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Cell Death & Differentiation
https://doi.org/10.1038/s41418-020-00624-8
REVIEW ARTICLE
Crosstalk between cGAS–STING signaling and cell death
Ambika M. V. Murthy 1●Nirmal Robinson 1●Sharad Kumar 1
Received: 2 July 2020 / Accepted: 7 September 2020
© The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare 2020
Abstract
Cytosolic nucleic acid sensors have a critical role in detecting endogenous nucleic acids to initiate innate immune responses
during microbial infections and/or cell death. Several seminal studies over the past decade have delineated the conserved
mechanism of cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) and the downstream signaling adapter stimulator of
interferon genes (STING) in mediating innate immune signaling pathways as a host defense mechanism. Besides the
predominant role in microbial infections and inflammatory diseases, there is an increased attention on alternative functional
responses of cGAS–STING-mediated signaling. Here we review the complexity of interactions between the cGAS–STING
signaling and cell death pathways. A better understanding of molecular mechanisms of this interplay is important with regard to
the development of new therapeutics targeting cGAS–STING signaling in cancer, infectious, and chronic inflammatory diseases.
Facts
●Cytosolic DNA stimulates various signaling pathways
that often lead to different modes of cell death.
●The cGAS–STING pathway recognizes cytosolic DNA
to initiate type I interferon production.
●Autophagy has a dual role in cGAS–STING signaling,
by initiating inflammatory responses and by targeting
STING for degradation.
Open questions
●What dictates the redundancy in cell death pathways
triggered by the cGAS–STING?
●What are the mechanisms by which the cGAS–STING
induces autophagy-dependent cell death?
●Can STING-mediated cell death be targeted for treating
cancer and other diseases?
Introduction
Cell death is indispensable for the development of tissues
and organs, and for eliminating infected, damaged, or
transformed cells to maintain organismal homeostasis. Not
that long-ago cell death was broadly categorized into just
two types, apoptosis (immunologically silent) and necrosis
(accidental or inflammatory cell death) [1,2]. Subsequent
studies uncovered various other cell death modalities
including (but not limited to) programmed necrosis, pyr-
optosis, ferroptosis, and autophagy-dependent cell death
(ADCD) [3]. In the last two decades, the ability of DNA to
initiate different types of cell death as a host protective
responsehaveemergedasakeyfeatureofinnateimmu-
nity in mammalian cells. Under normal circumstances
DNA is strictly confined to the nucleus or the mitochon-
drion of a eukaryotic cell. Therefore, the presence of self-
DNA in unusual sites such as cytoplasm or endosomes
and/or the presence of exogeneous DNA from microbial
challenge is considered as a threat. To counteract these
danger signals, cells are equipped with various DNA
sensors, such as toll-like receptor 9 (TLR9), absent
in melanoma 2 (AIM2), cyclic GMP-AMP synthase
(cGAS), RNA polymerase III, DNA-dependent activa-
tors of IRFs, interferon-inducible protein 16, DDX41,
LSm14A [4]. Of these, only TLR9, AIM2, and cGAS are
Edited by G. Melino
*Ambika M. V. Murthy
ambika.mosalevenkateshmurthy@unisa.edu.au
*Nirmal Robinson
nirmal.robinson@unisa.edu.au
*Sharad Kumar
sharad.kumar@unisa.edu.au
1Centre for Cancer Biology, University of South Australia, GPO
Box 2471, Adelaide, SA 5001, Australia
1234567890();,:
1234567890();,:
well characterized, whereas the rest of them are yet to be
validated by genetic approaches [5].
TLR9 was the first DNA-sensing receptor identified in
immune cells such as dendritic cells (DCs), B cells and
macrophages [6]. Under basal conditions, TLR9 is retained
in the endoplasmic reticulum (ER) [7,8]. However, detec-
tion of unmethylated cytosine-guanine (CpG)-rich motifs
that are relatively abundant in bacteria and DNA viruses,
occurs in early endosomes that activate NF-κB via MyD88
and type I interferon (IFN) production to initiate a cascade
of innate and adaptive immune responses [6,9,10]. Yet,
mitochondrial DNA (mtDNA) can also activate TLR9
marked by p38 mitogen-activated protein kinase activation
in human PMNs and immortalized podocytes [11,12]. This
is unsurprising as mitochondria evolved from bacteria that
are endosymbionts in mammalian cells and therefore,
mtDNA contains non-methylated CpG motifs [13,14].
Another sensor for cytosolic DNA is AIM2 that can bind
to DNA directly in a sequence-independent manner via C-
terminal HIN-200 domain, where an N-terminal pyrin domain
interacts with apoptosis-associated speck-like protein con-
taining a caspase activation and recruitment domain (ASC) to
initiate inflammasome formation [15]. AIM2 requires at least
80 bp of dsDNA to initiate inflammasome activation [16]
resulting in IL-1βand IL-18 release accompanied by
inflammatory cell death termed pyroptosis (discussed below)
to offer protection against cancer and infections caused by
DNA viruses, bacteria, and fungal pathogens [15].
