Caspase 8

Caspase 8 is an initiator cysteine-aspartic protease encoded by the CASP8 gene on chromosome 2q33-34, essential for mediating programmed cell death primarily through the extrinsic apoptosis pathway triggered by death receptors such as Fas (CD95), TNF receptor 1 (TNFR1), and TRAIL receptors.^[1] As a 55 kDa proenzyme consisting of 479 amino acids, it features two N-terminal death effector domains (DEDs) for protein interactions and a C-terminal catalytic region that forms large (p18/p20) and small (p10/p12) subunits upon activation.^[1] Beyond apoptosis, caspase 8 exhibits dual functions, inhibiting necroptosis by cleaving receptor-interacting protein kinases (RIPK1 and RIPK3) while contributing to pyroptosis and inflammasome activation in inflammatory contexts.^[2] Discovered in the mid-1990s through homology searches and cloning from human cell lines, caspase 8 was initially identified as a key mediator of TNF- and Fas-induced cell death, earning aliases such as MACH, FLICE, and Mch5.^[2] Its activation occurs via ligand-induced oligomerization of death receptors, recruiting the adaptor protein FADD to form the death-inducing signaling complex (DISC), where procaspase 8 dimerizes and undergoes autocleavage to generate the active heterotetramer.^[1] This process is tightly regulated by inhibitors like cFLIP, which can form heterodimers with caspase 8 to either enhance or suppress its activity depending on isoform and cellular context.^[2] Genetic knockout studies in mice reveal its indispensability, as Casp8-deficient embryos exhibit lethality around embryonic day 10.5 due to excessive necroptosis in hematopoietic and endothelial tissues.^[2] In apoptosis, activated caspase 8 directly cleaves and activates effector caspases such as caspase-3 and -7, amplifying the proteolytic cascade that dismantles cellular structures, while also processing BH3-interacting domain death agonist (Bid) to bridge extrinsic and intrinsic mitochondrial pathways.^[1] Its non-apoptotic roles extend to immune regulation, where it acts as a scaffold in the ripoptosome complex to suppress necroptosis and promote NF-κB signaling for cytokine production, including IL-1β via inflammasome modulation.^[2] Dysregulation of caspase 8 is implicated in diseases ranging from cancers (e.g., reduced expression in head and neck squamous cell carcinoma promoting survival) to inflammatory disorders like inflammatory bowel disease and sepsis, positioning it as a therapeutic target through specific inhibitors or activators such as Smac mimetics.^[1]

Structure and Properties

Domain Organization

Caspase 8 belongs to the cysteine-aspartic acid protease (caspase) family of enzymes, characterized by a catalytic cysteine residue and specificity for aspartate in substrate cleavage sites. The human CASP8 gene, situated on chromosome 2q33.1, encodes the 479-amino-acid proenzyme procaspase-8, which exists as an inactive zymogen with an approximate molecular weight of 55 kDa.^[3]^[4]^[5]^[6] The N-terminal region of procaspase-8 comprises two tandem death effector domains (DEDs), spanning approximately amino acids 1–160, that facilitate homotypic protein-protein interactions essential for its recruitment into oligomeric signaling complexes such as the death-inducing signaling complex (DISC).^[7]^[3] These DEDs, each approximately 70 amino acids long, enable binding to adaptor proteins like FADD, positioning procaspase-8 at the apex of extrinsic apoptosis pathways. The C-terminal portion houses the protease domain, which undergoes autoproteolytic processing to yield the active form. This domain consists of a large p18 subunit (amino acids 217–374) and a small p10 subunit (amino acids 385–479), which assemble into an active heterotetramer (p18/p10)₂. Within the p18 subunit lies the catalytic dyad, featuring the nucleophilic cysteine at position 360 (Cys360) and a histidine (His317) that activates it, conferring the enzyme's strict aspartate specificity at the P1 position of target substrates.^[3]^[8] Activation involves specific proteolytic cleavages at aspartate residues, primarily Asp374 to separate the p18 and p10 subunits, with Asp384 implicated in intermediate processing steps that generate forms like p41/p10 and enable full enzymatic competency.^[9] These events occur within proximity-induced dimers or oligomers, transitioning the zymogen to its mature, catalytically active state. The overall sequence of caspase 8 exhibits strong conservation across vertebrate species, with the catalytic domain showing over 80% identity, reflecting its evolutionarily preserved function in cell death regulation.^[10]

