Abstract 1234: G4 binders as potential immunostimulatory compounds for cancer therapy

Abstract

Abstract G-quadruplex (G4) is non-canonical nucleic acid structure involved in a plethora of fundamental biological processes. In past decades, it has been studied as a promising pharmaceutical target for anticancer therapy. However, no G4-targeting agent has shown significant clinical effects up to date. Pyridostatin (PDS), a well-known G4 binder, can induce double-stranded DNA breaks and genome instability. We have recently shown that PDS and other G4 binders can trigger micronuclei formation in cancer cells at non-cytotoxic concentrations [1]. As micronuclei can play a crucial role in linking genome instability to innate immunity, we have here wondered whether G4 binders can induce immune gene activation in human MCF-7 breast cancer cells. By using RNA-Seq technology, we show that non-cytotoxic doses of PDS can activate Interferon beta- and IRF3-dependent genes leading to an innate immune gene response in human cancer cells. In addition, immune gene activation follows the induction of micronuclei formation supporting cytoplasmic DNA as a trigger of immune gene activation. Immunofluorescence assays showed that cGAS (a cytoplasmic DNA sensor) can bind to and is activated by micronuclei. In addition, cytostatic PDS concentrations activate STING and IRF3 factors leading to activated immune gene expression. STING inhibition with siRNAs, CRISPR knockout or a chemical inhibitor resulted in a suppression of immune gene expression and Interferon-beta (IFN-B) production in human MCF-7 and murine B16 cell lines, showing that STING is required for PDS-induced immune gene activation in cancer cells. We have then extended the study to a structurally-unrelated G4 binder, PhenDC3, and the results show that PhenDC3 can induce micronuclei formation and IFN-B production in a STING-dependent manner. Then, using TCGA-BRCA dataset, we show that specific gene expression patterns mediated by PDS correlate with immunological hot features of human tumors, such as anti-tumor immune cell infiltration. We could also define a specific PDS gene signature that can predict a better survival of breast cancer patients, suggesting an impact of immunomodulation potential of PDS or other G4 binders in human patients. Thus, our results provide strong evidence that G4 binders can activate innate immune genes in human and murine cancer cells with therapeutic potentials, and that G4 binder-induced micronuclei accumulation can trigger the cGAS-STING signaling pathway. The findings may open to the development of novel immunotherapeutic anticancer strategies based on G4-targeting compounds.