Activation Of Receptor-interacting Protein Kinase Research Articles

A phase I randomized study to evaluate safety, pharmacokinetics, and pharmacodynamics of SIR2446M, a selective RIPK1 inhibitor, in healthy participants.

Activation of receptor-interacting protein kinase 1 (RIPK1), a broadly expressed serine/threonine protein kinase, by pro-inflammatory cytokines and pathogens can result in apoptosis, necroptosis, or inflammation. RIPK1 inhibition has been shown to reduce inflammation and cell damage in preclinical studies and may have therapeutic potential for degenerative and inflammatory diseases. SIR2446 is a potent and selective novel small molecule RIPK1 kinase inhibitor. This phase I, randomized, double-blind, placebo-controlled study in Australia (ACTRN12621001621808) evaluated the safety (primary objective), pharmacokinetics, and pharmacodynamics of single (3-600 mg) and multiple (5-400 mg for 10 days) ascending oral doses of SIR2446M (SIR2446 magnesium salt form) in healthy adults from Nov 24, 2021, until May 01, 2023. All treatment-emergent adverse events (TEAEs) were mild/moderate. The most reported TEAEs were vascular access site pain, headache, and rash morbilliform. SIR2446M plasma half-lives ranged from 11 to 19 h and there were no major deviations from dose proportionality for maximum concentration and area under the curve across doses. Renal excretion of unchanged SIR2446 was minimal. No marked accumulation was observed (mean accumulation ratio, 1.2-1.6) after multiple daily doses. A high-fat meal mildly reduced the exposure but was not considered clinically significant. SIR2446M had a rapid and sustained inhibitory effect on the activity of RIPK1, with an overall 90% target engagement at repeated doses ranging from 30 to 400 mg in peripheral blood mononuclear cells exvivo stimulated to undergo necroptosis. The favorable safety, pharmacokinetic, and pharmacodynamic profile of SIR2446M in healthy participants supports its further clinical development in patients with degenerative and inflammatory diseases.

Astrocytic RIPK3 exerts protective anti-inflammatory activity during viral encephalitis via induction of serpin protease inhibitors.

Flaviviruses pose a significant threat to public health due to their ability to infect the central nervous system (CNS) and cause severe neurologic disease. Astrocytes play a crucial role in the pathogenesis of flavivirus encephalitis through their maintenance of blood-brain barrier (BBB) integrity and their modulation of immune cell recruitment and activation within the CNS. We have previously shown that receptor interacting protein kinase-3 (RIPK3) is a central coordinator of neuroinflammation during CNS viral infection, a function that occurs independently of its canonical function in inducing necroptotic cell death. To date, however, roles for necroptosis-independent RIPK3 signaling in astrocytes are poorly understood. Here, we use mouse genetic tools to induce astrocyte-specific deletion, overexpression, and chemogenetic activation of RIPK3 to demonstrate an unexpected anti-inflammatory function for astrocytic RIPK3. RIPK3 activation in astrocytes was required for host survival in multiple models of flavivirus encephalitis, where it restricted neuropathogenesis by limiting immune cell recruitment to the CNS. Transcriptomic analysis revealed that, despite inducing a traditional pro-inflammatory transcriptional program, astrocytic RIPK3 paradoxically promoted neuroprotection through the upregulation of serpins, endogenous protease inhibitors with broad immunomodulatory activity. Notably, intracerebroventricular administration of SerpinA3N in infected mice preserved BBB integrity, reduced leukocyte infiltration, and improved survival outcomes in mice lacking astrocytic RIPK3. These findings highlight a previously unappreciated role for astrocytic RIPK3 in suppressing pathologic neuroinflammation and suggests new therapeutic targets for the treatment of flavivirus encephalitis.

Neuronal DAMPs exacerbate neurodegeneration via astrocytic RIPK3 signaling.

