Abstract
Brain tumors remain the leading cause of cancer-related deaths in children and often are associated with long-term sequelae among survivors of current therapies. Hence, there is an urgent need to identify actionable targets and to develop more effective therapies. Telomerase and telomeres play important roles in cancer, representing attractive therapeutic targets to treat children with poor-prognosis brain tumors such as diffuse intrinsic pontine glioma (DIPG), high-grade glioma (HGG), and high-risk medulloblastoma. We have previously shown that DIPG, HGG, and medulloblastoma frequently express telomerase activity. Here, we show that the telomerase-dependent incorporation of 6-thio-2′deoxyguanosine (6-thio-dG), a telomerase substrate precursor analogue, into telomeres leads to telomere dysfunction–induced foci (TIF) along with extensive genomic DNA damage, cell growth inhibition, and cell death of primary stem-like cells derived from patients with DIPG, HGG, and medulloblastoma. Importantly, the effect of 6-thio-dG is persistent even after drug withdrawal. Treatment with 6-thio-dG elicits a sequential activation of ATR and ATM pathways and induces G2–M arrest. In vivo treatment of mice bearing medulloblastoma xenografts with 6-thio-dG delays tumor growth and increases in-tumor TIFs and apoptosis. Furthermore, 6-thio-dG crosses the blood–brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. Together, our findings suggest that 6-thio-dG is a promising novel approach to treat therapy-resistant telomerase-positive pediatric brain tumors. Mol Cancer Ther; 17(7); 1504–14. ©2018 AACR.
This article is featured in Highlights of This Issue, p. 1353
Introduction
Telomeres are the physical ends of eukaryotic linear chromosomes and, in mammals, are composed of several kilobases of tandem TTAGGG repeats that are bound by the shelterin protein complex (1). Shelterin proteins protect telomeres from ATM and ATR-dependent DNA damage responses (DDR; ref. 1). We and others have previously shown that natural telomere shortening during replicative senescence or experimental telomere uncapping elicits ATM-dependent DDR triggered by telomere dysfunction (2, 3). The hallmark of telomere dysfunction is the formation of DNA damage foci localized at telomeres called TIFs (telomere dysfunction-induced foci). TIFs are focal accumulations of DDR factors such as ATM S1981-P, γH2AX, and 53BP1 at dysfunctional telomeres (4). Telomeres are maintained by telomerase activity in 73% to 90% of primary human cancers, whereas in most normal somatic cells, this activity is not detectable (5–7). Human telomerase consists of two essential components, the protein catalytic subunit (hTERT) and the RNA template (hTERC) that contribute to the synthesis of telomeric repeats, thereby maintaining telomeres. Telomerase activation, a feature of the vast majority of cancers, is essential for maintaining an immortal phenotype by conferring unlimited replicative potential.
