Advances in the prerequisite and consequence of STING downstream signalosomes

ER retention and ER exit

STING spans the ER membrane under basal conditions. Recent studies uncover that STING interacts with Ca²⁺ sensor stromal interaction molecule 1 (STIM1) [30] or Toll-interacting protein [31] in ER, preventing either its activation or degradation. In addition, transforming growth factor β-activated kinase 1 (TAK1) activation by STING recruits TAB1 in the ER, which in turn induces STING phosphorylation on S355, thereby facilitates later events of STEEP (STING ER exit protein) mediated STING trafficking [5]. STING(G158E) mutation causes an inability to dimerize and impairs TAK1 activation, indicating that STING dimerization is a prerequisite for TAK1 activation. STING oligomerization and trafficking are eliminated by the deletion of TAK1, STEEP, or a mutation in the CTT domain of human STING (S355A) (mouse STING S348A). Furthermore, ADP-ribosylation factor (ARF) GTPases and the Coat protein complex II complex are required for the translocation of activated STING to the Golgi and the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) [32].

Golgi-to-endosome trafficking

The trans-Golgi network (TGN) acts as a major sorting hub where cargos are sorted into several transport carriers for trafficking to different cell compartments [33]. Numerous investigations have utilized genetic ablation of secretion system cofactors to elucidate the intricate mechanism. Coat complex subunit alpha (COPA) deficiency impairs retrograde COPI vesicle trafficking, leaving activated STING accumulation in Golgi apparatus and triggering IFN mediated tissue pathology in mice [34, 35]. Through a genome-wide CRISPR-Cas9-mediated screening process in HeLa-Cas9 cells that are overexpressed varied STING mutants, STING (N154S/V155M), STING (R281M/R284M) and STING (N154S/V155M/R281M/R284M), Fang et al. have recently shown that lARMH3 interacts with STING in Golgi and recruited phosphatidylinositol 4-kinase beta (PI4KB) to synthesize phosphatidylinositol-4-phosphate (PI4P), which directs STING Golgi-to-endosome trafficking via PI4P-binding proteins adapter protein complex-1 (AP-1) and GGA2 and creates the lipid milieu that is required for STING activation through the reaction of PI4P binding proteins [36]. Similarly, Using the results of a spatiotemporal-resolved proximity labeling screen to define STING interactomes on various organelles along its trafficking route, Tu and his coworkers showed that trans-Golgi coiled-coil protein, GRIP And Coiled-Coil Domain Containing 2 (GCC2), and several RAB GTPases act as key regulators of STING post-Golgi trafficking [28]. The prolonged Golgi-dwell time boosted STING activation is evidenced by the delayed and sustained STING puncta production with a comparable peak observed in the Gcc2-deficiency MEFs. The mechanism is similar to COPA-deficiency while the overall phenotype in Gcc2 deficiency is less severe compared to COPA-deficiency. Importantly, loss of Golgi-to-lysosome STING cofactors, but not ER-to-Golgi cofactors, selectively activates tonic interferon signaling, as evidenced by around twice fold of nuclear phosphorylated IRF3 increases at its peak point. Interestingly, there is hardly any impact on the more ancient NF-κB and a slight change in autophagy. Primordial STING downstream activities, such as NF-κB and autophagy, might be relative earlier events that are perpetuated and occurred before STING Golgi exit, meaning that post-Golgi trafficking stoppage does not affect them [32, 37]. These suggest the uncoordinated perturbations in STING downstream networks and distinct underlying regulatory mechanisms.

STING degradation

Fine‐tuning STING activity is important for mounting a proper immune response. STING signaling is upregulated in the post-ER compartments and rapidly shifts off in the lysosome or proteasome to halt its activity [38] (Figure 1).

