Defective prelamin A processing promotes unconventional necroptosis driven by nuclear RIPK1.

Defects in the prelamin A processing enzyme caused by loss-of-function mutations in the ZMPSTE24 gene are responsible for a spectrum of progeroid disorders characterized by the accumulation of farnesylated prelamin A. Here we report that defective prelamin A processing triggers nuclear RIPK1-dependent signalling that leads to necroptosis and inflammation. We show that accumulated prelamin A recruits RIPK1 to the nucleus to facilitate its activation upon tumour necrosis factor stimulation in ZMPSTE24-deficient cells. Kinase-activated RIPK1 then promotes RIPK3-mediated MLKL activation in the nucleus, leading to nuclear envelope disruption and necroptosis. This signalling relies on prelamin A farnesylation, which anchors prelamin A to nuclear envelope to serve as a nucleation platform for necroptosis. Genetic inactivation of necroptosis ameliorates the progeroid phenotypes in Zmpste24-/- mice. Our findings identify an unconventional nuclear necroptosis pathway resulting from ZMPSTE24 deficiency with pathogenic consequences in progeroid disorder and suggest RIPK1 as a feasible target for prelamin A-associated progeroid disorders.

A guide to cell death pathways.

Regulated cell death mediated by dedicated molecular machines, known as programmed cell death, plays important roles in health and disease. Apoptosis, necroptosis and pyroptosis are three such programmed cell death modalities. The caspase family of cysteine proteases serve as key regulators of programmed cell death. During apoptosis, a cascade of caspase activation mediates signal transduction and cellular destruction, whereas pyroptosis occurs when activated caspases cleave gasdermins, which can then form pores in the plasma membrane. Necroptosis, a form of caspase-independent programmed necrosis mediated by RIPK3 and MLKL, is inhibited by caspase-8-mediated cleavage of RIPK1. Disruption of cellular homeostatic mechanisms that are essential for cell survival, such as normal ionic and redox balance and lysosomal flux, can also induce cell death without invoking programmed cell death mechanisms. Excitotoxicity, ferroptosis and lysosomal cell death are examples of such cell death modes. In this Review, we provide an overview of the major cell death mechanisms, highlighting the latest insights into their complex regulation and execution, and their relevance to human diseases.

Small molecule activators of TAK1 promotes its activity-dependent ubiquitination and TRAIL-mediated tumor cell death.

TAK1 is a key modulator of both NF-κB signaling and RIPK1. In TNF signaling pathway, activation of TAK1 directly mediates the phosphorylation of IKK complex and RIPK1. In a search for small molecule activators of RIPK1-mediated necroptosis, we found R406/R788, two small molecule analogs that could promote sustained activation of TAK1. Treatment with R406 sensitized cells to TNF-mediated necroptosis and RIPK1-dependent apoptosis by promoting sustained RIPK1 activation. Using click chemistry and multiple biochemical binding assays, we showed that treatment with R406 promotes the activation of TAK1 by directly binding to TAK1, independent of its original target Syk kinase. Treatment with R406 promoted the ubiquitination of TAK1 and the interaction of activated TAK1 with ubiquitinated RIPK1. Finally, we showed that R406/R788 could promote the cancer-killing activities of TRAIL in vitro and in mouse models. Our studies demonstrate the possibility of developing small molecule TAK1 activators to potentiate the effect of TRAIL as anticancer therapies.

Prolonged hypoxia alleviates prolyl hydroxylation-mediated suppression of RIPK1 to promote necroptosis and inflammation.

The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN-pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1-pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα-TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN-pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1.

Metabolic orchestration of cell death by AMPK-mediated phosphorylation of RIPK1.

Adenosine monophosphate-activated protein kinase (AMPK) activity is stimulated to promote metabolic adaptation upon energy stress. However, sustained metabolic stress may cause cell death. The mechanisms by which AMPK dictates cell death are not fully understood. We report that metabolic stress promoted receptor-interacting protein kinase 1 (RIPK1) activation mediated by TRAIL receptors, whereas AMPK inhibited RIPK1 by phosphorylation at Ser415 to suppress energy stress-induced cell death. Inhibiting pS415-RIPK1 by Ampk deficiency or RIPK1 S415A mutation promoted RIPK1 activation. Furthermore, genetic inactivation of RIPK1 protected against ischemic injury in myeloid Ampkα1-deficient mice. Our studies reveal that AMPK phosphorylation of RIPK1 represents a crucial metabolic checkpoint, which dictates cell fate response to metabolic stress, and highlight a previously unappreciated role for the AMPK-RIPK1 axis in integrating metabolism, cell death, and inflammation.