Brain tumors are the most common solid tumors of childhood and are the leading cause of cancer-related deaths in children (8). Diffuse intrinsic pontine glioma (DIPG) is a particularly poor-prognosis brain tumor with a median overall survival of less than 1 year (9). Hence, there is an urgent need to develop novel therapies that not only improve outcome, but mitigate long-term complications in children with these poor-prognosis brain tumors. We have previously shown that over 73% of DIPG and 50% of high-grade gliomas (HGG; ref. 10) demonstrate telomerase activity. We recently conducted a molecular biology and phase II study of imetelstat, a potent inhibitor of telomerase (11, 12), to estimate inhibition of tumor telomerase activity and efficacy in children with recurrent central nervous system (CNS) malignancies (13). The regimen proved intolerable, because of thrombocytopenia that led to bleeding. This toxicity prevented more frequent dosing of imetelstat to allow sustained telomerase inhibition. Because targeting telomerase directly, such as with imetelstat, would result in a significant lag period from the initiation of treatment until telomeres shortened sufficiently to reduce tumor burden, stopping therapy with imetelstat would result in rapid telomere regrowth. Thus, new approaches utilizing this almost universal cancer target are needed. Given the role played by telomerase reactivation in oncogenesis, telomeres and telomerase remain relevant therapeutic targets in this patient population (14–16). Recently, preclinical studies validated a telomere targeting strategy consisting of the incorporation of 6-thio-2′-deoxyguanosine (6-thio-dG), a telomerase substrate precursor nucleoside analogue, into telomeres by telomerase (17). Mender and colleagues have shown that telomerase-dependent incorporation of 6-thio-dG into telomeres is very effective and specific at targeting telomerase-positive cancer cells but not telomerase silent normal cells (17). Treatment with 6-thio-dG led to telomere damage and cell death in telomerase-positive cancer cell lines. Because this effect appears to be telomere length independent, the prediction using this novel approach is that treatment with 6-thio-dG will require a shorter time period to achieve a rapid effect on tumor growth and progression than direct telomerase inhibition-based therapy (18). This approach could be beneficial for patients with aggressive brain tumors, such as DIPG. In the current study, we tested the in vitro and in vivo effect of 6-thio-dG in telomerase-positive stem-like cells derived from poor-prognosis pediatric brain tumors and addressed the mechanistic aspect of 6-thio-dG–induced DNA damage response in telomerase-positive cancer and normal cells. Our findings suggest that 6-thio-dG is a promising novel approach to treat therapy-resistant pediatric brain tumors and provide a rationale for clinical testing of 6-thio-dG in children with brain tumors.
Materials and Methods
Cell lines and primary tumor cell culture
All patient specimens were collected after obtaining written informed consent from patients and families in accordance with approved Institutional Review Board (IRB) studies. The primary DIPG neurosphere line CCHMC-DIPG-1 was aseptically isolated by dissociating the brain tumor tissue postautopsy from a patient consented under the Pediatric Brain Tumor Repository (PBTR) study (IRB approved protocols 2013-1245 and 2013-5947) at Cincinnati Children's Hospital Medical Center (CCHMC, Cincinnati, OH). Primary patient-derived neurospheres high-risk group-3 medulloblastoma (MB004; refs. 19, 20), GBM (R0315-GBM), and DIPG [SU-DIPG-VI (21) and CCHMC-DIPG-1 (22)] were cultured in neurosphere stem cell media as described elsewhere (22, 23). Patient-derived medulloblastoma cell lines collected from the same patient at diagnosis D-425, and at recurrence D-458 (24, 25), were cultured in RPMI1640 (Gibco) supplemented with 15% FBS. Primary normal human foreskin fibroblast (HFF) strain (ATCC CRL-2091), the HeLa human cervical carcinoma cell line, the human osteosarcoma cell lines Saos-2 (ATCC HTB-85) and U2OS (ATCC HTB-96) were purchased from the ATCC. HFF cells were immortalized with hTERT (HFF + hTERT) by viral transduction as described previously (2). The source of other cell lines is referenced above. Commercially available cell lines were characterized at their original sources. All cells were expanded upon receipt or establishment for 2 to 3 passages and used within 1 to 2 months after thawing the cryopreserved cells without additional authentication. No testing was done by the authors for mycoplasma. However, we did not observe any evidence of their presence.
Telomerase activity assay
Telomerase activity was assayed using the TRAPeze Telomerase Detection Kit (Millipore). Cell extracts were prepared according to the manufacturer's protocol. A total of 50 to 100 ng of total protein was used to assess the telomerase activity by performing polyacrylamide gel (12.5%) electrophoresis.
Drug treatment
6-thio-dG (Metkinen Oy; provided by J.W. Shay) was dissolved in DMSO:water (1:1) to prepare a 10 mmol/L stock solution, aliquoted, and stored in −20°C. For in vitro treatments, 1 mmol/L final concentration was prepared in plain media. For in vivo studies, 6-thio-dG was prepared in a 5% DMSO solution. Kinase inhibitors of ATM (KU-55933; ref. 26) and ATR (VE-822; refs. 27, 28) were purchased from Selleckchem and reconstituted in DMSO. Imetelstat (GRN163L; Geron Corp.; ref. 29); was reconstituted (1 mg/mL) in PBS.
