The TERT copy number gain is sensitive to telomerase inhibitors in human melanoma.

Telomerase reverse transcriptase (TERT) copy number gain is frequently observed in Asian melanoma patients. Here, we explored the correlation between TERT copy number and the effect of telomerase inhibitors in melanoma. A total of 78 melanoma cases were enrolled in the study. The TERT copy number was examined by QuantiGenePlex DNA assay. The sensitivity to telomerase inhibitors was evaluated in cell lines and patient-derived xenograft (PDX) models with or without TERT copy number gain. Among the 78 patients, 33.3% showed TERT copy number gain, and the incidence of this gain in acral melanoma (61.5%) was higher than that in other melanoma subtypes (p=0.02). The telomerase inhibitors 6-Thio-dG and EGCG inhibited cell viability and repressed tumor growth in PDX models with TERT copy number gain.TERT copy number gain is frequently observed in Chinese patients with melanoma. Targeting telomerase may benefit melanoma patients with TERT copy number gain.


Background
With its fast-rising morbidity and high mortality, malignant melanoma (MM) is one of the most aggressive cancers worldwide 1,2 . Cutaneous melanoma is the predominant type of melanoma in Caucasians, while acral melanoma and mucosal melanoma are prevalent in Asian populations [3][4][5][6][7] . In recent years, immunotherapy and targeted therapy have improved the clinical outcome of patients with MM; however, patients with acral or mucosal MM achieve limited benefits. Blockade of programmed cell death (PD)-1 protein (also known as B7-H1), which functions as an immune checkpoint inhibitor, has been approved by Food and Drug Administration (FDA) as a first-line treatment for advanced melanoma and approved by the Chinese FDA (cFDA) as a second-line treatment for advanced melanoma [8][9][10][11] . In Caucasian cohorts, the overall efficacy rate of PD-1 blockade in melanoma was approximately 26% -44%, whereas the response rate in acral and mucosal melanoma was 32% and 23%, respectively 10,12,13 . In Asian cohorts, the overall efficacy rate of PD-1 blockade in melanoma was approximately 16.7% -20.7, whereas the response rate in acral and mucosal melanoma was 14.3% -15.8% and 0 -13%, respectively 14,15 .
The combination of BRAF inhibitor with MEK inhibitor was recommended as a first-line treatment for MM patients harboring the BRAF V600E mutation by the United States National Comprehensive Cancer Network (NCCN) treatment guideline (2016 Edition) for MM. However, the frequency of BRAF V600E is only 15.5% and 12.5% in acral and mucosal MM, respectively 16 . Furthermore, patients with acral or mucosal melanoma harboring the BRAF V600E mutation had lower response rates to corresponding inhibitors than patients with cutaneous melanomas with the same mutation 17,18 .
Telomerase is a ribonucleoprotein composed of telomerase reverse transcriptase (TERT) and RNA moieties. Telomerase maintains telomere length by adding telomeric repeats to chromosome ends [19][20][21] . It is commonly believed that progressive shortening of telomeres in normal cells weakens the replicative ability, which keeps the cell from engaging in tumor progression 22,23 . Dysregulated expression of telomerase is a key factor that drives the malignant transformation in many types of cancer 24 . As the most important element of telomerase, TERT functions as a specific reverse transcriptase and mature testing methods of it have been established 25 . Overexpression of TERT results in increased telomerase activity and aberrantly long telomeres, thus overcoming the proliferative barrier imposed by telomere shortening 26 . Besides, TERT also function independently on telomere 27-37 . In 2017, a study of 183 patients with melanoma (including 38 acral and 8 mucosal melanoma patients) revealed a high frequency of mutations at the TERT promoter 38 . A recent study on mucosal genomics confirmed again that mucosal melanoma has a high frequency of TERT gene variation 39 . These results indicate that the TERT pathway plays a key role in melanoma in Asian populations, but whether TERT can serve as an effective therapeutic target for melanoma is unclear.
In this study, we focused on TERT copy number variation and its association with clinicopathological parameters in 78 melanoma patients (including 30 with acral melanoma and 11 with mucosal melanoma) and evaluated the inhibitory effect of TERT inhibitors on melanoma cells harboring TERT copy number gain in cell lines and PDX models.  Table S1). This study was approved by the Medical Ethics Committee of the Beijing Cancer Hospital & Institute and was conducted according to the principles established by the Declaration of Helsinki. QuantiGene Plex DNA assay Tissue homogenates were prepared from formalin-fixed, paraffin-embedded (FFPE) tissues with a QuantiGene Sample Processing Kit (Affymetrix Panomics, Santa Clara, CA, USA) as described previously 40 . The tissue homogenate was used to test the TERT copy number by the branched DNA (bDNA) assay with a QuantiGene Plex DNA kit (Panomics). Measurement of the mean fluorescence intensities was performed in duplicate for all genes with the Bio-Plex 100 system (Bio-Rad). The geometric mean values of Rpph1, Rpp30 and Rplp0 served as the normalization control. For each tested gene, the normalized signal was divided by the signal of the reference human genomic DNA sample (G1521, Promega, Madison, America), and the values were multiplied by the known copy number (usually 2 copies) of each gene in the reference genome. The TERT copy numbers for the samples were identified as follows: copy number >2 was considered copy number gain, and copy number ≤ 2 was considered no copy number gain. DNA isolation and TaqMan copy number assay Total DNA was extracted from 5-8 FFPE sections using a QIAamp DNA FFPE Tissue Kit (56404, Qiagen, German). The copy number of TERT (Hs03078158_cn) was further quantified by TaqMan Copy Number Assays (Applied Biosystems of ThermoFisher). Quantitative real-time PCR was performed with an ABI 7500 FAST real-time PCR system (Applied Biosystems), and the relative copy number of TERT was calculated with CopyCaller v2.0 software (Applied Biosystems) using the ΔΔCt method. A relative copy number > 2.0 was defined as of TERT copy number gain, whereas a relative copy number equal to or less than 2.0 was defined as no TERT copy number gain. Immunohistochemistry Immunohistochemistry (IHC) was performed using rabbit monoclonal antibodies against Ki-67 (1:400; ab16667, Abcam, England) as described previously 41 . Three pathologists without prior knowledge of the TERT copy number independently evaluated all the slides. The Ki-67 staining score was marked by a positive rate (%).

