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Recently, necroptosis, as a programmed cell death pathway, has drawn much attention as it has been implicated in multiple pathologies, especially in the field of inflammatory diseases. Pseudokinase mixed lineage kinase domain-like protein (MLKL) serves as a terminal-known obligate effector in the process of necroptosis. To date, the majority of research on MLKL has focused on its role in necroptosis, and the prevailing view has been that the sole function of MLKL is to mediate necroptosis. However, increasing evidence indicates that MLKL can serve as a regulator of many diseases via its non-necroptotic functions. These functions of MLKL shed light on its functional complexity and diversity. In this review, we briefly introduce the current state of knowledge regarding the structure of MLKL, necroptosis signaling, as well as cross-linkages among necroptosis and other regulated cell death pathways, and we particularly highlight recent progress related to newly identified functions and inhibitors of MLKL. These discussions promote a better understanding of the role of MLKL in diseases, which will foster efforts to pharmacologically target this molecule in clinical treatments.

Significance A major yet perplexing question in the field of necroptosis is the role and involvement of necroptosis in cancer cells. Many cancer cells have protective mechanisms against necroptosis, but the underlying mechanism remains elusive. We report findings of cross-talk and a regulatory pathway that exist between MYC, a potent oncogene, and RIPK3, a pivotal factor in necroptosis. We find that MYC pathway is downregulated upon necroptotic, while MYC inhibits TNF-α–induced necroptosis. The inhibitory effect of MYC on necroptosis is unexpected because no transcriptional activity by MYC is required. Mechanistically, a direct interaction between MYC and RIPK3 takes place in the cytosol, preventing RIPK1–RIPK3 complex formation. Finally, MYC depletion enhances antitumor activity of necroptosis-inducing agents in a xenograft model., The underlying mechanism of necroptosis in relation to cancer is still unclear. Here, MYC, a potent oncogene, is an antinecroptotic factor that directly suppresses the formation of the RIPK1–RIPK3 complex. Gene set enrichment analyses reveal that the MYC pathway is the most prominently down-regulated signaling pathway during necroptosis. Depletion or deletion of MYC promotes the RIPK1–RIPK3 interaction, thereby stabilizing the RIPK1 and RIPK3 proteins and facilitating necroptosis. Interestingly, MYC binds to RIPK3 in the cytoplasm and inhibits the interaction between RIPK1 and RIPK3 in vitro. Furthermore, MYC-nick, a truncated form that is mainly localized in the cytoplasm, prevented TNF-induced necroptosis. Finally, down-regulation of MYC enhances necroptosis in leukemia cells and suppresses tumor growth in a xenograft model upon treatment with birinapant and emricasan. MYC-mediated suppression of necroptosis is a mechanism of necroptosis resistance in cancer, and approaches targeting MYC to induce necroptosis represent an attractive therapeutic strategy for cancer.

Necroptosis is a form of regulated cell death caused by formation of the necrosome complex. However, the factors modulating this process and the systemic pathophysiological effects of necroptosis are yet to be understood. Here, we identified that Beclin 1 functions as an anti-necroptosis factor by being recruited into the necrosome complex upon treatment with TNFα, Smac mimetic, and pan-caspase inhibitor and by repressing MLKL oligomerisation, thus preventing the disruption of the plasma membrane. Cells ablated or knocked-out for Beclin 1 become sensitised to necroptosis in an autophagy-independent manner without affecting the necrosome formation itself. Interestingly, the recruitment of Beclin 1 into the necrosome complex is dependent on the activation and phosphorylation of MLKL. Biochemically, the coiled-coil domain (CCD) of Beclin 1 binds to the CCD of MLKL, which restrains the oligomerisation of phosphorylated MLKL. Finally, Beclin 1 depletion was found to promote necroptosis in leukaemia cells and enhance regression of xenografted-tumour upon treatment with Smac mimetics and caspase inhibitors. These results suggest that Beclin 1 functions as a negative regulator in the execution of necroptosis by suppressing MLKL oligomerisation.