Cell growth and sphere formation assay
After dissociating primary neurospheres by TrypLE express (Gibco), single cells were seeded in respective growth media in 6-well plates and were incubated for one week at varying concentrations (0.5–10 μmol/L) of 6-thio-dG or DMSO. Fresh culture media were added every 3 days. Viable cell numbers were determined by Trypan blue exclusion method. For sphere formation assay, MB004 single cells were seeded in limited dilution (10 cells/well in 96-well plate) and treated with DMSO or 6-thio-dG. Sphere formation was monitored by microscopy.
Cell-cycle analysis
Following treatment with 6-thio-dG or DMSO, cells were collected in PBS, fixed with ice-cold 70% ethanol, and were kept in −20°C for at least an hour. Cells were then washed and stained with propidium iodide (PI) solution containing 25 μg/mL PI (Sigma), and 100 μg/mL Ribonuclease-A (Sigma) for 30 minutes in the dark. Flow cytometry was performed on BD FACSCanto II and cells were analyzed using FlowJo v.10 (FlowJo) software.
Immunofluorescence and telomere FISH assay
Cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes. Cells were then washed, permeabilized with 0.5% Triton-X-100 in PBS, blocked with 5% donkey serum and 0.3% Triton X-100 in TBS, and incubated with primary antibodies against γH2A.X (1:500, rabbit), or cleaved caspase-3 (1:400, rabbit; Cell Signaling Technology); and/or TRF2 (1:200; Mouse; NOVUS), as applicable, for overnight followed by TBST wash (three times) next day. Corresponding secondary antibodies were added (Alexa-Fluor 488- or 594-conjugated donkey anti-rabbit, or anti-mouse (1:500; Jackson ImmunoResearch) for 1 hour, and washed with TBS (three times) before mounting. For telomere-FISH, fixed and permeabilized cells were dehydrated with a graded ethanol concentration series, air dried, and covered with hybridization solution [70% formamide, 0.5% Blocking Reagent (Roche Diagnostics) diluted in 100 mmol/L maleic acid and 150 mmol/L sodium chloride, and 10 mmol/L Tris (pH 7.5)] with 300 ng/mL PNA(CCCTAA)3-Cy3 (Biosynthesis), and denatured for 6 minutes at 84°C followed by hybridization for at least 2 hours at room temperature. Cells were washed three times with 70% formamide and 10 mmol/L Tris (pH 7.5) and three times with TBS. Finally, they were embedded with mounting media with DAPI (Vector Laboratories H1200). Images were captured with 60× oil objective on Nikon Eclipse Ti confocal microscope.
Senescence assay
Senescence-associated β-galactosidase was detected as described previously (2). Cells were observed under the microscope until the development of the blue color, and the reaction was terminated. Images were captured, and stained and unstained cells were counted from multiple fields to quantitate the percent senescent cells.
Western blot analysis
Western blot analysis was performed as described previously (22). Antibodies used were against ATM-S1981P (R&D Systems); ATM (SIGMA); ATR-T1989P (GeneTex); and ATR, CHK1-S345P, CHK1, CHK2-T68P, CHK2, cleaved caspase-3, and β-actin (Cell Signaling Technology). Bands were visualized using ECL with Azurec500 imaging system (Azure Biosystems). Band intensities were quantified using ImageJ software (Ver. 1.49u, NIH, Bethesda, MD).
DNA extraction and agarose gel electrophoresis
Genomic DNA was extracted from DMSO or 6-thio-dG treated HFF, HFF + hTERT, and U2OS cells using Puregene Kit (Qiagen) following the manufacturer's protocol. A total of 100 ng of each sample was run on a 0.7% agarose gel followed by staining with GelRed Nucleic Acid Gel Stain (Biotium). Bands were visualized under UV illuminator.