Cell lines and primary cell culture
The A549 (catalog no. CCL-185), A2058 (catalog no. CRL-11147) and BJ fibroblast (catalog no. CRL-2522) cell lines were obtained from American Type Culture Collection and cultured at 37°C in a 5% CO 2 atmosphere in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen of ThermoFisher, MA, America) supplemented with 10% fetal bovine serum (HyClone of GE Healthcare, Logan, America) and 1% penicillin/streptomycin (Invitrogen of ThermoFisher, MA, USA). The cells were cultured in sterile culture flasks (Corning, America) and passaged every 2 days using 0.25% trypsin (Invitrogen of ThermoFisher, MA, America).
Human melanoma samples for the patient-derived xenograft (PDX) model were dissected and placed in tissue storage solution (Percans Oncology, cat. no. RUO-00101). After the samples were washed with cold PBS containing 1% penicillin-streptomycin, they were minced with surgical scissors and then subjected to enzyme digestion with a primary cell preparation kit (Percans Oncology, cat. no. RUO-00201) according to the manufacturer's instructions. The dissociated tumor cells were expanded using a modified conditional reprogramming (CR) technique named i-CR. Briefly, the cell suspension was filtered through a 100 m cell strainer to remove large pieces. After centrifugation, the cell pellet was collected and treated with red blood cell lysis buffer. The cells were then pelleted and washed three times with PBS. The isolated tumor cells were cultured in complete culture medium (Percans Oncology, cat. no. RUO-00601) at 37°C in 5% CO 2 .
The effects of the selected inhibitors on the i-CR primary tumor cells were measured using a cell quantification assay kit (Percans Oncology, RUO-00401). In brief, cells were continuously treated with the drugs or control for 7 days, and live cell staining was performed for the last 24 hours. The cells were fixed, stained with a nuclear dye to determine the total cell number and then scanned with an ArrayScan XTI 800. Images were captured and analyzed with the built-in BioApplication software package. The effect of each treatment regimen was quantified using the following formula: maximum inhibition (MI)=N 0 /N d , where N 0 and N d denote the number of proliferating tumor cells in wells treated with DMSO control and drug, respectively.

Immunoprecipitation and Western blotting
The protein expression level of TERT was detect by immunoprecipitation (IP) followed by Western blotting (WB) as reported 25 . IP was performed according to the instruction of Pierce™ Co-Immunoprecipitation Kit (26149, Thermo Scientific, America). Western blotting was performed using a standard method as previously described 42 . GAPDH antibody (1:3000; ab8245, Abcam, England) and telomerase antibody (1:1000; ab32020, Abcam, England) were used in accordance with the manufacturer's instructions. Image J software was used to analyzed the intensities of western bands.