Autophagy primarily regulates STING activity negatively, which operates via both canonical and non-canonical pathways [39]. The loss of Atg9a greatly enhances the assembly of STING and TBK1 by dsDNA, leading to aberrant activation of the innate immune response [40]. Furthermore, TBK1 phosphorylated p62/Sequestosome-1 facilitates STING degradation by autophagy‐controlled ubiquitination following DNA stimulation in murine embryonic fibroblasts and THP‐1 monocytes, where p62 knockouts display the bumped IFN responses to cytoplasmic DNA or pathogens. Furthermore, STING expression is negatively regulated by the antioxidant transcription factor NRF2, which transactivates the p62 gene promoter and promotes p62 expression in response to oxidative stress [41]. p62-mediated selective autophagy restricts cGAS-STING signaling by either specifically encouraging the degradation of p62 and STING [42] or by interacting of Beclin1 with cGAS to limit cGAMP synthesis [43]. In addition, AP-1 mediated delivery of phosphorylated STING from TGN46 positive Golgi apparatus into acidic endolysosome via clathrin-coated transport vesicles upon binding a highly conserved dileucine motif and the phosphorylated S366 residue in the STING CTT domain for sequential degradation, complementing the autophagy-mediated degradation of STING. L364F substitution enhances STING binding to AP-1 and speeds STING degradation kinetics, in accordance with a study that found a missense mutation p.LL363LF, located in the EXXXLI motif, which has been linked to human cancer [44]. Seventy-two human STING gene variations are found in a variety of malignancies, the majority of which are dominant-negative mutants. Further proximity-ligation proteomics and unbiased genetic screening reveal that the endosomal sorting complex is required for transport (ESCRT) complex, which includes Hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), Vacuolar protein sorting-associated protein 37A (VPS37A), and Ubiquitin associated protein 1(UBAP1), facilitates the degradation of STING [38]. ESCRT-dependent STING degradation inhibits steady-state and cGAMP-induced signaling. To enable a transient activation of immunity components, a structural mechanism of negative regulation of STING is established as signaling initiation and termination are intricately connected. The ESCRT complex functions as a homeostatic regulator of STING signaling while STING is susceptible to a tonic degradative flux.

While multiple mechanisms are reported to regulate the activated STING, it remains inconclusive how the ER’s nascent STING protein is homeostatically regulated. Recently, Pokatayev et al. revealed that the nascent STING protein level is strictly regulated by a constant tug-of-war between ‘stabilizer’ TOLLIP and ‘degrader’ inositol-requiring enzyme 1α (IRE1α)-lysosome that together preserves tissue immunological homeostasis [31]. In contrast, Ji et al. reported the ER-related degradation of SEL1L–HRD1 (suppressor of lin-12-like–HMG-CoA reductase degradation 1) protein complex ubiquitinates STING protein at the ER for proteasomal degradation [45]. This process uncouples from ER stress or its sensor IRE1α and controls the turnover and amount of the activable STING pool in the baseline state. More intriguingly, a recent study found that Sel1l ablation in CD8⁺ T-cell reduced the amount of its mature subpopulation and disrupted its peripheral homeostasis, which is worsened by IRE1α deletion and rescued by inhibition of PERK-ATF4-CHOP-Bim signaling [46]. Studying the involved role of STING turnover in controlling CD8⁺ T-cell biology is worthwhile given the well-documented role that causes T cell death [47], [48], [49], [50]. As a consequence, the maximum activation potential of STING is regulated. Further studies are needed to clarify this.

There are ample documentations on how the secretory system cofactor affects STING activation [38, 51]. Further research is necessary to determine whether the secretory route was responsible for programming and fully furnishing the spatiotemporal dynamics of STING signalosomes. More detailed description on the ratio of STING cellular localizations across organelles in various circumstances is needed.

Posttranslational events, including phosphorylation and dephosphorylation, nitro-alkylation, glycosylation, ubiquitylation and deubiquitylation, SUMOylation, carbonylation, oxidation, and palmitoylation, have been well documented to control STING activity [52]. STING activity is limited by its modification, spatial distribution or substrate. Although there has been a lot of effort in understanding the upstream events including protein modification during STING activation, how the reaction promoted by the posttranslational modification of STING protein proceeds specifically at the organelles remains poorly understood.