In recent years, cytosolic sensing of mislocalized DNA
by cGAS has emerged as a key event in cellular responses
to pathogen invasion, DNA damage, and mitochondrial
stress. cGAS recognizes DNA of various sizes in a
sequence-independent manner, with human cGAS able to
sense DNA sequences as short as 45 bp [17], however,
larger DNA fragments result in better immune response
[18]. cGAS-mediated stimulator of interferon genes
(STING) activation drives IFN responses and also activates
NF-κB (discussed in detail below). Historically, the nucleus
has been regarded as ‘immune-privileged’, however, this
paradigm was overturned by nuclear localization of cGAS
and its association with centromeric DNA in DCs [19], and
also the identification of heterogeneous nuclear ribonu-
cleoprotein A2B1 that can bind viral DNA in the nucleus
during herpes simplex virus-1 infections to drive IFN
responses [20]. Notably, nuclear localized cGAS is 200-fold
less efficient to nuclear DNA than extranuclear cGAS to
cytosolic DNA to induce IFN responses [19], which could
be due to suppression of cyclic guanosine monophosphate-
adenosine monophosphate (cGAMP) production by
nucleosomes [21]. In addition, positioning of cGAS to
plasma membrane could not be discounted [22]. This raises
the question if localization of the cGAS affects intensity of
the IFN responses. However, another persistent question is
why does any cell require multiple DNA sensors? One
possibility is that these sensors act as a safety checkpoints to
ensure any danger signal in the form of cytoplasmic DNA is
not missed in different cellular contexts. The other possi-
bility is that if one DNA-sensing pathway becomes non-
functional and/or inhibited by microbial or danger signals,
the other DNA sensors could act as a backup. Although,
mammalian cells are equipped with an array of DNA sen-
sors, cGAS–STING-mediated IFN response is gaining
increasing attention due its critical role in inflammatory and
infectious diseases, and cancer [23,24]. This is possibly due
to widespread STING expression in various cell-types and
the ability of cGAS–STING to regulate different pro-
grammed cell death pathways. In this review we discuss our
current understanding of the interplay between the
cGAS–STING signaling and different modes of cell death
such as apoptosis, necroptosis, and pyroptosis. Finally, we
summarize the new work linking the cGAS–STING path-
way to autophagy and ADCD.
The cGAS–STING signaling pathway
cGAS recognizes viral, bacterial, protozoal, mitochondrial,
and self-DNA from tumor or dead cells in the cytosol to
activate a cascade of signaling pathways leading to inflam-
mation [25–28](Fig.1). Binding of DNA to the cGAS indu-
ces conformational changes to cGAS, which in turn catalyzes
the formation of 2′,3′-cGAMP, a cyclic di-nucleotide (CDN)
with a unique phosphodiester linkage that uses adenosine
triphosphate (ATP) and guanosine triphosphate (GTP) [29].
DNA binding to cGAS induces liquidlike droplets that act as
a microreactor, enriching enzyme, and reactants to enhance
cGAMP production, as demonstrated by cGAS-DNA puncta
in both human fibroblast cell line and mouse embryonic
fibroblasts (MEF) [30]. The generated cGAMP acts as a
secondary messenger and activates STING (also known as
transmembrane protein 173 (TMEM173), N-terminal
methionine–proline–tyrosine–serine plasma membrane trans-
panner (MPYS), mediator of interferon regulatory factor 3
(IRF3) activation, and endoplasmic reticulum IFN stimulator)
[31]. Notably, cGAMP can transactivate STING in neigh-
boring cells [32] with the aid of gap junction proteins such as
connexins to amplify inflammatory responses [33]. In addi-
tion, bacterial CDNs such as cGAMP, c-di-AMP and c-di-
GMP can also activate STING directly [34–36].
In resting state, STING is contained in the ER as a
transmembrane homodimer by a Ca2+sensing transmem-
brane protein, stromal interaction molecule 1 (STIM1) [37].
Upon activation, STING undergoes a conformational
change and translocates to the perinuclear region via an
unknown mechanism, where it oligomerizes and recruit
tank-binding kinase 1 (TBK1) [38]. Oligomerized STING
A. M. V. Murthy et al.
offers a platform for trans-TBK1 autophosphorylation, and
in turn gets phosphorylated by TBK1 as delineated by
structural studies using human TBK1 complex with
cGAMP and full-length chicken STING [38]. STING can
interact with both TBK1 and the transcription factor IRF3,
therefore specifying IRF3 for phosphorylation by TBK1
[39,40]. Phosphorylated IRF3 dimerizes and translocates
into the nucleus to induce the expression of inflammatory
genes including IFN and IFN-stimulated genes (ISG). IFNs
are indispensable for antitumor immune responses in DCs
and CD8+T cells, and mediate adaptive immunity [41,42].
In addition, STING–TBK1 association also phosphorylates
IκB kinase, leading to noncanonical activation of NF-κB
that regulates the transcription of inflammatory cytokines,
including IL-6 and tumor necrosis factor (TNF) [25,26].
However, TBK1 and IκB kinase are dispensable for
STING-induced NF-κB activation in mouse myeloid cells
both in vivo and in vitro [43]. Notably, STING can be
degraded by autophagy (discussed later in this review) to
tightly regulate the cGAS–STING pathway, as aberrant
activation of this pathway causes chronic inflammatory
diseases such as systemic lupus erythematosus (SLE) [28].
However, IFN-independent functions of STING in antiviral
responses and T cell death to control immune evasion
cannot be discounted [44].