Expression and Localization

The CASP8 gene, which encodes caspase 8, is located on chromosome 2q33.1 in humans and spans approximately 54 kb, comprising 16 exons.^[4] Alternative splicing of CASP8 pre-mRNA generates multiple isoforms, including the full-length caspase-8/a (isoform 1), which retains the complete prodomain and catalytic regions, and caspase-8/b (isoform 2), which differs in a short N-terminal sequence within the prodomain but preserves the catalytic domain.^[4] Shorter isoforms, such as caspase-8/s (isoform 3, also known as MCH5-beta), arise from splicing events that exclude the catalytic domain, resulting in truncated proteins lacking protease activity.^[4]^[11] Caspase 8 exhibits ubiquitous expression across human tissues, with the highest levels observed in lymphoid organs such as the spleen, thymus, and lymph nodes, as well as in immune cells including T cells and B cells.^[3]^[12] In contrast, expression is notably lower in the brain and skeletal muscle, reflecting its prominent role in immune-related processes.^[12] Isoforms 1, 5, and 7 predominate in these expression patterns, while shorter variants like caspase-8/s show more restricted distribution.^[3] In its inactive proenzyme form, caspase 8 is primarily localized in the cytosol.^[13] Upon activation, it translocates to the plasma membrane or perinuclear regions through interactions mediated by its death effector domains (DEDs), facilitating recruitment to signaling complexes.^[14] Isoform-specific localization differs, with short isoforms such as caspase-8/s predominantly associating with mitochondria, where they may contribute to non-apoptotic functions.^[11] Post-translational modifications, including ubiquitination and SUMOylation, influence caspase 8 stability and localization without directly impacting activation. Ubiquitination by E3 ligases like cIAPs targets caspase 8 for proteasomal degradation, thereby regulating its protein levels and cytosolic abundance.^[15] SUMOylation, particularly on lysine residues in the prodomain, promotes nuclear translocation and enhances stability by preventing ubiquitination at overlapping sites.^[16]^[17] These modifications provide fine-tuned control over caspase 8 distribution in response to cellular cues.

Role in Cell Death Pathways

Initiation of Apoptosis

Caspase 8 serves as a key initiator caspase in the extrinsic apoptosis pathway, which is triggered by extracellular death signals binding to members of the tumor necrosis factor (TNF) receptor superfamily, including Fas (CD95) and TNFR1. Upon ligand binding, such as Fas ligand or TNF-α, these receptors trimerize and recruit the adaptor protein FADD through their death domains, leading to the assembly of the death-inducing signaling complex (DISC). Procaspase 8, the inactive zymogen form, is then recruited to the DISC via homotypic interactions between its N-terminal death effector domains (DEDs) and those of FADD, forming an oligomeric platform that promotes induced proximity for activation. Within the DISC, procaspase 8 undergoes auto-proteolytic processing through sequential cleavage events. Initial dimerization facilitates cleavage at Asp374, removing the intersubunit linker and prodomain to generate a partially active p43/41 intermediate, followed by cleavage at Asp384 to produce the mature active enzyme consisting of p18 and p10 subunits that form a heterotetramer. This activated caspase 8 then dissociates from the DISC and propagates the apoptotic signal by cleaving downstream targets, including effector caspases such as caspase 3 (at Asp175) and caspase 7, thereby initiating the execution phase of apoptosis. In type I cells, activated caspase 8 directly activates effector caspases at sufficient levels, while in type II cells, low caspase 8 activity requires amplification via the intrinsic pathway. Additionally, caspase 8 cleaves Bid at Asp60, generating truncated Bid (tBid) that translocates to mitochondria to amplify the signal via the intrinsic pathway by promoting Bax/Bak oligomerization and cytochrome c release.^[18] In apoptotic contexts, caspase 8 also processes RIPK1, limiting its kinase activity to favor apoptotic execution over alternative fates. The essential role of caspase 8 in apoptosis initiation is evidenced by genetic studies in mice, where homozygous knockout of the Casp8 gene results in embryonic lethality around day 10.5-11.5 post-coitum, characterized by impaired heart muscle development and excessive accumulation of trophoblast giant cells, primarily due to excessive necroptosis with contributions from defective apoptosis during embryogenesis. These findings underscore caspase 8's non-redundant function in developmental programmed cell death, as conditional ablation in specific tissues similarly disrupts apoptosis induction by death receptors.^[19]