Background: Previous studies have suggested that necroptosis, a programmed necrosis mechanism, is involved in the pathogenesis and outcomes of sepsis. Mixed lineage kinase domain-like protein (MLKL) is a final executor of necroptosis. However, association MLKL with severity and mortality of sepsis is not well known. Objectives: The aim of the study was to assess the association of plasma MLKL with severity and outcome of sepsis. Methods: We analyzed data of 188 prospectively-enrolled critically-ill patients admitted to the intensive care unit (ICU) of Samsung Medical Center. Plasma MLKL level was measured with the enzyme-linked immunosorbent assay. Results: Of 188 critically-ill patients, 58 (30.9%) and 84 (44.7%) patients were diagnosed with sepsis and septic shock, respectively. Plasma level of MLKL increased over the groups of control, sepsis, and septic shock (1.10 ng/mL vs. 2.03 ng/mL vs. 3.00 ng/mL, P for trend Conclusions: Plasma level of MLKL was associated with severity of sepsis and predictive of mortality.

Butylate hydroxyanisole (BHA) is a synthetic phenol that is widely utilized as a preservative by the food and cosmetic industries. The antioxidant properties of BHA are also frequently used by scientists to claim the implication of reactive oxygen species (ROS) in various cellular processes, including cell death. We report on the surprising finding that BHA functions as a direct inhibitor of RIPK1, a major signaling hub downstream of several immune receptors. Our in silico analysis predicts binding of 3-BHA, but not 2-BHA, to RIPK1 in an inactive DLG-out/Glu-out conformation, similar to the binding of the type III inhibitor Nec-1s to RIPK1. This predicted superior inhibitory capacity of 3-BHA over 2-BHA was confirmed in cells and using in vitro kinase assays. We demonstrate that the reported protective effect of BHA against tumor necrosis factor (TNF)-induced necroptotic death does not originate from ROS scavenging but instead from direct RIPK1 enzymatic inhibition, a finding that most probably extends to other reported effects of BHA. Accordingly, we show that BHA not only protects cells against RIPK1-mediated necroptosis but also against RIPK1 kinase-dependent apoptosis. We found that BHA treatment completely inhibits basal and induced RIPK1 enzymatic activity in cells, monitored at the level of TNFR1 complex I under apoptotic conditions or in the cytosol under necroptosis. Finally, we show that oral administration of BHA protects mice from RIPK1 kinase-dependent lethality caused by TNF injection, a model of systemic inflammatory response syndrome. In conclusion, our results demonstrate that BHA can no longer be used as a strict antioxidant and that new functions of RIPK1 may emerge from previously reported effects of BHA.

AIM: Granulovacuolar degeneration (GVD) in Alzheimer's disease (AD) involves the necrosome, which is a protein complex consisting of phosphorylated receptor-interacting protein kinase 1 (pRIPK1), pRIPK3 and phosphorylated mixed lineage kinase domain-like protein (pMLKL). Necrosome-positive GVD was associated with neuron loss in AD. GVD was recently linked to the C9ORF72 mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with transactive response DNA-binding protein (TDP-43) pathology (FTLD-TDP). Therefore, we investigated whether GVD in cases of the ALS-FTLD-TDP spectrum (ALS/FTLD) shows a similar involvement of the necrosome as in AD, and whether it correlates with diagnosis, presence of protein aggregates and cell death in ALS/FTLD. METHODS: We analysed the presence and distribution of the necrosome in post-mortem brain and spinal cord of ALS and FTLD-TDP patients (n = 30) with and without the C9ORF72 mutation, and controls (n = 22). We investigated the association of the necrosome with diagnosis, the presence of pathological protein aggregates and neuronal loss. RESULTS: Necrosome-positive GVD was primarily observed in hippocampal regions of ALS/FTLD cases and was associated with hippocampal TDP-43 inclusions as the main predictor of the pMLKL-GVD stage, as well as with the Braak stage of neurofibrillary tangle pathology. The central cortex and spinal cord, showing motor neuron loss in ALS, were devoid of any accumulation of pRIPK1, pRIPK3 or pMLKL. CONCLUSIONS: Our findings suggest a role for hippocampal TDP-43 pathology as a contributor to necrosome-positive GVD in ALS/FTLD. The absence of necroptosis-related proteins in motor neurons in ALS argues against a role for necroptosis in ALS-related motor neuron death. ispartof: NEUROPATHOLOGY AND APPLIED NEUROBIOLOGY vol:47 issue:2 pages:328-345 ispartof: location:England status: published

