Receptor-interacting Protein Kinase 1 Kinase Research Articles

Receptor interacting protein kinase 1 (RIPK1) pathways regulate innate B cell developmental checkpoints and delay effector function

Abstract Mutations in RIP kinases underlie the recently described human autoimmune syndrome, CRIA, characterized by lymphadenopathy, splenomegaly, and autoantibody production. While disease mechanisms for CRIA remain undescribed, RIP kinases work together with Caspase-8 to regulate cell death, which is critical for normal differentiation of many cell types. Here, we describe a key role for RIPK1 in facilitating innate B cell differentiation and subsequent activation. By comparing RIPK1, RIPK3, and Caspase8 triple deficient and RIPK3, Caspase 8 double deficient mice, we identified selective contributions of RIPK1 to an accumulation of murine splenic Marginal Zone (MZ) B cells and B1-b cells. We used mixed bone-marrow chimeras to determine that innate B cell commitment required B cell-intrinsic RIPK1, RIPK3, and Casp8 sufficiency. RIPK1 regulated MZ B cell differentiation rather than development and RIPK1 mediates its innate immune effects independent of the RIPK1 kinase domain. NP-KLH/alum and NP-Ficoll vaccination of RIPK3, Casp8, and RIPK1 triple deficient mice revealed uniquely delayed T-dependent and T-independent IgG responses, abnormal splenic germinal center architecture, and reduced extrafollicular plasmablast formation compared to double deficient and WT mice. RIPK1 deficiency was beneficial for survival following lethal, systemic Streptococcus pneumoniae infection, confirming the functionality of innate B cell populations which increased in the absence of RIPK1. Thus, RIPK1 orchestrates B cell fate, and delayed effector function, through a B cell-intrinsic mechanism.

RIPK1 activation mediates neuroinflammation and disease progression in multiple sclerosis.

SUMMARYReceptor interacting protein kinase 1 (RIPK1) mediates cell death and inflammatory signaling and is increased in multiple sclerosis (MS) brain samples. Here, we investigate the role of glial RIPK1 kinase activity in mediating MS pathogenesis. We demonstrate RIPK1 levels correlate with MS disease progression. We find microglia are susceptible to RIPK1-mediated cell death and identify an inflammatory gene signature that may contribute to the neuroinflammatory milieu in MS patients. We uncover a distinct role for RIPK1 in astrocytes in regulating inflammatory signaling in the absence of cell death and confirm RIPK1-kinase-dependent regulation in human glia. Using a murine MS model, we show RIPK1 inhibition attenuates disease progression and suppresses deleterious signaling in astrocytes and microglia. Our results suggest RIPK1 kinase activation in microglia and astrocytes induces a detrimental neuroinflammatory program that contributes to the neurodegenerative environment in progressive MS.

NF-κB inhibition in keratinocytes causes RIPK1-mediated necroptosis and skin inflammation.

Tumor necrosis factor receptor 1 (TNFR1) activates NF-κB-dependent pro-inflammatory gene expression, but also induces cell death by triggering apoptosis and necroptosis. Inhibition of inhibitor of NF-κB kinase (IKK)/NF-κB signaling in keratinocytes paradoxically unleashed spontaneous TNFR1-mediated skin inflammation in mice, but the underlying mechanisms remain poorly understood. Here, we show that TNFR1 causes skin inflammation in mice with epidermis-specific knockout of IKK2 by inducing receptor interacting protein kinase 1 (RIPK1)-dependent necroptosis, and to a lesser extent also apoptosis, of keratinocytes. Combined epidermis-specific ablation of the NF-κB subunits RelA and c-Rel also caused skin inflammation by inducing TNFR1-mediated keratinocyte necroptosis. Contrary to the currently established model that inhibition of NF-κB-dependent gene transcription causes RIPK1-independent cell death, keratinocyte necroptosis, and skin inflammation in mice with epidermis-specific RelA and c-Rel deficiency also depended on RIPK1 kinase activity. These results advance our understanding of the mechanisms regulating TNFR1-induced cell death and identify RIPK1-mediated necroptosis as a potent driver of skin inflammation.

