Activating tumor-intrinsic innate immune pathways can promote tumor antigen presentation and immune cell recruitment, thereby improving the immunosuppressive microenvironment and overcoming resistance to immune checkpoint blockade (ICB). However, the specific molecular targets remain insufficiently defined. Bioinformatic analysis revealed that Glycerol-3-phosphate acyltransferase 4 (GPAT4) is highly expressed in various tumors, including pancreatic cancer and breast cancer, and is significantly associated with low immunogenicity, poor immune-cell infiltration, and unfavorable prognosis. Syngeneic C57BL/6J and BALB/c tumor-bearing experiments showed that Gpat4-deficient pancreatic and breast tumors grew more slowly and exhibited increased infiltration of CD8+ T lymphocytes and enhanced CD8+ T cell effector-marker expression. Mechanistic studies indicated that GPAT4 deficiency leads to mitochondrial stress and the release of mitochondrial DNA (mtDNA) into the cytoplasm, thereby activating the cyclic GMP-AMP synthase (cGAS) signaling pathway. Interestingly, this process is independent of the metabolic enzyme activity of GPAT4. Single-cell analysis of breast and pancreatic tumors revealed that tumors with low GPAT4 expression exhibit higher levels of interferon response pathway activity and greater infiltration of immune cells, particularly CD8+ T cells and natural killer (NK) cells, which directly mediate tumor killing. In conclusion, our study identifies GPAT4 as a critical innate immune checkpoint molecule in tumor cells. Targeting this molecule can activate tumor-intrinsic innate immune pathways, thereby enhancing antitumor immune responses.

Upon infection, viral DNA/RNA is detected by cGAS/RIG-I-like receptors, triggering the adaptor MITA/STING- or VISA/MAVS-dependent innate antiviral immune response respectively. Both adaptors recruit the conserved TRAF3 and TRAF6 to activate TBK1-IRF3 and TAK1-NF-κB pathways respectively, leading to collaborative induction of antiviral effector genes. How the functions of TRAF3 and TRAF6 bifurcate in innate antiviral signaling remains enigmatic. We identified HTATSF1 as a positive regulator of virus-triggered innate antiviral response. Upon viral infection, HTATSF1 promotes HECTD3-catalyzed K63-linked polyubiquitination of TRAF3, leading to its recruitment of TBK1 and activation of IRF3. In contrast, HTATSF1 promotes recruitment of TAK1 to TRAF6 and activation of the TAK1-IKK-NF-κB axis independently of HECTD3. HTATSF1-deficiency impairs induction of downstream antiviral genes, and HTATSF1-deficient mice exhibit decreased cytokine production and increased mortality upon viral infection. Our findings demonstrate that HTATSF1 is an essential regulator of innate antiviral immune response by orchestrating the TRAF3-IRF3 and TRAF6-NF-κB pathways.

The cGAS-MITA/STING pathway plays critical roles in both host defense against DNA virus and intrinsic antitumor immunity by sensing viral genomic DNA or dis-located mitochondrial/cellular DNA. Whether carcinogenic metabolites can target the cGAS-MITA axis to promote tumorigenesis is unknown. In this study, we identified acetaldehyde, a carcinogenic metabolite, as a suppressor of the cGAS-MITA pathway. Acetaldehyde inhibits the DNA virus herpes simplex virus 1 (HSV-1)- and transfected DNA-triggered but not cGAMP-induced activation of downstream components and induction of downstream effector genes. Mechanistically, acetaldehyde impairs the binding of cGAS to DNA as well as the phase separation of the cGAS-DNA complex in cells. In mouse models, acetaldehyde inhibits antiviral cytokine production, promotes viral replication and lethality upon HSV-1 infection. In a colorectal tumor xenograft model, acetaldehyde promotes tumor growth and inhibits CD8+ T cell infiltration by targeting cGAS in both the tumor cells and immune cells in mice. Bioinformatic analysis indicates that expression of acetaldehyde dehydrogenase 2 (ALDH2), which converts acetaldehyde to acetic acid, is negatively correlated with stimulatory immune signatures in clinical colorectal tumors, and higher ALDH2 expression exhibits better prognosis of colorectal cancer patients. Collectively, our results suggest that acetaldehyde impairs cGAS activity to inhibit the cGAS-MITA axis, which contributes to its effects on carcinogenesis.