Objectives: During the advancement of atherosclerosis, plaque cellularity is governed by the influx of monocyte-derived macrophages and their turnover via apoptotic and nonapoptotic forms of cell death. Previous reports have demonstrated that programmed necrosis, or necroptosis, of plaque macrophages contribute to necrotic core formation. Knockdown or inhibition of the necrosome components RIPK1 (receptor-interacting protein kinase 1) and RIPK3 (receptor-interacting protein kinase 3) slow atherogenesis, and activation of the terminal step of necroptosis, MLKL (mixed lineage kinase domain-like protein), has been demonstrated in advanced human atherosclerotic plaques. However, whether MLKL directly contributes to lesion development and necrotic core formation has not been investigated. Approaches and Results: MLKL expression was knocked down in atherogenic Apoe -knockout mice via the administration of antisense oligonucleotides. During atherogenesis, Mlkl knockdown decreased both programmed cell death and the necrotic core in the plaque. However, total lesion area remained unchanged. Furthermore, treatment with the MLKL antisense oligonucleotide unexpectedly reduced circulating cholesterol levels compared with control antisense oligonucleotide but increased the accumulation of lipids within the plaque and in vitro in macrophage foam cells. MLKL colocalized with the late endosome and multivesicular bodies in peritoneal macrophages incubated with atherogenic lipoproteins. Transfection with MLKL antisense oligonucleotide increased lipid localization with the multivesicular bodies, suggesting that upon Mlkl knockdown, lipid trafficking becomes defective leading to enhanced lipid accumulation in macrophages. Conclusions: These studies confirm the requirement for MLKL as the executioner of necroptosis, and as such a significant contributor to the necrotic core during atherogenesis. We also identified a previously unknown role for MLKL in regulating endosomal trafficking to facilitate lipid handling in macrophages during atherogenesis.

Several non-apoptotic regulated cell death pathways have been recently reported. Necroptosis, a form of necrotic-regulated cell death, is characterized by the involvement of receptor-interacting protein kinases and/or the pore-forming mixed lineage kinase domain-like protein. Recent evidence suggests a key role for lipidic molecules in the regulation of necroptosis. The purpose of this mini-review is to outline the regulation of necroptosis by sphingolipids and phospholipids.

Necroptotic signaling converges in the assembly of a cytosolic signaling platform, the necrosome, with the activation of its downstream effector, MLKL. RIPK1 and RIPK3, key components of the necrosome, act as signaling intermediates for the activation of MLKL. We report that RIPK3 and MLKL continuously shuttle between the nucleus and the cytoplasm, whereas RIPK1 is constitutively present in both compartments. During TNF-induced necroptosis, nuclear RIPK1 becomes ubiquitinated, after which nuclear MLKL becomes phosphorylated and oligomerized. Pharmacological inhibition of the nuclear export machinery leads to retention of RIPK3 and MLKL in the nucleus, prevents the nucleation of cytosolic RIPK3/MLKL oligomerization, and reduces cell death. Our results suggest that passage of necroptotic signaling components through the nucleus is a mechanism for regulating cytosolic necrosome formation and consequently necroptotic cell death.

// Friederike Feldmann 1, 2, 3 , Barbara Schenk 1 , Sofie Martens 4, 5 , Peter Vandenabeele 4, 5 and Simone Fulda 1, 2, 3 1 Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany 2 German Cancer Consortium (DKTK), Partner Site, Frankfurt, Germany 3 German Cancer Research Center (DKFZ), Heidelberg, Germany 4 Inflammation Research Center, VIB, Ghent, Belgium 5 Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Correspondence to: Simone Fulda, email: simone.fulda@kgu.de Keywords: necroptosis, cell death, Sorafenib, Smac, leukemia Received: June 09, 2017 Accepted: July 25, 2017 Published: August 04, 2017 ABSTRACT Induction of necroptosis has emerged as an alternative approach to trigger programmed cell death, in particular in apoptosis-resistant cancer cells. Recent evidence suggests that kinase inhibitors targeting oncogenic B-RAF can also affect Receptor-interacting serine/threonine-protein kinase (RIP)1 and RIP3. Sorafenib, a multi-targeting kinase inhibitor with activity against B-RAF, is used for the treatment of acute leukemia. In the present study, we therefore investigated whether Sorafenib interferes with therapeutic induction of necroptosis in acute leukemia. Here, we report that Sorafenib inhibits necroptotic signaling and cell death in two models of necroptosis in acute leukemia. Sorafenib significantly reduces Second mitochondria-derived activator of caspases (Smac) mimetic-induced necroptosis in apoptosis-resistant acute myeloid leukemia (AML) cells as well as Smac mimetic/Tumor Necrosis Factor (TNF)α-induced necroptosis in FADD-deficient acute lymphoblastic leukemia (ALL) cells. Sub- to low micromolar concentrations of Sorafenib corresponding to its plasma levels reported in cancer patients are sufficient to inhibit necroptosis, emphasizing the clinical relevance of our findings. Furthermore, Sorafenib blocks Smac mimetic-mediated phosphorylation of mixed-lineage kinase domain-like protein (MLKL) that marks its activation, indicating that Sorafenib targets components upstream of MLKL such as RIP1 and RIP3. Intriguingly, Sorafenib reduces the Smac mimetic/TNFα-stimulated interaction of RIP1 with RIP3 and MLKL, demonstrating that it interferes with the assembly of the necrosome complex. Importantly, Sorafenib significantly protects primary, patient-derived AML blasts from Smac mimetic-induced necroptosis. By demonstrating that Sorafenib limits the anti-leukemic activity of necroptosis-inducing drugs in acute leukemia cells, our study has important implications for the use of Sorafenib in the treatment of acute leukemia.

