Telomerase-positive Cancer Cells Research Articles

Preparation and anticancer activity of telomerase inhibitor TAT-LPTS39 polypeptide.

Telomerase is activated in most cancer cells, and thus telomerase is an ideal target for cancer therapy. The human liver-associated candidate tumour suppressor LPTS/PinX1, is the only human protein reported to bind with the telomerase catalytic subunit telomerase reverse transcriptase (TERT) and inhibit telomerase activity. The C-terminal fragment of LPTS/PinX1 (LPTS/PinX1290-328) contains a telomerase inhibitory domain that is needed for inhibition of telomere elongation and induction of apoptosis. This study prepared the TAT-LPTS39 (TAT-LPTS/PinX1290-328) polypeptide and analysed its effect of the tumour growth. LPTS/PinX1290-328 was fused with TAT [11 amino acid (aa) peptide of the HIV transactivator of transcription protein] to generate the recombinant protein GST-TAT-LPTS39 and was transduced into cells. Telomerase activity was identified by the telomeric repeat amplification protocol (TRAP) and the relative telomere length (RTL) was measured by quantitative real-time polymerase chain reaction (qPCR). The effects of the TAT-LPTS39 protein on cell growth and death were evaluated by 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT), cell culture doubling time and flow cytometry assays. The cell derived xenograft (CDX) model was used to examine tumour growth inhibition effect of TAT-LPTS39 polypeptide in vivo. We successfully expressed and purified the recombinant protein GST-TAT-LPTS39 in vitro. The GST-TAT-LPTS39 protein was efficiently delivered into cells, inhibited telomerase activity and the growth of the telomerase-positive liver cancer cells BEL-7404 and QGY7701, and induced the senescence and apoptosis in telomerase-positive Hela, BEL-7404 and QGY7701 cells, but was ineffective to telomerase-negative cells in vitro. The TAT-LPTS39 polypeptide without the GST tag similarly inhibited the growth of telomerase-positive cancer cells Hela and PLC-PRF-5 in vitro, BEL-7404 CDX tumour in vivo and shortened telomere length. The TAT-LPTS39 polypeptide has the ability to inhibit telomerase activity and suppress the growth of all tested human telomerase-positive cancer cells in vitro and in vivo, suggesting a potential anticancer drug development.

Abstract 1639: Telomere dynamics in aging and cancer by nanopore long-read sequencing

Abstract Telomeres are the protective, nucleoprotein structure at the ends of linear eukaryotic chromosomes. The accurate measurement of both telomeric length and composition of individual telomeres in mammalian cells has been challenged by the length and repetitive nature of telomeres. With the advent of third generation sequencing technologies, it is now technically possible to sequence entire telomeres and map them to individual chromosome arms. Here, we report a reliable method to enrich, sequence and analyze human telomeres using Oxford Nanopore Technologies long-read sequencing. To enrich for telomeric sequences we combine the ligation of adapters complementary to the telomeric G-overhang with restriction enzyme digest to sequence the telomeric C-strand and part of the adjacent subtelomere. The subtelomeric information is harvested to map individual telomeric reads to specific chromosome arms and even alleles. We have measured bulk, chromosome-arm-specific telomere length dynamics during cellular aging of cultured primary cells and in a patient-derived aging cohort. To address the impact of the telomere maintenance mechanism on telomere length and composition, we have sequenced matched pairs of fibroblasts and induced pluripotent stem cells, as well as five well-established telomerase- and ALT-positive cancer cell lines. Our results suggest that based on nanopore telomere long-read sequencing ALT-positive cells can be easily discriminated from normal and telomerase-positive cancer cells. Further, telomere sequencing allows to evaluate the methylation status of the subtelomeric CpG islands adjacent to telomeres. In summary, nanopore telomere long-read sequencing allows to measure the length and composition of individual telomeres and their mapping to specific chromosome arms. Telomere long read sequencing methods will be valuable tools to study telomere biology during aging and cancer. Citation Format: Tobias T. Schmidt, Carly Tyer, Preeyesh Rughani, Candy Haggblom, Jeff Jones, Xiaoguang Dai, Kelly A. Frazer, Fred H. Gage, Sissel Juul, Scott Hickey, Jan Karlseder. Telomere dynamics in aging and cancer by nanopore long-read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1639.