Targeting RIPK1 kinase for modulating inflammation in human diseases.

Receptor-Interacting Serine/Threonine-Protein Kinase 1 (RIPK1) is a master regulator of TNFR1 signaling in controlling cell death and survival. While the scaffold of RIPK1 participates in the canonical NF-κB pathway, the activation of RIPK1 kinase promotes not only necroptosis and apoptosis, but also inflammation by mediating the transcriptional induction of inflammatory cytokines. The nuclear translocation of activated RIPK1 has been shown to interact BAF-complex to promote chromatin remodeling and transcription. This review will highlight the proinflammatory role of RIPK1 kinase with focus on human neurodegenerative diseases. We will discuss the possibility of targeting RIPK1 kinase for the treatment of inflammatory pathology in human diseases.

Myeloid-derived MIF drives RIPK1-mediated cerebromicrovascular endothelial cell death to exacerbate ischemic brain injury.

Macrophage migration inhibitory factor (MIF) is a multifaced protein that plays important roles in multiple inflammatory conditions. However, the role of MIF in endothelial cell (EC) death under inflammatory condition remains largely unknown. Here we show that MIF actively promotes receptor-interacting protein kinase 1 (RIPK1)-mediated cell death under oxygen-glucose deprivation condition. MIF expression is induced by surgical trauma in peripheral myeloid cells both in perioperative humans and mice. We demonstrate that MIF-loaded myeloid cells induced by peripheral surgery adhere to the brain ECs after distal middle cerebral artery occlusion (dMCAO) and exacerbate the blood-brain barrier (BBB) disruption. Genetic depletion of myeloid-derived MIF in perioperative ischemic stroke (PIS) mice with MCAO following a surgical insult leads to significant reduction in ECs apoptosis and necroptosis and the associated BBB disruption. The adoptive transfer of peripheral blood mononuclear cells (PBMC) from surgical MIFΔLyz2 mice to wild-type (WT) MCAO mice also shows reduced ECs apoptosis and necroptosis compared to the transfer of PBMC from surgical MIFf  l/f  l mice to MCAO recipients. The genetic inhibition of RIPK1 also attenuates BBB disruption and ECs death compared to that of WT mice in PIS. The administration of MIF inhibitor (ISO-1) and RIPK1 inhibitor (Nec-1s) can both reduce the brain EC death and neurological deficits following PIS. We conclude that myeloid-derived MIF promotes ECs apoptosis and necroptosis through RIPK1 kinase-dependent pathway. The above findings may provide insights into the mechanism as how peripheral inflammation promotes the pathology in central nervous system.

USP5-Beclin 1 axis overrides p53-dependent senescence and drives Kras-induced tumorigenicity.

Non-small cell lung cancers (NSCLC) frequently contain KRAS mutation but retain wild-type TP53. Abundant senescent cells are observed in premalignant but not in malignant tumors derived from the Kras-driven mouse model, suggesting that KRAS oncogenic signaling would have to overcome the intrinsic senescence burden for cancer progression. Here, we show that the nuclear Beclin 1-mediated inhibition of p53-dependent senescence drives Kras-mediated tumorigenesis. KRAS activates USP5 to stabilize nuclear Beclin 1, leading to MDM2-mediated p53 protein instability. KrasG12D mice lacking Beclin 1 display retarded lung tumor growth. Knockdown of USP5 or knockout of Becn1 leads to increased senescence and reduced autophagy. Mechanistically, KRAS elevates ROS to induce USP5 homodimer formation by forming the C195 disulfide bond, resulting in stabilization and activation of USP5. Together, these results demonstrate that activation of the USP5-Beclin 1 axis is pivotal in overriding intrinsic p53-dependent senescence in Kras-driven lung cancer development.

HOIP modulates the stability of GPx4 by linear ubiquitination.

LUBAC-mediated linear ubiquitination plays a pivotal role in regulation of cell death and inflammatory pathways. Genetic deficiency in LUBAC components leads to severe immune dysfunction or embryonic lethality. LUBAC has been extensively studied for its role in mediating TNF signaling. However, Tnfr1 knockout is not able to fully rescue the embryonic lethality of LUBAC deficiency, suggesting that LUBAC may modify additional key cellular substrates in promoting cell survival. GPx4 is an important selenoprotein involved in regulating cellular redox homeostasis in defense against lipid peroxidation-mediated cell death known as ferroptosis. Here we demonstrate that LUBAC deficiency sensitizes to ferroptosis by promoting GPx4 degradation and downstream lipid peroxidation. LUBAC binds and stabilizes GPx4 by modulating its linear ubiquitination both in normal condition and under oxidative stress. Our findings identify GPx4 as a key substrate of LUBAC and a previously unrecognized role of LUBAC-mediated linear ubiquitination in regulating cellular redox status and cell death.

