Patent Application
20240271138
The contents of the electronic sequence listing (B119570133US02-SEQ-TNG.txt; Size: 88,602 bytes; and Date of Creation: Dec. 7, 2023) is herein incorporated by reference in its entirety.
The Stimulator of Interferon Genes (STING) is an innate regulator of immune response against pathogen- or self-derived cytosolic DNA. The STING pathway consists of a cascade of molecular events that are essential for inducing protective immunity and can cause autoimmunity if dysregulated. STING acts as an intracellular sensor of cyclic di-nucleotides and as an adaptor for cyclic GMP-AMP (cGAMP) after its generation by the DNA sensor by cyclic GMP-AMP (cGAMP) Synthase (cGAS) (Ablasser et al., 2013; Gao et al., 2013; Sun et al., 2013; Wu et al., 2013). Homodimeric STING is localized at the endoplasmic reticulum (ER) (Ishikawa and Barber, 2008) and undergoes a cGAMP-dependent conformational switch that triggers its exit from the ER and trafficking to the Golgi. At the Golgi, STING palmitoylation is required for STING clustering and activation of type I IFN responses via Tank Binding Kinase 1 (TBK1) at the Trans-Golgi Network (TGN) (Mukai et al., 2016). After TBK1 phosphorylates STING at residue S366, phospho-STING forms a platform at the carboxy-terminal tail (CTT) for recruitment of IRF3 (Liu et al., 2015a). IRF3 is then phosphorylated by TBK1, homodimerizes and translocates to the nucleus resulting in activation of a type I IFN response. In addition to type I IFN induction, upon cDN ligation, STING induces autophagy independently of the classical macroautophagy machinery but dependent on ATG16L1, an ancestral and conserved function of the response (Fischer et al., 2020; Gui et al., 2019). Thus, STING intracellular trafficking and signaling activities are tightly connected. Recently, progress has been made in characterizing the pathways regulating STING trafficking and degradation. In particular, trafficking to the endolysosomal compartment is essential for STING degradation and signaling shutdown, but the proteins governing STING trafficking to the endosomal compartment and the signals triggering its degradation require further investigation (Ablasser and Hur, 2020; Gonugunta et al., 2017).
Further knowledge and identification of STING interacting genes as therapeutic points of intervention to modulate STING activation and innate immune responses would be of great use in the art to enhance STING signaling in cancer therapy, immunotherapy, pathogen defense, and the like.
Provided herein are compositions and methods for enhancing the activity of Stimulator of Interferon Genes (STING) with one or more agents for inhibiting the activity of one or more negative regulators of STING, including DNAJC13 and ESCRT. It is contemplated that the modulation of STING through the inhibition of the negative STING regulator proteins of this disclosure (e.g., DNAJC13 and ESCRT) can provide a therapeutic approach for treatment of a variety of diseases such as cancers, pathogen infection, autoimmune diseases or cellular senescence, by augmenting the innate immune response associated with the activation of STING signaling.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. For purposes of clarity, not every component may be labeled in every drawing. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure. In the drawings:
Activation of STING signaling is vital for mediating immune responses against a variety of intracellular pathogens, such as viruses and bacteria, in response to the accumulation of either cytosolic double stranded DNA or dinucleotides characteristic of these pathogens. STING is also highly implicated in immune responses to cancer, due to the recognition of cytosolic self-DNA. Due to its role in activating and recruiting a variety of immune cells, such as T-cells, to tumors, agents that enhance the activity of STING may be used to treat cancer or to augment the efficacy of current therapies. This disclosure provides compositions and methods for enhancing the activity of STING signaling, particularly through the inhibition of newly discovered STING interactors that negatively regulate STING signaling activity, including ESCRT and DNAJC13. The disclosure provides methods and compositions for targeted inhibition of negative regulators of STING signaling, including ESCRT and DNAJC13, such that STING signaling—and in turn, the innate immune response—becomes enhanced and/or augmented. Such methods and compositions can be used to enhance or otherwise augment STING activation and innate immune responses, thereby providing an approach to augmenting and/or enhancing cancer immunotherapies and the like.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art (e.g., the skilled artisan). The meaning and scope of the terms are clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this disclosure, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.
General terminology in cell and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (Eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9). General terminology definitions in molecular biology are also given in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (Eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.
Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present disclosure unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.
That the present disclosure may be more readily understood, select terms are defined below.
The singular forms “a”, “an”, and “the” include the plural unless the text clearly indicates otherwise. Similarly, the term “or” is intended to include “and” unless the text clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation “e.g.” is derived from the Latin phrase “exempli gratia” and is used herein to give a non-limiting example. For this reason, the abbreviation “for example (e.g.)” is synonymous with the term “for example”.
The terms “approximately” or “about,” as may be used interchangeably herein, and as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of (i.e., percentage greater than or percentage less than) the stated reference value unless otherwise stated or otherwise evident from the context (for example, when such number would exceed 100% of a possible value).
The term “cancer”, as may be used herein, refers to a cell or population of cells characterized by uncontrolled proliferation. The term “tumor”, as may be used herein, refers to a contiguous population of cancer cells. A cancer may be benign, meaning that it is localized to a single tissue, or malignant, meaning that it spreads to other parts of the body through the circulatory and/or lymphatic system. A cell or population of cells may be “pre-cancerous”, meaning that they share some characteristics of a cancer and risk developing into a cancer. Cells may become cancerous as a result of accumulated mutations in their genome. Examples of cancer include but are not limited to colorectal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, bladder cancer, kidney cancer, thyroid cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, esophageal cancer, stomach cancer, liver cancer, brain cancer, peritoneal cancer, lymphoma, leukemia, multiple myeloma, neuroblastoma, osteosarcoma, and soft tissue sarcoma.
The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all generally statistically significant herein. The terms are used to indicate a decrease in quantity, concentration, level, or the like. However, to prevent misunderstandings, “decreased”, “decreasing”, “decreasing”, or “inhibiting” is a reduction of at least 10% compared to the reference level, e.g., at least about 20% compared to the reference level. The % reduction may also be at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% reduction compared to the reference level. The reduction level may also be expressed in terms of fold-reduction, and includes at least a 2-fold reduction, or at least a 3-fold reduction, or at least a 3-fold reduction, or at least a 3-fold reduction, or at least a 3-fold reduction, or at least a 4-fold reduction, or at least a 5-fold reduction, or at least a 6-fold reduction, or at least a 7-fold reduction, or at least a 8-fold reduction, or at least a 9-fold reduction, or at least a 10-fold reduction, or at least a 11-fold reduction, or at least a 12-fold reduction, or at least a 13-fold reduction, or at least a 14-fold reduction, or at least a 15-fold reduction, or at least a 16-fold reduction, or at least a 17-fold reduction, or at least a 18-fold reduction, or at least a 19-fold reduction, or at least a 20-fold reduction, or at least a 25-fold reduction, or at least a 50-fold reduction, or more.
The terms “increased”, “increase”, “enhance” or “activate” are all generally statistically significant herein. The terms are used to indicate an increase in quantity, concentration, level, or the like. To prevent misunderstanding, the terms “increased”, “increase”, “enhance”, or “activate” is increase of at least 10% compared to the reference level, e.g., at least about 20% compared to the reference level. The % increase may also be at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% increase compared to the reference level. The increase level may also be expressed in terms of fold-increase, and includes at least a 2-fold increase, or at least a 3-fold increase, or at least a 3-fold increase, or at least a 3-fold increase, or at least a 3-fold increase, or at least a 4-fold increase, or at least a 5-fold increase, or at least a 6-fold increase, or at least a 7-fold increase, or at least a 8-fold increase, or at least a 9-fold increase, or at least a 10-fold increase, or at least a 11-fold increase, or at least a 12-fold increase, or at least a 13-fold increase, or at least a 14-fold increase, or at least a 15-fold increase, or at least a 16-fold increase, or at least a 17-fold increase, or at least a 18-fold increase, or at least a 19-fold increase, or at least a 20-fold increase, or at least a 25-fold increase, or at least a 50-fold increase, or more.
The term “substantially,” as may be used herein, when used to describe the degree or abundance of an activity, generally refers to the value of the activity as being an amount which is achievable without undue effort. As can be appreciated, this amount may vary depending on the activity being performed, with simpler activities requiring a higher threshold and more complex activities requiring a lower threshold.
As used herein, “subject” means a human or animal. Usually, the animal is a vertebrate such as a primate, rodent, livestock animal, or hunting animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques such as rhesus monkeys. Rodents include mice, rats, hamsters, rabbits, guinea pigs, squirrels, woodchucks, ferrets. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, cat species such as domesticated cats, dog species such as domesticated dogs, foxes, wolves, birds such as chickens, turkeys, ducks, geese, emus, ostriches, and fish such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms “individual”, “patient” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be conveniently used, for example, as subjects that represent animal models of cancer, e.g., a particular type of cancer, such as, lung cancer. The subject can be male or female. In various embodiment, the subject is a patient that has or is at risk of having a disease state, such as cancer, and is in need to being evaluated, e.g., by a liquid biopsy, to test for the risk of having or developing a disease, e.g., cancer. In other embodiments, that subject is a patient that has already been diagnosed or identified as having or having a disease in need of treatment (e.g., cancer), or one or more complications associated with such diseases. In other embodiments, a subject is a patient that has already been treated for a disease (e.g., cancer) or one or more complications associated with a disease, such as cancer. Alternatively, a subject can also be a patient that has not been previously diagnosed as having a disease (e.g., cancer) or one or more complications associated with the disease. For example, a subject can be a patient that exhibits one or more risk factors for a disease, or one or more complications associated with the disease (e.g., cancer), or a patient that does not exhibit a risk factor.
A “subject in need” of a diagnosis and/or treatment for a particular condition (e.g., cancer) can be a subject who has a condition, has been diagnosed with a condition, or is at risk of developing the condition.
As used herein, the term “agent” refers to any material which is capable of causing a specific outcome when applied to a biological setting. An agent can be a naturally occurring, semi-synthetic, or fully synthetic chemical compound. An agent can facilitate or interfere with one or more chemical reactions occurring in an organism, thereby having one or more biological effects. An agent having a biological effect is said to be bioactive. An agent may be administered to an organism with the intent of producing a desired biological effect. An agent may be administered to an organism in the presence of additional materials having no discernable biological effect, such as an excipient compound.
As used herein, the term “effective amount” refers to the amount of an administered agent that is sufficient to produce an intended biological effect in an organism that the agent is administered to. “Effective amount” is synonymous with the terms “effective dose” and “effective concentration”.
The term “protein degradation” is used to refer to the various processes by which protein and peptide species are hydrolyzed into smaller peptide fragments and/or individual amino acids. Protein degradation can occur as a result of enzymatic processes, such as those that are catalyzed by proteases. Protein degradation in a cell can occur in a variety of membrane-enclosed compartments (organelles), such as in lysosomes, or in the cytoplasm, as occurs within the cytoplasmic proteasome complex.
The term “small molecule”, as used herein, refers to molecule having a relatively low molecular weight. A small molecule may have a molecular weight of less than 500 daltons, less than 600 daltons, less than 700 daltons, less than 800 daltons, less than 900 daltons, or less than 1 kilodalton. A small molecule may diffuse freely across a cell membrane. A small molecule may be an effector that modulates the function of a macromolecule, such as a protein. A small molecule may be an agonist or an inhibitor of an enzyme.
The term “expression” is broadly used to refer to the processes by which one or more genes in a cell are transcribed by RNA polymerases to produce RNA transcripts which may be translated by ribosomes to produce one or more proteins. Expression may refer to either or both of the acts of transcribing genes to RNA and translating RNA into protein. Expression may be described in absolute terms, such as the number of a particular type of RNA transcript or protein present in a cell at a given time. Expression may also be described in terms that are relative, such as when cells are treated with one or more compounds that causes expression of one or more genes and/or proteins to increase or decrease.
The term “activity”, as is used herein, refers to the rate at which an enzyme catalyzes a particular chemical reaction. Activity may be described in absolute terms, such as the number of reactions an enzyme catalyzes on average per second. Activity may also be described in terms that are relative, such as when cells are treated with one or more compounds that causes the activity of one or more enzymes to increase or decrease.
The term “inhibit,” as is used herein, refers to the reduction of one or more particular chemical reactions in the presence of a particular compound, i.e., an inhibitor. Inhibition may be described in absolute terms, such as the number of reactions an enzyme catalyzes on average per second in the presence of the inhibitor, compared to the number of reactions the enzyme catalyzes on average per second in the absence of the inhibitor. Inhibition may also be described in terms that are relative, such as when cells are treated with one or more compounds that causes the activity of one or more enzymes to decrease. A compound that reduces the activity of an enzyme may interact the enzyme directly (e.g., through a physical interaction) or indirectly (e.g., by reducing the activity of one or more separate enzymes, the activity of which precede and are necessary for activity of the enzyme).
An agent may be delivered to a subject by one or more routes of administration. An agent may be enterally administered through the gastrointestinal tract, parenterally administered through any non-enteral route of administration, or topically administered to an external surface of the subject. Parenteral administration can be performed by injection of an agent into a specific location, tissue, or organ of a subject (e.g., intradermal, intravenous, subcutaneous, or intramuscular injection). Alternatively, parenteral administration can be inhalational, wherein the agent is administered by oral and/or nasal inhalation for uptake in the respiratory tract.