Immune-checkpoint blockers (ICBs) have revolutionized oncology and firmly established the subfield of immuno-oncology. Despite this renaissance, a subset of cancer patients remain unresponsive to ICBs due to widespread immuno-resistance. To "break" cancer cell-driven immuno-resistance, researchers have long floated the idea of therapeutically facilitating the immunogenicity of cancer cells by disrupting tumor-associated immuno-tolerance via conventional anticancer therapies. It is well appreciated that anticancer therapies causing immunogenic or inflammatory cell death are best positioned to productively activate anticancer immunity. A large proportion of studies have emphasized the importance of immunogenic apoptosis (i.e., immunogenic cell death or ICD); yet, it has also emerged that necroptosis, a programmed necrotic cell death pathway, can also be immunogenic. Emergence of a proficient immune profile for necroptosis has important implications for cancer because resistance to apoptosis is one of the major hallmarks of tumors. Putative immunogenic or inflammatory characteristics driven by necroptosis can be of great impact in immuno-oncology. However, as is typical for a highly complex and multi-factorial disease like cancer, a clear cause versus consensus relationship on the immunobiology of necroptosis in cancer cells has been tough to establish. In this review, we discuss the various aspects of necroptosis immunobiology with specific focus on immuno-oncology and cancer immunotherapy. ispartof: CELLS vol:9 issue:8 ispartof: location:Switzerland status: published

Alzheimer's disease (AD) is characterized by a specific pattern of neuropathological changes, including extracellular amyloid β (Aβ) deposits, intracellular neurofibrillary tangles (NFTs), granulovacuolar degeneration (GVD) representing cytoplasmic vacuolar lesions, synapse dysfunction and neuronal loss. Necroptosis, a programmed form of necrosis characterized by the assembly of the necrosome complex composed of phosphorylated proteins, i.e. receptor-interacting serine/threonine-protein kinase 1 and 3 (pRIPK1 and pRIPK3) and mixed lineage kinase domain-like protein (pMLKL), has recently been shown to be involved in AD. However, it is not yet clear whether necrosome assembly takes place in brain regions showing AD-related neuronal loss and whether it is associated with AD-related neuropathological changes. Here, we analyzed brains of AD, pathologically defined preclinical AD (p-preAD) and non-AD control cases to determine the neuropathological characteristics and distribution pattern of the necrosome components. We demonstrated that all three activated necrosome components can be detected in GVD lesions (GVDn+, i.e. GVD with activated necrosome) in neurons, that they colocalize with classical GVD markers, such as pTDP-43 and CK1δ, and similarly to these markers detect GVD lesions. GVDn + neurons inversely correlated with neuronal density in the early affected CA1 region of the hippocampus and in the late affected frontal cortex layer III. Additionally, AD-related GVD lesions were associated with AD-defining parameters, showing the strongest correlation and partial colocalization with NFT pathology. Therefore, we conclude that the presence of the necrosome in GVD plays a role in AD, possibly by representing an AD-specific form of necroptosis-related neuron death. Hence, necroptosis-related neuron loss could be an interesting therapeutic target for treating AD. ispartof: ACTA NEUROPATHOLOGICA vol:139 issue:3 pages:463-484 ispartof: location:Germany status: published

