Necroptotic Cell Death Pathway Research Articles

Metformin alleviates benzo[a]pyrene-induced alveolar injury by inhibiting necroptosis and protecting AT2 cells

Exposure to benzo[a]pyrene (B[a]P) has been linked to lung injury and carcinogenesis. Airway epithelial cells express the B[a]P receptor AHR, so B[a]P is considered to mainly target airway epithelial cells, whereas its potential impact on alveolar cells remains inadequately explored. Metformin, a first-line drug for diabetes, has been shown to exert anti-inflammatory and tissue repair-promoting effects under various injurious conditions. Here, we explored the effect of chronic B[a]P exposure on alveolar cells and the impact of metformin on B[a]P-induced lung injury by examining the various parameters including lung histopathology, inflammation, fibrosis, and related signal pathway activation. MLKL knockout (Mlkl-/-) and AT2-lineage tracing mice (SftpcCre-ERT2;LSL-tdTomatoflox+/-) were used to delineate the role of necroptosis in B[a]P-induced alveolar epithelial injury and repair. Mice receiving weekly administration of B[a]P for 6 weeks developed a significant alveolar damaging phenotype associated with pulmonary inflammation, fibrosis, and activation of the necroptotic cell death pathway. These effects were significantly relieved in MLKL null mice. Furthermore, metformin treatment, which were found to promote AMPK phosphorylation and inhibit RIPK3, as well as MLKL phosphorylation, also significantly alleviated B[a]P-induced necroptosis and lung injury phenotype. However, the protective efficacy of metformin was rendered much less effective in Mlkl null mice or by blocking the necroptotic pathway with RIPK3 inhibitor. Our findings unravel a potential protective efficacy of metformin in mitigating the detrimental effects of B[a]P exposure on lung health by inhibiting necroptosis and protecting AT2 cells.

Type I Interferon: Monkeypox/Mpox Viruses Achilles Heel?

Poxviruses are notorious for having acquired/evolved numerous genes to counteract host innate immunity. Chordopoxviruses have acquired/evolved at least three different inhibitors of host necroptotic death: E3, which blocks ZBP1-dependent necroptotic cell death, and vIRD and vMLKL that inhibit necroptosis downstream of initial cell death signaling. While this suggests the importance of the necroptotic cell death pathway in inhibiting chordopoxvirus replication, several chordopoxviruses have lost one or more of these inhibitory functions. Monkeypox/mpox virus (MPXV) has lost a portion of the N-terminus of its E3 homologue. The N-terminus of the vaccinia virus E3 homologue serves to inhibit activation of the interferon-inducible antiviral protein, ZBP1. This likely makes MPXV unique among the orthopoxviruses in being sensitive to interferon (IFN) treatment in many mammals, including humans, which encode a complete necroptotic cell death pathway. Thus, IFN sensitivity may be the Achille's Heel for viruses like MPXV that cannot fully inhibit IFN-inducible, ZBP1-dependent antiviral pathways.

Ponatinib and dabrafenib reduce the accumulation of necroptosis‐related proteins and neuronal loss in APP23xTAU58 mice.

AbstractBackgroundNeuronal death is the result of the neurodegenerative process in Alzheimer’s disease (AD). Both histopathological hallmark lesions of AD, i.e., amyloid plaques and neurofibrillary tangles, are associated with reduced numbers of neurons in the brain. To model AD‐like neuronal death, mouse models with transgenic overexpression of the amyloid precursor protein (APP) and the tau protein can be used. Recently, the necroptotic cell death pathway was found to be activated in the AD brain. In this context, the activated necrosome was found in granulovacuolar degeneration (GVD) lesions in neurons. Therefore, the question arises whether inhibition of the necroptosis pathway reduces neuron loss.MethodWe analyzed APP23xTAU58 double transgenic mice for the presence of the activated necrosome in GVD lesions in association with amyloid and tau pathology. APP23xTAU58 mice at 2, 6, and 12 months of age were studied (n = 5‐10 animals/group). Neuronal densities in the subiculum and the amygdala were compared with those of wildtype littermates. To clarify whether necroptosis inhibitors can interfere with neuron death in this model, we treated APP23xTAU58 mice from 2‐6 months either with dabrafenib or ponatinib (n = 5 animals/group) and compared neuronal densities with that of vehicle‐treated mice. Neuronal densities as well as the percentage of necroptosis markers exhibiting neurons in the subiculum and amygdala were assessed.ResultSix‐ and 12‐month‐old APP23xTAU58 mice showed significantly decreased neuronal densities in the subiculum and amygdala compared to wildtype mice (p < 0.005), as well as an increased number of neurons exhibiting the necroptosis executor pMLKL in GVD lesions (p < 0.0001). Treatment with the necroptosis inhibitors ponatinib and dabrafenib reduced neuronal loss in the subiculum and the amygdala compared to vehicle treatment (p ≤ 0.0002).ConclusionThese results show that neuron loss in the APP23xTAU58 model is associated with the accumulation of necroptosis‐related proteins. This neuron loss and the expression of necrosome components can be reduced by ponatinib or dabrafenib. Thus, it is tempting to speculate that necroptosis inhibition could be a promising strategy to prolong neuron survival and, by doing so, to slow down cognitive decline in AD patients. Funding: FWO

