TERT Expression Research Articles

Association of clinicopathological features and prognosis of TERT alterations in phyllodes tumor of breast

Phyllodes tumor (PT) of the breast is a rare but clinically important fibroepithelial tumor with potential risks of recurrence and metastasis. Recent studies identified recurrent TERT promoter mutations in PTs. However, the clinical significance of this alteration has not been fully examined. Two hundred and seven PTs from two intuitions were included. All cases were subjected to immunohistochemical analysis for TERT expression. Analysis of TERT promoter mutations was further performed by Sanger sequencing targeting the hotspot mutation region on cases from one of the involved institutions. The expression of TERT was correlated with clinicopathologic features, mutation status and recurrence. There was an association of TERT expression and its promoter mutation. Both stromal TERT expression and its promoter mutation correlated with PT grading and older patient age. Recurrence free survival (RFS) of PT patients with high stromal TERT expression was shorter if the excision margin was positive. Our findings suggested a possible pathogenic role of TERT alteration in PT malignancy. Currently there is no consensus for re-excision for PT patients with positive surgical margin, particularly for low grade cases. Stromal TERT expression could be potentially useful to guide management patients with benign PTs.

TERT regulates telomere-related senescence and apoptosis through DNA damage response in male germ cells exposed to BPDE invitro and to B[a]P invivo.

Increasing evidence shows that impaired telomere function is associated with male infertility, and various environmental factors are believed to play a pivotal role in telomerase deficiency and telomere shortening. Benzo[a]pyrene (B[a]P), a ubiquitous pollutant of polycyclic aromatic hydrocarbons (PAHs), can act as a reproductive toxicant; however, the adverse effect of B[a]P on telomeres in male reproductive cells has never been studied, and the related mechanisms remain unclear. In this study, we explored the effects of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), the active metabolite of B[a]P, on telomere dysfunction in mouse spermatocyte-derived cells (GC-2) and also the potential role of telomerase in BPDE-induced spermatogenic cell damage. The results showed that BPDE induced cell viability inhibition, senescence, and apoptosis in GC-2 cells in a dose-dependent manner. Shortened telomeres, telomere-associated DNA damage, reduced telomerase activity, and TERT expression were also observed in BPDE-treated cells, accompanied with the activation of DNA damage response pathway (ATM/Chk1/p53/p21). Moreover, by establishing the TERT knockdown and re-expression cell models, we found that TERT regulated telomere length and the expression of DNA damage response-related proteins to influence senescence and apoptosis in GC-2 cells. These invitro findings were further confirmed invivo in the testicular cells of rats orally administrated with B[a]P for 7 days. B[a]P treatment resulted in histological lesions, apoptosis, and senescence in the testes of rats, which were accompanied by shortened telomeres, reduced levels of TERT protein, and increased expression of DNA damage response-related proteins. In conclusion, it can be concluded that TERT-mediated telomere dysfunction contributes to B[a]P- and BPDE-induced senescence and apoptosis through DNA damage response in male reproductive cells.

Study of telomerase reverse transcriptase and uterine-ovarian-specific genes expression in the endometrial tissue of ovariectomized female Sprague-Dawley rats

BackgroundAn in vivo study was carried out to study of telomerase reverse transcriptase and Uterine-Ovarian-specific genes expression in the endometrial tissue of ovariectomized female Sprague-Dawley rats. Material and methodsTwenty-four female Sprague-Dawley rats divided into 4 groups of six rats. The first and second groups were ovariectomized and given tamoxifen and tamoxifen-loaded SLN respectively for six days continuously. Group 3 served as the untreated ovariectomized control group and group 4 was made up of untreated normal healthy rats. At the end of the study, the rats were sacrificed and study of the genes expression and serum zinc and copper were carried out. ResultsThe results showed that the expression of TERT in the group treated with tamoxifen, and tamoxifen-loaded solid lipid nanoparticles, significantly decreased (p<0.001) compared with ovariectomized control group. The results also revealed that the treatment with tamoxifen-loaded solid lipid nanoparticles increased expression of UO-44 gene compared to ovariectomized control group, while there was no difference between tamoxifen treated and control group. ConclusionsEncapsulation of tamoxifen in solid lipid nanoparticles increased its targeting efficiency and improved the impact of the drug on the serum levels of some trace elements.

Hexokinase 2 is a molecular bridge linking telomerase and autophagy.

