Mechanistic roles of R-loops and micronuclei in the innate immune response induced by anticancer G-quadruplex binders

Almost 1000 compounds are known to bind specifically to G-quadruplexes (G4), a type of non-canonical DNA structure. They were developed to discover new anticancer drugs, as non-canonical DNA structures are considered promising oncological targets. Nevertheless, few entered into clinical trials and none showed efficacy in patients. Recently, my group reported that PDS, a specific G4 ligand, stimulates micronuclei formation in human U2OS cells and that the mechanism of micronuclei generation involves an accumulation of double-strand DNA breaks (DSB) due to increased levels of R-loops, another type of non-canonical DNA/RNA structures. Micronuclei can be a source of cytoplasmatic DNAs that can activate Interferon-stimulated genes (ISG) and innate immunity response. As we have data supporting that PDS can indeed activate ISG in human cancer cells in a STING-dependent manner, my hypothesis is that ligand-stabilized G4s cause R-loopmediated DSBs, triggering micronuclei formation and ISG activation, which is likely mediated by cytoplasmic DNA sensors and the STING pathway. This may result in a significant stimulation of an immune response against the tumour. Here, I pursue a line of investigation to discover new anticancer G4 ligands that is not based on improving the cytotoxic potency, but rather on improving the ability to elicit immuno-stimulatory pathways in cancer cells. My specific aims are: - to define the roles of R-loops and DNA repair factors in nuclear PDS-triggered DNA repair mechanisms leading to micronuclei generation in cancer cells; - to establish the role of the cytoplasmic DNA sensors and related-pathways in PDS-induced immune response in human cancers and cell lines; - to discover new G4 ligand hits with optimised activity in inducing micronuclei/immune response in cancer cells. The project is divided into four work-packages, three addressing specific molecular and genetic aspects of the mechanism, and one focused on the discovery of effective G4 ligands as immune stimulation and anti-tumor agents. By using NGS and CRISPR technologies, we will investigate the mechanisms leading from DSBs to micronuclei, including the roles of replication and repair pathways, and then ISG activation in several cancer cell types. By bioinformatics, we will investigate nucleic acid immunity gene mutations in human cancers. Structure-activity studies will define structural determinants of immuno-stimulation activity of G4 ligands. We will use a murine tumor model to demonstrate the stimulation of the immune system against the tumor by G4 ligands. The results will demonstrate the role of R-loops in DSB formation at functional genomic regions, and the DNA repair factors in micronuclei generation. In particular, we will define the role played by BRCA1/2 gene in micronuclei formation. In addition, the findings will establish the main cytoplasmatic pathway that leads to the activation of ISG in cancer cells. Investigating a new pharmacophore, we expect to find more effective and potent G4 ligands in activating ISG in cancer cells If successful, the project will discover new ligand hits with the potential to optimise clinical immunotherapeutic protocols in unresponsive cancers. The results will establish a new rationale to discover G4 ligands, or other chemicals, as effective drugs in human cancer patients.