Inhibition of SIRT2 promotes death of human cytomegalovirus-infected peripheral blood monocytes via apoptosis and necroptosis

Peripheral blood monocytes are the cells predominantly responsible for systemic dissemination of human cytomegalovirus (HCMV) and a significant cause of morbidity and mortality in immunocompromised patients. HCMV establishes a silent/quiescent infection in monocytes, which is defined by the lack of viral replication and lytic gene expression. The absence of replication shields the virus within infected monocytes from the current available antiviral drugs that are designed to suppress active replication. Our previous work has shown that HCMV stimulates a noncanonical phosphorylation of Akt and the subsequent upregulation of a distinct subset of prosurvival proteins in normally short-lived monocytes. In this study, we found that SIRT2 activity is required for the unique activation profile of Akt induced within HCMV-infected monocytes. Importantly, both therapeutic and prophylactic treatment with a novel SIRT2 inhibitor, FLS-379, promoted death of infected monocytes via both the apoptotic and necroptotic cell death pathways. Mechanistically, SIRT2 inhibition reduced expression of Mcl-1, an Akt-dependent antiapoptotic Bcl-2 family member, and enhanced activation of MLKL, the executioner kinase of necroptosis. We have previously reported HCMV to block necroptosis by stimulating cellular autophagy. Here, we additionally demonstrate that inhibition of SIRT2 suppressed Akt-dependent HCMV-induced autophagy leading to necroptosis of infected monocytes. Overall, our data show that SIRT2 inhibition can simultaneously promote death of quiescently infected monocytes by two distinct death pathways, apoptosis and necroptosis, which may be vital for limiting viral dissemination to peripheral organs in immunosuppressed patients.

RIPK3 controls MAIT cell accumulation during development but not during infection

Cell death mechanisms in T lymphocytes vary according to their developmental stage, cell subset and activation status. The cell death control mechanisms of mucosal-associated invariant T (MAIT) cells, a specialized T cell population, are largely unknown. Here we report that MAIT cells express key necroptotic machinery; receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein, in abundance. Despite this, we discovered that the loss of RIPK3, but not necroptotic effector MLKL or apoptotic caspase-8, specifically increased MAIT cell abundance at steady-state in the thymus, spleen, liver and lungs, in a cell-intrinsic manner. In contrast, over the course of infection with Francisella tularensis, RIPK3 deficiency did not impact the magnitude of the expansion nor contraction of MAIT cell pools. These findings suggest that, distinct from conventional T cells, the accumulation of MAIT cells is restrained by RIPK3 signalling, likely prior to thymic egress, in a manner independent of canonical apoptotic and necroptotic cell death pathways.

Redox Status of Erythrocytes as an Important Factor in Eryptosis and Erythronecroptosis.