Mouse subcutaneous and orthotopic xenograft
Athymic Ncr-nu/nu female mice (6–7 weeks old) were subcutaneously injected with 1 × 104 MB004 cells. Mice were weighed and distributed in two groups (control and 6-thio-dG). After tumor establishment (day 23–24 postimplantation) with an average volume of 100 to 200 mm3, mice were injected intraperitoneally every 2 days with 6-thio-dG (2.5 mg/kg) or DMSO-PBS (vehicle) for 3 to 4 weeks until euthanization. Tumors were measured by slide calipers taking two longest tumor diameters (length and width) perpendicular to each other, and volumes were calculated by using the formula: (π/6) × d3, where d = mean diameter. For orthotopic xenograft, CCHMC-DIPG-1 luciferase-positive cells (1 × 104) were injected in the brain of NRG (NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ) mice. Briefly, mice were injected stereotactically with 2 μL medium containing 1 × 104 luciferase-positive cells. The coordinates for injection were 0.8–1 mm posterior to lambda suture and 3.5 mm deep, corresponding to the pons location in the brain. Tumors were visualized by luminescence using IVIS Spectrum CT in vivo imaging system (PerkinElmer). All animal procedures were approved by our Institutional Animal Care and Use Committee (IACUC; protocol #IACUC2015-0066, CCHMC).
IHC
Formalin-fixed paraffin-embedded (FFPE) sections were deparaffinized in xylene followed by rehydration through a graded ethanol concentration series. Heat-induced antigen retrieval was performed by steaming slides for 20 minutes in 10 mmol/L Citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 1% H2O2 followed by washing, blocking with 10% goat serum in TBST for an hour, and incubating with primary antibody Ki67 (1:1500; rabbit; Abcam), or cleaved caspase-3 (1:1,000; rabbit; Cell Signaling Technology) in 2% goat serum in TBST overnight at 4°C. Slides were washed with TBST (three times) and were treated with biotinylated anti-rabbit secondary antibody (1:500) and signal-amplified using ABC Kit (Vector Laboratories). Signal was visualized with DAB (Vector Laboratories) and counterstained with Harris Hematoxylin (Sigma). H&E staining was performed using hematoxylin-1 and eosin-Y (Thermo Fisher Scientific). Tissues were mounted with Permount (Thermo Fisher Scientific) and imaged by Nikon eclipse 80i microscope.
Tissue TIF assay
Tissue samples were prefixed with 4% PFA and were cryoprotected in 25% sucrose/PBS solution, and then embedded in OCT freezing molds with Neg-50 (VWR) in acetone and dry ice followed by cryosectioning. Heat-induced antigen retrieval and Telomere-FISH was performed as described above. The samples were incubated in blocking solution (5% donkey serum, 0.3% Triton X-100 in TBS) for 30 minutes, and treated with anti-53BP1 (rabbit 1:500; Novus Biologicals) for 1 hour at room temperature. After washing in TBST (three times), the samples were incubated with secondary antibody Alexa-Fluor 488–conjugated donkey anti-rabbit (1:400; Jackson ImmunoResearch), and washed in TBS (three times). The samples were embedded in mounting media with DAPI (Vector Laboratories H1200). Images were captured with 60× oil objective on Nikon Eclipse Ti confocal microscope.
Statistical analysis
The statistical analyses were performed by Student t test or multiple-way ANOVA as required using the GraphPad Prism (version 7.02). Each experiment was repeated at least twice. Error bars represent SD of at least three replicate wells or fields from one representative experiment considered as technical replicates, or from independent experiments or different animals for biological replicates. Differences were considered statistically significant at P < 0.05.