The term “pharmaceutical composition” refers to any composition or formulation that is suitable for administration to a subject. A pharmaceutical composition or a component thereof is said to be “pharmaceutically acceptable” if it is generally safe and non-toxic when administered to a subject.
The cGAS-STING Pathway
The methods and compositions described herein are based, at least in part, on the previously unrecognized utility of modulating the activity of specific negative regulators of STING for the purpose of modulating overall STING signaling in a cell or subject. These techniques substantially enhance current capabilities for treating subjects who are suspected of having, known to have, or known to have had a STING-related disease, such as a cancer, by specifically modifying the activity of STING in subjects' own cells.
The innate immune system encompasses a variety of pathways which detect and respond to the presence of intracellular and extracellular antigens, frequently through the upregulation of genes that encode one or more signaling proteins. Antigens may be either pathogen- or host-derived, and includes proteins, lipoproteins, and nucleic acids. One such innate immune signaling pathway is the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, referred to hereafter as the “STING pathway”. The STING pathway is an intracellular sensor of double-stranded DNA (dsDNA) which drives the expression of cytokines, particularly type 1 interferons, in response to cytosolic dsDNA.
Without wishing to be bound by theory, the STING pathway functions in four major steps, collectively referred to herein as “STING signaling”. First, cyclic GMP-AMP synthase (cGAS), a 522 amino acid protein localized to the cytosolic face of the plasma membrane, binds to dsDNA occurring in the cytoplasm irrespective of sequence, as a result of, for example, infection by a pathogen (e.g., a virus, bacteria, or intracellular parasite). In response to binding dsDNA, cGAS synthesizes 2′3′-cGAMP (cGAMP), a secondary messenger molecule that is released into the cytoplasm. Second, cGAMP is bound by the stimulator of interferon genes (STING), a 378-amino acid homodimeric protein localized to the endoplasmic reticulum membrane when inactive. Upon binding to cGAMP, STING undergoes a conformational change that triggers its movement (i.e., trafficking) from the endoplasmic reticulum to the Golgi network (referred to as “forward processing”). STING may also be activated by binding to a cyclic dinucleotide, such as c-di-GMP or c-di-AMP, that is secreted by an intracellular bacterial pathogen, thereby acting independently of cGAS activity. Third, Golgi-localized STING undergoes the covalent attachment of fatty acids, such as palmitic acid, to amino acid side chains (e.g., palmitoylation). Once palmitoylated, STING readily forms oligomers (complexes between multiple STING proteins) and subsequently recruits and activates type I IFN responses via tank binding kinase 1 (TBK1), which phosphorylates STING at serine 366. Finally, active phospho-STING recruits interferon regulatory factor 3 (IRF3), which is also phosphorylated by TBK1, homodimerizes, and traffics to the nucleus where it functions as a transcription factor to induce the expression of cytokines, namely type 1 interferons (IFNs, e.g., IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-τ, and IFN-ω).
Type 1 IFNs, such as IFN-α and IFN-β, are secreted by many cell types as a result of STING signaling, including fibroblasts, macrophages, and lymphocytes (e.g., B-cells, T-cells, and natural killer (NK) cells) in order to mediate immune signaling in other cells. Type 1 IFNs (e.g., IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-τ, and IFN-ω) bind to interferon receptors IFNAR1 and IFNAR2, which recruits Janus family kinase 1 (Jak1) and tyrosine kinase 2 (Tyk2). Jak1 and Tyk2 phosphorylate and activate IFNAR1 and IFNAR2, which in turn recruit and drive the phosphorylation of signal transducers and activators of transcription (STAT) proteins (collectively referred to as the Jak-STAT pathway). Phosphorylated STAT1 and STAT2, for example, associate with interferon regulatory factor 9 (IRF9) to form a complex termed the ISGF3 transcription factor, which binds to IFN stimulated response elements (ISREs) on interferon-stimulated genes (ISGs) to promote the expression of a milieu of proteins that collectively drive innate and adaptive immune responses.
STING signaling can also elicit the expression and secretion of pro-inflammatory cytokines, separately of IRF3, by for example stimulating the activity of other transcription factors, such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).
In addition to the detection of pathogen-derived dsDNA, such as that originating from viruses, bacteria, and parasites, the STING pathway also plays a key role in mediating innate immune responses to tumor cells. Just as cGAS produces cGAMP to trigger the forward processing and activation of STING in response to pathogen-derived dsDNA (e.g., viral, bacterial, or parasite dsDNA), so too does it respond to the presence of cytosolic self-DNA, as may occur during cancer. Cytosolic self-DNA may originate in a cell during cancer from any of several, non-mutually exclusive sources. First, genomic instability during cancer may trigger the release of cytosolic genomic DNA (cgDNA) from the nucleus of a cell. Errors occurring during genomic replication may also lead to the accumulation of cgDNA. Second, cytosolic DNA may also originate from mitochondrial DNA (mtDNA), due to mitochondrial instability during cancer. Third, dsDNA may accumulate in the cytoplasm of certain cell types (e.g., phagocytes, such as macrophages and dendritic cells) due to phagocytosis of tumor cells in various stages of apoptosis, or due to phagocytosis of cell free DNA (cfDNA) that occurs from dead or dying cells. Similar to detection of pathogen-derived dsDNA, detection of cgDNA, mtDNA, and/or phagocytosed tumor-derived DNA by cGAS triggers forward processing and activation of STING, which in turn induces expression and secretion of cytokines, such as type 1 IFNs. Without wishing to be bound by theory, STING signaling has been implicated in various components of cancer immunity, including triggering cancer cell apoptosis and antigen release, cancer antigen presentation by antigen presenting cells (APCs, e.g., dendritic cells (DCs) and macrophages), T-cell priming and activation by APCs, promotion of T-cell trafficking to tumors, T-cell infiltration into tumors, and antigen recognition and killing of cancer cells by cytotoxic T-cells and natural killer (NK) cells (see, e.g., Zhu, Y. et al. “STING: a master regulator in the cancer-immunity cycle”. Mol Cancer 18, 152 (2019); and Jiang, M., et al. “cGAS-STING, an important pathway in cancer immunotherapy.” J Hematol Oncol 13, 81 (2020), which are herein incorporated by reference). STING signaling has further been implicated in the immune response to a wide variety of cancers including, but not limited to, breast cancer, colorectal cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, and squamous cell carcinoma.
Due to the involvement of the STING pathway in mediating cancer immunity, cancer treatments would benefit from techniques that modulate STING activity. Previous efforts to modulate STING activity have focused on the development of STING agonists which can be administered to a cancer patient to activate STING signaling in a patient's cells. One STING modulator to be developed for cancer therapy is the small molecule 6-dimethylxanthenone-4-acetic acid (DMXAA) (see, e.g., Prantner D., et al. “5,6-Dimethylxanthenone-4-acetic acid (DMXAA) activates stimulator of interferon gene (STING)-dependent innate immune pathways and is regulated by mitochondrial membrane potential.” J Biol Chem. 2012; 287:39776-88, which is herein incorporated by reference) Although treatment with DMXAA induces type 1 IFN expression, it was ultimately determined to have substantially weaker binding to human STING in comparison to murine STING and therefor abandoned in clinical trials. Most STING agonists that have been developed subsequently have been cyclic dinucleotide analogs of cGAMP which are either naturally occurring or synthetically designed to bind and activate STING (see, e.g., Ding, C., et al. “Small molecules targeting the innate immune cGAS-STING-TBK1 signaling pathway.” Acta Pharm Sin B. 2020 December; 10(12):2272-2298, which is herein incorporated by reference). Cyclic dinucleotide compounds such as these typically require delivery by a liposome or nanoparticle however, as they are generally unable to cross cell membranes on their own. Despite the potential benefit that modulators of STING signaling might have for stimulating immune responses against cancer, to date no therapeutic agent for enhancing STING signaling has been approved for clinical use in the United States (see, e.g., Le Naour, J., et al. “Trial watch: STING agonists in cancer therapy.” Oncoimmunology. 2020 June; 9(1), 1777624, which is herein incorporated by reference).
Paradoxically however, STING has also been reported to impair cancer immunity in certain contexts. An increase in STING expression during cancer has been correlated with increased infiltration of certain immune-suppressing cell types into tumors, such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) (see, e.g., An X, et al. “An analysis of the expression and association with immune cell infiltration of the cGAS/STING pathway in pan-Cancer.” Mol Ther Nucleic Acids. 2018; 14:80-9, which is herein incorporated by reference). Moreover, agents that directly activate STING have been demonstrated to impair immunity, as treatment with DMXAA elicits stress pathways that induce apoptosis in T-cells (see, e.g., Larkin B, et al. “Cutting edge: activation of STING in T cells induces type I IFN responses and cell death.” J Immunol. 2017; 199:397-402, which is herein incorporated by reference). Similarly, treatment with exogenous cGAMP impairs T-cell proliferation and can cause T-cell death (see, e.g., Cerboni S, et al. “Intrinsic antiproliferative activity of the innate sensor STING in T lymphocytes.” J Exp Med. 2017; 214:1769-85, which is herein incorporated by reference). These discoveries indicate that, in spite of the potential benefit that modulation of STING signaling may have for cancer treatment, the use of agents that directly act upon STING risk limiting any potential benefit. Treatment with agents that modulate the activity of other proteins involved in STING signaling may be better suited to enhancing STING activity, without eliciting its immunosuppressive effects.
Cells must tightly regulate STING signaling in order to prevent improper activation of immune cells, as spontaneous activation of STING may lead to autoimmunity. The activity of factors (e.g., proteins) that promote STING activation, forward processing, and/or signaling (collectively referred to as “positive regulators”) is counterbalanced by factors (e.g., proteins) that impair STING activation, forward processing, and/or signaling (collectively referred to as “negative regulators”).
Negative regulators of STING include proteins involved in the turnover of active STING such as those involved in STING trafficking and degradation. Without wishing to be bound by theory, active STING is progressively trafficked to endosomes and modified with moieties of an 8.6 kDa protein called ubiquitin (i.e., ubiquitinated), marking it for protein degradation. In the late endosome, ubiquitinated STING recruits components of either the endosomal sorting complex required for transport-0 (ESCRT-0) complex or the ESCRT-1 complex, each of which contain ubiquitin-binding domains that bind to ubiquitinated proteins. Proteins, such as ubiquitinated STING, that are targeted by ESCRT-0 and/or ESCRT-1 are subsequently sorted into intralumenal vesicles (ILVs) in the endosomal lumen and degraded by proteases after fusion of the late endosome with lysosomes, which occurs, for example, through autophagy of late endosomes (endosomal microautophagy).
In some embodiments, a negative regulator of STING is hepatocyte growth factor-regulated tyrosine kinase substrate (HGS). Without wishing to be bound by theory, HGS (also referred to as “Hrs”) interacts with signal transducing adaptor molecule 1/2 (STAM1/2) to form the ESCRT-0 complex. HGS comprises a FYVE domain through which it interacts with the endosomal membrane, as well as a ubiquitin interacting motif through which it binds ubiquitinated proteins in the endosomal membrane (e.g., ubiquitinated STING). After binding to the endosomal membrane and ubiquitinated proteins (e.g., ubiquitinated STING), ESCRT-0 recruits the ESCRT-1 complex to initiate sorting of ubiquitinated proteins into ILVs for degradation.
The nucleotide sequence for an example human (Homo sapiens) gene encoding HGS is set forth as follows (Gene ID 9146; NM_004712.5):
The amino acid sequence for an example human (Homo sapiens) HGS protein is set forth as follows (NP_004703.1):
In some embodiments, a negative regulator of STING is vacuolar protein sorting-associated protein 37A (VPS37A). Without wishing to be bound by theory, VPS37A (also referred to as “HCRP1”, “PQBP2”, and “SPG53”) interacts with tumor susceptibility gene 101 (Tsg101), vacuolar protein sorting-associated protein 28 (Vps28), and multivesicular body sorting factor (Mvb12) to form the ESCRT-1 complex. VPS37A further interacts with ubiquitin associated protein 1 (UBAP1) as part of ESCRT-1 for binding and sequestration of ubiquitinated proteins in the endosomal membrane (e.g., ubiquitinated STING). ESCRT-1 subsequently recruits the ESCRT-II complex to ubiquitinated proteins (e.g., ubiquitinated STING), followed by the ESCRT-III complex, which together facilitate the formation of ILVs.
The nucleotide sequence for an example human (Homo sapiens) gene encoding VPS37A is set forth as follows (Gene ID 137492; NM_152415.3):
The amino acid sequence for an example human (Homo sapiens) VPS37A protein is set forth as follows (NP_689628.2):
Negative regulators of STING also include proteins involved in reducing the level of active STING in the Golgi and endosomes. Without wishing to be bound by theory, STING requires binding to cGAMP, modification with palmitoyl acid (i.e., palmitoylation), and phosphorylation to become active and induce expression of type 1 IFNs. Negative regulators of STING therefore include proteins which interfere any of these processes.
