Confirmatory dose-response cell survival assay for modulators of telomere damage signalling

Immortalisation is a hallmark of cancer commonly achieved by transcriptional reactivation of the telomerase reverse transcriptase gene TERT, leading to inappropriate telomere maintenance. Telomeres are capped by the multi-protein Shelterin complex which, along with multiple accessory proteins, regulates telomere end-processing, chromatin state, and prevents recognition of the chromosome ends as DNA damage. Telomere shortening is associated with normal cell division and loss of protection at critically shortened telomeres results in irreversible DNA damage signalling and replicative senescence. Targeting the telomere to induce senescence is a potential anti-cancer approach. However, mutational inactivation of components of the telomere complex also results in telomeropathies such as dyskeratosis which are associated with accelerated telomere shortening and premature aging syndromes. Agents that positively or negatively regulate telomere dysfunction pathways might have therapeutic application in these disease areas. To identify small molecule regulators of telomere signalling, an adenoviral vector expressing a mutant version of the human telomerase RNA gene was generated (Ad-hTR-mut). The vector is based on that reported by Kim et al (2001; Proc Natl Acad Sci U S A. 2001 Jul 3;98(14):7982-7). The native telomerase RNA sequence that ordinarily serves as template for reverse-transcription of telomere DNA is replaced with the sequence UAUAUAUAUAA. Expression of the RNA in human cells leads to telomerase-dependent synthesis of mutant telomere sequences lacking binding sites for proteins of the Shelterin complex and resulting in rapid telomere uncapping and cytotoxicity. Agents that modulate damage signalling from uncapped telomeres cause a shift in cytotoxicity after infection. Known modulators of telomere signalling suramin and BIBR1532 were tested in dose responses alongside CRT0063465, an optimised derivative of compound C430-0073, identified in screen TELO_01