TERT expression is associated with metastasis from thin primaries, exhausted CD4+ T cells in melanoma and with DNA repair across cancer entities

Christina Katharina Kuhn; Jaroslawna Meister; Sophia Kreft; Mathias Stiller; Sven-Holger Puppel; Anne Zaremba; Björn Scheffler; Vivien Ullrich; Torsten Schöneberg; Dirk Schadendorf; Susanne Horn

doi:10.1371/journal.pone.0281487

. 2023 Jul 7;18(7):e0281487. doi: 10.1371/journal.pone.0281487

TERT expression is associated with metastasis from thin primaries, exhausted CD4+ T cells in melanoma and with DNA repair across cancer entities

Christina Katharina Kuhn ^1,^*, Jaroslawna Meister ^1,², Sophia Kreft ³, Mathias Stiller ⁴, Sven-Holger Puppel ¹, Anne Zaremba ³, Björn Scheffler ⁵, Vivien Ullrich ⁵, Torsten Schöneberg ^1,⁶, Dirk Schadendorf ³, Susanne Horn ^1,^3,^*

Editor: Avaniyapuram Kannan Murugan⁷

¹Rudolf Schönheimer Institute of Biochemistry, University of Leipzig, Medical Faculty, Leipzig, Germany

²Institute for Clinical Diabetology, German Diabetes Centre, Leibniz Centre for Diabetes Research at Heinrich Heine University Düsseldorf, Düsseldorf, Germany

³Department of Dermatology, University Hospital Essen, University Duisburg-Essen, and German Cancer Consortium Partner Site Essen/Düsseldorf, Essen, Germany

⁴Institute of Pathology, University of Leipzig Medical Center, Leipzig, Germany

⁵DKFZ-Division Translational Neurooncology at the West German Cancer Center, University Hospital Essen/University of Duisburg-Essen, Essen, Germany

⁶School of Medicine, University of Global Health Equity, Kigali, Rwanda

⁷King Faisal Specialist Hospital and Research Center, SAUDI ARABIA

Competing Interests: The authors have declared that no competing interests exist.

^✉

* E-mail: Susanne.Horn@medizin.uni-leipzig.de (SH); Christina.Kuhn@medizin.uni-leipzig.de (CKK)

Roles

Christina Katharina Kuhn: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Jaroslawna Meister: Writing – original draft, Writing – review & editing

Sophia Kreft: Writing – original draft, Writing – review & editing

Mathias Stiller: Writing – original draft, Writing – review & editing

Sven-Holger Puppel: Data curation, Resources, Writing – review & editing

Anne Zaremba: Writing – original draft, Writing – review & editing

Björn Scheffler: Writing – original draft, Writing – review & editing

Vivien Ullrich: Writing – original draft, Writing – review & editing

Torsten Schöneberg: Funding acquisition, Project administration, Supervision

Dirk Schadendorf: Funding acquisition, Project administration

Susanne Horn: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Avaniyapuram Kannan Murugan: Editor

PMCID: PMC10328343 PMID: 37418389

Abstract

Telomerase reverse transcriptase (TERT) promoter mutations occur frequently in cancer, have been associated with increased TERT expression and cell proliferation, and could potentially influence therapeutic regimens for melanoma. As the role of TERT expression in malignant melanoma and the non-canonical functions of TERT remain understudied, we aimed to extend the current knowledge on the impact of TERT promoter mutations and expression alterations in tumor progression by analyzing several highly annotated melanoma cohorts. Using multivariate models, we found no consistent association for TERT promoter mutations or TERT expression with the survival rate in melanoma cohorts under immune checkpoint inhibition. However, the presence of CD4+ T cells increased with TERT expression and correlated with the expression of exhaustion markers. While the frequency of promoter mutations did not change with Breslow thickness, TERT expression was increased in metastases arising from thinner primaries. As single-cell RNA-sequencing (RNA-seq) showed that TERT expression was associated with genes involved in cell migration and dynamics of the extracellular matrix, this suggests a role of TERT during invasion and metastasis. Co-regulated genes found in several bulk tumors and single-cell RNA-seq cohorts also indicated non-canonical functions of TERT related to mitochondrial DNA stability and nuclear DNA repair. This pattern was also evident in glioblastoma and across other entities. Hence, our study adds to the role of TERT expression in cancer metastasis and potentially also immune resistance.

Introduction

Telomere maintenance mechanisms play a critical role in cellular survival as they are known to enhance the proliferative capacity of cancer cells [1]. Frequent mutations in the promoter region of the telomerase reverse transcriptase (TERT) gene were shown to be associated with the upregulation of telomerase expression [2–4]. TERT promoter mutations have been proposed as independent prognostic markers in non-acral cutaneous melanoma, since they correlated in a multivariate analysis with decreased overall survival (OS) without analysis of therapy [5]. The co-occurrence of BRAF or NRAS mutations with TERT promoter mutations was also prognostic for poor disease-free survival in multivariate analyses in primary melanoma [6]. TERT promoter mutations may serve as predictive biomarkers as they were independently associated with improved survival in patients receiving BRAF/MEK inhibitor therapy in univariate and multivariate analyses [7, 8]. Also, patients undergoing anti-CTLA4 therapy showed better overall survival with TERT promoter mutations, implying its potential as a predictive marker for immune checkpoint inhibitor (ICI) therapy [9]. Also, TERT expression has been suggested to be an independent prognostic marker associated with poor survival in human cancer patients beyond melanoma [10–12]. However, many variables, such as mutational load, tumor heterogeneity, and prior therapies, could be appended to existing multivariate models to test additional potentially stronger predictors.

The extent to which genetic alterations of TERT play a role in cancer progression and the molecular mechanisms affected by reactivation of TERT transcription vary widely among cancer entities, making it crucial to understand the underlying mechanisms [13]. One way cancer cells regain their proliferative ability is by forming a new ETS binding motif at the promoter mutation [2]. This binding motif attracts transcription factors, such as TCF1, Myc, Wnt/β-catenin, NF-kB, and GAPB, which are responsible for regulating numerous cellular processes including tumorigenesis [2, 14]. The canonical function of TERT is telomere maintenance, however, non-canonical functions of TERT have been discussed, e.g. related to reactive oxygen species (ROS), mitochondria and aging. TERT is imported into the mitochondria, where it binds mitochondrial DNA, protecting it from oxidative stress–induced damage, reducing apoptosis in human endothelial cells [15]. Furthermore, besides the full-length expressed TERT, alternatively spliced isoforms exist, which lack telomerase activity and may account for up to 10% of endogenous TERT. These are continuously expressed after telomerase silencing during human embryonic development [16] and are potentially associated with telomere-independent effects [17].