Astrocyte activation is a common feature of neurodegenerative diseases. However, the ways in which dying neurons influence the activity of astrocytes is poorly understood. Receptor interacting protein kinase-3 (RIPK3) signaling has recently been described as a key regulator of neuroinflammation, but whether this kinase mediates astrocytic responsiveness to neuronal death has not yet been studied. Here, we used the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine model of Parkinson's disease to show that activation of astrocytic RIPK3 drives dopaminergic cell death and axon damage. Transcriptomic profiling revealed that astrocytic RIPK3 promoted gene expression associated with neuroinflammation and movement disorders, and this coincided with significant engagement of damage-associated molecular pattern signaling. In mechanistic experiments, we showed that factors released from dying neurons signaled through receptor for advanced glycation endproducts to induce astrocytic RIPK3 signaling, which conferred inflammatory and neurotoxic functional activity. These findings highlight a mechanism of neuron-glia crosstalk in which neuronal death perpetuates further neurodegeneration by engaging inflammatory astrocyte activation via RIPK3.

RIPK3 activation promotes DAXX-dependent neuronal necroptosis after intracerebral hemorrhage in mice.

Necroptosis induced by receptor-interacting protein kinase 3 (RIPK3) is engaged in intracerebral hemorrhage (ICH) pathology. In this study, we explored the impact of RIPK3 activation on neuronal necroptosis and the mechanism of the death domain-associated protein (DAXX)-mediated nuclear necroptosis pathway after ICH. Potential molecules linked to the progression of ICH were discovered using RNA sequencing. The level of DAXX was assessed by quantitative real-time PCR, ELISA, and western blotting. DAXX localization was determined by immunofluorescence and immunoprecipitation assays. The RIPK3 inhibitor GSK872 and DAXX knockdown with shRNA-DAXX were used to examine the nuclear necroptosis pathway associated with ICH. Neurobehavioral deficit assessments were performed. DAXX was increased in patients and mice after ICH. In an ICH mouse model, shRNA-DAXX reduced brain water content and alleviated neurologic impairments. GSK872 administration reduced the expression of DAXX. shRNA-DAXX inhibited the expression of p-MLKL. Immunofluorescence and immunoprecipitation assays showed that RIPK3 and AIF translocated into the nucleus and then bound with nuclear DAXX. RIPK3 revitalization promoted neuronal necroptosis in ICH mice, partially through the DAXX signaling pathway. RIPK3 and AIF interacted with nuclear DAXX to aggravate ICH injury.

HSP70 attenuates neuronal necroptosis through the HSP90α-RIPK3 pathway following neuronal trauma.

Necroptosis, a newly defined regulatable necrosis with membrane disruption, has been demonstrated to participate in trauma brain injury (TBI) related neuronal cell death. Heat shock protein 70 (HSP70) is a stress protein with neuroprotective activity, but the potential protective mechanisms are not fully understood. Here, we investigated the effects of HSP70 regulators in a cellular TBI model induced by traumatic neuronal injury (TNI) and glutamate treatment. We found that necroptosis occurred in cortical neurons after TNI and glutamate treatment. Neuronal trauma markedly upregulated HSP70 protein expression within 24h. The results of immunostaining and lactate dehydrogenase release assay showed that necroptosis following neuronal trauma was inhibited by HSP70 activator TRC051384 (TRC), but promoted by the HSP70 inhibitor 2-phenylethyenesulfonamide (PES). In congruent, the expression and phosphorylation of receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) were differently regulated by HSP70. Furthermore, the expression of HSP90α induced by neuronal trauma was further promoted by PES but decreased by TRC. The data obtained from western blot showed that the phosphorylation of RIPK3 and MLKL induced by HSP70 inhibition were reduced by RIPK3 inhibitor GSK-872 and HSP90α inhibitor geldanamycin (GA). Similarly, inhibition of HSP90α with GA could partially prevented the increased necroptosis induced by PES. Taken together, HSP70 activation exerted protective effects against neuronal trauma via inhibition of necroptosis. Mechanistically, the HSP90α-mediated activation of RIPK3 and MLKL is involved in these effects.