Autophagy is systematically regulated by upstream factors and nutrients. Recent studies reported that telomerase and hexokinase 2 [HK2) regulate autophagy through mTOR and that telomerase has the capacity to bind to the HK2 promoter. However, the molecular linkage among telomerase, HK2, and autophagy is not fully understood. Here, we show that HK2 connects telomerase to autophagy. HK2 inhibition in HepG2 cells suppressed TERT-induced autophagy activation and further enhancement by glucose deprivation. The HK2 downstream factor mTOR was responsible for the TERT-induced autophagy activation under glucose deprivation, implying that TERT promotes autophagy through an HK2-mTOR pathway. TERC played a role similar to that of TERT, and simultaneous expression of TERT and TERC synergistically enhanced HK2 expression and autophagy. At the gene level, TERT bound to the HK2 promoter at a specific region harboring the telomerase-responsive sequence ‘TTGGG.’ Mutagenesis of TERC and the TERT-responsive element in the HK2 promoter revealed that TERC is required for the binding of TERT to the HK2 promoter. We demonstrate the existence of a telomerase-HK2-mTOR-autophagy axis and suggest that inhibition of the interaction between telomerase and the HK2 promoter diminishes glucose starvation-induced autophagy.

Regulation of mutant TERT by BRAF V600E/MAP kinase pathway through FOS/GABP in human cancer

The unique oncogene duet of coexisting BRAF V600E and TERT promoter mutations are widely found to be a robust genetic background promoting human cancer aggressiveness, but the mechanism is unclear. Here, we demonstrate that the BRAF V600E/MAP kinase pathway phosphorylates and activates FOS, which in turn acts as a transcription factor to bind and activate the GABPB promoter, increasing GABPB expression and driving formation of GABPA-GABPB complex; the latter selectively binds and activates mutant TERT promoter, upregulating TERT expression. Elevated TERT functions as a strong oncoprotein, robustly promoting aggressive behaviors of cancer cells and tumor development. We thus identify a molecular mechanism for the activation of mutant TERT by the BRAF V600E/MAP kinase pathway, in which FOS as a transcriptional factor of GABPB promoter plays a key role in functionally bridging the two oncogenes in cooperatively promoting oncogenesis, providing important cancer biological and clinical implications.

MiR-128 inhibits telomerase activity by targeting TERT mRNA.

Telomerase is a unique cellular reverse transcriptase (RT) essential for maintaining telomere stability and required for the unlimited proliferation of cancer cells. The limiting determinant of telomerase activity is the catalytic component TERT, and TERT expression is closely correlated with telomerase activity and cancer initiation and disease progression. For this reason the regulation of TERT levels in the cell is of great importance. microRNAs (miRs) function as an additional regulatory level in cells, crucial for defining expression boundaries, proper cell fate decisions, cell cycle control, genome integrity, cell death and metastasis. We performed an anti-miR library screen to identity novel miRs, which participate in the control of telomerase. We identified the tumor suppressor miR (miR-128) as a novel endogenous telomerase inhibitor and determined that miR-128 significantly reduces the mRNA and protein levels of Tert in a panel of cancer cell lines. We further evaluated the mechanism by which miR-128 regulates TERT and demonstrated that miR-128 interacts directly with the coding sequence of TERT mRNA in both HeLa cells and teratoma cells. Interestingly, the functional miR-128 binding site in TERT mRNA, is conserved between TERT and the other cellular reverse transcriptase encoded by Long Interspersed Elements-1 (LINE-1 or L1), which can also contribute to the oncogenic phenotype of cancer. This finding supports the novel idea that miRs may function in parallel pathways to inhibit tumorigenesis, by regulating a group of enzymes (such as RT) by targeting conserved binding sites in the coding region of both enzymes.

Accelerated decline in cardiac stem cell efficiency in Spontaneously hypertensive rat compared to normotensive Wistar rat.

