Abstract 5101: Common mutations in the hTERT promoter cause instability in quadruplex DNA which is overcome by quadruplex stabilizing agents and targeted oligonucleotides

Introduction: 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%) in a malignant melanoma and glioblastoma. These mutations occur at four sites in a G-rich region 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. 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. Four cancer cell lines obtained from ATCC were used to characterize the growth inhibitory effect of mutated and wild type oligonucleotides (A549, Calu1, A375, T24). The MTT assay was used for the cell proliferation experiments. 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 mutant sequences were 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 the wild type hTERT promoter sequence with the mutated or wild type oligonucleotides had very little effect on cell growth. However, treatment of cells with mutated hTERT promoter sequence resulted in significant growth inhibition that was time and concentration dependent. 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. The growth of cell lines containing the hTERT promoter mutations is inhibited by olig