Phylogenetic analysis has revealed that both cGAS and
STING homologs are present in mammals and the origin of
this pathway has been traced back to a unicellular eukaryote
Fig. 1 Activation of the
cGAS–STING signaling
pathway to drive innate
immune responses. a cGAS is
activated by mislocalized DNA
in the cytosol by various sources
such as self-DNA from damaged
mitochondrion or micronucleus,
or exogeneous DNA derived
from virus, dead cell, and
bacterium. Activated cGAS uses
ATP and GTP as substrates to
catalyze the formation of
cGAMP, which acts as a second
messenger to activate STING.
bCyclic dinucleotides secreted
from bacteria can also activate
STING directly. cActivated
STING dimerizes and
translocates from the ER to the
perinuclear region via Golgi to
recruit and promote TBK1-
IRF3-IκB assembly, which
subsequently drives activation of
IRF3 and NF-κB to produce IFN
and inflammatory cytokines
(TNF, IL-6), respectively.
Crosstalk between cGAS–STING signaling and cell death
Monosiga brevicollis, with the core CDN-binding function
of STING showing evolutionary conservation over 600
million years [45]. Interestingly, STING or cGAS alone is
unable to drive IFN-βexpression effectively as shown fol-
lowing Ectromelia Virus infections in HEK293T cells [46].
This is further supported by the fact that these two proteins
have coevolved, such that different species will either
contain homologs of both cGAS and STING or have lost
both, highlighting their crucial requirement to cooperatively
mediate the inflammatory responses [45].
Interactions between the cGAS–STING
pathway and apoptosis
Apoptosis is one of the most conserved and best studied cell
death pathways in terms of mechanism and regulation for its
vital role in development and homeostasis [47,48]. In brief,
apoptosis is mediated by caspases, a family of cysteine
proteases [49]. There are three major pathways that signal
caspase activation and apoptosis in mammalian cells, often
termed as intrinsic, extrinsic (or mitochondrial pathway),
and granzyme-mediated cell death pathway [50]. The
extrinsic pathway is initiated by binding of ‘death ligands’
such as TNF, FASL, TRAIL, to TNF superfamily receptors
such as TNF receptor 1 (TNFR1), FAS, death receptor 4/5,
respectively, to recruit cytoplasmic adapter proteins such as
Fas-associated death domain or TNF-associated death
domain. These adapters in-turn recruit and activate caspase-
8. Caspase-8 is the central protease in death receptor
induced apoptosis, and once activated, it mediates caspase-3
activation, either directly or through (BH3 Interacting
Domain Death Agonist) BID cleavage and engagement of
the mitochondrial pathway [51]. The intrinsic pathway is
centered on B-cell lymphoma-2 (BCL-2) family proteins
that comprises of both antiapoptotic and proapoptotic
members [47,52]. Cellular stress or danger signals can
either activate proapoptotic proteins such as BCL-2 asso-
ciated X protein (BAX) and BCL-2 homologous antagonist/
killer (BAK) or inhibit antiapoptotic proteins (BCL-2, BCL-
x, BCL-w, and MCL-1) through the activation of several
BH3 only domain family members (BIM, BID). This results
in mitochondrial outer membrane permeabilization, result-
ing in cytochrome c release, that binds to an adapter
apoptotic protease activating factor-1 (APAF1) to initiate
the formation of the apoptosome, a complex required for
recruitment and activation of caspase-9. Activated caspase-
9 cleaves and activates caspase-3 and -7, which targets a
number of cellular substrates to initiate apoptotic cell death
[53]. Last, in the granzyme pathway cytotoxic T lympho-
cytes kill target cells via secreted granular proteases and
perforins [54,55]. Despite the specific requirements of each
of these pathways, intrinsic, extrinsic, and granzyme B
converge on execution pathway (caspase-3 and -7 activa-
tion) of apoptosis [50]. It is however worth noting that
cytotoxic T lymphocytes can also induce non-apoptotic
types of death in target cells [56].
Apoptosis is generally regarded as silent cell death, where
apoptotic bodies are rapidly phagocytosed by neighboring
cells or macrophages to prevent the activation of inflamma-
tory pathways. Therefore, not surprisingly apoptotic caspase
activation has inhibitory effect on cGAS–STING inflamma-
tory pathways. For example, both in vitro and in vivo ISG is
constitutively expressed in caspase-9-deficient, and caspase-3/
7 double knockout MEFs. Furthermore, STING pathway is
constitutively activated in caspase-9 deficient cells [57].
Finally, mtDNA released from Bak/Bax activation is shown
to be the endogenous ligand that triggers the cGAS–STING
pathway activation, with caspases regulating the inflammatory
responses [57]. These findings are further corroborated by an
independent study that confirmed the role of mtDNA in
initiating cGAS–STING-mediated IFN responses and active
caspase-9 inhibiting these inflammatory responses in hema-
topoietic cells both in vivo and in vitro [58]. The mechanism
of apoptotic caspases downregulating inflammatory responses
was uncovered in a recent study, where active caspase-3
cleaved cGAS, and IRF3 in THP-1 cells during viral infec-
tions to prevent excessive IFN production [59]. Moreover,
defects in clearing apoptotic cells led to the formation of
apoptosis-derived membrane vesicles (ADMVs) that have
been associated with autoimmune diseases such as SLE [60].