Analysis of Signaling Pathways of Necroptotic and Pyroptotic Cell Death in the Hearts of Rats With Type 2 Diabetes Mellitus.

Diabetes mellitus is known to produce various cell-damaging events and thereby underlie heart dysfunction and remodeling. However, very little is known about its inflammation-associated pathomechanisms due to necrosis-like cell death. For this purpose, we aimed to investigate signaling pathways of necroptosis and pyroptosis, known to produce plasma membrane rupture with the resultant promotion of inflammation. One-year old Zucker diabetic fatty (ZDF) rats did not exhibit significant heart dysfunction as revealed by echocardiographic measurement. On the other hand, there was a decrease in heart rate due to diabetes. Immunoblotting analysis showed that the left ventricles of ZDF rats overexpress neither the main necroptotic proteins including receptor-interacting protein kinase 3 (RIP3) and mixed lineage domain kinase-like pseudokinase (MLKL), nor the pyroptotic regulators including NLR family pyrin domain containing 3 protein (NLRP3), caspase-1, interleukin-1 beta (IL-1beta and the N-terminal gasdermin D (GSDMD-N). On the other hand, the increased activation of the RIP3 kinase due to phosphorylation was found in such hearts. In summary, we showed for the first time that the activation of cardiac RIP3 is upregulated due to disturbances in glucose metabolism which, however, did not proceed to necrosis-like cell death. These data can indicate that the activated RIP3 might also underlie other pleiotropic, non-necroptotic signaling pathways under basal conditions.

Reduction of ovarian reserves and activation of necroptosis to in vivo air pollution exposures

ABSTRACT We investigated the association between air pollution and changes in ovarian follicles, anti-mullerian hormone (AMH) levels, the occurrence of necroptosis cell death by activation of receptor-interacting protein kinase 3 (RIPK3) and, the activation of mixed lineage kinase domain-like (MLKL) proteins. Forty-two female Wistar rats were divided into three groups of 14 each, which were exposed to real-ambient air, filtered air and purified air (control) in two periods of 3 and 5 months. The results showed that the number of ovarian follicles decreased in the group exposed to real-ambient air versus the control group (P < 0.0001). The trend of age-related AMH changes with respect to exposure to air pollutants was affected and its levels decreased after 3 months of exposure. The MLKL increased in the group exposed to the real-ambient air compared to the control group (P = 0.033). Apparently long-term exposure to air pollution can reduce ovarian reserves.

Necroptosis: A Pathogenic Negotiator in Human Diseases.

Over the past few decades, mechanisms of programmed cell death have attracted the scientific community because they are involved in diverse human diseases. Initially, apoptosis was considered as a crucial mechanistic pathway for programmed cell death; recently, an alternative regulated mode of cell death was identified, mimicking the features of both apoptosis and necrosis. Several lines of evidence have revealed that dysregulation of necroptosis leads to pathological diseases such as cancer, cardiovascular, lung, renal, hepatic, neurodegenerative, and inflammatory diseases. Regulated forms of necrosis are executed by death receptor ligands through the activation of receptor-interacting protein kinase (RIPK)-1/3 and mixed-lineage kinase domain-like (MLKL), resulting in the formation of a necrosome complex. Many papers based on genetic and pharmacological studies have shown that RIPKs and MLKL are the key regulatory effectors during the progression of multiple pathological diseases. This review focused on illuminating the mechanisms underlying necroptosis, the functions of necroptosis-associated proteins, and their influences on disease progression. We also discuss numerous natural and chemical compounds and novel targeted therapies that elicit beneficial roles of necroptotic cell death in malignant cells to bypass apoptosis and drug resistance and to provide suggestions for further research in this field.

Caspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis

The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)-dependent necroptosis invivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8- or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL (MlklFLAG/FLAG), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. Casp8-/-MlklFLAG/FLAG mice were viable and displayed phosphorylated MLKL in a variety of tissues, together with dramatically increased expression of ZBP1 compared to Casp8+/+ mice. Studies invitro revealed an increased expression of ZBP1 in cells lacking FADD or Caspase-8, which was suppressed by reconstitution of Caspase-8 or FADD. Ablation of ZBP1 in Casp8-/-MlklFLAG/FLAG mice suppressed spontaneous MLKL phosphorylation invivo. ZBP1 expression and downstream activation of RIPK3 and MLKL in cells lacking Caspase-8 or FADD relied on a positive feedback mechanism requiring the nucleic acid sensors cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and TBK1 signaling pathways. Our study identifies a molecular mechanism whereby Caspase-8 and FADD suppress spontaneous necroptotic cell death.

Feedback loop between fatty acid transport protein 2 and receptor interacting protein 3 pathways promotes polymorphonuclear neutrophil myeloid-derived suppressor cells-potentiated suppressive immunity in bladder cancer.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) promote tumor immune tolerance and cause tumor immunotherapy failure. In this study, we found that high PMN-MDSCs infiltration, overexpressed fatty acid transporter protein 2 (FATP2) and underexpressed receptor-interacting protein kinase 3 (RIPK3) existed in the mouse and human bladder cancer tissues. However, the related mechanisms remain largely unknown. Both FATP2 and RIPK3 expressions were associated with clinical stage. FATP2 knockout or up-regulating RIPK3 reduced the synthesis of prostaglandin E2 (PGE2) in PMN-MDSCs, attenuated the suppressive activity of PMN-MDSCs on CD8+ T cells functions and inhibited the tumor growth. There was a PGE2-mediated feedback loop between FATP2 and RIPK3 pathways, which markedly promoted the immunosuppressive activity of PMN-MDSCs. Combination therapy with inhibition of FATP2 and activation of RIPK3 can effectively inhibit tumor growth. This study demonstrated that a feedback loop between FATP2 and RIPK3 pathways in PMN-MDSCs significantly promoted the synthesis of PGE2, which severely impaired the CD8+ T cell functions. This study may provide new ideas for immunotherapy of human bladder cancer.

Rubiarbonol B induces RIPK1-dependent necroptosis via NOX1-derived ROS production.

The activation of receptor-interacting protein kinase 1 (RIPK1) by death-inducing signaling complex (DISC) formation is essential for triggering the necroptotic mode of cell death under apoptosis-deficient conditions. Thus, targeting the induction of necroptosis by modulating RIPK1 activity could be an effective strategy to bypass apoptosis resistance in certain types of cancer. In this study, we screened a series of arborinane triterpenoids purified from Rubia philippinesis and identified rubiarbonol B (Ru-B) as a potent caspase-8 activator that induces DISC-mediated apoptosis in multiple types of cancer cells. However, in RIPK3-expressing human colorectal cancer (CRC) cells, the pharmacological or genetic inhibition of caspase-8 shifted the mode of cell death by Ru-B from apoptosis to necroptosis though upregulation of RIPK1 phosphorylation. Conversely, Ru-B-induced cell death was almost completely abrogated by RIPK1 deficiency. The enhanced RIPK1 phosphorylation and necroptosis triggered by Ru-B treatment occurred independently of tumor necrosis factor receptor signaling and was mediated by the production of reactive oxygen species via NADPH oxidase 1 in CRC cells. Thus, we propose Ru-B as a novel anticancer agent that activates RIPK1-dependent cell death via ROS production, and suggest its potential as a novel necroptosis-targeting compound in apoptosis-resistant CRC.

LCK-Mediated RIPK3 Activation Controls Double-Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A-ERK Axis.

