Summary Inflammasomes are important sentinels of innate immune defense, sensing pathogens and inducing cell death in infected cells 1 . There are several inflammasome sensors that each detect and respond to specific pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) 1 . In contrast to this one PAMP/DAMP to one sensor specificity, during infection, live pathogens can induce the release of multiple PAMPs and DAMPs, which could contemporaneously engage multiple inflammasome sensors 2 – 5 . Here we discovered that AIM2 regulated the innate immune sensors Pyrin and ZBP1 to drive inflammatory signaling and inflammatory cell death, PANoptosis, and provide host protection during infections with herpes simplex virus 1 (HSV1) and Francisella novicida . We also observed that AIM2, Pyrin and ZBP1 were members of a large multi-protein complex along with ASC, caspase-1, caspase-8, RIPK3, RIPK1 and FADD that drove inflammatory cell death. Collectively, our findings define a previously unknown regulatory connection and molecular interaction among AIM2, Pyrin and ZBP1 that drives assembly of an AIM2-mediated multi-protein complex that involves multiple inflammasome sensors and cell death regulators. These results represent a new paradigm in understanding the functions of these molecules in innate immunity and inflammatory cell death, suggesting new therapeutic targets for AIM2-, ZBP1- and Pyrin-mediated diseases.

Cell death provides host defense and maintains homeostasis. Za-containing molecules are essential for these processes. Z-DNA binding protein 1 (ZBP1) activates inflammatory cell death, PANoptosis, whereas adenosine deaminase acting on RNA 1 (ADAR1) serves as an RNA editor to maintain homeostasis. Here, we identify and characterize ADAR1's interaction with ZBP1, defining its role in cell death regulation and tumorigenesis. Combining interferons (IFNs) and nuclear export inhibitors (NEIs) activates ZBP1-dependent PANoptosis. ADAR1 suppresses this PANoptosis by interacting with the Za2 domain of ZBP1 to limit ZBP1 and RIPK3 interactions. Adar1 fl/fl LysM cre mice are resistant to development of colorectal cancer and melanoma, but deletion of the ZBP1 Za2 domain restores tumorigenesis in these mice. In addition, treating wild-type mice with IFN-g and the NEI KPT-330 regresses melanoma in a ZBP1-dependent manner. Our findings suggest that ADAR1 suppresses ZBP1-mediated PANoptosis, promoting tumorigenesis. Defining the functions of ADAR1 and ZBP1 in cell death is fundamental to informing therapeutic strategies for cancer and other diseases.

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.

Highlights Cells exposed to TLR ligands and cytokines during infection activate PANoptosis. RIPK1-dependent PANoptosome is formed when cell survival signaling is inhibited. Influenza A virus Z-RNAs bind and activate ZBP1, promoting PANoptosome formation.

Resistance to cell death is a hallmark of cancer. Immunotherapy, particularly immune checkpoint blockade therapy, drives immunemediated cell death and has greatly improved treatment outcomes for some patients with cancer, but it often fails clinically. Its success relies on the cytokines and cytotoxic functions of effector immune cells to bypass the resistance to cell death and eliminate cancer cells. However, the specific cytokines capable of inducing cell death in tumors and the mechanisms that connect cytokines to cell death across cancer cell types remain unknown. In this study, we analyzed expression of several cytokines that are modulated in tumors and found correlations between cytokine expression and mortality. Of several cytokines tested for their ability to kill cancer cells, only TNF-a and IFN-g together were able to induce cell death in 13 distinct human cancer cell lines derived from colon and lung cancer, melanoma, and leukemia. Further evaluation of the specific programmed cell death pathways activated by TNF-a and IFN-g in these cancer lines identified PANoptosis, a form of inflammatory cell death that was previously shown to be activated by contemporaneous engagement of components from pyroptosis, apoptosis, and/or necroptosis. Specifically, TNF-a and IFN-g triggered activation of gasdermin D, gasdermin E, caspase-8, caspase-3, caspase-7, and MLKL. Furthermore, the intratumoral administration of TNF-a and IFN-g suppressed the growth of transplanted xenograft tumors in an NSG mouse model. Overall, this study shows that PANoptosis, induced by synergism of TNF-a and IFN-g, is an important mechanism to kill cancer cells and suppress tumor growth that could be therapeutically targeted.