Results
6-thio-dG selectively inhibits cell growth of telomerase-positive tumor cells
One of the major setbacks in oncology is the ability of certain cancers to recur after minimal or undetectable disease is achieved with aggressive therapies. Cancer stem-like cells have been proposed to represent a subpopulation of cells within a tumor that self-renew to promote tumor growth and recurrence. In the current study, primary stem-like cells were derived from DIPG, HGG, and medulloblastoma patients' tumor tissue and expanded in neurosphere stem cell media. Supplementary Table S1 indicates genetic features and subtypes of the cell lines. We tested 6-thio-dG in a panel of telomerase-positive pediatric brain tumor cells, including high-risk group-3 medulloblastoma (MB004), GBM (R0315-GBM), and DIPG (SU- DIPG-VI and CCHMC-DIPG-1) along with a panel of control cell lines, consisting of normal primary HFF (telomerase-negative), HFF-ectopically expressing hTERT (HFF + hTERT, telomerase-positive), HeLa cells (telomerase-positive), and osteosarcoma cells Saos-2 (telomerase-negative, alternative lengthening of telomeres or ALT-positive). Telomerase activity was verified by the gel-based TRAP assay (Supplementary Fig. S1). We and others previously reported that under serum-free culture conditions, HGG-, DIPG-, and medulloblastoma neurospheres expressed neural stem cell markers such as nestin, CD133, and olig2, and were capable of self-renewal and differentiation in the presence of serum (22, 23). These cancer stem-like cells are thought to be responsible for tumor recurrence (30). Moreover, we confirmed that these cells are able to establish tumors in mouse. The cells were treated with 0.5 to 10 μmol/L of 6-thio-dG every 3 days for one week. As expected, treatment with 6-thio-dG inhibited cell growth in a dose-dependent manner in all telomerase-positive cells, including brain tumor cells, with a minor to no effect in telomerase-negative cells HFF and Saos-2 cells up to 3 μmol/L (Fig. 1A and B). Interestingly, 6-thio-dG effectively inhibited cell growth of both patient-derived medulloblastoma cell lines collected from the same patient at diagnosis, D-425 cells (biopsy of cerebellar primary tumor of 6-year-old boy) and at recurrence D-458 cells (tumor cells in CSF following failure of radio- and chemotherapy; Fig. 1C; refs. 24, 31). All telomerase-positive cells including brain tumor cells were highly sensitive compared with telomerase-negative cells as evidenced by the IC50 ranging 0.14 to 1.45 μmol/L (Supplementary Table S2). Telomerase dependency of 6-thio-dG was further verified by using imetelstat. HFF and HFF + hTERT cells were treated with either 6-thio-dG or imetelstat, or in combination. The inhibition of telomerase activity by imetelstat treatment was confirmed by the TRAP assay (Supplementary Fig. S2A). As expected, the inhibition of cell growth by 6-thio-dG in the combination treatment was markedly reduced by imetelstat (Supplementary Fig. S2B and S2C), indicating that the 6-thio-dG effect is largely dependent on the presence of telomerase.
6-thio-dG specifically inhibits cell growth of telomerase-positive cells. A–C, Cell images (top) and corresponding viable cell counts (bottom). A, Telomerase-positive cells: HeLa and HFF + hTERT; telomerase-negative cells: HFF, and Saos-2 (ALT-positive). B, Telomerase-positive patient-derived pediatric brain tumor cells: MB004, R0315-GBM, SU-DIPG-VI, and CCHMC-DIPG-1. C, Matched pair of patient-derived medulloblastoma cells at biopsy (D-425, Primary) and at posttherapy failure (D-458, recurrence) treated with DMSO or 0.5 to 10 μmol/L of 6-thio-dG for 1 week. Error bars represent the SD from triplicates. Each experiment was performed at least twice.