ADMVs isolated from the sera of SLE patients contained
dsDNA that triggered cGAS–STING signaling pathway
leading to ISG expression in THP-1 cells [61]. Based on
these studies it can be argued that apoptosis prevents
cGAS–STING-mediated inflammatory responses (Fig. 2). By
contrast, emerging evidence suggests that ER stress associated
with STING activation can trigger apoptosis. For instance,
during mycobacterial infections, STING activation causes ER
stress leading to BAX activation and cytochrome c release
resulting in apoptosis in RAW.247 cells [62]. In addition, ER
stress-mediated STING activation in mouse hepatocytes
results in apoptosis in an early alcoholic liver disease model
[63]. It is possible that STING regulates calcium homeostasis
and ER stress, as STING gain-of-function mutant disrupts
calcium homeostasis causing ER stress that primes T cell
apoptosis [64]. Also, STING pathway activation by the
STING agonist 10-carboxymethyl-9-acridanone (CMA) trig-
gers apoptosis in primary and lymphoblastic leukemia murine
T cells by coordinated action of IRF3 and p53 but not in
murine DCs [65]. Similarly, the STING agonist cGAMP
initiates ER-stress-mediated apoptosis in normal and malig-
nant B cells, but not in MEF, B16 melanoma, Hepa 1–6
hepatoma, LL/2 Lewis lung cancer cell lines and wild-type
T cells from mice [66]. Murine-specific small molecule
STING agonists CMA and DMXAA also failed to trigger
A. M. V. Murthy et al.
apoptosis in MEF, B16 melanoma, Hepa 1–6 hepatoma, LL/2
Lewis lung cancer cell lines [66]. Inability of these small
molecules to activate human STING is attributed to dynamic
structural differences between mouse and human STING [67].
These findings also suggest that STING-mediated apoptosis is
cell- and/or context-dependent, which could be due to varia-
tion in endogenous STING expression amongst different cell
types as observed in some studies [65,66], or cell-specific
kinetics of STING regulation (phosphorylation or degrada-
tion). Further mechanistic insights on how STING regulates
calcium homeostasis, whether directly or indirectly by STIM1
or via other calcium signaling pathways, are required from the
therapeutic viewpoint of inhibiting ER stress to prevent
chronic STING activation in autoimmune diseases [64].
Interplay between the cGAS–STING signaling
and necroptosis
Necroptosis is a regulated form of necrosis, mediated by
receptor interacting protein kinases activation and its sub-
strate mixed lineage kinase like (MLKL) that leads to
Fig. 2 Interplay between the
cGAS–STING signaling
pathway and apoptosis. During
apoptosis signaling the
proapoptotic proteins such as
BAK/BAX cause mitochondrial
membrane permeabilisation and
release of cytochrome c and
mitochondrial DNA.
Mitochondrial DNA can trigger
cGAS–STING–TBK1 pathway
to drive IFN-mediated immune
responses. Whereas cytosolic
cytochrome c drives the
assembly of the Apaf1-
caspaspe-9 apoptosome that
subsequently activate the
initiator caspases (e.g. caspase-3
and caspase-7) to mediate
apoptosis. Caspase-3 negatively
regulates cGAS–STING
pathway resulting in cleavage of
IRF3 and STING. On the other
hand, STING activation causes
ER-stress that ultimately results
in apoptosis by via BAK/BAX
activation and cytochrome c
release. Red arrows indicate
negative regulation.
Crosstalk between cGAS–STING signaling and cell death
plasma membrane disruption [68–70]. When apoptosis is
inhibited by genetic defects, viral or bacterial infections, or
experimentally by caspase inhibitors, necroptosis is pro-
moted as a mode of regulated cell death that releases
DAMPS to initiate innate immune responses [71]. Impor-
tantly, IFN signaling is indispensable to trigger necroptosis
and IFN-αreceptor 1 (IFNAR1) deficient mice are resistant
to necroptosis during Salmonella Typhimurium infections
[72]. Also, in IFNAR1 deficient bone marrow-derived
macrophages (BMMs) necroptosis is inhibited, despite of
caspase inhibition and TLR induction by stimulation with
LPS, polyinosinic-polycytidilic acid, TNFα,orIFN-β[73].
Therefore, it is logical to deduce that the cGAS–STING
pathway could play a predominant role in triggering
necroptosis (Fig. 3). Along the same lines, cGAS–STING-
dependent TNF and IFN signaling triggers necroptosis in
response to cytosolic DNA, when apoptotic caspases are
inhibited in BMMs [74]. In addition, mtDNA activates
the STING pathway that subsequently enhances RIPK3/
MLKL expression to trigger necroptosis, when caspases are
inhibited in HT29 colon cancer cell line and MEFs [75].
Furthermore, constitutive IFN levels mediated by
cGAS–STING activation maintain MLKL at a threshold
level to predispose BMMs to necroptosis [76]. Yet, over-
expression of MLKL in BMMs from Ifnar−/−mice does not
sensitize cells to necroptosis [76,77]. This suggests the
presence of additional IFN-inducible necroptotic pathway
components, that are required for necroptosis, yet to iden-
tified. However, during Salmonella Typhimurium infec-
tions, IFN-mediated RIPK3 activation impairs antioxidative
responses that could sensitize BMMs to necroptosis [78].
