Abstract 675: Facilitating G-quadruplex formation in the KRAS promoter with polypurine reverse Hoogsteen oligonucleotides

Abstract KRAS is a GTPase involved in the proliferation signaling of a number of growth factors, such as epidermal growth factor. The protein is mutated or upregulated in an array of cancers, including pancreatic and ovarian cancers, where it correlates with more aggressive and chemoresistant disease. Within the promoter of KRAS lies a G-rich region capable of forming the higher order non-canonical DNA structure, the G-quadruplex (G4). Stabilization of G4 formation in the mid-region of the KRAS promoter downregulates transcription and facilitates decreased growth and chemo-sensitization of pancreatic and ovarian cancer cells with aberrant KRAS signaling. Our collaborative team designed Polypurine Reverse Hoogsteen (PPRH) hairpin oligonucleotides that establish Watson Crick bonds with the pyrimidine strand within the KRAS-mid-G4-forming region to enhance G4 formation, downregulate KRAS transcription and mediate an anti-proliferative effect. In particular, two PPRHs were designed to interact with the 3’ and 5’ ends of the G4-forming region, along with two correlating W:C, and one scramble, controls. The binding of these oligonucleotides to the G4-forming region was verified by gel electrophoresis, and the cell activity was examined in vitro both by luciferase and in pancreatic and ovarian cancer cells. Using a luciferase construct driven by the KRAS promoter, only the PPRHs, and none of the control oligonucleotides, significantly decreased activity. Cytotoxicity experiments in KRAS dependent and independent cell lines demonstrated that PPRHs, but not control oligonucleotides, selectively suppressed proliferation only in the dependent pancreatic and ovarian cancer cell lines. Correlation of the cytotoxic effects of these potential therapeutic PPRHs with transcriptional downregulation is under investigation. PPRH also demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound in KRAS-dependent pancreatic cells, and sensitization to standard chemotherapeutic regimens is also being studied. These designed PPRH oligonucleotides selectively stabilize G4 formation within the KRAS mid promoter region, and represent an innovative approach both for G4-stabilization and KRAS modulation with potential for development into novel therapeutics. Citation Format: Alexandra Maria Psaras, Simonas Valiuska, Veronique Noe, Carlos J. Ciudad, Tracy A. Brooks. Facilitating G-quadruplex formation in the KRAS promoter with polypurine reverse Hoogsteen oligonucleotides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 675.