Search

Necrosis of a cell is defined by the loss of its plasma membrane integrity. Morphologically, necrosis occurs in several forms such as coagulative necrosis, colliquative necrosis, caseating necrosis, fibrinoid necrosis, and others. Biochemically, necrosis was demonstrated to represent a number of genetically determined signalling pathways. These include (i) kinase-mediated necroptosis, which depends on receptor interacting protein kinase 3 (RIPK3)-mediated phosphorylation of the pseudokinase mixed lineage kinase domain like (MLKL); (ii) gasdermin-mediated necrosis downstream of inflammasomes, also referred to as pyroptosis; and (iii) an iron-catalysed mechanism of highly specific lipid peroxidation named ferroptosis. Given the molecular understanding of the nature of these pathways, specific antibodies may allow direct detection of regulated necrosis and correlation with morphological features. Necroptosis can be specifically detected by immunohistochemistry and immunofluorescence employing antibodies to phosphorylated MLKL. Likewise, it is possible to generate cleavage-specific antibodies against epitopes in gasdermin protein family members. In ferroptosis, however, specific detection requires quantification of oxidative lipids by mass spectrometry (oxylipidomics). Together with classical cell death markers, such as TUNEL staining and detection of cleaved caspase-3 in apoptotic cells, the extension of the arsenal of necrosis markers will allow pathological detection of specific molecular pathways rather than isolated morphological descriptions. These novel pieces of information will be extraordinarily helpful for clinicians as inhibitors of necroptosis (necrostatins), ferroptosis (ferrostatins), and inflammasomes have emerged in clinical trials. Anatomical pathologists should embrace these novel ancillary tests and the concepts behind them and test their impact on diagnostic precision, prognostication, and the prediction of response to the upcoming anti-necrotic therapies. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Objective The intestinal epithelium is a rapidly renewing tissue which plays central roles in nutrient uptake, barrier function and the prevention of intestinal inflammation. Control of epithelial differentiation is essential to these processes and is dependent on cell type-specific activity of transcription factors which bind to accessible chromatin. Here, we studied the role of SET Domain Bifurcated Histone Lysine Methyltransferase 1, also known as ESET (SETDB1), a histone H3K9 methyltransferase, in intestinal epithelial homeostasis and IBD. Design We investigated mice with constitutive and inducible intestinal epithelial deletion of Setdb1, studied the expression of SETDB1 in patients with IBD and mouse models of IBD, and investigated the abundance of SETDB1 variants in healthy individuals and patients with IBD. Results Deletion of intestinal epithelial Setdb1 in mice was associated with defects in intestinal epithelial differentiation, barrier disruption, inflammation and mortality. Mechanistic studies showed that loss of SETDB1 leads to de-silencing of endogenous retroviruses, DNA damage and intestinal epithelial cell death. Predicted loss-of-function variants in human SETDB1 were considerably less frequently observed than expected, consistent with a critical role of SETDB1 in human biology. While the vast majority of patients with IBD showed unimpaired mucosal SETDB1 expression, comparison of IBD and non-IBD exomes revealed over-representation of individual rare missense variants in SETDB1 in IBD, some of which are predicted to be associated with loss of function and may contribute to the pathogenesis of intestinal inflammation. Conclusion SETDB1 plays an essential role in intestinal epithelial homeostasis. Future work is required to investigate whether rare variants in SETDB1 contribute to the pathogenesis of IBD.

The linear-ubiquitin chain assembly complex (LUBAC) modulates signalling via various immune receptors. In tumour necrosis factor (TNF) signalling, linear (also known as M1) ubiquitin enables full gene activation and prevents cell death. However, the mechanisms underlying cell death prevention remain ill-defined. Here, we show that LUBAC activity enables TBK1 and IKKε recruitment to and activation at the TNF receptor 1 signalling complex (TNFR1-SC). While exerting only limited effects on TNF-induced gene activation, TBK1 and IKKε are essential to prevent TNF-induced cell death. Mechanistically, TBK1 and IKKε phosphorylate the kinase RIPK1 in the TNFR1-SC, thereby preventing RIPK1-dependent cell death. This activity is essential in vivo, as it prevents TNF-induced lethal shock. Strikingly, NEMO (also known as IKKγ), which mostly, but not exclusively, binds the TNFR1-SC via M1 ubiquitin, mediates the recruitment of the adaptors TANK and NAP1 (also known as AZI2). TANK is constitutively associated with both TBK1 and IKKε, while NAP1 is associated with TBK1. We discovered a previously unrecognized cell death checkpoint that is mediated by TBK1 and IKKε, and uncovered an essential survival function for NEMO, whereby it enables the recruitment and activation of these non-canonical IKKs to prevent TNF-induced cell death.

