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Purpose: Small cell lung cancer (SCLC) has an extremely poor prognosis. Despite high initial response rates to chemotherapy and modest survival improvements with the addition of immune checkpoint inhibitors (ICI), almost all patients experience relapse and fatal outcomes. Recent genomic insights uncovered extensive molecular heterogeneity in addition to the almost uniform loss of RB1 and TRP53. Additionally, defective DNA mismatch repair (MMR) has recently been described in some SCLC cases. Here, we generated a novel SCLC mouse model capturing MMR deficiency and assessed immunotherapy responses., Methods: We developed an MMR-deficient genetically engineered mouse model (GEMM) of SCLC by introducing a conditional Msh2 gene, crucial for maintaining MMR integrity, into the standard Rb1 fl/fl ;Trp53 fl/fl (RP) model. Genomic characteristics and preclinical therapy responses were evaluated by focusing on overall survival and whole exome sequencing (WES) analyses., Results: MMR-defective SCLC tumors (Rb1 fl/fl ;Trp53 fl/fl ;Msh2 fl/fl (RPM)) developed later than tumors in MMR-proficient mice. However, the time from tumor manifestation to death of the affected animals was substantially shortened (median survival 55 days in RP vs. 46.5 days in RPM), indicating increased aggressiveness of MMR-defective tumors. RPM tumors exhibited MMR deficiency, high tumor mutational burden (TMB), and an elevated load of candidate neoantigens, compared to RP lesions (p = 0.0106), suggesting increased immunogenicity. Importantly, the overall survival of RPM animals was significantly improved when exposed to ICI., Conclusion: We propose a novel RPM mouse model as a suitable system to mimic MMR-defective SCLC and tumors with high TMB. We provide in vivo evidence that Msh2 deficiency enhances ICI sensitivity. These findings could contribute to stratifying SCLC patients to immunotherapy, thereby improving treatment outcomes., Competing Interests: Declarations Conflict of interest H.C.R. received consulting and lecture fees from Abbvie, AstraZeneca, Vertex, and Merck. H.C.R. received research funding from AstraZeneca and Gilead Pharmaceuticals. H.C.R. is a co-founder of CDL Therapeutics GmbH. R.K.T. is a founder of Disco Pharmaceuticals GmbH, PearlRiver Bio (now part of Centessa), a shareholder of Centessa, founder and shareholder of Epiphanes Inc. and a consultant to PearlRiver Bio and Epiphanes Inc. R.K.T. has received research support from Roche. The remaining authors declare no competing financial interest. Animal ethics declaration Animal experiments in this study were approved by the local Ethics Committee of Animal Experiments authorities (LANUV, North Rhine-Westphalia, Germany) under license number 81-02.04.2019-A491. All mice were maintained according to FELASA recommendations and in compliance with the European Union and German guidelines., (© 2024. The Author(s).)