In some embodiments, a negative regulator of STING is DnaJ heat shock protein family member C13 (DNAJC13). DNAJC13 is contains a J-domain, through which it interacts with heat shock protein of 70 kDa (Hsp70) and heat shock cognate protein of 70 kDa (Hsc70), chaperone proteins that facilitate protein folding. Without wishing to be bound by theory, in the Golgi however, DNAJC13 interacts directly with STING, independently of Hsp70 and Hsc70. Through this interaction, DNAJC13 impedes palmitoylation of STING, preventing it from forming active oligomers.
The nucleotide sequence for an example human (Homo sapiens) gene encoding DNAJC13 is set forth as follows (Gene ID 23317; NM_001329126.2):
The amino acid sequence for an example human (Homo sapiens) DNAJC13 protein is set forth as follows (NP_001316055.1):
In one aspect, the present disclosure relates to the administration to a subject of an effective dose of an agent capable of inhibiting a negative regulator of STING. In some embodiments, an agent that is capable of inhibiting the activity of a negative regulator of STING inhibits the activity of the negative regulator of STING by up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 95%, up to 99%, or up to 100%. In some embodiments, an agent that is capable of inhibiting the activity of a negative regulator of STING enhances the level of STING signaling by up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 10-fold, up to 25-fold, up to 50-fold, up to 100-fold, up to 200-fold, up to 300-fold, up to 400-fold, up to 500-fold, up to 600-fold, up to 700-fold, up to 800-fold, up to 900-fold, or up to 1000-fold, as may be measured, for example, by the expression of STING, by the level of palmitoylated STING, the level of phosphorylated STING, or by the expression of one or more type 1 interferons (e.g. IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-τ, and IFN-ω).
In some embodiments, an agent that is capable of inhibiting the activity of a negative regulator of STING acts systemically. In other embodiments, an agent that is capable of inhibiting the activity of a negative regulator of STING acts only within a localized region of a subject, such as within a specific organ, tissue, or population of cells (e.g., a tumor).
In some embodiments, an agent that is capable of in inhibiting the activity of a negative regulator of STING is a small molecule inhibitor. In some embodiments, an agent that is capable of inhibiting the activity of a negative regulator of STING is a peptide inhibitor. In some embodiments, an agent that is capable of inhibiting the activity of a negative regulator of STING is a small molecule or peptide inhibitor of a component of ESCRT-1. In some embodiments, an agent that is capable of inhibiting the activity of a negative regulator of STING is a small molecule or peptide inhibitor of a component of HGS or VPS37A. In some embodiments, an agent that is capable of inhibiting the activity of a negative regulator of STING is a small molecule or peptide inhibitor of a J-domain protein. In some embodiments, an agent that is capable of inhibiting the activity of a negative regulator of STING is a small molecule or peptide inhibitor of DNAJC13.
In some embodiments, an agent capable of inhibiting the activity of a negative regulator of STING is an oligonucleotide capable of interfering with expression of one or more negative regulators of STING. In some embodiments, an agent capable of interfering with expression of one or more negative regulators of STING is a small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), or antisense oligonucleotide (ASO). In some embodiments, an agent capable of interfering with expression of one or more negative regulators of STING is an oligonucleotide capable of interfering with expression of a component of ESCRT-1. In some embodiments, an agent capable of interfering with expression of one or more negative regulators of STING is an oligonucleotide capable of interfering with expression of VPS37A, examples of which are commercially available or are otherwise well known within the art, each of which are incorporated by reference herein). In some embodiments, an agent capable of interfering with expression of one or more negative regulators of STING is an oligonucleotide capable of interfering with expression of HGS, examples of which are commercially available or are otherwise well known within the art, each of which are incorporated by reference herein). In some embodiments, an agent capable of interfering with expression of one or more negative regulators of STING is an oligonucleotide capable of interfering with expression of a J-domain protein. In some embodiments, an agent capable of interfering with expression of one or more negative regulators of STING is an oligonucleotide capable of interfering with expression of DNAJC13, examples of which are commercially available or are otherwise well known within the art, each of which are incorporated by reference herein).
In some embodiments, the agent is an oligonucleotide (e.g., siRNA, shRNA, miRNA, ASO) capable of interfering with expression of HGS that has a region of complementarity to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the agent is an oligonucleotide (e.g., siRNA, shRNA, miRNA, ASO) capable of interfering with expression of VPS37A that has a region of complementarity to the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the agent is an oligonucleotide (e.g., siRNA, shRNA, miRNA, ASO) capable of interfering with expression of DNAJC13 that has a region of complementarity to the nucleotide sequence of SEQ ID NO: 5.
In one aspect, the present disclosure relates to the administration to a subject of an effective dose of an agent that results in the inhibition of one or more negative regulators of STING (e.g., VPS37A, HGS, DNAJC13). As used herein, the term “effective dose” refers to a dose that is sufficient to bring about one or more desired biological effects within a particular subject. In some embodiments, the subject is a human.
In some embodiments, administration of an effective dose of an agent that is capable of inhibiting the activity of a negative regulator of STING inhibits the activity of the negative regulator of STING by up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 95%, up to 99%, or up to 100%, relative to the activity of the negative regulator of STING before the agent is administered. In some embodiments, administration of an effective dose of an agent that is capable of inhibiting the activity of a negative regulator of STING enhances the level of STING signaling by up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 10-fold, up to 25-fold, up to 50-fold, up to 100-fold, up to 200-fold, up to 300-fold, up to 400-fold, up to 500-fold, up to 600-fold, up to 700-fold, up to 800-fold, up to 900-fold, or up to 1000-fold, relative to the level of STING signaling before the agent is administered, as may be measured, for example, by the expression of STING, by the level of palmitoylated STING, the level of phosphorylated STING, or by the expression of one or more type 1 interferons (e.g. IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-τ, and IFN-ω)
In some embodiments, the administered agent is a small molecule or a peptide that inhibits the activity of VPS37A, HGS, and/or DNAJC13 by up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 95%, up to 99%, or up to 100%, relative to the activity before the agent is administered. In some embodiments, the administered agent is an oligonucleotide that inhibits the expression of VPS37A, HGS, and/or DNAJC13 by up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 95%, up to 99%, or up to 100%, relative to the activity before the agent is administered.
In some embodiments, the administered agent circulates in the bloodstream of the subject with a circulatory half-life of at least 10 minutes. In some embodiments, the administered agent circulates in the bloodstream of the subject with a circulatory half-life of up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours, up to 72 hours, or more than 72 hours.
In some embodiments, the agent is administered intravenously.
In some embodiments, the subject is a human patient. In some embodiments, the subject is a human patient that has, is suspected of having, or is at risk of having a disease which may be treated by eliciting or enhancing an immune response (e.g., an innate immune response). In some embodiments, the subject is a human patient that has, is suspected of having, or is at risk of having an infection caused by a pathogen (e.g., a virus, a bacterium, or a parasite). In some embodiments, the subject is a human patient that has, is suspected of having, or is at risk of having cancer.
In some embodiments, the administered agent is administered in the presence of one or more carriers or excipients. In some embodiments, the administered agent is administered in the presence of one or more agents for the treatment of cancer (e.g., a chemotherapeutic, an immunotherapeutic). In some embodiments, the administered agent is administered prior to the administration of one or more agents for the treatment of cancer (e.g., a chemotherapeutic, an immunotherapeutic). In some embodiments, the administered agent is administered following the administration of one or more agents for the treatment of cancer (e.g., a chemotherapeutic, an immunotherapeutic). In some embodiments, the administered agent is administered prior to, concurrently with, or following another therapy for cancer (e.g., radiotherapy, surgical intervention).
The agents and compositions of the disclosure may be administered to patients by any number of different routes, including enteral or parenteral routes. Enteral administration includes administration by the following routes: oral, sublingual, and rectal routes. Parenteral administration includes administration by the following routes: intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraocular, transepithelial, intraperitoneal, topical (including dermal, ocular, rectal, nasal, inhalation and aerosol (i.e., pulmonary)), and rectal systemic routes.
Administration can be performed e.g., by injection, or ballistically using a delivery gun to accelerate their transdermal passage through the outer layer of the epidermis. The nanoparticles may also be delivered in aerosols. This is made possible by the small size of the nanoparticles.
In another aspect, the present disclosure relates to pharmaceutical compositions for inhibiting the activity of one or more negative regulators of STING in cells of a subject. In some embodiments, the contemplated pharmaceutical compositions comprise one or more agents capable of inhibiting the activity of one or more negative regulators of STING, e.g., HGS, VPS38A, and/or DNAJC13.
The contemplated pharmaceutical compositions may be in any form suitable for administration to a subject, e.g., a liquid composition, a solid composition, a gel composition, or aerosolized compositions thereof. Other compositions are also contemplated, including those that may delivered by transdermal patches, emulsions, foams, granules, implants, pellets, pills, sprays, suppositories, suspensions, tablets, and the like, so long as agent(s) may be delivered and increase the concentration of phagocytosed DNA in phagocytes of one or more biological tissues or liquids. Such compositions will generally comprise a carrier of some sort, for example a solid carrier or a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may also be included. Such compositions and preparations generally contain at least 0.1 wt % of the active agent. Such formulations are well known in the art.
In some embodiments, the contemplated pharmaceutical compositions may comprise one or more of a pharmaceutically acceptable excipients, carriers, buffers, stabilizers, delivery agents, isotonicizing agents, preservatives or antioxidants, or other materials well known to those skilled in the art, in addition to one or more agents for increasing the concentration of tumor-derived DNA in a phagocyte, e.g., a macrophage. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the excipients, carriers, buffers, stabilizers, delivery agents, isotonicizing agents, preservatives or antioxidants, or other materials may depend on the route of administration, e.g., intravenously, orally, topically, or through inhalation.
For administration through inhalation, the contemplated pharmaceutical composition will be in the form of a liquid or dry powder that is capable of being aerosolized, e.g., by an inhalation device for oral and/or nasal inhalation for delivery to the lungs. In embodiments where the pharmaceutical composition is a liquid that can be aerosolized, it will be in the form of an acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability.
For intravenous, cutaneous, or subcutaneous injection, the contemplated pharmaceutical composition will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity, and stability. Those of relevant skill in the art are knowledgeable in means of preparing suitable solutions using, for example, solutions of containing the active agent(s) in, e.g., physiological saline, a dispersion prepared with glycerol, liquid polyethylene glycol, or oils.
In embodiments where the contemplated pharmaceutical composition is a liquid, the composition may be formulated to have a pH between about 3.0 and 9.0, or preferably between about 4.5 and 8.5. Ideally, a liquid pharmaceutical composition has a pH between about 5.0 and 8.0. The pH of a composition can be maintained by the use of a buffer such as acetate, citrate, phosphate, succinate, Tris or histidine, typically employed in the range from about 1 mM to 50 mM. The pH of compositions can otherwise be adjusted by using physiologically acceptable acids or bases.
The pharmaceutical compositions contemplated herein may also comprise preservatives. Preservatives are generally included in pharmaceutical compositions to retard microbial growth, thereby extending the shelf life of the compositions and allowing multiple use packaging. Examples of preservatives include phenol, meta-cresol, benzyl alcohol, parahydroxybenzoic acid and its esters, methyl paraben, propyl paraben, benzalconium chloride and benzethonium chloride. Preservatives are typically employed in the range of about 0.1 to 1.0% (w/v).
Compositions containing one or more agents for inhibiting the activity of one or more negative regulators of STING may also comprise one or more agents for the treatment of cancer.
Compositions containing one or more agents for inhibiting the activity of one or more negative regulators of STING are preferably administered to a subject in a sufficiently effective amount (i.e., for achieving an increase the concentration of phagocytosed DNA within phagocytes of the subject). In some embodiments, compositions containing one or more agents for inhibiting the activity of one or more negative regulators of STING are administered to a subject in a sufficiently effective amount concurrently, after, or prior to the administration of a sufficiently effective amount of one or more agents for the treatment of cancer. Examples of the techniques and protocols relevant for establishing the effective amount of a pharmaceutical composition can be found in Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA); Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994).
Intracellular DNA is a potent activator of innate immune responses via Stimulator of Interferon Genes (STING) (Ishikawa et al., 2009), which acts as an adaptor for cyclic GMP-AMP (cGAMP) after its generation by the DNA sensor by cyclic GMP-AMP (cGAMP) Synthase (cGAS) (Ablasser et al., 2013; Gao et al., 2013; Sun et al., 2013; Wu et al., 2013). In addition to the eukaryotic 2′3′-linked cGAMP, STING is also a sensor for 3′3′-linked cyclic-dinucleotides (cdNs) of bacterial origin (Burdette et al., 2011; Woodward et al., 2010). STING binding to its ligands induces both type I interferon (IFN) responses and autophagy, an ancestral and conserved activity important for clearance of intracellular pathogens (Gui et al., 2019). Activation of innate immune pathways by STING is highly conserved from metazoans to bacteria (Kranzusch et al., 2015; Morehouse et al., 2020). In addition to pathogen responses, the cGAS/STING axis is essential for antitumor immune responses, immune checkpoint therapy, development of autoimmune diseases and induction of cellular senescence (Motwani et al., 2019).