Necroptosis, an important form of programmed cell death (PCD), is a highly regulated caspase-independent type of cell death that plays a critical role in the pathophysiology of various inflammatory, infectious, and degenerative diseases. Currently, receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) have been widely recognized as critical therapeutic targets of the necroptotic machinery. Targeting RIPK1, RIPK3, and/or MLKL is a promising strategy for necroptosis-related diseases. Following the identification of the first RIPK1 inhibitor Nec-1 in 2005, the antinecroptosis field is attracting increasing research interest from multiple disciplines, including the biological and medicinal chemistry communities. Herein, we will review the functions of necroptosis in human diseases, as well as the related targets and representative small-molecule inhibitors, mainly focusing on research articles published during the past 10 years. Outlooks and perspectives on the associated challenges are also discussed.

Although necrosis and necroinflammation are central features of many liver diseases, the role of programmed necrosis in the context of inflammation-dependent hepatocellular death remains to be fully determined. Here, we have demonstrated that the pseudokinase mixed lineage kinase domain-like protein (MLKL), which plays a key role in the execution of receptor interacting protein (RIP) lcinase-dependent necroptosis, is upregulated and activated in human autoimmune hepatitis and in a murine model of inflammation-dependent hepatitis. Using genetic and pharmacologic approaches, we determined that hepatocellular necrosis in experimental hepatitis is driven by an MLKL-dependent pathway that occurs independently of RIPK3. Moreover, we have provided evidence that the cytotoxic activity of the proinflammatory cytokine IFN-gamma in hepatic inflammation is strongly connected to induction of MLKL expression via activation of the transcription factor STAT1. In summary, our results reveal a pathway for MLKL-dependent programmed necrosis that is executed in the absence of RIPK3 and potentially drives the pathogenesis of severe liver diseases.

Mixed lineage kinase domain-like protein (MLKL) is the proposed executioner of necroptosis. Our recent findings identify a novel inhibitor necroptosis-blocking compound 1 (NBC1) which specifically conjugates to two cysteines of heat shock protein 70 (HSP70) to block its function. Importantly, HSP70 promotes MLKL polymerization to activate necroptosis.

International audience; Cells may die from accidental cell death (ACD) or regulated cell death (RCD). ACD is a biologically uncontrolled process, whereas RCD involves tightly structured signaling cascades and molecularly defined effector mechanisms. A growing number of novel non-apoptotic forms of RCD have been identified and are increasingly being implicated in various human pathologies. Here, we critically review the current state of the art regarding non-apoptotic types of RCD, including necroptosis, pyroptosis, ferroptosis, entotic cell death, netotic cell death, parthanatos, lysosome-dependent cell death, autophagy-dependent cell death, alkaliptosis and oxeiptosis. The in-depth comprehension of each of these lethal subroutines and their intercellular consequences may uncover novel therapeutic targets for the avoidance of pathogenic cell loss.

Cancer immunotherapy can induce durable antitumor responses. However, many patients poorly respond to such therapies. Here we describe a generic antitumor therapy that is based on the intratumor delivery of mRNA that codes for the necroptosis executioner mixed lineage kinase domain-like (MLKL) protein. This intervention stalls primary tumor growth and protects against distal and disseminated tumor formation in syngeneic mouse melanoma and colon carcinoma models. Moreover, MLKL-mRNA treatment combined with immune checkpoint blockade further improves the antitumor activity. MLKL-mRNA treatment rapidly induces T cell responses directed against tumor neo-antigens and requires CD4+ and CD8+ T cells to prevent tumor growth. Type I interferon signaling and Batf3-dependent dendritic cells are essential for this mRNA treatment to elicit tumor antigen-specific T cell responses. Moreover, MLKL-mRNA treatment blunts the growth of human lymphoma in mice with a reconstituted human adaptive immune system. MLKL-based treatment can thus be exploited as an effective antitumor immunotherapy., Necroptosis has immunogenic cell death properties. Here, the authors show that the intra-tumor delivery of mRNA that codes for the necroptosis effector MLKL triggers neo-epitope-specific anti-tumor T cell responses and inhibits primary tumor growth and lung metastasis.

Necroptosis is a caspase-independent form of programmed cell death that is induced by a variety of different signalling cascades-all culminating in the activation of the pseudokinase mixed lineage kinase domain-like (MLKL). TNF-induced necroptosis is the most intensively studied of these pathways. Here we describe reagents and cell-based techniques that can be used to investigate TNF-mediated necroptosis in the lab.