Background: Patient-derived colorectal cancer (CRC) organoids (PDOs) solely consisting of malignant cells led to major advances in the understanding of cancer treatments. Yet, a major limitation is the absence of cells from the tumor microenvironment, thereby prohibiting potential investigation of treatment responses on immune and structural cells. Currently there are sparse reports describing the interaction of PDOs, cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in complex primary co-culture assay systems., Methods: Primary PDOs and patient matched CAF cultures were generated from surgical resections. Co-culture systems of PDOs, CAFs and monocytic myeloid cells were set up to recapitulate features seen in patient tumors. Single-cell transcriptomics and flow cytometry was used to show effects of culture systems on TAM populations in the co-culture assays under chemotherapeutic and oncolytic viral treatment., Results: In contrast to co-cultures of tumor cells and monocytes, CAF/monocyte co-cultures and CAF/monocyte/tumor cell triple cultures resulted in a partial differentiation into macrophages and a phenotypic switch, characterized by the expression of major immunosuppressive markers comparable to TAMs in CRC. Oxaliplatin and 5-fluorouracil, the standard-of-care chemotherapy for CRC, induced polarization of macrophages to a pro-inflammatory phenotype comparable to the immunogenic effects of treatment with an oncolytic virus. Monitoring phagocytosis as a functional proxy to macrophage activation and subsequent onset of an immune response, revealed that chemotherapy-induced cell death, but not virus-mediated cell death, is necessary to induce phagocytosis of CRC cells. Moreover, CAFs enhanced the phagocytic activity in chemotherapy treated CRC triple cultures., Conclusions: Primary CAF-containing triple cultures successfully model TAM-like phenotypes ex vivo and allow the assessment of their functional and phenotypic changes in response to treatments following a precision medicine approach., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Background: The phase 2 ASUNCTIS study assessed the efficacy and safety of asunercept, a fully human CD95 (Fas) ligand-binding protein, in hospitalised patients with moderate-to-severe coronavirus disease (COVID-19) to assess the clinical benefit of CD95 ligand inhibition in this viral disease., Methods: In this open-label, multicentre, randomised, controlled, phase 2 trial, patients with COVID-19-induced pneumonia and respiratory deterioration were randomly assigned (1:1:1:1) in 12 Russian and Spanish hospitals using an interactive web-response system to receive standard of care (SOC) or SOC plus weekly asunercept 25 mg, 100 mg, or 400 mg, administered intravenously for up to 4 weeks, or until hospital discharge or death. The randomisation was stratified according to the respiratory support methods at the time of enrolment, corresponding to categories 4-6 of a clinical severity assessment scale comprising 9 levels that was recommended by the World Health Organization (WHO) at the time of the study. The main inclusion criterion was laboratory confirmed infection with SARS-CoV-2 OR typical radiological signs of SARS-CoV-2 infection. The primary endpoint was time from randomisation to clinical improvement on two consecutive days of at least one category on a WHO clinical severity assessment scale in the modified intent-to-treat population. All patients were subjected to regular safety analyses. This trial is registered with EudraCT (2020-001887-27) and ClinicalTrials.gov (NCT04535674)., Findings: Between October 9, 2020, and September 24, 2021, 438 patients were randomly assigned to SOC (n = 110) or SOC plus asunercept 25 mg (n = 109), 100 mg (n = 109), or 400 mg (n = 110). The primary endpoint, time to sustained clinical improvement of one WHO category on two consecutive days from randomization, was in median [95% confidence interval]: 9 [6-12], 8 [7-12], 8 [7-11] and 13 [9-20] days for the 400 mg, 100 mg, 25 mg asunercept and SOC groups, respectively. The standard deviations for the 400 mg, 100 mg, 25 mg asunercept and SOC groups were 5.3, 4.9, 4.7 and 5 days, respectively. The observed differences between groups failed to reach statistical significance (one-sided p-value = 0.041). In total, 290 adverse events (AE) were registered in 145 patients who received at least one dose of the study treatment: 77 AEs in 37 (33.6%) patients in the SOC group, 80 AEs in 38 (34.9%) patients in the 25 mg group, 61 AEs in 35 (32.7%) patients in the 100 mg group and 72 AEs in 35 (32.1%) patients in the 400 mg group. There was no treatment-related death reported. In summary, asunercept was well tolerated at all doses tested and no specific safety signals were detected., Interpretation: The primary endpoint of time to sustained clinical improvement for distinct asunercept arms compared to SOC failed to meet statistical significance. The compound was safe and well tolerated., Funding: Apogenix GmbH, Heidelberg, Germany., Competing Interests: C Schoergenhofer and FH received honoraria from the sponsor for participation in the DSMB. CD is an employee of Saarmetrics GmbH which received payments from Apogenix GmbH for the analysis of the influence of asunercept on lymphocyte counts described in the manuscript. TL is a stakeholder of Saarmetrics GmbH which received payments from Apogenix GmbH for the analysis of the influence of asunercept on lymphocyte counts described in the manuscript. Saarmetrics received consulting fees for CD's and TL's work as consultants for Apogenix GmbH. AK and C Straub were employed by Apogenix GmbH at the time the study was conducted. JRPP, C Schoergenhofer, MB and BJ received funding for clinical trials from the sponsor. MK is an employee of Cytel Inc. and worked as a statistical consultant for Apogenix GmbH. HF, HW and TH are co-founders and shareholders of Apogenix GmbH. HW also worked as a consultant for Apogenix GmbH. JP, DdM and MT declare that they have no conflicts of interest., (© 2024 The Author(s).)