Homodimeric STING is localized at the endoplasmic reticulum (ER) (Ishikawa and Barber, 2008) and undergoes a cGAMP-dependent conformational switch that triggers its exit from the ER and trafficking to the Golgi. STING palmitoylation is required for STING clustering and activation of type I IFN responses via Tank Binding Kinase 1 (TBK1) at the Trans-Golgi Network (TGN) (Mukai et al., 2016). After TBK1 phosphorylates STING at residue S366, phospho-STING forms a platform at the carboxy-terminal tail (CTT) for recruitment of IRF3 (Liu et al., 2015a). IRF3 is then phosphorylated by TBK1, homodimerizes and translocates to the nucleus resulting in activation of a type I IFN response. In addition to type I IFN induction, upon CDN ligation, STING induces autophagy independently of the classical macroautophagy machinery but dependent on ATG16L1, an ancestral and conserved function of the response (Fischer et al., 2020; Gui et al., 2019). Thus, STING intracellular trafficking and signaling activities are tightly connected.
Recently, progress has been made in characterizing the pathways regulating STING trafficking and degradation. ER-to-Golgi STING translocation is facilitated by coatomer protein complex II (COPII) vesicles that rely on STING association with SEC24C (Gui et al., 2019). Other proteins have been shown to regulate this trafficking step, such as STEEP, or to stabilize STING on the ER membrane, such as STIM1 or TOLLIP (Pokataycv et al., 2020; Srikanth et al., 2019; Zhang et al., 2020). STING can also traffic from the ER to LC3B positive vesicles, although the mechanisms of this step are less clear (Gui et al., 2019). Interestingly, while proteins of the autophagy machinery have been shown to regulate STING signaling, their impact on STING degradation is ambiguous. Knockout of ATG5, ATG9A, ATG16L1 or ULK1/2 do not seem to regulate STING degradation, while ATG3 has been both shown to regulate STING degradation in human monocytic cells or to be dispensable in mouse embryonic fibroblasts (Fischer et al., 2020; Gonugunta et al., 2017; Gui et al., 2019; Prabakaran et al., 2018; Saitoh et al., 2009). Post-Golgi trafficking to the endolysosomal compartment is essential for STING degradation and signaling shutdown, but the proteins governing these processes and the signals triggering STING degradation require further investigation (Ablasser and Hur, 2020; Gonugunta et al., 2017). Overall, the hierarchy of STING interactions with the intracellular trafficking and autophagy machineries and how these pathways are integrated to regulate STING degradation and signaling shutdown remain to be clarified. Finally, the physiological role of trafficking of this innate immune sensor to distinct intracellular compartments remains unknown.
Presented herein is a systems approach to systematically and unbiasedly identify genes involved in STING cell biology and to uncover the physiological significance of these intracellular trafficking pathways. Based on proximity-ligation proteomics, a time-resolved map of STING trafficking and potential interactors in distinct intracellular compartments was created. Combining proteomics with a genome-wide CRISPR screen, an endosomal sorting complex required for trafficking (ESCRT) complex was identified including Hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), Vacuolar Protein Sorting 37A (VPS37A) and Ubiquitin-associated protein 1 (UBAP1) as required for STING degradation and as a signal coordinator between STING trafficking to the endosome and autophagy resolution at this organelle. Similar to the phosphorylation of STING that creates a platform for IRF3 recruitment and type I IFN signaling at the TGN, it is shown that ubiquitination of STING creates a platform for ESCRT recruitment at the late endosome, therefore identifying a role for this post-translational modification in regulating STING post-Golgi trafficking. By using a targeted library for ubiquitin and autophagy related proteins, a role for Ubiquitin-conjugating enzyme E2 N (UBE2N) was also identified in STING degradation and its association with ESCRT. Ubiquitination mediated association of STING with ESCRT at the endosome drives autophagy resolution through phagophore closure and amphisome formation and is responsible for STING degradation, therefore clarifying how autophagy and STING trafficking to endosomes are integrated. Finally, it is shown that disruption of ESCRT function through a human pathogenic Ubiquitin-Associated Protein 1 (UBAP1) mutant leads to constitutive STING dependent type I IFN signaling in fibroblasts and monocyte derived dendritic cells at steady-state with an intracellular post-Golgi accumulation of activated STING. Based on these findings, a revised model of STING trafficking was proposed, in which the sensor is subject to a homeostatic degradative flux. Physiologically, ESCRT-dependent STING removal at the endosome acts as a gatekeeper to curtail spontaneous induction of STING activation at steady state and control the magnitude of STING responses to ligands.
Identification of Subcellular Compartments and Proteins in Proximity to STING During a Time Course of cGAMP Stimulation
To establish the hierarchy of molecular mechanisms governing STING trafficking and to identify proteins that interact with STING during this process, recently developed proximity ligation technologies that overcome the limitations of classical co-immunoprecipitation in identifying native interactions were used. STING was fused to the biotin ligase TurboID (
To map protein-protein interactions, proximity labeling was performed followed by quantitative mass spectrometry analysis. STING-TurboID expressing cells were stimulated with cGAMP, then were biotin-labeled for 30 minutes at different time points post-stimulation (
Out of the ˜2000 proteins identified, statistical analysis led to filtering the dataset down to a network of 132 proteins that were differentially biotinylated between time-points. The inferred localization and trafficking pattern of this network of proteins was consistent with the analyses performed on the full dataset (
This dataset provides a time-resolved map of potential STING interactors and the basis to identify the hierarchy of events involved in STING trafficking and degradation.
To separately identify proteins required for STING trafficking and degradation, a genome wide CRISPR screen was performed based on activity of a STING protein reporter that undergoes degradation. A system was optimized to follow STING degradation with flow cytometry by fusing STING to the bright green fluorescent protein mNeonGreen (mNG) under control of a weak promoter. Stimulation of 293T cells stably expressing the reporter with the stable cGAMP analog 2′3′-cGAMP(pS)2 led to a decrease in STING-mNG signal (
To facilitate the CRISPR screen, a cell line stably expressing spCas9 and STING-mNG (
To determine which screen hits likely interact with STING, significant hits from the CRISPR screen were intersected with the full dataset of STING-TurboID-labelled proteins (
When intersected with the filtered 132 proteins from the TurboID dataset, only 2 genes were identified, HGS and VPS37A, which are components of the ESCRT machinery (
The ESCRT machinery is involved in many cellular processes that entail inverse membrane involution and formation of vesicles. Related to intracellular protein trafficking, ESCRT has been characterized to be required for intraluminal vesicles (ILVs) formation at late endosomes and more recently for resolution of particular forms of autophagy (Victri et al., 2020a). In addition to HGS (ESCRT-0) and VPS37A (ESCRT-I), the proteomics dataset identified other components of the ESCRT machinery: STAM (ESCRT-0), two subunits of the ESCRT-I heterotetrameric complex, TSG101 and UBAP1, and the Bro1 domain protein PTPN23 which has been shown to bridge ESCRT-I to ESCRT-III (Christ et al., 2017) (
Attention was focused on HGS and VPS37A because they were identified at the intersection of the proteomics and genetics data. When knocked out with two independent sgRNAs per gene, KO STING-mNG reporter cells showed a reduction in STING degradation, confirming the screen results (
Since STING functions are dependent on its intracellular trafficking and degradation, it was asked if HGS and VPS37A KO would impact STING signaling. CRISPR KO human primary fibroblasts (BJ1) and monocytic U937 cell lines, with two independent sgRNAs per gene, were generated. KO of HGS or VPS37A increased STING signaling in the two cell types for both genes (
Taken together, these data suggest that in addition to being required for STING degradation, ESCRT is also responsible for STING signaling shutdown
Interestingly, HGS and VPS37A KO in fibroblasts not only blocked STING degradation but also increased lipidated LC3B levels, an autophagy marker that accumulates in cells with defective resolution of autophagy (
The co-localization of STING and ESCRT with the late endosomal marker CD63 and the autophagy markers p62 and LC3B prompted further investigation to determine if STING could be degraded through endosomal microautophagy, a process that involves selective autophagy of late endosomal cargo. Endosomal microautophagy requires acidification of lysosomes for selective degradation of cargo, shows co-localization of selective autophagy receptors with late endosomal markers, and requires the ESCRT subunits VPS4A/B for resolution (Mejlvang et al., 2018; Tekirdag and Cuervo, 2018). STING degradation was already shown to be dependent on lysosomal acidification, and the screen confirmed this result (
The co-localization of STING and ESCRT in proximity of the late endosomal marker CD63 and the autophagy markers p62 and LC3B prompted the thought that if resolution of STING-dependent autophagy could happen at the endosome in the process of amphisome formation. Amphisomes are hybrid degradative organelles resulting from the fusion of autophagosomes and endosomes. Therefore, STING expressing cells were stained for p62 and CD63 (
To test if STING degradation was VPS4A/B dependent, the VPS4A E228Q dominant negative (DN) mutant fused to mScarlet was overexpressed in a STING-HA reporter cell line (VPS4A DN was used in lieu of genetic deletion to block both VPS4A and VPS4B since they have redundant functions). When stimulated with cGAMP, there were comparable levels of STING degradation between non transfected cells and cells transfected with a control plasmid (
Taken together, these data suggest that activated STING is sorted to endosomes where its degradation is coordinated with autophagy resolution through ESCRT mediated phagophore sealing on this organelle.
ESCRT-0 and ESCRT-I associate with ubiquitinated cargo at the endosome for formation of ILVs. HGS and VPS37A both contain a ubiquitin binding motif (Vietri et al., 2020a). It was asked if STING was ubiquitinated upon cGAMP stimulation and if ubiquitination could drive its association with ESCRT. To block ubiquitination of STING, the Ubiquitin Activating enzyme 1 (UBA1) inhibitor MLN7243 was used (Hyer et al., 2018). MLN7243 blocked STING ubiquitination upon cGAMP stimulation (
It was predicted that ubiquitination at multiple lysine residues would drive STING degradation, since HGS contains 3 ubiquitin binding sites (Vietri et al., 2020a). When lysine conservation was examined in human STING, K20, K150, K236, K289, K338 and K370 were identified as highly conserved, among 9 lysine residues (
Since ubiquitination is driving STING association with ESCRT, it was asked if the UBA1 inhibitor MLN7243 would recapitulate HGS and VPS37A KO effects on STING signaling, degradation and autophagy resolution. Indeed, treatment with MLN7243 abrogated STING degradation in fibroblasts, increased phospho-STING signaling and also induced accumulation of lipidated LC3B (
Next, the impact of UBA1 inhibition on STING subcellular localization and its co-localization with the late endosome marker CD63, autophagy markers p62 and LC3B and ESCRT was identified. When treated with MLN7243, HGS foci co-localizing with STING were completely lost (
Taken altogether, analogous to phosphorylation that creates a platform at the TGN for type I IFN signaling, ubiquitinated STING traffics to and decorates the endosome creating an organizing platform that coordinates STING degradation and autophagy resolution through ESCRT. ESCRT drives phagophore sealing and fusion with the endosome and consequent autophagy of endosomal content. Since VPS37A has been shown to be required for phagophore closure, and ubiquitinated STING assembles a specific VPS37A containing ESCRT complex, VPS37A acts as a bridge between endosomal sorting of STING and phagophore closure on this organelle.