Upon DNA virus infection, cGAS senses viral DNA and triggers MITA (also called STING)-dependent induction of type I interferons (IFN-Is) and other cytokines/chemokines. IFN-Is further activate STAT1/2 to induce interferon-stimulated genes (ISGs) and the innate antiviral response. How the innate antiviral response is silenced in uninfected cells and efficiently mounts upon viral infection is not fully understood. In this study, we found that FBXW7, a substrate recognition component of the SCF E3 ubiquitin ligase complex, is a multifaceted regulator of the innate immune response to DNA viruses. In uninfected cells, FBXW7 mediates the polyubiquitination and degradation of GSK3α/β-phosphorylated SLC35B2/3 at the Golgi apparatus. This leads to the downregulation of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus and the inactivation of MITA in uninfected cells. In addition, FBXW7 mediates the degradation of GSK3α/β-phosphorylated MYC, which is a repressor of STAT1/2, leading to proper STAT1/2 levels in uninfected cells. The differential regulation of FBXW7 on MITA and STAT1/2 ensures inactivation but is ready for fast mount of the innate immune response in uninfected cells. Infection with DNA viruses activates the PI3K‒AKT axis, which inactivates GSK3α/β and inhibits FBXW7-mediated polyubiquitination and degradation of SLC35B2/3, leading to increased production of sGAGs, activation of MITA and rapid onset of the innate antiviral response. Consistently, gene disruption experiments indicate that FBXW7 modulates the innate antiviral response in human THP-1 and mouse BMDM cells. These findings suggest that FBXW7 functions as a versatile regulator of the innate immune response to DNA viruses by differentially regulating upstream and downstream components of the type I interferon induction loop.

Expression of major histocompatibility complex I (MHC-I) on tumor cells is extremely important for the antitumor immune response for its essential role in activating various immune cells, including tumor-specific CD8+ T cells. Cancers of lower MHC-I expression commonly exhibit less immune cell infiltration and worse prognosis in clinic. In this study, we conducted bioinformatic-experimental screening to identify potential gene targets to enhance MHC-I expression in breast cancer (BRCA). Through a combination of MHC-I scoring, gene expression correlation analysis, survival prognostication, and Cibersort tumor-infiltrated lymphocytes (TILs) scoring, we identify 144 genes negatively correlated with both MHC-I expression and TILs in breast cancer. Furthermore, we verified partially according to KEGG functional enrichment or gene-dependency analysis and figured out multiple genes, including PIP5K1A, NCKAP1, CYFIP1, DIS3, TBP, and EXOC1, as effective gene targets for increasing MHC-I expression in breast cancer. Mechanistically, knockout of each of these genes activated the intrinsic interferon response in breast cancer cells, which not only promoted MHC-I expression but also caused immunogenic cell death of breast cancer. Finally, the scRNA-seq confirmed the negative correlation of PIP5K1A et al. with TILs in breast cancer patients. Collectively, we identified multiple gene targets for an increase in MHC-I expression in breast cancer in this study.

Estrogen receptor α (ERα) is an important driver and therapeutic target in ∼70% of breast cancers. How ERα drives breast carcinogenesis is not fully understood. In this study, we show that ERα is a negative regulator of type I interferon (IFN) response. Activation of ERα by its natural ligand estradiol inhibits IFN-β-induced transcription of downstream IFN-stimulated genes (ISGs), whereas ERα deficiency or the stimulation with its antagonist fulvestrant has opposite effects. Mechanistically, ERα induces the expression of the histone 2A variant H2A.Z to restrict the engagement of the IFN-stimulated gene factor 3 (ISGF3) complex to the promoters of ISGs and also interacts with STAT2 to disrupt the assembly of the ISGF3 complex. These two events mutually lead to the inhibition of ISG transcription induced by type I IFNs. In a xenograft mouse model, fulvestrant enhances the ability of IFN-β to suppress ERα+ breast tumor growth. Consistently, clinical data analysis reveals that ERα+ breast cancer patients with higher levels of ISGs exhibit higher long-term survival rates. Taken together, our findings suggest that ERα inhibits type I IFN response via two distinct mechanisms to promote breast carcinogenesis.

