Battle of the hottest: STING and Inflammasome - Heating up the tumors
Development of the next wave of rational clinical combination strategies will require combination partners to immune checkpoint blockers (ICB) propelling the battle of the immune system's capabilities to fight cancer and counteract the immune-suppressive processes that promote tumor growth.
Until recently, the trials have mostly focused on up-regulating the adaptive immune system — a slow-starting but long-lasting protective network defending against non-self threats. Recently, however, a few companies have started to explore whether the broader acting, rapid-onset innate immune system might provide the alternative and/or complementary anticancer agents that everyone is looking for.
A January News & Analysis recapped the ongoing activities in the field (Mullard, 2018):
- While Merck & Co., launched a trial early in 2017 of its pro-inflammatory STING agonist MK‑1454 alone and in combination with the approved checkpoint PD1 blocker pembrolizumab, in September, Novartis and partner Aduro Biotech likewise launched a phase I trial that combines Aduro’s STING agonist ADU‑S100 with Novartis’s experimental PD1 inhibitor PDR001.
- In related activity, Bristol-Myers Squibb (BMS) acquired the biotech IFM Therapeutics in August to get their hands on preclinical agonists of STING and of NLRP3.
- Just a month later, Merck bought Rigontec to access the phase I drug candidate RGT100, an agonist of another innate immune system target known as RIG-I.
Figure 1. Selected precision innate immune system agonists (Mullard, 2018)
The idea is to use these agents to convert the uninflamed 'a.k.a. cold' tumors into immune cell-infiltrated tumors 'a.k.a. hot' cancers. To date, immunotherapies have been particularly effective in the therapy of hot tumors that are already infiltrated with CTLs.
What is a Hot and what is a Cold tumor ?
Histologically, tumors can be broadly categorized as inflamed or noninflamed. Inflamed tumors are characterized by the presence of tumor-infiltrating lymphocytes (TIL), high density of IFNγ-producing CD8+ T cells, expression of PD-L1 in tumor-infiltrating immune cells, possible genomic instability, and the presence of a preexisting antitumor immune response. In contrast, noninflamed tumors are immunologically ignorant and are poorly infiltrated by lymphocytes, rarely express PD-L1, and at the antipodal end of the tumor immunity continuum, are characterized by highly proliferating tumors with low mutational burden and low expression of antigen presentation machinery markers including MHC class I (Fig. 2).
Figure 2. The tumor immunity continuum. Representative images of tumor CD8 IHC show three patterns of T cells associated with tumor cells. Tumors with preexisting immunity are represented by abundance of TILs, dense functional CD8+ T-cell infiltration reflected by increased IFNγ signaling, expression of checkpoint markers, including PD-L1, and high mutational burden. These characteristics reflect highly inflamed tumors. Despite high mutational burden, tumors with the excluded infiltrate or stromal T-cell phenotype are represented by increased influence of immunosuppressive reactive stroma, myeloid-derived suppressor cells (MDSC), and angiogenesis, all of which prevent infiltration of T cells into the tumors or suppress activation of T cells in the tumor milieu. Finally, immunologically ignorant tumors that contain very low infiltration of T cells are genomically stable with highly proliferating tumor cells. These are representative of noninflamed tumors.
Strategies to promote the accumulation of CTLs in tumor lesions
Intratumoral accumulation of CTLs is an independent prognostic factor for survival of patients with different cancer types and is required for the clinical effectiveness of checkpoint blockade therapies.
Several strategies have been shown to activate endogenous innate immunity, in particularly antigen-presenting cells (APCs) and other tumor microenvironment (TME) components, resulting in enhanced cross-priming of tumor-antigen-specific CD8+ T cells, augmented local chemokine production, and enhanced CTL accumulation. Productive cross-priming of CD8+ T cells in vivo involves early innate immune recognition of cancer cells and induction of type-1 interferons (IFNs) in DCs (Fuertes et al., 2013).