Here, we test if TERT promoter mutations and TERT expression are associated with OS and progression free survival (PFS) under ICI, as well as clinicopathological and demographic parameters in multiple highly annotated melanoma cohorts with exome- and transcriptome sequencing. To examine the non-canonical functions of TERT, we investigate associations with ROS-related genes, hypoxia, chromothripsis, and TERT isoform expression. Using molecular pathway analyses, we replicate known and identify new signaling pathways regulated with genetically modified TERT in bulk tumors, cell lines, and single-cells. Tumor immune signatures are estimated to elucidate the role of TERT during tumor rejection. Finally, our analyses are extended to additional cancer entities from the Pan-Cancer Analysis of Whole Genomes (PCAWG) cohort and glioblastoma (GBM) to determine the signaling pathways co-regulated with TERT across entities.

Materials and methods

Next generation sequencing data analysis

We analyzed several bulk RNA-seq cohorts with respective survival data (Table 1, Transcriptomics in S1 File). These included five melanoma cohorts with samples taken prior to or early-on ICI therapy (Gide, Hugo, Liu, Riaz, Van Allen cohorts), one melanoma cohort without ICI therapy (Skin Cutaneous Melanoma cohort, SKCM), two cross-entity datasets: cell lines from the Cancer Cell Line Encyclopedia (CCLE), and bulk tumors of the Pan-Cancer Analysis of Whole Genomes (PCAWG). We further included melanoma single-cell RNA-seq (scRNA-seq) and glioblastoma tumors in our workflow (S1 Figin S1 File). In brief, we extended the available gene expression levels with additional metadata, as described below and in the S1 File. We annotated published TERT promoter mutations in the RNA-seq datasets of two melanoma cohorts (Van Allen, SKCM) and two cross-entity datasets (CCLE and PCAWG, TERT promoter mutation calls in S1 Material). We added immune signatures for the six melanoma bulk RNA-seq cohorts using TIMER 2.0 [18] (Immune signatures in S1 Material). Isoform analysis was performed for the Van Allen, SKCM and PCAWG cohorts. To analyze mitochondria-related features in the PCAWG and SKCM cohorts, we further integrated measurements for hypoxia, chromothripsis [19] and mitochondrial mutations [19, 20]. Hypoxia scores for PCAWG were determined as in Buffa et al. 2010 [21], and hypoxia scores for the SKCM cohort were obtained from the cBioPortal (www.cbioportal.org/study/clinicalData?id=skcm_tcga_pan_can_atlas_2018). We reanalyzed the MSK IMPACT Clinical Sequencing Cohort (dataset “MSKCC, Nat Med 2017”) [9] as obtained from www.cbioportal.org annotating the corresponding therapy data from Samstein [22].

Table 1. Analyzed cohorts.

Name	Datatype	Expression Unit	Source	TERT Promoter Status known	Cancer Entities	Immune Checkpoint Blockade Therapy	PMID	Mito data
SKCM	Bulk RNA-Seq	TPM	Tissue	Subset	Melanoma	No	26091043	yes
Gide	Bulk RNA-Seq	TPM	Tissue	No	Melanoma	Yes	30753825	no
Riaz	Bulk RNA-Seq	TPM	Tissue	No	Melanoma	Yes	29033130	no
Van Allen	Bulk RNA-Seq	TPM	Tissue	Yes	Melanoma	Yes	26359337	no
Liu	Bulk RNA-Seq	TPM	Tissue	No	Melanoma	Yes	31792460	no
Hugo	Bulk RNA-Seq	TPM	Tissue	No	Melanoma	Yes	26997480	no
Li	Panel Seq	not applicable, mutations	Tissue	Yes	Multiple	Yes	32810393	no
Jerby-Arnon	Single-Cell RNA-Seq	TPM	Tissue	No	Melanoma	Yes	30388455	no
PCAWG	Bulk RNA-Seq	uqv2 FPKM for genes, TPM for isoforms	Tissue	Subset	Multiple	No	32025007	yes
CCLE	Bulk RNA-Seq	TPM	Cell Culture	Subset	Multiple	No	31068700	no
Klughammer	Bulk RNA-Seq	RPKM	Tissue	No	Glioblastoma	No	30150718	no

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TPM = transcripts per million; mito data = mitochondrial data available; Uqv2 FPKM = upper quartile normalized fragments per kilobase of transcript per million mapped reads; RPKM = reads per kilobase of transcript per million mapped reads

Enrichment analysis

Enrichment analysis of top genes associated with TERT promoter mutation and TERT expression using bulk RNA-seq cohorts was conducted using the gene set enrichment analysis tool https://maayanlab.cloud/Enrichr/ [23] with default settings. For enrichment analysis in the melanoma single-cell context, the top 100 genes associated with TERT expression in the scRNA-seq dataset were queried with Enrichr using default settings. TERT promoter mutation status was not available for single-cell data. The top ten results of each enrichment set from gene ontology pathways, ontologies and cell types were then used for discussion of the most frequently occurring terms.

TERT isoform analysis

Short names, coding information, biotype, and transcript lengths of TERT isoforms were collected from Ensembl (GRCh38.p13) using ENSG gene identifiers (TERT isoform information in S1 Material). The presence of the mitochondrial targeting protein sequence ‘MPRAPRCRAVRSLLRSHYRE’ [24] in the translated isoforms was analyzed using the UCSC Human Genome Browser. In detail, we verified whether transcripts included the targeting sequence by aligning the protein sequence with the genome (GRCh38.p13) and comparing the start/stop positions with the isoform exons. The isoform expressed in normal control tissues was assessed using the GTEx portal (https://gtexportal.org/home/gene/TERT#gene-transcript-browser-block, S2 Fig in S1 File). We then summed over the measured transcripts per million (TPM) of all isoforms and calculated the proportion of isoforms with mitochondrial targeting sequence. Data on isoforms lacking the targeting sequence were available only for the CCLE and Van Allen cohorts.