PP6 negatively modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL to promote TNFα-mediated cell death.

Activation of TNFR1 by TNFα induces the formation of a membrane-associated, intracellular complex termed complex I. Complex I orchestrates a complex pattern of modifications on key regulators of TNF signaling that collectively determines the cell fate by activating pro-survival or executing cell death programs. However, the regulatory mechanism of complex I in cell-fate decision is not fully understood. Here we identify protein phosphatase-6 (PP6) as a previously unidentified component of complex I. Loss of PP6 protects cells from TNFα-mediated cell death. The role of PP6 in regulating cell death requires its phosphatase activity and regulatory subunits. Further mechanistic studies show that PP6 modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL to promote RIPK1 activation and c-FLIPL degradation. We also show that melanoma-associated PP6 inactivating mutants offer resistance to cell death due to the loss of sensitivity to TNFα. Thus, our study provides a potential mechanism by which melanoma-related PP6 inactivating mutations promote cancer progression.

SARS-CoV-2 promotes RIPK1 activation to facilitate viral propagation.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the ongoing global pandemic that poses substantial challenges to public health worldwide. A subset of COVID-19 patients experience systemic inflammatory response, known as cytokine storm, which may lead to death. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important mediator of inflammation and cell death. Here, we examined the interaction of RIPK1-mediated innate immunity with SARS-CoV-2 infection. We found evidence of RIPK1 activation in human COVID-19 lung pathological samples, and cultured human lung organoids and ACE2 transgenic mice infected by SARS-CoV-2. Inhibition of RIPK1 using multiple small-molecule inhibitors reduced the viral load of SARS-CoV-2 in human lung organoids. Furthermore, therapeutic dosing of the RIPK1 inhibitor Nec-1s reduced mortality and lung viral load, and blocked the CNS manifestation of SARS-CoV-2 in ACE2 transgenic mice. Mechanistically, we found that the RNA-dependent RNA polymerase of SARS-CoV-2, NSP12, a highly conserved central component of coronaviral replication and transcription machinery, promoted the activation of RIPK1. Furthermore, NSP12 323L variant, encoded by the SARS-CoV-2 C14408T variant first detected in Lombardy, Italy, that carries a Pro323Leu amino acid substitution in NSP12, showed increased ability to activate RIPK1. Inhibition of RIPK1 downregulated the transcriptional induction of proinflammatory cytokines and host factors including ACE2 and EGFR that promote viral entry into cells. Our results suggest that SARS-CoV-2 may have an unexpected and unusual ability to hijack the RIPK1-mediated host defense response to promote its own propagation and that inhibition of RIPK1 may provide a therapeutic option for the treatment of COVID-19.

Caspase inhibition prolongs inflammation by promoting a signaling complex with activated RIPK1.

Activation of inflammation by lipopolysaccharide (LPS) is an important innate immune response. Here we investigated the contribution of caspases to the LPS-mediated inflammatory response and discovered distinctive temporal roles of RIPK1 in mediating proinflammatory cytokine production when caspases are inhibited. We propose a biphasic model that differentiates the role of RIPK1 in early versus late phase. The early production of proinflammation cytokines stimulated by LPS with caspase inhibition is mediated by the NF-κB pathway that requires the scaffold function of RIPK1 but is kinase independent. Autocrine production of TNFα in the late phase promotes the formation of a novel TNFR1-associated complex with activated RIPK1, FADD, caspase-8, and key mediators of NF-κB signaling. The production of proinflammatory cytokines in the late phase can be blocked by RIPK1 kinase inhibitor Nec-1s. Our study demonstrates a mechanism by which the activation of RIPK1 promotes its own scaffold function to regulate the NF-κB-mediated proinflammatory cytokine production that is negatively regulated by caspases to restrain inflammatory signaling.

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.

Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners.