Lymphotoxin β receptor (LTβR), a member of the TNF receptor superfamily (TNFR-SF), is essential for development and maturation of lymphoid organs. In addition, LTβR activation promotes carcinogenesis by inducing a proinflammatory secretome. Yet, we currently lack a detailed understanding of LTβR signaling. In this study we discovered the linear ubiquitin chain assembly complex (LUBAC) as a previously unrecognized and functionally crucial component of the native LTβR signaling complex (LTβR-SC). Mechanistically, LUBAC-generated linear ubiquitin chains enable recruitment of NEMO, OPTN and A20 to the LTβR-SC, where they act coordinately to regulate the balance between canonical and non-canonical NF-κB pathways. Thus, different from death receptor signaling, where LUBAC prevents inflammation through inhibition of cell death, in LTβR signaling LUBAC is required for inflammatory signaling by enabling canonical and interfering with non-canonical NF-κB activation. This results in a LUBAC-dependent LTβR-driven inflammatory, protumorigenic secretome. Intriguingly, in liver cancer patients with high LTβR expression, high expression of LUBAC correlates with poor prognosis, providing clinical relevance for LUBAC-mediated inflammatory LTβR signaling., (© 2024. The Author(s).)

Objective: Receptor-interacting protein kinase 1 (RIPK1) orchestrates the decision between cell survival and cell death in response to tumor necrosis factor (TNF) and other cytokines. Whereas the scaffolding function of RIPK1 is crucial to prevent TNF-induced apoptosis and necroptosis, its kinase activity is required for necroptosis and partially for apoptosis. Although TNF is a proinflammatory cytokine associated with β-cell loss in diabetes, the mechanism by which TNF induces β-cell demise remains unclear., Methods: Here, we dissected the contribution of RIPK1 scaffold versus kinase functions to β-cell death regulation using mice lacking RIPK1 specifically in β-cells (Ripk1 β-KO mice) or expressing a kinase-dead version of RIPK1 (Ripk1 D138N mice), respectively. These mice were challenged with streptozotocin, a model of autoimmune diabetes. Moreover, Ripk1 β-KO mice were further challenged with a high-fat diet to induce hyperglycemia. For mechanistic studies, pancreatic islets were subjected to various killing and sensitising agents., Results: Inhibition of RIPK1 kinase activity (Ripk1 D138N mice) did not affect the onset and progression of hyperglycemia in a type 1 diabetes model. Moreover, the absence of RIPK1 expression in β-cells did not affect normoglycemia under basal conditions or hyperglycemia under diabetic challenges. Ex vivo, primary pancreatic islets are not sensitised to TNF-induced apoptosis and necroptosis in the absence of RIPK1. Intriguingly, we found that pancreatic islets display high levels of the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) and low levels of apoptosis (Caspase-8) and necroptosis (RIPK3) components. Cycloheximide treatment, which led to a reduction in cFLIP levels, rendered primary islets sensitive to TNF-induced cell death which was fully blocked by caspase inhibition., Conclusions: Unlike in many other cell types (e.g., epithelial, and immune), RIPK1 is not required for cell death regulation in β-cells under physiological conditions or diabetic challenges. Moreover, in vivo and in vitro evidence suggest that pancreatic β-cells do not undergo necroptosis but mainly caspase-dependent death in response to TNF. Last, our results show that β-cells have a distinct mode of regulation of TNF-cytotoxicity that is independent of RIPK1 and that may be highly dependent on cFLIP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Necroptosis is a caspase-independent modality of cell death implicated in many inflammatory pathologies. The execution of this pathway requires the formation of a cytosolic platform that comprises RIPK1 and RIPK3 which, in turn, mediates the phosphorylation of the pseudokinase MLKL (S345 in mouse). The activation of this executioner is followed by its oligomerisation and accumulation at the plasma-membrane where it leads to cell death via plasma-membrane destabilisation and consequent permeabilisation. While the biochemical and cellular characterisation of these events have been amply investigated, the study of necroptosis involvement in vivo in animal models is currently limited to the use of Mlkl -/- or Ripk3 -/- mice. Yet, even in many of the models in which the involvement of necroptosis in disease aetiology has been genetically demonstrated, the fundamental in vivo characterisation regarding the question as to which tissue(s) and specific cell type(s) therein is/are affected by the pathogenic necroptotic death are missing. Here, we describe and validate an immunohistochemistry and immunofluorescence-based method to reliably detect the phosphorylation of mouse MLKL at serine 345 (pMLKL-S345). We first validate the method using tissues derived from mice in which Caspase-8 (Casp8) or FADD are specifically deleted from keratinocytes, or intestinal epithelial cells, respectively. We next demonstrate the presence of necroptotic activation in the lungs of SARS-CoV-infected mice and in the skin and spleen of mice bearing a Sharpin inactivating mutation. Finally, we exclude necroptosis occurrence in the intestines of mice subjected to TNF-induced septic shock. Importantly, by directly comparing the staining of pMLKL-345 with that of cleaved Caspase-3 staining in some of these models, we identify spatio-temporal and functional differences between necroptosis and apoptosis supporting a role of RIPK3 in inflammation independently of MLKL versus the role of RIPK3 in activation of necroptosis., (© 2024. The Author(s).)