Statistics

Correlation of TERT mRNA expression, isoform expression and clinical variables was tested using Spearman correlations. For comparison between samples with or without TERT promoter mutation, two-sided Wilcoxon rank tests were applied. The correlates associated with TERT expression or TERT promoter mutations were ranked by p-value with false discovery rate control, and overlaps of the gene lists from several cohorts were performed. For survival analysis, the cohorts were divided into low vs. high TERT expression groups, with approximately one-third of the patients showing high TERT expression. If available, cohorts (SKCM and Van Allen cohort) were also divided into two groups by TERT promoter mutation absence/presence. Kaplan-Meier survival curves, log-rank tests, multivariate Cox Proportional-Hazards (Cox PH) analyses, and odds ratios were computed using R v.4.0.1 [25].

Data availability statement

The data used in this study are available within this manuscript and its supplementary files. Previously published, processed next generation sequencing data can be accessed at https://tools.hornlab.org/cru337phenotime/.

Results

In each of the following sections, we start at the genomic level by analyzing the role of TERT promoter mutations in malignant melanoma and extend the analysis to the transcriptomic level by examining the impact of TERT expression on clinical and molecular associations.

Association of TERT promoter mutation status and TERT expression with survival

TERT promoter mutations and TERT expression are proposed as prognostic biomarkers in melanoma and TERT promoter mutations might be predictive under anti-CTLA4 therapy [5, 6, 9, 26, 27]. Therefore we investigated the impact of both TERT alterations using univariate and multivariate survival analyses based on six bulk RNA-seq cohorts of metastatic melanoma treated with ICI (Gide, Riaz, Van Allen, Liu, Hugo cohorts) and without targeted therapy or ICI (SKCM). Our analysis showed that TERT promoter mutations and TERT expression were not associated with survival (S3-S5 Figs, S1 Table). PFS under anti-PD1 immunotherapy with pembrolizumab appeared decreased with higher TERT expression in only one analyzed cohort with small sample size (n = 8 vs. 24, Gide cohort, PFS log rank p = 0.038), however, it was not significantly associated in multivariate Cox PH including age, sex and BRAF mutation status (p = 0.071, S1A Table).

In contrast to our finding, an association of TERT promoter mutations with survival under anti-CTLA4 therapy was described previously [9], however, no multivariate model was attempted. Therefore, we reanalyzed the melanoma subset of the MSK Impact 2017 cohort [9] and the corresponding therapy data and included tumor mutational burden (TMB), sex, and BRAFV600 variants in a multivariate model. The previously described association of TERT promoter mutations with survival still holds, albeit with reduced significance (p = 0.015 compared to the previous univariate analysis p = 0.0007, S6A Fig in S1 File).

TERT expression and increased intratumoral CD4+ T helper type 1 cells

To investigate the role of TERT during tumor rejection, we correlated immune signatures with TERT expression. The CD4+ T helper type 1 cell signature repeatedly increased with TERT in five of the six cohorts analyzed here (Fig 1A, S2 Table). However, within a patient population with high CD4+ T helper type 1 cell estimates, survival did not differ with elevated TERT expression (S7 Fig in S1 File). To find a possible interaction of CD4+ T cells, we tested the presence of potential exhaustion markers [28]. Indeed, CTLA4, PDCD1IPD1, HAVCR2/TIM3, and TIGIT were expressed at higher levels (Fig 1B) in CD4+ T cells of tumors with high TERT expression of malignant cells.

Fig 1 — (A) Five out of six cohorts showed correlation of *TERT* expression and a CD4+ Th1 T cell immune signature (xCell assay). (B) Expression of T cell exhaustion markers in single CD4+ T cells of patients with low vs. high *TERT* expression in malignant tumor cells (</>0.05 TPM). Jerby-Arnon cohort, pre-therapy samples. Th1: T-helper type 1; TME: tumor microenvironment; TPM: transcripts per million; xCell: xCell cell types enrichment analysis. The figure was created with BioRender.com.

Primary tumor thickness

Our examination of clinical variables in association with TERT promoter mutations and expression was based on the two largest melanoma cohorts, the SKCM and Liu cohorts (S3A–S3C Table). There was no obvious difference in the primary tumor thickness between metastatic samples with vs. without TERT promoter mutations (Wilcoxon p = 0.508, Fig 2A). Notably, TERT expression was higher in metastases of tumors arising from thinner primaries (Spearman p = 0.01, coeff = -0.16, Fig 2A; Wilcoxon p = 0.011, Breslow >2 mm: 8.95 TPM vs. Breslow <2 mm: 11.89 TPM, Fig 2B).

Fig 2 — (A) Breslow depth of primaries with *TERT* promoter mutations and *TERT* expression measured in corresponding metastatic tumors of the SKCM cohort (without one outlier; *TERT* expression >600, for data including outliers see S8 Fig in S1 File; also see S3D Table). (B) *TERT* expression in metastatic tumors arising from thin (Breslow depth <2 mm) and thick tumors (>2 mm). Information on *TERT* alterations in thin primaries are not available. *TERT*pm: *TERT* promoter mutations; TPM: transcripts per million. The figure was created with BioRender.com.

TERT in melanoma subtypes

As the mitogen-activated protein kinase (BRAF-MAPK) signaling pathway is frequently activated in melanoma [29], we analyzed TERT in melanoma subtypes with hotspot mutations in BRAF, RAS (NRAS, KRAS, HRAS) and NF1 genes in the SKCM cohort. Melanomas with hotspot mutations carried TERT promoter mutations more often than triple wild-type melanomas (odds ratios >20, S4A Table). With respect to TERT expression, we found significantly lower TERT expression in BRAF hotspot mutants than in RAS hotspot mutants (Wilcoxon p = 0.0004, S4B Table). The group of patients with TERT promoter mutations showed down-regulated TERT expression in BRAF mutants and up-regulated TERT expression in RAS mutants (mutated RAS: Wilcoxon p = 0.024, mutated BRAF: Wilcoxon p = 0.004, S4C Table).

Mutational load

A common pattern of TERT promoter mutations and TERT expression was the association with more nuclear mutational load. In the entire SKCM cohort, samples with TERT promoter mutations showed higher overall mutation counts (Wilcoxon p = 0.014, Fig 3A, not significant for the metastatic subgroup) and higher TERT expression was also associated with mutation counts (Spearman p = 0.002, coeff = 0.16, S3B Fig in S1 File, metastatic subgroup: Spearman p = 0.017, coeff = 0.14, Fig 3A), however, not in the Liu cohort (Spearman: 0.194, coeff = 0.12, S3A and S3B Table).

