Human TERT Lentivirus (CMV, Hygro)

Telomeres are DNA sequences that cap the ends of chromosomes and become shorter as cells divide. Telomerase maintains telomere length so that cells can continue to divide. Overcoming replicative senescence is an essential step in tumorigenesis, and reactivation of TERT through promoter mutations is a common mechanism. Approximately 75% of melanoma patients acquire TERT promoter mutations, but these are not sufficient to maintain telomeres, suggesting that additional mutations are required. Researchers have identified a cluster of variants in the ACD promoter, which encodes the shelterin component TPP1. Approximately 5% of cutaneous melanomas harbor ACD promoter variants that co-occur with TERT promoter mutations. The two most common somatic variants create or modify binding sites for the E-twenty-six (ETS) transcription factor, similar to mutations in the TERT promoter. These variants increase TPP1 expression and work together with TERT to synergistically lengthen telomeres. These findings suggest that TPP1 promoter variants work in concert with TERT activation to enhance telomere maintenance and immortalization in melanoma.

The study found that introduction of either the –75C>T or –108C>T variants significantly increased TPP1 expression (Figure 1A). The increase in TPP1 expression levels from the modified endogenous promoter was greater than that observed in the luciferase assay (Figure 1C), suggesting that other factors may contribute to TPP1 expression in melanoma. Telomeres in MEL624 and LOX cells are extremely long (>20 kb), precluding detection of length changes using Southern blot analysis. Therefore, fluorescence in situ hybridization (FISH) was used to detect modified telomeric sequences as a surrogate for telomerase activity in vivo (Figure 1B). The researchers used telomerase RNA encoding the variant telomeric repeat TTAGGT, which can be incorporated into telomeres and localized using a peptide nucleic acid fluorescent probe. Wild-type or genome-edited MEL624 or LOX cell lines with the most common promoter variants were co-transduced with lentivirus expressing the variant telomerase RNA and hTERT. Using FISH to determine the percentage of variant repeat sequences in telomeres, the study found that cells with modified TPP1 promoters incorporated more TTAGGT variant repeat sequences at telomeres (Figure 1C and D). These findings suggest that TPP1 promoter mutations cooperate with hTERT to increase telomere repeat addition in melanoma cells.

Figure 1. TPP1 promoter mutations increase expression of endogenous transcripts and occur simultaneously with TERT promoter mutations. (Chun-On P, et al., 2022)