RIPK1 is a death-domain (DD) containing kinase involved in regulating apoptosis, necroptosis and inflammation. RIPK1 activation is known to be regulated by its DD-mediated interaction and ubiquitination, though underlying mechanisms remain incompletely understood. Here we show that K627 in human RIPK1-DD and its equivalent K612 in murine RIPK1-DD is a key ubiquitination site that regulates the overall ubiquitination pattern of RIPK1 and its DD-mediated interactions with other DD-containing proteins. K627R/K612R mutation inhibits the activation of RIPK1 and blocks both apoptosis and necroptosis mediated by TNFR1 signaling. However, Ripk1K612R/K612R mutation sensitizes cells to necroptosis and caspase-1 activation in response to TLRs signaling. Ripk1K612R/K612R mice are viable, but develop age-dependent reduction of RIPK1 expression, spontaneous intestinal inflammation and splenomegaly, which can be rescued by antibiotic treatment and partially by Ripk3 deficiency. Furthermore, we show that the interaction of RIPK1 with FADD contributes to suppressing the activation of RIPK3 mediated by TLRs signaling. Our study demonstrates the distinct roles of K612 ubiquitination in mRIPK1/K627 ubiquitination in hRIPK1 in regulating its pro-death kinase activity in response to TNFα and pro-survival activity in response to TLRs signaling.

Reduction of mNAT1/hNAT2 Contributes to Cerebral Endothelial Necroptosis and Aß Accumulation in Alzheimer's Disease.

The contribution and mechanism of cerebrovascular pathology in Alzheimer's disease (AD) pathogenesis are still unclear. Here, we show that venular and capillary cerebral endothelial cells (ECs) are selectively vulnerable to necroptosis in AD. We identify reduced cerebromicrovascular expression of murine N-acetyltransferase 1 (mNat1) in two AD mouse models and hNat2, the human ortholog of mNat1 and a genetic risk factor for type-2 diabetes and insulin resistance, in human AD. mNat1 deficiency in Nat1-/- mice and two AD mouse models promotes blood-brain barrier (BBB) damage and endothelial necroptosis. Decreased mNat1 expression induces lysosomal degradation of A20, an important regulator of necroptosis, and LRP1ß, a key component of LRP1 complex that exports Aß in cerebral ECs. Selective restoration of cerebral EC expression of mNAT1 delivered by adeno-associated virus (AAV) rescues cerebromicrovascular levels of A20 and LRP1ß, inhibits endothelial necroptosis and activation, ameliorates mitochondrial fragmentation, reduces Aß deposits, and improves cognitive function in the AD mouse model.

Hepatocyte-specific TAK1 deficiency drives RIPK1 kinase-dependent inflammation to promote liver fibrosis and hepatocellular carcinoma.

Transforming growth factor ß-activated kinase1 (TAK1) encoded by the gene MAP3K7 regulates multiple important downstream effectors involved in immune response, cell death, and carcinogenesis. Hepatocyte-specific deletion of TAK1 in Tak1ΔHEP mice promotes liver fibrosis and hepatocellular carcinoma (HCC) formation. Here, we report that genetic inactivation of RIPK1 kinase using a kinase dead knockin D138N mutation in Tak1ΔHEP mice inhibits the expression of liver tumor biomarkers, liver fibrosis, and HCC formation. Inhibition of RIPK1, however, has no or minimum effect on hepatocyte loss and compensatory proliferation, which are the recognized factors important for liver fibrosis and HCC development. Using single-cell RNA sequencing, we discovered that inhibition of RIPK1 strongly suppresses inflammation induced by hepatocyte-specific loss of TAK1. Activation of RIPK1 promotes the transcription of key proinflammatory cytokines, such as CCL2, and CCR2+ macrophage infiltration. Our study demonstrates the role and mechanism of RIPK1 kinase in promoting inflammation, both cell-autonomously and cell-nonautonomously, in the development of liver fibrosis and HCC, independent of cell death, and compensatory proliferation. We suggest the possibility of inhibiting RIPK1 kinase as a therapeutic strategy for reducing liver fibrosis and HCC development by inhibiting inflammation.

Casein kinase-1γ1 and 3 stimulate tumor necrosis factor-induced necroptosis through RIPK3.