Oxidative guanine base damage plays a dual role in regulating productive ALT-associated homology-directed repair

Cancer cells maintain telomeres by upregulating telomerase or alternative lengthening of telomeres (ALT) via homology-directed repair at telomeric DNA breaks. 8-Oxoguanine (8oxoG) is a highly prevalent endogenous DNA lesion in telomeric sequences, altering telomere structure and telomerase activity, but its impact on ALT is unclear. Here, we demonstrate that targeted 8oxoG formation at telomeres stimulates ALT activity and homologous recombination specifically in ALT cancer cells. Mechanistically, an acute 8oxoG induction increases replication stress, as evidenced by increased telomere fragility and ATR kinase activation at ALT telomeres. Furthermore, ALT cells are more sensitive to chronic telomeric 8oxoG damage than telomerase-positive cancer cells, consistent with increased 8oxoG-induced replication stress. However, telomeric 8oxoG production in G2 phase, when ALT telomere elongation occurs, impairs telomeric DNA synthesis. Our study demonstrates that a common oxidative base lesion has a dual role in regulating ALT depending on when the damage arises in the cell cycle.

Smart Telomerase-Gated DNA cage for precise siRNA release in cancer cells

Oligonucleotide nanoparticles (ONPs) have been utilized as a small interfering RNA (siRNA) delivery system because of their multiple advantages. However, the release of siRNA by ONPs inner cells mainly relies on acidic environments and high concentrations of ATP. These conditions do not allow the specific release of siRNA in tumor cells, which would prevent the side effects that result from the release of siRNA in normal cells. In this study, a prism oligonucleotide nanoparticle (pONP)-based delivery system was designed, which targeted with telomeric primers (TS) that can be extended by cancer-specific telomerase for specifically releasing siRNA inside telomerase-positive cancer cells. In vitro tests confirmed that this cage facilitated the release of siRNA specifically into telomerase-positive HeLa and MDA-MB-231 tumor cells. The release was so precise that almost no siRNA was released into telomerase-negative cells. In vivo tests also confirmed that this cage could release sibcl-2 specifically in tumor cells in an MDA-MB-231 orthotopic breast cancer mouse model. Moreover, the cage has a long half-life (e.g., 2.83 h in FBS) in the circulatory system and protects the cargo from nuclease degradation; thus, the released sibcl-2 effectively silences the translation of target cancer-related mRNA (bcl-2), similar to the sibcl-2 delivered by Lipo6000 in the telomerase-positive environment of cancer cells. pONP/TS/siRNA is readily prepared using a simple sequence design together with one-step self-assembly. This nanoparticle cage is a promising delivery method for precise siRNA release into targeted telomerase-positive cancer cells.

SAMHD1 restricts the deoxyguanosine triphosphate pool contributing to telomere stability in telomerase-positive cells.

SAMHD1 (Sterile alpha motif and histidine/aspartic acid domain-containing protein 1) is a dNTP triphosphohydrolase crucial in the maintenance of balanced cellular dNTP pools, which support genome integrity. In SAMHD1 deficient fibroblasts isolated from Aicardi-Goutières Syndrome (AGS) patients, all four DNA precursors are increased and markedly imbalanced with the largest effect on dGTP, a key player in the modulation of telomerase processivity. Here, we present data showing that SAMHD1, by restricting the dGTP pool, contributes to telomere maintenance in hTERT-immortalized human fibroblasts from AGS patients as well as in telomerase positive cancer cell lines. Only in cells expressing telomerase, the lack of SAMHD1 causes excessive lengthening of telomeres and telomere fragility, whereas primary fibroblasts lacking both SAMHD1 and telomerase enter normally into senescence. Telomere lengthening observed in SAMHD1 deficient but telomerase proficient cells is a gradual process, in accordance with the intrinsic property of telomerase of adding only a few tens of nucleotides for each cycle. Therefore, only a prolonged exposure to high dGTP content causes telomere over-elongation. hTERT-immortalized AGS fibroblasts display also high fragility of chromosome ends, a marker of telomere replication stress. These results not only demonstrate the functional importance of dGTP cellular level but also reveal the critical role played by SAMHD1 in restraining telomerase processivity and safeguarding telomere stability.