Regulation of Necroptosis and Pyroptosis

Caspase 8 plays a critical dual role in regulating necroptosis, a form of programmed necrotic cell death, by acting as a suppressor in the absence of active apoptosis. Upon activation by death receptors such as TNFR1, Caspase 8 forms a complex with RIPK1 and FADD, where it cleaves RIPK1 and RIPK3 to prevent the formation of the necrosome complex. This cleavage inhibits the kinase activity of RIPK3, thereby blocking downstream phosphorylation and activation of MLKL, which would otherwise lead to plasma membrane rupture and inflammatory cell death. In Caspase 8-deficient models, such as embryonic mice lacking Caspase 8, unchecked RIPK1/RIPK3 activity results in embryonic lethality due to excessive necroptosis, underscoring its essential suppressive function.^[19]^[20] The regulatory influence of Caspase 8 on necroptosis is highly context-dependent, serving as a molecular switch that can pivot between apoptotic and necroptotic outcomes. When Caspase 8 activity is inhibited, for instance by viral proteins like those from cowpox virus (e.g., CrmA) or human cytomegalovirus, it fails to cleave RIPK1/RIPK3, allowing the necrosome to form and necroptosis to proceed as a backup mechanism to evade pathogen-induced apoptosis. This switch ensures cellular elimination in scenarios where apoptosis is blocked, highlighting Caspase 8's role in balancing cell death pathways during infection. Studies in knockout models confirm that combined inhibition of Caspase 8 and necroptosis effectors like RIPK3 restores viability, illustrating the pathway's evolutionary adaptation for host defense.^[21]^[22] In pyroptosis, an inflammatory form of lytic cell death, Caspase 8 contributes indirectly through crosstalk with inflammasome pathways, linking extrinsic death signals to the processing of gasdermin D (GSDMD) and IL-1β release. In cells deficient in canonical pyroptotic caspases like Caspase 1, Caspase 8 can be recruited to the NLRP3 or AIM2 inflammasome, where it cleaves GSDMD at an alternative site to induce pore formation and cytokine secretion, albeit less efficiently than Caspase 1. This alternative activation occurs particularly when primary inflammasome responses are suppressed, such as during certain bacterial infections, allowing Caspase 8 to drive a hybrid pyroptotic response that amplifies innate immunity.^[23]^[24] Recent post-2020 research has further elucidated Caspase 8's role in innate immunity, where it limits excessive inflammation by modulating NLRP3 inflammasome activity. For example, Caspase 8-mediated cleavage of RIPK3 restricts NLRP3-dependent pyroptosis and IL-1β production in scenarios involving inhibitors of apoptosis proteins, preventing hyperinflammation during infection or stress. A 2024 study demonstrated that this regulation is crucial in macrophages, where Caspase 8 dampens NLRP3-driven responses to maintain immune homeostasis. These findings emphasize Caspase 8's broader function in fine-tuning inflammasome outputs beyond direct cell death execution.^[25]^[26] The regulatory functions of Caspase 8 in necroptosis and pyroptosis exhibit evolutionary conservation across non-mammalian species, reflecting an ancient mechanism for balancing cell death and inflammation. Caspase-8 function in apoptosis is evolutionarily conserved across vertebrates, including in teleost fish.^[27] This conservation highlights Caspase 8's fundamental role in integrating apoptotic and inflammatory signaling to prevent pathological tissue damage across vertebrates.

Activation and Regulation

Activation Mechanisms

Caspase 8 activation is primarily triggered by extrinsic signals through death receptors such as Fas (CD95) and TRAIL receptors. Ligand binding, for example by FasL or TRAIL, induces trimerization of the receptor, which recruits the adaptor protein FADD via death domain interactions. FADD then binds procaspase-8 through death effector domain (DED) homotypic interactions, forming the death-inducing signaling complex (DISC) and promoting procaspase-8 oligomerization.^[28] Within the DISC, activation occurs via proximity-induced dimerization of procaspase-8 molecules. This dimerization induces a conformational change that exposes the active site cleft, enabling catalytic activity without requiring allosteric regulation. The process relies on the stable homophilic interactions between DEDs, which position the catalytic domains in close proximity to facilitate initial proteolysis.^[29]^[30] Following dimerization, a proteolytic cascade ensues through auto-cleavage at the interdomain linker, specifically after Asp384. This cleavage dissociates the procaspase-8 into the p18 (large) and p10 (small) subunits, which then assemble into a heterotetrameric structure ((p18)₂(p10)₂). The fully processed heterotetramer exhibits significantly enhanced catalytic activity compared to the monomeric zymogen form, due to optimized active site geometry and substrate access.^[31] Active caspase-8 can amplify the apoptotic signal intrinsically by cleaving Bid at Asp60, generating truncated Bid (tBid). tBid translocates to the mitochondria, where it promotes Bax/Bak oligomerization, leading to cytochrome c release and subsequent activation of caspase-9 in the apoptosome.^[32] Structurally, caspase-8's specificity for aspartate residues in substrates arises from its S1 pocket, which accommodates the P1 Asp side chain through salt bridges with Arg260 and Arg413, as well as hydrogen bonds with Gln358. Kinetic studies indicate Km values for peptide substrates like Ac-IETD-pNA in the micromolar range, reflecting its role as an initiator caspase with moderate substrate affinity.^[33]