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The dysregulated immune response and inflammation resulting in severe COVID-19 are still incompletely understood. Having recently determined that aberrant death-ligand-induced cell death can cause lethal inflammation, we hypothesized that this process might also cause or contribute to inflammatory disease and lung failure following SARS-CoV-2 infection. To test this hypothesis, we developed a novel mouse-adapted SARS-CoV-2 model (MA20) that recapitulates key pathological features of COVID-19. Concomitantly with occurrence of cell death and inflammation, FasL expression was significantly increased on inflammatory monocytic macrophages and NK cells in the lungs of MA20-infected mice. Importantly, therapeutic FasL inhibition markedly increased survival of both, young and old MA20-infected mice coincident with substantially reduced cell death and inflammation in their lungs. Intriguingly, FasL was also increased in the bronchoalveolar lavage fluid of critically-ill COVID-19 patients. Together, these results identify FasL as a crucial host factor driving the immuno-pathology that underlies COVID-19 severity and lethality, and imply that patients with severe COVID-19 may significantly benefit from therapeutic inhibition of FasL., (© 2024. The Author(s).)

Mitochondrial outer membrane permeabilisation (MOMP) is often essential for apoptosis, by enabling cytochrome c release that leads to caspase activation and rapid cell death. Recently, MOMP has been shown to be inherently pro-inflammatory with emerging cellular roles, including its ability to elicit anti-tumour immunity. Nonetheless, how MOMP triggers inflammation and how the cell regulates this remains poorly defined. We find that upon MOMP, many proteins localised either to inner or outer mitochondrial membranes are ubiquitylated in a promiscuous manner. This extensive ubiquitylation serves to recruit the essential adaptor molecule NEMO, leading to the activation of pro-inflammatory NF-κB signalling. We show that disruption of mitochondrial outer membrane integrity through different means leads to the engagement of a similar pro-inflammatory signalling platform. Therefore, mitochondrial integrity directly controls inflammation, such that permeabilised mitochondria initiate NF-κB signalling., (© 2024. The Author(s).)

The ability of cells to mount an interferon response to virus infections depends on intracellular nucleic acid sensing pattern recognition receptors (PRRs). RIG-I is an intracellular PRR that binds short double-stranded viral RNAs to trigger MAVS-dependent signalling. The RIG-I/MAVS signalling complex requires the coordinated activity of multiple kinases and E3 ubiquitin ligases to activate the transcription factors that drive type I and type III interferon production from infected cells. The linear ubiquitin chain assembly complex (LUBAC) regulates the activity of multiple receptor signalling pathways in both ligase-dependent and -independent ways. Here, we show that the three proteins that constitute LUBAC have separate functions in regulating RIG-I signalling. Both HOIP, the E3 ligase capable of generating M1-ubiquitin chains, and LUBAC accessory protein HOIL-1 are required for viral RNA sensing by RIG-I. The third LUBAC component, SHARPIN, is not required for RIG-I signalling. These data cement the role of LUBAC as a positive regulator of RIG-I signalling and as an important component of antiviral innate immune responses., (© 2023. The Author(s).)

