pyridostatin and Breast-Neoplasms

G-quadruplexes (G4s) are non-canonical nucleic acid structures involved in fundamental biological processes. As G4s are promising anticancer targets, in past decades the search for effective anticancer G4 binders aimed at the discovery of more cytotoxic ligands interfering with specific G4 structures at oncogenes or telomeres. Here, we have instead observed a significant activation of innate immune genes by two unrelated ligands at non-cytotoxic concentrations. The studied G4 binders (pyridostatin and PhenDC3) can induce an increase of micronuclei triggering the activation of the cytoplasmic STING (stimulator of interferon response cGAMP interactor 1) signaling pathway in human and murine cancer cells. Ligand activity can then lead to type I interferon production and innate immune gene activation. Moreover, specific gene expression patterns mediated by a G4 binder in cancer cells correlate with immunological hot features and better survival in human TCGA (The Cancer Genome Atlas) breast tumors. The findings open to the development of cytostatic G4 binders as effective immunomodulators for combination immunotherapies in unresponsive tumors.

Topics: Aminoquinolines; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line; Cytostatic Agents; Female; Fused-Ring Compounds; G-Quadruplexes; Humans; Immunity, Innate; Interferon Regulatory Factor-3; Interferon-beta; MCF-7 Cells; Melanoma, Experimental; Membrane Proteins; Mice; Micronuclei, Chromosome-Defective; Nucleotidyltransferases; Picolinic Acids; Transcriptional Activation

Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy.

Topics: Aminoquinolines; Animals; Breast Neoplasms; Cell Line; CRISPR-Cas Systems; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Polymerase theta; DNA-Directed DNA Polymerase; HEK293 Cells; Humans; Mice; Mitomycin; Picolinic Acids

Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here we show that the G-quadruplex-interacting drug pyridostatin promotes growth arrest in human cancer cells by inducing replication- and transcription-dependent DNA damage. A chromatin immunoprecipitation sequencing analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein abundance and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target of this drug. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions.

Topics: Aminoquinolines; Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Proliferation; DNA; DNA Damage; Drug Screening Assays, Antitumor; G-Quadruplexes; Humans; Molecular Weight; Picolinic Acids; Proto-Oncogene Mas; Structure-Activity Relationship; Tumor Cells, Cultured