A RIPK1-regulated inflammatory microglial state in amyotrophic lateral sclerosis

Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.

Ring closure strategy leads to potent RIPK3 inhibitors.

Necroptosis is a form of regulated necrotic cell death that is independent of caspases. Receptor-interacting protein kinase 3 (RIPK3) has been identified as a key regulator for necroptosis, and has been proposed as a potential therapeutic target for the treatment of diseases associated with necroptosis. In this report, we describe the design, synthesis, and evaluation of a series of novel RIPK3 inhibitors. The lead compound 38 exhibited potent activity (EC50=0.42μM) in blocking TNFα, Smac mimetic and z-VAD (TSZ) induced cell death in HT-29cells. Mechanistic studies showed that compound 38 bound to RIPK3 with high affinity (Kd=7.1nM), and inhibited RIPK3 kinase activity in a ADP-Glo functional assay. In addition, compound 38 displayed good selectivity over another necroptosis regulator RIPK1 (Kd=6000nM). Furthermore, compound 38 demonstrated excellent invitro safety profiles with minimal inhibition of CYP isozymes and hERG potassium channel. Lastly, compound 38 efficiently blocked hypothermia and death in mice in the TNFα-induced systemic inflammatory response syndrome model.

RIP3 Blockade Drives Myeloid-Derived Suppressor Cell-Mediated Immune Tolerance in Immune-Mediated Hepatitis

Background: Excessive and uncontrolled immune activation plays a deleterious role in liver damage of autoimmune hepatitis (AIH), thus, it`s of great significance to identify the regulation of immune tolerance. Receptor-interacting protein kinase 3 (RIP3) has been found to be down-regulated in tumor tissues and mediates tumor immune evasion. However, the role of RIP3 in AIH remains elusive. We aimed to investigate the role of RIP3 in the regulation of inflammation and immune tolerance in immune-mediated hepatitis (IMH). Methods: RIP3 expression was evaluated in the livers of AIH patients and mice models. Mice were treated with GSK872, a RIP3 specific inhibitor, or glucocorticoid dexamethasone before concanavalin A (ConA)-induced IMH. Real-time PCR, western blotting, and flow cytometry served to explore the molecular mechanisms involved. The depleting anti-mouse Gr-1 antibody and isotype control were used for further determine the role of MDSCs in GSK872 treatment. Findings: AIH patients and mice model displayed pronounced RIP3 activation in the liver tissues. RIP3 blockade by GSK872 prevented ConA-induced 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+ Gr-1+ MDSCs with immunoregulatory properties in the livers, 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. Interpretation: RIP3 blockade prevents ConA-induced IMH through a MDSC-dependent mechanism. Inhibition of RIP3 kinase may be a novel therapeutic avenue for AIH treatment. Funding Statement: The study was supported by the National Natural Science Foundation of China (81860109). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: This study was approved by the Ethics Committee of Tianjin Medical University General Hospital, and all participants provided signed informed consents. The animal use protocol has been reviewed and approved by the Animal Ethical and Welfare Committee of Tianjin Medical University, Approval No. IRB2020-WZ-175.

MIND bomb 2 prevents RIPK1 kinase activity-dependent and -independent apoptosis through ubiquitylation of cFLIPL

Mind bomb 2 (MIB2) is an E3 ligase involved in Notch signalling and attenuates TNF-induced apoptosis through ubiquitylation of receptor-interacting protein kinase 1 (RIPK1) and cylindromatosis. Here we show that MIB2 bound and conjugated K48– and K63–linked polyubiquitin chains to a long-form of cellular FLICE-inhibitory protein (cFLIPL), a catalytically inactive homologue of caspase 8. Deletion of MIB2 did not impair the TNF-induced complex I formation that mediates NF-κB activation but significantly enhanced formation of cytosolic death-inducing signalling complex II. TNF-induced RIPK1 Ser166 phosphorylation, a hallmark of RIPK1 death-inducing activity, was enhanced in MIB2 knockout cells, as was RIPK1 kinase activity-dependent and -independent apoptosis. Moreover, RIPK1 kinase activity-independent apoptosis was induced in cells expressing cFLIPL mutants lacking MIB2-dependent ubiquitylation. Together, these results suggest that MIB2 suppresses both RIPK1 kinase activity-dependent and -independent apoptosis, through suppression of RIPK1 kinase activity and ubiquitylation of cFLIPL, respectively.

Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

Acetaminophen (APAP)-induced liver necrosis is a form of regulated cell death (RCD) in which APAP activates the mitogen-activated protein kinases (MAPKs) and specifically the c-Jun-N-terminal kinase (JNK) pathway, leading to necrotic cell death. Previously, we have shown that receptor interacting protein kinase-1 (RIPK1) knockdown is also protective against APAP RCD upstream of JNK. However, whether the kinase or platform function of RIPK1 is involved in APAP RCD is not known. To answer this question, we used genetic mouse models of targeted hepatocyte RIPK1 knockout (RIPK1HepCKO) or kinase dead knock-in (RIPK1D138N) and adult hepatocyte specific knockout of the cytoprotective protein A20 (A20HepCKO), known to interact with RIPK1, to study its potential involvement in MAPK signaling. We observed no difference in injury between WT and RIPK1D138N mice post APAP. However, RIPK1HepCKO was protective. We found that RIPK1HepCKO mice had attenuated pJNK activation, while A20 was simultaneously upregulated. Conversely, A20HepCKO markedly worsened liver injury from APAP. Mechanistically, we observed a significant upregulation of apoptosis signal-regulating kinase 1 (ASK1) and increased JNK activation in A20HepCKO mice compared with littermate controls. We also demonstrated that A20 coimmunoprecipitated (co-IP) with both RIPK1 and ASK1, and that in the presence of RIPK1, there was less A20-ASK1 association than in its absence. We conclude that the kinase-independent platform function of RIPK1 is involved in APAP toxicity. Adult RIPK1HepCKO mice are protected against APAP by upregulating A20 and attenuating JNK signaling through ASK1, conversely, A20HepCKO worsens injury from APAP.

Cell-specific activation of RIPK1 and MLKL after intracerebral hemorrhage in mice

Receptor-interacting protein kinase-1 (RIPK1) is a master regulator of cell death and inflammation, and mediates programmed necrosis (necroptosis) via mixed-lineage kinase like (MLKL) protein. Prior studies in experimental intracerebral hemorrhage (ICH) implicated RIPK1 in the pathogenesis of neuronal death and cognitive outcome, but the relevant cell types involved and potential role of necroptosis remain unexplored. In mice subjected to autologous blood ICH, early RIPK1 activation was observed in neurons, endothelium and pericytes, but not in astrocytes. MLKL activation was detected in astrocytes and neurons but not endothelium or pericytes. Compared with WT controls, RIPK1 kinase-dead (RIPK1D138N/D138N) mice had reduced brain edema (24 h) and blood-brain barrier (BBB) permeability (24 h, 30 d), and improved postinjury rotarod performance. Mice deficient in MLKL (Mlkl-/-) had reduced neuronal death (24 h) and BBB permeability at 24 h but not 30d, and improved post-injury rotarod performance vs. WT. The data support a central role for RIPK1 in the pathogenesis of ICH, including cell death, edema, BBB permeability, and motor deficits. These effects may be mediated in part through the activation of MLKL-dependent necroptosis in neurons. The data support development of RIPK1 kinase inhibitors as therapeutic agents for human ICH.

Enhanced RIPK3 kinase activity-dependent lytic cell death in M1 but not M2 macrophages