Overall, reactive oxygen species (ROS) signalling significantly contributes to initiation and mo-dulation of multiple regulated cell death (RCD) pathways. Lately, more information has become available about RCD modalities of erythrocytes, including the role of ROS. ROS accumulation has therefore been increasingly recognized as a critical factor involved in eryptosis (apoptosis of erythrocytes) and erythro-necroptosis (necroptosis of erythrocytes). Eryptosis is a Ca2+-dependent apoptosis-like RCD of erythrocytes that occurs in response to oxidative stress, hyperosmolarity, ATP depletion, and a wide range of xenobiotics. Moreover, eryptosis seems to be involved in the pathogenesis of multiple human diseases and pathological processes. Several studies have reported that erythrocytes can also undergo necroptosis, a lytic RIPK1/RIPK3/MLKL-mediated RCD. As an example, erythronecroptosis can occur in response to CD59-specific pore-forming toxins. We have systematically summarized available studies regarding the involvement of ROS and oxidative stress in these two distinct RCDs of erythrocytes. We have focused specifically on cellular signalling pathways involved in ROS-mediated cell death decisions in erythrocytes. Furthermore, we have summarized dysregulation of related erythrocytic antioxidant defence systems. The general concept of the ROS role in eryptotic and necroptotic cell death pathways in erythrocytes seems to be established. However, further studies are required to uncover the complex role of ROS in the crosstalk and interplay between the survival and RCDs of erythrocytes.

Suppression of the necroptotic cell death pathways improves survival in Smn 2B/- mice.

Spinal muscular atrophy (SMA) is a monogenic neuromuscular disease caused by low levels of the Survival Motor Neuron (SMN) protein. Motor neuron degeneration is the central hallmark of the disease. However, the SMN protein is ubiquitously expressed and depletion of the protein in peripheral tissues results in intrinsic disease manifestations, including muscle defects, independent of neurodegeneration. The approved SMN-restoring therapies have led to remarkable clinical improvements in SMA patients. Yet, the presence of a significant number of non-responders stresses the need for complementary therapeutic strategies targeting processes which do not rely solely on restoring SMN. Dysregulated cell death pathways are candidates for SMN-independent pathomechanisms in SMA. Receptor-interacting protein kinase 1 (RIPK1) and RIPK3 have been widely recognized as critical therapeutic targets of necroptosis, an important form of programmed cell death. In addition, Caspase-1 plays a fundamental role in inflammation and cell death. In this study, we evaluate the role of necroptosis, particularly RIPK3 and Caspase-1, in the Smn2B/− mouse model of SMA. We have generated a triple mutant (TKO), the Smn2B/−; Ripk3−/−; Casp1−/− mouse. TKO mice displayed a robust increase in survival and improved motor function compared to Smn2B/− mice. While there was no protection against motor neuron loss or neuromuscular junction pathology, larger muscle fibers were observed in TKO mice compared to Smn2B/− mice. Our study shows that necroptosis modulates survival, motor behavior and muscle fiber size independent of SMN levels and independent of neurodegeneration. Thus, small-molecule inhibitors of necroptosis as a combinatorial approach together with SMN-restoring drugs could be a future strategy for the treatment of SMA.

ZBP1 promotes inflammatory responses downstream of TLR3/TLR4 via timely delivery of RIPK1 to TRIF

ZBP1 is widely recognized as a mediator of cell death for its role in initiating necroptotic, apoptotic, and pyroptotic cell death pathways in response to diverse pathogenic infection. Herein, we characterize an unanticipated role for ZBP1 in promoting inflammatory responses to bacterial lipopolysaccharide (LPS) or double-stranded RNA (dsRNA). In response to both stimuli, ZBP1 promotes the timely delivery of RIPK1 to the Toll-like receptor (TLR)3/4 adaptor TRIF and M1-ubiquitination of RIPK1, which sustains activation of inflammatory signaling cascades downstream of RIPK1. Strikingly, ZBP1-mediated regulation of these pathways is important in vivo, as Zbp1−/− mice exhibited resistance to LPS-induced septic shock, revealed by prolonged survival and delayed onset of hypothermia due to decreased inflammatory responses and subsequent cell death. Further findings revealed that ZBP1 promotes sustained inflammatory responses by mediating the kinetics of proinflammatory “TRIFosome” complex formation, thus having a profound impact downstream of TLR activation. Given the well-characterized role of ZBP1 as a viral sensor, our results exemplify previously unappreciated crosstalk between the pathways that regulate host responses to bacteria and viruses, with ZBP1 acting as a crucial bridge between the two.