G-quadruplex stabilizer Tetra-Pt(bpy) disrupts telomere maintenance and impairs FAK-mediated migration of telomerase-positive cells

G-quadruplex regulates a wide spectrum of biological processes, including telomere maintenance, DNA replication and transcription. The development of small molecules to selectively target G-quadruplex and their application remain hotspots in cancer therapy. Here, we explored the biological effect of G-quadruplexes stabilizer Tetra-Pt(bpy) in telomerase-positive cancer cells. Telomere maintenance was evaluated by telomerase repeat amplification protocol, chromosome orientation fluorescence in situ hybridization and telomere restriction fragment assays. We found that Tetra-Pt(bpy) accelerates telomere shortening through dual inhibition of telomerase activity and telomere sister chromatin exchanges mediated by telomeric G-quadruplexes. Consequently, Tetra-Pt(bpy)-treated cancer cells became enriched with extremely short telomeres and produced a strong telomeric DNA damage response following long-term treatment, leading to cell proliferation inhibition and senescence. Experimental evidence from RNA seq and cell migration-related assays showed that Tetra-Pt(bpy) decreased cell-matrix adhesion and inhibited the migration of non-senescent tumor cells. Mechanistically, Tetra-Pt(bpy) induced the formation of G-quadruplexes in focal adhesion kinase (FAK)-encoding gene PTK2, resulting in FAK transcription inhibition. Tetra-Pt(bpy) reduced xenograft tumor formation and inhibited tumor cell growth and migration in mice. This study further elucidates the function of G-quadruplexes in the human genome and reveals the potential of Tetra-Pt(bpy) as a novel chemotherapeutic agent for targeting telomerase-positive cancer cells.

An inducible CRISPR/Cas9 screen identifies DTX2 as a transcriptional regulator of human telomerase

SummaryMost tumor cells reactivate telomerase to ensure unlimited proliferation, whereas the expression of human telomerase reverse transcriptase (hTERT) is tightly regulated and rate-limiting for telomerase activity maintenance. Several general transcription factors (TFs) have been found in regulating hTERT transcription; however, a systematic study is lacking. Here we performed an inducible CRISPR/Cas9 KO screen using an hTERT core promoter-driven reporter. We identified numerous positive regulators including an E3 ligase DTX2. In telomerase-positive cancer cells, DTX2 depletion downregulated hTERT transcription and telomerase activity, contributing to progressive telomere shortening, growth arrest, and increased apoptosis. Utilizing BioID, we characterized multiple TFs as DTX2 proximal proteins, among which NFIC functioned corporately with DTX2 in promoting hTERT transcription. Further analysis demonstrated that DTX2 mediated K63-linked ubiquitination of NFIC, which facilitated NFIC binding to the hTERT promoter and enhanced hTERT expression. These findings highlight a new hTERT regulatory pathway that may be exploited for potential cancer therapeutics.

The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity.

Simple SummaryA clinical test for alternative-lengthening-of-telomeres (ALT) could assist with cancer diagnosis and monitoring of disease progression. ALT-targeted anticancer treatments are being developed; however, there is no appropriate companion ALT diagnostic. The C-Circle biomarker is the only known ALT specific molecule and the C-Circle Assay the only quantitative ALT assay that is amenable to clinical use. We show here that C-Circles are secreted by ALT+ cancer cell lines inside the exosomes and are protected from nucleases. We also show that secreted C-Circles, like intracellular C-Circles, are an ALT-specific biomarker, and in high-risk neuroblastoma, the blood-based C-Circle Assay has the potential to be an accurate diagnostic for ALT cancer activity. Therefore, the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and, potentially, a clinical diagnostic for ALT activity, which is required for the development of ALT-targeted therapies as well as for the diagnosis and monitoring of ALT+ cancer.C-Circles, self-primed telomeric C-strand templates for rolling circle amplification, are the only known alternative-lengthening-of-telomeres (ALT)-specific molecule. However, little is known about the biology of C-Circles and if they may be clinically useful. Here we show that C-Circles are secreted by ALT+ cancer cells inside exosomes, and that a blood-based C-Circle Assay (CCA) can provide an accurate diagnostic for ALT activity. Extracellular vesicles were isolated by differential centrifugation from the growth media of lung adenocarcinoma, glioblastoma, neuroblastoma, osteosarcoma, and soft tissue sarcoma cell lines, and C-Circles were detected in the exosome fraction from all eleven ALT+ cancer cell lines and not in any extracellular fraction from the eight matching telomerase positive cancer cell lines or the normal fibroblast strain. The existence of C-Circles in ALT+ exosomes was confirmed with exosomes isolated by iodixanol gradient separation and CD81-immunoprecipitation, and C-Circles in the exosomes were protected from nucleases. On average, 0.4% of the total ALT+ intracellular C-Circles were secreted in the exosomes every 24 h. Comparing the serum-based and tumor-based CCAs in 35 high risk neuroblastoma patients divided randomly into ALT+ threshold derivation and validation groups, we found the serum-based CCA to have 100% sensitivity (6/6), 70% specificity (7/10), and 81% concordance (13/16). We conclude that the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and a potential clinical diagnostic for ALT activity.

