Abstract 1469: T-oligo mediates DNA damage responses by modulating telomere associated proteins and telomerase

Abstract

Abstract T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3′ overhang of telomeres, elicits potent DNA-damage responses (DDRs) in cancer cells. However, the detailed molecular mechanism of T-oligo in cancer cells remains elusive. Recent studies confirmed that T-oligo can form intermolecular G-quadruplexes (G4), which are stabilized by hydrogen bonding of guanine residues, and cause disruption of the protective shelterin complex of telomeres. We hypothesize that single-stranded (SS) T-oligo and G4 T-oligo may modulate the shelterin proteins TRF2 and POT2 and thus induce DDRs. In this study, we utilized a pull-down assay using T-oligo, showing that T-oligo is co-localized with TRF2 and POT1 and indicating that T-oligo may interact with these telomeric proteins. We further investigated the modulation of these proteins by western blotting, showing that T-oligo treatment upregulates TRF2 by 2.2 and 3.0-fold (p<0.01) at 48h and 72h, respectively, and POT1 by 3.0-fold (p<0.02) both at 48h and 72h in melanoma cells (MM-AN). Immunofluorescence studies confirmed upregulation of TRF2 (2.4-fold) and POT1 (2.0-fold). Additionally we found that T-oligo can co-localize with telomere binding proteins TRF2 (88.4±4.5%) and POT1 (84.5±8%) using immunofluorescence. Using qRT-PCR, we found that T-oligo inhibited mRNA expression of hTERT, a catalytic subunit of telomerase, by 50%. It has been suggested that JNK activation may lead to downregulation of hTERT, hence we 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. Recently, it has been reported that the novel drug 6-Thio-2'-Deoxyguanosine (6-thio-dG), a nucleoside analogue of the approved drug 6-thioguanine, inhibits growth of cancer cells by its incorporation into the telomere via telomerase followed by subsequent shelterin disruption. Furthermore, this study aims to investigate the potential of 6-thio-dG in combination with T-oligo as an anticancer therapeutic. We first studied the concentrations of 6-thio-dG that are effective in inhibiting the growth of melanoma cells. We found that 1.25μM and 2.5μM 6-thio-dG significantly inhibited growth of melanoma cells by 1.6 and 3.8-fold, respectively (p<0.01). However, treatment of melanoma cells in combination with both 10μM T-oligo and 6-thio-dG (1.25μM or 2.5μM) did not significantly inhibit cell growth in comparison to T-oligo alone. T-oligo may downregulate hTERT, which is required for telomerase activity, and/or disrupt the shelterin complex, both of which are necessary for 6-thio-dG mediated inhibition of growth of melanoma cells. These results indicate that T-oligo and 6-thio-dG may induce their effects by a similar mechanism of action. Citation Format: Nabiha Haleema Khan, Zachary Schrank, Joseph Kellen, Sanjana Singh, Chike Osude, Neelu Puri, Gagan Chhabra. T-oligo mediates DNA damage responses by modulating telomere associated proteins and telomerase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1469.