A cell-based screen identifies HDAC inhibitors and PLK inhibitor as activators of RIG-I signaling

Enhancing the immune microenvironment in cancer by targeting the nucleic acid sensors is becoming a potent therapeutic strategy. Among the nucleic acid sensors, activation of the RNA sensor Retinoic Acid-inducible Gene (RIG-I) using small hairpin RNAs has been shown to elicit powerful innate and adaptive immune responses. Given the challenges inherent in pharmacokinetics and delivery of RNA based agonists, we set out to discover small molecule agonists of RIG-I using a cell-based assay. To this end, we established and validated a robust high throughput screening assay based on a commercially available HEK293 reporter cell line with a luciferase reporter downstream of tandem interferon stimulated gene 54 (ISG54) promoter elements. We first confirmed that the luminescence in this cell line is dependent on RIG-I and the interferon receptor using a hairpin RNA RIG-I agonist. We established a 96-well and a 384-well format HTS based on this cell line and performed a proof-of-concept screen using an FDA approved drug library of 1200 compounds. Surprisingly, we found two HDAC inhibitors Entinostat, Mocetinostat and the PLK1 inhibitor Volasertib significantly enhanced ISG-luciferase activity. This luminescence was substantially diminished in the null reporter cell line indicating the increase in signaling was dependent on RIG-I expression. Treatment of tumor cell lines with Entinostat, Mocetinostat or Volasertib induced interferon signature genes and increased RIG-I induced cell death in a mammary carcinoma cell line. Taken together, our data indicates an unexpected role for HDAC1,-3 inhibitors and PLK1 inhibitors in enhancing RIG-I signaling and highlight potential opportunities for therapeutic combinations.

Nucleic Acid Sensing in the Tumor Vasculature

Endothelial cells form a powerful interface between tissues and immune cells. In fact, one of the underappreciated roles of endothelial cells is to orchestrate immune attention to specific sites. Tumor endothelial cells have a unique ability to dampen immune responses and thereby maintain an immunosuppressive microenvironment. Recent approaches to trigger immune responses in cancers have focused on activating nucleic acid sensors, such as cGAS-STING, in combination with immunotherapies. In this review, we present a case for targeting nucleic acid-sensing pathways within the tumor vasculature to invigorate tumor-immune responses. We introduce two specific nucleic acid sensors—the DNA sensor TREX1 and the RNA sensor RIG-I—and discuss their functional roles in the vasculature. Finally, we present perspectives on how these nucleic acid sensors in the tumor endothelium can be targeted in an antiangiogenic and immune activation context. We believe understanding the role of nucleic acid-sensing in the tumor vasculature can enhance our ability to design more effective therapies targeting the tumor microenvironment by co-opting both vascular and immune cell types.

Increased presence of nuclear DNAJA3 and upregulation of cytosolic STAT1 and of nucleic acid sensors trigger innate immunity in the ClpP-null mouse

Mitochondrial dysfunction may activate innate immunity, e.g. upon abnormal handling of mitochondrial DNA in TFAM mutants or in altered mitophagy. Recent reports showed that also deletion of mitochondrial matrix peptidase ClpP in mice triggers transcriptional upregulation of inflammatory factors. Here, we studied ClpP-null mouse brain at two ages and mouse embryonal fibroblasts, to identify which signaling pathways are responsible, employing mass spectrometry, subcellular fractionation, immunoblots, and reverse transcriptase polymerase chain reaction. Several mitochondrial unfolded protein response factors showed accumulation and altered migration in blue-native gels, prominently the co-chaperone DNAJA3. Its mitochondrial dysregulation increased also its extra-mitochondrial abundance in the nucleus, a relevant observation given that DNAJA3 modulates innate immunity. Similar observations were made for STAT1, a putative DNAJA3 interactor. Elevated expression was observed not only for the transcription factors Stat1/2, but also for two interferon-stimulated genes (Ifi44, Gbp3). Inflammatory responses were strongest for the RLR pattern recognition receptors (Ddx58, Ifih1, Oasl2, Trim25) and several cytosolic nucleic acid sensors (Ifit1, Ifit3, Oas1b, Ifi204, Mnda). The consistent dysregulation of these factors from an early age might influence also human Perrault syndrome, where ClpP loss-of-function leads to early infertility and deafness, with subsequent widespread neurodegeneration.

Nucleic Acid Sensing in the Tumor Vasculature

Endothelial cells form a powerful interface between tissues and immune cells. In fact, one of the underappreciated roles of endothelial cells is to orchestrate immune attention to specific sites. Tumor endothelial cells have a unique ability to dampen the immune responses and thereby maintain an immunosuppressive microenvironment. Recent approaches to trigger immune responses in cancers have focused on activating nucleic acid sensors such as cGAS/STING in combination with immunotherapies. In this review, we present a case for targeting nucleic acid sensing pathways within the tumor vasculature to invigorate tumor immune responses. We introduce two specific nucleic acid sensors, the DNA sensor TREX1 and the RNA sensor RIG-I and discuss their functional roles in the vasculature. Finally, we present perspectives on how these nucleic acid sensors in the tumor endothelium can be targeted in an antiangiogenic and immune activation context. We believe understanding the role of nucleic acid sensing in the tumor vasculature can enhance our ability to design more effective therapies targeting the tumor microenvironment.

Cellular Metabolites Regulate Central Nucleic Acid Sensing Pathways

Detection of pathogen-derived DNA or RNA species by cellular nucleic acid sensors prompts release of anti-microbial interferons and cytokines. In contrast to their protective anti-microbial functions, inappropriate or excessive activation of nucleic acid sensors can cause inflammatory diseases. Nucleic acid sensing is therefore tightly controlled by regulatory factors acting through both transcriptional and post-transcriptional mechanisms. Recently, it has become clearer that metabolic pathways—previously thought to be unconnected with immune responses—can influence nucleic acid sensing. This regulation can be observed when immune system cells undergo metabolic reprogramming in response to stimulation with pathogen-associated molecular patterns such as lipopolysaccharide from gram negative bacteria. Metabolic reprogramming leads to accumulation and secretion of metabolites, which have been mostly viewed as end-products of processes providing cellular energy and building blocks. However, metabolites have now been identified as important regulators of nucleic acid sensing. This mini-review aims to outline current knowledge on regulation of central nucleic acid sensing pathways by metabolites during metabolic reprogramming.

Application strategies of peptide nucleic acids toward electrochemical nucleic acid sensors

Peptide nucleic acids (PNAs) have attracted tremendous interest in the fabrication of highly sensitive electrochemical nucleic acid biosensor due to their higher stability and increased sensitivity than common DNA probes....

Hybridizing clinical translatability with enzyme-free DNA signal amplifiers: recent advances in nucleic acid detection and imaging

Strand amplifying HCR and CHA are adaptable with signalers for novel and clinically translatable nucleic acid sensors and imaging agents.