6-thio-dG induces sustained G2–M cell-cycle arrest in telomerase-positive cells
Next, we investigated the mechanistic aspect of 6-thio-dG-induced DDR. Cell growth inhibition caused by 6-thio-dG treatment prompted us to check its effect on cell-cycle progression. Three-day treatment with 3 μmol/L of 6-thio-dG caused G2–M arrest in telomerase-positive cells HFF + hTERT and HeLa. In contrast, this effect was negligible in telomerase-negative cells HFF (Fig. 2A). Furthermore, we assessed the long-term effect of 6-thio-dG on the cell-cycle profile as illustrated in Fig. 2B. In continuous treatment, 6-thio-dG–induced G2–M arrest was sustained and more pronounced in telomerase-positive normal cells (HFF + hTERT) along with an increase in >4n cell population compared with telomerase-negative HFF cells (Fig. 2C). For HeLa cells, continuous treatment caused a sharp increase in G2–M, >4n, and sub-G1 cell populations, indicating aneuploidy and cell death. G2–M fraction increased after prolonged treatment of HFF cells with 6-thio-dG, likely due to the accumulation of genomic damage. In contrast to telomerase-positive cells, no increase in sub-G1 and >4n population was noticed. Interestingly, even when the drug was removed from the media (Fig. 2B), 6-thio-dG effect on cell-cycle progression persisted several days after drug withdrawal in HeLa cells (Fig. 2D). In HFF + hTERT cells, G2–M and >4n accumulation decreased over time and the cell-cycle profile partially reverted to that of the vehicle control. These results indicate that 6-thio-dG treatment has limited effect in normal cells (HFF) and causes G2–M arrest, aneuploidy, and cell death in HeLa cells. This effect was sustained several days after drug withdrawal in cancer cells but declined in normal telomerase-positive cells (HFF + hTERT).
6-thio-dG induces persistent G2–M cell-cycle arrest in telomerase-positive cells. A, Cell-cycle analysis of HFF, HFF + hTERT, and HeLa cells treated with DMSO or with 3 μmol/L of 6-thio-dG for 3 days. B, Schematic of the experimental design of continuous and wash-off treatment. C, Cell-cycle plots of DMSO controls and 6-thio-dG–treated HFF, HFF + hTERT, and HeLa cells at days 5, 6, 7, and 8. D, Cell-cycle plots of pretreated HFF + hTERT and HeLa with DMSO or 3 μmol/L of 6-thio-dG for 3 days at day 1 to 5 after drug removal. The percentage of cells in sub-G1, G1, S, G2–M, and >4n is indicated in each plot. Bottom, percent distribution of cell-cycle phases as a function of time. The experiments were conducted at least twice, and the results shown are representative of the replicates.
6-thio-dG induces persistent telomere dysfunction in telomerase-positive cells
Previous studies have shown that 6-thio-dG treatment leads to TIFs in telomerase-positive cells but not in telomerase-negative cells (17). We visualized TIFs by FISH combined staining using γH2AX colocalization with a telomere-specific PNA probe. As expected, 6-thio-dG caused an acute increase of the number of cells with TIFs (∼25%) in telomerase-positive cells, HFF + hTERT, after 2 days (Fig. 3A). TIF formation was limited in telomerase-negative cells HFF with less than 3% after 2- or 5-day treatment. This effect was amplified over time; approximately 34% of cells were TIF-positive at 5-day continuous treatment (Fig. 3A). Interestingly, approximately 31% of cells were still TIF-positive in telomerase-positive cells 3 days after drug withdrawal while TIFs completely disappeared in telomerase-negative cells. Furthermore, imetelstat treatment inhibited 6-thio-dG–induced TIF formation in HFF + hTERT (Supplementary Fig. S2D), confirming telomerase dependency of 6-thio-dG–induced TIFs. As observed in the previous study (17), 6-thio-dG also caused a modest increase in genomic DNA damage in telomerase-negative cells. This general damage was more evident in telomerase-positive cells (Fig. 3A). Importantly, TIF-negative cells treated with 6-thio-dG displayed markedly reduced genomic DNA damage, suggesting that TIFs exacerbate genomic DNA damage (Supplementary Fig. S3A). Indeed, the inhibition of telomerase using imetelstat reduced the genomic DNA damage in cells treated with 6-thio-dG (Supplementary Fig. S3B).