Cell death in lung transplantation. The roles of apoptosis, necroptois, and pyroptosis

Abstract Objective For patients with end stage lung disease, lung transplantation (Ltx) is the unique treatment option. Nevertheless, a large number of lung allografts fail, because of acute or chronic rejection. Innate immune responses following Ltx play a critical role in the development of primary graft dysfunction, and ultimately promote the development of chronic lung allograft dysfunction, leading to increased mortality after lung transplantation. Here, we focus on the mechanisms responsible for disruption of the cells membranes integrity, resulting in the release of Damage Associated Molecular Patterns (DAMPs) and activation of cell death pathways triggering cell injury in lungs with different preservation status. Methods Rat lungs were exposed to cold ischemia (CI group, n=9) or warm ischemia (WI group, n=9). In CI group, after Perfadex flushing, and hypothermic preservation (4 h) left lung was transplanted. In WI group, after 1 h of in situ WI, flashing and 3 h cold preservation the left lung was transplanted. Lung mechanics were determined during reperfusion. Recipients were sacrificed at 30, 60 and 120 min after Ltx. Bronchoalveolar lavage (BAL) was performed on the grafts to measure markers of cellular injury, and cytokines. Cell death markers were determined in graft tissue. Biological samples were kept at -80°C for additional analysis. Results Compared to CI, WI group displayed significantly decreased graft function associated with increased activation of pyroptotic cell death (NLRP3, caspase1, IL-1β) pathway, whereas apoptotic (caspas3 and caspase7) and necroptotic (RIP1, RIP3, MLKL) cell death pathways activation were similar in both groups. WI group showed more important cellular damage with DAMPs release (LDH, sRAGE, HMGB) and inflammation (CXCL1, IL1-β, IL-33). Conclusion Our results suggest that pyroptosis could play a critical role in the development of early graft dysfunction in lungs subjected to warm ischemia. Inhibition of pyroptotic pathway could be a strategy to improve damaged donor lungs.

Z-DNA binding protein 1 mediates necroptotic and apoptotic cell death pathways in murine astrocytes following herpes simplex virus-1 infection

BackgroundThe mechanisms by which glia respond to viral central nervous system (CNS) pathogens are now becoming apparent with the demonstration that microglia and astrocytes express an array of pattern recognition receptors that include intracellular RNA and DNA sensors. We have previously demonstrated that glia express Z-DNA binding protein 1 (ZBP1) and showed that this cytosolic nucleic acid sensor contributes to the inflammatory/neurotoxic responses of these cells to herpes simplex virus-1 (HSV-1). However, the relative contribution made by ZBP1- to HSV-1-mediated cell death in glia has not been determined.MethodsWe have investigated the relative contribution made by ZBP1- to HSV-1-mediated cell death in primary astrocytes derived from mice genetically deficient in this sensor. We have used capture ELISAs and immunoblot analysis to assess inflammatory cytokine production and ZBP1 and phosphorylated mixed lineage kinase domain-like protein (MLKL) expression levels, respectively, following HSV-1 challenge. Furthermore, we have used a commercially available cell viability assay to determine the proportion and rate of cell death in cells following infection with laboratory and neuroinvasive clinical strains of HSV-1, and pharmacological inhibitors of necroptotic and apoptotic pathway components to assess the relative role of each.ResultsWe show that the loss of ZBP1 in astrocytes results in an increase in the number of viral particles released following HSV-1 infection. Importantly, we have confirmed that HSV-1 induces necroptosis in astrocytes and have established the ability of ZBP1 to mediate this cell death pathway. Interestingly, while ZBP1 is best known for its role in necroptotic signaling, our findings indicate that this sensor can also contribute to virally induced apoptosis in these glia.ConclusionsOur findings indicate that ZBP1 serves as a restriction factor for HSV-1 infection and is associated with the induction of both necroptotic and apoptotic cell death pathways in primary murine astrocytes. While it remains to be seen whether ZBP1-mediated activation of cell death in astrocytes contributes significantly to host protection or, rather, exacerbates HSV-1 encephalitis pathology, the identification of such a role in resident CNS cells may represent a novel target for therapeutic intervention to reduce HSV encephalitis-associated morbidity and mortality.

Impairment of Mitochondrial ATP Synthesis Induces RIPK3-dependent Necroptosis in Lung Epithelial Cells During Lung Injury by Lung Inflammation.