The discovery of frequent 8p11-p12 amplifications in squamous cell lung cancer (SQLC) has fueled hopes that FGFR1, located inside this amplicon, might be a therapeutic target. In a clinical trial, only 11% of patients with 8p11 amplification (detected by FISH) responded to FGFR kinase inhibitor treatment. To understand the mechanism of FGFR1 dependency, we performed deep genomic characterization of 52 SQLCs with 8p11-p12 amplification, including 10 tumors obtained from patients who had been treated with FGFR inhibitors. We discovered somatically altered variants of FGFR1 with deletion of exons 1-8 that resulted from intragenic tail-to-tail rearrangements. These ectodomain-deficient FGFR1 variants (ΔEC-FGFR1) were expressed in the affected tumors and were tumorigenic in both in vitro and in vivo models of lung cancer. Mechanistically, breakage-fusion-bridges were the source of 8p11-p12 amplification, resulting from frequent head-to-head and tail-to-tail rearrangements. Generally, tail-to-tail rearrangements within or in close proximity upstream of FGFR1 were associated with FGFR1 dependency. Thus, the genomic events shaping the architecture of the 8p11-p12 amplicon provide a mechanistic explanation for the emergence of FGFR1-driven SQLC. Specifically, we believe that FGFR1 ectodomain-deficient and FGFR1-centered amplifications caused by tail-to-tail rearrangements are a novel somatic genomic event that might be predictive of therapeutically relevant FGFR1 dependency.

Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. Cellular FLICE-like inhibitory protein (cFLIP) is a crucial regulator of cell death and a substrate of Caspase-8. However, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we found an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, Cflip D377A , exhibited increased sensitivity to severe acute respiratory syndrome coronavirus (SARS-CoV)-induced lethality, impaired skin wound healing, and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to tumor necrosis factor(TNF)-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on glutamine-469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress to ensure the execution of repair programs.

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., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Background: Symptoms lasting longer than 12  weeks after severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection are called post-coronavirus disease (COVID) syndrome (PCS). The identification of new biomarkers that predict the occurrence or course of PCS in terms of a post-viral syndrome is vital. T-cell dysfunction, cytokine imbalance, and impaired autoimmunity have been reported in PCS. Nevertheless, there is still a lack of conclusive information on the underlying mechanisms due to, among other things, a lack of controlled study designs., Methods: Here, we conducted a prospective, controlled study to characterize the humoral and cellular immune response in unvaccinated patients with and without PCS following SARS-CoV-2 infection over 7 months and unexposed donors., Results: Patients with PCS showed as early as 6 weeks and 7 months after symptom onset significantly increased frequencies of SARS-CoV-2-specific CD4 + and CD8 + T-cells secreting IFNγ, TNF, and expressing CD40L, as well as plasmacytoid dendritic cells (pDC) with an activated phenotype. Remarkably, the immunosuppressive counterparts type 1 regulatory T-cells (TR1: CD49b/LAG-3 + ) and IL-4 were more abundant in PCS + ., Conclusion: This work describes immunological alterations between inflammation and immunosuppression in COVID-19 convalescents with and without PCS, which may provide potential directions for future epidemiological investigations and targeted treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Augustin, Heyn, Ullrich, Sandaradura de Silva, Albert, Linne, Schlotz, Schommers, Pracht, Horn, Suarez, Simonis, Picard, Zoufaly, Wenisch, Fätkenheuer, Gruell, Klein, Hallek, Walczak, Rybniker, Theobald and Lehmann.)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine produced and secreted by immune cells in response to an infection, often in response to interferon (IFN) stimulation. In cancer, it has also been shown that IFN stimulates the production of TRAIL, and it has been proposed that this TRAIL can induce apoptosis in an autocrine or paracrine manner in different cancer cells. Yet, the mechanism mediating TRAIL upregulation and the implications of TRAIL as an apoptotic molecule in cancer cells are still poorly understood. We show here that in certain cancer cells, TRAIL is upregulated by enhancer clusters, potent genomic regulatory regions containing densely packed enhancers that have combinatorial and additive activity and that are usually found to be associated with cancer-promoting genes. Moreover, we found that TRAIL upregulation by IFNα is mediated by these enhancer clusters in breast and lung cancer cells. Surprisingly, IFNα stimulation leads to the intracellular accumulation of TRAIL protein in these cancer cells. Consequently, this TRAIL is not capable of inducing apoptosis. Our study provides novel insights into the mechanism behind the interferon-mediated upregulation of TRAIL and its protein accumulation in cancer cells. Further investigation is required to understand the role of intracellular TRAIL or depict the mechanisms mediating its apoptosis impairment in cancer cells.