Autophagy

The autophagy apparatus is present in all eukaryotes and plays a crucial role in the recycling and degradation of cellular components in response to a variety of stimuli [57, 58]. ERGIC vesicles coated with ligand-binding STING are membrane source for modification by the ubiquitin-like protein LC3, a crucial stage in autophagosome biogenesis [32]. It has been determined that the human STING residues L333, R334, and the corresponding tiny area (residues 330–334) are crucial for autophagy [32]. It is interesting to note that this small region is contained inside the STING unfolded protein response (UPR) motif (residues 322–343), which was first linked to STING-activated ER stress and subsequent T cell death [49]. LC3B lipidation is significantly influenced by the STING-TMD following STING translocation. cGAMP induced LC3 lipidation through a mechanism which is dependent on WIPI2 and ATG5 but independent of the ULK and VPS34–beclin kinase complexes [32]. Further, STING activation causes LC3B lipidation onto single-membrane perinuclear vesicles mediated via non-canonical autophagy factors ATG16L1 via its WD40 domain [59], bypassing the necessity for canonical upstream autophagy machinery. Selective autophagy can be initiated by p62 (mouse S405 and S409, equivalent to human S403 and S407), phosphorylated by CK2 (Casein Kinase II), or TBK1 and the key autophagy-related kinase ULK1 downstream of the STING. Oligomeric p62 detects ubiquitin stress and causes the targets to be degraded by attaching ubiquitinated proteins or intracellular bacterial or viral particles to ATG8 (referred to as LC3 in mammals) presented on the autophagosome membrane. P62-Ub interaction together with p62 phosphorylation and oligomerization are required for the formation and functions of p62-mediated selective autophagy. It is interesting to note that activated ULK1 preferentially suppresses IRF3/IFN signaling and causes immunosuppression by targeting STING on serine 366 after its transcriptional activities [60]. Another autophagy upstream factor CK2 downregulates IRF3 and TBK1 phosphorylation and unidentified intermediate molecules respectively, while it promotes NF-κB activation and proinflammatory cytokine production [61]. Compared to porcine WT STING, IFN-defective mutants such as △CTT, S365A, and pLxIS sub all elicited similar levels of lipid-bound LC3 (LC3-II), indicating that STING-mediated autophagy and p-p62 were not dependent on IFN. Additionally, autophagy positively influenced porcine STING-induced NF-κB activity, suggesting that these two may be inherently linked [62]. These findings imply that autophagy is in opposition to the IRF3 signal while in tandem with the NF-κB signal.

NF-κB

The NF-κB pathway, a crucial transcription factor of STING downstream signalosome, activates a wide range of genes, including inflammatory cytokines (Figure 1). TBK1 and IκB kinase ε (IKKε) act redundantly to elicit NF-κB activation STING downstream in myeloid cells, providing a view that monotherapy inhibiting TBK1 kinase may not entirely block STING-driven pathology [63]. Importantly, IKKb and TAK1/MAP3K7 complexes drive canonical NF-κB responses and mitogen-activated protein kinases (MAPK) p38 and JNK signaling pathway induced proinflammatory cytokines production [5]. STING-activated TAK1 regulates STING trafficking prior to STING dimerization in ER. These findings indicate that NF-κB signaling is an early STING signaling event that occurs before ER exit. In a mouse allograft tumor model, TAK1 activation by monophosphoryl lipid A (MPLA), a TLR4 agonist, boosts cGAMP-induced antitumor immunity reliant on STING phosphorylation, consistent with a study from another individual laboratory. Studies indicates that STING activation is boosted by NF-κB activation caused by a wide range of factors such as Toll-like receptor (TLR), interleukin-1 receptor (IL-1R), tumor necrosis factor receptor (TNFR), growth factor receptor (GF-R), or protein kinase C (PKC) [29]. These highlight a valuable connection between STING activation and other PRRs. Thus, the activation of NF-κB signaling through STING activation is ensured by the sequential action of two molecular machinery in different organelles. Non-canonical NF-κB signaling was associated with higher levels of chromosomal instability and worse clinical prognosis and a lower survival rate by increasing cancer cell fitness [64], [65], [66]. STING activates TRAF3-dependent non-canonical activation of NF-κB (Figure 1), which entails NF-κB-inducing kinase (NIK) stabilization following TRAF3 degradation [67]. In TRAF3-deficient cells, the increase of NIK, a crucial enzyme regulating non-canonical NF-κB activation, leads to deregulation of the canonical NF-κB pathway. A genetic study in human inborn errors of the alternative NF-κB pathway (autosomal-dominant p52LOF/IκBδGOF NF-κB2 disorders, autosomal-recessive NIK, or autosomal-recessive RELB) has shown that these disorders are associated with a higher risk of developing life-threatening COVID-19 pneumonia as well as the development of neutralizing autoantibodies against IFN-I [68]. This suggests that the alternative NF-κB pathway p52-RELB activation is necessary to prevent central T cell tolerance toward IFN-I in human. The coordinated effect of NF-κB signaling and IRF3/IFN-I downstream of STING activation to T cell tolerance remains unclear. Additionally, a study by Hou et al. raises the possibility that radiotherapy dampens the therapeutic benefit by activating non-canonical NF-κB pathways and retarding the IRF3/IFNβ cascade downstream of the STING signal [69]. These results indicate non-canonical NF-κB coordinates perturbation of both canonical NF-κB and IRF3 activation downstream of the STING signal.