N6-methyladenosine (m6A) methylation of RNA by the methyltransferase complex (MTC), with core components including METTL3-METTL14 heterodimers and Wilms’ tumor 1-associated protein (WTAP), contributes to breast tumorigenesis, but the underlying regulatory mechanisms remain elusive. Here, we identify a novel cleaved form METTL3a (residues 239-580 of METTL3). We find that METTL3a is required for the METTL3-WTAP interaction, RNA m6A deposition, as well as cancer cell proliferation. Mechanistically, we find that METTL3a is essential for the METTL3-METTL3 interaction, which is a prerequisite step for recruitment of WTAP in MTC. Analysis of m6A sequencing data shows that depletion of METTL3a globally disrupts m6A deposition, and METTL3a mediates mTOR activation via m6A-mediated suppression of TMEM127 expression. Moreover, we find that METTL3 cleavage is mediated by proteasome in an mTOR-dependent manner, revealing positive regulatory feedback between METTL3a and mTOR signaling. Our findings reveal METTL3a as an important component of MTC, and suggest the METTL3a-mTOR axis as a potential therapeutic target for breast cancer.

Various cellular stress conditions trigger mitochondrial DNA (mtDNA) release from mitochondria into the cytosol. The released mtDNA is sensed by the cGAS-MITA/STING pathway, resulting in the induced expression of type I interferon and other effector genes. These processes contribute to the innate immune response to viral infection and other stress factors. The deregulation of these processes causes autoimmune diseases, inflammatory metabolic disorders and cancer. Therefore, the cGAS-MITA/STING pathway is a potential target for intervention in infectious, inflammatory and autoimmune diseases as well as cancer. In this review, we focus on the mechanisms underlying the mtDNA-triggered activation of the cGAS-MITA/STING pathway, the effects of the pathway under various physiological and pathological conditions, and advances in the development of drugs that target cGAS and MITA/STING.

35 N 6 -methyladenosine (m 6 A) methylation of RNA by the methyltransferase complex (MTC), with core 36 components including METTL3-METTL14 heterodimers and Wilms’ tumor 1-associated protein 37 (WTAP), contributes to breast tumorigenesis, but the underlying regulatory mechanisms remain elusive. 38 Here, we identify a novel cleaved form METTL3a (residues 239-580 of METTL3). We find that 39 METTL3a is required for the METTL3-WTAP interaction, RNA m 6 A deposition, as well as cancer cell 40 proliferation. Mechanistically, we find that METTL3a is essential for the METTL3-METTL3 interaction, 41 which is a prerequisite step for recruitment of WTAP in MTC. Analysis of m 6 A sequencing data shows 42 that depletion of METTL3a globally disrupts m 6 A deposition, and METTL3a mediates mTOR activation 43 via m 6 A-mediated suppression of TMEM127 expression. Moreover, we find that METTL3 cleavage is 44 mediated by proteasome in an mTOR-dependent manner, revealing positive regulatory feedback between 45 METTL3a and mTOR signaling. Our findings reveal METTL3a as an important component of MTC, 46 and suggest the METTL3a-mTOR axis as a potential therapeutic target for breast cancer. 47

Mitochondria play essential roles in cancer cell adaptation to hypoxia, but the underlying mechanisms remain elusive. Through mitochondrial proteomic profiling, we here find that the prolyl hydroxylase EglN1 (PHD2) accumulates on mitochondria under hypoxia. EglN1 substrate-binding region in the β2β3 loop is responsible for its mitochondrial translocation and contributes to breast tumor growth. Furthermore, we identify AMP-activated protein kinase alpha (AMPKα) as an EglN1 substrate on mitochondria. The EglN1-AMPKα interaction is essential for their mutual mitochondrial translocation. After EglN1 prolyl-hydroxylates AMPKα under normoxia, they rapidly dissociate following prolyl-hydroxylation, leading to their immediate release from mitochondria. In contrast, hypoxia results in constant EglN1-AMPKα interaction and their accumulation on mitochondria, leading to the formation of a Ca2+ /calmodulin-dependent protein kinase 2 (CaMKK2)-EglN1-AMPKα complex to activate AMPKα phosphorylation, ensuring metabolic homeostasis and breast tumor growth. Our findings identify EglN1 as an oxygen-sensitive metabolic checkpoint signaling hypoxic stress to mitochondria through its β2β3 loop region, suggesting a potential therapeutic target for breast cancer.

Upon viral infection, cytoplasmic pattern recognition receptors detect viral nucleic acids and activate the adaptor protein VISA/MAVS- or MITA/STING-mediated innate antiviral response. Whether and how the innate antiviral response is regulated by neuronal endocrine functions is unclear. Here, we show that viral infection reduced the serum levels of the β-adrenergic hormones epinephrine and norepinephrine as well as the cellular levels of their receptors ADRB1 and ADRB2. We further show that an increase in epinephrine/norepinephrine level inhibited the innate antiviral response in an ADRB1-/2-dependent manner. Mechanistically, epinephrine/norepinephrine stimulation activated the downstream kinase PKA, which catalyzed the phosphorylation of MITA at S241, S243 and T263, inhibiting MITA activation and suppressing the innate immune response to DNA virus. In addition, phosphorylation of VISA at T54 by PKA antagonized the innate immune response to RNA virus. These findings reveal the regulatory mechanisms of innate antiviral responses by epinephrine/norepinephrine and provide a possible explanation for increased host susceptibility to viral infection in stressful and anxiety-promoting situations.