Acute kidney injury (AKI) is characterized by necrotic tubular cell death and inflammation. The TWEAK/Fn14 axis is a mediator of renal injury. Diverse pathways of regulated necrosis have recently been reported to contribute to AKI, but there are ongoing discussions on the timing or molecular regulators involved. We have now explored the cell death pathways induced by TWEAK/Fn14 activation and their relevance during AKI. In cultured tubular cells, the inflammatory cytokine TWEAK induces apoptosis in a proinflammatory environment. The default inhibitor of necroptosis [necrostatin-1 (Nec-1)] was protective, while caspase inhibition switched cell death to necroptosis. Additionally, folic acid-induced AKI in mice resulted in increased expression of Fn14 and necroptosis mediators, such as receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage domain-like protein (MLKL). Targeting necroptosis with Nec-1 or by genetic RIPK3 deficiency and genetic Fn14 ablation failed to be protective at early time points (48 h). However, a persistently high cell death rate and kidney dysfunction (72-96 h) were dependent on an intact TWEAK/Fn14 axis driving necroptosis. This was prevented by Nec-1, or MLKL, or RIPK3 deficiency and by Nec-1 stable (Nec-1s) administered before or after induction of AKI. These data suggest that initial kidney damage and cell death are amplified through recruitment of inflammation-dependent necroptosis, opening a therapeutic window to treat AKI once it is established. This may be relevant for clinical AKI, since using current diagnostic criteria, severe injury had already led to loss of renal function at diagnosis.

The Mediator complex is a key regulator of gene transcription and several studies demonstrated altered expressions of particular subunits in diverse human diseases, especially cancer. However a systematic study deciphering the transcriptional expression of the Mediator across different cancer entities is still lacking. We therefore performed a comprehensive in silico cancer vs. benign analysis of the Mediator complex subunits (MEDs) for 20 tumor entities using Oncomine datasets. The transcriptional expression profiles across almost all cancer entities showed differentially expressed MEDs as compared to benign tissue. Differential expression of MED8 in renal cell carcinoma (RCC) and MED12 in lung cancer (LCa) were validated and further investigated by immunohistochemical staining on tissue microarrays containing large numbers of specimen. MED8 in clear cell RCC (ccRCC) associated with shorter survival and advanced TNM stage and showed higher expression in metastatic than primary tumors. In vitro, siRNA mediated MED8 knockdown significantly impaired proliferation and motility in ccRCC cell lines, hinting at a role for MED8 to serve as a novel therapeutic target in ccRCC. Taken together, our Mediator complex transcriptome proved to be a valid tool for identifying cancer-related shifts in Mediator complex composition, revealing that MEDs do exhibit cancer specific transcriptional expression profiles.

Renal tubules represent an intercellular unit and function as a syncytium. When acute tubular necrosis was first visualized to occur through a process of synchronized regulated necrosis (SRN) in handpicked primary renal tubules, it became obvious that SRN actually promotes nephron loss. This realization adds to our current understanding of acute kidney injury (AKI)-chronic kidney disease (CKD) transition and argues for the prevention of AKI episodes to prevent CKD progression. Because SRN is triggered by necroptosis and executed by ferroptosis, 2 recently identified signaling pathways of regulated necrosis, a combination therapy employing necrostatins and ferrostatins may be beneficial for protection against nephron loss. Clinical trials in AKI and during the process of kidney transplantation are now required to prevent SRN. Additionally, necrotic cell death drives autoimmunity and necroinflammation and therefore represents a therapeutic target even for the prevention of antibody-mediated rejection of allografts years after the transplantation process.