Fig 3 — (A) *TERT* alterations and exome mutation load in the SKCM cohort. (B) *TERT* expression and DNA repair genes associated across all cohorts. (C) *TERT* alterations, mitochondrial mutation load and hypoxia (PCAWG cohort). TPM: transcripts per million; FPKM: fragments per kilobase of transcript per million fragments mapped; nsSNP: non-synonymous mitochondrial SNP; *TERT*pm: *TERT* promoter mutations. The figure was created with BioRender.com.

Moreover, in the SKCM cohort we did not find TERT promoter mutations associated with Breslow depth or age at diagnosis, and we did not find TERT expression associated with sex (SKCM, Liu), age at diagnosis (SKCM), or various clinicopathological variables of the Liu cohort, such as heterogeneity or total neoantigens (S3 Table).

Gene expression associated with TERT alterations

At first, we replicated the previously described association of higher TERT expression levels in the presence of TERT promoter mutations in three independent cohorts (S5A Table). Since alternative splicing may result from mutation of the TERT promoter [17], we analyzed the expression of TERT isoforms. However, none of the isoforms were consistently regulated with TERT promoter mutation (S5A Table, S9 Fig in S1 File).

Next, we aimed to identify other cellular signaling pathways that may be driven by or co-regulated with either TERT promoter mutations (S6 Table) or TERT expression (S7 Table) followed by an enrichment analysis of associated gene expression. Associations with the presence of TERT promoter mutations revealed an overlap of 30 genes in the three bulk RNA-seq cohorts (in melanoma and across entities), of which six genes were consistently up- or down-regulated (up: ARL17A, FBF1, KAZALD1, PRAF2; down: PLEKHB2, RASGRP3; S6 Table). One gene (PRAF2) showed a consistent up-regulation also in the cell line data comprising other cancer entities (CCLE cohort). Enrichment analysis of the 30 genes associated with TERT promoter mutations showed overlaps with genes involved in ‘cilium organization’; ‘plasma membrane bound cell projection assembly’ and ‘actin cytoskeleton’ (S7A Table). To elucidate whether our findings are transferable to brain tumors, we extended our analyses to glioblastoma, in which TERT promoter mutations are highly recurrent [3] and diagnostic biomarkers [30]. Analysis of genes associated with TERT promoter mutations in 28 bulk tumor samples from the glioblastoma subcohort in PCAWG showed that of the six genes consistently associated with TERT promoter mutations in all bulk RNA-Seq cohorts, only ARL17A was also upregulated in glioblastoma (S6A Table).

In the context of TERT expression, 17 genes were consistently upregulated with TERT expression in all seven bulk RNA-seq cohorts, comprising melanoma and a cross-entity cancer cohort, as well as a cross-entity cell line cohort (Table 2, S7 Table). We confirmed that these TERT correlates were mostly tumor cell-specific genes within melanoma, as 15 genes were predominantly expressed in malignant tumor cells in our scRNA-seq data (Table 2, S7A and S7H Table). Enrichment analysis of the 17 genes upregulated with TERT expression revealed ‘DNA repair’, ‘double-strand break repair’, and ‘replication fork processing’ as relevant processes (Fig 3B, S8A Table). We replicated most of these associations in glioblastoma (Klughammer cohort) where 14 of 17 genes were also positively associated with TERT expression, including four genes related to DNA repair (XRCC2, MSH5, TRAIP, BRIP1, S6 Table).

Table 2. Genes repeatedly associated with TERT mRNA in 9 cohorts (S7 Table).

Overlap of bulk RNA-seq and CCLE cohort	Overlap with malignant single-cell	Consistent up or downregulated^a	Function
17	15
FBXW9	FBXW9	Up	Substrate-recognition component of the SCF (SKP1-CUL1-F-box protein)-type E3 ubiquitin ligase complex
KIF14	KIF14	Up	Microtubule motor protein that binds to microtubules with high affinity through each tubulin heterodimer and has an ATPase activity (By similarity), plays a role in many processes like cell division, cytokinesis and also in cell proliferation and apoptosis
KLHL23	KLHL23	Up	Kelch-Like Family Member 23 (no function available)
MSH5	MSH5	Up	Involved in DNA mismatch repair and meiotic recombination processes
PUS7	PUS7	Up	Pseudouridylate synthase that catalyzes pseudouridylation of RNAs, acts as a regulator of protein synthesis in embryonic stem cells by mediating pseudouridylation of RNA fragments derived from tRNAs
SMARCA4	SMARCA4	Up	Involved in transcriptional activation and repression of selected genes by chromatin remodeling (alteration of DNA-nucleosome topology), component of SWI/SNF chromatin remodeling complexes
STIL	STIL	Up	Immediate-early gene, plays an important role in embryonic development as well as in cellular growth and proliferation, its long-term silencing affects cell survival and cell cycle distribution as well as decreases CDK1 activity correlated with reduced phosphorylation of CDK1
TMEM201	TMEM201	Up	Involved in nuclear movement during fibroblast polarization and migration
TRAIP	TRAIP	Up	E3 ubiquitin ligase required to protect genome stability in response to replication stress
TTK	TTK	Up	Phosphorylates proteins on serine, threonine, and tyrosine, probably associated with cell proliferation
UCKL1	UCKL1	Up	May contribute to UTP accumulation needed for blast transformation and proliferation
XRCC2	XRCC2	Up	Involved in the homologous recombination repair (HRR) pathway of double-stranded
SMG5	SMG5	Up	Plays a role in nonsense-mediated mRNA decay
DNA2	DNA2	Up	Key enzyme involved in DNA replication and DNA repair in nucleus and mitochondriaS1
ALKBH2	ALKBH2	Up	Dioxygenase that repairs alkylated DNA and RNA containing 1-methyladenine and 3-methylcytosine by oxidative demethylation
BRIP1		Down	DNA-dependent ATPase and 5’ to 3’ DNA helicase required for the maintenance of chromosomal stability
ARGHGEF39		Up	Rho Guanine Nucleotide Exchange Factor 3

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TERT = telomerase reverse transcriptase; CCLE = cancer cell line encyclopedia

Note: Genes are sorted alphabetically and the first 12 genes were also found in glioblastoma

^aConsistent up- or down-regulation: similar direction of correlation with TERT expression in all bulk, single-cell, and cell line cohorts; up = positively correlation, down = negative correlation

To further elucidate tumor cell-specific signaling pathways, we used single-cell RNA-seq data and analyzed the top 100 genes co-expressed with TERT mRNA and performed an enrichment analysis with pathways, ontologies and cell type enrichment sets (S8C, S8D Table). Several cellular expression programs were repeatedly enriched, including the extracellular matrix, integrins, stromal cells, and smooth muscle cells (S8B Table).