Patient-derived xenograft model and treatment
Patient-derived melanoma tissues with or without TERT copy number gain were cut into pieces and then subcutaneously inoculated into NOD/SCID female mice (4 to 6 weeks old, 18-22 g weight) to establish the PDX model. When the tumor volume reached approximately 1 cm 3 , the mice were sacrificed by cervical dislocation, and tumor tissues were separated and reinoculated into recipient mice; this procedure was repeated for 3 passages. Eight groups of PDX model mice were finally established, of which four harbored TERT copy number gain.
When the tumor volume reached approximately 100 mm 3 , mice were treated with either control vehicle or the telomerase inhibitors EGCC (i.p. 50 mg/kg, every other day) or 6-thio-dG (i.p. 10 mg/kg, daily). Tumor volume was measured every 3 days and was calculated according to the following formula: volume = length * width 2 /2. The tumor growth inhibitory rate (IR) = (volume of treatment armthe volume of control arm)/ volume of control arm*100%. Fourteen days after the first treatment, the mice were sacrificed, and the tumors were fixed in 10% formalin for IHC analysis. The above experiments were repeated twice. All procedures involving animals were performed in compliance with the Animal Care Ethics guidelines and were approved by the Medical Ethics Committee of the Beijing Cancer Hospital & Institute. And all the animal work took place in the experimental animal house of Beijing Cancer Hospital & Institute. Statistical analysis All statistical analyses were performed using SPSS 19.0 software (SPSS Inc.). Continuous data distributed normally are expressed as the mean value ± mean standard deviation (SD). Chi-square test or Fisher's exact test was used to analyze the correlations between TERT gain and clinical characteristics. The statistical significance of differences between the control and experimental groups was assessed by Student's t test and one-way ANOVA or the nonparametric test. The comparison of the tumor growth curves was done with the repeated measure variance analysis. A p-value < 0.05 was considered significant. Data and materials available All materials will be made available to the scientific community.

TERT copy number is gained in melanoma patients
To investigate the copy number of TERT in melanoma, 78 patients were enrolled in this study. A total of 26 cases (33.3%) showed gain of TERT copy number, among which 2-3 copies accounted for 65.4%.
Detailed information for each patient, such as gender, age, thickness, ulceration, TNM stage, TERT status and melanoma subtype, is described in Supplementary Table S1. The mean age (50.08 vs. 49.73) and gender distribution were comparable between patients with TERT gain and those with a normal TERT copy number ( Table 1). The median thickness of samples with TERT gain was 4 mm (range: 1.3-20 mm), and that of samples with a normal TERT copy number was 3.75 mm (range: 1.2-9 mm); this difference was not significant between the two groups (p=0.764) ( Table 1). The ulceration rate and TNM stage were not significantly different between patients with TERT gain and those with a normal TERT copy number ( Table 1). We also analyzed the correlation of TERT copy number gain and melanoma subtype. Among the 26 patients with TERT gain, patients with acral, mucosal, CSD, non-CSD and unknown primary subtype comprised 61.5% (16), 3.8% (1), 3.8% (1), 23.1% (6) and 7.7% (2) of the subgroup, respectively, which was significantly different from the frequencies in the normal TERT copy number subgroup (p =0.02) ( Table 1). The rate of TERT copy number gain is high in acral melanoma, and TERT copy number gain is more commonly observed in acral melanoma patients.
Cells with TERT gain are more sensitive to telomerase inhibitors Next, we explored the correlation between TERT copy number and the effect of telomerase inhibitors on cell proliferation. To this aim, we searched the Broad Institute Cancer Cell Line Encyclopedia (CCLE) database (https://portals.broadinstitute.org/ccle/about) for the TERT copy number of different cell lines. A549 (lung cancer cells, positive control) and A2058 (melanoma cells) cells were selected as cancer cells with TERT copy number gain and BJ fibroblast cells as control cells with a normal TERT copy number. The TERT copy number of the three cell lines detected by TaqMan PCR was 2, 5 and 8 in BJ fibroblasts, A549 cells and A2058 cells, respectively (Figure 1A), which is consistent with the CCLE database. Further IP and Western blotting also showed that the TERT protein expression level in A549 and A2058 was higher than that in BJ fibroblasts ( Figure 1B). Then, cell proliferation assays were performed to explore the effects of telomerase inhibitors (Cos, 6-Thio-dG, RHPS, EGCG, and BIBR) on cells with or without TERT copy number gain. ECGC and 6-thio-dG exhibited a higher inhibitory effect in A549 and A2058 cells than in BJ fibroblast cells ( Figure 1C). However, the inhibitory efficiency of RHPS, Cos and BIBR was not specific for A549 or A2058 cells ( Figure  1C). Therefore, we measured the protein expression of telomerase in these cells cultured in the presence or absence of the inhibitors. We found that telomerase protein expression was significantly decreased in A549 and A2058 cell lines but not in BJ cells when treated with ECGC or 6-thio-dG ( Figure 1D). In contrast, the protein expression of telomerase did not change when treated with Cos, RHPS or BIBR ( Figure 1D). These data indicate that EGCG and 6-thio-dG have specific inhibitory effects on cancer cells with TERT copy number gain.