Cardiac hypertrophy is recognized as an independent risk factor for cardiac failure. Efficient management of hypertensive heart disease requires identification of factors that can possibly mediate the transition from hypertrophy to failure. Resident cardiac stem cells have a prominent role in the maintenance of cardiac tissue homeostasis. Decline in the proportion of healthy cardiac stem cells (CSCs) can affect tissue regeneration. In pathological conditions, apart from natural aging, an adverse microenvironment can lead to decrease in efficiency of CSCs. A systematic analysis of cardiac stem cell characteristics in pathological conditions has not been reported so far. Therefore, this study was designed with the objective of examining the age associated variation in stem cell attributes of Spontaneously hypertensive rat (SHR) in comparison with normotensive Wistar rat. Spontaneously hypertensive rat was used as the experimental model since the cardiac remodeling resembles the clinical course of hypertensive heart disease. CSCs were isolated from atrial explants. Stem cell attributes were assessed in 1-week, 6, 12 and 18-month-old male SHR, in comparison with age matched Wistar rats. In 1-week-old pups, stem cell attributes of SHR and Wistar were comparable. Migration potential, proliferative capacity, TERT expression, telomerase activity and the proportion of c-kit+ cells decreased with age, both in SHR and Wistar. DNA damage and the proportion of senescent CSCs increased with age both in SHR and Wistar rats. Age associated increase was observed in the oxidative stress of stem cells, possibly mediated by the enhanced oxidative stress in the microenvironment. The changes were more pronounced in SHR, and as early as six months of age, there was significant decrease in efficiency of CSCs of SHR compared to Wistar. The density of healthy CSCs determined as a fraction of the differentiated cells was remarkably low in 18-month-old SHR. Age associated decrease in functionally efficient CSCs was therefore accelerated in SHR. Considering the vital role of CSCs in the maintenance of a healthy myocardium, decrease in functionally efficient CSCs can be a precipitating factor in pathological cardiac remodeling. Elevated ROS levels in CSCs of SHR lends scope for speculation that decrease in efficiency of CSCs is mediated by oxidative stress; and that modulation of the microenvironment by therapeutic interventions can restore a healthy stem cell population and facilitate maintenance of cardiac homeostasis and prevent cardiac decompensation.

MYC drives overexpression of telomerase RNA (hTR/TERC) in prostate cancer.

Telomerase consists of at least two essential elements, an RNA component hTR or TERC that contains the template for telomere DNA addition and a catalytic reverse transcriptase (TERT). While expression of TERT has been considered the key rate-limiting component for telomerase activity, increasing evidence suggests an important role for the regulation of TERC in telomere maintenance and perhaps other functions in human cancer. By using three orthogonal methods including RNAseq, RT-qPCR, and an analytically validated chromogenic RNA in situ hybridization assay, we report consistent overexpression of TERC in prostate cancer. This overexpression occurs at the precursor stage (e.g. high-grade prostatic intraepithelial neoplasia or PIN) and persists throughout all stages of disease progression. Levels of TERC correlate with levels of MYC (a known driver of prostate cancer) in clinical samples and we also show the following: forced reductions of MYC result in decreased TERC levels in eight cancer cell lines (prostate, lung, breast, and colorectal); forced overexpression of MYC in PCa cell lines, and in the mouse prostate, results in increased TERC levels; human TERC promoter activity is decreased after MYC silencing; and MYC occupies the TERC locus as assessed by chromatin immunoprecipitation (ChIP). Finally, we show that knockdown of TERC by siRNA results in reduced proliferation of prostate cancer cell lines. These studies indicate that TERC is consistently overexpressed in all stages of prostatic adenocarcinoma and that its expression is regulated by MYC. These findings nominate TERC as a novel prostate cancer biomarker and therapeutic target. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

TERT structural rearrangements in metastatic pheochromocytomas.

Pheochromocytomas (PC) and paragangliomas (PGL) are endocrine tumors for which the genetic and clinicopathological features of metastatic progression remain incompletely understood. As a result, the risk of metastasis from a primary tumor cannot be predicted. Early diagnosis of individuals at high risk of developing metastases is clinically important and the identification of new biomarkers that are predictive of metastatic potential is of high value. Activation of TERT has been associated with a number of malignant tumors, including PC/PGL. However, the mechanism of TERT activation in the majority of PC/PGL remains unclear. As TERT promoter mutations occur rarely in PC/PGL, we hypothesized that other mechanisms - such as structural variations - may underlie TERT activation in these tumors. From 35 PC and four PGL, we identified three primary PCs that developed metastases with elevated TERT expression, each of which lacked TERT promoter mutations and promoter DNA methylation. Using whole genome sequencing, we identified somatic structural alterations proximal to the TERT locus in two of these tumors. In both tumors, the genomic rearrangements led to the positioning of super-enhancers proximal to the TERT promoter, that are likely responsible for the activation of the normally tightly repressed TERT expression in chromaffin cells.

The TERT hypermethylated oncologic region predicts recurrence and survival in pancreatic cancer.