Macrophages play a crucial role in host innate immune defense against infection and tissue injury. Macrophages are highly plastic cells and their subtypes have been characterized as M1 (also termed classically activated) and M2 (alternatively activated). Although the M1/M2 paradigm has been well documented, less is known regarding the role of macrophage activation/polarization in inflammation-associated necrotic cell death. To address this gap in current knowledge, we prepared bone marrow-derived macrophages, induced them to M1 or M2 subtypes, and then investigated the expression of necroptosis signaling molecules and macrophage subtype-dependent responses to different necroptosis inducers. We found that necroptosis effector mixed lineage kinase domain-like protein (MLKL) and the key necroptosis regulator Z-DNA/RNA binding protein 1 were predominantly induced in M1 but not M2 macrophages. Interestingly, the protein but not mRNA levels of receptor-interacting protein kinase-3 (RIPK3) were also upregulated in M1 macrophages. We further found that macrophage necrotic cell death, the releases of lactate dehydrogenase and dead cell proteases as well as MLKL phosphorylation at Ser345 in response to various necroptosis inducers were greatly augmented in M1 but not M2 macrophages, and the accelerated effects were blocked by two structurally distinct specific RIPK3 inhibitors GSK872 or GSK843. Thus, our findings demonstrate that M1 but not M2 subtypes of macrophages are more susceptible to inflammation-related lytic cell death in an RIPK3 kinase activity-dependent manner.

ICP6 Prevents RIP1 Activation to Hinder Necroptosis Signaling.

Necroptosis is a type of programmed necrosis which depends on the activation of receptor-interacting protein kinase 3 (RIP3). Herpes simplex virus type 1 (HSV-1) is known to block necroptosis by the viral protein ICP6 in human cells, but its specific inhibitory mechanism is not fully understood. Here we reported that ICP6 could promote rather than suppress the formation of necrosome, the necroptosis signaling complex containing RIP3 and upstream regulator receptor-interacting protein kinase 1 (RIP1), but blocked RIP3 activation. Moreover, ICP6 could reduce the necroptosis-specific auto-phosphorylation of RIP1 regardless of the presence of RIP3. These results indicate that ICP6 block necroptosis through preventing RIP1 activation dependent signal transduction in necrosome.

TRIM24-RIP3 axis perturbation accelerates osteoarthritis pathogenesis

ObjectivesRecently, necroptosis has attracted increasing attention in arthritis research; however, it remains unclear whether its regulation is involved in osteoarthritis (OA) pathogenesis. Since receptor-interacting protein kinase-3 (RIP3) plays a pivotal...

Autophosphorylation at serine 166 regulates RIP kinase 1-mediated cell death and inflammation

Receptor interacting protein kinase 1 (RIPK1) regulates cell death and inflammatory responses downstream of TNFR1 and other receptors, and has been implicated in the pathogenesis of inflammatory and degenerative diseases. RIPK1 kinase activity induces apoptosis and necroptosis, however the mechanisms and phosphorylation events regulating RIPK1-dependent cell death signaling remain poorly understood. Here we show that RIPK1 autophosphorylation at serine 166 plays a critical role for the activation of RIPK1 kinase-dependent apoptosis and necroptosis. Moreover, we show that S166 phosphorylation is required for RIPK1 kinase-dependent pathogenesis of inflammatory pathologies in vivo in four relevant mouse models. Mechanistically, we provide evidence that trans autophosphorylation at S166 modulates RIPK1 kinase activation but is not by itself sufficient to induce cell death. These results show that S166 autophosphorylation licenses RIPK1 kinase activity to induce downstream cell death signaling and inflammation, suggesting that S166 phosphorylation can serve as a reliable biomarker for RIPK1 kinase-dependent pathologies.

3-O-acetylrubianol C (3AR-C) induces RIPK1-dependent programmed cell death by selective inhibition of IKKβ.

In tumor necrosis factor (TNF) signaling, phosphorylation and activation of receptor interacting protein kinase 1 (RIPK1) by upstream kinases is an essential checkpoint in the suppression of TNF-induced cell death. Thus, discovery of pharmacological agents targeting RIPK1 may provide new strategies for improving the therapeutic efficacy of TNF. In this study, we found that 3-O-acetylrubianol C (3AR-C), an arborinane triterpenoid isolated from Rubia philippinesis, promoted TNF-induced apoptotic and necroptotic cell death. To identify the molecular mechanism, we found that in mouse embryonic fibroblasts, 3AR-C drastically upregulated RIPK1 kinase activity by selectively inhibiting IKKβ. Notably, 3AR-C did not interfere with IKKα or affect the formation of the TNF receptor1 (TNFR1) complex-I. Moreover, in human cancer cells, 3AR-C was only sufficient to sensitize TNF-induced cell death when c-FLIPL expression was downregulated to facilitate the formation of TNFR1 complex-II and necrosome. Taken together, our study identified a novel arborinane triterpenoid 3AR-C as a potent activator of TNF-induced cell death via inhibition of IKKβ phosphorylation and promotion of the cytotoxic potential of RIPK1, thus providing a rationale for further development of 3AR-C as a selective IKKβ inhibitor to overcome TNF resistance in cancer therpay.

Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease.

Receptor Interacting Protein Kinase 1 (RIPK1) is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is post-translationally regulated by well characterised ubiquitylation and phosphorylation events, as well as caspase-8 mediated cleavage1–7. The physiological relevance of this cleavage remains unclear, though it is believed to inhibit activation of RIPK3 and necroptosis8. Here we show that heterozygous missense mutations p.D324N, p.D324H and p.D324Y prevent caspase cleavage of RIPK1 in humans and result in early-onset periodic fever episodes and severe intermittent lymphadenopathy, a condition we designate ‘Cleavage-resistant RIPK1-Induced Autoinflammatory’ (CRIA) syndrome. To define the mechanism for this disease we generated a cleavage-resistant Ripk1D325A mutant mouse strain. While Ripk1-/- mice die postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by combined loss of Casp8 and Ripk3 but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, however the mice died before weaning from multi organ inflammation in a RIPK3 dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3 dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but maintains inflammatory homeostasis throughout life.

K63-linked ubiquitination regulates RIPK1 kinase activity to prevent cell death during embryogenesis and inflammation

Receptor-interacting protein kinase 1 (RIPK1) is a critical regulator of cell death through its kinase activity. However, how its kinase activity is regulated remains poorly understood. Here, we generate Ripk1K376R/K376R knock-in mice in which the Lys(K)63-linked ubiquitination of RIPK1 is impaired. The knock-in mice display an early embryonic lethality due to massive cell death that is resulted from reduced TAK1-mediated suppression on RIPK1 kinase activity and forming more TNFR1 complex II in Ripk1K376R/K376R cells in response to TNFα. Although TNFR1 deficiency delays the lethality, concomitant deletion of RIPK3 and Caspase8 fully prevents embryonic lethality of Ripk1K376R/K376R mice. Notably, Ripk1K376R/- mice are viable but develop severe systemic inflammation that is mainly driven by RIPK3-dependent signaling pathway, indicating that K63-linked ubiquitination on Lys376 residue of RIPK1 also contributes to inflammation process. Together, our study reveals the mechanism by which K63-linked ubiquitination on K376 regulates RIPK1 kinase activity to control cell death programs.

Autophagy genes in myeloid cells counteract IFNγ-induced TNF-mediated cell death and fatal TNF-induced shock

Host inflammatory responses must be tightly regulated to ensure effective immunity while limiting tissue injury. IFN gamma (IFNγ) primes macrophages to mount robust inflammatory responses. However, IFNγ also induces cell death, and the pathways that regulate IFNγ-induced cell death are incompletely understood. Using genome-wide CRISPR/Cas9 screening, we identified autophagy genes as central mediators of myeloid cell survival during the IFNγ response. Hypersensitivity of autophagy gene-deficient cells to IFNγ was mediated by tumor necrosis factor (TNF) signaling via receptor interacting protein kinase 1 (RIPK1)- and caspase 8-mediated cell death. Mice with myeloid cell-specific autophagy gene deficiency exhibited marked hypersensitivity to fatal systemic TNF administration. This increased mortality in myeloid autophagy gene-deficient mice required the IFNγ receptor, and mortality was completely reversed by pharmacologic inhibition of RIPK1 kinase activity. These findings provide insight into the mechanism of IFNγ-induced cell death via TNF, demonstrate a critical function of autophagy genes in promoting cell viability in the presence of inflammatory cytokines, and implicate this cell survival function in protection against mortality during the systemic inflammatory response.