Inhibitory and Modulatory Factors

One key endogenous inhibitor of caspase-8 is cFLIP (also known as CFLAR), which heterodimerizes with procaspase-8 at the death-inducing signaling complex (DISC), thereby blocking its proteolytic cleavage and activation. The cFLIP protein exists in multiple isoforms, with the long isoform (cFLIP-L) featuring a caspase-like domain that allows partial activity but ultimately inhibits full caspase-8 processing, while the short isoform (cFLIP-S) lacks catalytic activity and dominantly suppresses caspase-8 by competing for binding to adaptor proteins like FADD.^[34] These isoforms fine-tune caspase-8 activity to balance apoptotic signaling and prevent excessive cell death during immune responses or stress.^[35] Phosphorylation serves as a critical modulatory mechanism for caspase-8, with specific sites altering its activation threshold. For instance, phosphorylation at Ser387 by cyclin-dependent kinase 1 (Cdk1) during mitosis inhibits caspase-8 processing and activity, promoting cell survival in dividing cells; conversely, dephosphorylation at this site by protein phosphatase 2A (PP2A) enhances caspase-8 activation and sensitivity to death signals. This reversible modification ensures temporal control, as seen in contexts like neutrophil survival where dephosphorylation correlates with increased caspase-8-mediated apoptosis.^[36] Ubiquitination further modulates caspase-8 stability and function through distinct chain linkages. K48-linked polyubiquitination, mediated by inhibitors of apoptosis proteins such as cIAP1, targets caspase-8 for proteasomal degradation, thereby suppressing its pro-apoptotic effects during TNF receptor signaling.^[37] In contrast, K63-linked ubiquitination of associated proteins like RIPK1, also facilitated by cIAP1, promotes non-degradative signaling that indirectly modulates caspase-8 by stabilizing anti-apoptotic complexes.^[38] These ubiquitin modifications establish a threshold for caspase-8 activation, preventing unintended cell death in inflammatory settings. Viruses have evolved modulators that mimic host inhibitors to evade apoptosis, exemplified by vFLIP from Kaposi's sarcoma-associated herpesvirus (KSHV). vFLIP structurally resembles cFLIP and binds directly to procaspase-8, inhibiting its dimerization and cleavage at the DISC to promote viral persistence in endothelial cells and lymphocytes.^[39] This viral strategy not only blocks apoptosis but also sustains infected cell survival, contributing to KSHV-associated malignancies like Kaposi's sarcoma.^[40] Feedback loops involving caspase-8 auto-cleavage provide intrinsic self-regulation to limit prolonged activity. Upon initial activation, caspase-8 undergoes further auto-proteolytic processing that cleaves its own catalytic domains, reducing enzymatic longevity and preventing over-amplification of death signals while allowing resolution of the apoptotic response.^[41] This mechanism maintains homeostasis by balancing initiation and termination of caspase-8-driven pathways, as disruptions in auto-cleavage can shift toward necroptosis or chronic inflammation.^[31]