We explore the biomarker potential of the TERT hypermethylated oncologic region (THOR) in pancreatic cancer. We assessed the methylation status of THOR using the cancer genome atlas data on the cohort of pancreatic cancer (n=193 patients). THOR was significantly hypermethylated in pancreatic tumor tissue when compared with the normal tissue used as control (p<0.0001). Also, THOR hypermethylation could distinguish early stage I disease from normal tissue and was associated with worse prognosis. We found that THOR is hypermethylated in pancreatic tumor tissue when compared with normal tissue and that THOR methylation correlates with TERT expression in tumor samples. Our preliminary findings support the diagnostic and prognostic values of THOR in pancreatic cancer.

The extracts of bredigite bioceramics enhanced the pluripotency of human dental pulp cells.

Biomaterials have a profound effect on tissue engineering and regenerative medicine, but few studies have reported the role of extracts from bioceramics in the regulation of stem cell pluripotency. The present study investigated the effects of bioceramics extracts, including silicate bredigite (Ca7 MgSi4 O16 ) and conventional β-tricalcium phosphate (β-TCP), on the pluripotency and the multilineage differentiation potential of human dental pulp cells (hDPCs). Basic fibroblast growth factor (bFGF), which is a known regulator of hDPCs pluripotency, was used as a reference. Bredigite extracts significantly promoted cell growth, proliferation, TERT expression and maintained hDPCs in a presenescent state. The extracts of bredigite significantly up-regulated the expression of pluripotency-related genes such as Stro1, Oct4 and Sox2, and further promoted the multilineage differentiation of hDPCs after odontogenic/adipogenic induction. The stimulation of bredigite extracts on hDPCs pluripotency was comparable to that of bFGF, whereas β-TCP extracts lacked these properties. Our results suggested for the first time that bredigite extracts enhance the pluripotency of dental-derived stem cells, paving the way for extended applications in regenerative medicine. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3465-3474, 2017.

Abstract 4881: Dissecting telomere maintenance mechanisms in neuroblastoma

Abstract Background. Telomere maintenance is a major cancer hallmark and has recently attracted attention as an oncogenic mechanism in the pediatric malignancy, neuroblastoma (NBL), due to detection of frequent structural rearrangements near the telomerase (TERT) gene. NBLs display substantial clinical and molecular heterogeneity; tumors with MYCN amplification and TERT-associated rearrangements define separate groups of high-risk patients with active TERT. ATRX mutations are frequent in non-TERT active high-risk tumors, indicating use of alternative lengthening of telomeres (ALT) as a maintenance mechanism. Furthermore, the 4S low-risk NBLs display an elevated rate of spontaneous regression and overall good prognosis; it has been hypothesized that lacking a telomere maintenance mechanism (“telomere crisis”) may be behind this phenomenon. Methods. To gain mechanistic insight behind these phenomena, we introduce an integrative analysis of a cohort comprising 104 high-risk (32 MYCN-amplified) and 23 4S tumors with matched blood/tumor whole genome sequencing, CpG methylation, and RNA-seq data from the TARGET consortium. Here, we preformed structural variant (SV), allelic specific expression (ASE), and differential CpG methylation analyses. Additionally, we introduce a novel approach to measure relative telomere DNA abundance from short-read WGS measured as the ratio between tumor and blood telomeric reads (containing canonical repeats TTAGGG/CCCTAA) per million (TBrpm). Results. We identified 26 tumors harboring TERT rearrangements (25% of all high risk tumors) and 3 tumors with ATRX mutations. We define 4 groups of high-risk NBL with different telomere maintenance mechanisms: 1) Tumors with MYCN amplification or elevated MYC/N activity, TERT gene body hyper-methylation (β-value≅0.8), and expression of biallelic TERT; 2) Tumors harboring TERT associated SVs, hemi-methylation (β-value≅0.5), and expression of mono-allelic TERT, 3) Tumors without TERT expression, including ATRX mutants with hypo-methylated TERT (β-value≅0.3), 4) tumors with abnormally high telomeric DNA abundance (TBrpm &amp;gt; 1.5), hypo-methylation (β-value≅0.3), and no expression of TERT. Intriguingly, while groups 1-3 show telomeric loss (TBrpm ≅ 0.7-0.9), 4S tumors show conservation of telomere abundance between tumor and blood (TBrpm ≅ 1); hence no indication of telomere crisis, perhaps due to early timing of biopsy at diagnosis. Conclusion. We have used ASE status and gene body methylation of TERT in order to understand and validate mechanisms underlying TERT activation; we extend MYCN-driven TERT activation to tumors with high MYC/MYCN activity in the absence of MYCN amplification. Our telomere analysis suggests that different ALT mechanisms might take place in NBLs. Defining the mechanism of 4S NBL spontaneous regression requires further investigation. Citation Format: Gonzalo Lopez, Karina Conkrite, Kendra Hong, Jo Lynne Harenza, John M. Maris, Sharon Diskin. Dissecting telomere maintenance mechanisms in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4881. doi:10.1158/1538-7445.AM2017-4881

