zα domain. The three compounds were shown to interact with Lys169, Lys170, Asn173, and Tyr177 of the Zα domain-like helical backbone of Z-RNA. The study provides a comprehensive and novel insights of repurposes drugs for the inhibition of ADAR1 function.

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.

Enrichment of Zα domains at cytoplasmic stress granules is due to their innate ability to bind to nucleic acids.

Zα domains recognize the left-handed helical Z conformation of double-stranded nucleic acids. They are found in proteins involved in the nucleic acid sensory pathway of the vertebrate innate immune system and host evasion by viral pathogens. Previously, it has been demonstrated that ADAR1 (encoded by ADAR in humans) and DAI (also known as ZBP1) localize to cytoplasmic stress granules (SGs), and this localization is mediated by their Zα domains. To investigate the mechanism, we determined the interactions and localization pattern for the N-terminal region of human DAI (ZαβDAI), which harbours two Zα domains, and for a ZαβDAI mutant deficient in nucleic acid binding. Electrophoretic mobility shift assays demonstrated the ability of ZαβDAI to bind to hyperedited nucleic acids, which are enriched in SGs. Furthermore, using immunofluorescence and immunoprecipitation coupled with mass spectrometry, we identified several interacting partners of the ZαβDAI-RNA complex in vivo under conditions of arsenite-induced stress. These interactions are lost upon loss of nucleic acid-binding ability or upon RNase treatment. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to SGs is mainly mediated by the nucleic acid-binding ability of their Zα domains. This article has an associated First Person interview with Bharath Srinivasan, joint first author of the paper.

First person – Bharath Srinivasan

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Bharath Srinivasan is co-first author on ‘ Enrichment of Zα domains at cytoplasmic stress granules is due to their innate ability to bind to nucleic acids’, published in JCS. Bharath conducted the research described in this article while a Marie Sklodowska-Curie Individual Fellow in Alekos Athanasiadis's lab at the Instituto Gulbenkian de Ciência, Oeiras, Portugal. He is now a senior research scientist at AstraZeneca in Cambridge, UK, investigating mechanistic biology and enzymology.

Systematic Approach to Find the Global Minimum of Relaxation Dispersion Data for Protein-Induced B-Z Transition of DNA.

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in μs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZαADAR1)–DNA complex. The global search results indicated that a α-helical segment of hZαADAR1 provided the main contribution to the three-state conformational changes of a hZαADAR1—DNA complex with a slow B–Z exchange process. The two global exchange rate constants, kex and kZB, were found to be 844 and 9.8 s−1, respectively, in agreement with two regimes of residue-dependent chemical shift differences—the “dominant oscillatory regime” and “semi-oscillatory regime”. We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.

Solution NMR backbone assignments of the N-terminal Zα-linker-Zβ segment from Homo sapiens ADAR1p150.

Adenosine-to-inosine (A-to-I) editing of a subset of RNAs in a eukaryotic cell is required in order to avoid triggering the innate immune system. Editing is carried out by ADAR1, which exists as short (p110) and long (p150) isoforms. ADAR1p150 is mostly cytoplasmic, possesses a Z-RNA binding domain (Zα), and is only expressed during the innate immune response. A structurally homologous domain to Zα, the Zβ domain, is separated by a long linker from Zα on the N-terminus of ADAR1 but its function remains unknown. Zβ does not bind to RNA in isolation, yet the binding kinetics of the segment encompassing Zα, Zβ and the 95-residue linker between the two domains (Zα-Zβ) are markedly different compared to Zα alone. Here we present the solution NMR backbone assignment of Zα-Zβ from H. Sapiens ADAR1. The predicted secondary structure of Zα-Zβ based on chemical shifts is in agreement with previously determined structures of Zα and Zβ in isolation, and indicates that the linker is intrinsically disordered. Comparison of the chemical shifts between the individual Zα and Zβ domains to the full Zα-Zβ construct suggests that Zβ may interact with the linker, the function of which is currently unknown.

Dual conformational recognition by Z-DNA binding protein is important for the B-Z transition process.

Left-handed Z-DNA is radically different from the most common right-handed B-DNA and can be stabilized by interactions with the Zα domain, which is found in a group of proteins, such as human ADAR1 and viral E3L proteins. It is well-known that most Zα domains bind to Z-DNA in a conformation-specific manner and induce rapid B–Z transition in physiological conditions. Although many structural and biochemical studies have identified the detailed interactions between the Zα domain and Z-DNA, little is known about the molecular basis of the B–Z transition process. In this study, we successfully converted the B–Z transition-defective Zα domain, vvZαE3L, into a B–Z converter by improving B-DNA binding ability, suggesting that B-DNA binding is involved in the B–Z transition. In addition, we engineered the canonical B-DNA binding protein GH5 into a Zα-like protein having both Z-DNA binding and B–Z transition activities by introducing Z-DNA interacting residues. Crystal structures of these mutants of vvZαE3L and GH5 complexed with Z-DNA confirmed the significance of conserved Z-DNA binding interactions. Altogether, our results provide molecular insight into how Zα domains obtain unusual conformational specificity and induce the B–Z transition.