Considering that loss of apoptosis is a hallmark of cancer
[79], it is rational to trigger secondary cell death pathway
such as necroptosis that is mechanistically distinct.
Based on some studies (as discussed above) indicating
cGAS–STING signaling enhances expression of necroptotic
apparatus to trigger robust cell death upon specific stimuli
[75,76], it is reasonable to consider using STING agonists
to trigger necroptosis in cancer cells. However, the use of
the STING agonist DMXAA combined with caspase inhi-
bition in vivo increases TNF and IFN levels subsequently
leading to necroptosis, but this resulted in increased mor-
tality due to septic shock [74]. Although, DMXAA is a
failure in clinics due to its inability to effectively activate
human STING, small molecule STING agonist amido-
benzimidazole (ABZI) has been reported to bind and acti-
vate both human and mouse STING [80,81]. Therefore,
such STING agonists in clinical trials will need to be
assessed for their roles in triggering necroptosis without
extensive secondary effects. This conclusion is reinforced
by the observation that necroptotic rather than apoptotic cell
death is efficient in DC-mediated cross-priming of CD8+
T cells to achieve antitumor immunity [82]. Despite the
clinical interest in use of STING agonists as anticancer
drug, surprisingly little is known about the role of necrop-
tosis during STING activation, however, this is clearly an
area of ongoing investigations. On the other hand, it would
be of interest to examine if STING antagonists can be used
Fig. 3 Initiation of necroptosis by cGAS–STING signaling. Acti-
vation of the cGAS–STING pathway by mitochondrial DNA results in
production of IFN and TNF. Binding of IFN to IFNAR1 and TNF to
TNFR1 can results in RIPK1–RIPK3 activation, when caspase-8 is
inhibited. RIPK1–RIPK3 activates MLKL to execute necroptosis.
Moreover, IFN upregulates RIPK3 and MLKL expression. It would be
interesting to understand if STING agonists can initiate necroptosis to
limit tumor growth when apoptotic caspases are usually inhibited in
cancer cells. Green arrow indicate upregulation.
A. M. V. Murthy et al.
to treat septic shock caused by bacterial infections. This will
require further understanding of the mechanism and role of
necroptosis in inducing septic shock in the context of bac-
terial infections.
Crosstalk between the cGAS–STING pathway
and pyroptosis
Pyroptosis is an inflammatory form of cell death initiated by
inflammasomes, eventually leading to gasdermin-D (or E)
cleavage to generate the N-terminal domain, which forms a
pore in the cell membrane [83–85]. In brief, inflammasomes
are large multiprotein complexes comprised of germline-
encoded pattern recognition receptors of the Nod-like receptor
(NLR) family (NLR family pyrin domain-containing 3,
NLRP3; NLR family CARD domain-containing protein 4,
NLRC4; and AIM2), the adapter ASC and procaspase-1. In
response to pathogenic or physiological perturbation in the
cytosol, procaspase-1 undergoes inflammasome-assisted
proximity-induced autoproteolysis resulting in active
caspase-1 [86]. Active caspase-1 converts pro-IL-1βand pro-
IL-18 into its mature form and also cleaves gasdermin-D to
initiate pyroptosis [87]. A considerable body of literature
highlights the critical role of the AIM2 inflammasome in the
host defense mechanism, and AIM2 deficient mice are highly
susceptible to infections with Francisella tularensis,Myco-
bacterium tuberculosis,Staphylococcus aureus, and display
higher mortality and bacterial burden in comparison to wild-
type mice [88–90]. Nonetheless, the role of the
cGAS–STING in these scenarios are yet to be examined.
A key question is whether both cGAS and AIM2 func-
tion concurrently or act sequentially to initiate immune
responses? To address this, a study demonstrated that
cGAS–STING-IFN1 pathway activation amplified AIM2
protein levels to induce robust innate immune responses
during murine cytomegaloviral infections in BMMs [91].
Similarly, cGAS-mediated IFN responses increase caspase-
1 and caspase-11 expression, which in turn increased IL-1β
release and pyroptotic cell death during Chlamydia tra-
chomatis infections [92]. Therefore, robust activation of
both canonical and noncanonical inflammasomes are
dependent on early IFN priming of inflammasome compo-
nents. With these observations it can be proposed that
AIM2 activation follows STING signaling, and the activa-
tion of these signaling pathways amplify the host defense
response (Fig. 4). However, cGAS–STING-NLRP3 acti-
vation replaces the AIM2 function in human myeloid cells
in response to DNA, as STING activation induces lysoso-
mal cell death leading to K+efflux, which in turn activates
the NLRP3 inflammasome [93]. This finding raises the
question as to whether STING is more sensitive to mis-
localized DNA than AIM2? It is possible that
cGAS–STING can detect lower levels of DNA in the
cytosol than AIM2. Another possible explanation is that
there is still an unknown factor that could determine which
DNA sensor to be activated depending on the levels of
DNA in the cytosol, that would correspond to the degree of
the danger signal or localization of DNA.