Abstract 4877: Transcriptome sequencing of pediatric hepatocellular carcinoma reveals genomic events involving APC and TERT

Abstract Background: The goal of this project is to molecularly characterize a cohort of pediatric HCCs in order to understand the spectrum of genetic drivers for this disease. Hepatocellular carcinoma (HCC) is a rare pediatric liver tumor with a poor prognosis. The fibrolamellar variant of HCC (FL-HCC) has a characteristic DNAJB1-PRKACA gene fusion. In non-FL-HCC a genetic cause has not been identified. We hypothesize that alterations in known cancer genes play a role in non-FL-HCC tumorigenesis. Methods: We have used RNA sequencing (RNA-seq) on fresh frozen tissue to survey a cohort of 8 FL-HCCs, 4 pediatric HCCs, and 6 normal liver samples for chimeric transcripts. Fusion transcripts in the tumor samples were detected using deFuse on FASTQ files followed by subtraction of fusions called in the normal liver dataset. Fusions were verified using BLAST and validated by RT-PCR. On average the deFuse algorithm predicted 150 fusion transcripts per tumor; filtering by normal liver reduced the number of calls in the tumors by ~60%. As expected, DNAJB1-PRKACA fusions were identified in the FL-HCC cohort with no additional fusions. Filtering by the COSMIC Cancer Gene Census list reduced the total number of fusion calls to 5 for the HCC dataset (3 unique events, 2 of which were confirmed by RT-PCR). Results: The first event is an inversion disrupting APC, resulting in two in-frame fusion transcripts between APC and AP3B1. This fusion was confirmed and the intronic breakpoints were mapped in tumor and germline DNA through WGS by 10X Genomics®. Variant calling by Platypus on tumor RNA-seq reads did not identify additional variants in APC. Reports of inversion of APC are rare, however we have identified an additional published case of APC inversion in a patient with pediatric HCC. We will perform WGS on germline DNA from this patient to determine if the APC inversion breakpoint is recurrent. The second event is a deletion encompassing the TERT promoter and results in an in-frame fusion between TERT and LPCAT1. An increase in TERT expression is seen in this tumor as compared to both normal liver and other pediatric HCCs. Conclusions: The detection of chimeric transcripts by RNA-seq has allowed us to identify two structural variants involving known cancer genes in two tumors, including an inversion of APC which have been historically difficult to detect. These events present a possible genetic cause for these tumors and provide genes for further analysis in the remaining cases with no identified fusions. These results will inform future studies as we expand this cohort to include 18 additional pediatric HCCs which we plan to characterize through NGS and targeted sequencing. Citation Format: Katherine Haines, Angshumoy Roy, Stephen Sarabia, Linghua Wang, Pavel Sumazin, Kyle Covington, Harsha Doddapaneni, Oliver Hampton, Donna M. Muzny, Dolores Lopez-Terrada, David A. Wheeler, Sharon E. Plon, D. Williams Parsons. Transcriptome sequencing of pediatric hepatocellular carcinoma reveals genomic events involving APC and TERT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4877. doi:10.1158/1538-7445.AM2017-4877

Abstract 5715: Gene body methylation and expression of SHOX2 gene are potent prognostic markers for survival in intermediate grade gliomas and renal cancers

