Detection of Alternative Lengthening of Telomeres in Telomerase-Positive Cancers

Abstract

Immortalisation is a hallmark of cancer and requires activation of a telomere lengthening mechanism (TLM) to counteract natural telomere shortening. There are two TLMs: telomerase, reported in 85% of cancers, and Alternative Lengthening of Telomeres (ALT), reported in 13% of cancers. Because most somatic tissues do not have TLM activity, TLMs are widely regarded as targets for the development of anti-cancer therapies. Preliminary data from the Reddel laboratory indicated that in non-small cell lung carcinoma (NSCLC), ALT was more common than previously reported but mostly at very low levels, and together with telomerase activity. A panel of NSCLCs were examined for ALT and telomerase activity by the C-Circle Assay (CCA) and TRAP assay, respectively. ALT (at least a low level) and telomerase coexisted in 18% of the panel, suggesting that ALT is a more important therapeutic target in NSCLC than previously appreciated. ALT+/telomerase+ tumours could result from intratumoral heterogeneity, or from individual cancer cells being dual-positive. It was hypothesised that ALT and telomerase may be activated spontaneously in the same cancer cell. As functional studies are not possible in human tumours, a panel of 384 cancer cell lines was examined to determine whether some telomerase+ lines have ALT markers. Three cell lines positive for CCA and TRAP activity were subcloned, each of which were positive for both TLMs, indicating that both TLMs may occur spontaneously in a cancer cell. One of the dual-positive lines (LOX IMVI) was used for functional studies. A DNA tag inserted into one of its telomeres was copied on to other telomeres; this is regarded as the most definitive empirical evidence for the presence of a functional ALT mechanism. Therefore, telomere lengthening is occurring via ALT activity. The functional significance of telomerase activity was also addressed by CRISPR/Cas9-mediated knockout of the TERC gene. A blood-based diagnostic for ALT could be important for patient management and could be provided by the CCA because C-Circles have been reported in the blood of patients with ALT+ osteosarcomas. It was demonstrated that C-Circles are secreted by cancer cells in vesicles, which may be lost during plasma isolation. This knowledge may allow correct implementation of the CCA as a blood-based diagnostic for ALT activity.