Investigating Mechanism Of Action Of Oligonucleotides Homologous To The Telomere Overhang In Melanoma

Telomeres have become a popular target for novel cancer therapeutics because activation of telomerase is associated with development of immortality in nearly 90% of all cancers. It has been shown that administration of an oligonucleotide homologous to the telomere overhang called T-oligo (5’-GTTAGGGTTAG-3’) induces potent DNA damage responses (DDRs) in several cancers, including melanoma, and causes minimal toxicity to normal cells. Our study focuses on T-oligo which has the guanine rich character of the telomere 3’ overhang. The folding and stabilization of G-quadruplexes (G4) which are G-rich structures at the end of telomeres may be involved in regulation of telomere length and cell cycle regulation. To date, T-oligo has not been shown to form G4, although evidence suggests it has a G4-mediated mechanism of action. In this study, we investigate T-oligo9s ability to form G4 structures in vitro. We hypothesize the additional secondary structure of G4 configured T oligo may confer increased stability, resistance to nucleases in serum and, initiate DDRs. G4 formation is a potential target since G4 presence can cause telomere uncapping leading to DDRs. In our studies, G4 T-oligo was formed by heating various concentrations of T-oligo in KCl solutions to 90°C and incubating at 60°C for 24-36 hours. G4 T-oligo formation was verified using native PAGE and NMR spectroscopy before subjecting it to DNase1 degradation and analyzing with native PAGE. We also analyzed the uptake and anti-proliferative ability of G4 T-oligo using FACS analysis and MTT assay respectively. The change in expression of TRF2 and JNK activity was performed using western blot analysis since T-oligo may induce activation of SAPK/JNK pathway. We further investigated the interaction between T-oligo and TRF2 using immunofluorescence (IF) with FITC labeled T-oligo in MM-AN melanoma cells. Densitometric analysis after nuclease digestion demonstrated a 52.6% decrease in T-oligo and a 20% decrease in G4 T-oligo as well as a 96% increase of single stranded T-oligo within the G4 T-oligo sample after 90 minutes, indicating nuclease resistance of G4-T-oligo. FACS analysis revealed G4 T-oligo and T-oligo had similar cellular uptake but G4 T-oligo was shown to be less effective in inhibiting cell proliferation suggesting the reduced anti-proliferative activity of G4 T-oligo is not likely attributed to cellular uptake. Western blot results showed upregulation of both p-JNK and total JNK by 4.0- and 2.0-fold respectively after 24 hour treatment. JNK has previously been demonstrated to be activated by G4 stabilizing ligands, providing more evidence that T-oligo may induce telomere dysfunction and upregulation of JNK activity. IF showed 80% co-localization of T-oligo and TRF2 in the nucleus suggesting T oligo may induce dissociation of TRF2 from the telomere shelterin complex causing DDRs. In conclusion, formation of G4 species by T-oligo may induce DDRs and future studies may provide insights into its mechanism of action and suggests new avenues for research. Citation Format: Mark Frakes, Luke Wojdyla, Marko Ivancich, Perlina Fortinberry, Pooja Vinay, Priyanka Pandey, Gagan Chhabra, Neelu Puri. Investigating mechanism of action of oligonucleotides homologous to the telomere overhang in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-048.