Selective Apoptosis and Growth Impairment of Cancer Cells Induced by Human Telomerase Reverse Transcriptase (hTERT) Targeting Artificial MicroRNA

Human telomerase reverse transcriptase (hTERT) is a promising cancer target, and amiRNA particle displays the siRNA’s specificity and miRNA’s safety, suggesting that cancers can be treated more effective and safely by hTERT targeting amiRNA particles. Hela, NCI-H446, U2-OS and Huvec cells were transfected by hTERT targeting amiRNA particles. hTERT expression, telomerase activity and cell viability were evaluated by quantitative reverse transcription-PCR (qRT-PCR), western blot (WB), telomeric repeat amplification protocol (TRAP) assays, MTT method, transwell protocol, fluorescence-activated cell sorting (FACS) technologies, angiogenesis assay, and xenograft tumor models. Results: hTERT expression and telomerase activity in Hela and NCIH446 were significantly inhibited by amiRNA. Anti-proliferation and pro-apoptosis effects were only observed in transfected Hela and NCI-H446 cells, but anti-migration and anti-angiogenesis effects were presented in transfected Huvec cells. More interestingly, low to 1.56 nM amiRNA can inhibit the proliferation of Hela cells by 80.99±5.24%. Conclusion: amiRNA selectively and effectively impairs the growth, and assists the apoptosis of telomerase-positive cancer cells.

A scalable and reproducible preparation for the antitumor protein TLC, a human-derived telomerase inhibitor

Telomerase, which is overexpressed in approximately 90% of liver cancer cells, is an ideal target for anti-liver cancer therapy. LPTS, a putative liver tumor suppressor, is the only human-derived protein that can bind telomerase directly and inhibit the extension of telomere activity. Our previous studies demonstrated that TAT-LPTS-LC (TLC), a recombinant protein fused by the C-terminal 133–328 fragment of LPTS and TAT peptides, could be delivered into cells to inhibit telomerase-positive hepatoma cell growth in vitro and in vivo with very low toxicity. In the present study, E. coli strains which expressed TLC in abundance were screened and cultured in a laboratory bioreactor. A reproducible protein separation process was built, and this process was suitable for industrial amplification. The yields of TLC protein were up to 184 mg in one batch with a purity of approximately 95%. The purified TLC protein had a similar inhibitory effect on telomerase activity in vitro compared with those purified by Ni-affinity chromatography. Furthermore, TLC protein could be delivered into the cell nucleus to increase the doubling time of the cell and suppress cell growth in telomerase-positive liver cancer cell lines. Cell growth inhibition was negatively correlated with telomere length, suggesting that TLC is a highly targeted telomerase-telomere anticancer agent. These results will contribute to future preclinical studies of the TLC protein.

Detection of Telomeric DNA:RNA Hybrids Using TeloDRIP-qPCR.

Because of their intrinsic characteristics, telomeres are genomic loci that pose significant problems during the replication of the genome. In particular, it has been observed that telomeres that are maintained in cancer cells by the alternative mechanism of the lengthening of telomeres (ALT) harbor higher levels of replicative stress compared with telomerase-positive cancer cells. R-loops are three-stranded structures formed by a DNA:RNA hybrid and a displaced ssDNA. Emerging evidence suggests that controlling the levels of R-loops at ALT telomeres is critical for telomere maintenance. In fact, on the one hand, they favor telomere recombination, but on the other, they are a source of detrimental replicative stress. DRIP (DNA:RNA immunoprecipitation) is the main technique used for the detection of R-loops, and it is based on the use of the S9.6 antibody, which recognizes preferentially DNA:RNA hybrids in a sequence-independent manner. The detection of DNA:RNA hybrids in repetitive sequences such as telomeres requires some additional precautions as a result of their repetitive nature. Here, we share an optimized protocol for the detection of telomeric DNA:RNA hybrids, and we demonstrate its application in an ALT and in a telomerase-positive cell line. We demonstrate that ALT telomeres bear higher levels of DNA:RNA hybrids, and we propose this method as a reliable way to detect them in telomeres.