Whether and how innate antiviral response is regulated by humoral metabolism remains enigmatic. We show that viral infection induces progesterone via the hypothalamic-pituitary-adrenal axis in mice. Progesterone induces downstream antiviral genes and promotes innate antiviral response in cells and mice, whereas knockout of the progesterone receptor PGR has opposite effects. Mechanistically, stimulation of PGR by progesterone activates the tyrosine kinase SRC, which phosphorylates the transcriptional factor IRF3 at Y107, leading to its activation and induction of antiviral genes. SARS-CoV-2-infected patients have increased progesterone levels, and which are co-related with decreased severity of COVID-19. Our findings reveal how progesterone modulates host innate antiviral response, and point to progesterone as a potential immunomodulatory reagent for infectious and inflammatory diseases.

The current view of nucleic acid-mediated innate immunity is that binding of intracellular sensors to nucleic acids is sufficient for their activation. Here, we report that endocytosis of virus or foreign DNA initiates a priming signal for the DNA sensor cyclic GMP-AMP synthase (cGAS)-mediated innate immune response. Mechanistically, viral infection or foreign DNA transfection triggers recruitment of the spleen tyrosine kinase (SYK) and cGAS to the endosomal vacuolar H+ pump (V-ATPase), where SYK is activated and then phosphorylates human cGASY214/215 (mouse cGasY200/201) to prime its activation. Upon binding to DNA, the primed cGAS initiates robust cGAMP production and mediator of IRF3 activation/stimulator of interferon genes-dependent innate immune response. Consistently, blocking the V-ATPase-SYK axis impairs DNA virus- and transfected DNA-induced cGAMP production and expression of antiviral genes. Our findings reveal that V-ATPase-SYK-mediated tyrosine phosphorylation of cGAS following endocytosis of virus or other cargos serves as a priming signal for cGAS activation and innate immune response.

Anti-PD-1/PD-L1 immunotherapy has achieved impressive therapeutic outcomes in patients with multiple cancer types. However, the underlined molecular mechanism(s) for moderate response rate (15-25%) or resistance to PD-1/PD-L1 blockade remains not completely understood. Here, we report that inhibiting the deubiquitinase, USP8, significantly enhances the efficacy of anti-PD-1/PD-L1 immunotherapy through reshaping an inflamed tumor microenvironment (TME). Mechanistically, USP8 inhibition increases PD-L1 protein abundance through elevating the TRAF6-mediated K63-linked ubiquitination of PD-L1 to antagonize K48-linked ubiquitination and degradation of PD-L1. In addition, USP8 inhibition also triggers innate immune response and MHC-I expression largely through activating the NF-κB signaling. Based on these mechanisms, USP8 inhibitor combination with PD-1/PD-L1 blockade significantly activates the infiltrated CD8+ T cells to suppress tumor growth and improves the survival benefit in several murine tumor models. Thus, our study reveals a potential combined therapeutic strategy to utilize a USP8 inhibitor and PD-1/PD-L1 blockade for enhancing anti-tumor efficacy.

SARS-CoV-2 infection induces imbalanced immune response such as hyperinflammation in patients with severe COVID-19. Here, we studied the immunometabolic regulatory mechanisms for the pathogenesis of COVID-19. We depicted the metabolic landscape of immune cells, especially macrophages, from bronchoalveolar lavage fluid of patients with COVID-19 at single-cell level. We found that most metabolic processes were upregulated in macrophages from lungs of patients with mild COVID-19 compared to cells from healthy controls, whereas macrophages from severe COVID-19 showed downregulation of most of the core metabolic pathways including glutamate metabolism, fatty acid oxidation, citrate cycle, and oxidative phosphorylation, and upregulation of a few pathways such as glycolysis. Rewiring cellular metabolism by amino acid supplementation, glycolysis inhibition, or PPARγ stimulation reduces inflammation in macrophages stimulated with SARS-CoV-2. Altogether, this study demonstrates that metabolic imbalance of bronchoalveolar macrophages may contribute to hyperinflammation in patients with severe COVID-19 and provides insights into treating COVID-19 by immunometabolic modulation.