Programmed necrosis, also known as necroptosis, is a form of regulated necrotic cell death that is mediated by receptor-interacting protein kinases RIP1 (or RIPK1), RIP3 (or RIPK3), and the mixed lineage kinase domain-like protein, MLKL. Following the induction of programmed necrosis, MLKL is phosphorylated by RIP3 and oligomerizes and then the protein translocates to cell plasma membrane in order to execute programmed necrosis. Here, we describe a detailed protocol to detect MLKL oligomerization in necroptotic cells by Western blotting analysis under nonreducing condition. Therefore, we established the method to detect the activation of programmed necrotic pathway.

We report the development of novel Mixed Lineage Kinase Domain-Like protein (MLKL) inhibitors with single nanomolar potency (compound 15 is also named as TC13172). Using the converting biochemistry to chemistry activity-based protein profiling (BTC-ABPP) method, we were able to determine that the inhibitors covalently bind to Cysteine86 (Cys-86) of MLKL. This is the first example of the use of LC-MS/MS to identify the binding site of an MLKL inhibitor. The novel MLKL inhibitors provide powerful tools to study the biological function of MLKL and demonstrate that MLKL should be viewed as a druggable target.

In this issue of Blood, Wagner and colleagues implicate Rip1 kinase (Ripk1)–mediated necroptosis in myelodysplastic syndrome (MDS)-like disease in mice and detect increased RIPK1 expression and mixed lineage kinase domain-like pseudokinase (MLKL) activation in human MDS samples.1

SummaryAlthough mixed lineage kinase domain-like (MLKL) protein has emerged as a specific and crucial protein for necroptosis induction, how MLKL transduces the death signal remains poorly understood. Here, we demonstrate that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death. Moreover, we found that a patch of positively charged amino acids on the surface of the 4HBD binds to phosphatidylinositol phosphates (PIPs) and allows recruitment of MLKL to the plasma membrane. Importantly, we found that recombinant MLKL, but not a mutant lacking these positive charges, induces leakage of PIP-containing liposomes as potently as BAX, supporting a model in which MLKL induces necroptosis by directly permeabilizing the plasma membrane. Accordingly, we found that inhibiting the formation of PI(5)P and PI(4,5)P2 specifically inhibits tumor necrosis factor (TNF)-mediated necroptosis but not apoptosis.

Through high-throughput screening of 200 000 compounds and subsequent structure–activity relationship (SAR) studies we identified necrosulfonamide (NSA) as a potent small molecule inhibitor for necroptosis, induced by a combination of TNF-a, Smac mimetic, and z-VAD-fmk (T/S/Z). Applying a forward chemical genetic approach, we utilized an NSA based chemical probe to further reveal that NSA selectively targeted the Mixed Lineage Kinase Domain-like Protein (MLKL) to block the necrosome formation.

Cell death and inflammation in the proximal tubules are the hallmarks of acute kidney injury (AKI), but the underlying mechanism has not been fully elucidated. Recent evidence has shown that necroptosis, a type of programmed necrosis, plays an important role in AKI. The necrosis-inducing signaling complex is called the necrosome, which contains receptor-interacting protein 1, receptor-interacting protein 3, and mixed lineage kinase domain-like protein. Studies have found that inhibition of the core components of the necroptotic pathway by gene knockout, RNA interference, or a chemical inhibitor diminished proximal tubule damage, showing that necroptosis is a major contributor to AKI. This review focuses on the functional roles of the necrosome in regulating renal tubular cell necroptosis, and the physiological and pathologic roles of necrosome in AKI.

Necroptosis was initially identified as a backup cell death program when apoptosis is blocked. However, it is now recognized as a cellular defense mechanism against infections and is presumed to be a detrimental factor in several pathologies driven by cell death. Necroptosis is a prototypic form of regulated necrosis that depends on activation of the necrosome, which is a protein complex in which receptor interacting protein kinase (RIPK) 3 is activated. The RIP homotypic interaction motif (RHIM) is the core domain that regulates activation of the necrosome. To date, three RHIM-containing proteins have been reported to activate the kinase activity of RIPK3 within the necrosome: RIPK1, Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF), and DNA-dependent activator of interferon regulatory factors (DAI). Here, we review and discuss commonalities and differences of the increasing number of activators of the necrosome. Since the discovery that activation of mixed lineage kinase domain-like (MLKL) by RIPK3 kinase activity is crucial in necroptosis, interest has increased in monitoring and therapeutically targeting their activation. The availability of new phospho-specific antibodies, pharmacologic inhibitors, and transgenic models will allow us to further document the role of necroptosis in degenerative, inflammatory and infectious diseases.