Association of TERT and mitochondrial variables

To further elucidate the alternative role of TERT beyond telomere elongation in the molecular biology of mitochondria and reactive oxygen species (ROS) [15, 31], we first tested associations of several ROS-related candidate genes with the presence of genetic alterations in TERT. However, we did not observe consistent up- or downregulation off the examined genes NOX1-5, DUOX1/2, SOD1/2, OPA1, MFN1/2, PINK1, FUNDC1, and GPX4 in correlation with TERT promoter mutations or expression (S5B and S5C Table). Yet, within our discovery approach, we identified a consistent upregulation of DNA2 with higher TERT expression in several bulk tumor cohorts, potentially pointing to a role of TERT in DNA repair (Table).

We then analyzed the mutational load specifically in the mitochondria using the cross-entity PCAWG cohort (n = 1200 with data on mitochondrial mutations, S3C and S3E Table). TERT promoter mutation status was not associated with nonsynonymous mitochondrial or other mitochondrial mutations (S3C and S3E Table). However, TERT expression correlated with fewer, especially nonsynonymous mitochondrial mutations (Wilcoxon p = 0.007, Spearman p = 0.049, coeff = -0.06, S3D Table, Fig 3C) in stark contrast to the increased overall, and mainly nuclear, mutations assessed above (in SKCM metastases, n = 363 with data on overall mutations, Fig 3B). We further tested whether the expression of the mitochondrial targeting sequence, which is required for the transport of TERT into mitochondria [24], could be affected by TERT promoter mutations. Here, we did not detect a change in the proportion of expressed isoforms with mitochondrial targeting sequence (S5A Table) and conclude that TERT promoter mutations do not promote or restrain transcripts with mitochondrial targeting (Fig 3C).

TERT alterations in hypoxia and chromothripsis

We next tested whether TERT promoter mutations and TERT expression correlated with hypoxia and chromothripsis in the PCAWG and SKCM cohorts (S3B–S3E Table). Ragnum hypoxia scores in melanoma patients (SKCM, Wilcoxon p = 0.024, S3C Table) and Buffa scores across entities (PCAWG, Wilcoxon p = 4.678e-21, Fig 3C) were higher in samples carrying a TERT promoter mutation. We also observed upregulation of TERT expression with all hypoxia scores in the melanoma cohort (S3B Table) and with Buffa hypoxia scores across entities (Wilcoxon p = 7.43e-22). We observed significantly more chromothripsis in samples carrying TERT promoter mutations and a higher median TERT expression in samples with chromothripsis (PCAWG, S3D, S3E Table).

Discussion

TERT and ICI therapy

TERT promoter mutations have been discussed as potential prognostic factors in metastatic melanoma [5, 6, 8, 26, 27] and as potentially predictive most recently under BRAF/MEK [7] and anti-CTLA4 therapy [9]. TMB is discussed as a biomarker for ICI success [32, 33] and TERT promoter mutations showed association with higher TMB [9], however, multivariate approaches controlling for TMB were missing. Our analyses show that, when controlling for mutational load and prior MAPK inhibitor therapy, the association of TERT promoter mutations in the tested anti-CTLA4 and non-ICI cohorts was weak or absent. Hence, we believe that the extent to which TERT promoter mutations govern survival under anti-CTLA4 therapy is not statistically supported [7, 8]. In addition, since elevated TERT expression was not associated with survival, we conclude that melanoma differs from other cancer entities that showed an association between TERT levels and prognosis in lung cancer, oral squamous cell carcinoma, and colorectal carcinoma [10–12]. To our best knowledge, TMB, however, was not assessed in these studies, and could possibly be improved by multivariate analyses. A striking correlation of TERT expression in our study was a higher estimate of tumor-infiltrating CD4+ T cells, which were previously associated with improved clinical outcome of ICI therapy [34]. There is evidence for a baseline TERT-specific CD4+ T cell immune response, occurring in over 50% of melanoma patients, rising to 80% in ICI responders [35, 36]. Consequently, a higher CD4+ T cell response, possibly explained by an anti-TERT T-helper type 1 cell response, could define a subgroup of patients with better ICI therapy response. However, our analyses of single-cell expression showed that, if malignant cells expressed higher levels of TERT, the corresponding CD4+ T cells from these tumors expressed exhaustion markers at higher levels (Fig 1B). Therefore, in TERT-high hypoxic melanoma, dysfunctional CD4+ T cells may accumulate employing these immuno-suppressive checkpoints [37]. As previously suspected, this could argue for a defect in anti-TERT CD4+ response during melanoma progression [36] and may explain why in our analysis, patients with high CD4+ T cell estimates and increased TERT expression did not show a survival benefit.

TERT expression in metastases from thin primaries

TERT promoter mutations were previously described to upregulate TERT expression [38, 39] involving the formation of ETS/TCF transcription factor binding sites [2, 38], and have been found in primary and metastatic melanoma. Here, metastatic melanomas showed higher TERT expression if they originated from thinner primaries, prompting the question of increased TERT expression in efficient early metastasis. This is especially relevant as TERT expression has been shown to be involved in most steps of the invasion-metastasis cascade [40]. The local invasion of primary melanoma cells might be regulated through increased TERT expression affecting cellular programs, such as NF-kB or metalloproteinases in addition to canonical telomerase activity [41, 42]. As the gene expression enrichment of specifically malignant melanoma cells revealed that TERT expression is co-regulated with integrins, TGF-beta, and interleukins, a TERT expression shift may be involved in remodeling the extracellular matrix, which is seen as a driving force for cancer stemness [43] and would aid invasion. It remains unclear whether TERT expression was elevated already in the thinner primary tumors corresponding to the metastases measured for RNA (Fig 2B). Higher Breslow depth was previously shown to be associated with the presence of TERT mutations in primary tumors [5, 26]. However, this association was not apparent in our analysis, probably because only thicker primaries were selected for the RNA-seq experiment in the SCKM cohort.