Mitochondrial stress (mitostress) triggered by viral infection or mitochondrial dysfunction causes the release of mitochondrial DNA (mtDNA) into the cytosol and activates the cGAS-mediated innate immune response. The regulation of mtDNA release upon mitostress remains uncharacterized. Here, we identified mitochondria-associated vaccinia virus-related kinase 2 (VRK2) as a key regulator of this process. VRK2 deficiency inhibited the induction of antiviral genes and caused earlier and higher mortality in mice after viral infection. Upon viral infection, VRK2 associated with voltage-dependent anion channel 1 (VDAC1) and promoted VDAC1 oligomerization and mtDNA release, leading to the cGAS-mediated innate immune response. VRK2 was also required for mtDNA release and cGAS-mediated innate immunity triggered by nonviral factors that cause Ca2+ overload but was not required for the cytosolic nucleic acid-triggered innate immune response. Thus, VRK2 plays a crucial role in the mtDNA-triggered innate immune response and may be a potential therapeutic target for infectious and autoimmune diseases associated with mtDNA release.

The cyclic GMP-AMP synthase (cGAS) is a widely used DNA sensor, which detects cytosolic DNA species without a preference of self or non-self microbial DNA in interphase to initiate innate immune response. How cGAS is regulated to avoid self-DNA sensing upon nuclear envelope breakdown (NEBD) during mitosis remains enigmatic. Here we show that cGAS is mostly localized in the cytoplasm in interphase and rapidly translocated to chromosomes upon NEBD in mitosis. The major mitotic kinase CDK1-cyclin B complex phosphorylates human cGAS at S305 or mouse cGAS at S291, which inhibits its ability to synthesize cGAMP upon mitotic entry. The type 1 phosphatase PP1 dephosphorylates cGAS upon mitotic exit to enable its DNA sensing ability. Our findings reveal a mechanism on how the DNA sensor cGAS is post-translationally regulated by cell cycle-dependent enzymes to ensure its proper activation for host defense of cytosolic DNA in interphase and inert to self-DNA in mitosis.

Significance cGAS is a protein which senses the infected viral DNA in the cytosol. Sensing of viral DNA by cGAS signals an innate immune response to virus. How cGAS is regulated by posttranslational modifications is still not fully understood. In this study, we found that the acetyltransferase KAT5 mediated acetylation of the N terminus of cGAS after infection of DNA virus, such as herpes simplex virus 1. This modification of cGAS causes its higher affinity to viral DNA and therefore promotes innate antiviral response. This study helps to understand the delicate regulatory mechanisms of innate antiviral response. Manipulation of these mechanisms may help to develop novel therapeutics for infectious and inflammatory diseases.

Upon detection of viral DNA, the cytoplasmic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) utilizes GTP and ATP as substrates to synthesize the second messenger molecule 2′3′cyclic GMP-AMP (cGAMP), which binds to the ER-associated adaptor protein MITA/STING to signal innate antiviral response to DNA virus. How the cGAS-MITA pathways are post-translationally regulated is not fully understood. In this study, we identified the tyrosine kinase CSK as a positive regulator of cGAS-MITA mediated innate antiviral response. CSK-deficiency inhibits DNA virus-triggered induction of downstream antiviral effector genes. Following DNA virus infection, CSK phosphorylates MITA at Y240 and Y245, which is important for its activation. These results suggest that CSK plays a role in modulating innate immune response to DNA virus.

The mechanisms on metabolic regulation of immune responses are still elusive. We show here that viral infection induces immediate-early NF-κB activation independent of viral nucleic acid-triggered signaling, which triggers a rapid transcriptional induction of bile acid (BA) transporter and rate-limiting biosynthesis enzymes as well as accumulation of intracellular BAs in divergent cell types. The accumulated intracellular BAs activate SRC kinase via the TGR5-GRK-β-arrestin axis, which mediates tyrosine phosphorylation of multiple antiviral signaling components including RIG-I, VISA/MAVS, MITA/STING, TBK1 and IRF3. The tyrosine phosphorylation of these components by SRC conditions for efficient innate antiviral immune response. Consistently, TGR5 deficiency impairs innate antiviral immunity, whereas BAs exhibit potent antiviral activity in wild-type but not TGR5-deficient cells and mice. Our findings reveal an intrinsic and universal role of intracellular BA metabolism in innate antiviral immunity.