Characterization of nuclear and mitochondrial G-Quadruplex binding compounds in human Liposarcoma cell lines

G-quadruplexes (G4s) are non-canonical DNA secondary structures that can form in G-rich sequences, which are not randomly distributed along the genome but clustered in defined regions according to recent bioinformatic analyses. The fact that the majority of G4s were found around transcription start sites (TSS) led to the hypothesis that G4s take part in gene regulation. Recent findings confirmed that G4s are implicated in the transcription of genes involved in differentiation, cancer progression and metabolic regulation. In the first part of the study, I focused on characterizing unreported G4s present at relevant genes associated with Liposarcoma (LPS), in particular CDK4 gene. Being LPS a class of soft tissue sarcomas particularly resistant to current chemotherapy, new therapeutic strategies are strongly needed. The most frequent subtypes of LPS are well- and de-differentiated liposarcomas (WD/DDLS). Both WD/DDLS are characterized by the amplification of chromosome segment 12q13-15, which carries the oncogenes MDM2 and CDK4. In the promoters of MDM2 and CDK4, our group has identified G4s by Chromatin Immunoprecipitation with an anti-G-Quadruplex antibody (G4 ChIP). Here we evaluated the activity of the recently emerged G4-binder, named SOP1812, against a panel of Liposarcoma cell lines. SOP1812 displayed an antiproliferative activity in the nanomolar concentrations and induced significant down-regulation of MDM2 and CDK4 transcription and protein expression levels. Intriguingly, the decrease of MDM2 inhibited the ubiquitin-mediated degradation of the tumor suppressor p53, restoring the signal of programmed cell death. Thus, SOP1812 was able to counteract the effects of MDM2 and CDK4 amplification in WD/DDLS, acting at G4s in their promoters. This work presents an efficient compound and highlights a possible new therapeutic strategy against WD/DDLS. Formation of G4 hybrid structure was identified within the mitochondrial DNA non-coding regulatory region, which takes part in the premature termination of mitochondrial RNA polymerase at the conserved sequence box II (CSBII), necessary for the switch between mitochondrial DNA (mtDNA) transcription and replication. In the second part of the project, we developed and evaluated a novel mitochondrial G4-ligand (mtPhamPEG) characterized by a well-known G4-ligand moiety functionalized with the (triphenylphosphonium) TPP+ mitochondrial moiety. We first validated the mitochondrial localization of mtPhamPEG into mitochondria and its cytotoxicity in a panel of cancer and non-cancer cells. mtPhamPEG induced significant reduction of cancer cells growth without affecting normal cells growth. In addition, we observed that the stabilization of mitochondrial CSB II-G4 by mtPhamPEG led to decrease of both mitochondrial transcription and replication in different cancer cells. In conclusion, here we present an alternative antiproliferative strategy based on dysregulation of mitochondrial metabolism of cancer cells.