In conclusion, our data indicate that TERT expression may contribute to early metastasis from thin primaries, potentially through extracellular matrix remodeling, without a clear role for promoter mutations in this respect.

Hotspot mutations, mutational load and DNA repair

Initially, we confirmed the known co-occurrence of TERT promoter mutations and hotspot mutations in BRAF, RAS and NF1 as previously described for melanoma [5]. While, thus a co-mutation of the TERT promoter and members of the MAPK pathway is evident, TERT expression varied significantly between mutants, showing lower TERT expression in BRAF mutants compared to RAS mutants. Moreover, our finding that BRAF mutants exhibited lower TERT expression compared with non-BRAF mutants in TERT promoter-mutated samples somewhat contradicts the proposed model of TERT reactivation in BRAF-mutated melanoma samples through acquisition of TERT promoter mutations, subsequent binding of TCF, and thus increased TERT expression [44]. These contradicting findings are also of great interest for thyroid tumors, as the status of TERT promoter mutations together with BRAF mutations have been shown to be associated with clinicopathological aggressiveness [45–47]. As our analysis of co-mutations were only available in one cohort, further investigations may improve our understanding of the MAPK pathway and TERT activation via TERT promoter mutations.

As a source of hotspot mutations, mutational load is also correlated with TERT expression in metastatic melanomas here and previously described [9]. The increased co-expression of DNA repair-related genes suggests that TERT expression may be involved in establishing genomic stability under replication stress since TERT expression regulates the chromatin state and DNA damage responses in normal human fibroblasts [48]. Beyond melanoma, co-expression of TERT and DNA repair genes was shared in various cancer entities including GBM, however, common gene expression patterns were not observed in association with TERT promoter mutations. To date, studies of TERT alterations in GBM have focused on the fairly frequent promoter mutations. Hence, our results may spark interest in assessing TERT expression levels in GBM to test sensitivity to DNA damage [49]. This could be relevant in the context of chemo- and radiotherapy independent of TERT promoter mutations, since TERT expression is also mediated via rearrangements and hypermethylation [50]. Moreover, our results support the view that this non-canonical function of TERT could be especially relevant under hypoxic conditions and after chromothripsis, where TERT expression was increased. In line with the proposed concept of a two-step activation of TERT [38], the up-regulation of TERT expression in later stages of melanoma development could aid the survival of melanoma cells in hypoxic tumors and metastasis to other sites.

TERT and mitochondrial stability

In cancer cells exposed to hypoxia, ROS levels produced by mitochondria are increased [51]. Moreover, TERT has previously been shown to be involved in the protection of cells from ROS-mediated oxidative stress, delaying aging processes supposedly via TERT activity in mitochondria [15]. As these effects were described predominantly in non-cancer tissues, we aimed to identify potential mitochondrial correlates of TERT expression in cancer and, specifically, in melanoma. While at first, we did not find associations of TERT with a set of fifteen ROS-related candidate genes, our less-biased discovery approach identified a consistently upregulated DNA2, known to be involved in DNA repair in the nucleus and mitochondria [52]. This is in line with TERT translocation into mitochondria to protect mitochondrial DNA from damage and implicates a dedicated repair gene in the described process [15, 24]. The correlation between TERT expression and fewer nonsynonymous mitochondrial mutations further supports this rationale. It contrasts though, with the fact that TERT expression and TERT promoter mutations were associated with more overall mutations in the nuclear genome. Therefore, DNA repair via TERT and possibly DNA2 may be mitochondria-specific. Interestingly, differing mutational processes have already been described in colorectal and gastric cancers, where microsatellite instability was observed in the nuclear, but not in the mitochondrial genome [53]. Thus, it would be highly interesting to explore the cellular localization of TERT and DNA2 in various cancers and in the context of hypoxia.

A potential limitation of our study is that different subtypes of TERT promoter mutations may differentially affect TERT expression and, therefore clinical factors. However, individual promoter mutations were only available for the SKCM and CCLE cohorts. Indeed, in both cohorts, the C288T mutation is the only mutation showing association with increased TERT expression (S5D Table). However, analyzing the different promoter mutations individually, whenever possible, did not reveal any differences in our results (S10 Fig). We strongly believe that, as the assessment of mutation types is currently part of routine testing, the different effects of the subtypes of TERT promoter mutations could be taken into account in future studies.

In conclusion, our analyses did not support a significant association between TERT genetic alterations and survival in melanoma patients. Hence, its presumed role as a biomarker, especially for anti-CTLA4 or MAPK inhibitor therapy, needs further validation. In addition, our data indicate dysfunctional CD4+ T cells in TERT high hypoxic melanoma. Since melanoma metastases with high TERT expression were shown to have originated from thinner primary tumors, we conclude that upregulation of TERT expression may contribute to metastasis. This is also supported by the involvement of TERT expression in the dynamics of the extracellular matrix. We add support to the non-canonical function of TERT expression related to mitochondrial and nuclear DNA stability even across cancer entities, especially under hypoxic conditions and after chromothripsis. In summary, our results demonstrate the possible role of TERT expression in metastasis formation and invasion in melanoma and highlight its role in DNA repair mechanisms across cancer entities.

Supporting information

S1 File. Supplementary material including the supplementary methods, description of supplementary tables and S1-10 Figs.

(DOCX)

Click here for additional data file.^{(2.4MB, docx)}

S1 Table. Multivariate survival analysis.

Multivariate analysis using Cox proportional hazard models for progression free survival (PFS) (S1A Table) and overall survival (OS) (S1B Table) subsetted for ICI therapy type in cohorts Liu, Gide, Hugo, Riaz, Van Allen and SKCM.

(XLSX)

Click here for additional data file.^{(56.8KB, xlsx)}

S2 Table. TERT expression and immune signatures.

Significant immune signatures in SKCM associated with TERT expression and comparison against other bulk RNA-seq cohorts (Gide, Hugo, Liu, Riaz, Van Allen).