Conversely, to prevent overactivation of inflammatory
responses, the innate immune system maintains a balance
between different DNA sensors expression and/or activa-
tion. For instance, gasdermin-D activated by the AIM2
inflammasome inhibits IFN-βresponses to cytoplasmic
DNA via K+efflux in BMM [94]. In addition, AIM2
deficient BMMs and bone marrow-derived DCs display
elevated IFN-βlevels in response to cytoplasmic DNA-
mediated cGAS–STING activation [95]. Moreover, to pre-
vent deleterious effects of inflammatory responses not only
the DNA-sensing AIM2 inflammasomes, but also other
components of inflammasomes can negatively regulate the
cGAS–STING pathway. For example, the NLRC3 interacts
directly with STING to prevent its translocation into the
perinuclear region and binding to TBK1, that ultimately
hinders IFN responses and NF-κB activation [96]. Also,
caspase-1 limits cytosolic DNA-mediated cGAS–STING
activation by directly cleaving cGAS during canonical
inflammasome activation [97]. Similarly, during LPS-
induced noncanonical inflammasome activation caspase-4,
-5 (human), and -11 (mouse) can cleave human and mouse
cGAS, respectively [97]. Furthermore, NLRP4 can recruit
the ubiquitin ligase DTX4 to TBK1 for ubiquitination,
which leads to TBK1 degradation and thereby regulation of
DNA-mediated IFN responses [98]. By contrast, IFN
inhibited NLRP3 inflammasome activation in STAT1-
dependent manner, thereby preventing caspase-1 depen-
dent IL-1βrelease [99]. Collectively, these studies highlight
the sophisticated interplay between cGAS–STING and
inflammasome components to prevent hyper-inflammation.
cGAS–STING and autophagy
The evolutionarily conserved catabolic process of autop-
hagy is essential for cellular health and organismal home-
ostasis [100,101]. Autophagy involves a complex
machinery that targets damaged or long-lived organelles
and/or proteins packaged in double membrane vesicles
termed autophagosome, to lysosomes for degradation and/
or recycling. Based on the mode of cargo delivery to
lysosomes, autophagy can be broadly classified into mac-
roautophagy (cytoplasmic contents are delivered to lyso-
some by autophagosomes), microautophagy (direct
lysosomal membrane invagination), and chaperone-
mediated (direct translocation across lysosomal mem-
brane), which is a non-conserved process only observed in
Crosstalk between cGAS–STING signaling and cell death
mammalian cells [102]. Macroautophagy, henceforth
referred to as autophagy, involves several steps: initiation,
nucleation or phagophore formation, elongation of phago-
phore membrane to form autophagosome, fusion of autop-
hagosome with the lysosomes, and degradation of
autophagosome contents. Each of these steps involves a
series of autophagy-related gene (Atg) products. Briefly,
autophagy initiation is assisted by the Atg1/ULK1 serine-
threonine complex comprising ULK1, ATG13, ATG101,
and FAK-family interacting protein (FLIP2000) [103]. The
class III phosphatidylinositol 3-kinase (PI3K) complex,
consisting of ATG15, vacuolar protein sorting (VPS) 15,
VPS 34, and Beclin1-related proteins mediate nucleation by
generating phosphatidylinositol 3-phosphate (PI3P) [104].
ATG9 vesicles offer a portion of the autophagosomal
membrane, whilst WIPI proteins and its interacting partners
(ATG2a or ATG2b), and two transmembrane ER proteins,
vacuole membrane protein 1 and TMEM41 B are necessary
to form isolation membrane [105–108]. Two unique
ubiquitin-like protein conjugation systems, ATG12-ATG5-
ATG16L complex and ubiquitin-like microtubule-asso-
ciated protein 1 light chain 3 (MAP1LC3B/ATG8) are
conjugated to a lipid molecule phosphatidylethanolamine to
aid in elongation [109]. LC3 is synthesized in its precursor
form as pro-LC3, which is cleaved by ATG4 to LC3-I
[110,111]. ATG3 and ATG7 convert LC3-I to LC3-II,
which aids in elongating autophagosome membrane
[112–114]. While the autophagosome closure remains
Fig. 4 Interplay between the
cGAS–STING signaling
pathway and pyroptosis. A
model that proposes DNA
concentration could determine
type of DNA sensor and level of
innate immune responses
required to maintain
homeostasis. During low
concentration of DNA in the
cytosol, cGAS–STING could be
activated to drive IFN-mediated
immune responses. Whereas,
when high concentrations of
DNA accumulate in the cytosol,
the AIM2 inflammasome could
be activated to intensify the
innate immune responses by IL-
1βrelease, and pyroptosis
mediated by active caspase-1
and gasdermin cleavage.
Therefore, IFN release from
cGAS–STING activation primes
AIM2 inflammasome
components as marked by
increased expression of AIM2,
procaspase-1/11/4/5.
Conversely, to prevent
hyperactivation of the immune
responses, K+efflux mediated
by gasdermin-d cleavage
inhibits cGAS, and active
caspase-1/11/4/5 can cleave
cGAS directly. Red arrows
indicate negative regulation, and
green arrows indicate positive
regulation.
A. M. V. Murthy et al.
poorly understood [115], autophagosomes fusion with the
lysosomes requires specific SNARE proteins, small GTPa-
ses and their effectors [116] to initiate degradation of cargo
by a series of lysosomal enzymes. Although, autophagy is
regarded as an unselective process, selective autophagy is
mediated by ubiquitin-binding proteins (p62, autophagy
receptor optineurin, and NDP52), or proteins with trans-
membrane domains such as Nix (NIP3-like protein X as
mitophagy receptor) [117,118].