Herpes simplex virus 1 ICP6 impedes TNF receptor 1–induced necrosome assembly during compartmentalization to detergent-resistant membrane vesicles

Receptor-interacting protein (RIP) kinase 3 (RIPK3)-dependent necroptosis directs inflammation and tissue injury, as well as anti-viral host defense. In human cells, herpes simplex virus 1 (HSV1) UL39-encoded ICP6 blocks RIP homotypic interacting motif (RHIM) signal transduction, preventing this leakage form of cell death and sustaining viral infection. TNF receptor 1 (TNFR1)-induced necroptosis is known to require the formation of a RIPK1-RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL) signaling complex (necrosome) that we find compartmentalizes exclusively to caveolin-1-associated detergent-resistant membrane (DRM) vesicles in HT-29 cells. Translocation proceeds in the presence of RIPK3 kinase inhibitor GSK'840 or MLKL inhibitor necrosulfonomide but requires the kinase activity, as well as RHIM signaling of RIPK1. ICP6 impedes the translocation of RIPK1, RIPK3, and MLKL to caveolin-1-containing DRM vesicles without fully blocking the activation of RIPK3 or phosphorylation of MLKL. Consistent with the important contribution of RIPK1 RHIM-dependent recruitment of RIPK3, overexpression of RHIM-deficient RIPK3 results in phosphorylation of MLKL, but this does not lead to either translocation or necroptosis. Combined, these data reveal a critical role of RHIM signaling in the recruitment of the MLKL-containing necrosome to membrane vesicle-associated sites of aggregation. A similar mechanism is predicted for other RHIM-containing signaling adaptors, Z-nucleic acid-binding protein 1 (ZBP1) (also called DAI and DLM1), and TIR domain-containing adapter-inducing interferon-β (TRIF).

Kinase domain dimerization drives RIPK3-dependent necroptosis.

Necroptosis, an inflammatory form of cell death, is initiated by the activation of receptor-interacting protein kinase 3 (RIPK3), which depends on its interaction with RIPK1. Although catalytically inactive, the RIPK3 mutant D161N still stimulates RIPK1-dependent apoptosis and embryonic lethality in RIPK3 D161N homozygous mice. Whereas the absence of RIPK1 rescues RIPK3 D161N homozygous mice, we report that the absence of RIPK1 leads to embryonic lethality in RIPK3 D161N heterozygous mice. This suggested that the kinase domain of RIPK3 had a noncatalytic function that was enhanced by a conformation induced by the D161N mutation. We found that the RIPK3 kinase domain homodimerized through a surface that is structurally similar to that of the RAF family members. Mutation of residues at the dimer interface impaired dimerization and necroptosis. Kinase domain dimerization stimulated the activation of RIPK3 through cis-autophosphorylation. This noncatalytic, allosteric activity was enhanced by certain kinase-deficient mutants of RIPK3, including D161N. Furthermore, apoptosis induced by certain RIPK3 inhibitors was also dependent on the kinase dimerization interface. Our studies reveal that the RIPK3 kinase domain exhibits catalytically independent function that is important for both RIPK3-dependent necroptosis and apoptosis.

RIP1 protects melanoma cells from apoptosis induced by BRAF/MEK inhibitors

Many recent studies have uncovered the necessary role for the receptor-interacting protein kinase 1 (RIP1) in regulating apoptosis and necrosis that cells undergo in response to various cellular stresses. However, the functional significance of RIP1 in promoting cancer cell survival remains poorly understood. Here, we report that RIP1 was upregulated and contributed to both intrinsic and acquired resistance of melanoma cells to BRAF/MEK inhibitors through activation of NF-κB. Strikingly, Snail1-mediated suppression of CYLD played a crucial role in promoting RIP1 expression upon ERK activation, particularly, in melanoma cells with acquired resistance to BRAF inhibitors. In addition, RIP1 kinase activity was not required for melanoma cells to survive BRAF/MEK inhibition as RIP1 mediated NF-κB activation through its intermediate domain. Collectively, our findings reveal that targeting RIP1 in combination with BRAF/MEK inhibitors is a potential approach in the treatment of the disease.