The multiple faces of cGAS-STING in antitumor immunity: prospects and challenges.

As a key sensor of double-stranded DNA (dsDNA), cyclic GMP-AMP synthase (cGAS) detects cytosolic dsDNA and initiates the synthesis of 2'3' cyclic GMP-AMP (cGAMP) that activates the stimulator of interferon genes (STING). This finally promotes the production of type I interferons (IFN-I) that is crucial for bridging innate and adaptive immunity. Recent evidence show that several antitumor therapies, including radiotherapy (RT), chemotherapy, targeted therapies and immunotherapies, activate the cGAS-STING pathway to provoke the antitumor immunity. In the last decade, the development of STING agonists has been a major focus in both basic research and the pharmaceutical industry. However, up to now, none of STING agonists have been approved for clinical use. Considering the broad expression of STING in whole body and the direct lethal effect of STING agonists on immune cells in the draining lymph node (dLN), research on the optimal way to activate STING in tumor microenvironment (TME) appears to be a promising direction. Moreover, besides enhancing IFN-I signaling, the cGAS-STING pathway also plays roles in senescence, autophagy, apoptosis, mitotic arrest, and DNA repair, contributing to tumor development and metastasis. In this review, we summarize the recent advances on cGAS-STING pathway's response to antitumor therapies and the strategies involving this pathway for tumor treatment.

Implication of 99mTc-sum IL-2 SPECT/CT in immunotherapy by imaging of tumor-infiltrating T cells.

BACKGROUND: Although immune checkpoint blockade (ICB) and adoptive T cell transfer (ACT) therapy have achieved impressive clinical outcomes, majority of patients do not respond to immunotherapy. Tumor-infiltrating T cells, a critical factor to immunotherapy, is dynamically changing. Therefore, a reliable real-time in vivo imaging system for tumor-infiltrating T cells, but not immunohistochemical analyses, will be more valuable to predict response and guide immunotherapy. In this study, we developed a new SPECT/CT imaging probe 99mTc-sum IL-2 targeting the IL-2Rß/IL-2Rγ (CD122/CD132) receptor on tumor-infiltrating T cells, and evaluated its application in predicting the immune response to anti-PD-L1 (αPD-L1) therapy as well as tracking infused T cells in ACT therapy. METHODS: The binding affinity of the super mutated IL-2 (sum IL-2) in various T cell subtypes was measured. Sum IL-2 was subsequently labeled with 99mTc through Sortase-A mediated site-specific transpeptidation. SPECT/CT imaging and biodistribution studies of 99mTc-sum IL-2 were performed in a MC38 mouse model. Wild type IL-2 (IL-2) was used as control in the above studies. Finally, we evaluated 99mTc-sum IL-2 SPECT/CT for the detection of tumor-infiltrating T cells in the context of αPD-L1 immunotherapy and ACT therapy. RESULTS: Sum IL-2 preferentially bound to CD8+ T cells, especially activated CD8+ T cells, while IL-2 showed biased binding to Treg cells. As a result, 99mTc-sum IL-2 could detect tumor-infiltrating T cells. In the MC38 tumor model, SPECT/CT imaging showed the increased tumor uptake of 99mTc-sum IL-2 after αPD-L1 treatment, suggesting that the treatment significantly increased tumor-infiltrating T cells, resulting in a correspondingly significant curative effect. In addition, 99mTc-sum IL-2 SPECT/CT could also track the infiltration of antigen-specific cytotoxic CD8+ T cells during ACT therapy. CONCLUSION: 99mTc-sum IL-2 has great clinical potential for non-invasive and specific SPECT/CT imaging of tumor-infiltrating T cells as well as for timely prediction and evaluation of the therapeutic efficacy of ICB and ACT therapy.

RNF213 modulates γ-herpesvirus infection and reactivation via targeting the viral Replication and Transcription Activator.