Abstract Background: Previous data indicated that low level of promoter methylation of the SHOX2 gene was a prognostic marker for worse progression-free survival for non-small cell lung cancer. Purpose: (a) to determine the relationship between SHOX2 gene methylation and expression; b) to determine whether SHOX2 gene methylation and expression were prognostic markers for other cancers. Methods: We examined multiple methylation and expression datasets available from The Cancer Genome Atlas (TCGA) and other previously published studies. Data from a total of more than 1000 intermediate grade gliomas and 1100 renal cancer cases were examined, along with smaller number of corresponding non-malignant tissues. We performed Kaplan-Meier survival analyses and univariate and multivariate Cox regression model analyses. Results: SHOX2 gene contains three CpG islands encompassing the promoter region and body. Methylation of various regions of SHOX2 gene body was significantly positively correlated with its expression in gliomas and renal cancer. Aberrantly high methylation and expression of SHOX2 gene, which were significantly higher than the corresponding nonmalignant tissues, were identified in subsets of gliomas and renal cancers. Survival analysis of patients indicated that the methylation and expression of SHOX2 gene in gliomas and renal cancer (both clear cell and papillary cell carcinomas) were highly potent markers for poor survival. We further investigated the combination of SHOX2 with other known clinically relevant glioma markers (IDH genotype status, TERT expression, 1p/19q chromosome co-deletion, MGMT methylation, ATRX mutation and NES expression). When combined with SHOX2 expression, we identified subsets of glioma patients with significantly favorable survival outcomes, especially in the subgroup (i.e., IDH wild-type) associated with worse prognosis for each individual marker. Multivariate analysis demonstrated that SHOX2 was a potent independent survival marker for gliomas. Conclusion: Gene body methylation and expression of SHOX2 gene are tightly positively correlated and are potent new markers for overall survival prognosis for intermediate grade gliomas and renal cancer. The combination of IDH or other relevant prognostic markers with SHOX2 identified intermediate grade glioma subsets with improved survival outcomes. Citation Format: Yu-An Zhang, Yunyun Zhou, Xin Luo, Guanghua Xiao, Adi F. Gazdar. Gene body methylation and expression of SHOX2 gene are potent prognostic markers for survival in intermediate grade gliomas and renal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5715. doi:10.1158/1538-7445.AM2017-5715

Abstract 3117: Eribulin is a novel TERT-targeting inhibitor that penetrates into intracerebrally grown glioblastomas and suppresses their growth in mice

Abstract [Introduction] No effective molecular targeting therapy has been clinically developed for patients with glioblastoma (GBM) despite of intense genome analysis. Approximately 60-80% of GBM harbor mutations in the promoter region of TERT that leads to its upregulation, making it the most common single genetic abnormalities in GBM. TERT, a reverse-transcriptase subunit of telomerase, has been an attractive candidate for therapy target because of their observed upregulation in a wide variety of cancers, including GBMs, a phenomenon that presumably helps maintain telomeres for their indefinite proliferation. It has been reported recently that TERT has an RNA-dependent RNA polymerase (RdRP) activity, with which TERT is involved in maintenance of stem cell phenotype and mitotic progression. We have previously identified eribulin as a specific inhibitor of RdRP activity of TERT through drug screening and shown that eribulin suppressed growth of ovarian cancers with high TERT expression. In order to investigate an efficacy of eribulin as a novel TERT-targeted therapy against GBM, we investigated the antitumor effect of eribulin in cultured cell and brain tumor model using GBM cell lines. [Methods] The TERT promoter status was examined in 20 glioma cell lines. Seven cell lines were subjected to in vitro cytotoxicity assay. U87MG was either subcutaneously or intracranially transplanted in athymic mice. Tissue or plasma concentration of eribulin was measured by LC-MS/MS. [Results] All GBM cell lines tested that harbored TERT promoter mutations including U87MG, U251MG, U118MG as well as several patient-derived glioblastoma sphere culture cells were highly sensitive to eribulin, IC50 below 1nM. Intraperitoneal administration of eribulin completely suppressed the growth of U87MG subcutaneous tumor in nude mice, and the RdRP activities in treated tumors were significantly decreased in a dose-dependent manner. In the brain tumor model, a high concentration of eribulin was detected in the brain tumor tissues as early as 15 minutes after intravenous injection of eribulin, which remained stable even 24 hours later when the plasma concentration of eribulin became undetectable. Finally, intraperitoneal administration of eribulin significantly prolonged the survival of mice with intracerebrally transplanted U87MG xenograft (p&amp;lt;0.001). [Conclusion] Our results showed that eribulin efficiently transfers into brain tumor tissues and has a strong antitumor effect against GBM cells through inhibition of RdRP activity. Eribulin thus appears to be a promising novel TERT-targeting therapeutic agent against GBM. A clinical trial is being scheduled. Citation Format: Masamichi Takahashi, Shunichiro Miki, Yuko Matsushita, Kohei Fukuoka, Yoshiko Maida, Mami Yasukawa, Mitsuhiro Hayashi, Akinobu Hamada, Akitake Mukasa, Ryo Nishikawa, Kenji Tamura, Yoshitaka Narita, Kenkichi Masutomi, Koichi Ichimura. Eribulin is a novel TERT-targeting inhibitor that penetrates into intracerebrally grown glioblastomas and suppresses their growth in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3117. doi:10.1158/1538-7445.AM2017-3117

