TPP1 promoter mutations cooperate with TERT promoter mutations to lengthen telomeres in melanoma

Abstract

Overcoming replicative senescence is an essential step during oncogenesis, and the reactivation of TERT through promoter mutations is a common mechanism. TERT promoter mutations are acquired in about 75% of melanomas but are not sufficient to maintain telomeres, suggesting that additional mutations are required. We identified a cluster of variants in the promoter of ACD encoding the shelterin component TPP1. ACD promoter variants are present in about 5% of cutaneous melanoma and co-occur with TERT promoter mutations. The two most common somatic variants create or modify binding sites for E-twenty-six (ETS) transcription factors, similar to mutations in the TERT promoter. The variants increase the expression of TPP1 and function together with TERT to synergistically lengthen telomeres. Our findings suggest that TPP1 promoter variants collaborate with TERT activation to enhance telomere maintenance and immortalization in melanoma.

Fig. 1.. Identification of a cluster of somatic promoter variant in TPP1.

(A) The genomic locus of the ACD gene is depicted from UCSC genome browser data with dark blue rectangles indicating the exon for TPP1-S and TPP1-L respectively. The red bars below the gene tract show the location of the somatic variants identified in the ICGC database with taller bars corresponding to the number of melanomas found with a specific variant. RNA-seq data (GSE153592) is shown above the gene tract in purple along with vertebrate conservation and H3K27 acetylation marks from multiple cell lines, indicating the location of likely regulatory regions. (B) HeLa stable cell lines expressing a C-terminally FLAG-tagged TPP1 were stained for the shelterin component TRF2 and the FLAG epitope. Colocalization of TPP1 with TRF2 suggest that the C-terminal FLAG-tag does not disrupt localization of TPP1 to the telomere. (C) Western blot of HEK293, LOX, and MEL624 cells transfected with plasmids encoding the cDNAs for TPP1-S and TPP1-L, and TPP1-L-M87A that is incapable of expressing TPP1-S together with plasmids expressing the entire genomic locus of TPP1 with and without the most common promoter variants.

Fig. 2.. ETS transcription factors activate the variant TPP1 promoter.

(A) Luciferase assays were performed with a 285 base pair fragment of the TPP1 proximal promoter in melanoma and non-melanoma cell lines. The TPP1 promoter variants had little effect on the transcriptional activity in non-melanoma cells lines (HEK293), but increased reporter activity in two melanoma derived lines. (B) Quantitative PCR examining the levels of three ETS transcription factor family members in non-melanoma (HeLa, HEK293, and BJ fibroblast; n=3) and melanoma cell lines (MEL624 and LOX) and short-term primary cultures (n=6–7). Medians are shown and groups were compared using Mann-Whitney test. (C) Western blot showing high expression of ETS transcription factors in LOX melanoma and MEL624 lines. (D) Luciferase assays comparing activity of the TPP1 promoter reporter in the presence of three transfected ETS transcription factors in HEK293 cells. Cells were co-transfected with a pGL4 reporter and pCDNA3.1 expression plasmid with one of the three ETS transcription factors. Mean and standard deviation are shown from at least three independent experiments in (A) and (D) and groups were compared with a one-way ANOVA followed by Tukey’s multiple comparison test for pairwise comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3.. TPP1-S over expression causes telomere lengthening and is synergistic with TERT overexpression.

(A) Southern blot of telomeres in HeLa cell lines that stably expressed TPP1-S or TPP1-L for 90 days. Two independent clones of each are shown. P – parental cell line that was used to establish each of the modified clones. (B) Growth curves of cumulative population doublings of BJ fibroblasts expressing TPP1-S or TERT (average of 3 independent transduction for each group). (C) Western blot showing expression of each of the transgenes in cells collected from (B). (D) Southern blot of telomere lengths of BJ fibroblasts in (B) 15 passages after transduction showing synergistic telomere lengthening in cells exogenously overexpressing TPP1 and TERT.

Fig. 4.. TPP1 promoter mutations increase expression of the endogenous transcript and co-occur with TERT promoter mutations.

(A) Quantitative PCR of TPP1 expression following introduction of promoter mutations in LOX and MEL624 cells. Labels below the graph indicate the presumed zygosity based on sequencing. The media is shown from three independent measurements from each clone and groups were compared using one-way ANOVA followed by Dunnett’s multiple comparison test. (B) Schematic of the experimental approach to measure telomerase activity in genetically modified cells. Cells are transduced with a TERT-expressing lentivirus to increase the rate of variant telomere incorporation. Following introduction of the mutant telomerase RNA (encoding TTAGGT), cells are passaged and the canonical and variant telomeres are quantitated. (C) Fluorescent in situ hybridization for the WT (TTAGGG; red) and variant (TTAGGT; green) in parental or genome edited MEL624 cells. Images were taken 7 days after transduction with lentiviruses. (D) Quantitation of the fraction of telomeres that had both TTAGGG and TTAGGT signals from a single clone. Groups were compared using ANOVA with Dunnett’s correction for multiple comparison. **P<0.01 and ****P < 0.0001. (E) Proportion of cutaneous melanomas that had TERT, TPP1, or TERT+TPP1 variants from Hayward et. al. (25) (F) Model of telomere length dynamics in melanoma progression. TERT promoter variants likely occur early and slow telomere attrition but are not sufficient to prevent telomere shortening (blue dashed lines in model). Telomere shortening continues until cells enter crisis (red dashed line). Additional mutations, like the TPP1 promoter, are likely required to fully maintain telomeres and escape crisis (2^nd hit).