Dramatic enhancement of NF-κB signaling was elicited via zebrafish not human STING-CTT [37]. The canonical TRAF6 signaling complex is directly recruited by the zebrafish STING CTT with an additional c-terminal module DPVETTDY, which is absent from human and mammalian STING alleles, to activate NF-κB signaling [37, 70]. STING alleles in humans and mice produced a relatively moderate NF-κB response but an elevated IRF3 response. An evolving substitution the atypical I365 (human STING) with F372 (zebrafish STING) in the IRF3 binding motif obtains IRF3-mediated signaling while a NF-κB hyperactivating module is fine-tuned [37]. These findings show that the STING-CTT is modularly organized and that modifications that modify STING downstream innate immune responses have a low evolutionary threshold. Over the course of evolution, the acquisition of modular motifs in STING-CTT regulates the specificity and intensity of downstream effects, thereby tailoring cGAS-STING immune responses to species-specific pathogen loads. It has been demonstrated that the human STING pathway is more selective and less reactive compared to mice.

Thus, understanding the evolutionary origins of the downstream consequences of STING pathway may provide insight into how this critical pathway has evolved to balance these risks. STING-CTT mediated signaling, including TBK1/IRF3/IFN-I axis and heightened NF-κB responses, is actually a relatively recent innovation. Finding out how pre-existing NF-κB affects latecomer IRF3/IFN-I or the coupling IRF3/IFN-I on top of ancestral NF-κB pathway is of significance, providing a mechanism for how STING accommodates these transcriptional signalosomes.

NF-κB synergizes with IRF3 to induce high amounts of IFN-I and proinflammatory cytokines [29, 71, 72]. At the transcriptional level, the promoter of IFNβ gene is activated by recruiting transcription factors, such as IRF3, NF-κB, and ATF-2-c-Jun [71]. At the posttranslational level, NF-κB activation prevents STING degradation by limiting microtubule-mediated STING trafficking from the Golgi apparatus to lysosomes, prolonging and increasing STING signaling and IFN production [29]. Consequently, this coordinated effect strengthens host antiviral defenses and STING-mediated IFN responses.

IRF3

It is widely known that TBK1 phosphorylates IRF3, which causes it to dimerize and depart to the nucleus, triggering the transcription of IFN-I and other IFN-stimulated genes (ISGs) as well as antigen-specific immune responses against tumor and viral infection [53, 54] (Figure 1). TLR ligands and IL-1β activated TBK1 do not, however, cause IRF3 phosphorylation, suggesting that particular adapter proteins – TRIF for TLR3 and TLR4, MAVS for RIG-I and MDA5, and STING for the DNA-sensing pathways – are involved in signaling pathways that result in IRF3 activation [55, 73]. It has been shown that late endosomes play a significant role as vesicle compartments in STING-mediated IRF3 phosphorylation [5, 74, 75]. Wang et al. and colleagues reported that STING dimerizes upon ligand-binding and interacts with membrane-bound epidermal growth factor receptor (EGFR), which autophosphorylates and provides the platform for the recruitment of cytoplasmic Syk to the signaling complex and its activation. Activated Syk phosphorylates Tyr240 of STING, followed by phosphorylation of Tyr245 by EGFR [75]. The later modification in the ER is required for STING translocation to the late endosome where it binds IRF3. Alternatively, activated STING translocates to the autophagosome in the absence of EGFR with no IRF3 activation, IFN production and antiviral activity in cells and mice. This finding suggests potential therapeutic and experimental strategies for the selective modulation of STING functions [74].