Dysfunction of mitochondrial metabolism is implicated in cellular injury and cell death. While mitochondrial dysfunction is associated with lung injury by lung inflammation, the mechanism by which the impairment of mitochondrial ATP synthesis regulates necroptosis during acute lung injury (ALI) by lung inflammation is unclear. Here, we showed that the impairment of mitochondrial ATP synthesis induces receptor interacting serine/threonine kinase 3 (RIPK3)-dependent necroptosis during lung injury by lung inflammation. We found that the impairment of mitochondrial ATP synthesis by oligomycin, an inhibitor of ATP synthase, resulted in increased lung injury and RIPK3 levels in lung tissues during lung inflammation by LPS in mice. The elevated RIPK3 and RIPK3 phosphorylation levels by oligomycin resulted in high mixed lineage kinase domain-like (MLKL) phosphorylation, the terminal molecule in necroptotic cell death pathway, in lung epithelial cells during lung inflammation. Moreover, the levels of protein in bronchoalveolar lavage fluid (BALF) were increased by the activation of necroptosis via oligomycin during lung inflammation. Furthermore, the levels of ATP5A, a catalytic subunit of the mitochondrial ATP synthase complex for ATP synthesis, were reduced in lung epithelial cells of lung tissues from patients with acute respiratory distress syndrome (ARDS), the most severe form of ALI. The levels of RIPK3, RIPK3 phosphorylation and MLKL phosphorylation were elevated in lung epithelial cells in patients with ARDS. Our results suggest that the impairment of mitochondrial ATP synthesis induces RIPK3-dependent necroptosis in lung epithelial cells during lung injury by lung inflammation.

The palladacycle, BTC2, exhibits anti-breast cancer and breast cancer stem cell activity

In women globally, breast cancer is responsible for most cancer-related deaths and thus, new effective therapeutic strategies are required to treat this malignancy. Platinum-based compounds like cisplatin are widely used to treat breast cancer, however, they come with limitations such as poor solubility, adverse effects, and drug resistance. To overcome these limitations, complexes containing other platinum group metals such as palladium have been studied and some have already entered clinical trials. Here we investigated the anti-cancer activity of a palladium complex, BTC2, in MCF-7 oestrogen receptor positive (ER+) and MDA-MB-231 triple negative (TN) human breast cancer cells as well as in a human breast cancer xenograft chick embryo model. BTC2 exhibited an average IC50 value of 0.54 μM, a desirable selectivity index of >2, inhibited the migration of ER+ and TN breast cancer cells, and displayed anti-cancer stem cell activity. We demonstrate that BTC2 induced DNA double strand breaks (increased levels of γ-H2AX) and activated the p-ATM/p-CHK2 and p-p38/MAPK pathways resulting in S- and G2/M-phase cell cycle arrests. Importantly, BTC2 sensitised breast cancer cells by triggering the intrinsic (cleaved caspase 9) and extrinsic (cleaved caspase 8) apoptotic as well as necroptotic (p-RIP3 and p-MLKL) cell death pathways and inhibiting autophagy and its pro-survival role. Furthermore, in the xenograft in vivo model, BTC2 displayed limited toxicity and arrested the tumour growth of breast cancer cells over a 9-day period in a manner comparable to that of the positive control drug, paclitaxel. BTC2 thus displayed promising anti-breast cancer activity.

The necroptotic cell death pathway operates in megakaryocytes, but not in platelet synthesis

Necroptosis is a pro-inflammatory cell death program executed by the terminal effector, mixed lineage kinase domain-like (MLKL). Previous studies suggested a role for the necroptotic machinery in platelets, where loss of MLKL or its upstream regulator, RIPK3 kinase, impacted thrombosis and haemostasis. However, it remains unknown whether necroptosis operates within megakaryocytes, the progenitors of platelets, and whether necroptotic cell death might contribute to or diminish platelet production. Here, we demonstrate that megakaryocytes possess a functional necroptosis signalling cascade. Necroptosis activation leads to phosphorylation of MLKL, loss of viability and cell swelling. Analyses at steady state and post antibody-mediated thrombocytopenia revealed that platelet production was normal in the absence of MLKL, however, platelet activation and haemostasis were impaired with prolonged tail re-bleeding times. We conclude that MLKL plays a role in regulating platelet function and haemostasis and that necroptosis signalling in megakaryocytes is dispensable for platelet production.