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) can induce apoptosis in a wide variety of cancer cells, both in vitro and in vivo, importantly without killing any essential normal cells. These findings formed the basis for the development of TRAIL-receptor agonists (TRAs) for cancer therapy. However, clinical trials conducted with different types of TRAs have, thus far, afforded only limited therapeutic benefit, as either the respectively chosen agonist showed insufficient anticancer activity or signs of toxicity, or the right TRAIL-comprising combination therapy was not employed. Therefore, in this review we will discuss molecular determinants of TRAIL resistance, the most promising TRAIL-sensitizing agents discovered to date and, importantly, whether any of these could also prove therapeutically efficacious upon cancer relapse following conventional first-line therapies. We will also discuss the more recent progress made with regards to the clinical development of highly active non-immunogenic next generation TRAs. Based thereupon, we next propose how TRAIL resistance might be successfully overcome, leading to the possible future development of highly potent, cancer-selective combination therapies that are based on our current understanding of biology TRAIL-induced cell death. It is possible that such therapies may offer the opportunity to tackle one of the major current obstacles to effective cancer therapy, namely overcoming chemo- and/or targeted-therapy resistance. Even if this were achievable only for certain types of therapy resistance and only for particular types of cancer, this would be a significant and meaningful achievement., (© 2022. The Author(s).)

The development of immune checkpoint inhibitors (ICI) offers novel treatment possibilities for solid cancers, with the crucial benefit of providing higher cure rates. These agents have become part of standard treatments in the metastatic and adjuvant setting for select cancers, such as melanoma, non-small cell lung cancer (NSCLC) or urological malignancies. Currently, there is ample clinical interest in employing ICI in a neoadjuvant setting with a curative intent. This approach is especially supported by the scientific rationale that ICI primarily stimulate the host immune system to eradicate tumor cells, rather than being inherently cytotoxic. Aside from tumor downstaging, neoadjuvant immunotherapy offers the potential of an in situ cancer vaccination, leading to a systemic adjuvant immunological effect after tumor resection. Moreover, preclinical data clearly demonstrate a synergistic effect of ICI with radiotherapy (RT), chemoradiotherapy (CRT) or chemotherapy (ChT). This review harmonizes preclinical concepts with real world data (RWD) in the field of neoadjuvant ICI in gastrointestinal (GI) cancers and discusses their limitations. We believe this is a crucial approach, since up to now, neoadjuvant strategies have been primarily developed by clinicians, whereas the advances in immunotherapy primarily originate from preclinical research. Currently there is limited published data on neoadjuvant ICI in GI cancers, even though neoadjuvant treatments including RT, CRT or ChT are frequently employed in locally advanced/oligometastatic GI cancers (i.e. rectal, pancreatic, esophagus, stomach, etc.). Utilizing established therapies in combination with ICI provides an abundance of opportunities for innovative treatment regimens to further improve survival rates., Competing Interests: Competing interests All authors declare no competing interest in the context of the present review., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Durable cell-mediated immune responses require efficient innate immune signaling and the release of pro-inflammatory cytokines. How precisely mRNA vaccines trigger innate immune cells for shaping antigen specific adaptive immunity remains unknown. Here, we show that SARS-CoV-2 mRNA vaccination primes human monocyte-derived macrophages for activation of the NLRP3 inflammasome. Spike protein exposed macrophages undergo NLRP3-driven pyroptotic cell death and subsequently secrete mature interleukin-1β. These effects depend on activation of spleen tyrosine kinase (SYK) coupled to C-type lectin receptors. Using autologous cocultures, we show that SYK and NLRP3 orchestrate macrophage-driven activation of effector memory T cells. Furthermore, vaccination-induced macrophage priming can be enhanced with repetitive antigen exposure providing a rationale for prime-boost concepts to augment innate immune signaling in SARS-CoV-2 vaccination. Collectively, these findings identify SYK as a regulatory node capable of differentiating between primed and unprimed macrophages, which modulate spike protein-specific T cell responses., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Anticancer drug development campaigns often fail due to an incomplete understanding of the therapeutic index differentiating the efficacy of the agent against the cancer and its on-target toxicities to the host. To address this issue, we established a versatile preclinical platform in which genetically defined cancers are produced using somatic tissue engineering in transgenic mice harboring a doxycycline-inducible short hairpin RNA against the target of interest. In this system, target inhibition is achieved by the addition of doxycycline, enabling simultaneous assessment of efficacy and toxicity in the same animal. As proof of concept, we focused on CDK9—a cancer target whose clinical development has been hampered by compounds with poorly understood target specificity and unacceptable toxicities. We systematically compared phenotypes produced by genetic Cdk9 inhibition to those achieved using a recently developed highly specific small molecule CDK9 inhibitor and found that both perturbations led to robust antitumor responses. Remarkably, nontoxic levels of CDK9 inhibition could achieve significant treatment efficacy, and dose-dependent toxicities produced by prolonged CDK9 suppression were largely reversible upon Cdk9 restoration or drug withdrawal. Overall, these results establish a versatile in vivo target validation platform that can be employed for rapid triaging of therapeutic targets and lend support to efforts aimed at advancing CDK9 inhibitors for cancer therapy.

