Abstract 3679: Nanotechnology delivery and mechanism of action of telomere oligonucleotides in melanoma

Abstract

Abstract In nearly 90% of cancer cell types, telomere ends are stabilized by inappropriate activation of telomerase, thereby preventing telomeres from sequential shortening and establishing conditions which promote malignant transformation and immortality. As a result, telomeres and telomerase have recently become attractive targets in the development of novel anticancer therapeutics. Administration of an 11-mer oligonucleotide homologous to the 3′ overhang of the telomere, T-oligo, is presumed to mimic or signal for damaged telomeric DNA, thereby inducing inherent telomere-based DNA damage responses. Exposure to T-oligo induces cell cycle arrest, senescence, apoptosis, and differentiation in a cancer cell-type specific manner. Intravenous administration of T-oligo drastically reduces tumor burden in mice. Remarkably, T-oligo has minimal side-effects on normal cells, and has shown no detectable toxicity in in vivo mouse studies. However, as an oligonucleotide, T-oligo has limited stability due to serum and intracellular nucleases. We have previously improved the delivery of T-oligo by complexing it with a novel cationic alpha helical peptide, PVBLG-8, which improved its efficacy both in vitro and in vivo. Moreover, the T-oligo-PVBLG (TOP) nanocomplex demonstrated increased charge stability and diameter in comparison to T-oligo. In this study we have further optimized the TOP¬¬¬ nanocomplex, thereby improving the physiochemical properties of T-oligo for delivery and bringing T-oligo closer to clinical fruition as an innovative, anticancer therapeutic. The binding efficiency of T-oligo and PVBLG was optimized by measuring the amount of naked DNA under a range of nitrogen/phosphorus (N/P) ratios. We then compared the biodistribution of both T-oligo and TOP in vivo at various N/P ratios. T-oligo was conjugated with FITC and intravenously administered to the tail vein of mice in the presence or absence of PVBLG and monitored using molecular imaging. We found that T-oligo distribution was isolated to the kidneys, indicating that its small size makes it susceptible to renal filtration. In contrast, the TOP complex, which has a larger diameter, was able to bypass renal filtration and enter the liver and/or lungs. We also found that PVBLG was able to protect T-oligo from nucleolytic degradation in vitro. ¬¬¬¬In addition, to further elucidate T-oligo's mechanism of action, we investigated its ability to form G-quadruplexes, since increasing G-quadruplex stability is known to induce apoptosis. G-quadruplex formation by T-oligo was confirmed by NMR and native-PAGE. Furthermore, since increasing G-quadruplex stability is known to activate the SAPK/JNK signaling pathway, we also investigated T-oligo's effect on the SAP/JNK pathway using immunoblotting. Collectively, these studies will significantly advance the therapeutic utility of T-oligo against melanoma. Citation Format: Luke Wojdyla, Neelu Puri. Nanotechnology delivery and mechanism of action of telomere oligonucleotides in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3679. doi:10.1158/1538-7445.AM2015-3679