In recent years growing evidence has indicated interac-
tions between autophagy machinery and the cGAS–STING
pathway. Over 10 years ago Akira et al. [119] identified that
ATG9a and LC3-II co-localize with STING in vesicles after
dsDNA stimulation in MEFs. In addition, deletion of
ATG9a increases STING–TBK1 assembly upon sensing
dsDNA, suggesting the possible roles of autophagy proteins
in STING regulation [119]. Subsequent studies demon-
strated interactions between autophagy and cGAS–STING
using both genetic and biochemical approaches [120–122].
It is possible that STING activation can initiate autophagy
and follow the key steps of autophagy pathway (discussed
above), or STING–TBK1 can be translocated directly to
autophagosome via an unknown mechanism independent of
p62 (Fig. 5). As an example, cGAS-Beclin-1 interaction
initiates autophagy by releasing the autophagy negative
regulator, Rubicon to allow PI3K complex activation upon
dsDNA recognition during HSV infections [123]. Further-
more, STING-induced autophagy requires WIPI2 and
ATG5 proteins [122], ATG3, p62 and increase in LC3-II
conversion followed by degradation of STING via con-
ventional autophagy [121]. On the other hand, STING
interacts directly with LC3 leading to degradation of STING
itself and pTBK1, highlighting unconventional means of
autophagy activation [120]. Undoubtedly, there is a high
degree of complexity associated with re-localization of
STING with different autophagy proteins. It should be
noted that most of the STING-autophagy studies used V-
ATPase inhibitors such as Bafilomycin or concanamycin A,
or chloroquine to inhibit autophagy pathway that could
affect other signaling pathways. Also, studies involving
genetic manipulation of autophagy should account for the
non-autophagic role of autophagy proteins in cell survival,
maintenance, and death [124].
During Mycobacterium tuberculosis infection bacterial
DNA-mediated STING–TBK1 activation targets a sub-
population of bacteria to autophagosomes in BMMs [125]
that is likely to attenuate virulence and pathogenicity in
murine lungs [126]. However, during Mycobacterium tuber-
culosis infections, NF-κB-induced expression of the DNA
damage-regulated autophagy modulator DRAM1, which then
localized with p62 and STING in zebrafish and human
macrophages initiating autophagic defense responses [127]. It
is possible that autophagy prevents sustained STING
phosphorylation but not NF-κB activation [128]. Although,
the NF-κB activation is dependent on MyD88, the role of
STING in NF-κB activation cannot be overlooked [127].
Although p62 was demonstrated to be crucial for trans-
location of STING to lysosome-associated compartments
for STING degradation, it should be noted that p62
knockout MEFs and THP-1 cells show a delay rather than
abrogation of STING degradation in comparison to wild-
type cells [121]. This suggests that ubiquitination by other
ubiquitin ligases such as RNF5 and TRIM30 can also target
STING for proteasomal degradation [129,130]. Additional
studies are required to understand the role of ubiquitin
ligases in autophagy-dependent STING activation and reg-
ulation. Collectively, STING activation initiates autophagy
followed by its own degradation. However, cell-type, con-
centration of cytoplasmic DNA and the strength of
inflammatory responses may dictate how and whether
autophagy components mediate STING pathway signaling.
Importantly, it is possible that STING-mediated autophagy
can be a potential way to clear infections effectively, and
further studies are warranted to understand the role of
STING agonists to treat bacterial infections.
Some studies have reported that rather than STING, it is
cGAS that interacts with autophagy components, thereby
increasing the complexity of interactions between the
cGAS–STING pathway and autophagy. In keeping with this
concept, cGAS−/−mice display increased susceptibility to
Mycobacterium tuberculosis than their wild-type or STING-
deficient counterparts [131]. In addition, cGAS-induced
autophagy protects hepatocytes that lack STING [132]from
hypoxia-induced ischemia-reperfusion injury, independent of
Beclin-1 [133]. Overall, it could be envisaged that cGAS can
activate STING-dependent and -independent autophagy as
host protective response depending on the intensity of the
threat posed, and the degree of inflammatory responses
required to clear danger signals. One possibility is that
STING, but not its activation is required for basal autophagy
as STING-dependent autophagy occurs independent of TBK1
activation and IFN production [122]. In contrast,
cGAS–STING pathway activation leading to inflammatory
responses as a host defense mechanism is required to coun-
teract a severe threat. Alternatively, there could be an
unknown sensor that licenses STING-dependent versus
STING-independent autophagy in a cell-type or cytosolic
DNA concentration context [134].
ADCD following cGAS–STING activation
As autophagy removes damaged organelles and protects
cells under nutrient-limiting conditions, it is primarily a
cytoprotective process. However, autophagy often accom-
panies cell death and has been shown to be a driver of cell
Crosstalk between cGAS–STING signaling and cell death
death in specific contexts [135]. ADCD is defined by The
Nomenclature Committee of Cell Death as ‘a form of
regulated cell death that mechanistically depends on the
autophagy components’[3]. To be termed as ADCD,
genetic or chemical inhibition of autophagy should prevent
cell death [102,136]. Also, more than one autophagy pro-
teins must be involved in triggering cell death [135].