Receptor-interacting protein kinases 1 and 3 (RIPK1/3) have best been described for their role in mediating a regulated form of necrosis, referred to as necroptosis. During this process, RIPK3 phosphorylates mixed lineage kinase domain-like (MLKL) to cause plasma membrane rupture. RIPK3-deficient mice have recently been demonstrated to be protected in a series of disease models, but direct evidence for activation of necroptosis in vivo is still limited. Here, we sought to further examine the activation of necroptosis in kidney ischemia-reperfusion injury (IRI) and from TNFα-induced severe inflammatory response syndrome (SIRS), two models of RIPK3-dependent injury. In both models, MLKL-ko mice were significantly protected from injury to a degree that was slightly, but statistically significantly exceeding that of RIPK3-deficient mice. We also demonstrated, for the first time, accumulation of pMLKL in the necrotic tubules of human patients with acute kidney injury. However, our data also uncovered unexpected elevation of blood flow in MLKL-ko animals, which may be relevant to IRI and should be considered in the future. To further understand the mode of regulation of cell death by MLKL, we screened a panel of clinical plasma membrane channel blockers and we found phenytoin to inhibit necroptosis. However, we further found that phenytoin attenuated RIPK1 kinase activity in vitro, likely due to the hydantoin scaffold also present in necrostatin-1, and blocked upstream necrosome formation steps in the cells undergoing necroptosis. We further report that this clinically used anti-convulsant drug displayed protection from kidney IRI and TNFα-induces SIRS in vivo. Overall, our data reveal the relevance of RIPK3-pMLKL regulation for acute kidney injury and identifies an FDA-approved drug that may be useful for immediate clinical evaluation of inhibition of pro-death RIPK1/RIPK3 activities in human diseases.

Death of renal cells is central to the pathophysiology of acute tubular necrosis, autoimmunity, necrotizing glomerulonephritis, cystic kidney disease, urosepsis, delayed graft function and transplant rejection. By means of regulated necrosis, immunogenic damage-associated molecular patterns (DAMPs) and highly reactive organelles such as lysosomes, peroxisomes and mitochondria are released from the dying cells, thereby causing an overwhelming immunologic response. The rupture of the plasma membrane exhibits the “point of no return” for the immunogenicity of regulated cell death, explaining why apoptosis, a highly organized cell death subroutine with long-lasting plasma membrane integrity, elicits hardly any immune response. Ferroptosis, an iron-dependent necrotic type cell death, results in the release of DAMPs and large amounts of lipid peroxides. In contrast, anti-inflammatory cytokines are actively released from cells that die by necroptosis, limiting the DAMP-induced immune response to a surrounding microenvironment, whereas at the same time, inflammasome-associated caspases drive maturation of intracellularly expressed interleukin-1β (IL-1β). In a distinct setting, additionally interleukin-18 (IL-18) is expressed during pyroptosis, initiated by gasdermin-mediated plasma membrane rupture. As all of these pathways are druggable, we provide an overview of regulated necrosis in kidney diseases with a focus on immunogenicity and potential therapeutic interventions., Key Points Regulated necrosis is a genetically determined process that contributes to acute kidney injury and causes antibody-mediated rejection (ABMR).Necroptosis, ferroptosis and pyroptosis are the best-studied pathways of regulated necrosis in acute kidney injury and transplantation.Necrotic cell death results in the release of DAMPs and is often prone to elicit an immune response.Failure to efficiently remove necrotic debris by LC3-associated phagocytosis (LAP) results in autoimmunity.During the process of kidney transplantation, necrotic cells are capable of priming memory B cells that may drive ABMR years after transplantation.Necroptosis, ferroptosis and pyroptosis can be therapeutically interfered with by small molecules.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) constitutes life-threatening autoimmune diseases affecting every organ, including the kidneys, where they cause necrotizing crescentic glomerulonephritis. ANCA activates neutrophils and activated neutrophils damage the endothelium, leading to vascular inflammation and necrosis. Better understanding of neutrophil-mediated AAV disease mechanisms may reveal novel treatment strategies. Here we report that ANCA induces neutrophil extracellular traps (NETs) via receptor-interacting protein kinase (RIPK) 1/3- and mixed-lineage kinase domain-like (MLKL)-dependent necroptosis. NETs from ANCA-stimulated neutrophils caused endothelial cell (EC) damage in vitro. This effect was prevented by (i) pharmacologic inhibition of RIPK1 or (ii) enzymatic NET degradation. The alternative complement pathway (AP) was recently implicated in AAV, and C5a inhibition is currently being tested in clinical studies. We observed that NETs provided a scaffold for AP activation that in turn contributed to EC damage. We further established the in vivo relevance of NETs and the requirement of RIPK1/3/MLKL-dependent necroptosis, specifically in the bone marrow-derived compartment, for disease induction using murine AAV models and in human kidney biopsies. In summary, we identified a mechanistic link between ANCA-induced neutrophil activation, necroptosis, NETs, the AP, and endothelial damage. RIPK1 inhibitors are currently being evaluated in clinical trials and exhibit a novel therapeutic strategy in AAV.