Necroptotic movers and shakers: cell types, inflammatory drivers and diseases.

The necroptotic cell death pathway has received significant attention for its ability to trigger inflammatory responses and its potential involvement in related conditions. Recent insights into the essential membrane damaging necroptotic pseudokinase effector, Mixed lineage kinase domain like (MLKL), have revealed a number of diverse MLKL functions that contribute to the inflammatory nature of necroptosis. Here we review distinct MLKL signalling roles and document the immunogenic molecules released by necroptosis. We discuss specific in vivo MLKL-driven responses, the activation of inflammasome complexes and innate lymphoid cells, which have been documented to drive disease. Finally, we list necroptotic competent cell types and their involvement in MLKL-driven cell death-associated and inflammatory-associated conditions.

Human cytomegalovirus protein pUL36: A dual cell death pathway inhibitor

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of intrinsic, innate, and adaptive viral immune evasion. Here, we employed multiplexed tandem mass tag-based proteomics to characterize host proteins targeted for degradation late during HCMV infection. This approach revealed that mixed lineage kinase domain-like protein (MLKL), a key terminal mediator of cellular necroptosis, was rapidly and persistently degraded by the minimally passaged HCMV strain Merlin but not the extensively passaged strain AD169. The strain Merlin viral inhibitor of apoptosis pUL36 was necessary and sufficient both to degrade MLKL and to inhibit necroptosis. Furthermore, mutation of pUL36 Cys131 abrogated MLKL degradation and restored necroptosis. As the same residue is also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of procaspase-8, we define pUL36 as a multifunctional inhibitor of both apoptotic and necroptotic cell death.

Cyclin-Dependent Kinase 9 Inhibition Suppresses Necroptosis and Pyroptosis in the Progress of Endotoxemia.

The host innate immune response stands at the first line of defense against the outburst of pathogen invasion and their byproduct release. The balanced and coordinated expression of genes in normal immune responses is compromised in the progress of endotoxemia with exacerbated inflammation and massive cell death. In the present study, we identified cyclin-dependent kinase 9 (CDK9), the functional subunit of the positive transcription elongation factor b, as a master regulator of inflammatory gene transcription in the process of promoter-proximal pausing to productive elongation. Therapeutic pharmacological inhibition of CDK9 by flavopiridol (FVD) rescued mice from death in experimental models of fatal endotoxemia. In addition to alleviation of the cytokine storm in the circulation system following lethal endotoxin injection, FVD treatment significantly dampened the onset of inflammation in the livers and lungs and reduced the necroptosis and pyroptosis in livers. Moreover, CDK9 inhibition reduced inflammatory cytokine release and decreased cell death in the pro-inflammatory pyroptotic and necroptotic cell death pathway in monocytes in responses to lipopolysaccharide. In conclusion, CDK9 inhibition may affect the progress of endotoxemia by dampening inflammation and cell death including necroptosis and pyroptosis.

Assessment of necroptosis in the retina in a repeated primary ocular blast injury mouse model

Primary blast injury (caused by the initial rapid increase in pressure following an explosive blast) to the retina and optic nerve (ON) causes progressive visual loss and neurodegeneration. Military personnel are exposed to multiple low-overpressure blast waves, which may be in quick succession, such as during breacher training or in combat. We investigated the necroptotic cell death pathway in the retina in a mouse repeated primary ocular blast injury (rPBI) model using immunohistochemistry. We further evaluated whether intravitreal injections of a potent necroptosis inhibitor, Necrostatin-1s (Nec-1s), protects the retina and ON axons by retinal ganglion cells (RGC) counts, ON axonal counting and optical coherence tomography (OCT) analysis of vitreous haze. Receptor interacting protein kinase (RIPK) 3, increased in the inner plexiform layer 2 days post injury (dpi) and persisted until 14 dpi, whilst RIPK1 protein expression did not change after injury. The number of degenerating ON axons was increased at 28 dpi but there was no evidence of a reduction in the number of intact ON axons or RNA-binding protein with multiple splicing (RBPMS)+ RGC in the retina by 28 dpi in animals not receiving any intravitreal injections. But, when intravitreal injections (vehicle or Nec-1s) were given there was a significant reduction in RBPMS+ RGC numbers, suggesting that rPBI with intraocular injections is damaging to RGC. There were fewer RGC lost after Nec-1s than vehicle injection, but there was no effect of Nec-1s or vehicle treatment on the number of degenerating axons. OCT analysis demonstrated no effect of rPBI on vitreous haze, but intravitreal injection combined with rPBI increased vitreous haze (P = 0.004). Whilst necroptosis may be an active cell death signalling pathway after rPBI, its inhibition did not prevent cell death, and intravitreal injections in combination with rPBI increased vitreous inflammation and reduced RBPMS+ RGC numbers, implying intravitreal injection is not an ideal method for drug delivery after rPBI.

