Abstract 1436: T-oligo's mechanism of action in melanoma cells
Authors: Agnihotri Archit , Rager Taylor , Fan Kelly , Alaraj Hyatt , Miller Lawrence , Puri Neelu 
Published: June 2023

Abstract
Telomeres have become an attractive target for anticancer therapeutics and in 2022, an estimated 99,780 new cases and 7,650 people are expected to die of melanoma. T-oligo, a hexanucleotide tandem repeat oligonucleotide that is homologous to the 3′ overhang of telomeres has been shown to have potent anticancer effects in-vitro by inhibiting the growth of cancer cells. There are three mechanisms of action proposed for T-oligo, which are shelterin dissociation model, exposed telomere mimicry model, and G-quadruplex formation. Previous studies utilizing NMR spectroscopy and immunofluorescence assays have shown that T-oligo forms G-quadruplexes in the nucleus of the melanoma cells. We hypothesize that formation of G-quadruplexes can be detected with the help of FRET analysis and T-oligo could be delivered using lipid nanoparticles. Immunofluorescence assays and an acceptor photobleaching method were used to detect FRET and confirm the formation of G-quadruplexes in the nucleus of melanoma cells. Melanoma cells were plated and grown until 70% confluent in 8-well glass chamber slides and were treated with single-labeled FITC-T-oligo (11-mer), and Cy3-T-oligo (11-mer), and double labeled T-oligo 11-mer (T-11) and 22-mer (T-22) labeled with FITC on the 5′ end and Cy3 on the 3′ end. Slides were mounted and cells were observed under a fluorescent microscope. For FRET detection, we used the acceptor photobleaching method in which the acceptor (Cy3) was completely photobleached and any corresponding increase in donor’s intensity was detected and quantified. Any significant increase in donor intensity was considered to be due to FRET. We also performed some preliminary experiments with lipid nanoparticles to study whether these lipid nanoparticles are a viable delivery system for T-oligo. We encapsulated T-oligo in lipid nanoparticles using a microfluidic mixer and studied the encapsulation efficiency, uptake, and efficacy of encapsulated T-11 and T-22 on MM-AN melanoma cells. We found a significant increase of 40% - 150% in donor intensity after acceptor photobleaching in multiple melanoma cell lines. This may be due to G-quadruplex formation causing FRET thus indicating that T-oligo may be utilizing this mechanism of action for its anticancer effects. Preliminary results indicate that lipid nanoparticles encapsulated T-oligo with an efficiency of 72%. Uptake experiments showed that encapsulated T-11 and T-22 were taken up by the cells after an incubation time of 6hrs and the encapsulated T-22 also inhibited cell proliferation by 92%. In conclusion, three melanoma cell lines showed a significant increase in donor intensity after acceptor photobleaching upon treatment with T-22 and T-11 and lipid nanoparticles might be an effective delivery system for T-oligo.
Citation Format: Archit Agnihotri, Taylor Rager, Kelly Fan, Hyatt Alaraj, Lawrence Miller, Neelu Puri. T-oligo's mechanism of action in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1436.