Purpose: The Mediator complex is a multiprotein assembly, which serves as a hub for diverse signaling pathways to regulate gene expression. Because gene expression is frequently altered in cancer, a systematic understanding of the Mediator complex in malignancies could foster the development of novel targeted therapeutic approaches. Experimental Design: We performed a systematic deconvolution of the Mediator subunit expression profiles across 23 cancer entities (n = 8,568) using data from The Cancer Genome Atlas (TCGA). Prostate cancer–specific findings were validated in two publicly available gene expression cohorts and a large cohort of primary and advanced prostate cancer (n = 622) stained by immunohistochemistry. The role of CDK19 and CDK8 was evaluated by siRNA-mediated gene knockdown and inhibitor treatment in prostate cancer cell lines with functional assays and gene expression analysis by RNAseq. Results: Cluster analysis of TCGA expression data segregated tumor entities, indicating tumor-type–specific Mediator complex compositions. Only prostate cancer was marked by high expression of CDK19. In primary prostate cancer, CDK19 was associated with increased aggressiveness and shorter disease-free survival. During cancer progression, highest levels of CDK19 and of its paralog CDK8 were present in metastases. In vitro, inhibition of CDK19 and CDK8 by knockdown or treatment with a selective CDK8/CDK19 inhibitor significantly decreased migration and invasion. Conclusions: Our analysis revealed distinct transcriptional expression profiles of the Mediator complex across cancer entities indicating differential modes of transcriptional regulation. Moreover, it identified CDK19 and CDK8 to be specifically overexpressed during prostate cancer progression, highlighting their potential as novel therapeutic targets in advanced prostate cancer. Clin Cancer Res; 23(7); 1829–40. ©2016 AACR.

Head and neck squamous cell carcinoma (HNSCC) remains a clinical challenge and identification of novel therapeutic targets is necessary. The receptor tyrosine kinase AXL has been implicated in several tumor entities and a selective AXL small molecule inhibitor (BGB324) is currently being tested in clinical trials for patients suffering from non-small cell lung cancer or acute myeloid leukemia. Our study investigates AXL expression during HNSCC progression and its use as a potential therapeutic target in HNSCC. AXL protein expression was determined in a HNSCC cohort (n = 364) using immunohistochemical staining. For functional validation, AXL was either overexpressed or inhibited with BGB324 in HNSCC cell lines to assess proliferation, migration and invasion. We found AXL protein expression increasing during tumor progression with highest expression levels in recurrent tumors. In HNSCC cell lines in vitro, AXL overexpression increased migration as well as invasion. Both properties could be reduced through treatment with BGB324. In contrast, proliferation was neither affected by AXL overexpression nor by inhibition with BGB324. Our patient-derived data and in vitro results show that, in HNSCC, AXL is important for the progression to more advanced tumor stages. Moreover, they suggest that AXL could be a target for precision medicine approaches in this dismal tumor entity.

Although head and neck squamous cell carcinoma (HNSCC) is the sixth most common tumour entity worldwide, it remains a clinical challenge. Large-scale explorative genomic projects have identified several genes as potential targets for therapy, including fibroblast growth factor receptor 3 (FGFR3). The aim of this study was to investigate the biological significance of wild-type and mutated FGFR3 to evaluate its potential as a novel therapeutic target in HNSCC. FGFR3 protein expression was analysed in a large HNSCC tissue cohort (n = 536) and FGFR3 mRNA expression from The Cancer Genome Atlas (TCGA; n = 520). Moreover, FGFR3 wild-type and mutant versions were overexpressed in vitro, and both proliferation and migration was assessed with and without BGJ398 (a specific FGFR1-3 inhibitor) treatment. Although FGFR3 expression for both cohorts decreased during tumour progression, high FGFR3 expression levels were observed in a small subset of patients. In vitro, FGFR3 overexpression led to increased proliferation, whereas migration was not altered. Moreover, FGFR3-overexpressing cells were more sensitive to BGJ398. Cells overexpressing FGFR3 mutant versions showed increased proliferation compared to wild-type FGFR3 under serum-reduced conditions and were largely as sensitive as the wild-type protein to BGJ398. Taken together, the results of this study demonstrate that although FGFR3 expression decreases during HNSSC progression, it plays an important role in tumour cell proliferation and thus may be a potential target for therapy in selected patients suffering from this dismal tumour entity.