Upon necroptosis activation, receptor interacting serine/threonine kinase (RIPK)1 and RIPK3 form a necrosome complex with pseudokinase mixed lineage kinase-like (MLKL). Although protein phosphorylation is a key event for RIPK1 and RIPK3 activation in response to a necroptosis signal, relatively little is known about other factors that might regulate the activity of these kinases or necrosome formation. Through a gain-of-function screen with 546 kinases and 127 phosphatases, we identified casein kinase 1 gamma (CK1γ) as a candidate necroptosis-promoting factor. Here, we show that the decreased activity or amounts of CK1γ1 and CK1γ3, either by treatment with a chemical inhibitor or knockdown in cells, reduced TNFα-induced necroptosis. Conversely, ectopic expression of CK1γ1 or CK1γ3 exacerbated necroptosis, but not apoptosis. Similar to RIPK1 and RIPK3, CK1γ1 was also cleaved at Asp343 by caspase-8 during apoptosis. CK1γ1 and CK1γ3 formed a protein complex and were recruited to the necrosome harboring RIPK1, RIPK3 and MLKL. In particular, an autophosphorylated form of CK1γ3 at Ser344/345 was detected in the necrosome and was required to mediate the necroptosis. In addition, in vitro assays with purified proteins showed that CK1γ phosphorylated RIPK3, affecting its activity, and in vivo assays showed that the CK1γ-specific inhibitor Gi prevented abrupt death in mice with hypothermia in a model of TNFα-induced systemic inflammatory response syndrome. Collectively, these data suggest that CK1γ1 and CK1γ3 are required for TNFα-induced necroptosis likely by regulating RIPK3.

TAM Kinases Promote Necroptosis by Regulating Oligomerization of MLKL.

Necroptosis, a cell death pathway mediated by the RIPK1-RIPK3-MLKL signaling cascade downstream of tumor necrosis factor α (TNF-α), has been implicated in many inflammatory diseases. Members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases are known for their anti-apoptotic, oncogenic, and anti-inflammatory roles. Here, we identify an unexpected role of TAM kinases as promoters of necroptosis, a pro-inflammatory necrotic cell death. Pharmacologic or genetic targeting of TAM kinases results in a potent inhibition of necroptotic death in various cellular models. We identify phosphorylation of MLKL Tyr376 as a direct point of input from TAM kinases into the necroptosis signaling. The oligomerization of MLKL, but not its membranal translocation or phosphorylation by RIPK3, is controlled by TAM kinases. Importantly, both knockout and inhibition of TAM kinases protect mice from systemic inflammatory response syndrome. In conclusion, this study discovers that immunosuppressant TAM kinases are promoters of pro-inflammatory necroptosis, shedding light on the biological complexity of the regulation of inflammation.

Targeting RIPK1 for the treatment of human diseases.

RIPK1 kinase has emerged as a promising therapeutic target for the treatment of a wide range of human neurodegenerative, autoimmune, and inflammatory diseases. This was supported by extensive studies which demonstrated that RIPK1 is a key mediator of apoptotic and necrotic cell death as well as inflammatory pathways. Furthermore, human genetic evidence has linked the dysregulation of RIPK1 to the pathogenesis of ALS as well as other inflammatory and neurodegenerative diseases. Importantly, unique allosteric small-molecule inhibitors of RIPK1 that offer high selectivity have been developed. These molecules can penetrate the blood-brain barrier, thus offering the possibility to target neuroinflammation and cell death which drive various neurologic conditions including Alzheimer's disease, ALS, and multiple sclerosis as well as acute neurological diseases such as stroke and traumatic brain injuries. We discuss the current understanding of RIPK1 regulatory mechanisms and emerging evidence for the pathological roles of RIPK1 in human diseases, especially in the context of the central nervous systems.

Chaperone-mediated autophagy is involved in the execution of ferroptosis.

Necroptosis and ferroptosis are two distinct necrotic cell death modalities with no known common molecular mechanisms. Necroptosis is activated by ligands of death receptors such as tumor necrosis factor-α (TNF-α) under caspase-deficient conditions, whereas ferroptosis is mediated by the accumulation of lipid peroxides upon the depletion/or inhibition of glutathione peroxidase 4 (GPX4). The molecular mechanism that mediates the execution of ferroptosis remains unclear. In this study, we identified 2-amino-5-chloro-N,3-dimethylbenzamide (CDDO), a compound known to inhibit heat shock protein 90 (HSP90), as an inhibitor of necroptosis that could also inhibit ferroptosis. We found that HSP90 defined a common regulatory nodal between necroptosis and ferroptosis. We showed that inhibition of HSP90 by CDDO blocked necroptosis by inhibiting the activation of RIPK1 kinase. Furthermore, we showed that the activation of ferroptosis by erastin increased the levels of lysosome-associated membrane protein 2a to promote chaperone-mediated autophagy (CMA), which, in turn, promoted the degradation of GPX4. Importantly, inhibition of CMA stabilized GPX4 and reduced ferroptosis. Our results suggest that activation of CMA is involved in the execution of ferroptosis.