Targeting melanoma cells through G-quadruplex formation: Mechanism and enhanced efficacy of T-oligo in combination therapy.

e21605 Background: Melanoma is among the deadliest skin cancers, responsible for an estimated 7,990 annual deaths. Telomerase binds to the 3' overhang of telomeres, enabling elongation and cell survival. T-oligo, a guanine-rich oligonucleotide homologous to the telomere overhang, targets this 3' region, inducing apoptosis in melanoma cells. As a guanine-rich oligonucleotide, T-oligo can form G-quadruplexes (G4), secondary nucleic acid structures detectable using FRET microscopy. Current melanoma treatments include vemurafenib and trametinib. This study also explored the efficacy of T-oligo in combination with these agents compared to monotherapy. We hypothesize that T-oligo forms G-quadruplexes in the nucleus of melanoma cells and that combination therapy of vemurafenib/trametinib with T-oligo enhances efficacy compared to monotherapy. Methods: T-oligo was evaluated in melanoma cell lines. MM-AN cells were plated in triplicates in 6-well plates and treated with single- or double-labeled T-oligo of 11 mer or 22 mer length, with FITC on the 5'-end and Cy3 on the 3'-end. Fluorescence microscopy assessed nuclear localization, and FRET microscopy (acceptor photobleaching) detected G4 formation. MTT assays evaluated the efficacy of T-oligo, vemurafenib, or trametinib alone or in combination in WM35 and 451Lu cell lines. Cells were plated (2,000 cells/well in 96-well plates), adhered for 48 hours, and treated with diluent, T-oligo, vemurafenib, trametinib, or their combinations for 96 hours before MTT analysis. Results: Approximately 90% of T-oligo localized to the nucleus in melanoma cells. FRET microscopy using the acceptor photobleaching method showed a 40%-150% increase in donor (FITC) intensity after Cy3 photobleaching, indicating G-quadruplex formation. IC50 values from MTT analysis demonstrated that combining T-oligo with vemurafenib or trametinib had an additive or greater effect in all tested cell lines. Conclusions: FRET microscopy confirmed G-quadruplex formation in T-oligo, suggesting it may utilize this mechanism for anticancer effects. Lower IC50 values in combination therapy confirmed that T-oligo enhances the efficacy of vemurafenib or trametinib, supporting its potential as an adjunctive melanoma treatment.