Interferons (IFNs) and the products of interferon-stimulated genes (ISGs) play crucial roles in host defense against virus infections. Although many ISGs have been characterized with respect to their antiviral activity, their target specificities and mechanisms of action remain largely unknown. Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that is linked to several human malignancies. Here, we used the genetically and biologically related virus, murine gammaherpesvirus 68 (MHV-68) and screened for ISGs with anti-gammaherpesvirus activities. We found that overexpression of RNF213 dramatically inhibited MHV-68 infection, whereas knockdown of endogenous RNF213 significantly promoted MHV-68 proliferation. Importantly, RNF213 also inhibited KSHV de novo infection, and depletion of RNF213 in the latently KSHV-infected iSLK-219 cell line significantly enhanced lytic reactivation. Mechanistically, we demonstrated that RNF213 targeted the Replication and Transcription Activator (RTA) of both KSHV and MHV-68, and promoted the degradation of RTA protein through the proteasome-dependent pathway. RNF213 directly interacted with RTA and functioned as an E3 ligase to ubiquitinate RTA via K48 linkage. Taken together, we conclude that RNF213 serves as an E3 ligase and inhibits the de novo infection and lytic reactivation of gammaherpesviruses by degrading RTA through the ubiquitin-proteasome pathway.

Peripheral immune factors aiding clinical parameter for better early recurrence prediction of hepatocellular carcinoma after thermal ablation.

OBJECTIVES: Current predictors are largely unsatisfied for early recurrence (ER) of hepatocellular carcinoma (HCC) after thermal ablation. We aimed to explore the prognostic value of peripheral immune factors (PIFs) for better ER prediction of HCC after thermal ablation. METHODS: Patients who received peripheral blood mononuclear cells (PBMCs) tests before thermal ablation were included. Clinical parameters and 18 PIFs were selected to construct ModelClin, ModelPIFs and the hybrid ModelPIFs-Clin. Model performances were evaluated using area under the curve (AUC), and recurrence-free survival (RFS) were analyzed by Kaplan-Meier analysis and log-rank tests. RESULTS: 244 patients were included and were randomly divided in 3:1 ratio to discovery and validation cohorts. Clinical parameters including tumor size and AFP, and PIFs including neutrophils, platelets, CD3+CD16+CD56+ NKT and CD8+CD28- T lymphocytes were selected. The ModelPIFs-Clin showed increase in predictive performance compared with ModelClin, with the AUC improved from 0.664 (95%CI:0.588-0.740) to 0.801 (95%CI:0.734-0.867) in discovery cohort (p < 0.0001), and from 0.645 (95%CI:0.510-0.781) to 0.737(95%CI:0.608-0.865) in validation cohort (p = 0.1006). ModelPIFs-Clin enabled ER risk stratification of patients. Patients predicted in ModelPIFs-Clin high-risk subgroup had a poor RFS compared with those predicted as ModelPIFs-Clin low-risk subgroup, with the median RFS was 18.00 month versus 100.78 month in discovery cohort (p < 0.0001); and 24.00 month versus 60.35 month in validation cohort (p = 0.288). Patients in different risk subgroups exhibited distinct peripheral immune contexture. CONCLUSIONS: Peripheral immune cells aiding clinical parameters boosted the prediction ability for ER of HCC after thermal ablation, which be helpful for pre-ablation ER risk stratification.

Zinc cyclic di-AMP nanoparticles target and suppress tumours via endothelial STING activation and tumour-associated macrophage reinvigoration.

The clinical utility of stimulator of interferon genes (STING) agonists has been limited due to poor tumour-targeting and unwanted toxicity following systemic delivery. Here we describe a robust tumour-targeted STING agonist, ZnCDA, formed by the encapsulation of bacterial-derived cyclic dimeric adenosine monophosphate (CDA) in nanoscale coordination polymers. Intravenously injected ZnCDA prolongs CDA circulation and efficiently targets tumours, mediating robust anti-tumour effects in a diverse set of preclinical cancer models at a single dose. Our findings reveal that ZnCDA enhances tumour accumulation by disrupting endothelial cells in the tumour vasculature. ZnCDA preferentially targets tumour-associated macrophages to modulate antigen processing and presentation and subsequent priming of an anti-tumour T-cell response. ZnCDA reinvigorates the anti-tumour activity of both radiotherapy and immune checkpoint inhibitors in immunologically 'cold' pancreatic and glioma tumour models, offering a promising combination strategy for the treatment of intractable human cancers.

AXL targeting restores PD-1 blockade sensitivity of STK11/LKB1 mutant NSCLC through expansion of TCF1+ CD8 T cells.

Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB), and introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype recapitulated in human STK11/LKB1 mutant NSCLCs. Systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expanded tumor-associated TCF1+PD-1+CD8 T cells, restoring therapeutic response to PD-1 ICB in KPL tumors. This was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. NSCLC-affected individuals with identified STK11/LKB1 mutations receiving bemcentinib and pembrolizumab demonstrated objective clinical response to combination therapy. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC.