Molecular Interactions

Key Protein Partners

Caspase 8, an initiator caspase in the extrinsic apoptosis pathway, engages in direct interactions with several key protein partners through its death effector domains (DEDs) and catalytic subunits, facilitating signal transduction in cell death mechanisms. One primary partner is Fas-associated death domain (FADD), where the death domain (DD) of FADD binds the DEDs of procaspase 8 with high affinity, measured at a dissociation constant (Kd) of 38 nM via surface plasmon resonance assays, enabling recruitment to the death-inducing signaling complex (DISC).^[42] This interaction is mediated by specific residues, such as F122 on caspase 8 DED2 docking into the α1/α4 groove of FADD DED, and is essential for DISC assembly, as mutations disrupting this contact abolish binding and downstream signaling.^[43] Another critical partner is cellular FLICE-like inhibitory protein (cFLIP), which forms heterodimers with procaspase 8 primarily through DED-DED contacts, exhibiting structural mimicry where the pseudo-catalytic p12 subunit of cFLIP resembles that of caspase 8.^[44] Cryo-EM structures of these binary complexes reveal stable stoichiometries, such as 6:4 or 9:4 (cFLIP:caspase 8), with type III-II-III composite self-assembling sites driving the interaction and limiting full caspase 8 activation to promote cell survival.^[44] This heterodimerization occurs independently of FADD in some contexts, modulating apoptotic threshold by competing for binding sites within the DISC. Receptor-interacting protein kinase 1 (RIPK1) interacts with caspase 8 in dual modes: proteolytic cleavage at Asp324 in necroptosis-prone conditions, separating the kinase domain from the death domain to inhibit RIPK1 activation, and non-cleaved binding within the TNFR1 signaling complex, where caspase 8 associates with uncleaved RIPK1, FADD, and NF-κB components to regulate inflammation.^[45] Mutations preventing cleavage at Asp324 enhance RIPK1-dependent proinflammatory signaling, highlighting the regulatory role of this interaction.^[45] Caspase 8 also directly processes BH3-interacting domain death agonist (Bid) by cleavage between Asp60 and Gly62, generating truncated Bid (tBid) that translocates to the mitochondrial outer membrane to induce permeabilization and cytochrome c release.^[46] This cleavage is a key link between extrinsic and intrinsic apoptosis pathways, with tBid promoting caspase-independent mitochondrial clustering and subsequent apoptotic execution.^[46] Among other direct partners, apoptosis-associated speck-like protein containing a CARD (ASC) recruits caspase 8 into NLRP3 inflammasomes via CARD-CARD interactions, as evidenced by co-immunoprecipitation and colocalization studies showing caspase 8 processing within ASC-enriched complexes to drive IL-1β maturation in caspase-1-deficient cells.^[47] Similarly, TNF receptor-associated factor 2 (TRAF2) binds caspase 8 at the DISC to serve as an E3 ubiquitin ligase scaffold, mediating K48-linked polyubiquitination at lysines K224, K229, and K231 on the p18 subunit, which tags activated caspase 8 for proteasomal degradation and sets an apoptotic threshold, confirmed through co-immunoprecipitation and ubiquitination assays.^[48] These interactions, often quantified via co-IP, underscore caspase 8's role in balancing death and survival signals without forming larger assemblies.

Involvement in Signaling Complexes

Caspase 8 serves as a central component in the death-inducing signaling complex (DISC), a multi-protein assembly triggered by ligation of death receptors such as Fas (CD95). The DISC comprises the death receptor Fas, the adaptor protein FADD, procaspase-8, and regulatory factors including cFLIP isoforms, which modulate the activation threshold.^[49] Upon Fas trimerization, FADD is recruited via death domain interactions, followed by binding of procaspase-8 through death effector domain (DED) homotypy, leading to induced proximity and autocatalytic activation of caspase 8.30526-3) Cryo-EM studies reveal that the tandem DEDs of caspase 8 form a helical filament structure within the DISC, incorporating approximately 5-10 caspase 8 molecules per complex, which facilitates efficient dimerization and proteolytic maturation while being dynamically regulated by cFLIP integration to prevent excessive activation.^[50] In response to genotoxic stress or toll-like receptor (TLR) stimulation, caspase 8 participates in the ripoptosome, an intracellular platform that integrates apoptotic and necroptotic signals. The ripoptosome consists of RIPK1, FADD, caspase 8, and cFLIP, assembling upon depletion of inhibitor of apoptosis proteins (IAPs) such as XIAP and cIAP1/2, which normally ubiquitinate and stabilize RIPK1.^[51] DNA damage, exemplified by etoposide treatment, induces ripoptosome formation by promoting IAP degradation, allowing RIPK1 deubiquitination and recruitment of FADD and procaspase-8 to initiate caspase 8 activation.00420-5) Similarly, TLR3 or TLR4 signaling via TRIF platforms triggers ripoptosome assembly, where caspase 8 balances apoptosis through substrate cleavage or necroptosis if inhibited, thereby preventing inflammatory cytokine overproduction. TNF receptor 1 (TNFR1) signaling involves caspase 8 recruitment during a phase transition from pro-survival to pro-death complexes. In the initial TNFR1 complex I, ligand binding recruits RIPK1 and TRAF2 to activate kinase-dependent pathways like NF-κB via IKK and MAPK signaling.00521-X) Upon disassembly—often due to IAP depletion or TAK1 inhibition—RIPK1 translocates to the cytosol, forming complex IIa with FADD and procaspase-8, which drives caspase 8-mediated apoptosis through death domain and DED interactions.^[52] This transition is spatially and temporally regulated, with complex IIa filaments visualized by structural analyses showing coordinated recruitment of 4-6 caspase 8 units to amplify death signaling while avoiding necroptotic shift via RIPK3.^[53] Caspase 8 also engages in crosstalk with inflammasomes, particularly a non-canonical NLRP3 platform that processes IL-1β independently of caspase 1. In this complex, NLRP3 and ASC recruit caspase 8 via FADD upon TLR priming and secondary stimuli like fungal β-glucans via Dectin-1, enabling direct cleavage of pro-IL-1β to its mature form.^[54] This pathway operates in parallel to the canonical NLRP3 inflammasome, where caspase 8 modulates IL-1β secretion in macrophages and dendritic cells without requiring gasdermin D pore formation for pyroptosis.42157-X/fulltext) Structural insights indicate that caspase 8 integrates into the ASC filament via DED interactions, sustaining low-level activation for cytokine maturation under non-lethal conditions.^[55] The stability of these caspase 8-containing complexes is finely tuned by ubiquitination, which influences assembly dynamics and outcome decisions. K63-linked ubiquitin chains on RIPK1, mediated by cIAP1/2 in complex I or ripoptosome precursors, promote pro-survival signaling, whereas their removal by deubiquitinases like CYLD facilitates caspase 8 recruitment and complex II formation.^[56] Polyubiquitination of caspase 8 itself, via CUL3-based E3 ligases at the DISC, enhances aggregation with p62 for signal amplification, while linear ubiquitin chains from LUBAC stabilize RIPK1 to suppress necroptosis.^[57] Recent cryo-EM structures from 2021 onward elucidate oligomeric interfaces, revealing how ubiquitin-binding motifs on FADD and caspase 8 DEDs dictate filament stability and prevent aberrant activation in immune cells.^[58]

