Htert G-Quadruplex-Targeted Oligonucleotides Inhibit Glioblastoma Cell Growth

Glioblastoma is one of the most common and deadly forms of brain cancer, representing roughly 75% of all brain malignancies. These tumors generally have poor prognoses and are resistant to conventional therapy. It has recently been shown that as many as 80% of all glioblastomas contain mutations in a G-rich 68 base pair region of the hTERT promoter. hTERT is the catalytic subunit of telomerase, a holoenzyme responsible for lengthening the ends of chromosomes, thereby conferring immortality to cells. Normally, hTERT is not expressed in somatic cells and its expression is tightly controlled in stem cells. However, hTERT is upregulated in up-to 95% of human tumors and is considered a key activator of cancer progression and a sign of poor clinical outcome. Therefore, hTERT has been under investigation for the past decade as a potential therapeutic target. We have shown that the mutations in hTERT promoter occur in a G-rich region that is part of a silencer element which forms a secondary G-quadruplex structure required for function, and that these mutations destabilize the G-quadruplex structure, allowing hTERT expression. We have also demonstrated that oligonucleotides encoding the G-quadruplex forming sequence in the c-MYC promoter can stabilize the G-quadruplex structure and downregulate c-MYC expression. Therefore, we hypothesized that oligonucleotides targeted to the G-quadruplex of the hTERT promoter could downregulate this gene expression and inhibit glioblastoma cell proliferation in a similar manner. We designed several G-quadruplex-forming oligonucleotides covering the mutated sites in the hTERT promoter either in totality (68 nucleotides) or separately (25 nucleotides) to stabilize the G-quadruplex region. Two glioblastoma cell lines (A172 and U87) and one neuroblastoma cell line (CHP134) were exposed to these oligonucleotides and evaluated for growth inhibition using the MTT assay and for gene expression by QRT-PCR. All oligonucleotides tested were found to induce between 40 to 90% growth inhibition in the 3 cell lines. The cell growth inhibition was both time and dose dependent and showed effectiveness as early as 3 days suggesting that this effect is not solely due to telomere shortening. Four oligonucleotides with the most consistent efficacy in growth inhibition were evaluated for their effect on hTERT gene expression in the 3 cell lines at 4 day exposure and revealed that two of the G-quadruplex forming oligonucleotides significantly decreased hTERT expression compared to untreated cells. In conclusion, we have defined G-quadruplex oligonucleotides targeted to the hTERT promoter that downregulate hTERT gene expression and are effective growth inhibitors in glioblastoma cells. Our findings indicate that downregulation of hTERT with targeted oligonucleotides affects non-canonical functions of hTERT conferring an advantage to this therapeutic approach. Note: This abstract was not presented at the meeting. Citation Format: Alex West, Francine Rezzoug, Shelia D. Thomas, Donald M. Miller. hTERT G-quadruplex-targeted oligonucleotides inhibit glioblastoma cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1521. doi:10.1158/1538-7445.AM2017-1521