Whether mixed lineage kinase domain-like (MLKL) mediates necroptosis by forming ion channel, none-selective pore, or simply by disrupting plasma membrane is unclear. In a paper published in recent issue of Cell Research, Xia et al. showed that MLKL functions as a novel class of cation channel.

Our current knowledge of the molecular mechanisms regulating the signaling pathways leading to cell survival, cell death, and inflammation has shed light on the tight mutual interplays between these processes. Moreover, the fact that both apoptosis and necrosis can be molecularly controlled has greatly increased our interest in the roles that these types of cell death play in the control of general processes such as development, homeostasis, and inflammation. In this review, we provide a brief update on the different cell death modalities and describe in more detail the intracellular crosstalk between survival, apoptotic, necroptotic, and inflammatory pathways that are activated downstream of death receptors. An important concept is that the different cell death processes modulate each other by mutual inhibitory mechanisms, serve as alternative back-up death routes in the case of a defect in the first-line cell death response, and are controlled by multiple feedback loops. We conclude by discussing future perspectives and challenges in the field of cell death and inflammation research.

Necroptosis contributes to the pathophysiology of several inflammatory, infectious and degenerative disorders. TNF-induced necroptosis involves activation of the receptor-interacting protein kinases 1 and 3 (RIPK1/3) in a necrosome complex, eventually leading to the phosphorylation and relocation of mixed lineage kinase domain like protein (MLKL). Using a high-content screening of small compounds and FDA-approved drug libraries, we identified the anti-cancer drug Sorafenib tosylate as a potent inhibitor of TNF-dependent necroptosis. Interestingly, Sorafenib has a dual activity spectrum depending on its concentration. In murine and human cell lines it induces cell death, while at lower concentrations it inhibits necroptosis, without affecting NF-κB activation. Pull down experiments with biotinylated Sorafenib show that it binds independently RIPK1, RIPK3 and MLKL. Moreover, it inhibits RIPK1 and RIPK3 kinase activity. In vivo Sorafenib protects against TNF-induced systemic inflammatory response syndrome (SIRS) and renal ischemia–reperfusion injury (IRI). Altogether, we show that Sorafenib can, next to the reported Braf/Mek/Erk and VEGFR pathways, also target the necroptotic pathway and that it can protect in an acute inflammatory RIPK1/3-mediated pathology.

Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomain-helicase-DNA binding protein 5, chromodomain-helicase-DNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation, and should be validated in future studies.

The pseudokinase domain of the necroptosis effector mixed lineage kinase domain-like (MLKL) functions as a latch to restrain the unleashing of its N-terminal 4-helix bundle (4HB) domain. Cell death mediated by the 4HB domain relies on membrane association and oligomerization, which can be inhibited by an ATP-mimetic small molecule that binds the pseudokinase domain of MLKL.

In human cells, the RIPK1-RIPK3-MLKL-PGAM5-Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types. ispartof: Cell Death & Disease vol:5 issue:1 ispartof: location:England status: published

Endoplasmic reticulum (ER) stress-induced cellular dysfunction and death is associated with several human diseases. It has been widely reported that ER stress kills through activation of the intrinsic mitochondrial apoptotic pathway. Here we demonstrate that ER stress can also induce necroptosis, an receptor-interacting protein kinase 1 (RIPK1)/RIPK3/mixed lineage kinase domain-like protein (MLKL)-dependent form of necrosis. Remarkably, we observed that necroptosis induced by various ER stressors in L929 cells is dependent on tumor necrosis factor receptor 1 (TNFR1), but occurs independently of autocrine TNF or lymphotoxin α production. Moreover, we found that repression of either TNFR1, RIPK1 or MLKL did not protect the cells from death but instead allowed a switch to ER stress-induced apoptosis. Interestingly, while caspase inhibition was sufficient to protect TNFR1- or MLKL-deficient cells from death, rescue of the RIPK1-deficient cells additionally required RIPK3 depletion, indicating a switch back to RIPK3-dependent necroptosis in caspase-inhibited conditions. The finding that ER stress also induces necroptosis may open new therapeutic opportunities for the treatment of pathologies resulting from unresolved ER stress.