To date, the most robust genetic evidence for ADCD
comes from studies involving the degradation of larval
midgut during larval-pupal transition in Drosophila
[137,138]. Genetic studies have demonstrated that larval
midgut degradation can occur in the complete absence of
the apoptosis machinery in Drosophila but has an essential
requirement for autophagy induction [131,134,135]. Stu-
dies in mammalian and other model systems have also
identified instances of ADCD in specific developmental or
pathophysiological context [135]. For example, autosis is a
specific form of ADCD that involves the Na+/K+-ATPase
[139]. In another instance, the removal of interdigital web
cells during embryonic development is further delayed in
Atg5/Bax/Bak triple deficient mice, compared to Bax/Bak
double knockouts, suggesting a role for autophagy in
removing interdigital web in mammals [140]. Notably,
invertebrates lack C-terminal tail of STING that recruits
Fig. 5 Complexity of
interactions between
autophagy machinery and the
cGAS–STING pathway.
Autophagy process involves five
key steps, (1) initiation, (2)
nucleation, (3) elongation, (4)
autophagosome formation, and
(5) degradation, where each step
is regulated by specific ATG
proteins as highlighted, or
explicit proteins such as ULK1,
Beclin-1, P13P, LC3
conjugation system as
mentioned. cGAS–STING
activation can initiate autophagy
and follow the five key steps
triggering its own degradation,
however the requirement of
specific autophagy components
at each step is ambiguous. In
addition, cGAS–STING
activation mediated by
cGAS–STING–TBK1–IRF3
assembly complex can
translocate directly to
autophagosomes via an
unknown mechanism. Also,
cGAS can interact directly with
Beclin-1 to initiate autophagy
independent and/or absence of
STING. Furthermore, ULK1 can
phosphorylate STING and
prevent its activation.
A. M. V. Murthy et al.
TBK1 and IRF3 and can still mediate ADCD [141]. How-
ever, recent studies link self-DNA stimulated cGAS–
STING to excessive autophagy leading to ADCD
[139,140]. One study demonstrated that cell crisis due to
telomeric damage results in release of DNA fragments into
the cytoplasm, that trigger autophagy as observed by
increased in LC3-II or decrease in p62 [142]. Inhibition of
autophagy or STING pathway components prevented cell
crisis resulting in unstable genome and chromosomal
aberrations, suggesting that this pathway is involved in
deleting damaged and potentially premalignant cells [142].
In other recent study the infection by Burkholderia pseu-
domallei, that causes host cell fusion, was shown to activate
cGAS–STING activation, independent of bacterial ligands
[140]. Infection induced cell fusion lead to aberrant mitosis,
formation of micronuclei, which colocalised with cGAS,
followed by the activation of cGAS–STING pathway and
ADCD [140]. These two studies demonstrate that cell crisis
or bacterial-mediated cell fusion results in cGAS–STING-
mediated autophagy and that subsequent ADCD limits
accumulation of damaged cells [142,143]. Thus, these
studies suggest that one of the functions of cGAS–STING-
dependent ADCD is to prevent tumorigenesis. However,
autophagy is known to have dual role in cancer [144]
and therefore, further mechanistic studies are required to
understand context-dependent ADCD. Although, excessive
autophagy can clearly trigger cell death, further work is
warranted to establish if different molecular factors are
involved in developmental versus stress-induced ADCD.
Conclusion and perspectives
Activation of the cGAS–STING pathway is a major
responder to viral and bacterial infections. However, this
pathway can be activated by self-DNA and can act as a
double-edged sword, where it can be beneficial or detri-
mental to the host depending on the context of its activation
(self-DNA, infection, and chronic inflammation), cell-type,
cell death pathways activated, and the magnitude and
duration of activation. Structural studies of STING have
shown that a closed conformational change in the ‘lid’loop
of STING upon binding to CDN is necessary for its acti-
vation. However, studies with the newly identified ABZI
STING agonist demonstrated that the activation can occur
in an open conformation. Identification of such novel small
molecules also gives a perspective that structures other than
CDN with distinct physiochemical and biological properties
could bypass cGAS to activate STING and provides more
options for the clinical development of STING agonists.
Considering that multiple cell death pathways can be trig-
gered following cGAS–STING signaling, a better under-
standing of the regulatory mechanism distinct to cell-types,
context and/or stimulus-dependency will require further
investigations. Moreover, the role of STING in the co-
existence of various cell death pathways such as the newly
described PANopoptosis [145] and switch between cell
death pathways remain to be studied. Further knowledge
will help answer questions such as what dictates the
redundancy in cell death pathways triggered by the
cGAS–STING. Any direct role of cGAS–STING signaling
in ADCD and the mechanisms of ADCD also require fur-
ther investigations. More importantly, answers to these
questions will eventually open new avenues to target
STING-mediated cell death pathways for treating cancer
and infectious diseases.
Acknowledgements The work in our laboratories is supported by the
National Health & Medical Research Council (NHMRC) project
grants (1144500, 1156601), a NHMRC Senior Principal Research
Fellowship (1103006) to SK, and the University of South Australia
internal research support to SK and NR.
Author contributions AMVM drafted, edited, and revised the manu-
script text and prepared figures, NR provided intellectual input and
edited the manuscript, SK conceptualized, edited, and revised the
manuscript, and contributed intellectual input.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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