The stimulator of interferon genes (STING) pathway detects the presence of a tumor and can drive DC activation and induction of T-cell immunity against tumor-associated antigens (TAAs) in vivo (Woo et al., 2014). RIG-I and NLRP3 offer alternative means of activating the innate immune response and warming up cold tumours. The RIG-I pathway is physiologically activated by viral RNA and also triggers type I inteferon signalling. By contrast, NLRP3 triggers include stress signals, such as reactive oxygen species. These lead to upregulation of proinflammatory interleukin (IL)‑1β and IL‑18. Additional alternative strategies involve targeting exogenous antigens and adjuvants to DCs (reviewed in Kreutz et al. 2013), or exposing tumor tissues to combinatorial chemokine modulatory adjuvants involving exogenous type I IFNs (Yang et al., 2014) in combination with Toll-like receptor (TLR) ligands or interleukin (IL)-18, or targeted delivery of CTL-attracting chemokine genes to tumors.
Pioneers in the class: the struggle of TLR agonists
While ICB have been demonstrating the ability of the immune system to control tumor growth, Toll-like receptor (TLR) agonists have hit several setbacks and eventually lost the battle in cancer immunotherapy due to several - at that time uncomprehensive - reasons. They were not used in the right way (Guha, 2012).
Most importantly it seems that in contrast to ICB which are effective in inflammed tumors as monotherapies, TLR agonists are unlikely to prove themselves in most cancers as monotherapies and need to be combined with either therapeutic vaccines or drugs that kill cancer cells and thereby release tumor antigens, and/or agents that counter the immunosuppressive TME. A most likely explanation for this being that TLR agonists themselves induce a negative feedback mechanism that limits the uncontrolled inflammation that could otherwise arise. Therefore they still offer promise if they are better combined with anticancer drugs that kill cancer cells and thereby release tumour antigens that stimulate an immune response, or if they are used as therapeutic cancer vaccine adjuvant, together with ICB.
Amongst the most high-profile failures of TLR agonists, Idera Pharmaceuticals’ top-line Phase II results of the oligonucleotide-based TLR9 agonist IMO‑2055 showed that the drug did not improve progression-free survival in second-line recurrent or metastatic head and neck cancer and Pfizer’s 2007 discontinuation of its oligonucleo‑ tide-based TLR9 agonist CPG 7909 (also known as ProMune) after the drug failed to demonstrate efficacy when used in combination with chemotherapy in Phase III trials in non-small-cell lung cancer (NSCLC).
Because members of the TLR family each trigger the release of a different spectrum of inflammatory cytokines, there is also uncertainty over which targets to agonize to evoke the most potent antitumour immune responses. The field has been focused on TLR9 and TLR7 agonists because of the antitumour activity of TLR9 in various preclinical models, whereas TLR7 agonists were pursued because of imiquimod's success (the drug was found to act through TLR7 after its clinical efficacy was demonstrated). The Cancer Vaccine Collaborative — a joint programme of the Cancer Research Institute and the Ludwig Institute for Cancer Research — has conducted a series of parallel, early-stage trials evaluating TLR3, TLR4, TLR7 and TLR9 agonists as vaccine adjuvants in combination with the cancer–testis antigen NY-ESO-1. It found that the TLR3 agonist poly-ICLC elicited the most favourable immune response, as assessed by antigen-specific antibody levels as well as CD4 and CD8 T cell responses.
Figure 3. Mammalian TLR signalling pathways. TLR5, TLR11, TLR4, and the heterodimers of TLR2–TLR1 or TLR2–TLR6 bind to their respective ligands at the cell surface, whereas TLR3, TLR7–TLR8, TLR9 and TLR13 localize to the endosomes, where they sense microbial and host-derived nucleic acids. TLR4 localizes at both the plasma membrane and the endosomes. (O'Neill et al., 2013)
STING (a.k.a. Tmem173, MITA, ERIS, or MPYS)
cGMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) sensing has emerged as a key regulator of innate immune responses to both exogenous and endogenous DNA. Various nucleic acid sensors converge on the STING adaptor protein, which induces type I IFN expression via IRF3 and NFκB nuclear translocation and, ultimately, IFN-stimulated gene (ISG) expression, potentially in both cancer cells and neighboring cells. Among the variety of intracellular DNA sensors, the nucleotidyltransferase cGAS appears to be the primary, non-dispensable sensor of cytosolic double-stranded DNA (dsDNA). The cGAS–STING axis is unusual in that signaling is mediated not through protein–protein interactions but rather through a soluble second messenger, the cyclic dinucleotide cyclic 2′3′-GMP–AMP (cGAMP) synthesized by cGAS from ATP and GTP, that then directly binds STING. Concordantly, cGAS–STING agonists, including STING-binding molecules and cGAMP derivatives, have been developed and explored in the context of monotherapy, combination therapy, and vaccine adjuvanticity (see below).
