Genomic instability, inflammatory signaling and response to cancer immunotherapy

Genomic and chromosomal instability are hallmarks of cancer and shape the genomic composition of cancer cells, thereby determining their behavior and response to treatment. Various genetic and epigenetic alterations in cancer have been linked to genomic instability, including DNA repair defects, oncogene-induced replication stress, and spindle assembly checkpoint malfunction. A consequence of genomic and chromosomal instability is the leakage of DNA from the nucleus into the cytoplasm, either directly or through the formation and subsequent rupture of micronuclei. Cytoplasmic DNA subsequently activates cytoplasmic DNA sensors, triggering downstream pathways, including a type I interferon response. This inflammatory signaling has pleiotropic effects, including enhanced anti-tumor immunity and potentially results in sensitization of cancer cells to immune checkpoint inhibitors. However, cancers frequently evolve mechanisms to avoid immune clearance, including suppression of inflammatory signaling. In this review, we summarize inflammatory signaling pathways induced by various sources of genomic instability, adaptation mechanisms that suppress inflammatory signaling, and implications for cancer immunotherapy

The cell-intrinsic innate immune response to DNA damage in human cells

The innate immune response has evolved to detect DNA from viruses and intracellular bacteria and differentiate this from our almost identical self-DNA. DNA detected in the cytoplasm is thought to be identified as foreign due to its location, and bacterial DNA in endosomes in recognised by its hypomethylated motifs. However, it is not clear how foreign DNA in the nucleus is differentiated from self-DNA, or what happens when the appearance of self-DNA is altered as it is in the case of DNA damage, a regular cellular occurrence. DNA damage has been shown to induce an immune response in tissues exposed to chemical mutagens – this has implications in the clearance of cancer cells after chemotherapy-induced DNA damage, with evidence that the immune response mounted by patients may determine chemotherapy responsiveness. To investigate the immune events following DNA damage, we analysed human keratinocytes, fibroblasts, and monocytes, and found that Etoposide, an inducer of double strand breaks, induced an early innate immune response, characterised by Type-I interferon and inflammatory cytokine production. This innate immune response to DNA damage was particularly potent in keratinocytes. This response required components of the cytoplasmic DNA sensing pathway, the predominantly nuclear sensor IFI16 and the cytoplasmic adaptor protein STING, but was independent of the enzyme cGAS, which is essential for the immune response to cytosolic DNA. The transcriptional and signalling profiles of the Etoposide-induced response differ from those of the classical pathogen sensing pathway, indicating that these two stimuli have overlapping but distinct pathways. This cell-intrinsic innate immune response also involved DNA damage response factors ATM and p53, suggesting that DDR components may interact with immune components to alert the cell to damage. The innate immune response that we observe may play a role in the inflammatory phenotype and immune clearance of cancer cells after DNA damage-inducing chemotherapy

R-loops and G4-structures: from DNA damage to innate immune response gene activation

Non-B DNA structures like R-loops and G-quadruplexes play a pivotal role in several cellular vital processes like DNA transcription regulation. Misregulation of said non-canonical DNA structures can often lead to genome instability, DNA damage, and, eventually, to the activation of an innate immune response. For such reasons they have been studied as adjuvants in anticancer therapies. Here we studied drugs targeting R-loops (Top1 poisons) and G4s (hydrazone derivatives) in order to observe their effects in terms of DNA damage induction and, subsequently, activation of innate immune response. We studied how non-cytotoxic doses of ampthotecin and LMP-776 impact on genome instability, are capable to induce DNA damage and micronuclei, and, eventually lead to an innate immune gene response via the cGAS/STING pathway. G-quadruplexes are another ubiquitous, non-canonical DNA structure, more abundant in telomeric regions, demonstrating a marked relation with the impairment of telomerase and the regulation of DNA replication and transcription. Furthermore, we investigated the properties of new-synthesized molecules belonging to the highly promising class of hydrazone derivatives, in terms of cytotoxicity, ability to stabilize G4-structures, induce DNA damage, and activate interferon-B production. Both Top1 poisons and G4-stabilizers possess several features that can be very useful in clinical applications, in light of their ability to stimulate innate immune response factors and exert a certain cell-killing power, plus they offer a broad and diverse range of treatment options in order to face a variety of patient treatment needs. It is for these very reasons that it is of uttermost importance that further studies are conducted on these compounds, in order to synthesize new and increasingly powerful and flexible ones, with fewer side effects to customize therapies on specific cancers’ and patients’ features