Sensitivity of PDX models and primary melanoma cells to telomerase inhibitors
To evaluate the efficacy of EGCG and 6-thio-dG on tumor growth in cells with normal TERT copy number and TERT copy number gain, we established PDX models from four patients harboring TERT copy number gain (PDX-J01, 02, 03 and 04) and another four patients harboring normal TERT copy number (PDX-J05, 06, 07 and 08). Compared with the control treatment, EGCG and 6-thio-dG significantly suppressed the tumor growth of PDX-J01, 02, 04 and 07, three of which were from the group with TERT copy number gain (Figure 2A). The tumor growth IR was significantly higher in these models ( Figure 2B). The effective rates of the two telomerase inhibitors in models with TERT copy number gain and normal TERT copy number were 75% and 25%, respectively. The mean tumor growth IR of telomerase inhibitor-sensitive models of the two groups was 51% and 56%, respectively. In addition, primary cells from patients PDX-J01 and PDX-J02 were separated and cultured successfully. EGCG and 6-thio-dG significantly inhibited the in vitro viability of these primary cells with TERT copy number gain (Figure 3 and Supplementary Table 2).
Next, we subjected tumor tissues to IHC staining of Ki-67. The positive rate of Ki-67 staining in PDX-J01, 02 and 03 decreased after treatment with EGCG or 6-thio-dG, but this decrease was not observed in tissues from the other model patients, including PDX-J05, 06, 07 and 08 (Figure 4). Collectively, these data show that EGCG and 6-thio-dG exhibited better inhibitory effects on melanoma with TERT copy number gain than melanoma without TERT copy number gain in vivo.