A single factor elicits multilineage reprogramming of astrocytes in the adult mouse striatum.

SignificanceOutside the neurogenic niches, the adult brain lacks multipotent progenitor cells. In this study, we performed a series of in vivo screens and reveal that a single factor can induce resident brain astrocytes to become induced neural progenitor cells (iNPCs), which then generate neurons, astrocytes, and oligodendrocytes. Such a conclusion is supported by single-cell RNA sequencing and multiple lineage-tracing experiments. Our discovery of iNPCs is fundamentally important for regenerative medicine since neural injuries or degeneration often lead to loss/dysfunction of all three neural lineages. Our findings also provide insights into cell plasticity in the adult mammalian brain, which has largely lost the regenerative capacity.

Rejuvenation of tumour-specific T cells through bispecific antibodies targeting PD-L1 on dendritic cells.

Bispecific T-cell engagers (BiTEs) preferentially targeting tumour-associated antigens and stimulating CD3-mediated signalling are being used in patients to treat acute B-cell lymphoblastic leukemia. However, the potency of BiTEs in solid tumours is limited by their short half-life and their severe toxicity at relevant therapeutic doses. Here we report the design and in vivo performance of a bispecific antibody that simultaneously targets the murine T-cell co-receptor CD3ε and the murine immune checkpoint programmed-death ligand 1 (PD-L1). In multiple syngeneic tumour models, the bispecific antibody generated higher antitumour immune responses than conventional BiTEs targeting tumour-associated antigens and CD3ε. We found that the durable antigen-specific T-cell responses resulted from the rejuvenation of CD8 T cells, owing to the blockade of PD-L1 on dendritic cells (but not on tumour cells) and co-stimulation by B7-1&2 (a peripheral membrane protein on dendritic cells). Bispecific T-cell engagers targeting dendritic cells rather than tumour cells may represent a general means of T-cell rejuvenation for durable cancer immunotherapy.

The AIM2 and NLRP3 inflammasomes trigger IL-1-mediated antitumor effects during radiation.

The inflammasome promotes inflammation-associated diseases, including cancer, and contributes to the radiation-induced tissue damage. However, the role of inflammasome in radiation-induced antitumor effects is unclear. We observed that tumors transplanted in Casp1-/- mice were resistant to radiation treatment compared with tumors in wild-type (WT) mice. To map out which molecule in the inflammasome pathway contributed to this resistant, we investigated the antitumor effect of radiation in several inflammasome-deficient mice. Tumors grown in either Aim2-/- or Nlrp3-/- mice remained sensitive to radiation, like WT mice, whereas Aim2-/-Nlrp3-/- mice showed radioresistance. Mechanistically, extracellular vesicles (EVs) and EV-free supernatant derived from irradiated tumors activated both Aim2 and Nlrp3 inflammasomes in macrophages, leading to the production of interleukin-1ß (IL-1ß). IL-1ß treatment helped overcome the radioresistance of tumors growing in Casp1-/- and Aim2-/-Nlrp3-/- mice. IL-1 signaling in dendritic cells (DCs) promoted radiation-induced antitumor immunity by enhancing the cross-priming activity of DCs. Overall, we demonstrated that radiation-induced activation of the AIM2 and NLRP3 inflammasomes coordinate to induce some of the antitumor effects of radiation by triggering IL-1 signaling in DCs, leading to their activation and cross-priming.

Personalized Ultrafractionated Stereotactic Adaptive Radiotherapy (PULSAR) in Preclinical Models Enhances Single-Agent Immune Checkpoint Blockade.

