Abstract PR02: Specific inhibition of hTERT expression in melanoma by targeting common promoter mutations which cause quadruplex DNA instability

Abstract

Abstract Background: Telomerase reverse transcriptase (hTERT) is a catalytic subunit of the enzyme telomerase. It has recently been shown that the hTERT promoter is commonly mutated (>75% of cases) in malignant melanoma. These mutations occur at four sites in a G-rich region of the promoter which has previously been shown to form quadruplex DNA and to downregulate hTERT expression. We have tested the hypothesis that mutations in the quadruplex-forming region of the hTERT promoter destabilize quadruplex-formation resulting in increased hTERT expression and cellular proliferation, thus providing a novel therapeutic target for melanoma. Materials and Methods: Quadruplex formation by the mutated and wild type hTERT promoter oligonucleotides was determined by circular dichroism. Analytical ultracentrifugation was used for sedimentation equilibrium analysis. Thermal denaturation was used to characterize the relative stability of the mutated and wild type oligonucleotides. The growth inhibitory effect of mutated and wild type oligonucleotides were determined in four cell lines which contained mutated or wild type hTERT promoter sequences. Cell proliferation was characterized by MTT and cell counting. Results: In order to characterize the effects of the hTERT mutations, the biophysical properties of structures formed by wild-type and mutant TERT sequences were explored by several methods. Circular dichroism and thermal denaturation studies showed that all sequences formed quadruplex structures but that those formed by the mutated sequences were markedly less stable than the wild-type. Analytical ultracentrifugation showed that all sequences formed one major unimolecular folded form but that mutant sequences had a greater tendency to form misfolded aggregated species than the wild-type. Addition of the quadruplex binder TmPyP4 to the mutant sequences lessened that amount of such aggregates and resulted in sedimentation profiles that closely resembled the wild-type sequences. Treatment of cells with mutated hTERT promoter sequence with oligonucleotides encoding the mutated or wild type sequence resulted in significant growth inhibition that was time and concentration dependent. DNA crosslinking studies indicate that the G-rich oligonucleotide is binding to the C-rich strand by Watson-Crick base pairing suggesting that it inhibits transcription initiation by strand invasion. A similar growth inhibitory effect was seen in cells exposed to quadruplex stabilizing agents. Conclusions: The common mutations in the hTERT promoter destabilize quadruplex formation and likely prevent quadruplex-mediated transcriptional silencing. This instability can be overcome by quadruplex-binding drugs and/or oligonucleotides encoding this sequence. The growth of cell lines containing the hTERT promoter mutations is inhibited by oligonucleotides encoding the wild type and mutated sequences. These oligonucleotides stabilize the mutated genomic quadruplex structure, resulting in transcriptional silencing. This approach has obvious clinical potential. This abstract is also being presented as Poster A07. Citation Format: Donald M. Miller, Alexandra Sokolova, Francine Rezzoug, Shelia Thomas, Jonathan Chaires, Jonathan Chaires, William Dean, John Trent. Specific inhibition of hTERT expression in melanoma by targeting common promoter mutations which cause quadruplex DNA instability. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr PR02.