Autoinflammatory diseases are a heterogenous group of disorders defined by fever and systemic inflammation suggesting involvement of genes regulating innate immune responses. Patients with homozygous loss-of-function variants in the OTU-deubiquitinase OTULIN suffer from neonatal-onset OTULIN-related autoinflammatory syndrome (ORAS) characterized by fever, panniculitis, diarrhea, and arthritis. Here, we describe an atypical form of ORAS with distinct clinical manifestation of the disease caused by two new compound heterozygous variants (c.258G>A (p.M86I)/c.500G>C (p.W167S)) in the OTULIN gene in a 7-year-old affected by a life-threatening autoinflammatory episode with sterile abscess formation. On the molecular level, we find binding of OTULIN to linear ubiquitin to be compromised by both variants; however, protein stability and catalytic activity is most affected by OTULIN variant p.W167S. These molecular changes together lead to increased levels of linear ubiquitin linkages in patient-derived cells triggering the disease. Our data indicate that the spectrum of ORAS patients is more diverse than previously thought and, thus, supposedly asymptomatic individuals might also be affected. Based on our results, we propose to subdivide the ORAS into classical and atypical entities., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Primary or acquired therapy resistance is a major obstacle to the effective treatment of cancer. Resistance to apoptosis has long been thought to contribute to therapy resistance. We show here that recombinant TRAIL and CDK9 inhibition cooperate in killing cells derived from a broad range of cancers, importantly without inducing detectable adverse events. Remarkably, the combination of TRAIL with CDK9 inhibition was also highly effective on cancers resistant to both, standard-of-care chemotherapy and various targeted therapeutic approaches. Dynamic BH3 profiling revealed that, mechanistically, combining TRAIL with CDK9 inhibition induced a drastic increase in the mitochondrial priming of cancer cells. Intriguingly, this increase occurred irrespective of whether the cancer cells were sensitive or resistant to chemo- or targeted therapy. We conclude that this pro-apoptotic combination therapy has the potential to serve as a highly effective new treatment option for a variety of different cancers. Notably, this includes cancers that are resistant to currently available treatment modalities., (© 2021. The Author(s).)

Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell-induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the γ-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies., Competing Interests: Disclosures: I. Sagi reported a patent (no. US 10,933,122 B2) issued. D. Schmidt-Arras reported personal fees from Mestag Therapeutics Ltd. outside the submitted work. No other disclosures were reported., (© 2021 Bolik et al.)