Protein-induced B-Z transition of DNA duplex containing a 2′-OMe guanosine

Structural transformation of the canonical right-handed helix, B-DNA, to the non-canonical left-handed helix, Z-DNA, can be induced by the Zα domain of the human RNA editing enzyme ADAR1 (hZαADAR1). To characterize the site-specific preferences of binding and structural changes in DNA containing the 2′-O-methyl guanosine derivative (mG), titration of the imino proton spectra and chemical shift perturbations were performed on hZαADAR1 upon binding to Z-DNA. The structural transition between B–Z conformation as the changing ratio between DNA and protein showed a binding affinity of the modified DNA onto the Z-DNA binding protein similar to wild-type DNA or RNA. The chemical shift perturbation results showed that the overall structure and environment of the modified DNA revealed DNA-like properties rather than RNA-like characteristics. Moreover, we found evidence for two distinct regimes, “Z-DNA Sensing” and “Modification Sensing”, based on the site-specific chemical shift perturbation between the DNA (or RNA) binding complex and the modified DNA–hZαADAR1 complex. Thus, we propose that modification of the sugar backbone of DNA with 2′-O-methyl guanosine promotes the changes in the surrounding α3 helical structural segment as well as the non-perturbed feature of the β-hairpin region.

The Zα2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development

Z-DNA-binding protein 1 (ZBP1) is an innate immune sensor of nucleic acids that regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating receptor-interacting Ser/Thr kinase 3 (RIPK3), caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate immune sensors because of its N-terminal Zα1 and Zα2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Zα domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we generated Zbp1ΔZα2/ΔZα2 mice that express ZBP1 lacking the Zα2 domain and demonstrate that this domain is critical for influenza A virus-induced PANoptosis and underlies perinatal lethality in mice in which the RIP homotypic interaction motif domain of RIPK1 has been mutated (Ripk1mRHIM/mRHIM). Deletion of the Zα2 domain in ZBP1 abolished influenza A virus-induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Zα2 domain of ZBP1 was sufficient to rescue Ripk1mRHIM/mRHIM mice from perinatal lethality caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Zα2 domain of ZBP1 in several physiological functions and establish a link between Z-RNA sensing via the Zα2 domain and promotion of influenza-induced PANoptosis and perinatal lethality.

Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation.

Z-DNA and Z-RNA are left-handed double helix nucleic acid structures with poorly understood biological function1–3. Z-DNA binding protein 1 (ZBP1, also known as DAI or DLM-1) is a nucleic acid sensor containing two Zα domains that bind Z-DNA4,5 and Z-RNA6–8. ZBP1 mediates host-defence against certain viruses6,7,9–14 by sensing viral nucleic acids6,7,10. RIPK1 deficiency or mutation of its RIP homotypic interaction motif (RHIM) triggers ZBP1-dependent necroptosis and inflammation in mice15,16, however, the mechanisms inducing ZBP1 activation in the absence of viral infection remain elusive. Here we show that Zα-dependent sensing of endogenous ligands induces ZBP1-mediated perinatal lethality in mice expressing RIPK1 with mutated RHIM (Ripk1mR/mR) and skin inflammation in mice with epidermis-specific RIPK1 deficiency (RIPK1E-KO), as well as colitis in mice with intestinal epithelial-specific FADD deficiency (FADDIEC-KO). Consistently, functional Zα domains were required for ZBP1-induced necroptosis in fibroblasts that express RIPK1 with mutated RHIM or were treated with caspase inhibitors. Moreover, inhibition of nuclear export triggered Zα-dependent activation of RIPK3 in the nucleus resulting in cell death, suggesting that ZBP1 may recognise Z-form nucleic acids (Z-NA) in the nucleus. We found that ZBP1 constitutively bound cellular double stranded RNA (dsRNA) in a Zα-dependent manner. Furthermore, endogenous retroelement (ERE)-derived complementary reads were detected in epidermal RNA, suggesting that ERE-derived dsRNA may act as Zα domain ligand triggering ZBP1 activation. Collectively, our results provide evidence that sensing of endogenous Z-NA by ZBP1 triggers RIPK3-dependent necroptosis and inflammation, which could underlie the development of chronic inflammatory conditions particularly in patients with mutations in the RIPK1 and CASPASE-8 genes17–20.