Mutations in UBA1 have been recently identified in patients, leading to an autoinflammatory disease named Vacuoles E1 enzyme X-linked Autoinflammatory Somatic (VEXAS) syndrome (Beck et al., 2020). VEXAS syndrome patients express a de novo hypomorphic UBA1 isoform in the myeloid compartment. Since these patients show type I IFN and proinflammatory cytokine production at steady state, it was wondered if UBA1 inhibition in CD14+ monocytes could lead to an increase in STING signaling. After cGAMP stimulation, CD14+ monocytes from healthy donors showed a strong increase in phospho-STING levels when UBA1 was inhibited by MLN7243 treatment (
To specifically identify ubiquitin related genes involved in STING degradation, a targeted CRISPR screen was performed in both the STING-mNG and STING-HA reporter cell lines. The targeted library contained guides targeting 669 E3 and adaptors (compiled from Medvar B et al and Li W et al), 40 E2 from Interpro, 7 E1, 28 Autophagy core proteins and 10 positive controls from the genome-wide CRISPR screen. While the STING-mNeonGreen performed better than the STING-HA screen, both recovered positive controls as required for STING degradation (SEC24C, ATP6V1G1, ATP6V0C, HGS, VPS37A, UBAP1) and STING as the top depleted gene (
UBE2N is responsible for K63 polyubiquitination of proteins that prompts them for degradation through the endolysosomal compartment and has been shown to play a regulatory role in multiple innate immune sensing pathways. Consistent with a possible role for UBE2N in STING biology, STING has been shown in multiple studies to be K63 polyubiquitinated upon activation. To validate the results of the screens, UBE2N was knocked out with two individual sgRNAs in 293T STING-mNG and stimulated the cells with cGAMP. UBE2N KO cells showed reduced STING degradation (
Next, proteins in the dataset were investigated to determine whether proteins in the dataset have established roles in human disease, potentially suggesting altered STING signaling as part of the underlying pathophysiology. Mutations in the ESCRT-I subunits VPS37A and UBAP1 have been shown to induce the neurodegenerative disease Hereditary Spastic Paraplegia (HSP) (Farazi Fard et al., 2019; Gu et al., 2020; Lin et al., 2019; Zivony-Elboum et al., 2012). Interestingly, a subset of patients with mutations in the Aicardi-Goutiére Syndrome genes ADAR1, IFIH1 and RNASEH2B, that present with constitutive induction of type I IFN, have been shown to develop HSP (Crow et al., 2014). Since UBAP1 is an endosome specific ESCRT subunit that does not play a role in cytokinetic abscission, it was asked if disease causing mutations in this gene could lead to dysfunctional STING degradation and exacerbate STING dependent signaling. Consistent with the mass spectrometry (
Stable UBAP1DN expressing fibroblasts were generated and stimulated them with cGAMP. UBAP1DN expressing fibroblasts showed a marked increase in STING signaling, reduction in STING degradation and lack of autophagy resolution shown by accumulation of lipidated LC3B (
Mutations in genes regulating STING trafficking, specifically COPA, have been recently shown to induce STING dependent spontaneous type I IFN production due to accumulation of STING at the Golgi (Deng et al., 2020; Lepelley et al., 2020; Mukai et al., 2020; Steiner et al., 2020; Uematsu et al., 2020). Similar to COPA, it was reasoned that activation of constitutive type I IFN in UBAP1DN expressing cells could be the result of impaired homeostatic STING trafficking, specifically post-Golgi, leading to intracellular accumulation of activated STING. Consistent with this hypothesis, UBAP1DN expressing fibroblast showed accumulation of phospho-STING that co-localized partially with the Golgi marker GM130 and the early endosome marker EEA1 (
To test if induction of ISGs in UBAP1DN expressing fibroblasts was due to accumulation of activated STING, CRISPR KO was used. When STING was deleted, UBAP1DN expressing fibroblasts showed a drastic reduction of MX1 induction (
cGAS has been shown to induce tonic ISG transcription at steady-state in vitro and in vivo, regardless of presence of exogenous DNA, suggesting that cGAS produces a low level of cGAMP in cells to prime this response. To test if cGAS was required for priming STING trafficking at steady state and contributed to the phenotype shown by pathogenic UBAP1 expressing cells, cGAS was knocked out in UBAP1DN expressing fibroblasts. cGAS KO reduced induction of the ISG MX1, suggesting that cGAMP production at steady state is responsible for priming the constitutive flux of STING trafficking (
Taken together, these data suggest that cells expressing a pathogenic mutant of UBAP1 have a heightened response to cGAMP through impaired ESCRT mediated STING degradation and autophagy resolution. Additionally, mutant UBAP1 leads to intracellular accumulation of post-Golgi activated STING with consequent STING-dependent constitutive type I IFN activation.
Patients with COPA mutations show accumulation of STING at the Golgi at steady state with consequent constitutive STING activation (Lepelley et al., 2020). While it has been hypothesized that COP-I acts in recycling STING from the Golgi to the ER at steady state, COP-I has been also shown to be required for proper early endosome function and autophagy maturation from the endosome (Popoff et al., 2011; Razi et al., 2009). Loss of COP-I leads to defects in endosomal trafficking and inhibition of autophagosome maturation at the endosome with consequent accumulation of ubiquitinated proteins, including p62 (Razi et al., 2009). COP-I acts on STING sorting with the adaptor SURF4 (Deng et al., 2020), identified in both the proteomics and CRISPR screen as required for STING degradation (
Therefore, it is proposed that cGAS primes constant trafficking of STING between the ER and the endosome for degradation at steady state, and functionality of an endosome specific VPS37A/UBAP1 containing ESCRT complex prevents STING accumulation with consequent spontaneous activation of downstream signaling. Mutations in genes regulating STING trafficking could represent a general disease sensitizing mechanism leading to either a lowered threshold for STING activation or directly inducing STING dependent responses via disruption of the homeostatic STING degradative flux.
While some of the factors regulating STING exit from the ER have been characterized, the signals and genes regulating STING post-Golgi trafficking remained to be identified. To address this gap and unbiasedly identify genes involved in STING trafficking, a time-resolved map of STING neighboring proteins was generated at different intracellular compartments post cGAMP activation and a genome-wide CRISPR screen for regulators of STING degradation was carried out, providing a basis for the studies presented herein as well as resource for the field. By focusing on mechanisms of STING post-Golgi trafficking, ubiquitin was identified as the post-Golgi signal regulating STING degradation and signaling shutdown via association with an endosome-specific VPS37A/UBAP1-containing ESCRT complex. ESCRT association to ubiquitinated STING induces coordinated degradation of the sensor and resolution of autophagy in the endosomal compartment. A targeted CRISPR screen identified UBE2N as a driver of STING association with VPS37A, therefore regulating STING degradation and signaling shutdown. Similar to the signaling platform created at the TGN by phosphorylated STING for activation of IRF3 and induction of type I IFN, ubiquitinated STING in the endosomal compartment creates a platform for ESCRT recruitment driving both STING degradation and autophagy resolution (
It is becoming increasingly clear that defects in genes regulating STING trafficking can lead to human disease. Mutations in COPA lead to STING accumulation in the Golgi with spontaneous activation of the sensor. Similarly, mutations in STIM1 lead to spontaneous type I IFN activation in patients' peripheral blood mononuclear cells by releasing an ER retention signal for STING. Moreover, NPC1 deficiency has also been linked to constitutive STING activation through blockage of SREBP induced STING flux to the lysosome. It is shown that expression of a UBAP1 mutant found in patients with hereditary spastic paraplegia leads to post-Golgi accumulation of activated STING with consequent constitutive induction of type I IFN. Based on all this evidence, proposed herein is an updated model of STING trafficking in which tonic cGAMP production primes a basal flux of STING trafficking from the ER to the lysosome with consequent constant degradation. Inactivating mutations in genes controlling STING trafficking and degradation represent a generalized mechanism for inducing constitutive STING-dependent responses that could lead to disease. A similar mechanism blocking steady-state TLR7 activation has been characterized, by which UNC93B1-Syntenin1 mediated trafficking of the receptor to MVBs is required for reduction of TLR7 intracellular levels and blunting of signaling at steady state. STING is highly conserved and it is shown that accumulation of STING in the endosomal compartment is sufficient to induce STING responses. It is tempting to speculate that an ancestral function of STING in anti-pathogen defense could be sensing of perturbation of intracellular trafficking pathways due to pathogen invasion. Interestingly, STING has been shown to be activated by HSV, influenza and HIV-1 viral entry independently of nucleic acids.
Activation of exacerbated type I IFN responses has been shown to drive neuronal cell death in both cell intrinsic and extrinsic manners. Post-Golgi accumulation and constitutive type I IFN activation in cells expressing the UBAP1 mutant responsible for the development of spastic paraplegia opens the possibility that STING is involved in neurodegeneration in these patients. In addition to UBAP1, mutations in the ESCRT subunit VPS37A and the vesicular trafficking regulator AP4M1, identified in the proteomics, have also lead to HSP and been identified in patients with spastic paraplegia (Abou Jamra et al., 2011; Zivony-Elboum et al., 2012). Moreover, mutations in the ATPase domain of the ESCRT subunit VPS4A lead to a multisystem disease with abnormal neurodevelopment. As for UBAP1, it is plausible that mutations in these genes could lead to spontaneous STING dependent type I IFN activation. How defects in genes regulating STING trafficking might lead to neurodegeneration remain to be explored. Based on our model, mechanisms for disease development could include lowering the threshold with consequent increase in the magnitude of STING responses, or inducing STING-dependent type I IFN production at steady state by impairment of the homeostatic STING flux.
293T (CRL-3216), hTert-BJ1 (BJ-5ta-CRL-4001) and U-937 (CRL-1593.2) were from ATCC. 293T were cultured in DMEM (Corning) supplemented with 10% FBS (VWR), 1× GlutaMax (Thermo Fisher) and 1× Penicillin/Streptomycin (Corning). hTert-BJ1 were cultured in a 4:1 mixture of DMEM (Corning): Medium 199 (Lonza) supplemented with 10% FBS (VWR), 1× GlutaMax (Thermo Fisher) and 1× Penicillin/Streptomycin (Corning). U-937 were cultured in RPMI (Thermo Fisher) supplemented with 10% FBS (VWR), 1× GlutaMax (Thermo Fisher) and 1× Penicillin/Streptomycin (Corning).
CD14+ monocytes were isolated from peripheral adult human blood as previously described 1. CD14+ monocytes were cultured in RPMI (Gibco) supplemented with 10% FBS (VWR), 1× GlutaMax (Thermo Fisher), 50 μg/mL Gentamicin (Thermo Fisher) and 1× Penicillin/Streptomycin (Corning). Human Monocyte Derived Dendritic Cells were differentiated as described in Gentili et al., 20192. Medium was replaced 1 days after transfection and cells were selected with 2 μg/mL Puromycin (Invivogen). Cells were replated at 0.15×10{circumflex over ( )}6c/w in a 96 well plate 5 days post differentiation, rested for 1 hour in the incubator and then stimulated with direct addition of cGAMP in the medium or with pI:C complexed with Lipofectamine.
The plasmids psPAX2 (#12260) and pCMV-VSV-G (#8454) were from Addgene. pSIV3+ was previously described. pTRIP-hPGK-Blast-2A was cloned from pTRIP-CMV-STING-GFP (kind gift of Nicolas Manel) by Gibson assembly of PCR amplified hPGK from pCW57-MCS1-2A-MCS2 (Addgene #71782) and a gBlock (IDT) for Blasticidin resistance. pTRIP-UbC-Blast-2A-STING-mNconGreen was cloned from Gibson assembly of PCR amplified UbC promoter from FUGW, PCR amplified Blast, PCR amplified STING and a gBlock for humanized mNconGreen described in (Tanida-Miyake et al., 2018). pTRIP-hPGK-STING-TurboID was cloned by Gibson assembly of PCR amplified TurboID from V5-TurboID-NES_pCDNA3 (Addgene #107169) and STING from pTRIP-CMV-STING-GFP. pTRIP-hPGK-Blast-2A-STING-HA/STING V155M-HA/STING R284S-HA/were cloned by Gibson assembly from PCR amplification or PCR mutagenesis. pXPR101-Hygro was cloned by Gibson assembly of a gBlock for Hygromycin resistance into pXPR101 (kind gift of Broad GPP). CROPseq-guide-Puro was a kind gift of Paul Blaincy. pXPR_BRD023 (lentiCRISPR v2) was a kind gift of the Broad GPP platform. sgRNAs were cloned by gateway cloning into the respective vectors of annealed primers. pTRIP-SFFV-mNeonGreen and pTRIP-SFFV-Blast-2A-STING-mNeonGreen were obtained by Gibson assembly. pTRIP-SFFV-Hygro-2A-mScarlet, pTRIP-SFFV-Hygro-2A-mScarlet-HGS and pTRIP-SFFV-Hygro-2A-mScarlet-VPS37A were obtained by Gibson assembly of PCR amplified Hygro from pXPR101-Hygro, PCR amplified HGS or VPS37A from U937 cDNA and PCR amplified mScarlet from pmScarlet_C1 (Addgene #85042) into pTRIP-SFFV-EGFP-NLS (Addgene #86677). pTRIP-SFFV-Hygro-2A-mScarlet-VPS4A E228Q was obtained by Gibson assembly of PCR amplified VPS4A E228Q from pEGFP-VPS4-E228Q (Addgene #80351). pTRIP-SFFV-Hygro-2A-mScarlet-UBAP1DN and pTRIP-SFFV-Puro-2A-mScarlet-UBAP1DN (truncated mutant at residue 97-mutation G98X) were obtained by Gibson assembly of a gBlock for UBAP1DN. pTRIP-hPGK-Hygro-2A-FLAG-Ubiquitin was obtained by Gibson assembly of PCR amplified Hygro from pXPR101-Hygro and a gBlock for FLAG-Ubiquitin. pTRIP-hPGK-Blast-2A-TMEM192-STING-HA was obtained by TMEM192 PCR amplification from 293T cDNA, PCR amplification of STING (aa139-379) and Gibson assembly.
Lentiviruses were produced as described in (Gentili et al., 2019). Briefly, 0.8 million/well 293T in a 6 well plate were transfected with 1 μg psPAX, 0.4 μg pCMV-VSV-G and 1.6 μg of viral genomic DNA with TransIT-293 (Mirus) and left O/N. To generate SIV-VLPs, cells were transfected with 2.6 μg pSIV3+ and 0.4 μg pCMV-VSV-G. Medium was then changed to 3 mL of fresh medium corresponding to the cell line to be transduced. Supernatants were harvested 30-34 hours after medium changed and filtered at 0.45 μm. 0.5 million 293T, hTert-BJ1 or U937 were infected with 2 mL of fresh virus in presence of 8 μg/mL Protamine (Millipore Sigma). To generate transduced MDDCs, 2×10{circumflex over ( )}6 freshly isolated CD14+ monocytes were transduced in a 6 well plate with 1 mL of lentivirus and 1 mL of SIV-VLPs with 8 μg/mL protamine.
