Multiomic characterization of the effects of G-quadruplex binders on R-loop homeostasis and innate immune response in human cancer cells

Abstract

R-loops are non-canonical DNA structures consisting of a DNA-RNA hybrid and a displaced ssDNA. R-loops can be structurally compatible with G-quadruplexes, that are secondary DNA structures composed by three stacked tetrads of guanosine. G4s stabilization and R-loops accumulation in cells have been associated with genomic instability and DNA damage. Here, through bioinformatic analysis of genomic R-loop maps and the development of a specific tool to better annotate R-loop regions to gene, we propose that stabilization of G4 structures with specific ligands leads to accumulation of DNA damage and genome instability in cancer cells and that this process is mediated by R-loop stabilization. Moreover, we found that G4 ligand pyridostatin stimulates formation of micronuclei, a hallmark of genome instability. Micronuclei accumulation has been associated with innate immune response triggering through cGAS/STING pathway activation. Activation of this pathway leads to the expression of cytokines, interferons and interferon-stimulated genes. Innate immune system modulation has been proposed as a promising therapeutic strategy for cancer treatment. Here, through analysis of RNA-seq data, we demonstrate that pyridostatin may induce an innate immune stimulation through micronuclei induction in cancer cells. Moreover, to clarify the role of cGAS/STING pathway and the effects of its perturbation in human tumor tissues, we analyzed mutations and expression levels of genes involved in this pathway across 31 cancer types and ~7800 tumor samples from The Cancer Genome Atlas (TCGA). Alterations in mutation status or expression in these genes have been related with innate immune response activation and patient survival and other immune tumor microenvironment features. Our findings indicate that these genes are rarely mutated in human cancers, while their expression may affect the interaction of the tumor with host immune cells affecting disease progression and patient survival.