Radiolabeled Oligonucleotides Targeting the RNA Subunit of Telomerase Inhibit Telomerase and Induce DNA Damage in Telomerase-Positive Cancer Cells.

Telomerase is expressed in the majority (>85%) of tumors, but has restricted expression in normal tissues. Long-term telomerase inhibition in malignant cells results in progressive telomere shortening and reduction in cell proliferation. Here we report the synthesis and characterization of radiolabeled oligonucleotides that target the RNA subunit of telomerase, hTR, simultaneously inhibiting enzymatic activity and delivering radiation intracellularly. Oligonucleotides complementary (Match) and noncomplementary (Scramble or Mismatch) to hTR were conjugated to diethylenetriaminepentaacetic dianhydride (DTPA), allowing radiolabeling with the Auger electron-emitting radionuclide indium-111 (111In). Match oligonucleotides inhibited telomerase activity with high potency, which was not observed with Scramble or Mismatch oligonucleotides. DTPA-conjugation and 111In-labeling did not change telomerase inhibition. In telomerase-positive cancer cells, unlabeled Match oligonucleotides had no effect on survival, however, 111In-labeled Match oligonucleotides significantly reduced clonogenic survival and upregulated the DNA damage marker γH2AX. Minimal radiotoxicity and DNA damage was observed in telomerase-negative cells exposed to 111In-Match oligonucleotides. Match oligonucleotides localized in close proximity to nuclear Cajal bodies in telomerase-positive cells. In comparison with Match oligonucleotides, 111In-Scramble or 111In-Mismatch oligonucleotides demonstrated reduced retention and negligible impact on cell survival. This study indicates the therapeutic activity of radiolabeled oligonucleotides that specifically target hTR through potent telomerase inhibition and DNA damage induction in telomerase-expressing cancer cells and paves the way for the development of novel oligonucleotide radiotherapeutics targeting telomerase-positive cancers. SIGNIFICANCE: These findings present a novel radiolabeled oligonucleotide for targeting telomerase-positive cancer cells that exhibits dual activity by simultaneously inhibiting telomerase and promoting radiation-induced genomic DNA damage.

Abstract 2724: A combination of short-read and long-read RNA sequencing reveals NOVA1’s role in telomere biology