Abstract 4879: Genomic and epigenomic profiling of high-stage neuroblastoma

Abstract Neuroblastoma (NB) is the most common pediatric extracranial solid tumor with a wide range of clinical phenotypes from spontaneous regression to highly resistant to chemotherapy. Despite intense multi-modal therapy, NB remains the second most common cause of cancer deaths among children. MYCN amplification is the most prominent poor prognosis factor for NB, however, certain subgroup of high stage tumor without MYCN amplification also shows low survival rate in terms of long-term follow-up (10 years survival rate was 51%, n=161). To elucidate the molecular basis of malignant subtype of NB, integrated genomic and epigenomic analyses were performed. Array CGH of 537 tumors indicated that genomic subgroups of 17q gain, with 1p loss and/or 11q loss, exhibited aggressive characteristics even without MYCN amplification. Whole exome sequencing of 92 high stage, MYCN-non-amplified primary tumors (SureSelect XT Human All Exon V4, mean depth CDS:138, x20 coverage:96%) identified 1,735 non-silent somatic mutations, among which 53 were significant mutations observed more than once in the sample set. Aggressive tumors (died of disease, n=48) had several notable features, with mutations in chromatin remodeling/modulating genes (27%, cf. ATRX), in DNA-damage response (30%), in TP53 (28%) and RB (22%)-related gene networks, as well as with high TERT expression (38%). By hierarchical clustering of methylome signature (Infinium HumanMethylation450 BeadChip), the 92 tumors were subdivided into four clusters. Unfavorable cluster-2 and -4 exhibited hyper-methylation phenotypes in CpG islands and the former was enriched by MYCN-amplified tumors. These two clusters (53%) showed strong correlation with patient prognosis (p=0.0055). Thus, these prognosis-related genomic features could be useful markers for risk classification and help understanding molecular mechanism for aggressive phenotypes of MYCN-non-amplified NBs. Citation Format: Miki Ohira, Yasutoshi Tatsumi, Yohko Nakamura, Kenji Tatsuno, Shuichi Tsutsumi, Shogo Yamamoto, Genta Nagae, Claire Renard-Guillet, Ryuichi Sugino, Hiroki Nagase, Takehiko Kamijo, Hiroyuki Aburatani, Akira Nakagawara. Genomic and epigenomic profiling of high-stage neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4879. doi:10.1158/1538-7445.AM2017-4879

Lack of micro-RNA 128A expression as a novel prognostic marker in neuroblastoma patients and combination with TERT hyperexpression to define patient outcomes.

e22014 Background: Rearrangements of TERT gene with its correspondent hyperexpression were identified as adverse prognostic markers in neuroblastoma (NB) patients, while significance of microRNAs expression is undiscovered. Methods: RNA from 103 fresh-frozen NB tissues was subjected to reverse transcription qPCR for miR128A and TERT expression evaluation. Copy number variations were determined by MLPA and FISH. Correspondence of miR128A and TERT expression levels to event-free survival (EFS) was proved by ROC-analysis and established threshold levels (TL) were utilized for group separation in subsequent survival analysis. Median of follow-up time achieved 5.8 years. Results: Abundant TERT expression and lack of miR128A expression resulted in superior frequency of adverse events (p = 0.027, TL = 4,7E-3 and p = 0.004, TL = 4.6E-2 respectively). EFS in group of patients with TERT expression above 4,7E-3 (group TERT) was 0.66SE0.07, in patients with miR128A expression below 4.6E-2 (group miR128A) was 0.64SE0.15, patients harboring both TERT overexpression and lack of miR128A expression (group miR128/TERT) had dismal outcome: EFS 0.29SE0.11, comparing to patients without these abnormalities (group "neither"): EFS 0.92SE0.06, p &lt; 0.001. Analogously, cumulative incidence of progression differed significantly between all these groups, p &lt; 0.001. MYCN amplified (MNA) samples were accumulated in the groups TERT and miR128/TERT (p = 0.061), while chromosome 17 gain, 9p and 14q deletions prevailed in the group "neither" (p = 0.014, 0.043 and 0.017 accordingly). MNA and 14q deletion had prognostic significance in the correspondent groups (p &lt; 0.001, p = 0.022). The classifier was proposed for distinguishing patients with unequal outcome: MNA EFS = 0.25SE0.11, MYCN single copy patients divided into groups miR128A/TERT (0.40SE0.15), miR128A (0.60SE0.15) and TERT (0.74SE0.08). Group "neither" was separated basing on presence (0.71SE0.17) and absence (EFS = 1.00) of 14q deletion, p &lt; 0.001. Conclusions: Levels of miR128A and TERT expression together with cytogenetic data allow discriminating patients into groups with significantly different outcome.