Squamous cell carcinoma of the head and neck (HNSCC) is the tenth most common tumor entity in men worldwide. Nevertheless therapeutic options are mostly limited to surgery and radio-chemotherapy resulting in 5-year survival rates of around 50%. Therefore new therapeutic options are urgently needed. During the last years, targeting of receptor tyrosine kinases has emerged as a promising strategy that can complement standard therapeutical approaches. Here, we aimed at investigating if the receptor tyrosine kinase DDR2 is a targetable structure in HNSCC. DDR2 expression was assessed on a large HNSCC cohort (554 patients) including primary tumors, lymph node metastases and recurrences and normal mucosa as control. Subsequently, DDR2 was stably overexpressed in two different cell lines (FaDu and HSC-3) using lentiviral technology. Different tumorigenic properties such as proliferation, migration, invasion, adhesion and anchorage independent growth were assessed with and without dasatinib treatment using in-vitro cell models and in-vivo zebrafish xenografts. DDR2 was overexpressed in all tumor tissues when compared to normal mucosa. DDR2 overexpression led to increased migration, invasion, adhesion and anchorage independent growth whereas proliferation remained unaltered. Upon dasatinib treatment migration, invasion and adhesion could be inhibited in-vitro and in-vivo whereas proliferation was unchanged. Our data suggest treatment with dasatinib as a promising new therapeutic option for patients suffering from DDR2 overexpressing HNSCC. Since dasatinib is already FDA-approved we propose to test this drug in clinical trials so that patients could directly benefit from this new treatment option.

// Anne von Massenhausen 1, 2, 3, * , Christine Sanders 1, 2, 3, * , Britta Thewes 1, 2, 3 , Mario Deng 4, 5 , Angela Queisser 1, 2, 3 , Wenzel Vogel 4, 5 , Glen Kristiansen 2, 3 , Stefan Duensing 6 , Andreas Schrock 3, 7 , Friedrich Bootz 3, 7 , Peter Brossart 3, 8 , Jutta Kirfel 2, 3 , Lynn Heasley 9 , Johannes Bragelmann 1, 3, 8, * , Sven Perner 4, 5 * 1 Section of Prostate Cancer Research, University Hospital of Bonn, Bonn, Germany 2 Institute of Pathology, University Hospital of Bonn, Bonn, Germany 3 Center for Integrated Oncology Cologne/Bonn, University Hospital of Bonn, Bonn, Germany 4 Pathology of the University Hospital of Luebeck, Luebeck, Germany 5 Leibniz Research Center Borstel, Borstel, Germany 6 Department of Urology, University of Heidelberg, Heidelberg, Germany 7 Department of Otorhinolaryngology/Head and Neck Surgery, University Hospital of Bonn, Bonn, Germany 8 Department of Hematology/Oncology, University Hospital of Bonn, Bonn, Germany 9 Department of Craniofacial Biology, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA * These authors have contributed equally to this work Correspondence to: Sven Perner, email: Sven.Perner@uksh.de Keywords: head and neck cancer, MERTK, targeted therapy Abbreviations: HNSCC, head and neck squamous cell carcinoma; MERTK, MER proto-oncogene tyrosine kinase; FAK, focal adhesion kinase; TCGA, The Cancer Genome Atlas Received: January 07, 2016 Accepted: March 28, 2016 Published: April 13, 2016 ABSTRACT Although head and neck cancer (HNSCC) is the sixth most common tumor entity worldwide therapy options remain limited leading to 5-year survival rates of only 50 %. MERTK is a promising therapeutic target in several tumor entities, however, its role in HNSCC has not been described yet. The aim of our study was to investigate the biological significance of MERTK and to evaluate its potential as a novel therapeutic target in this dismal tumor entity. In two large HNSCC cohorts (n=537 and n=520) we found that MERTK is overexpressed in one third of patients. In-vitro, MERTK overexpression led to increased proliferation, migration and invasion whereas MERTK inhibition with the small molecule inhibitor UNC1062 or MERTK knockdown reduced cell motility via the small GTPase RhoA. Taken together, we are the first to show that MERTK is frequently overexpressed in HNSCC and plays an important role in tumor cell motility. It might therefore be a potential target for selected patients suffering from this dismal tumor entity.