Abstract 2965: Induction of DNA damage responses by T-oligo and 6-thio-dG via modulating telomere associated proteins and telomerase

Abstract

Abstract T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3′ telomeric overhang, has been shown to elicit potent DNA-damage responses (DDRs) in numerous cancer cell lines. However, the detailed molecular mechanism by which T-oligo induces these responses in cancer cells remains elusive. Additionally, it has recently 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. This study aims to investigate the mechanism of action of T-oligo and 6-thio-dG at the telomere, as well as to assess the therapeutic potential of T-oligo in combination with 6-thio-dG. Finally, we also investigated the therapeutic efficacy of T-oligo in combination with Vemurafenib, an FDA-approved BRAF inhibitor, in BRAF-mutant melanoma cells as a potential combinatorial therapy for aggressive BRAF-mutant melanoma. Previous studies have shown that T-oligo is able to reduce expression of hTERT, the catalytic subunit of telomerase, by 50% in MM-AN melanoma cells. Results from this study showed that 2.5µM of 6-thio-dG increased expression of hTERT by 50% in this cell line, as seen by qPCR. This suggests that the mechanism of action of 6-thio-dG and T-oligo may involve modulation of telomerase in different ways. Treatment of MM-AN melanoma cells with 2.5μM 6-thio-dG induced a 1.54- and 1.75-fold upregulation of TRF2 at 48 and 72 hours, respectively, and an upregulation of POT1 by 1.63- and 2.15-fold at 48 and 72 hours, respectively, as seen by immunoblotting. These results suggest that 6-thio-dG may induce shelterin dissociation events similar to T-oligo, which has been shown to upregulate TRF2 and POT1 in this cell line. Our studies showed 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), by MTT assay. However, treatment of melanoma cells with 10μM T-oligo in combination with 6-thio-dG (1.25μM or 2.5μM) did not significantly inhibit cell growth in comparison to T-oligo alone, suggesting that these drugs may induce DDRs by similar mechanisms. Preliminary results indicate that, in MU melanoma cells, which are positive for the V600E BRAF mutation, treatment with T-oligo and Vemurafenib induced an additive effect in comparison to Vemurafenib or T-oligo alone. These results suggest that T-oligo and Vemurafenib may be potential candidates for combinatorial therapy for aggressive BRAF-mutant melanomas. Citation Format: Zachary Schrank, Nabiha Khan, Joseph Kellen, Sanjana Singh, Chike Osude, Neelu Puri. Induction of DNA damage responses by T-oligo and 6-thio-dG via modulating telomere associated proteins and telomerase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2965.