Abstract Dysregulated alternative splicing underlies many aspects of cancer cell biology including tumor cell telomere maintenance. We recently described a role for the neuronal splicing factor, NOVA1, in non-small cell lung cancer in the regulation of full-length hTERT splicing, telomerase activity, maintenance of telomere length, and general tumor growth characteristics. We wanted to further define the role of NOVA1 in telomere biology. Cells that maintain telomeres with different mechanisms were studied to be able to decipher telomerase specific versus alternative lengthening of telomeres (ALT) specific perturbations by NOVA1. Both short-read (Illumina) and long-read (Pacific Biosciences) sequencing of control and NOVA1 siRNA knockdown cells in H920 non-small cell lung cancer cells (telomerase positive) and U2OS cells (ALT positive cells) were performed to detail the transcriptomes of these cells. A combination of rMATS analysis of the short-read sequencing, SQANTI analysis of the long-read sequencing, and pathway analysis was performed to identify genes related to telomere biology and alternative splicing related to telomerase. Skipped exon events were the primary focus of this study as this is NOVA1’s main role in alternative splicing. rMATS analysis identified 4,530 significant events mapping to 2,950 genes in H920 cells and 2,534 significant events in 1,863 genes in U2OS cells. SQANTI analyzed 15,443 full-length transcripts (7,530 genes) in control and 14,463 transcripts (7,240 genes) in NOVA1 siRNA treated samples of the H920 cells and 12,133 transcripts (6,618 genes) in control and 10,126 transcripts (5,959 genes) in NOVA1 siRNA treated U2OS cells. There were 2,762 and 2,470 novel transcripts in control and NOVA1 siRNA treated H920 cells, respectively. In U2OS cells there were 2,178 novel transcripts in control and 1,688 novel transcripts in NOVA1 siRNA treated cells. The pathway analysis of rMATS cross-referenced with SQANTI revealed five top candidates that were specifically altered in NOVA1 knockdown cells and related telomere length maintenance by telomerase (POLD1, SUN2, ERCC1, SRSF2, and PCBP2). Interestingly, SRSF2 and PCBP2 were identified in our recent hTERT minigene loss of function screen for splicing regulatory genes of hTERT and have binding sites in hTERT near NOVA1s motif in intron 8. Long-read sequencing determined the full-length nature of these splicing events pointed out by the rMATS analysis. These data indicate that telomerase positive cancer cells with dysregulated expression of NOVA1 utilize different isoforms compared to ALT cells with NOVA1 to maintain telomeres. These new observations reveal that alternative splicing regulated in part by NOVA1 plays a larger and unexpected role in telomere biology. Manipulation of alternative splicing to shorten/deprotect telomeres should be pursued as a means to inhibit survival and growth of cancer cells. Citation Format: Andrew T. Ludlow, Mohammed E. Sayed, Aaron L. Slusher, Mark Ribick, Anisha Pancholi, Brian Sereni, Yu Qui, Elizabeth Tseng, Meredith Ashby, David C. Corney. A combination of short-read and long-read RNA sequencing reveals NOVA1’s role in telomere biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2724.

Repression of TERRA Expression by Subtelomeric DNA Methylation Is Dependent on NRF1 Binding.

Chromosome ends are transcribed into long noncoding telomeric repeat-containing RNA (TERRA) from subtelomeric promoters. A class of TERRA promoters are associated with CpG islands embedded in repetitive DNA tracts. Cytosines in these subtelomeric CpG islands are frequently methylated in telomerase-positive cancer cells, and demethylation induced by depletion of DNA methyltransferases is associated with increased TERRA levels. However, the direct evidence and the underlying mechanism regulating TERRA expression through subtelomeric CpG islands methylation are still to establish. To analyze TERRA regulation by subtelomeric DNA methylation in human cell line (HeLa), we used an epigenetic engineering tool based on CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats – dead CRISPR associated protein 9) associated with TET1 (ten-eleven 1 hydroxylase) to specifically demethylate subtelomeric CpG islands. This targeted demethylation caused an up-regulation of TERRA, and the enhanced TERRA production depended on the methyl-sensitive transcription factor NRF1 (nuclear respiratory factor 1). Since AMPK (AMP-activated protein kinase) is a well-known activator of NRF1, we treated cells with an AMPK inhibitor (compound C). Surprisingly, compound C treatment increased TERRA levels but did not inhibit AMPK activity in these experimental conditions. Altogether, our results provide new insight in the fine-tuning of TERRA at specific subtelomeric promoters and could allow identifying new regulators of TERRA.

A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2′-deoxyriboside 5′-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and T-motif in the RNA-binding domain of the telomerase reverse transcriptase. In vitro data show that 5-MeCITP inhibits telomerase with a similar potency as the clinically administered nucleoside analog reverse transcriptase inhibitor azidothymidine (AZT). In addition, cell-based studies show that treatment with the cell-permeable nucleoside counterpart of 5-MeCITP leads to telomere shortening in telomerase-positive cancer cells, while resulting in significantly lower cytotoxic effects in telomerase-negative cell lines when compared with AZT treatment.

Characterization of human telomerase reverse transcriptase promoter methylation and transcription factor binding in differentiated thyroid cancer cell lines.

