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Regulated cell death (RCD) relies on activation and recruitment of pore-forming proteins (PFPs) that function as executioners of specific cell death pathways: apoptosis regulator BAX (BAX), BCL-2 homologous antagonist/killer (BAK) and BCL-2-related ovarian killer protein (BOK) for apoptosis, gasdermins (GSDMs) for pyroptosis and mixed lineage kinase domain-like protein (MLKL) for necroptosis. Inactive precursors of PFPs are converted into pore-forming entities through activation, membrane recruitment, membrane insertion and oligomerization. These mechanisms involve protein-protein and protein-lipid interactions, proteolytic processing and phosphorylation. In this Review, we discuss the structural rearrangements incurred by RCD-related PFPs and describe the mechanisms that manifest conversion from autoinhibited to membrane-embedded molecular states. We further discuss the formation and maturation of membrane pores formed by BAX/BAK/BOK, GSDMs and MLKL, leading to diverse pore architectures. Lastly, we highlight commonalities and differences of PFP mechanisms involving BAX/BAK/BOK, GSDMs and MLKL and conclude with a discussion on how, in a population of challenged cells, the coexistence of cell death modalities may have profound physiological and pathophysiological implications.

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.

Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells. ispartof: ISCIENCE vol:25 issue:10 ispartof: location:United States status: published

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.

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.

// 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.

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.

Background: Mixed lineage kinase 3 (MLK3) opposes pathologic cardiac remodeling, but its role in blood pressure (BP) has not been studied. MLK3 activates JNK signaling through kinase-dependent effects, and opposes RhoA activation through kinase-independent mechanisms, but the relevance of these mechanisms to BP is unknown. We investigated the effect of genetic deletion of MLK3 on BP. Methods and Results: Using ambulatory telemetric monitoring in 3 month old male mice, MLK3 -/- mice had significant hypertension compared to wild type (WT) littermates (WT systolic BP 121 ± 2 mmHg, MLK3-/- 162 ± 5 mmHg; n=3; p Summary and Conclusions: These data demonstrate that MLK3 deletion leads to hypertension with increased arterial stiffness, reduced distensibility and eutrophic remodeling of resistance vessels, but retained BP responsiveness to downstream ROCK inhibition. Together with previous work, these findings support that MLK3 modulates cardiac remodeling through a kinase dependent mechanism while modulating blood pressure through kinase-independent effects. Because hypotension limits many heart failure therapies, delineating vascular versus cardiac mechanisms of MLK3 signaling has the potential to suggest novel approaches to heart failure treatment.

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.

The dual leucine zipper kinase (DLK, MAP3K12) is essential for neuronal development and has been shown to mediate axon regeneration. On the other hand, DLK is involved in the pathogenesis of neurodegenerative disease and diabetes mellitus. Several patents have been published claiming to modulate or inhibit DLK by various approaches including ATP competitive inhibitors. In addition, two publications describe SAR of highly selective DLK inhibitors with efficacy in distinct mouse models of neurodegeneration.This review summarized patents claiming to modulate DLK activity published between 2010 and 2015. Peer-reviewed publications related to the patents and additional peer-reviewed publications are included. This article describes 18 patents from three pharmaceutical companies and three academic research groups.Several methods are proposed to modulate DLK activity, some of them very experimental and not suitable for easy application in patients. ATP competitive kinase inhibitors exert high affinity, but for the majority, no information about their selectivity is available. To date, two inhibitors have been tested in mice. Given the controversial findings that DLK is required for neurodegeneration and for axon regeneration, more research is needed to further elucidate the regulation and the function of this kinase in diverse organs/tissues and under physiological and pathological conditions.

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.

Background Many anti-remodeling signaling molecules also modulate blood pressure. Thus, therapeutic modulation of these potential therapeutic targets has been limited by hypotension. Mixed lineage kinase 3 (MLK3) opposes pathologic cardiac remodeling, but its role in blood pressure (BP) has not been studied. MLK3 activates JNK signaling through kinase dependent effects, and opposes RhoA activation through kinase-independent mechanisms, but the relevance of these mechanisms to BP is unknown. We investigated the effect of genetic deletion of MLK3 on BP. Methods and Results Using ambulatory telemetric monitoring in 3 month old male mice, MLK3 -/- mice had significant hypertension compared to wild type (WT) littermates (WT systolic BP 121 ± 2 mmHg, MLK3-/- 162 ± 5 mmHg; n=3; p Conclusions These data demonstrate that MLK3 deletion leads to hypertension with increased arterial stiffness, reduced distensibility and eutrophic remodeling of resistance vessels, but retained BP responsiveness to downstream ROCK inhibition. Together with previous work, these findings support that MLK3 modulates cardiac remodeling through a kinase dependent mechanism while modulating blood pressure through kinase-independent effects. Because hypotension limits many heart failure therapies, delineating vascular versus cardiac mechanisms of MLK3 signaling has the potential to suggest novel approaches to heart failure treatment.