Identification of the PANoptosome: A Molecular Platform Triggering Pyroptosis, Apoptosis, and Necroptosis (PANoptosis).

Programmed cell death plays crucial roles in organismal development and host defense. Recent studies have highlighted mechanistic overlaps and extensive, multifaceted crosstalk between pyroptosis, apoptosis, and necroptosis, three programmed cell death pathways traditionally considered autonomous. The growing body of evidence, in conjunction with the identification of molecules controlling the concomitant activation of all three pathways by pathological triggers, has led to the development of the concept of PANoptosis. During PANoptosis, inflammatory cell death occurs through the collective activation of pyroptosis, apoptosis, and necroptosis, which can circumvent pathogen-mediated inhibition of individual death pathways. Many of the molecular details of this emerging pathway are unclear. Here, we describe the activation of PANoptosis by bacterial and viral triggers and report protein interactions that reveal the formation of a PANoptosome complex. Infection of macrophages with influenza A virus, vesicular stomatitis virus, Listeria monocytogenes, or Salmonella enterica serovar Typhimurium resulted in robust cell death and the hallmarks of PANoptosis activation. Combined deletion of the PANoptotic components caspase-1 (CASP1), CASP11, receptor-interacting serine/threonine-protein kinase 3 (RIPK3), and CASP8 largely protected macrophages from cell death induced by these pathogens, while deletion of individual components provided reduced or no protection. Further, molecules from the pyroptotic, apoptotic, and necroptotic cell death pathways interacted to form a single molecular complex that we have termed the PANoptosome. Overall, our study identifies pathogens capable of activating PANoptosis and the formation of a PANoptosome complex.

Smac mimetics can provoke lytic cell death that is neither apoptotic nor necroptotic.

Smac mimetics, or IAP antagonists, are a class of drugs currently being evaluated as anti-cancer therapeutics. These agents antagonize IAP proteins, including cIAP1/2 and XIAP, to induce cell death via apoptotic or, upon caspase-8 deficiency, necroptotic cell death pathways. Many cancer cells are unresponsive to Smac mimetic treatment as a single agent but can be sensitized to killing in the presence of the cytokine TNFα, provided either exogenously or via autocrine production. We found that high concentrations of a subset of Smac mimetics could provoke death in cells that did not produce TNFα, despite sensitization at lower concentrations by TNFα. The ability of these drugs to kill did not correlate with valency. These cells remained responsive to the lethal effects of Smac mimetics at high concentrations despite genetic or pharmacological impairments in apoptotic, necroptotic, pyroptotic, autophagic and ferroptotic cell death pathways. Analysis of dying cells revealed necrotic morphology, which was accompanied by the release of lactate dehydrogenase and cell membrane rupture without prior phosphatidylserine exposure implying cell lysis, which occurred over a several hours. Our study reveals that cells incapable of autocrine TNFα production are sensitive to some Smac mimetic compounds when used at high concentrations, and this exposure elicits a lytic cell death phenotype that occurs via a mechanism not requiring apoptotic caspases or necroptotic effectors RIPK3 or MLKL. These data reveal the possibility that non-canonical cell death pathways can be triggered by these drugs when applied at high concentrations.