Physiological and Pathological Roles

Functions in Development and Immunity

Caspase 8 plays a critical role in embryonic development, as evidenced by the embryonic lethality observed in Casp8 knockout mice, which die around embryonic day 10.5 (E10.5) due to defects in heart muscle development, yolk sac vascularization, hematopoietic processes, and neural tube abnormalities.^[59] These phenotypes arise from disrupted vascular, cardiac, and hematopoietic processes, highlighting Caspase 8's necessity for proper embryogenesis beyond its apoptotic functions.^[60] Ex vivo culture studies of Caspase 8-deficient embryos further demonstrate that supplementation can normalize aberrant neural tube and heart development, underscoring its direct involvement in tissue morphogenesis.^[61] In the immune system, Caspase 8 is essential for T-cell homeostasis and function, where its conditional deletion in T cells results in reduced peripheral T-cell numbers and impaired proliferation in response to antigenic stimulation.^[62] It also regulates activation-induced cell death (AICD) in T cells, preventing excessive lymphocyte expansion while maintaining immune responsiveness, as seen in Caspase 8-deficient T cells that exhibit defective AICD following repeated stimulation.^[63] In macrophages, Caspase 8 limits excessive proinflammatory cytokine production, such as IL-1β, by suppressing RIPK3-dependent inflammasome activation independently of its catalytic activity.^[64] Beyond cell death pathways, Caspase 8 exhibits non-apoptotic roles, including nuclear translocation to influence gene regulation; for instance, it facilitates the activation of NF-κB signaling by promoting the nuclear import of the p65 subunit through phosphorylation at serine 536.^[65] Additionally, Caspase 8 contributes to cellular processes like migration and differentiation, as demonstrated in progenitor cells where its absence impairs adhesion and homing, thereby affecting tissue repair and development.^[66] In innate immunity, Caspase 8 suppresses excessive antiviral responses by cleaving IRF3, which promotes its proteasomal degradation and thereby attenuates type I interferon production during RNA virus infections.^[67] Recent studies have further linked Caspase 8 to the modulation of type I IFN pathways, showing that its activation in influenza A-infected cells enhances antiviral signaling by promoting type I interferon production.^[68] This regulatory function helps balance innate immune activation against viral threats. Caspase 8 maintains epithelial tissue homeostasis by inhibiting necroptosis, particularly in the intestinal barrier, where its deficiency in epithelial cells leads to TNF-α-induced necroptotic cell death, compromising barrier integrity and promoting inflammation. In this context, Caspase 8 acts as a checkpoint to prevent uncontrolled RIPK3/MLKL-mediated necroptosis, ensuring the survival and functional cohesion of epithelial layers during microbial challenges.^[69]