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.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ?accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. "Regulated cell death" (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Nomenclature Committee on Cell Death. Equipe 11 Apoptose, Cancer et Immunité. Centre de Recherche des Cordeliers; Francia

Leucine-rich repeat kinase 2 (LRRK2) has attracted considerable interest as a therapeutic target for the treatment of Parkinson's disease. Compounds derived from a 2-aminopyridine screening hit were optimised using a LRRK2 homology model based on mixed lineage kinase 1 (MLK1), such that a 2-aminopyridine-based lead molecule 45, with in vivo activity, was identified.

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

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.

Hepatocellular carcinoma (HCC) is one of the leading cause of cancer-related deaths worldwide. Of the estimated 40,710 new cases of liver cancers diagnosed in 2017, about three-fourths were HCCs. Surgery, liver transplantation, tumor ablation and chemotherapy are available treatment options if detected at an early stage. However, the only therapeutic option currently available for advanced HCCs is Sorafenib which is only effective for a few months. There is thus an urgent need for the development of safe and more efficacious treatment options for HCC. MLK3 is a member of the mitogen-activated protein kinase (MAPK) family that can activate JNK pathway to induce cell death in neuronal and non-neuronal cells. Several studies have shown that MLK3 plays a significant role in cancer invasion, migration and metastasis. Earlier studies from our laboratory have demonstrated that MLK3 can induce the expression of β-catenin and regulate β-catenin downstream signaling. The current studies were designed to obtain further mechanistic insight on MLK3-β-catenin crosstalk and elucidate the detailed mechanism. To address this, our first goal was to map the domain(s) of β-catenin that mediate MLK3-induced stabilization and interaction by creating β-catenin deletion constructs with N terminal or C-terminal deletions. Overexpression studies designed with these revealed that AA 51-130 of β-catenin was important to mediate MLK3-induced stabilization. In addition, initial Immunoprecipitation and Immunoblotting combination studies also suggest this domain of β-catenin might be mediating the interaction with MLK3. Since our earlier studies suggested that MLK3-induced stabilization of β-catenin was dependent on its kinase activity, studies are currently underway to identify potential phosphorylation sites that might be mediating this stabilization of β-catenin. To obtain a broader picture on the potential effects of MLK3 in HCC disease progression, studies were performed with HCC cell lines following treatment with a pan MLK inhibitor CEP-1347 (CEP). These studies revealed a decrease in PCNA and c-myc expression along with increased PARP cleavage upon CEP treatment, in a time-dependent manner, indicative of a role in antagonizing cell proliferation and induction of cell death respectively. Cell cycle analysis revealed that CEP arrests HCC cells at G2/M phase of cell cycle. Flow cytometric studies also revealed a time-dependent increase in cell apoptosis and increased mitochondrial depolarization following treatment with CEP, associated with increased cleavage of Caspases 3, 8 and PARP. Further validation following siRNA-mediated knockdown of MLK3 showed a potentiation of the apoptotic response following CEP treatment. Taken together these findings indicate that MLK3 plays a major role in HCC progression and CEP-induced targeting of this axis might be efficacious in the management of HCC. Citation Format: Karan S. Saini, Taher Gheewala, Subhasis Das, Gautam Sondarva, Ajay Rana, Basabi Rana. Elucidating the role of mixed lineage kinase 3 (MLK3)-β-catenin axis in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3450.

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.

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.

We are pleased to review current and future strategies being developed to modulate neuroinflammation while reducing residual viral burden in the central nervous system. This has been realized by targeted long-acting antiretroviral nano and adjunctive therapies being developed for HIV-infected people. Our ultimate goal is to eliminate virus from its central nervous system reservoirs and, in so doing, reverse the cognitive and motor dysfunctions.Herein, we highlight our laboratories' development of adjunctive and nanomedicine therapies for HIV-associated neurocognitive disorders. An emphasis is placed on drug-drug interactions that target both the viral life cycle and secretory proinflammatory neurotoxic factors and signaling pathways.Antiretroviral therapy has improved the quality and duration of life for people living with HIV-1. A significant long-term comorbid illness is HIV-associated neurocognitive disorders. Symptoms, although reduced in severity, are common. Disease occurs, in part, through continued low-level viral replication, inducing secondary glial neuroinflammatory activities. Our recent works and those of others have seen disease attenuated in animal models through the use of adjunctive and long-acting reservoir-targeted nanoformulated antiretroviral therapy. The translation of these inventions from animals to humans is the focus of this review.

The mixed lineage kinase (MLK) family is a recently described protein kinase family. The MLKs contain a kinase domain followed by a dual leucine zipper-like motif. We previously reported the molecular cloning of LZK (leucine zipper-bearing kinase), a novel MLK, and that LZK activated the c-Jun NH2 terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway through MKK7 in cells. Here, we reveal that LZK forms dimers/oligomers through its dual leucine zipper-like motif, and that this is necessary for activation of the JNK/SAPK pathway. We also identify the C-terminal functional region of LZK, which is indispensable for the activation of SEK1, but not that of MKK7.