Receptor-interacting protein kinase 3 (RIPK3) is the primary regulator of necroptotic cell death. RIPK3 expression is often silenced in various cancer cells, which suggests that it may have tumor suppressor properties. However, the exact mechanism by which RIPK3 negatively regulates cancer development and progression remains unclear. This report indicates that RIPK3 acts as a potent regulator of the homeostatic proliferation of CD4+ CD8+ double-positive (DP) thymocytes. Abnormal proliferation of RIPK3-deficient DP thymocytes occurs independently of the well-known role for RIPK3 in necroptosis (upstream of MLKL activation), and is associated with an incidental thymic mass, likely thymic hyperplasia. In addition, Ripk3-null mice develop increased thymic tumor formation accompanied by reduced host survival in the context of an N-ethyl-N-nitrosourea (ENU)-induced tumor model. Moreover, RIPK3 deficiency in p53-null mice promotes thymic lymphoma development via upregulated extracellular signal-regulated kinase (ERK) signaling, which correlates with markedly reduced survival rates. Mechanistically, lymphocyte-specific protein tyrosine kinase (LCK) activates RIPK3, which in turn leads to increases in the phosphatase activity of protein phosphatase 2 (PP2A), thereby suppressing hyper-activation of ERK in DP thymocytes. Overall, these findings suggest that a RIPK3-PP2A-ERK signaling axis regulates DP thymocyte homeostasis and may provide a potential therapeutic target to improve thymic lymphoma therapies.

RIPK3 inhibitor-AZD5423 alleviates acute kidney injury by inhibiting necroptosis and inflammation

Acute kidney injury (AKI) is a clinical syndrome that is defined as a sudden decline in renal function and characterized by inflammation and programmed cell death of renal tubular epithelial cells. Necroptosis is a form of regulated cell death that requires activation of receptor interacting protein kinase 3 (RIPK3) and its phosphorylation of the substrate MLKL. RIPK3 plays an important role in acute kidney injury, and hence developing its inhibitors is considered as one of the promising strategies aimed at prevention and treatment of AKI. Recently, we discovered AZD5423 as a novel potent RIPK3 inhibitor using a computer-aided hybrid virtual screening strategy according to three-dimensional structure of RIPK3. Our findings revealed that AZD5423 strongly inhibits activation of RIPK3, and MLKL phosphorylation upon cisplatin-, hypoxia/reoxygenation (H/R)- and TNF-α stimuli as compared with GSK872, which is a previously identified RIPK3 inhibitor. Importantly, AZD5423 exerts effective protection against cisplatin- and ischemia/reperfusion (I/R)-induced AKI mouse model. The results of cellular thermal shift assay and experiments in RIPK3 knockout cells indicated that AZD5423 could directly target RIPK3 to inhibit RIPK3 kinase activity. Mechanistically, the docking of AZD5423 and RIPK3 suggested that the kinase domain of RIPK3 for Lys50, Arg313, Lys29, Arg37 might form hydrogen bonds with AZD5423. Site-directed mutagenesis further revealed that AZD5423 reduces injury response via interacting with the key RIPK3 amino acid residues of Lys50 and Arg313. In conclusion, our study has demonstrated that AZD5423 may serve as a potent inhibitor of RIPK3 kinase and a promising clinical candidate for AKI treatment.

Conjugation of the Fn14 Ligand to a SMAC Mimetic Selectively Suppresses Experimental Squamous Cell Carcinoma in Mice

The mimetic of SMAC induced cell death in cancers by depleting the inhibitor of apoptosis proteins. Recent studies showed that Fn14 is overexpressed in the cells of squamous cell carcinoma (SCC), providing a promising candidate target for selective antitumor therapy. In this study, we conjugated a small-molecule SMAC mimetic MV1 to the ligand of Fn14, TWEAK. Our results showed that TWEAK‒MV1 conjugate retained adequate binding specificity to Fn14-positive SCC cells invitro and accumulated selectively in tumor tissue of cutaneous SCC xenografts mice after intraperitoneal administration. This conjugation compound exhibited remarkable effectiveness in suppressing tumor growth and extending overall survival without causing significant side effects in SCC xenograft mice. Moreover, TWEAK‒MV1 conjugate greatly enhanced both apoptotic and necroptotic cell death both invitro and invivo, accompanied by a cellular inhibitor of apoptosis proteins degradation as well as activation of receptor-interacting protein kinase. Taken together, our preclinical data suggested that the designed conjugation compound of TWEAK and MV1 might provide a potential therapeutic strategy for cutaneous SCC with improved antitumor efficacy and negligible toxicity.