Associations with Diseases

Caspase 8 dysregulation plays a pivotal role in oncogenesis, particularly through epigenetic silencing via promoter hypermethylation, which occurs frequently in neuroblastoma tumors and leads to reduced expression and impaired apoptosis. In neuroblastoma, CASP8 promoter hypermethylation has been observed in approximately 60% of primary tumors, contributing to resistance against death receptor-mediated apoptosis and promoting tumor survival. Additionally, somatic mutations such as D302H in the CASP8 gene have been identified in non-Hodgkin lymphomas, where this variant acts as a genetic risk factor by impairing caspase 8 activation and enzymatic function, thereby enhancing lymphomagenesis. In autoimmune diseases, genetic variations in CASP8 influence susceptibility and progression. Although specific associations with systemic lupus erythematosus (SLE) remain under investigation, polymorphisms in the caspase family, including CASP8, have been implicated in altered apoptotic thresholds that exacerbate autoimmunity in SLE through dysregulated immune cell clearance. In rheumatoid arthritis (RA), caspase 8 activity in synovial fibroblasts contributes to persistent inflammation; variant G of caspase 8 promotes aggressive behavior in RA fibroblast-like synoviocytes by enhancing cell adhesion, migration, and invasion, independent of its catalytic function, thereby driving synovial hyperplasia and joint destruction. Dysfunction of caspase 8 is central to inflammatory bowel diseases (IBD), where reduced expression or activity in intestinal epithelial cells triggers necroptosis and compromises barrier integrity. In Crohn's disease models, caspase 8 deficiency leads to TNF-α-induced necroptosis of epithelial cells, resulting in terminal ileitis, increased permeability, and chronic inflammation reminiscent of human pathology. Recent analyses, including those from 2025, highlight caspase 8's role in modulating epithelial cell death during IBD flares, where its impairment exacerbates necroptotic signaling and perpetuates mucosal damage in inflammatory episodes. As of 2025, studies highlight caspase 8's potential as a therapeutic target in IBD by restoring epithelial barrier function and suppressing necroptotic flares.^[1] In neurodegenerative disorders, partial loss-of-function of caspase 8 accelerates motor neuron degeneration through unchecked necroptosis. In amyotrophic lateral sclerosis (ALS) models, both sporadic and familial forms, astrocytes from ALS patients induce necroptotic death of motor neurons via receptor-interacting protein kinase 3 (RIPK3)-dependent pathways, which are normally suppressed by caspase 8; inhibition of necroptosis protects neurons, underscoring caspase 8's protective role against progressive motor neuron loss. Caspase 8 is also targeted by viral proteins to evade host apoptosis and ensure persistence in infectious diseases. In HIV infection, the Tat protein impairs caspase 8 activation dynamics in T cells, delaying Fas-mediated apoptosis and allowing survival of infected cells, which facilitates viral latency and chronic persistence.

Therapeutic Implications

Targeting in Cancer

Caspase 8 serves as a key initiator in the extrinsic apoptosis pathway, making it a promising target for enhancing tumor cell death in cancers where intrinsic apoptosis is impaired. Strategies to activate Caspase 8 primarily leverage death receptor signaling, particularly through tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) agonists, which selectively induce apoptosis in cancer cells while sparing normal tissues. Recombinant TRAIL and agonistic antibodies bind to TRAIL receptors (TRAIL-R1/DR4 or TRAIL-R2/DR5), leading to death-inducing signaling complex (DISC) formation and Caspase 8 activation in tumor cells resistant to mitochondrial-mediated apoptosis.^[70] For instance, mapatumumab, a monoclonal antibody targeting TRAIL-R1, has demonstrated Caspase 8 activation and apoptosis induction in various solid tumors, including non-small cell lung cancer and hematologic malignancies, through cleavage of downstream effectors like Caspase 3.^[71] However, phase II clinical trials of mapatumumab as monotherapy or in combination have shown limited efficacy, with no significant clinical benefit in cancers such as non-small cell lung cancer, colorectal cancer, and multiple myeloma due to challenges in response rates and tumor heterogeneity.^[72]^[73] Another approach involves Smac mimetics, which are inhibitors of inhibitor of apoptosis proteins (IAPs) that indirectly promote Caspase 8 activity by disrupting survival signaling. These bivalent compounds, such as birinapant, antagonize cIAP1 and XIAP, leading to cIAP1 auto-ubiquitination and degradation, which relieves suppression of Caspase 8 and facilitates ripoptosome assembly—a Caspase 8-containing complex that amplifies apoptotic signaling in cancer cells.^[74] Birinapant has exhibited antitumor effects in preclinical models of breast, pancreatic, and myeloid cancers by promoting Caspase 8-dependent cell death, often synergizing with TRAIL to overcome resistance.^[75] Phase I/II trials of birinapant have reported manageable toxicity and evidence of Caspase 8 pathway engagement, though development has been inactive as of May 2025, limiting its advancement for cancers with high IAP expression.^[76] Gene therapy represents an emerging strategy to restore Caspase 8 function in tumors where its expression is epigenetically silenced, such as through promoter methylation in hepatocellular carcinoma (HCC). Viral vectors, including adenoviral systems, deliver wild-type CASP8 to re-express the protein, sensitizing cells to apoptotic stimuli and inhibiting tumor growth in preclinical HCC models.^[77] Combination therapies further enhance Caspase 8 activation by integrating it with conventional chemotherapeutics, particularly in p53-mutant cancers where mitochondrial apoptosis is defective. For example, doxorubicin sensitizes tumor cells to extrinsic signals by upregulating TRAIL receptors and promoting Bid cleavage by Caspase 8, which bridges the extrinsic pathway to the mitochondrial amplification loop and restores apoptosis in resistant p53-mutant lines like colon and breast cancers.^[78] This synergy has been validated in preclinical studies, where doxorubicin-induced DNA damage leads to Caspase 8-mediated tBid formation, amplifying effector Caspase activation and tumor cell killing without excessive reliance on p53.^[79] Despite these advances, targeting Caspase 8 in cancer therapy faces significant challenges, including off-target toxicity to normal cells due to ubiquitous death receptor expression, which can cause hepatotoxicity or cytokine storms in TRAIL-based regimens.^[70] Additionally, variable Caspase 8 expression across tumors necessitates biomarkers for patient selection; hypermethylation of the CASP8 promoter, detectable via methylation-specific PCR, identifies responsive subsets in neuroblastomas and other malignancies, guiding therapy to avoid ineffective treatments.^[80] Ongoing research aims to refine delivery methods and combinations to mitigate these issues while maximizing therapeutic index.