Discussion
During the past decade, targeted and immune-based therapies have shown efficacy against cancers. Melanoma has been at the forefront of rapid development in both targeted and immune-based therapies. For example, studies on BRAF and KIT mutations have provided evidence for therapies of melanoma [43][44][45][46][47] . However, in the Asian population, the incidence of BRAF and KIT mutations is only 25.5% and 10.8%, respectively, which is too low to meet the demands for effective clinical therapy 16,41 . PD-l blockade exhibits a surprising antitumor effect on melanoma, but more than half of patients with the acral or mucosal subtypes are resistant to PD-1 blockade 13,46 . Previously, we proved that the copy number variation (CNV) of NRAS and CDK4 predicted the sensitivity to MEK and CDK4/6 inhibitors preclinically, respectively, which suggested discovering new driver CNVs may help to supply more choice for target therapy 40,48 .
TERT is located on chromosome 5p15.33, and variations in this gene were perceived to contribute to tumorigenesis 49 . The induction of telomerase activation by TERT confers immortal potential to cancer cells by stabilizing telomere length. Besides, TERT also plays important role in tumor progression independently on telomere, including regulation mitochondrial and ubiquitin-proteasomal function, DNA damage repair, gene transcription, and microRNA expression 27-37 . What's more, TERT is also a key molecular downstream of the RAS-ERK pathway, which is known to be one of the key oncogenic pathways that drive melanoma progression 50,51 . One previous study revealed that the frequency of mutations in the TERT promoter regions was abnormally increased in some solid tumors, especially in glioblastoma and cutaneous melanoma 52 . In glioma, TERT promoter mutation is a poor prognostic factor and susceptibility locus [53][54][55] . Two recent genomic landscape studies of melanoma revealed that the TERT pathway is likely to be an important driving pathway in melanoma, particularly in mucosal melanoma, the telomere status of which may be influenced by TERT copy number 38,39 . This study cohort of 78 patients showed that the incidence of TERT gain reached up to 33.3% and was specifically higher in acral melanoma (61.5%), which is consistent with a previous study that reported rates from 44.9% to 75% 56,57 .
Several inhibitors targeting TERT or telomerase were tested in preclinical and clinical studies. In the ClinicalTrials.gov database (https://clinicaltrials.gov/), most inhibitor studies were in the early phase, and no clinical study was reported in melanoma. In basic science studies, TERT inhibitors were reported to exert cancer inhibitory effects in leukemia, breast cancer, uterus carcinoma, colon cancer, lung cancer cells, etc. [58][59][60][61] . However, no study exploring the specificity of TERT or telomerase inhibitors has been reported. In studies on melanoma, some TERT/telomerase inhibitors were shown to inhibit melanoma cell line growth in vitro and in vivo through the ERK1/2 or TRAF6 pathways [62][63][64][65] . However, the relationship of TERT copy number gain and the effect of TERT/telomerase inhibitors has not been reported; here, we provide the first evidence of this relationship. We selected 5 inhibitors and observed that EGCG and 6-thio-dG appeared to decrease the TERT expression level in cancer cell lines with a high TERT copy number and showed comparable antitumor effects in the PDX model, which were greater in the TERT copy number gain group. Thus, TERT copy number gain seems to be an indicator for the effectiveness of telomerase inhibitor therapy. EGCG and 6-thio-dG are both indirect/direct telomerase inhibitors. EGCG is the major polyphenolic compound commonly found in green tea and shows beneficial effects on the treatment of cancer, cardiovascular diseases, diabetes, neurodegenerative diseases and viral infection [66][67][68][69][70][71][72][73][74] . Previous study has showed that EGCG could modulate multiple signal transduction pathways, including inhibition of NF-κB [75][76][77] . Meanwhile NF-κB is a direct regulator of TERT expression 78 . So ECGC can inhibit TERT expression through inhibition of NF-κB at least. 6-Thio-dG is a nucleoside analog and telomerase substrate. Previously, 6-thio-dG was shown to induce telomere dysfunction in telomerase-positive human cells, resulting in rapid cell death of cancer cell lines but not of normal human fibroblasts 79 . In this work, we are the first to report the specific efficiency of these two inhibitors in melanoma with TERT copy number gain.
There are some limitations and potential perspectives of our study. As we can see specific case (PDX-J03) with TERT copy number gain was not sensitive to inhibitors. Because other gene variation, transcript and post-transcript level were not analyzed, Downloaded from https://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20190890/865607/cs-2019-0890.pdf by guest on 14 January 2020 and other interference factor was unknown. Besides, PDX-J04 has a very low positive rate of ki67 staining but also sensitive to the inhibitors. That is mainly due to the immense cross talk of TERT between other pathways. As mentioned above, TERT playing its role doesn't only dependent on telomeric function but also dependent on non-canonical function, including anti-apoptosis, gene transcription, such as activation of RNA polymerase Ⅲ etc. 37,80,81 Indeed cells with more TERT will be affected more as these cells with increased copy number of TERT are also more addicted to non-canonical functions of TERT. Therefore, supplement sequencing of transcriptome and epigenome will supply more evidence.
In summary, our study revealed that a group (33.3%) of Chinese melanoma patients harbors TERT copy number gain. The growth of melanoma containing TERT copy number gain was inhibited by EGCG and 6-thio-dG in vitro and in vivo. Therefore, targeting telomerase may benefit melanoma patients with TERT copy number gain, which need to be validated in clinical trials.

Clinical perspectives
TERT pathway plays a key role in melanoma in Asian populations, but whether TERT can serve as an effective therapeutic target for melanoma is unclear.
In this study, we revealed TERT gain is more frequently in acral melanoma. Telomerase inhibitors, 6-Thio-dG and EGCG could reduce TERT expression in cells with TERT gain and also show better growth inhibition effect on cell with TERT gain in vitro and vivo.
In conclusion, a group of Chinese melanoma patients harbors TERT copy number gain. Targeting telomerase may benefit melanoma patients with TERT copy number gain.   (A) TERT copy number was detected by the TaqMan copy number assay in BJ fibroblast, A549, and A2058 cell lines. (B) TERT protein expression was examined by IP followed by western blotting in BJ fibroblast, A549, and A2058 cell lines. (C) BJ fibroblast, A549, and A2058 cell lines were treated with the indicated concentrations of telomerase inhibitors. Cell viability was evaluated by the Cell Titer-Glo Luminescent Cell Viability Assay. The statistical significance of the growth curves (as compared with the BJ fibroblast growth curve) was evaluated by repeated measure variance analysis. (D) TERT protein levels in BJ fibroblasts, A549 cells, and A2058 cells cultured in the presence or absence of the indicated inhibitors were detected by IP followed by western blotting (upper), and the quantitative data were analyzed (lower). * p<0.05 vs. control, **p<0.01 vs. control.    Abbreviations: CSD, melanoma on skin with chronic sun-induced damage; non-CSD, melanoma on skin without sun-induced damage; TNM stage, tumor-node-metastasis stage.