Abstract 1958: T-oligo mechanism of action and delivery with an alpha-helical peptide

Abstract

Abstract When telomeres are critically short, the telomere overhang is exposed which induces cell senescence or apoptosis. T-oligo, an oligonucleotide homologous to the telomere overhang, induces DNA damage responses similar to those induced after exposure of the telomere overhang, and is an effective, cancer-specific therapy both in vitro and in vivo. However, T-oligo's mechanism of action is unclear, and as an oligonucleotide, its inherent instability severely limits its clinical use. Hence, our studies focus on its mechanism of action, stabilization and improved delivery. To understand T-oligo's mechanism of action, we studied the role of the telomere specific poly(ADP-ribose) polymerase, tankyrase-1, in T-oligo induced DNA damage responses. In normal cells, tankyrase-1 PARsylates TRF1 which causes disruption of the shelterin complex and exposure of the T-loop. This exposure activates telomerase resulting in elongation of telomeres leading to possible cell immortality. This mechanism has made tankyrase-1 inhibition a potential therapy for telomerase inhibitor resistant cancers where tankyrase-1 is found to be upregulated. Since several studies suggest that tankyrase-1 and T-oligo may have important roles in p53 mediated DNA damage responses to critically shortened telomeres, we measured total and phospho-p53 in MU melanoma cells after treatment with tankyrase-1 inhibitors by immunoblot analysis. We found that cells treated with T-oligo and tankyrase-1 inhibitors, 3AB and XAV939, exhibited a 4 and 2 fold down regulation of phospho-p53, and 2.5 and 2 fold down regulation of total p53, respectively, compared to T-oligo alone which increased phospho-p53 and total p53 by 4 and 2 fold, respectively. Furthermore, XAV939, when given with T-oligo, completely abrogated T-oligo induced inhibition of cell proliferation, indicating that tankyrase-1 may play a role in T-oligo induced DNA damage responses. To improve T-oligo's delivery, we combined T-oligo with a nano-sized cationic α-helical polypeptide (PVBLG150-8) which stabilizes the negatively charged T-oligo. We complexed and delivered T-oligo with PVBLG150-8 into H358 lung cancer and AN melanoma cells. We found that in vitro delivery of the T-oligo and peptide (0.025-.25 mg/ml) complex inhibited AN melanoma cell growth in a dose responsive manner from 4.5-8.3 fold compared to untreated controls, while T-oligo alone inhibited growth by only 3 fold. Similarly, when H358 cells were given T-oligo complexed with PVBLG150-8 (0.0125-.25 mg/ml), their growth was inhibited in a dose dependent manner from 3.3-6.6 fold, compared to T-oligo alone (2 fold). Our studies indicate that T-oligo has great potential as an anti-cancer therapy, and due to its unique mechanism of action, it could be used in patients with upregulated tankyrase-1 activity. Additionally, T-oligo delivery and efficacy can be greatly improved when complexed with a cationic α-helical polypeptide. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1958. doi:1538-7445.AM2012-1958