IRF3 phosphorylation is restricted by multiple machineries. According to recent reports from two separate groups, STING ubiquitination has been shown to specifically control IRF3 signal transcriptomes [76, 77]. The results of the mass spectrometry approach in 293T expressing STING reveal that K224 substitution nearly eliminates STING ubiquitination by E3 ligase MUL1 (mitochondrial E3 ubiquitin protein ligase 1) in the presence of cytosolic DNA. Blocking K224 ubiquitination specifically prevents IRF3 but not NF-κB activation [77]. Furthermore, Stempel et al. revealed that m152, a highly conserved protein among various MCMV strains, interacts with murine STING (but not human STING) via their respective ER-luminal domains and traffic together from the ER to the Golgi compartment, leading to a declined STING trafficking and delayed IFN response, however, NF-κB response remains intact [76]. K288R mutation sequesters STING protein in ER after stimulation, the NF-κB pathway is inducible while IFN-I response is invalid. A similar phenomenon is observed in TBK1‐deficient cells. Further study demonstrates that STING activates NF-κB-dependent signaling in ER that is independent of the kinase TBK1, a beneficial outcome for early MCMV transcription [76]. Thus, they uncovered that murine CMV has evolved an attack strategy to specifically antagonize the STING-mediated antiviral IFN response, while preserving its pro-viral NF-κB response, giving it an edge in the establishment of an infection. Importantly, K244 and K288 ubiquitination restricts IRF3 cascade. STING-mediated NF-κB responses appear to be independent of them, indicating that these responses may occur prior to Golgi re-localization. This implies that STING trafficking is not required to stimulate the signaling pathways like NF-κB pathway and uncoupling of IFN-I and NF-κB signaling can occur.

On the opposite to heightened IRF3/IFN-I cascade facilitated by activated NF-κB pathway, IRF3 regulates NF-κB signal by multiple mechanisms at diverse layers in different inflammatory settings. IRF3 restricts kinase IKKβ upstream of the NF-κB signal in the context of metabolic diseases [63], restraining chronic high-fat diet (HFD)-induced hepatic insulin resistance and steatosis through preserving glucose and lipid homeostasis [78]. Furthermore, in cellular and mouse models of Sendai virus infection, IRF3 directly binds to the p65 subunit during nuclear translocation of NF-κB, sequestering it in the cytosol and suppressing lung inflammation [79]. A comparable function of IRF3 in the cytoplasmic sequestration of β-catenin has been documented in relation to colon cancer tumorigenesis [80]. In the context of HFD-induced liver injury, the nontranscriptional function of IRF3-mediated anti-inflammatory protection, as illustrated in Irf3^S1/S1 mice with alanine substitutions at Ser-388 and Ser-390 are primarily associated retardation of nuclear translocation of p65 and decreased expression of NF-κB-dependent inflammatory cytokines [78]. The multi-face of IRF3 in the liver is representative as IRF3 activation by viral infection in vivo greatly enhances bile acid- and aspirin-induced hepatotoxicity via stimulating transcriptional suppressor Hes1 to reduce RXRα level [81]. In contrast to this finding of the transcription-independent role of IRF3 on NF-κB repression, two individual groups show that STING^S365A mutants, which substitute the docking site for IRF3, result in precisely abolished transcriptional activity and subsequent IFN-dependent response, also show enhanced NF-κB signal transduction in 293T [62], as well as in primary T cells [82]. Nonetheless, Yan et al. observed that IRF3 promotes MyD88-mediated NF-κB signaling pathway in fish [83]. Thus, uncertainty surrounds the equilibrium between IRF3 and NF-κB transcriptional signalosomes downstream of the STING signal. Further, whether IRF3 regulation of NF-κB signal could reappear in other disease contexts is of interest to examine. TBK1/IRF3 axis can be activated by different adapter proteins downstream of respective innate sensors including RIG-I, cGAS, and TLRs [73]. Given that a range of factors can disrupt both signals, it is noteworthy to investigate the role of STING in maintaining equilibrium between these two downstream signal node molecules. IRF3 also exerts non-transcriptional activation as evidenced by IRF3 with modification of a linear polyubiquitination, activated by the RIG-I pathway recruited TBK1/TRAF2/TRAF6 complex and sequential linear ubiquitin chain assembly complex binding, serves as a proapoptotic factor, as a mechanism to limit virus spread within the host. Thus, the cellular control of IRF3 by posttranslational modifications regulates its activation, activity and stability. IRF3 acts as a network node regulated by multiple mechanisms to be devoid of inflammation and autoimmunity.