Sequences of non-specific dsDNA species greater than 30 bp have been reported to stimulate cGAS activity, with a single CDN generated by cGAS binding to two molecules of STING in the ER. Potent activators of the STING pathway include self-DNA that has leaked from the nucleus of the host cell, perhaps following cell division or as a consequence of DNA damage. Mitochondrial stress can cause mtDNA leakage into the cytosol that can also activate the STING pathway and the production of cytokines.
Figure 3. Stimulator of interferon genes (STING) is activated by cyclic dinucleotides (CDNs) produced by certain bacteria or by cyclic GMP–AMP synthase (cGAS), which in the presence of ATP and GTP catalyses the production of a type of CDN referred to as cGAMP (cyclic GMP–AMP) following binding to cytosolic DNA species (from viruses or bacteria, or self -DNA from the nucleus or mitochondria). STING is associated with the endoplasmic reticulum (ER) and, following binding to CDNs, STING forms a complex with TANK-binding kinase 1 (TBK1). This complex traffics to the perinuclear Golgi via pre-autophagosomal-like structures — a process resembling autophagy — to deliver TBK1 to endolysosomal compartments where it phosphorylates the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB). Stimulation of the IRF3 and NF-κB signalling pathways leads to the induction of cytokines and proteins, such as the type I interferons (IFNs), that exert anti-pathogen activity. c-di-AMP, cyclic di-AMP; dsDNA, double-stranded DNA; ISGF3, interferon-stimulated gene factor 3; JAK, Janus kinase; STAT, signal transducer and activator of transcription; TYK, tyrosine kinase. (Barber, 2015)
One hypothesis for the underlying mechanism is that CD8α+ DCs engulf necrotic tumour cells, and the tumour cell-derived DNA triggers STING signalling in the DC. The resultant type I IFNs, functioning in a paracrine or autocrine manner, may induce the production of additional proteins in the DC that facilitates cross-presentation and T cell activation
Figure 4. STING-dependent antitumour cytotoxic T lymphocyte (CTL) priming. Dying tumour cells are engulfed by antigen-presenting cells such as CD8α+ dendritic cells (DCs). DNA from the engulfed cell triggers STING-dependent cytokine production in the phagocyte, which facilitates cross-presentation and antitumour CTL responses. Agonists of STING have been shown to exert potent antitumour activity. (Barber, 2015)
STING signalling appears to be independent of other DNA sensing pathways such as the Toll like receptor 9 (TLR9) pathway, which is activated by binding to unmethylated CpG dinucleotides of approximately 21 bases long. Thus, the TLR9 and STING signalling pathways have evolved to sense different types of DNA species, although both pathways use IRF3 and NF-κB to predominantly control gene induction. Furthermore, TLR9 is mainly expressed in pDCs and B cells, whereas STING is more broadly expressed. The STING pathway is also independent of AIM2 (absent in melanoma 2), which also interacts with non-specific dsDNA species but triggers caspase 1-mediated cleavage of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 from their precursor proteins (see below). However, STING signalling is known to potently influence the expression of the precursor proteins (but not their processing), and therefore it is possible that STING may act in concert with the AIM2 pathway.
Targeting cGAS-STING in the clinic
Insight into the role of STING in facilitating antitumour T cell responses has stimulated interest in evaluating whether STING agonists could be useful therapies to treat cancer.
Although chemotherapeutic and radiotherapeutic approaches were not designed to target cGAS–STING, it is important to note that these agents induce DNA damage, potentially activating the cGAS–STING pathway and promoting immune responses that enhance tumor cell death and rejection. For example, the current chemotherapeutic agents cisplatin and etoposide are known to induce cGAS–STING via DNA damage and cytosolic leakage. Further, doxorubicin and daunorubicin are able to potently activate cGAS–STING via inhibition of topoisomerase II and dsDNA breaks.