Generation of cells and stimulation. 293T cells were transduced with pTRIP-hPGK-Blast-2A-STING-TurboID and selected with 15 μg/mL Blasticidin (Invivogen) for one week. To test the construct via pull-down, 0.8 million cells were seeded in a 6 well plate. The following day, cells were permeabilized with 300 μl/well of cGAMP permeabilization buffer [50 mM HEPES (Corning), 100 mM KCl (Thermo Fisher), 3 mM MgCl2 (Thermo Fisher), 0.1 mM DTT (Thermo fisher), 85 mM Sucrose (Thermo Fisher), 0.5 mM ATP (Cayman chemicals), 0.1 mM GTP (Cayman Chemicals), 0.2% BSA (Seracare), 0.001% Digitonin (Promega)| containing 1 μg/mL 2′3′-cGAMP (Invivogen) or water for 10 minutes at 37° C., washed with 3 mL of warm medium and then medium was replaced. For mass-spec, 6 million cells were seeded in a 10 cm dish for each condition. Cells were stimulated with 2.8 mL of cGAMP permeabilization buffer containing 6 μg total of cGAMP per dish. Cells were left stimulating for the desired times and 500 μM biotin (Cayman chemicals) was added in each well 30 minutes prior to harvest. Cells were harvested by trypsinization, washed 3 times in cold PBS and pellets were frozen until processing. For experiments in 6 well plates, 3 wells per condition were harvested. For experiments in 10 cm dishes, one dish per condition was harvested.
Pull-down. Cells were lysed on ice in 550 μL (6 well plates) or 1 mL (10 cm dishes) of RIPA buffer (Boston Bioproducts) in presence of complete, Mini, EDTA-free Protease Inhibitor Cocktail (Millipore Sigma) and PhosSTOP (Millipore Sigma) for 10 minutes. Lysates were cleared by centrifugation at 16000 g for 10 minutes at 4° C. 10% of the lysed cells was set aside as input. set aside as input. Pull-down and washes were performed as in (Branon et al., 2018). Briefly, lysates were mixed with Pierce Streptavidin Magnetic Beads (Thermo Fisher) at a ratio of 100 μl beads/4 million cells. Lysates were incubated with beads with constant rotation for one hour at room temperature and then overnight (O/N) at 4° C. Beads were then applied to a magnet and subjected to the following washes: 2 times with 1 mL of RIPA, 1 time with 1M 1 mL of KCl (Thermo Fisher), 1 time with 1 mL of 0.1M Na2CO3 (VWR), 1 time with 1 mL of freshly prepared 2M Urea (VWR) resuspended in 10 mM Tris-HCl pH 8.0 (Thermo Fisher) and 2 times with RIPA. Proteins were eluted from beads by adding 150 μl (6 well plates) or 500 μl (10 cm dish) of non-reducing Laemmli (Boston bioproducts) containing 20 mM DTT (Thermo Fisher) and 2 mM biotin (Cayman chemicals) and boiled for 20 minutes. Input was diluted with 2× sample buffer (Sigma). For mass spectrometry, beads were processed as follows.
Sample processing for mass spectrometry. Co-IP was performed using 2.2 mg of HEK293T cells expressing hPGK-Blasticidin-P2A-STING TurboID, 200 μL of Streptavidin beads, in duplicates, at 5 time points: not-stimulated, 30 minutes, 1, 2 and 6 hours.
Samples were received in duplicates, each in 1 mL RIPA lysis buffer. Beads were washed with 50 mM Tris HCL (200 μL, pH 7.5, 2×) and transferred to fresh 1.5 mL Eppendorf tubes. Beads were further washed with 2 M Urea/50 mM Tris HCL (200 μL, pH 7.5, 2×). Proteins were digested with trypsin (5 μg/mL, 80 μL) in 2 M urea/50 mM Tris HCL/1 mM dithiothreitol (DTT)) at 25° C. for 1 hr). Following a brief centrifugation step using a table-top centrifuge (5-10 seconds), supernatants were transferred to clean 1.5 mL Eppendorf tubes. Beads were washed once with 2 M urea/50 mM Tris HCL (60 μL, pH 7.5, 2×) and supernatants were combined with respective supernatants from the first centrifugation step. Combined supernatants were centrifuged at 5000 g for 30 s to pellet remaining beads and the supernatants were transferred to clean 1.5 mL Eppendorf tubes.
Samples were reduced with DTT (4 mM) using a shaker (1000 rpm) for 30 minutes at 25° C.) and alkylated with iodoacetamide (IAA, 10 mM) for 45 minutes at 25° C. in the dark. Proteins were digested overnight with trypsin (0.5 μg in trypsin buffer) at 25° C. using a shaker (700 rpm). Samples were acidified with formic acid (FA, 1%, 200 μL pH<3) and peptides were desalted using C18 stage tips (2 punches) following standard protocol (Mertins et al., 2018). Briefly, stage tips were activated with 50% ACN, 0.1% FA (50 μL, 1500 rcf) and conditioned with 0.1% FA (50 μL, 1500 rcf, 2×). Samples (350 μL) were loaded on the tips and spun at 1500 rcf until all volume flowed through completely without drying the stage tips. Samples were washed with 0.1% FA (50 μL, 2×, 1500 rcf), eluted with 50% ACN/0.1% FA (50 μL, 1500 rcf) and lyophilized. Peptides were subsequently reconstituted in fresh HEPES (50 mM, 95.3 μL, pH 7.5) for TMT labeling. Samples were labeled with TMT as follows: Non-Stimulated (126, 129N), 0.5 hr (127N, 129C), 1 h (127C, 130N), 2 h (128N, 130C), 6 hr (128C, 131). TMT labeling (240 μg per sample) occurred for 1 hr at room temperature with shaking (800 rpm), following standard protocol. Samples were quenched with 5% hydroxylamine (8 μL, 20° C., 700 rpm), all channels were combined in one vial and lyophilized. The combined samples were reconstituted in 0.5% acetic acid (100 μL) and fractionated following standard protocol (Rappsilber et al., 2007). Briefly, 3 SCX discs (polytetrafluoroethylene (PTFE) material) were placed in 200 μL pipette tips followed by 2×C18 discs on top. Tips were conditioned with methanol (100 μL, 3500 g, 1 minute), followed by 0.5% acetic acid/80% ACN (100 μL, 3500 g, 1 minute) and 0.5% acetic acid (100 μL, 3500 g, 1 min). Tips were equilibrated with 0.5% acetic acid (100 μL, 3500 g, 1 minute), 500 mM NH4AcO/20% ACN (100 μL, 3500 g, 1 min) and 0.5% acetic acid (100 μL, 3500 g, 1 minute) prior to sample loading (100 μL, 3500 g). The sample was washed twice with 0.5% acetic acid (100 μL, 3500 g, 1 minute, 2×), followed by 0.5% acetic acid/80% ACN (100 μL, 3500 g, 1 minute). A stepwise elution occurred using 50 mM NH4AcO/20% ACN (pH 5.15, 50 μL, 3500 g, 1 minute, fraction 1), 50 mM NH4HCO3/20% ACN (pH 8.25, 50 μL, 3500 g, 1 minute, fraction 2) and 0.1% NH4OH/20% ACN (pH 10.3, 50 μL, 3500 g, 1 minute, fraction 3). Acetic acid (0.5%, 200 μL) was added to each eluate to reduce ACN concentration to <5%. Fractions were subsequently desalted using 2 punch C18 stage tips following the protocol described above and eluted with 80% ACN/0.5% acetic acid (60 μL, 1500 rcf). Samples were lyophilized and re-suspended in 3% ACN/0.1% FA (10 μL) for nanoLC-MS/MS analysis.
MS Analysis. Fractionated samples were analyzed on an Orbitrap Q-Exactive HF Plus MS (Thermo Fisher Scientific) equipped with a nanoflow ionization source and coupled to a nanoflow Proxcon EASY-nLC 1000 UHPLC system (Thermo Fisher Scientific). Acquisition occurred in positive ion mode. Samples were injected on an in-house packed column (22 cm×75 μm diameter C18 silica picofrit capillary column) heated at 50° C. The mobile phase flow rate was 250 nL/minutes of 3% ACN/1% FA (solvent A) and 90% ACN/0.1% FA (solvent B). Peptides were separated using the following LC gradient: 0-6% B in 1 minute, 6-30% B in 85 minutes, 30-60% B in 9 minutes, 60-90% B in 1 minute, stay at 90% B for 5 minutes, 90-50% B in 1 minute, and stay at 50% B for 5 minutes. Data was acquired in centroid mode for both MS1 and MS2 scans. Samples were analyzed in data dependent analysis (DDA) mode using a Top-12 method. Ion source parameters were: spray voltage 2 kV, source temperature 250° C. Full MS scans were acquired in the m/z range 200-2000, with an AGC target 3e6, maximum IT 10 ms and resolution 70,000 (at m/z 200). MS/MS parameters were as follows: AGC target 1e5, maximum IT 50 ms, loop count 10, isolation window 1.6 m/z, isolation offset 0.3 m/z, NCE 31, resolution 17,500 (at m/z 200) and fixed first mass 100 m/z; unassigned and singly charged ions were excluded from MS/MS.
Proteomic Data Analysis. Raw MS data were analyzed using Spectrum Mill Proteomics Workbench (prerelease version B.06.01.202, Agilent Technologies). A trypsin-specific enzyme search was performed against 2017 uniprot human fasta file (UniProt.human.20171228.RISnrNF.553smORFs.264contams) containing 65095 entries. Peptide and fragment tolerances were at 20 ppm, minimum matched peak intensity 40% and peptide false discovery rates (FDR) were calculated to be <1% using the target-decoy approach (Elias and Gygi, 2007). Fixed modifications were carbamidomethylation, TMT 10 (N-term, K) and variable modifications were Acetyl (ProN-term), Oxidized methionine (M), Pyroglutamic acid (N-termQ) and Deamidation (N). Spectra with a score<4 were filtered out. Peptides were validated using the following parameters: for charge states 2-4, a FDR of 1.2 was applied to each run and for charge state 5, a FDR of 0.6 was applied across all runs. Results were further validated at the protein level and proteins with a score of 20 or higher were accepted as valid. Reporter ion correction factors, specific to the TMT batch, were applied. A protein/peptide summary was generated using the median across all TMT channels as the denominator. Shared peptides were assigned to the protein with the highest score (SGT).
Bioinformatics Analysis. Calculated ratios at the protein level were imported into Protigy v0.8.X.X for normalization and features selection (github.com/broadinstitute/protigy). To account for variability between samples, log ratios were normalized by centering using the sample Median and scaled using the sample Median Absolute Deviation (Median MAD). A moderated F-test (Ritchie et al., 2015) was performed to identify proteins whose expression changed significantly across time points. Temporal profiles of significant proteins (FDR p-value<0.05) were z-scored and further subjected to fuzzy c-means clustering implemented in the e1071 R package. The number of clusters was set to three upon visual inspection of temporal profiles. The optimal fuzzification parameter m was determined as described in (Schwämmle and Jensen, 2010). Gene Ontology (GO) overrepresentation analysis of proteins in the resulting clusters was performed with the gProfiler R-package (Raudvere et al., 2019).
Network representation and analysis. The filtered dataset on adj p.value<0.07 was uploaded on STRING to generate a network. The network was then imported in Cytoscape (v3.8.1) and analyzed with stringApp (v1.6.0). Subcellular compartments were assigned by filtration in stringApp. Maps representing enrichments at different time-points were plotted by using the Hierarchical Clustering function in clusterMaker.
GO and Reactome enrichments. GO and Reactome enrichments present in
Generation of cells and screen. 293T were transduced with pTRIP-UbC-Blast-2A-STING-mNeonGreen(mNG) and selected with 15 μg/mL of Blasticidin (Invivogen) for one week. mNGhi cells were sorted on a Sony SH-800. Cells were then transduced with pXPR 101-Hygro to introduce spCas9, and kept under selection with 320 μg/mL Hygromicin for the time of culturing. Cells were then transduced with the human targeting genome-wide sgRNA library Brunello (Doench et al., 2016) at MOI 0.3 at a 1000× coverage (80 million cells). The library in lentiviral form was obtained from the Broad GPP. Cells were then selected in 2 μg/mL Puromycin and passaged maintaining 1000× representation of the library for one week. The day prior to the stimulation, cells were plated at 20 million/T225. The cells were then stimulated by adding 40 ml of fresh medium containing 1 μg/mL of 2′3′-cGAM(PS)2 (Invivogen) for 24 hours. Cells were then lifted, resuspended in MACS buffer (0.5% BSA, 2 mM EDTA in PBS) and sorted on two Sony SH-800 at a 4000× coverage (320 million total cells sorted). Cells were then pelleted, washed with PBS, and pellets were frozen until DNA extraction. DNA was extracted with DNeasy Blood & Tissue Kit (Qiagen) following manufacturer's recommendations.
Sequencing and screen analysis. Extracted DNA was submitted to the Broad Genetic Perturbation Platform (GPP) for Next Generation Sequencing. After deconvolution, reads per barcode were analyzed with the GPP Pooled Screen Analysis Tool using the Hypergeometric method (portals.broadinstitute.org/gpp/public/analysis-tools/crispr-gene-scoring).
