Abstract 2743: Telomere transcripts improve synthetic inhibitors of telomerase

Abstract

Most tumors express telomerase for infinite cell growth capacity. Telomerase was recognized recently as central regulator of all of the hallmarks of cancer and synthetic inhibitors of telomerase, such as Imetelstat blocking the RNA subunit of telomerase TERC, are in clinical trials. RNA transcripts from telomeres called TERRA were identified to block telomerase activity (TA) potentially via direct binding to TERC. We established recombinant expression systems to modulate TERRA transcripts in cell culture cells and study in vitro cell growth and viability directly and in combination with telomerase inhibitors. Adeno- and lentivirus constructs (AV and LV) were established for transient and stable recombinant TERRA expression in TA tumor cell lines (N=3) and mortal fibroblast cells as control. AV and LV express TERRA under control of hH1 promoter with around 120 units of UUAGGG repeats in sense (S) or anti-sense (AS) orientation. Similarly, AV and LV express polyadenylated form of TERRA under control of CMV promoter and polyadenylation signaling sites. Telomere length (TL), endogenous and recombinant TERRA expression was analyzed by qPCR, TA by TRAP assay. Population doubling (PD) times were calculated from cell growth numbers of LV clones. Small molecule telomerase inhibitors and AVs expressing dominant-negative telomerase and shRNA against hTERT were used in MTT cell viability and clonogenicity assays. TERRA expression was modulated transiently and stably by recombinant AVs and LVs as compared to virus controls expressing eGFP. Moderate 1.5-1.7 fold elevation of recombinant TERRA-S transcripts caused reduction of TA to 23-38%. In contrast, AS expression reduced TERRA 3-8 fold without reduction of TA. TL, PD time, cell viability and clonogenicity were not affected by TERRA-S and -AS expression up to 3 weeks in culture. Single cell clones were isolated from cells infected by LVs. Significant improve of telomerase targeting approaches was identified as IC-50 values decreased 2.0-2.6 fold in LV cell clones expressing moderate recombinant TERRA-S compared to TERRA-AS and eGFP. Similarly, clone formation capacity decreased 1.3-1.7 fold. Human fibroblasts showed 15 fold increased TERRA expression compared to tumor cells and were not affected by treatments applied. Preliminary data with modified RNA oligonucleotides containing TERRA sequences indicate that up-take by cells was efficient. Furthermore, IC-50 values of TA inhibitor MST-312 in combination with 3 nM TERRA oligonucleotides decreased 1.9-3.9 fold compared to mismatch controls. Effective concentrations were 1000 fold lower compared to DNA oligonucleotide telomerase inhibitors currently in clinical trials. We demonstrated that recombinant expression of TERRA significantly improved telomerase targeting approaches in tumor cells but not in mortal fibroblasts. TERRA oligonucleotides are candidates for in vivo application in combination with telomerase targeting approaches or other tumor therapies. Citation Format: Sandra Sampl, Doris Mejri, Christian Stern, Hui Wang, Klaus Holzmann. Telomere transcripts improve synthetic inhibitors of telomerase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2743. doi:10.1158/1538-7445.AM2014-2743

Epigenetic Regulation of Telomere Maintenance for Therapeutic Interventions in Gliomas

“…We recently identified glioma cell lines with long telomeres and absence or low TA [ 78 ]. Candidate ALT cell lines were screened for ALT associated C-Circles and one TA negative cell line YTBO was identified as ALT GBM cell model [ 83 ]. This cell line was established from a patient derived glioma tissue of a distinct morphological subset of GBMs and was cultivated in medium containing serum.…”