NLRP3 inflammasome

NLRP3 functions as a guardian of intracellular homeostasis and monitors a range of endogenous cues [84], such as the efflux of potassium ions (K⁺) or chloride ions (Cl⁻), the flux of calcium ions (Ca²⁺), ROS, cytosolic mtDNA and cardiolipin, mitochondrial damage, lysosomal disruption, ER stress, trans-Golgi disassembly, intracellular kinase signaling, and lipid uptake and accumulation. Recently, the role of STING in perturbing NLRP3 inflammasome has gained increasing attention (Figure 1). Inflammasome activation requires two signals, priming and activation, both of which have differential requirements for STING [85]. A recent report described the histone methylation mediated by WDR5/DOT1L plays a crucial role in augmenting IRF3 binding to the Nlrp3 promoter and facilitating STING-induced Nlrp3 transcription [86]. NF-κB primes the NLRP3-inflammasome for activation by promoting pro-IL-1β and NLRP3 expression. However, it also hinders NLRP3-inflammasome activation by causing a delayed accumulation of the autophagy receptor p62/SQSTM1[87]. The “NF-κB-p62-mitophagy” pathway is an intrinsic regulatory loop in macrophages that allows NF-κB to control its own proinflammatory activity and triggers a self-limiting host response that preserves homeostasis and promotes tissue repair [87]. During HSV-1 infection, STING recruits and interacts with NLRP3 on ER, attenuating K48- and K63-linked polyubiquitination of NLRP3, thereby facilitating NLRP3 inflammasome formation [88]. It has been discovered recently that NLRP3 inflammasome activation in Golgi vesicles is triggered by STING’s proton channel activity [85, 89]. The mechanism remains unclear. It is noteworthy that two individual groups reported PI4P-binding proteins mediate STING or NLRP3 Golgi-to-endosome trafficking and the importance of PI4P in mediating STING activation and NLRP3 inflammasome aggregation [90]. It is unknown if the coupling protein contact occurred during trafficking. The coordinated effect mediates hepatocyte pyroptosis involving oxidative stress and metabolic reprogramming [86]. cGAMP induction of IFN-I precedes inflammasome activation, which then occurs when IFN-I is waning. cGAMP activates the inflammasome in addition to IFN-I, and both are required to prevent DNA virus infection.

STING activation by intercellular transmission

STING traffics across organelles through the secretory pathway. Recently, a study revealed that STING is a secreted protein. RAB22A-mediated non-canonical autophagosome, which originated from the ER and is released as an extracellular vesicle, allows activated STING to shuttle across cells to spread antitumor immunity [115]. These studies provided a rationale for adopting extracellular vesicles as a form of delivery for activated STING. Natural STING agonist cGAMP shuttles through gap junctions, receptor-based transport, membrane fusion, and viral particles. Mice receiving immune checkpoint inhibition saw increased tumor-specific T cell responses and antitumor effects when combined with virus-like particles delivering cGAMP. Nanotechnology has been employed to solve the aforementioned issues by altering the pharmacokinetics, biodistribution and cytosolic delivery of CDN therapy, resulting in moderate impact in preclinical models [18]. Apart from the cGAMP-PC7A polymeric nanoparticles above [18], CDN–Mn²⁺ nanoparticle particle, a self-assembled Mn²⁺ coordinated with CDN STING agonists, effectively delivers STING agonists to immune cells and offers a new platform for local and systemic cancer treatments [116, 117]. A similar nanoscale coordination polymer encapsulated with CDA, ZnCDA [118], also mediates robust antitumor effects in a diverse set of preclinical cancer models. These investigations highlight the enormous potential of coordinated nanomedicine in the field of metalloimmunotherapy. A formulation of CDN-conjugated poly (β-amino ester) nanoparticles that targets TME and secondary lymphoid organs was recently developed [119]. The proximal immune cells in the TME were gradually stimulated by the drug internalized by cancer cells. The differentiation of immunological memory is aided by nanoparticles that gathered in the spleen. These results demonstrate that APCs may benefit more from the selectively organ-targeted STING agonists, particularly in poorly immunogenic tumors. ADC exert with highly specific targeting capabilities is leading a new era of targeted cancer therapy. CRD5500 (Takeda) and XMT-2056 (Mersana), the leading candidates of HER2-targeted STING-agonist ADC [92], are undergoing clinical research, awaiting the effect results of efficacy and estimated toxicity.