Targeting the p62-ZZ/N-End Rule Pathway in Multiple Myeloma Overcomes Proteasome Inhibitor-Resistance Via Induction of Necroptosis and Enhances the Bone Anabolic Effects of Proteasome Inhibitors

Multiple myeloma (MM) is incurable and 80% of MM patients develop MM bone disease (MMBD). MMBD lesions do not heal due to the persistent suppression of bone formation, which markedly increases mortality and contributes to MM drug resistance. Current treatments for MMBD, such as bisphosphonates and denosumab, target bone destruction but do not result in new bone formation. Although proteasome inhibitors (PIs) have greatly improved survival of MM patients, they only have transient bone anabolic effects. Further, development of drug resistance to PIs remains a major clinical problem. We previously showed that the ZZ domain of p62 (sequestosome-1),plays an important role in both MM growth and suppression of osteoblast (OB) differentiation in the MM microenvironment, by regulating multiple signaling pathways and acting as a cargo-receptor for autophagy. Recently, our collaborators showed that the p62-ZZ is a high-affinity N-recognin of the N-end rule pathway (NERP). p62-ZZ also serves as the molecular switch for necroptotic versus apoptotic cell death pathways. We previously reported that MM cells or TNFα prevent OB differentiation by inducing persistent epigenetic repression of the Runx2-P1 promoter in MM patient bone marrow stromal cells (BMSCs), via the transcriptional repressor Gfi1. We found that blocking p62-ZZ by saturating it with a novel synthetic p62-ZZ/NERP competing ligand, XRK3F2, prevented and reversed MM-induced Gfi1 occupancy at the Runx2-P1 promoter, allowing BMSCs to increase OB marker gene expression and to mineralize. These results suggest that targeting the p62-ZZ/N-end rule pathway would enhance the bone anabolic effects of PIs. To test this hypothesis, we first exposed normal OBs to different doses of bortezomib (Btz) or XRK3F2 or their combination. The combination significantly increased OB differentiation markers Runx2 (60%), Osterix (20%) and ATF4 (60%), and induced mineral deposition compared with either drug alone. The combination also blocked TNFα up-regulation of Gfi1 and suppression of OB differentiation. Interestingly, none of the concentrations tested decreased OB viability. Studies with MM patient-BMSCs showed that XRK3F2 reversed suppression of OB differentiation induced by MM cells, allowing them to mineralize. Importantly, our preliminary in vivo data showed that administration of XRK3F2 to mice with established MM induced dramatic cortical bone formation in MM-containing bones but had no effect on tumor burden. We and others previously showed that MM cells subjected to sustained proteasomal inhibition, rely on p62-mediated autophagic degradation to reduce the proteotoxic load caused by excessive immunoglobulin synthesis. We recently found that targeting the p62-ZZ domain in human MM cells, increases Btz-induced MM cell death, independently of their p53 status. The combination also significantly reduced cell viability in Btz resistant cells although no caspase 3 activation was observed, suggesting a caspase-3 independent cell death. To determine the mechanism(s) responsible for MM cell death induced by the combination, we pretreated MM cell lines and primary CD138+ MM cells with Z-DEVD (20μM), bafilomycin (Baf, 40nM), or necrostatin1 (NEC1, 50μM). The anti-MM effects of XRK3F2 or Btz+XRK3F2 were fully blocked by NEC1, an inhibitor of necroptosis, but not by inhibitors of caspase-dependent apoptosis (Z-DEVD) or autophagy (Baf), supporting that p62-ZZ regulates necroptosis in MM cells. RNAseq analysis of the additive effect of Btz+XRK3F2 on gene expression showed a total of 583 differentially regulated genes, including 374 significantly down-regulated and 209 significantly upregulated. GO term analysis of up-regulated DEGs identified an enrichment in the endoplasmic reticulum (ER) stress and ER unfolded protein response, and regulation of transcription in response to stress and autophagy. In summary, our results demonstrate that targeting the p62-ZZ/N-end rule pathway in combination with PIs in MM significantly reduces MM cell viability by activating multiple death pathway and overcomes PI-resistance of MM cells. In addition, targeting the p62-ZZ in OBs potentiates the bone anabolic action of PIs and reverses the persistent OB suppression induced by MM cells to allow bone formation. Thus, p62-ZZ plays a critical role in MM and bone cells and identifies p62-ZZ as an important molecular target for the treatment of MMBD. Disclosures Xie: Oxis Biotech: Consultancy; ID4Pharma: Other: Founder. Roodman:Amgen: Membership on an entity's Board of Directors or advisory committees.