Applications in Inflammatory and Autoimmune Disorders

Inflammatory and autoimmune disorders often involve dysregulated Caspase 8 activity, which contributes to excessive necroptosis, pyroptosis, and inflammatory signaling in immune cells and tissues. Therapeutic strategies targeting Caspase 8 aim to modulate these pathways, reducing cytokine storms, tissue damage, and autoimmunity without broadly suppressing apoptosis. These approaches include small-molecule inhibitors of downstream necroptotic effectors and biologics that disrupt upstream signaling complexes, with emerging targeted therapies showing promise in preclinical models.^[81] Necroptosis inhibitors, such as small molecules targeting RIPK1 like necrostatin-1 and GSK2982772, indirectly enhance Caspase 8's suppressive effects on MLKL-mediated necroptosis in intestinal epithelial cells, thereby alleviating inflammation in inflammatory bowel disease (IBD). In murine models of colitis, necrostatin-1 treatment reduced weight loss, colon shortening, and mucosal damage by blocking RIPK1-RIPK3-MLKL signaling, which is hyperactive in Caspase 8-deficient states that promote IBD-like pathology.^[82] Clinical translation includes RIPK1 inhibitors like GSK2982772, which advanced to phase II trials for ulcerative colitis, demonstrating safety but no significant efficacy in reducing disease activity; as of 2025, no phase III evaluations are underway due to lack of benefit in earlier studies.^[83]^[84]^[85] These therapies compensate for impaired Caspase 8 function in IBD by preventing epithelial barrier breakdown and uncontrolled inflammation. In rheumatoid arthritis (RA), high levels of cFLIP inhibit caspase-8 activation, protecting synovial fibroblasts from TNF-induced apoptosis and promoting inflammation. Strategies to downregulate cFLIP, such as through NF-κB pathway modulation, sensitize these cells to death receptor signaling, reducing synovial inflammation and joint destruction in preclinical models. Preclinical studies indicate that cFLIP inhibition diminishes joint erosion and proinflammatory responses, with compounds targeting related pathways like ferroptosis and pyroptosis offering potential to limit RA progression. These approaches provide a selective means to enhance caspase-8-dependent apoptosis in RA without broad immunosuppressive effects.^[81]^[86] Anti-TNF biologics, such as infliximab, promote caspase-8-dependent apoptosis in Crohn's disease by neutralizing TNF-α and inducing death in lamina propria monocytes and T cells, thereby reducing excessive inflammatory signaling and aiding mucosal healing. By disrupting TNFR1 complex formation, infliximab activates caspase pathways in pathogenic immune cells, leading to decreased cytokine production and reduced disease flares in clinical practice. This approach highlights Caspase 8's role in TNF-driven autoinflammation and supports its established use in managing Crohn's disease complications.^[87]^[88]^[89] In systemic lupus erythematosus (SLE), caspase-8 contributes to inflammatory cell death pathways like PANoptosis, exacerbated by type I interferons. While gene silencing techniques targeting related pathways show preclinical potential in curbing pyroptosis and autoantibody production, specific CASP8-targeted siRNA has not demonstrated efficacy in SLE models as of 2025. Studies in SLE murine models link reduced inflammasome activity to attenuated skin lesions and systemic autoimmunity, positioning PANoptosis modulation as a promising area for targeted therapy.^[90]^[91] As of 2025, no PROTACs specifically targeting caspase-8 have advanced for autoinflammatory syndromes like familial Mediterranean fever (FMF), where inflammasome dysregulation primarily involves caspase-1. Ongoing preclinical research into degraders for caspase family members may offer future options to inhibit necroptotic and pyroptotic pathways in such conditions.^[81]^[92]