By utilizing the delivery routes, the encapsulated STING agonists are able to overcome the barriers of poor stability, deliverability, and unwanted effects of cell heterogeneity consequences after systemic delivery. However, tumors can easily evade this suppression by downregulation or loss of endogenous STING due to epigenetic silencing or missense mutations in TME [44].

Established mimics imitate STING actions independent of endogenous expression

Take advantage of ultra-light-sensitive CRY2-clustering system CRY2clust as STING oligomerization inducible inducer, further conjunction with STING-CTT as the scaffold for interactions with downstream TBK1 and IRF3, which together constitutively recapitulate STING activation [25]. The platform confers antitumor immune response with high spatiotemporal precision and minimizes adverse effects. The innovation relies on and is restricted to the adoptive cell transfer of DC expressing CRY2clust/STING (Table 2). In an investigation regarding to a ribonucleoprotein STING agonist [120], STING lacking the TMD complexed with cGAMP efficiently initiates STING signaling even in STING-deficient cell lines, which is striking since the STING-TMD associated oligomerization is prerequisite for its activation [22] (Table 2). This ribonucleoprotein self-assembled a tetrameric form under physiological conditions. The molecular mechanism is still unknown. It requires cGAMP presence for the preassembly of the STING/cGAMP protein complex, and therefore represents a challenge for intracellular delivery. In 2023, the groundbreaking work on the mRNA vaccines against COVID-19 by Drs. Katalin Karikó and Drew Weissman is recognized by the Nobel Prize, sparking a new area of medicine with broad therapeutic possibilities. An mRNA-encoded constitutively active STING mutant (V155M) mobilizes immune response without dependence of endogenous STING expression in TME [121] (Table 2). This mutant, localizing STING to the ERGIC, activates downstream signaling including IRF3/IFN-I and pro-inflammatory NF-κB/p65/IL-6 that consequently initiates undesired dose-limiting side toxicity as described above. To tackle this, it is necessary to consider uncoupling beneficial and detrimental signalosomes for cancer therapeutics design.

Uncoupling STING signalosomes for cancer therapeutics

Sustained activation of inflammatory autophagy and NF-κβ signal or suppressed IFN are hallmarks of immunosuppressive tumors [44, 122]. Respective mechanisms described above or unknown machinery regulate these STING downstream signalosomes. A recent study revealed an attempt for uncoupling STING signalosomes to treat cancer. GB1275, a CD11b agonist, enters the phase I clinical trial of advanced solid tumors (NCT040603420), elicits robust T cell specific responses by uncoupling two signaling pathways in macrophages, including NF-κB p65/IL-1 inhibition and STING/IRF3/IFN-I/CXCL axis activation [123]. Specifically, through an axis known as focal adhesion kinase (FAK)/sirtuin-3 (SIRT3)/ROS, GB1275 promotes STING-dependent IFN-I production. Overproduction of ROS causes damage to the mitochondria, which allows mtDNA to be released into the cytosol and activate STAT1-dependent antitumor immunity and cGAS/STING signaling. More importantly, CD11b agonists induce p65 degradation to repress NF-κB/IL-1 signaling dependent on proteasome activity, which lessens detrimental STING effects. Further research and drug development that potentially uncouple the STING downstream pathways are needed. Recent reports implicate poly (ADP-ribose) polymerase-1 (PARP-1) in the activation of NF-κB [124, 125]. Cytosolic PARP1 suppresses antiviral immunity through PARylating human cGAS on D191 or mouse cGAS on E176 to terminate its DNA binding. Furthermore, the transglutaminase TG2 expression has been linked to constitutive activation of NF-κB, autophagy, and chemotherapy resistance in lymphoma cells, while inhibition of its transamidating activity facilitates the TBK1-IRF3 interaction in the cytosol correlated with an increase in the IFN-I transcripts in human melanoma [126]. Repressing TG2 may enhance the effect of STING pathway-based cancer therapeutics. These initiatives, such as targeting PARP-1 or TG2, provide a theoretical basis for the proposal to decouple STING signalosomes for cancer therapeutics.