Screen validation. 293T STING-mNG spCas9 were transduced with SEC24C or ATP6V1G1 sgRNAs cloned in CROPseq-Guide-Puro and selected with 2 μg/mL Puromycin (Invivogen) for one week. 0.016 million cells/well were plated in a 96 well plate the day prior to the stimulation, and then stimulated with 100 μL of fresh medium containing 2′3′-cGAM(PS)2 (Invivogen) for 24 hours. For HGS and VPS37A, 293T STING-mNG cells were transduced with pXPR023 (lentiCRISPR v2) expressing sgRNAs for each of the genes and selected on Puromycin for one week. 0.125 million cells/well were plated in a 24 well plate the day prior to stimulation, and then stimulated with 500 μL of fresh medium containing 2′3′-cGAM(PS)2 (Invivogen) for 24 hours.
The library contained guides targeting 669 E3 and adaptors (compiled from Medvar et al., 201613 and Li et al., 200814), 40 E2 from Interpro, 7 E1, 28 Autophagy core proteins and 10 positive controls from the genome-wide CRISPR screen and was synthetized and cloned by the Broad GPP. Cells were generated as for the genome wide CRISPR screen. STING-mNG cells were sorted without fixation while STING-HA cells were sorted after fixation and staining as described in the flow cytometry paragraph. Both cell lines were sorted in 4 bins (top 5%, second top 5%, bottom 5%, second bottom 5%) at 4000× coverage. For STING-mNG DNA was extracted as for the genome-wide CRISPR screen. For STING-HA DNA was extracted with Quick-DNA FFPE kit (Zymo). Sequencing was performed as described in Fulco et al., 201615. Analysis was performed as for the genome-wide CRISPR screen.
293T were transduced with either pTRIP-hPGK-Blast-2A, pTRIP-hPGK-Blast-2A-STING-HA, pTRIP-hPGK-Blast-2A-STING V155M-HA or pTRIP-hPGK-Blast-2A-STING R284S-HA and selected with 15 μg/mL Blasticidin for one week. Cells were then plated at 0.8 million cells/well in a 6 well plate and transfected with either pTRIP-SFFV-Hygro-2A-mScarlet-HGS or pTRIP-SFFV-Hygro-2A-mScarlet-VPS37A with TransIT-293 (Mirus) (3 μg DNA/well). 24 hours post-transfection, 3 wells per condition were harvested via trypsinization. Cells were washed with PBS and lysed 550 μL of Co-IP buffer (20 mM Tris-HCl PH 7.5, 150 mM NaCl, 0.5% NP-40 on ice for 30 minutes and cleared by centrifugation at 16000 g for 20 minutes). 10% of the lysate was saved as input. The lysates were then incubated with Pierce Anti-HA Magnetic Beads (Thermo Fisher) at a concentration of 100 μL beads/4 million cells O/N at 4° C. Beads were washed 5 times with Co-IP buffer and proteins were eluted by adding 150 μL of non-reducing Laemmli (Boston bioproducts) containing 20 mM DTT (Thermo Fisher) and boiled for 20 minutes. Input was diluted with 2× sample buffer (Sigma).
U937 were transduced with either pTRIP-SFFV-mNeonGreen or pTRIP-SFFV-Blast-2A-STING-mNeonGreen. For immunoprecipitation, 5 million cells/well of a 6-well plate were seeded for each condition and stimulated with medium containing 5 μg/mL Digitonin (Promega) with or without 20 μg/mL cGAMP for 4 hours. Cells were washed with ice-cold PBS once and lysed in 500 μL Co-IP buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.2% NP-40) with rotation at 4° C. for 10 minutes and cleared by centrifugation at 20,000 g for 10 minutes. 10% of the lysate was saved as input. The lysates were then incubated with 30 L/sample Co-IP buffer pre-washed mNeongreen-trap Magnetic Beads (Chromotek) for 1 hour at 4° C. Beads were washed 3 times with Co-IP buffer and proteins were eluted by adding 40 μL of 2× reducing Laemmli (Sigma) and boiled for 5 minutes. Input was diluted with 2× sample buffer (Sigma).
293T cells were transduced with pTRIP-hPGK-Blast-2A-STING-HA and pTRIP-hPGK-Hygro-2A-FLAG-Ubiquitin and selected with 15 μL Blasticidin (Invivogen) and 320 μg/mL Hygromycin (Invivogen) for one week. 1 million cells/well were plated in a 6 well plate the day prior to the stimulation. Cells were then stimulated with 300 μL cGAMP permeabilization buffer containing 1 μg/mL cGAMP for 10 minutes, washed with 3 mL of warm medium, and the medium was replaced. 3 wells per condition were harvested 2 hours post stimulation, washed with PBS and lysed with 550 μL of RIPA buffer for 10 minutes on ice. Lysates were cleared at 16000 g for 10 minutes at 4° C. 10% of the lysate was saved as input. The remaining lysates were incubated with 150 μL Pierce Anti-DYKDDDDK Magnetic beads (Thermo Fisher) O/N at 4° C. with constant rotation. Beads were washed 3 times with a buffer containing 10 mM Tris-HCl pH7.5 (Thermo Fisher), 2 mM EDTA (Thermo Fisher), 1% Nonidet-P40 Substitute (Roche) and 50 mM NaCl (Thermo Fisher), and 2 times with RIPA buffer. Proteins were eluted by adding 150 μL of non-reducing Laemmli (Boston bioproducts) containing 20 mM DTT (Thermo Fisher) and boiled for 20 minutes. Input was diluted with 2× sample buffer (Sigma).
U937 and hTert-BJ1 Stimulation for Western Blotting
To obtain KO U937 and hTert-BJ1 for HGS and VPS37A, cells were transduced with pXPR023 expressing the corresponding guides and selected with 2 μg/mL Puromycin for one week. In regard to HGS, U937 were transduced with HGS_g01 and HGS_g02, while hTert-BJ1 were transduced with HGS_g01 and HGS_g04 (more efficient than HGS_g02). To stimulate U937, 0.2 million cells/well were seeded in a 96 well plate U bottom in 100 μL and stimulated by adding 100 μL of fresh medium containing cGAMP to a final concentration of 20 μg/mL for 6 hours. Two wells per condition were harvested 6 hours post-stimulation, washed with PBS and pellets were frozen. For hTert-BJ1, 0.25 million cells/well were seeded in a 6 well plate the day before stimulation. Cells were then stimulated with 300 μL of cGAMP permeabilization buffer containing cGAMP at 0.5 μg/mL for 10 minutes, washed with 3 mL of warm medium, and then medium was replaced. Cells were harvested at the indicated time-points post stimulation, washed with PBS, and pellets were frozen.
0.2×10{circumflex over ( )}6 cells per well were plated in a 96 well plate (U bottom) and stimulated with direct addition of 20 μg/mL cGAMP in the medium. Cells were stimulated for 24 hours. Half the cells were then recovered and used for Cell Titer Glo assay (Promega). Half the cells were stained with Annexin V Apoptosis Detection Kit (Biolegend) following manufacturer's instructions.
293T cells were transduced with pTRIP-hPGK-Blast-2A-STING-HA and selected with 15 μg/mL Blasticidin (Invivogen) for one week. 0.08 million cells/well were seeded in a 24 well plate and transfected with TransIT-293 (Mirus) with 0.5 μg/well of either pTRIP-SFFV-Hygro-2A-mScarlet, pTRIP-SFFV-Hygro-2A-mScarlet-COPA K230N, pTRIP-SFFV-Hygro-2A-mScarlet-VPS4A E228Q or pTRIP-SFFV-Hygro-2A-mScarlet-UBAP1DN and medium was replaced after O/N incubation. Cells were stimulated 40 hours post-transfection with 200 μL/well of cGAMP permeabilization buffer containing 2 μg/mL 2′3′-cGAMP for 10 minutes. Cells were then washed with 2 mL of medium and medium was replaced. Cells were lifted and stained as indicated in the Flow Cytometry paragraph.
Treatments with Drugs
MLN7243 (Selleckchem) was used at 0.5 μM in all experiments. All cell lines were pre-treated for one hour before cGAMP stimulation. 293T STING-mNeonGreen were plated the day before stimulation in a 24 well plate at 0.2 million cells/well and were stimulated by adding 4 μg/mL 2′3′-cGAM(PS) in the medium for 6 hours. hTert-BJ1 were seeded the day before stimulation in a 6 well plate at 0.25 million cells/wells. Cells were stimulated with 300 μL cGAMP permeabilization buffer containing 0.5 μg/mL 2′3′-cGAMP for 10 minutes, washed with 3 mL of medium, and medium was then replaced. MLN7243 was added again after medium replacement. Cells were stimulated for the indicated times. Bortezomib final concentration was 1 μM, MG-132 2 μM, BafA1 100 nM.
For flow cytometry analysis of 293T STING-mNG, cells were lifted with TrypLE (Thermo Fisher), washed in medium and resuspended in FACS buffer (1% BSA, 1 mM EDTA, 0.01% NaN3 in PBS). For experiments involving intracellular staining of HA, 293T expressing HA-tagged WT or STING mutants, cells were lifted with TrypLE (Thermo Fisher), washed with PBS and stained using BD Cytofix/Cytoperm (BD Biosciences). Cells were fixed in Cytofix for 1 hours, washed twice with Cytoperm, and stained with Alexa Fluor 647 anti-HA.11 Epitope Tag Antibody (BioLegend) for one hour. Cells were then washed twice with Cytoperm and resuspended in FACS buffer for flow cytometry. Acquisition was performed on a Cytoflex S or Cytoflex LX (Beckman Coulter). Data was analyzed with FlowJo (BD).
293T STING-TurboID were seeded directly on coverslips. 293T STING-HA were seeded on Fibronectin bovine plasma (Sigma-stock: 100×) coated coverslips. Cells were seeded the day before stimulation at 0.1 million cells/well density in 24 well plates. Cells were stimulated with cGAMP permeabilization buffer containing 1 μg/mL cGAMP for 10 minutes, washed with warm medium, and incubated for the indicated times. hTert-BJ1 mScarlet or mScarlet-UBAP1DN were seeded Fibronectin bovine plasma coated coverslips at 0.05 million cells/well density in a 24 well plate and fixed 6 hours post seeding. Cells were then fixed with 2% Paraformaldehyde (Electron Microscopy Sciences) in PHEM buffer (Electron Microscopy Sciences) for 30 minutes at 37° C., washed three times with PBS and quenched with freshly prepared 0.1M Glycine for 10 minutes. Coverslips were then permeabilized and blocked with 10% goat serum (Thermo Fisher) in PBS, 0.5% BSA (Seracare), 0.05% Saponin from Quillaja bark (Sigma) for 30 minutes. Coverslips were then stained with primary antibodies for 1-2 hours at room temperature in PBS, 0.5% BSA (Seracare), 0.05% Saponin from Quillaja bark (Sigma) with 10% goat serum, washed 5 times, and then stained with secondary antibodies in PBS, 0.5% BSA (Seracare), 0.05% Saponin from Quillaja bark (Sigma) for 1-2 hours. Coverslips were then washed 5 times, mounted with Fuoromont-G, with DAPI (Thermo Fisher) and dried at 37° C. for one hour. Images were acquired on an Olympus IX83 using an Olympus PlanApo N 60×1.42NA oil immersion objective controlled by Fluoview software. Images were analyzed in FiJi. TMEM192-STING images in
Samples for pull-downs or immunoprecipitations were treated as described in the corresponding paragraphs. For experiments involving seeding of cells in 6 well plates or 24 well plates (293 Ts and hTert-BJ1), one well per condition was harvested, lysed in RIPA buffer (Boston Bioproducts) containing complete, Mini, EDTA-free Protease Inhibitor Cocktail (Millipore Sigma) and PhosSTOP (Millipore Sigma) for 10 minutes on ice. Lysates were cleared by centrifugation at 16000 g for 10 minutes at 4° C. and Laemmli 6×, Sample buffer, SDS, and reducing agents (Boston Bioproducts) were added prior to loading. Samples were run on NuPAGE 4 to 12%, Bis-Tris Gels (Thermo Fisher) and transferred on nitrocellulose membrane with an iBlot2 (Thermo Fisher). Membranes were blocked in 5% non-fat milk in TBS Tween. Antibodies against phospho-proteins were incubated in 5% BSA TBS tween. ECL signal was recorded on a ChemiDoc Biorad Imager. Data was analyzed with ImageLab (Biorad).