Functional characterization of a multi-cancer risk locus on chr5p15.33 reveals regulation of TERT by ZNF148

Genome wide association studies (GWAS) have mapped multiple independent cancer susceptibility loci to chr5p15.33. Here, we show that fine-mapping of pancreatic and testicular cancer GWAS within one of these loci (Region 2 in CLPTM1L) focuses the signal to nine highly correlated SNPs. Of these, rs36115365-C associated with increased pancreatic and testicular but decreased lung cancer and melanoma risk, and exhibited preferred protein-binding and enhanced regulatory activity. Transcriptional gene silencing of this regulatory element repressed TERT expression in an allele-specific manner. Proteomic analysis identifies allele-preferred binding of Zinc finger protein 148 (ZNF148) to rs36115365-C, further supported by binding of purified recombinant ZNF148. Knockdown of ZNF148 results in reduced TERT expression, telomerase activity and telomere length. Our results indicate that the association with chr5p15.33-Region 2 may be explained by rs36115365, a variant influencing TERT expression via ZNF148 in a manner consistent with elevated TERT in carriers of the C allele.

Genetic and Epigenetic Alterations of TERT Are Associated with Inferior Outcome in Adolescent and Young Adult Patients with Melanoma

Progression of melanoma to distant sites in adolescents and young adults (AYAs) is not reliably predicted by clinicopathologic criteria. TERT promoter mutations when combined with BRAF/NRAS mutations correlate with adverse outcome in adult melanoma. To determine the prognostic value of TERT alterations in AYA melanoma, we investigated the association of TERT promoter mutations, as well as promoter methylation, an epigenetic alteration also linked to TERT upregulation, with TERT mRNA expression and outcome using a well-characterized cohort of 27 patients with melanoma (ages 8–25, mean 20). TERT mRNA expression levels were significantly higher in tumors harboring TERT promoter mutation and/or hypermethylation than those without either aberration (P = 0.046). TERT promoter mutations alone did not predict adverse outcomes (P = 0.50), but the presence of TERT promoter methylation, alone or concurrent with promoter mutations, correlated with reduced recurrence-free survival (P = 0.001). These data suggest that genetic and epigenetic alterations of TERT are associated with TERT upregulation and may predict clinical outcomes in AYA melanoma. A more exhaustive understanding of the different molecular mechanisms leading to increased TERT expression may guide development of prognostic assays to stratify AYA melanoma patients according to clinical risk.

Identification of a molecular mechanism underlying estrogen‐mediated neuroprotection in neuronal cells against oxidative stress

Estrogen (17β‐estradiol; E2) has neuroprotective effects in the setting of CNS injury and neurodegenerative disorders however, the mediator for neuroprotection mechanisms by E2 remains largely unknown. Recent studies have demonstrated that E2 might be potent modulator of both expression and activity of telomerase, which is a ribonucleoprotein complex involved in maintenance of the chromosome end and DNA repair. To examine whether E2 treatment exerts cytoprotective effects in neuronal cells, mouse origin neuroblastoma Neuro‐2a (N2a) cells were differentiated into cholinergic phenotypes, and used as cell culture model following exposure to hydrogen peroxide to induce oxidative stress. Neuronal cell death was induced and morphologic changes were observed following oxidative stress. In addition, apoptotic changes including Bax:Bcl‐2 ratio and apoptosis‐related proteases caspase‐3 were increased in neuronal cells rendered by oxidative stress. E2 treatment attenuated these changes, as well as increased cell viability. Our data demonstrated that E2 treatment significantly increased TERT expression in a dose‐dependent manner in neuronal cells. To confirm whether the estrogen signaling is associated with TERT, selective siRNA targeted against both ERα and ERβ were transfected to neuronal cells. TERT mRNA expression was down‐regulated in both ERα and ERβ‐silenced neuronal cells in particular, this change was more distinctive in knockdown of ERα. Taken together, these findings indicate that the telomerase enzyme may be an important mediator of the neuroprotective effects of E2.Support or Funding InformationNRF‐2013R1A2A2A01067169 to Y.H., NRF‐2014R1A1A3051724 to S.P., Republic of Korea.