PURPOSE: Harnessing the immune-stimulatory effects of radiation by combining it with immunotherapy is a promising new treatment strategy. However, more studies characterizing immunotherapy and radiation dose scheduling for the optimal therapeutic effect is essential for designing clinical trials. METHODS AND MATERIALS: A new ablative radiation dosing scheme, personalized ultrafractionated stereotactic adaptive radiation therapy (PULSAR), was tested in combination with α-PD-L1 therapy in immune-activated and resistant syngeneic immunocompetent mouse models of cancer. Specifically, tumor growth curves comparing immunotherapy and radiation therapy dose sequencing were evaluated in immunologically cold and hot tumor models. The response relative to cytotoxic killer T cells was evaluated using an α-CD8 depleting antibody, and immunologic memory was tested by tumor rechallenge of cured mice. RESULTS: We report that both radiation and immunotherapy sequencing, as well as radiation therapy fraction spacing, affect the combination treatment response. Better tumor control was achieved by giving α-PD-L1 therapy during or after radiation, and spacing fractions 10 days apart (PULSAR) achieved better tumor control than traditional daily fractions. We showed that CD8+ depleting antibody abrogated tumor control in the PULSAR combination treatment, and certain treatment schedules induced immunologic memory. CONCLUSIONS: These results illustrate that radiation therapy dosing and scheduling affect tumor control, in combination with checkpoint blockade therapies. PULSAR-style radiation dosing is more complementary in combination with single-agent immunotherapy than traditional daily fractions in this preclinical model. Preclinical investigation could prove helpful in designing clinical trials investigating combination therapy.

DNA Sensing in Mismatch Repair-Deficient Tumor Cells Is Essential for Anti-tumor Immunity.

Increased neoantigens in hypermutated cancers with DNA mismatch repair deficiency (dMMR) are proposed as the major contributor to the high objective response rate in anti-PD-1 therapy. However, the mechanism of drug resistance is not fully understood. Using tumor models defective in the MMR gene Mlh1 (dMLH1), we show that dMLH1 tumor cells accumulate cytosolic DNA and produce IFN-ß in a cGAS-STING-dependent manner, which renders dMLH1 tumors slowly progressive and highly sensitive to checkpoint blockade. In neoantigen-fixed models, dMLH1 tumors potently induce T cell priming and lose resistance to checkpoint therapy independent of tumor mutational burden. Accordingly, loss of STING or cGAS in tumor cells decreases tumor infiltration of T cells and endows resistance to checkpoint blockade. Clinically, downregulation of cGAS/STING in human dMMR cancers correlates with poor prognosis. We conclude that DNA sensing within tumor cells is essential for dMMR-triggered anti-tumor immunity. This study provides new mechanisms and biomarkers for anti-dMMR-cancer immunotherapy.

Small molecular drugs reshape tumor microenvironment to synergize with immunotherapy.

Recently, immune checkpoint blockade (ICB), especially anti-programmed death 1 (anti-PD-1) and anti-programmed death-ligand 1 (anti-PD-L1) therapy, has become an increasingly appealing therapeutic strategy for cancer patients. However, only a small portion of patients responds to anti-PD treatment. Therefore, treatment strategies are urgently needed to reverse the ICB-resistant tumor microenvironment (TME). It has become clear that the TME has diminished innate sensing that is critical to activate adaptive immunity. In addition, tumor cells upregulate various immunosuppressive factors to diminish the immune response and resist immunotherapy. In this review, we briefly update the current small molecular drugs that could synergize with immunotherapy, especially anti-PD therapy. We will discuss the modes of action by those drugs including inducing innate sensing and limiting immunosuppressive factors in the TME.

Author Correction: Tumor cells suppress radiation-induced immunity by hijacking caspase 9 signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ß-Catenin regulates tumor-derived PD-L1.

In this issue of JEM, Du et al. (https://doi.org/10.1084/jem.20191115) report that enhancement of the ß-catenin signaling by Wnt or EGF treatment increases the expression of PD-L1 in an AKT and ß-catenin-dependent manner, and blocking the AKT pathway synergizes with anti-PD-1 in a glioblastoma model.

Telomere Stress Potentiates STING-Dependent Anti-tumor Immunity.

Telomerase is an attractive target for anti-tumor therapy as it is almost universally expressed in cancer cells. Here, we show that treatment with a telomere-targeting drug, 6-thio-2'-deoxyguanosine (6-thio-dG), leads to tumor regression through innate and adaptive immune-dependent responses in syngeneic and humanized mouse models of telomerase-expressing cancers. 6-thio-dG treatment causes telomere-associated DNA damages that are sensed by dendritic cells (DCs) and activates the host cytosolic DNA sensing STING/interferon I pathway, resulting in enhanced cross-priming capacity of DCs and tumor-specific CD8+ T cell activation. Moreover, 6-thio-dG overcomes resistance to checkpoint blockade in advanced cancer models. Our results unveil how telomere stress increases innate sensing and adaptive anti-tumor immunity and provide strong rationales for combining telomere-targeting therapy with immunotherapy.