Novel ZBP1-IRF3 Dependency in Multiple Myeloma Mediated By IRF3-Driven Regulation of Cell Cycle Genes

ZBP1 is an inducible nucleic acid (NA) sensor that is activated when pathogen NA bind to its Zα and Zβ domains. ZBP1 is required for TBK1-dependent phosphorylation of the transcription factor IRF3 (pIRF3) followed by its direct activation of type I interferon genes. However, the role, if any, of ZBP1 in tumour biology is not known. By searching for genes selectively expressed in multiple myeloma (MM) we identified ZBP1 mRNA expressed in 29 MM cell lines (MMCL) but not in >1000 other cancer cell lines (CCLE dataset); ZBP1 was expressed in all 766 patient myeloma PC (CoMMpass dataset) but not in normal blood cells (Blueprint) or 53 healthy tissues (GTex). We confirmed expression of ZBP1 mRNA and/or protein in MMCL, primary human and murine germinal centre B (GCB) and plasma cells (PC) as well as in myeloma PC. By inducing T cell-dependent humoral immune responses after ip alum-NP-KLH immunisation, we explored the role of selective and constitutive expression of Zbp1 in GCB to PC transition. We found no differences in the frequency of splenic GCB cells and PC between control WT and Zbp1-/- mice and in GCB cell frequency between immunised WT and Zbp1-/- mice. However, compared to WT, the increase in PC frequency in immunised Zbp1-/- mice was 50% lower (n=10/group, p<0.0001) commensurate with a 40% (n=6/group, p<0.01), lower increase in NP-KLH-specific IgG but not IgM levels in Zbp1-/- mice. These findings suggest that although Zbp1 is not required for GCB cell and PC development it is required for optimal, T cell-dependent humoral immune responses. To explore the function of ZBP1 in MM we depleted by 2 lentiviral shRNAs either isoform 1 (contains both Zα and Zβ domains; shRNA1) or both isoform 1 and isoform 2 (latter lacks Zα domain; shRNA2). Both shRNAs were toxic to all 5 MMCL tested suggesting that isoform 1 but not isoform 2 is essential for myeloma cell survival. This effect was specific because survival of K562 cells, which lack expression of ZBP1, was not affected by either shRNA and exogenous ZBP1 cDNA rescued cell death of ZBP1-depleted myeloma cells. Dox-induced ZBP1 depletion was toxic to MMCL in vitro and significantly inhibited myeloma cell growth in a subcutaneous NSG model of the MMCL H929 and MM.1S. Together, these findings reveal a novel myeloma cell-specific ZBP1 dependency. Transcriptome analysis of ZBP1-depleted H929 and MM.1S cells showed amongst the significantly downregulated genes enrichment for the cell cycle control and DNA repair pathways consistent with a critical role of ZBP1 in promoting myeloma cell proliferation. Flow-cytometric analysis of ZBP1-depleted MMCL as well as of patient-derived myeloma PC revealed cell cycle arrest at the G0/1 phase and increasing apoptosis. Exploring potential links with IRF3, we first observed that unlike in non-malignant cells, IRF3 was constitutively phosphorylated in MMCL. Using protein-co-immunoprecipitation we found that endogenous ZBP1 interacts with IRF3 and TBK1 while upon co-transfection with different ZBP1 deletion mutants, ZBP1-IRF3 interaction required primarily the ZBP1, RHIM domain-containing, C-terminus. Further, while in ZBP1-depleted myeloma cells total IRF3 and TBK1 levels were not altered, pIRF3 and pTBK1 levels decreased thus showing a post-translational dependency of constitutive pIRF3 and pTBK1 on ZBP1. Finally, pharmacological inhibition of TBK1 resulted in decrease of pIRF3 without affecting total IRF3. Importantly, shRNA-mediated IRF3 depletion resulted in cell cycle arrest and death of MMCL. By integrating histone mark and in-house IRF3 ChiP-seq with transcriptome of IRF3-depleted MM.1S cells we identified 770 down- and 330 up-regulated genes predicted to be directly regulated by IRF3. Pathway enrichment analysis confirmed cell cycle as the most highly regulated by IRF3. Notably, we observed no direct or indirect regulation of the interferon genes (e.g., IFNA1, IFNB1) by IRF3. As well as the IRF3 motif, IRF3 cistrome analysis revealed significant enrichment for the distinct IRF4 motif. Integration of the IRF3/IRF4 cistromes identified >80% IRF3 binding regions are co-occupied by IRF4 and co-regulation of cell cycle genes. Further we validated IRF3-IRF4 interaction at the IRF4 super-enhancer by ChIP-re-ChIP. These data show a novel dependency in MM comprising constitutive activation of the ZBP1-IRF3 pathway and regulation of cell cycle and proliferation by IRF3 thus providing opportunities for therapeutic targeting. Disclosures Caputo: GSK: Research Funding. Auner:Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy; Karyopharm: Consultancy. Karadimitris:GSK: Research Funding.