(XLSX)

Click here for additional data file.^{(19.9KB, xlsx)}

S3 Table. TERT with respect to clinical variables.

Wilcoxon tests for group comparisons and Spearman correlations of clinical variables against TERT expression and TERT promoter mutation in Liu, SKCM and PCAWG cohorts (S3A–S3E Table).

(XLSX)

Click here for additional data file.^{(73.6KB, xlsx)}

S4 Table. TERT in melanoma subtypes.

Mutation subtypes against TERT expression (S4A Table) and TERT promoter mutation status (S4B Table) in the SKCM melanoma cohort. Effect of hotspot mutations on TERT expression in TERT promoter mutated samples (S4C Table).

(XLSX)

Click here for additional data file.^{(10.5KB, xlsx)}

S5 Table. TERT isoforms and mutation subtypes and influence of TERT alterations on ROS.

Influence of TERT promoter mutation on TERT isoform on mitochondrial targeting sequence (S5A Table), associations of ROS-related genes with TERT expression levels (S5B Table), and TERT promoter mutation status (S5C Table) in Gide, Riaz, Hugo, Liu, Van Allen, SKCM, PCAWG, CCLE cohorts, and single-cell. Difference on TERT promoter mutation subtypes on TERT expression on SKCM and CCLE cohorts (S5D Table).

(XLSX)

Click here for additional data file.^{(16.9KB, xlsx)}

S6 Table. Genetic correlates with TERT promoter mutations.

Genes associated with TERT promoter mutation status for the Van Allen, SKCM, PCAWG, and CCLE cohorts, and overlap of cohorts (S6A–S6E Table).

(XLSX)

Click here for additional data file.^{(9.9MB, xlsx)}

S7 Table. Genetic correlates with TERT expression.

Genes associated with TERT expression in each cohort (Gide, Riaz, Van Allen, Hugo, Liu, SKCM, PCAWG, CCLE, single-cell, and Klughammer) and overlap of cohorts (S7A–S7K Table).

(XLSX)

Click here for additional data file.^{(16.6MB, xlsx)}

S8 Table. Enrichment analysis.

Enrichment analysis of 17 genes associated with TERT expression and 30 genes associated with TERT promoter mutation in bulk tumors and cell lines (S8A Table), and for the top 100 genes associated with TERT expression in single-cells (S8B Table).

(XLSX)

Click here for additional data file.^{(178.2KB, xlsx)}

Acknowledgments

We thank Udo Stenzel for help with the data analysis.

Abbreviations

anti-CTLA4: Antibodies for cytotoxic T-lymphocyte-associated protein 4
BRAF: V-raf murine sarcoma viral oncogene homolog B1
CCLE: Cancer cell line encyclopedia
CD4+/CD8+: Cluster of differentiation 4/8
Cox PH: Cox proportional-hazard ratio
DNA2: DNA replication helicase/nuclease 2
ETS: Erythroblast transformation specific (transcription factor)
FPKM: Fragments per kilobase of transcript per million
GAPB: GA-binding protein
GBM: Glioblastoma multiforme
GTEx: Genotype-tissue expression project
HAVCR2/TIM3: Hepatitis A virus cellular receptor 2/T-cell immunoglobulin and mucin-domain containing-3
HRAS: Harvey rat sarcoma viral oncogene
ICI: Immune checkpoint inhibition
KRAS: Kirsten rat sarcoma viral oncogene
MAPK: Mitogen-activated protein kinase
MEK: Mitogen-activated kinase
Myc: Myelocytomatosis, MYC proto-oncogene, bHLH transcription factors
NF-kB: Nuclear factor kappa B (transcriptions factor)
NF1: Neurofibromatose type 1
NRAS: Neuroblastoma ras viral oncogene homolog
OS: Overall survival
PCAWG: Pan-cancer analysis of whole genomes cohort
PDCD1/PD1: Programmed cell death protein 1
PFS: Progression free survival
ROS: Reactive oxygen species
scRNA-seq: Single-cell RNA-sequencing
SKCM: Skin cutaneous melanoma cohort
TCF: Ternary complex factor
TERT: Telomerase reverse transcriptase
TGF-beta: Transforming growth factor beta
TIGIT: T cell immunoreceptor with Ig and ITIM domains
TMB: Tumor mutational burden
TPM: Transcripts per million
Wnt/β-catenin: "Wingless-related integration site" Wnt/beta-catenin pathway

Data Availability

The data used in this study are available within this paper and its supplementary files. Previously published, processed NGS data can be accessed at https://tools.hornlab.org/cru337phenotime/.

Funding Statement

This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, HO 6389/2-2, SCHA 422/17-2, 'KFO 337' - 405344257). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0281487.r001

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PONE-D-23-01824TERT expression is associated with metastasis from thin primaries, exhausted CD4+ T cells in melanoma and with DNA repair across cancer entitiesPLOS ONE

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Additional Editor Comments;

1. Raised critiques to be addressed in a point-by-point manner before consideration, this would also substantially improve the manuscript.

2. Care is necessary in addressing particularly the reviewer #2 and the same is critical for publication.

3. Consider discussing if mutation in TERT could have any similar impact as expression and discuss the same with melanoma and others including thyroid by citing these articles PMID: 23766237; PMID: 24617711; PMID: 25024077.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: It would help to appreciate the importance of the identified TERT mRNA gene associations and the directions (up/down regulated) better if the authors could add the corresponding gene expression levels (raw expression counts normalized to logCPM (counts per million)) and the logFC (fold changes).

Reviewer #2: In this manuscript, Kuhn et al. claimed that TERT alterations were not consistently

associated with survival in melanoma cohorts under immune checkpoint inhibition. The presence of CD4+ T cells increased with TERT expression and correlated with the expression of exhaustion markers. While the frequency of promoter mutations did not change with Breslow thickness, TERT expression was increased in metastases arising from thinner primaries. Increased TERT expression was related to mitochondrial DNA stability and nuclear DNA repair during chromothripsis (PCAWG cohort) and under hypoxic conditions (PCAWG and SKCM cohorts). This should be a clear story, but authors tried to fit all the data into one story and made it so hard to understand and audiences were keeping lost in the irrelevant description.

Major points:

1. TERT promoter mutations were not always be associated with TERT expression in all cohorts. This made things much more complicated and confusing. If some mutations led to high TERT expression, while other mutations led to low TERT expression or irrelevant, authors should take this into account and some non-significant differences may actually significant.

