Abstract 4128: Mechanism of action of G-quadruplex forming oligonucleotide homologous to the telomere overhang in melanoma
Authors: Gagan Chhabra, Luke Wojdyla, Ankita Sanjali, Mark Frakes, Marko Ivancich, Pooja Vinay, Zachary Schrank, Benjamin E. Ramirez, Neelu Puri
Journal: Cancer Research (2017)

Abstract T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3'-telomeric overhang of telomeres, elicits potent DNA-damage responses (DDRs) in cancer cells. However, the detailed molecular mechanism of action of T-oligo in cancer cells is largely unknown. Recent studies suggest that GROs can form G-quadruplexes (G4) which are stabilized by the hydrogen-bonding of guanine residues. This study aims to examine the G4-forming capabilities of T-oligo in vitro and investigates the molecular mechanism of single-stranded (SS) and G4-T-oligo induced DDRs in melanoma cells (MM-AN). G4-formation by T-oligo was confirmed using the SS-T-oligo and G4-T-oligo on a polyacrylamide gel under non-denaturing conditions. NMR studies for T-oligo in KCl confirmed that T-oligo forms G4 structures. Immunofluorescence studies conducted with an anti-G-quadruplex antibody (BG4), a G4 detecting antibody, showed 88.4% co-localization of T-oligo and BG4 in the nuclei of melanoma cells confirming the ability of T-oligo to form G-quadruplexes inside melanoma cells. While G4-T-oligo was found more stable in nuclease degradation assay by DNase I, it had a decreased anti-proliferative effects compared to SS-T-oligo. However, G4-T-oligo had similar cellular uptake as SS-T-oligo. Further, two shelterin complex proteins TRF2 and POT1 which are mainly found at the telomere ends were found to be upregulated (2.0 fold) by T-oligo suggesting TRF2 and POT1 mediated telomere overhang dissociation. We also found that T-oligo can co-localize with telomere binding proteins TRF2 and POT1 by 88.4±4.5% (n=12) and 84.5±8% (n=10) respectively. Western blot analysis results also showed upregulation of both p-JNK and total JNK by 4.0- and 2.0-fold respectively. To further confirm the involvement of p-JNK in T-oligo mediated apoptosis we used a specific JNK inhibitor SP600125. Western blot analysis showed that T-oligo mediated upregulation of p-JNK was reversed in presence of SP600125. Results from an MTT assay showed a 73.8% decrease in cell viability after T-oligo treatment alone; however, cell viability was decreased to 45.8%, and 25.3% when SP600125 was present at concentrations of 10 μM, and 12 μM respectively, in comparison to diluent. T-oligo also inhibited mRNA expression of hTERT; a catalytic subunit of telomerase by 50% .We further investigated the effect of the JNK inhibitor SP600125 on hTERT expression and found that treatment with SP600125 in presence of T-oligo partially reversed the downregulation of hTERT. We found a 16% decrease in hTERT expression in comparison to 50% reduction by T-oligo treatment alone. In conclusion, these studies demonstrate that T-oligo can form G-quadruplexes and the anti-proliferative mechanism of T-oligo may be mediated through POT1 and TRF2 as well as via JNK-activation inducing hTERT-inhibition in melanoma cells. Citation Format: Gagan Chhabra, Luke Wojdyla, Ankita Sanjali, Mark Frakes, Marko Ivancich, Pooja Vinay, Zachary Schrank, Benjamin E. Ramirez, Neelu Puri. Mechanism of action of G-quadruplex forming oligonucleotide homologous to the telomere overhang in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4128. doi:10.1158/1538-7445.AM2017-4128