DLK/MUK/ZPK is a serine/threonine kinase that belongs to the mixed-lineage (MLK) subfamily of protein kinases. As is the case for most members of this family, relatively little is known about the physiological role of DLK/MUK/ZPK in mammalian cells. Because analysis of subcellular distribution may provide important clues concerning the potential in vivo function of a protein, an antiserum was generated against the amino terminal region of murine DLK/MUK/ZPK and used for localization studies in wild-type NIH 3T3 cells. Light microscopic immunocytochemistry experiments performed with the antiserum revealed that DLK/MUK/ZPK was specifically localized in a juxtanuclear structure characteristic of the Golgi complex. In support of this, treatment of cells with brefeldin A, a drug known to disintegrate the Golgi apparatus, caused disruption of DLK/MUK/ZPK perinuclear staining. Ultrastructural observation of NIH 3T3 cells also confirmed this localization, showing that most of the immunoreactivity was detected on membranes of the stacked Golgi cisternae. Consistent with localization studies, biochemical analyses revealed that DLK/MUK/ZPK was predominantly associated with Golgi membranes on fractionation of cellular extracts and was entirely partitioned into the aqueous phase when membranes were subjected to Triton X-114 extraction. On the basis of these findings, we suggest that DLK/MUK/ZPK is a peripheral membrane protein tightly associated with the cytoplasmic face of the Golgi apparatus. (J Histochem Cytochem 47:1287-1296, 1999)

Hypoxic-ischemic brain injury is often delayed and involves both apoptotic and immunoregulatory mechanisms. In this study, we used a neonatal model of hypoxia-ischemia to examine the effect of the mixed lineage kinase (MLK) inhibitor CEP-1347 on brain damage, apoptosis and inflammation. The tissue volume loss was reduced by 28% (p = 0.019) in CEP-1347-treated versus vehicle-treated rats and CEP-1347 significantly attenuated microgliosis at 7 days (p = 0.038). CEP-1347 decreased TUNEL-positive staining as well as cleaved caspase 3 immunoreactivity. CEP-1347 did not affect the expression of pro-inflammatory cytokines IL-1β, IL-6 and MCP-1, nor did it affect the expression of OX-42 (CR3) and OX-18 (MHC I) 24 h after the insult. In conclusion, the MLK inhibitor CEP-1347 has protective effects in a neonatal rat model of hypoxia-ischemia, which is mainly related to reduced apoptosis.

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 p21-GTPase activated kinase, PAK1, and the mixed lineage kinase, MLK2, have been implicated in the activation of jun N-terminal kinase (JNK). However, the role of PAK1 in JNK activation is still not understood. Here we show that over-expression of the SH3-SH2 adapter Nck ‘squelches’ JNK activation but this squelching is relieved by over-expression of PAK1. In turn, PAK1 squelches activation of JNK by MLK2 and these kinases interact via their catalytic domains. The data suggest that PAK1 recruits MLK2 to an activated receptor via the adapter Nck, but cannot itself induce activation of the JNK cascade.

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.

Recent developments in preparation and structure determination of proteins have made analysis possible at a sensitivity and with a throughput that was hardly anticipated only a few years ago. In particular, efficient tools for screening and characterization of gene products are important. Protocols and combinations involving sequencer analysis and mass spectrometry have in this regard a high potential (Bergman et al., 1998).

Ling He,1 Kuan Peng,2 Yizhi Liu,1 Jing Xiong,1 Fu-fan Zhu1 1Department of Gynecology and Obstetrics, The Second Xiangya Hospital of Central South University, 2Institute of Biomedical Engineering, School of Info-Physics and Geomatics Engineering, Central South University, Changsha, People’s Republic of China Background: Mixed lineage kinase domain-like protein (MLKL) was initially identified as a key receptor interacting protein 3 downstream component of tumor-necrosis-factor-induced necrosis. In this study, we characterized the expression of MLKL in ovarian carcinomas and evaluated the prognostic value of MLKL in patients with ovarian cancer. Materials and methods: The ovarian cancer tissue specimens were collected from 153 patients diagnosed as primary ovarian cancer after operation at The Second Xiangya Hospital from January 2005 to December 2008. Immunohistochemistry was performed for MLKL and the protein expression score was quantified using an established scoring system. Kaplan–Meier survival curves were generated for disease-free survival (DFS) and overall survival (OS) for all patients. MLKL expression levels were correlated with DFS and OS using univariate and multivariate Cox regression analysis. Results: Seventy-five patients (49%) were defined as having high MLKL expression and 67 patients (43.7%) had >80% of cells staining for MLKL. Remarkably, low MLKL expression was significantly associated with decreased DFS (median 40 months versus 25 months, P=0.0282) and OS (median 43 months versus 28 months, P=0.0032). In multivariate analysis, retained significance was also observed. Conclusion: Low MLKL expression was significantly associated with both decreased DFS and OS in patients with primary ovarian cancer. MLKL expression may serve as a potential prognostic marker in patients with ovarian cancer. Keywords: MLKL, ovarian cancer, prognostic value

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.