2. It is so hard to understand the results parts and figures. They seem completely separated. I can’t even find where the figures were mentioned in the result parts (fig. 1B was even only in the discussion part). My suggestion is following the figures, which are much clearer than the results description.

Minor points:

1. There should be an abbreviation chart or explain every abbreviation when they appear first time.

2. Page 6, single-cell RNA-seq data were mentioned. However, authors failed to describe the results.

3. Page 7, in first line, “was not associated with nonsynonymous”, is that a typo or the result in page 6 was wrong?

4. Discussion should mainly focus on authors’ data.

Reviewer #3: The manuscript-“TERT expression is associated with metastasis from thin primaries, exhausted CD4+ T cells in melanoma, and with DNA repair across cancer entities” by Kuhn et al, is an interesting study adding new insights into melanoma metastasis that is attributed to TERT expression. The manuscript is well-written, and the methodology is adequately described. The overall results presented in the manuscript are logically described. The choice of melanoma cohorts, cell lines as well as single-cell RNA seq data analysis look appropriate in context to TERT mutations. Overall, the results are clear and compelling with a well-structured possible role of TERT in mitochondrial and nuclear DNA stability.

I would recommend this manuscript for publication.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PLoS One. 2023 Jul 7;18(7):e0281487. doi: 10.1371/journal.pone.0281487.r002

Author response to Decision Letter 0

25 Apr 2023

We would like to thank the editorial team and the reviewers for their valuable comments and in-depth review of our manuscript. We are convinced that the critiques have had a great impact on the strengthening of the quality of our work. We have carefully addressed all the criticisms, especially those of reviewer #2, in the attached file "Response to Reviewers".

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(391KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0281487.r003

Decision Letter 1

Avaniyapuram Kannan Murugan

20 Jun 2023

TERT expression is associated with metastasis from thin primaries, exhausted CD4+ T cells in melanoma and with DNA repair across cancer entities

PONE-D-23-01824R1

Dear Dr. Katharina Kuhn,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Avaniyapuram Kannan Murugan, M.Phil., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

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PLoS One. doi: 10.1371/journal.pone.0281487.r004

Acceptance letter

Avaniyapuram Kannan Murugan

27 Jun 2023

PONE-D-23-01824R1

TERT expression is associated with metastasis from thin primaries, exhausted CD4+ T cells in melanoma and with DNA repair across cancer entities

Dear Dr. Kuhn:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Avaniyapuram Kannan Murugan

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. Supplementary material including the supplementary methods, description of supplementary tables and S1-10 Figs.

(DOCX)

Click here for additional data file.^{(2.4MB, docx)}

S1 Table. Multivariate survival analysis.

(XLSX)

Click here for additional data file.^{(56.8KB, xlsx)}

S2 Table. TERT expression and immune signatures.

Significant immune signatures in SKCM associated with TERT expression and comparison against other bulk RNA-seq cohorts (Gide, Hugo, Liu, Riaz, Van Allen).

(XLSX)

Click here for additional data file.^{(19.9KB, xlsx)}

S3 Table. TERT with respect to clinical variables.

Wilcoxon tests for group comparisons and Spearman correlations of clinical variables against TERT expression and TERT promoter mutation in Liu, SKCM and PCAWG cohorts (S3A–S3E Table).

(XLSX)

Click here for additional data file.^{(73.6KB, xlsx)}

S4 Table. TERT in melanoma subtypes.

(XLSX)

Click here for additional data file.^{(10.5KB, xlsx)}

S5 Table. TERT isoforms and mutation subtypes and influence of TERT alterations on ROS.

(XLSX)

Click here for additional data file.^{(16.9KB, xlsx)}

S6 Table. Genetic correlates with TERT promoter mutations.

Genes associated with TERT promoter mutation status for the Van Allen, SKCM, PCAWG, and CCLE cohorts, and overlap of cohorts (S6A–S6E Table).

(XLSX)

Click here for additional data file.^{(9.9MB, xlsx)}

S7 Table. Genetic correlates with TERT expression.

Genes associated with TERT expression in each cohort (Gide, Riaz, Van Allen, Hugo, Liu, SKCM, PCAWG, CCLE, single-cell, and Klughammer) and overlap of cohorts (S7A–S7K Table).

(XLSX)

Click here for additional data file.^{(16.6MB, xlsx)}

S8 Table. Enrichment analysis.

(XLSX)

Click here for additional data file.^{(178.2KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(391KB, docx)}

Data Availability Statement

The data used in this study are available within this paper and its supplementary files. Previously published, processed NGS data can be accessed at https://tools.hornlab.org/cru337phenotime/.

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PERMALINK

TERT expression is associated with metastasis from thin primaries, exhausted CD4+ T cells in melanoma and with DNA repair across cancer entities

Christina Katharina Kuhn

Jaroslawna Meister

Sophia Kreft

Mathias Stiller

Sven-Holger Puppel

Anne Zaremba

Björn Scheffler

Vivien Ullrich

Torsten Schöneberg

Dirk Schadendorf

Susanne Horn

Roles

Abstract

Introduction

Materials and methods

Next generation sequencing data analysis

Table 1. Analyzed cohorts.

Enrichment analysis

TERT isoform analysis

Statistics

Data availability statement

Results

Association of TERT promoter mutation status and TERT expression with survival

TERT expression and increased intratumoral CD4+ T helper type 1 cells

Fig 1. TERT expression and immune signatures.

Primary tumor thickness

Fig 2. Primary tumor thickness and TERT in corresponding metastases.

TERT in melanoma subtypes

Mutational load

Fig 3. Genetic associates of TERT alterations.

Gene expression associated with TERT alterations

Table 2. Genes repeatedly associated with TERT mRNA in 9 cohorts (S7 Table).

Association of TERT and mitochondrial variables

TERT alterations in hypoxia and chromothripsis

Discussion

TERT and ICI therapy

TERT expression in metastases from thin primaries

Hotspot mutations, mutational load and DNA repair

TERT and mitochondrial stability

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Avaniyapuram Kannan Murugan

Roles

Author response to Decision Letter 0

Decision Letter 1

Avaniyapuram Kannan Murugan

Roles

Acceptance letter

Avaniyapuram Kannan Murugan

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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