Although it was not known to target STING at the time, 5,6-dimethylxanthenone-4-acetic acid (DMXAA) was a highly promising chemotherapeutic agent lauded for its antitumor effects in advanced lung, prostate, and breast cancers. Following development by Antisoma and Novartis, DMXAA failed in Phase III trials due to the fact that DMXAA–STING interactions are restricted to mice and thus have minimal effect on human STING. Since then, much attention has focused on the development of clinically relevant STING agonists, including a Phase I clinical trial on direct intratumoral injection of MIW815 (Novartis, reference NCT02675439) alone or in conjunction with immune checkpoint blockade in solid malignancies. Along the lines of DMXAA, these efforts may be confounded by the fact that various human STING alleles have been identified displaying altered functionality; however, rationally designed synthetic cyclic dinucleotide derivatives have been synthesized that activate all known human STING alleles, which may be worth pursuing in the clinic. These synthetic derivatives have been demonstrated to strongly induce IFNb in both murine BMDMs and primary human cells, decrease tumor volume on injection into murine melanoma, colon, and breast models, and induce antitumor immunological memory following tumor regression.
Cyclic dinucleotides appear to enhance the effect of various current and prospective cancer therapies, including vaccines, CD47 blockade, and fluorouracil. Most strikingly, in some instances, STING agonists were shown to be effective against tumours that were resistant to programmed cell death protein 1 (PD1) blockade. Such agonists have also been shown to be experimentally useful as adjuvants in anticancer vaccine studies. Furthermore, the ability of checkpoint inhibitors to stimulate T cell responses was also abrogated in STING deficient mice, indicating a role for STING in the efficacy of checkpoint inhibitors.
Table 1. . Current and Potential Therapeutic Mechanisms Enhance or Target cGAS–STING Signaling. (Ng et al., 2018)
NLRP3 Inflammasome
The discovery of the inflammasome protein complex in 2002 was a breakthrough in our understanding of how the immune system triggers inflammation. Martinon, et al. published a paper (2002) that solved the puzzle of how a key component of inflammation is activated. Caspase 1 is a member of an evolutionarily conserved family of protease enzymes that cleave proteins that regulate cell death and inflammation. Martinon et al. discovered that a multiprotein complex is needed to activate caspase 1, and they named this complex the inflammasome.
Figure 5. The inflammasome. Before 2002, it was known that, in response to infection by a bacterial pathogen, the enzyme caspase 1 can promote the release of inflammatory signalling molecules, such as interleukin-1β (IL-1β) and IL-18, and that caspase 1 also has a role in pyroptosis, a type of cell death that can eliminate immune cells. However, how caspase 1 is activated and induces pyroptosis was unknown. In 2002, Martinon, Burns and Tschopp discovered that innate-immune receptor proteins from the NLR protein family and the adaptor protein ASC assemble into an inflammasome complex that recruits and activates caspase 1. Diverse inflammasome complexes have been identified and the innate-immune receptors in some inflammasomes are not NLRs but are instead AIM2 or pyrin proteins. Another key advance was the identification of the non-canonical inflammasome pathway, in which caspase 4, 5 or 11 act upstream of caspase 1 activation. Caspase mediated cleavage of the protein gasdermin D is the mechanism that enables inflammatory caspase activation to induce pyroptosis in both the canonical and non-canonical inflammasome pathways. (Lamkanfi and Dixit, 2017)
Inflammasomes are cytosolic multi-protein complexes that form in response to diverse cellular insults to promote proximity-induced autoprocessing of caspase-1 (Lamkanfi and Dixit, 2014). Each complex is assembled by sensor proteins that respond to particular pathogenic conditions. Inflammasomes composed of a particular NLR respond to certain microbe-derived molecules known as pathogen-associated molecular patterns (PAMPs), or to environmental and host-derived cellular stress signals called damage-associated molecular patterns (DAMPs). The identification of inflammasomes that contain the proteins AIM2 or pyrin indicated that caspase 1 can also be activated by innate-immune receptors other than NLR proteins.