Telomerase reverse transcriptase (TERT) activation plays an important role in cancer development by enabling the immortalization of cells. TERT regulation is multifaceted, and its promoter methylation has been implicated in controlling expression through alteration in transcription factor binding. We have characterized TERT promoter methylation, transcription factor binding, and TERT expression levels in five differentiated thyroid cancer (DTC) cell lines and six normal thyroid tissue samples by targeted bisulfite sequencing, ChIP-qPCR, and qRT-PCR. DTC cell lines express varying levels of TERT and exhibit TERT promoter methylation patterns similar to patterns seen in other telomerase positive cancer cell lines. The minimal promoter immediately surrounding the transcription start site is hypomethylated, while further upstream portions show dense methylation. In contrast, the TERT promoter in normal thyroid tissue is largely unmethylated throughout and expresses TERT minimally. Transcription factor binding is also affected by TERT mutation status. The E-twenty-six (ETS) factor GABPA exhibits TERT binding in the TERT mutant DTC cells only, and allele-specific methylation patterns at the minimal promoter were observed as well, which may indicate allele-specific factor recruitment at the minimal promoter. Furthermore, we identified binding sites for activators MYC and GSC in the hypermethylated upstream region, pointing to its possible importance in TERT regulation. Overall, TERT expression and telomerase activity depend on the interplay of multiple regulatory mechanisms including TERT promoter methylation, mutation status, and recruitment of transcription factors. This work explores of the interplay between these regulatory mechanisms and offers insight into cellular control of active telomerase in human cancer.

Cell-based chemical fingerprinting identifies telomeres and lamin A as modifiers of DNA damage response in cancer cells

Telomere maintenance by telomerase activity supports the infinite growth of cancer cells. MST-312, a synthetic telomerase inhibitor, gradually shortens telomeres at non-acute lethal doses and eventually induces senescence and apoptosis of telomerase-positive cancer cells. Here we report that MST-312 at higher doses works as a dual inhibitor of telomerase and DNA topoisomerase II and exhibits acute anti-proliferative effects on cancer cells and xenografted tumours in vivo. Our cell-based chemical fingerprinting approach revealed that cancer cells with shorter telomeres and lower expression of lamin A, a nuclear architectural protein, exhibited higher sensitivity to the acute deleterious effects of MST-312, accompanied by formation of telomere dysfunction-induced foci and DNA double-strand breaks. Telomere elongation and lamin A overexpression attenuated telomeric and non-telomeric DNA damage, respectively, and both conferred resistance to apoptosis induced by MST-312 and other DNA damaging anticancer agents. These observations suggest that sufficient pools of telomeres and a nuclear lamina component contribute to the cellular robustness against DNA damage induced by therapeutic treatment in human cancer cells.

Transient Telomerase Inhibition with Imetelstat Impacts DNA Damage Signals and Cell-Cycle Kinetics.

Telomerase is the ribonucleoprotein reverse transcriptase that catalyzes the synthesis of telomeres at the ends of linear chromosomes and contributes to proper telomere-loop (T-loop) formation. Formation of the T-loop, an obligate step before cell division can proceed, requires the generation of a 3'-overhang on the G-rich strand of telomeric DNA via telomerase or C-strand specific nucleases. Here, it is discovered that telomerase activity is critical for efficient cell-cycle progression using transient chemical inhibition by the telomerase inhibitor, imetelstat. Telomerase inhibition changed cell cycle kinetics and increased the proportion of cells in G2-phase, suggesting delayed clearance through this checkpoint. Investigating the possible contribution of unstructured telomere ends to these cell-cycle distribution changes, it was observed that imetelstat treatment induced γH2AX DNA damage foci in a subset of telomerase-positive cells but not telomerase-negative primary human fibroblasts. Chromatin-immunoprecipitation with γH2AX antibodies demonstrated imetelstat treatment-dependent enrichment of this DNA damage marker at telomeres. Notably, the effects of telomerase inhibition on cell cycle profile alterations were abrogated by pharmacological inhibition of the DNA-damage-repair transducer, ATM. Also, imetelstat potentiation of etoposide, a DNA-damaging drug that acts preferentially during S-G2 phases of the cell cycle, depends on functional ATM signaling. Thus, telomerase inhibition delays the removal of ATM-dependent DNA damage signals from telomeres in telomerase-positive cancer cells and interferes with cell cycle progression through G2Implications: This study demonstrates that telomerase activity directly facilitates the progression of the cell cycle through modulation of transient telomere dysfunction signals. Mol Cancer Res; 16(8); 1215-25. ©2018 AACR.