369-OR: Defining the Role of RIPK3 in Beta Cells and the Mechanism of Its Activation by ER Stress

Receptor interacting protein kinase 3 (RIPK3) can activate and be activated by ER stress. Islet inflammation associated with underlying activation of RIPK3 is conserved from zebrafish to humans. However, the role of RIPK3 in mammalian beta cells and the mechanism by which ER stress activates RIPK3 are not well defined. To investigate the role of RIPK3 in beta cell function, we generated beta cell specific Ripk3 knockout mice (Ripk3ßKO) in C57BL/6J background with or without the Akita mutation (Ins2C96Y/+, which causes ER stress induced beta cell apoptosis and early onset diabetes) (Ripk3ßKOAkita) . We found that the glucose tolerance was worsened in Ripk3ßKO mice than wildtype controls after 16 weeks of high fat diet. Similarly, Ripk3ßKOAkita mice had higher random blood glucose, lower circulating insulin, and worse glucose tolerance than Akita C57BL/6J mice as early as 4 weeks of age. Unexpectedly, isolated islets from Ripk3ßKO and Ripk3ßKOAkita mice have comparable glucose stimulated insulin secretion (GSIS) and are more resilient to palmitate-induced lipotoxicity than their control counterparts. Similarly, pharmacological inhibition of RIPK3 protected human islets from palmitate-induced impairment of GSIS ex vivo. We are currently investigating the role of specific islet inflammation stages in the divergent GSIS phenotypes between in vivo and ex vivo. To investigate how ER stress activates RIPK3 in beta cells, we tested the effect of inhibiting classic UPR branches on RIPK3 activation in MIN6 cells, a mouse beta cell line. We found that IRE1a activation appears to play an essential role in RIPK3 activation. Interestingly, Ripk3 knockdown in MIN6 cells also decreased IRE1a levels. We next seek to define the mechanism underlying the crosstalk between RIPK3 and IRE1a signaling pathways. Disclosure L.Yang: None. B.Yang: None. X.Tong: None. C.Dai: None. Z.Tang: None. W.Chen: None. Funding National Institutes of Health (DK117147)

Prognosis and Therapeutic Efficacy Prediction of Adrenocortical Carcinoma Based on a Necroptosis-Associated Gene Signature.

Background Adrenocortical carcinoma (ACC) is a rare and poor prognosis malignancy. Necroptosis is a special type of cell apoptosis, which is regulated in caspase-independent pathways and mainly induced through the activation of receptor-interacting protein kinase 1, receptor-interacting protein kinase 3, and mixed lineage kinase domain-like pseudokinase. A precise predictive tool based on necroptosis is needed to improve the level of diagnosis and treatment. Method Four ACC cohorts were enrolled in this study. The Cancer Genome Atlas ACC (TCGA-ACC) cohort was used as the training cohort; three datasets (GSE19750, GSE33371, and GSE49278) from Gene Expression Omnibus (GEO) platform were combined as the GEO testing cohort after removing of batch effect. Forty-nine necroptosis-associated genes were obtained from a prior study and further filtered by least absolute shrinkage and selection operator Cox regression analysis; corresponding coefficients were used to calculate the necroptosis-associated gene score (NAGs). Patients in the TCGA-ACC cohort were equally divided into two groups with the mean value of NAGs. We investigated the associations between NAGs groups and clinicopathological feature distribution and overall survival (OS) in ACC, the molecular mechanisms, and the value of NAGs in therapy prediction. A nomogram risk model was established to quantify risk stratification for ACC patients. Finally, the results were confirmed in the GEO-combined cohort. Result Patients in the TCGA-ACC cohort were divided into high and low NAGs groups. The high NAGs group had more fatal cases and advanced stage patients than the low NAGs group (P < 0.001, hazard ratio (HR) = 13.97, 95% confidence interval (95% CI): 4.168–46.844; survival rate: low NAGs, 7.69% vs. high NAGs, 61.53%). NAGs were validated to be negatively correlated with OS (R = −0.48, P < 0.001) and act as an independent factor in ACC with high discriminative efficacy (P < 0.001, HR = 11.76, 95% CI: 2.86–48.42). In addition, a high predictive efficacy nomogram risk model was established combining NAGs with tumor stage. Higher mutation rates were observed in the high NAGs group, and the mutation of TP53 may lead to a high T cell infiltration level among the NAGs groups. Patients belonged to the high NAGs are more sensitive to the chemotherapy of cisplatin, gemcitabine, paclitaxel, and etoposide (all P < 0.05). Ultimately, the same statistical algorithms were conducted in the GEO-combined cohort, and the crucial role of NAGs prediction value was further validated. Conclusion We constructed a necroptosis-associated gene signature, revealed the prognostic value between ACC and it, systematically explored the molecular alterations among patients with different NAGs, and manifested the value of drug sensitivity prediction in ACC.