Molecular mechanisms and cellular functions of cGAS-STING signalling

The cGAS–STING signalling axis, comprising the synthase for the second messenger cyclic GMP–AMP (cGAS) and the cyclic GMP–AMP receptor stimulator of interferon genes (STING), detects pathogenic DNA to trigger an innate immune reaction involving a strong type I interferon response against microbial infections. Notably however, besides sensing microbial DNA, the DNA sensor cGAS can also be activated by endogenous DNA, including extranuclear chromatin resulting from genotoxic stress and DNA released from mitochondria, placing cGAS–STING as an important axis in autoimmunity, sterile inflammatory responses and cellular senescence. Initial models assumed that co-localization of cGAS and DNA in the cytosol defines the specificity of the pathway for non-self, but recent work revealed that cGAS is also present in the nucleus and at the plasma membrane, and such subcellular compartmentalization was linked to signalling specificity of cGAS. Further confounding the simple view of cGAS–STING signalling as a response mechanism to infectious agents, both cGAS and STING were shown to have additional functions, independent of interferon response. These involve non-catalytic roles of cGAS in regulating DNA repair and signalling via STING to NF-κB and MAPK as well as STING-mediated induction of autophagy and lysosome- dependent cell death. We have also learnt that cGAS dimers can multimerize and undergo liquid–liquid phase separation to form biomolecular condensates that could importantly regulate cGAS activation. Here, we review the molecular mechanisms and cellular functions underlying cGAS–STING activation and signalling, particularly highlighting the newly emerging diversity of this signalling pathway and discussing how the specificity towards normal, damage-induced and infection-associated DNA could be achieved

Potentiation of Cancer Therapy by Novel Pharmacological Inhibitors of DNA Repair

DNA-dependent protein kinase (DNA-PK) has a central role in the repair of DNA damage induced by radiotherapy of cancer. As such, the combination of DNA-PK inhibitors and radiotherapy are under investigation in cancer clinical trials. While much of this efficacy is attributed to cancer-cell intrinsic mechanisms, little is known about the immunological ramifications. This thesis evaluates the immunological effects of a novel inhibitor of DNA-PK, M3814 (peposertib), in combination with radiotherapy. Here, M3814 is shown to radiosensitise a panel of cancer cell lines. Furthermore, in KP.B6.F1 cells, a murine non-small cell lung carcinoma model, this cell death is accompanied by markers of immunogenic cell death including translocation of calreticulin to the cell-surface membrane and release of high mobility group box 1 (HMGB1). Additionally, treatment with radiotherapy and M3814 in MC38 cells increased expression of Interferon-β (Ifnβ), an anti-viral cytokine induced by the cGAS-STING pathway. In contrast, KP.B6.F1 cells failed to express IFNβ, despite evidence of increase formation of cGAS positive micronuclei following combination treatment. This was found to be due to a deficiency of STING which could be restored by the hypomethylating agent decitabine. In a syngeneic murine tumour model, addition of decitabine to combination treatment improved KP.B6.F1 tumour control and increased survival. Analysis of the immune compartments of KP.B6.F1 tumours determined increased infiltration of active T effector cells, active CD8+ T cells, inflammatory monocytes and M1 macrophages in mice treated with a combination of decitabine, M3814 radiotherapy. Together, this work demonstrates that addition of M3814 to radiotherapy not only increases cancer cell death but also increases the immunogenicity of tumour cells. Furthermore, it provides a potential therapeutic strategy to overcome cGAS-STING downregulation in cancer

An in silico analysis identifies drugs potentially modulating the cytokine storm triggered by SARS-CoV-2 infection

The ongoing COVID-19 pandemic is one of the biggest health challenges of recent decades. Among the causes of mortality triggered by SARS-CoV-2 infection, the development of an inflammatory “cytokine storm” (CS) plays a determinant role. Here, we used transcriptomic data from the bronchoalveolar lavage fluid (BALF) of COVID-19 patients undergoing a CS to obtain gene-signatures associated to this pathology. Using these signatures, we interrogated the Connectivity Map (CMap) dataset that contains the effects of over 5000 small molecules on the transcriptome of human cell lines, and looked for molecules which effects on transcription mimic or oppose those of the CS. As expected, molecules that potentiate immune responses such as PKC activators are predicted to worsen the CS. In addition, we identified the negative regulation of female hormones among pathways potentially aggravating the CS, which helps to understand the gender-related differences in COVID-19 mortality. Regarding drugs potentially counteracting the CS, we identified glucocorticoids as a top hit, which validates our approach as this is the primary treatment for this pathology. Interestingly, our analysis also reveals a potential effect of MEK inhibitors in reverting the COVID-19 CS, which is supported by in vitro data that confirms the anti-inflammatory properties of these compounds.Open access funding provided by Karolinska Institute.S

Homologous recombination-deficient cancers: approaches to improve treatment and patient selection