Long-range telomere regulation of gene expression: Telomere looping and telomere position effect over long distances (TPE-OLD)

The human cellular reverse transcriptase, telomerase, is very tightly regulated in large long-lived species. Telomerase is expressed during early human fetal development, is turned off in most adult tissues, and then becomes reactivated in almost all human cancers. However, the exact mechanism regulating these switches in expression are not known. We recently described a phenomenon where genes are regulated by telomere length dependent loops (telomere position effects over long distances; TPE-OLD). The hTERT gene is ~ 1.2Mb from the human chromosome 5p end. We observed that when telomeres are long hTERT gene expression is repressed and a probe next to the 5p telomere and the hTERT locus are spatially co-localized. When telomeres are short at least one of the hTERT alleles is spatially separated from the telomere, developing more active histone marks and changes in DNA methylation in the hTERT promoter region. These findings have implications for how cells turn off telomerase when telomeres are long during fetal development and how cancer cells reactivate telomerase in cells that have short telomeres. In addition to TPE-OLD, in proliferating stem cells such as activated T-lymphocytes, telomerase can be reversibly activated and silenced by telomere looping. In telomerase positive cancer cells that are induced to differentiate and downregulate telomerase, telomere looping correlates with silencing of the hTERT gene. These studies and others support a role of telomeres in regulating gene expression via telomere looping that may involve interactions with internal telomeric sequences (ITS). In addition to telomere looping, TPE-OLD may be one mechanism of how cells time changes in physiology without initiating a DNA damage response.

Abstract 3412: Functional knockout of ATRX or DAXX permits the alternative lengthening of telomeres (ALT) mechanism in prostate cancer cells

Abstract A key hallmark of cancer is unlimited replication, which requires cancer cells to evade replicative senescence induced by short telomeres. The majority of cancers overcome this critical replication barrier by upregulating the telomerase enzyme, a telomere-specific reverse transcriptase. However, a subset of cancers lack telomerase, and telomeres are maintained by employing the Alternative Lengthening of Telomeres (ALT) pathway, which is dependent on homologous recombination. Across a variety of tumor types, our laboratory and others have reported a robust correlation between ALT and recurrent cancer-associated somatic inactivating mutations in the ATRX-DAXX chromatin remodeling complex. In a previous comprehensive cancer survey of ALT, we reported that ALT was highly prevalent in some tumor types (e.g. astrocytomas, sarcomas, pancreatic neuroendocrine tumors), but we did not observe any ALT-positive primary prostate cancer (out of 1,176 analyzed). However, we subsequently found a subset of metastatic prostate cancers that harbor ALT, suggesting that mutations giving rise to ALT in this disease are unique to metastatic prostate cancer. Here, we have created the first prostate cancer cell lines exhibiting ALT, with the explicit purpose of molecularly characterizing ALT in prostate cancer and identifying promising therapeutic targets for men with ALT-positive lethal metastatic prostate cancer. Inactivating mutations in either ATRX or DAXX using the CRISPR/Cas9n system were introduced into the genetically well-characterized, telomerase-positive (ALT-negative) prostate cancer cell lines, LAPC-4 and CWR22Rv1. Resulting mutant subclones were compared to their parental (or empty vector) counterparts. In these new models, abolishing ATRX expression was sufficient to induce the ALT phenotype in both LAPC-4 and CWR22Rv1, as assessed by multiple biomarkers of ALT, including the presence of bright telomeric FISH foci, ALT-associated PML bodies (ABPs), and extrachromosomal telomere c-circles. Interestingly, abolishing DAXX expression induced ALT in only a subset (3/5) of LAPC-4 DAXX KO clones, and a subset (4/19) of CWR22Rv1 DAXX KO clones. We have successfully activated the ALT telomere maintenance phenotype in two prostate cancer cell lines through CRISPR-mediated targeted gene deletion. We are currently utilizing these isogenic cell lines to further characterize and elucidate the underlying biology of cancers harboring ALT, with the goal of pharmacologically targeting the molecular features unique to the ALT phenotype (e.g. ATR inhibition). The identification of ALT-specific drugs may pave the way for the development of new targeted treatments for the subset of men with ALT-positive lethal metastatic prostate cancer, and more broadly, other ALT-positive cancers. ALT is easily detected in clinical tissue samples, and thus would serve as a predictive biomarker for personalized medicine. Citation Format: Mindy K. Graham. Functional knockout of ATRX or DAXX permits the alternative lengthening of telomeres (ALT) mechanism in prostate cancer cells [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 3412. doi:10.1158/1538-7445.AM2017-3412