Salt-inducible kinases inhibitor HG-9-91-01 targets RIPK3 kinase activity to alleviate necroptosis-mediated inflammatory injury

Receptor-interacting protein kinase 3 (RIPK3) functions as a central regulator of necroptosis, mediating signaling transduction to activate pseudokinase mixed lineage kinase domain-like protein (MLKL) phosphorylation. Increasing evidences show that RIPK3 contributes to the pathologies of inflammatory diseases including multiple sclerosis, infection and colitis. Here, we identified a novel small molecular compound Salt-inducible Kinases (SIKs) inhibitor HG-9-91-01 inhibiting necroptosis by targeting RIPK3 kinase activity. We found that SIKs inhibitor HG-9-91-01 could block TNF- or Toll-like receptors (TLRs)-mediated necroptosis independent of SIKs. We revealed that HG-9-91-01 dramatically decreased cellular activation of RIPK3 and MLKL. Meanwhile, HG-9-91-01 inhibited the association of RIPK3 with MLKL and oligomerization of downstream MLKL. Interestingly, we found that HG-9-91-01 also trigger RIPK3-RIPK1-caspase 1-caspase 8-dependent apoptosis, which activated cleavage of GSDME leading to its dependent pyroptosis. Mechanistic studies revealed that SIKs inhibitor HG-9-91-01 directly inhibited RIPK3 kinase activity to block necroptosis and interacted with RIPK3 and recruited RIPK1 to activate caspases leading to cleave GSDME. Importantly, mice pretreated with HG-9-91-01 showed resistance to TNF-induced systemic inflammatory response syndrome. Consistently, HG-9-91-01 treatment protected mice against Staphylococcus aureus-mediated lung damage through targeting RIPK3 kinase activity. Overall, our results revealed that SIKs inhibitor HG-9-91-01 is a novel inhibitor of RIPK3 kinase and a potential therapeutic target for the treatment of necroptosis-mediated inflammatory diseases.

RIP3 blockade prevents immune-mediated hepatitis through a myeloid-derived suppressor cell dependent mechanism.

Autoimmune hepatitis (AIH) is an immune-mediated chronic inflammatory liver disease, and its pathogenesis is not fully understood. Our previous study discovered that receptor interacting protein kinase 3 (RIP3) is correlated with serum transaminase levels in AIH patients. However, its role and underlying mechanism in AIH are poorly understood. Here, we detected the increased expression and activation of RIP3 in livers of patients and animal models with AIH. The inhibition of RIP3 kinase by GSK872 prevented concanavalin A (ConA)-induced immune-mediated hepatitis (IMH) by reduced hepatic proinflammatory cytokines and immune cells including Th17 cells and macrophages. Further experiments revealed that RIP3 inhibition resulted in an increase in CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) with immunoregulatory properties in the liver, spleen, and peripheral blood. Moreover, the depletion of Gr-1+ MDSCs abrogated the protective effect and immune suppression function of GSK872 in ConA-induced IMH. Altogether, our data demonstrate that RIP3 blockade prevents ConA-induced IMH through promoting MDSCs infiltration. Inhibition of RIP3 kinase may be a novel therapeutic avenue for AIH treatment.