NLRP3 inflammasome responds to the broadest array of medically relevant PAMPs, DAMPs, and insults. Indeed, DAMPs like extracellular ATP and hyaluronic acid, medically relevant crystals such as alum, CCPD, MSU, silica, and asbestos, ionophores such as nigericin, and β-fibrils such as β-amyloid can all engage the NLRP3 inflammasome . Moreover, the major component of the outer membrane of Gram-negative bacteria, LPS, activates NLRP3 through a noncanonical pathway involving caspase-11. The inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) connects sensor components within the inflammasome to caspase-1 and is observed in a micrometer-sized supramolecular fibril structure in the stimulated cell named the “ASC speck.” Caspase-1 proteolytically matures IL-1β and IL-18, two highly potent inflammatory cytokines that also act as co-stimulators of T cell functions. Caspase-1 and the related inflammatory caspases 4, 5, and 11 also cleave gasdermin D to induce pyroptosis, which is a proinflammatory form of regulated necrosis that is intrinsically associated with the passive release of IL-1β and IL-18 into the extracellular milieu along with DAMPs such as IL-1α, HMGB1, and ATP.
Associate Professor Kate Schroder’s Laboratory recently revised the field’s understanding of inflammasome signalling (Boucher et al. 2018) and summarized their findings in the following video: https://youtu.be/-FNFZ9F1eB4 .
The NLRP3 inflammasome uniquely requires a two-step mechanism for activation: transcriptional up-regulation of NLRP3 and pro-IL-1β, for example by TLR signaling, serves as a priming step for subsequent activation by NLRP3 agonists (Bauernfeind et al., 2009).
Figure 5. Activation of the NLRP3 inflammasome and its inhibition by CY-09 and sulfonylurea compounds. Activation of the NLRP3 inflammasome involves two steps. First, TLR4 stimulation induces transcriptional up-regulation of NLRP3 and the inflammasome substrate proIL-1β. In the second activation step, NLRP3 agonists such as ATP, the ionophore nigericin, pore-forming toxins and internalized crystals, and β-fibrils trigger NLRP3 oligomerization, ASC speck formation, and inflammasome-mediated caspase-1 autoactivation. Caspase-1 cleaves its cytokine substrates IL-1β and IL-18, and it induces pyroptosis through cleavage of gasdermin D, which promotes the passive release of IL-1β and IL-18 along with DAMPs such as IL-1α and HMGB1. CY-09 inhibits NLRP3 inflammasome assembly by blocking ATP/dATP binding in the central NACHT domain, whereas the target and mechanism of action of sulfonylurea compound MCC950/CRID3 are unknown. (Lamkanfi and Dixit, 2017)
A comprehensive lecture by Dixit V on inflammasomes is embedded below.
IL-1-neutralizing treatments dominate this field, but as the understanding of the mechanisms that regulate caspase-1 activation becomes increasingly sophisticated, researchers are exploring other treatment approaches. Previous efforts to block capase-1 enzymatic activity directly have mainly failed, so researchers are now trying to intervene pharmacologically at points further upstream in the caspase-activation pathway. Selective inflammasome inhibition might well be within reach — the drug glyburide, (an anti-diabetic), has been found to selectively block activation of the NLRP3-containing inflammasome, thus setting the stage for the identification of additional anti-inflammatory agents.
Currently, MCC950/CRID3 and related diarylsulfonylurea compounds that are structurally related to glyburide are the most potent reported inhibitors of the NLRP3 inflammasome with IC50 values around 20 nM (Perregaux et al., 2001; Coll et al., 2015; Primiano et al., 2016). CY-09 as a novel inhibitor of the NLRP3 inflammasome that acts as a competitive inhibitor of nucleotide binding to the central NLRP3 NACHT domain. Binding of ATP/dATP to NLRP3 was shown previously to be required for NLRP3-dependent IL-1β secretion. Several compounds that interfere with the intrinsic ATPase activity of NLRP3 have been reported, but they target additional proteins with roles in innate immunity, suggesting that their selectivity profile would need significant optimization before widespread adoption as inflammasome-targeting agents.
BMS with IFM Therapeutics plan to start phase I trials of their candidate early this year.
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