RNA Isolation. 0.25 million cells/well in a 6 well plate were plated the day before harvesting in triplicates. Cells were left in culture for 24 hours before harvesting. RNA was isolated using the AllPrep DNA/RNA Mini Kit (Qiagen #80204). Following total RNA extraction, mRNA was purified using Dynabeads® Oligo(dT)25 (ThermoFisher #61005). Generation of RNA-seq libraries. Bulk RNA-seq libraries were assembled from purified mRNA using the Smart-Seq3 workflow (Hagemann-Jensen et al., 2020). The reactions were scaled 8-fold per sample, using 15 ng input mRNA. First-strand synthesis and template switch reactions were performed by combining RT mix 1 and RT mix 2 and incubated in the thermocycler conditions noted in table First-strand synthesis. cDNA amplification was performed using 20 μL of the first-strand synthesis reaction as input in the cDNA PCR mix and thermocycled as noted in table cDNA amplification. Amplified cDNA was cleaned up using 0.8× concentration of Ampure XP beads (Beckman Coulter #A63881). Following clean up, cDNA samples underwent tagmentation and subsequent final library amplification. Briefly, 4 ul of tagmentation mix was combined with 400 pg of cDNA sample diluted in 4 μL of H2O. The tagmentation reaction was incubated at 55° C. for 10 minutes, with reaction being stopped with addition of 2 μL 0.2% SDS. Tagmented samples were then added to the final library mix and amplified in the conditions noted in table Final library amplification. Final libraries were cleaned using 0.8× concentration of Ampure XP beads (Beckman Coulter #A63881) and quantified using the Agilent Bioanalyzer High Sensitivity DNA (Agilent #5067-4626) system.
RNA-Seq Analysis. Libraries were sequenced on an Illumina NextSeq 500 with paired-end reads at a depth of 10-15 million reads per sample. Reads were extracted and demultiplexed using bc12fastq2 (v. 2.20.0) and sequencing quality was assessed with FastQC (v. 0.11.9), after which adapter and quality filtering was performed with Cutadapt (v. 3.1) (Martin, 2011). Reads were mapped with Salmon (v. 1.4.0) (Patro et al., 2017) using a whole genome decoy-aware transcriptome index built from GENCODE GRCh38 release 36, and mapping rates for all samples were between 90-93%. Differential expression analysis was performed in R with DESeq2 using a standard workflow. The criterion for identifying DEGs was padj≤0.01. Volcano plots were plotted with VolcaNoseR (Goedhart and Luijsterburg, 2020). GO enrichment plots were calculated using the Functional Enrichment Analysis in STRING.
List of sgRNAs
STING is the sensor of cyclic di-nucleotides of bacterial origins (Burdette et al. 2011) and 2′3′-cGAMP produced inside mammalian cells by dsDNA sensor cGAS (Sun et al. 2013; Wu et al. 2013; Ablasser et al. 2013). As a transmembrane protein, STING localizes on the ER surface as a homodimer form. Upon binding with agonists, the STING dimer goes through a 180-degree rotation to its activated conformation and translocates from ER through Golgi apparatus to the endosome and degrades at the lysosome (Shang et al. 2019). Upon STING trafficking out of ER, it activates TBK1-IRF3 pathway for IFN production (Tanaka and Chen 2012) and induces autophagy (Gui et al. 2019). Localization of STING is crucial for STING activity. The blockade of STING ER exit by BFA or bacteria derived protein VirA inhibits STING activity (Dobbs et al. 2015) while the blockade of STING degradation at the endolysosome further boost its activity (Gonugunta et al. 2017). In vitro biochemistry and structure analysis provide invaluable information about details about the STING conformation switch upon ligand binding, oligomerization interface and demonstrate that TBK1 needs to trans-phosphorylation STING (Shang et al. 2019; Zhang et al. 2019; Zhao et al. 2019).
While there is no difference of STING TBK1 binding with/without cGAMP stimulation through Co-IP and STING can be activated by TBK1 upon cGAMP stimulation in vitro (Zhang et al. 2019; Zhao et al. 2019), these did not reflect STING behaviors inside the intact cells. STING without agonists or blockade of STING trafficking out of ER upon stimulation failed to recruit TBK1 and blocked STING activation, suggesting a unique localization dependent STING activity controlling mechanism in the intact living cells (Dobbs et al. 2015; Zhang et al. 2019; Zhao et al. 2019). Moreover, while agonist binding is required for oligomerization, agonist-independent STING activation has also been observed in different settings. While STING overexpression is sufficient to drive its activation (Ishikawa and Barber 2008; Sun et al. 2009; Zhong et al. 2008), mislocalization of STING protein has also been shown to activate the TBK1-IRF3 pathway without cGAMP in COPA mutant patient cells (Deng et al. 2020; Lepelley et al. 2020; Mukai et al. 2021), further challenging the classic model that agonist driven oligomerization process is essential for the actual TBK1 activation step.
At the same time, given the importance of STING translocation, the driving force of STING translocation is still highly debatable. Agonist binding or mutants in the connector helix induce STING dimer rotation, triggering STING trafficking and STING oligomerization (Shang et al. 2019). Given that palmitoylation inhibition at TGN impairs STING oligomerization dramatically without influencing STING ER translocation (Haag et al. 2018), it is unclear if oligomerization drives translocation or translocation drives oligomerization. Moreover, SAVI mutants outside the STING connector helix can also translocate and induce STING activation, further complicating the interpretation but also providing vital information for discovering the actual driving force of STING translocation.
For a long time, agonist independent STING activation and SAVI mutants outside connector helix were “outliers” of the classic STING activation process. As a result of the information provided by these outlier phenomena, three questions were sought to be answered: 1. How does localization regulate STING activity?, 2. Which factor controls STING translocation?, and 3. What genes control STING activity and the mechanism of the regulation?
STING consists of N terminal 4 transmembrane domains and a C-terminal LBD (ligand binding domain). STING activation depends on its conserved PLPLRT/SD TBK1 binding motif and STING phosphorylation site in the C terminal tail region (CTT) (Zhang et al. 2019; Zhao et al. 2019). The structure of the STING-TBK1 complex provides clear evidence of a “trans-phosphorylation model”, that TBK1 needs to phosphorylate neighboring STING rather than the “anchoring” STING it binds due to close distance of TBK1 binding site and phosphorylation site on STING. TBK1 binds with STING directly on its CTT site. However, the affinity is ˜100 μM, suggesting a very unstable binding (Zhao et al. 2019). Combined with the fact that STING can only be phosphorylated after translocation from ER, it was hypothesized that STING activation depends on its concentration on the membrane (
The dynamic nature of the trafficking process upon STING activation makes it hard to precisely perturb STING trafficking in the living cells before. In order to test this, the STING transmembrane domain was truncated and human STING LBD (139-376) was fused with different organelle targeting sequences on its N terminal (
While STING density change is driven by its translocation, it is unclear what is the driven force of the translocation process. Ligand binding or SAVI mutants at the connector helix induces STING dimer rotation and oligomerization (Shang et al. 2019). STING oligomerization is well conserved even to STING analogue in bacteria (Morehouse et al. 2020). STING oligomerization was explored to determine whether it was the cause of STING translocation.
The cryo-EM chicken STING tetramer structure illustrates the packing pattern of the STING dimer and provides important insights into unresolved tetramer interface through modeling with STING dimer structure. Modeling using the chicken STING inactive dimer and active dimer illustrate the importance of movement of the oligomerization motif between LBDα2 and LBDα3 for mediating the oligomerization process (Shang et al. 2019). While the details of the interface are still not clear, a similar trend of cGAMP binding induced oligomerization motif movement in human STING (
Mutations that push the lower loop further outward or induce upper further inward should theoretically tense the oligomerization motif and block oligomerization even with agonist binding. As expected, mutating Ala277 in the inhibitory loop to Glu together with Glu273 in the oligomerization motif to alanine (AQQA) inhibited STING activation and also its oligomerization. Interestingly, the AQQA mutant was also incapable of trafficking out of ER (
Next, oligomerization was explored to determine whether it was sufficient to induce STING translocation starting from understanding SAVI mutants outside the connector helix. SAVI mutants outside the connector helix like Arginine284 mutants theoretically should not induce dimer rotation as connector helix mutants but have been reported to oligomerize. While the mechanism of R284 SAVI mutant is still debated, paper reports CTT release in R284 mutant is the cause of the oligomerization and translocation (Ergun et al. 2019). STING (1-341) (CTT deletion mutant) did not show automatic translocation like STING R284S and only translocated upon agonist stimulation (
Given the importance of the oligomerization motif for mediating STING oligomerization, perturbing the oligomerization motifs was explored to determine whether they were the reason for R284S's constitutive activity (
While STING oligomerization is required for its dramatic translocation, blockade of STING retrograde trafficking in COPA mutation patients induced STING accumulation at TGN, indicating STING going through a low level of constitutive trafficking out of ER. Upon agonist binding, STING binds to its trafficking adaptor Sec24C stronger (Gui et al. 2019), inducing a dramatic trafficking out of ER and induce the downstream signaling. The enhanced Sec24C binding is likely to come from the increased avidity of STING oligomer.
Starting from the principle for controlling STING signaling and trafficking, the genetic network regulating STING activity through these processes was explored. It was found that monocytic cell line U937 dies upon STING activation (
Interestingly, DNAJC13 showed up as one of the strongest hits in the cell death enhancing arm in both lethal dose and sublethal dose (
DNAJC13 is a member of the HSP40 family (J domain protein) which is known to resolve protein aggregate and oligomerization (Wentink et al. 2020). From the discussion above, oligomerization is a conserved property of STING and plays an important role in its trafficking. It was hypothesized that DNAJC13 influences STING trafficking and activity through influencing its oligomerization state. Through native gel, sgDNAJC13 U937 cells were observed to form more STING oligomers than sgCTRL cells upon STING agonist stimulation (
From a published IP-MS data for STING interaction proteins (Lee et al. 2013), it was discovered that DNAJC13 is a STING interaction protein suggesting DNAJC13 regulates STING activity through biochemical binding (
A large portion of cancer cells have a tonic level of STING activation due to reasons like DNA damage. Amplifying the tonic STING signal can be beneficial for tumor immunotherapy (Liu et al. 2019). Given the strong negative regulation effect of DNAJC13, DNAJC13 perturbation was explored to determine whether it could amplify this tonic STING activation in these cells. Indeed, there was a dramatic elevation of ISG overexpression (MX1, IFITM3) in these cells with DNAJC13 KO (
While a lot of HSP40 proteins execute the function in the cytosol (Wentink et al. 2020), DNAJC13 has a conserved PI3P binding domain, it was wondered whether the endosome localization is important for its function in inhibiting STING activity. Interestingly, while the WT guide1-resistant DNAJC13 (glrDNAJC13 WT) rescued the sgDNAJC13 effect, PI3P binding site mutant glrDNAJC13 K17A was unstable (Xhabija and Vacratsis 2015) and failed to rescue the sgDNAJC13 KO effect (
Presented herein is a STING density dependent activation model that explains the importance of localization in STING activation in the living cells, and also explains why STING mislocalization (in COPA patients) and STING overexpression trigger agonist-independent STING activation. Consistently, the blockade of STING palmitoylation, which is believed to concentrate STING on TGN, inhibits STING activity upon cGAMP binding (Haag et al. 2018) or in COPA mutation (Mukai et al. 2021), further supporting that concentration is the key for STING activity. The model presented herein takes advantage of structural analysis to show how ligand binding or mutation on different sites of STING induce the same oligomerization property change to induce STING trafficking and activation, providing mechanistic insights of STING auto-inhibition through Ser272-Arg284 lock which further explain the molecular details of constitutive activation properties of SAVI mutants.
Moreover, through genome-wide screening, J domain protein DNAJC13 was identified as a negative regulator for STING oligomerization to inhibit STING activation. DNAJC13's function depends on its J domain activity and endosome localization through ER-endosome contact sites. Furthermore, DNAJC13's role in helping resolve STING oligomers to promote its degradation was also found. Combined with DNAJC13's role in inhibiting STING trafficking, DNAJC13 inhibits STING translocation and speeds up STING degradation through disassembling STING oligomers, preventing its accumulation in TGN and endosome to control STING activation threshold.
While DNAJC13 has been shown to inhibit STING activity in different cells, the effect is much stronger in U937 and Thp1 compared to BJ1 and 293T. It is unclear if this difference comes from difference in DNAJC13 or STING protein amount or cell structural difference like ER-endosome contacts level. Clearly, the DNAJC13 protein serves as a way to control STING sensitivity in different types and the physiological relevance still needs to be further explored.
In addition to the embodiments expressly described herein, it is to be understood that all of the features disclosed in this disclosure may be combined in any combination (e.g., permutation, combination). Each element disclosed in the disclosure may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, and can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
In the articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Embodiments or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claims that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the embodiments. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any embodiment, for any reason, whether or not related to the existence of prior art.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended embodiments. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following embodiments.
This application is a national stage filing under 35 U.S.C. § 371 of International PCT Application PCT/US2022/033081, filed Jun. 10, 2022, which claims the benefit under 35 U.S.C. § 119(c) of U.S. Provisional Application No. 63/209,316, filed Jun. 10, 2021, entitled “METHODS AND COMPOSITIONS FOR MODULATING STING SIGNALING AND INNATE IMMUNE RESPONSES,” and U.S. Provisional Application No. 63/304,554, filed Jan. 28, 2022, entitled “METHODS AND COMPOSITIONS FOR MODULATING STING SIGNALING AND INNATE IMMUNE RESPONSES,” the entire disclosures of each of which are hereby incorporated by reference in their entireties.
This invention was made with government support under Grant Nos. AI133524, AI158495, and EB025854 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/033081 | 6/10/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63304554 | Jan 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 63209316 | Jun 2021 | US |
| Child | 18568797 | US |