Activation of UQCRC2-dependent mitophagy by tetramethylpyrazine inhibits MLKL-mediated hepatocyte necroptosis in alcoholic liver disease

Hepatocyte necroptosis is a core pathogenetic event during alcoholic liver disease. This study was aimed to explore the potential of tetramethylpyrazine (TMP), an active hepatoprotective ingredient extracted from Ligusticum Wallichii Franch, in limiting alcohol-triggered hepatocyte necroptosis and further specify the molecular mechanism. Results revealed that TMP reduced activation of receptor-interacting protein kinase 1 (RIPK1)/RIPK3 necrosome in ethanol-exposed hepatocytes and phosphorylation of mixed-lineage kinase domain-like protein (MLKL), which thereby diminished necroptosis and leakage of damage-associated molecular patterns. Suppression on mitochondrial translocation of p-MLKL by TMP contributed to recovery of mitochondrial function in ethanol-damaged hepatocytes. TMP also disrupted necroptotic signal loop by interrupting mitochondrial reactive oxygen species (ROS)-dependent positive feedback between p-MLKL and RIPK1/RIPK3 necrosome. Further, TMP promoted clearance of impaired mitochondria in ethanol-incubated hepatocytes via restoring PINK1/parkin-mediated mitophagy. Ubiquinol-cytochrome c reductase core protein 2 (UQCRC2) was downregulated in ethanol-exposed hepatocytes, which was restored after TMP treatment. In vitro UQCRC2 knockdown lowered the capacities of TMP in reducing mitochondrial ROS accumulation, relieving mitochondria damage, and enhancing PINK1/parkin-mediated mitophagy in ethanol-exposed hepatocytes. Analogously, systematic UQCRC2 knockdown interrupted the actions of TMP to trigger autophagic signal, repress necroptotic signal, and protect against alcoholic liver injury, inflammation, and ROS overproduction. In conclusion, this work concluded that TMP rescued UQCRC2 expression in ethanol-challenged hepatocytes, which contributed to necroptosis inhibition by facilitating PINK1/parkin-mediated mitophagy. These findings uncovered a potential molecular pharmacological mechanism underlying the hepatoprotective action of TMP and suggested TMP as a promising therapeutic candidate for alcoholic liver disease.

RIP1 kinase inactivation protects against acetaminophen-induced acute liver injury in mice

Many studies have investigated the role of receptor-interacting protein 1 (RIP1) kinase in acetaminophen (APAP) overdose-induced acute liver injury. However, the results were not consistent and there still remain controversies. Importantly, in these previous studies, the usage of DMSO to dissolve the RIP1 kinase inhibitor Nec-1, resulted in misleading conclusion. Our study aimed to determine the role of RIP1 kinase in APAP-induced liver injury, via genetically or pharmaceutically inhibition of RIP1 kinase activity. Our results indicated that APAP-induced liver injury was significantly attenuated in RIP1 kinase-dead (Rip1K45A/K45A) mice compared to WT control. High dosage of APAP-induced mortality was also rescued by RIP1 kinase inactivation. In agreement, RIP1 kinase inhibitor, Nec-1 which was formulated with PEG400, could efficiently alleviate APAP-induced hepatotoxicity. For the underlying mechanism, our results suggested that RIP1 kinase inactivation did not influence the hepatic GSH depletion, but significantly reduced the hepatic cell death and inflammation induced by APAP treatment. Using bone marrow transplantation model, we also demonstrated that it was RIP1 kinase activity in tissue-resident hepatic cells other than hematopoietic-derived cells mainly responsible for APAP-induced liver injury. Our study confirmed the important role of RIP1 kinase activity in APAP-induced acute liver failure.