In order for cells to divide, all the DNA in a cell must be copied and divided into two new cells. However, DNA in our cells is constantly dealing with different types of damage, either from factors outside (eg UV rays in sunlight) or inside the body (eg due to errors that occur during the copying of the DNA). To ensure that this damage does not lead to permanent changes, cells have DNA damage repair mechanisms. An important mechanism is homologous recombination (HR) that repairs double-stranded DNA breaks. Without this mechanism, cells cannot survive. However, some cancers have a defect in HR. This is a paradox, because healthy cells do not survive without HR, while these cancer cells apparently survive without HR. BRCA1 and BRCA2 are two important genes in HR and a BRCA1/2 mutation is associated with an increased risk to develop breast and ovarian cancer. In this thesis, models are used in which a BRCA1/2 defect is induced to study an HR defect in cancer. Since 2013, PARP inhibitors have been approved for the treatment of patients with BRCA1/2-mutated breast and ovarian cancer. However, a defect in HR can also be caused by other gene mutations and these patients could also benefit from PARP inhibitors. In this thesis, a test is validated to select the right patients for PARP inhibitor treatment. The working mechanisms of PARP inhibitors are also being investigated to make treatment even more effective. In addition, the immune system plays an important role in cancers with an HR defect. Some of these mechanisms are described and investigated

Host DNA damage responses to the typhoid toxin of Salmonella enterica

The typhoid toxin is a virulence factor of the bacterial pathogen Salmonella enterica, which causes typhoid fever. The toxin has been shown to cause a DNA damage response in intoxicated human cells and to promote infection (Ibler et al., 2019). DNA damage responses have been shown to activate innate immune pathways via leakage of self-DNA into the cytosol and activation of the cGAS-STING pathway (Wolf et al., 2016). This thesis shows that purified typhoid toxin upregulates a type-I interferon-like response, including the antiviral ubiquitin-like interferon-stimulated gene 15 (ISG15), in a STING-dependent manner. ISG15 was upregulated in response to toxigenic Salmonella infection and overexpression of ISG15 reduced Salmonella burden, suggesting a role in host defence. Chronic Salmonella infection has been linked to gallbladder cancer (Di Domenico et al., 2017), and ISG15 has been implicated as a regulator of P53 and thus tumour suppression in response to DNA damage (Park et al., 2016). The toxin induces cell death in wild-type MEFs, whereas ISG15 KO MEFs survive and proliferate despite hallmarks of genomic instability such as micronuclei. This suggests that ISG15 may protect the host from pathogen-induced genomic instability. Taken together, this thesis provides new insights into host responses to the typhoid toxin, and the findings may be applicable to other bacterial genotoxins

Chemical modifications of DNA activate the cGAS/STING-signaling pathway even in the presence of the cytosolic exonuclease TREX1

To recognize pathogen threats, the innate immune system is equipped with pattern recognition receptors (PRRs) that bind to and are activated by pathogen-associated molecular patterns (PAMPs). Most PAMPs are conserved across species of microbes but at the same time not present in the host, allowing for the efficient discrimination between endogenous and foreign material. However, viruses rely on the host transcriptional and translational machinery to produce every viral component, and therefore do not really contain foreign molecules. It has become apparent that viruses instead are mainly detected via their nucleic acid genomes in the endosomes or cytosol of the host cell. However, virus sensing based on their nucleic acids comes at the risk of erroneous recognition of self-DNA - a process that leads to autoinflammation and possibly autoimmune disease. In particular, the receptor cGAMP synthase (cGAS) detects the mere presence of any DNA in the cytosol by binding its sugar phosphate backbone, and thus shows no apparent preference for sequence or specific molecular structures. Within this work, evidence is provided that specific damage-associated DNA modifications strongly enhance cGAS-dependent innate immune activation. DNA modifications occurring after UV irradiation, incubation with cytostatic agents, ROS exposure or as a consequence of neutrophil extracellular trap (NET) release were shown to potentiate the interferon (IFN) release in response to cytosolic DNA. However, this differential immune response was not due to higher affinity binding of the modified DNA to cGAS itself, but rather due to an impaired degradation by the cytosolic exonuclease TREX1. Resistance to TREX1 promoted an accumulation of the modified DNA in the cytosol, leading to a prolonged activation of the cGAS/STING-signaling pathway and the release of type I IFN. One well-known autoimmune disease driven by autoantibodies recognizing double-stranded DNA is lupus erythematosus (LE). Using the lupus-prone mouse model MRL/lpr, UV-damaged DNA (UV-DNA) was shown to be able to induce lupus-like lesions. Thus, UV-DNA could be a potential cause for the phototoxicity often observed in LE patients. Moreover, intravenous administration of UV-DNA induced a type I IFN response in MRL/lpr mice, which could be linked to F4/80-positive monocytes/macrophages. Together, these data show that under certain conditions self-DNA is transformed into a damage-associated molecular pattern (DAMP) that provides an additional layer of information to distinguish danger and damage from healthy state