Tumor cells suppress radiation-induced immunity by hijacking caspase 9 signaling.

High-dose radiation activates caspases in tumor cells to produce abundant DNA fragments for DNA sensing in antigen-presenting cells, but the intrinsic DNA sensing in tumor cells after radiation is rather limited. Here we demonstrate that irradiated tumor cells hijack caspase 9 signaling to suppress intrinsic DNA sensing. Instead of apoptotic genomic DNA, tumor-derived mitochondrial DNA triggers intrinsic DNA sensing. Specifically, loss of mitochondrial DNA sensing in Casp9-/- tumors abolishes the enhanced therapeutic effect of radiation. We demonstrated that combining emricasan, a pan-caspase inhibitor, with radiation generates synergistic therapeutic effects. Moreover, loss of CASP9 signaling in tumor cells led to adaptive resistance by upregulating programmed death-ligand 1 (PD-L1) and resulted in tumor relapse. Additional anti-PD-L1 blockade can further overcome this acquired immune resistance. Therefore, combining radiation with a caspase inhibitor and anti-PD-L1 can effectively control tumors by sequentially blocking both intrinsic and extrinsic inhibitory signaling.

Targeting innate sensing in the tumor microenvironment to improve immunotherapy.

The innate immune sensing pathways play critical roles in the defense against pathogen infection, but their roles in cancer immunosurveillance and cancer therapies are less defined. We propose that defective innate immune sensing inside the tumor microenvironment might limit T-cell responses to immunotherapy. A recent mechanistic understanding of conventional therapies revealed that both innate immune sensing and T-cell responses are essential for optimal antitumor efficacy. T-cell-based immunotherapy, particularly immune checkpoint blockade, has achieved great success in reactivating antitumor immune responses to lead to tumor regression, but only in a small fraction of patients. Therefore, incorporating conventional therapy that can increase innate sensing and immunotherapy should lead to promising strategies for cancer patients. Here, we review the innate sensing pathways related to cancer initiation/progression and therapies, summarize the recent key findings in innate immune sensing related to conventional therapies, evaluate current combination strategies, and highlight the potential issues of combinational therapies in terms of antitumor efficacy and toxicities.

Hypofractionated EGFR tyrosine kinase inhibitor limits tumor relapse through triggering innate and adaptive immunity.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are a first-line therapy for rapidly killing tumors such as those associated with non-small cell lung cancer by blocking oncogenic receptor signaling, but tumor relapse often occurs. Here, we have observed that hypofractionated EGFR TKI treatment (HypoTKI) is more potent than standard hyperfractionated EGFR TKI treatment (HyperTKI), and its antitumor effect associated with preventing tumor relapse depends on T cells. HypoTKI triggers greater innate sensing for type I IFN and CXCL10 production through the Myd88 signaling pathway to enhance tumor-specific T cell infiltration and reactivation. We also demonstrate that timely programmed cell death ligand-1 (PD-L1) blockade can synergize with HypoTKI to control advanced large tumors and effectively limit tumor relapse without severe side effects. Our study provides evidence for exploring the potential of a proper combination of EGFR TKIs and immunotherapy as a first-line treatment for treating EGFR-driven tumors.

NQO1 targeting prodrug triggers innate sensing to overcome checkpoint blockade resistance.

Lack of proper innate sensing inside tumor microenvironment (TME) limits T cell-targeted immunotherapy. NAD(P)H:quinone oxidoreductase 1 (NQO1) is highly enriched in multiple tumor types and has emerged as a promising target for direct tumor-killing. Here, we demonstrate that NQO1-targeting prodrug ß-lapachone triggers tumor-selective innate sensing leading to T cell-dependent tumor control. ß-Lapachone is catalyzed and bioactivated by NQO1 to generate ROS in NQO1high tumor cells triggering oxidative stress and release of the damage signals for innate sensing. ß-Lapachone-induced high mobility group box 1 (HMGB1) release activates the host TLR4/MyD88/type I interferon pathway and Batf3 dendritic cell-dependent cross-priming to bridge innate and adaptive immune responses against the tumor. Furthermore, targeting NQO1 is very potent to trigger innate sensing for T cell re-activation to overcome checkpoint blockade resistance in well-established tumors. Our study reveals that targeting NQO1 potently triggers innate sensing within TME that synergizes with immunotherapy to overcome adaptive resistance.