6-thio-dG causes sustained telomere damage in telomerase-positive cells. A, HFF and HFF + hTERT cells were cultured with 3 μmol/L of 6-thio-dG for 2 days (initial treatment), then continuously for another 3 days (continuous treatment) or 3 days after drug removal (drug wash-off). The number of TIFs per cell was counted for each treatment (∼100 cells/treatment). Bottom, representative images of FISH-immunofluorescence using a telomere-specific PNA probe (red) and γH2AX staining (green). White arrows indicate colocalization of γH2AX and telomere signals (yellow), indicative of TIFs. DAPI (blue) indicates nucleus staining. Average values of at least three fields per cell per condition were evaluated. P values are indicated, **, P < 0.01; ***, P < 0.001. B, Cell-cycle plots of MB004 treated with DMSO or 3 μmol/L of 6-thio-dG for 3 days. Percent events of cell-cycle phases are indicated. C, Cell images of neurosphere formation assay at day 3 and day 7 posttreatment with DMSO or 3 μmol/L of 6-thio-dG. Right, quantification of the number of spheres at day 3 and 7. Error bars represent the SD generated from triplicates. D, Relative growth of MB004 cells treated with 6-thio-dG to DMSO control. The cells were initially treated with 3 μmol/L of 6-thio-dG for 2 days followed by 3 days of continuous or discontinuous treatment. Bottom, representative IF-FISH images. Telomeres (red), γH2AX (green), and TIFs (yellow) are depicted. DAPI (blue) indicates nucleus staining. Error bars represent the SD obtained from triplicates.
Next, we evaluated the effect of 6-thio-dG treatment in telomerase-positive primary high-risk group 3 medulloblastoma stem-like cells MB004. Treatment with 3 μmol/L of 6-thio-dG for 3 days resulted in an increase in sub-G1 and G2–M cell populations and a total abolition of sphere formation ability at day 7 (Fig. 3B and C). Of note, at day 3, cells treated with 6-thio-dG were able to form small spheres or “spherelets” containing 4 to 10 cells. At day 7, these “spherelets” completely disappeared with 6-thio-dG treatment, while the DMSO-treated spherelets continued to grow. We then evaluated the effect of continuous and discontinuous exposure to 6-thio-dG after an initial treatment of 2 days. Both treatment schemes showed a robust growth inhibition and sustained TIF formation, indicating that the effect of 6-thio-dG treatment persists several days after drug withdrawal (Fig. 3D). These data demonstrate the telomerase-dependent induction of TIFs and their persistence even after the drug is removed, providing a possible explanation for the sustained G2–M arrest. Together, these results indicate that 6-thio-dG effect on cell growth is dependent on telomerase-induced TIFs probably in conjunction with genomic DNA damage, and this compound is active in brain tumor cells derived from therapy-resistant patients' tumors.
Sequential activation of ATR and ATM pathways in response to 6-thio-dG–induced telomere damage
ATM and ATR kinases are master regulators of DDR signaling. We and others have shown that telomere dysfunction induces ATM-dependent DDR (2, 3). To extend our characterization of 6-thio-dG–induced telomere damage, we investigated ATM and ATR DDR in telomerase-positive cells HFF + hTERT and in matched telomerase-negative cells HFF. Both ATM and ATR signaling pathways were engaged in response to 6-thio-dG treatment in HFF + hTERT as evidenced by the accumulation of ATR-T1989 and ATM-S1981 phosphorylation (Fig. 4A), whereas 6-thio-dG treatment in telomerase-negative cells correlated with the activation of the ATR pathway, probably due to genomic DNA damage. Timing-wise, the ATR pathway was first activated then progressively inhibited (Fig. 4A). The decrease in ATR signaling overlapped with ATM pathway activation. Although at day 1, we did not observe TIF formation, the number of TIFs per cell and the number of cells with TIFs markedly increased from day 1 to day 3 in the HFF + hTERT cells (Fig. 4B). This increase correlated with a sustained increase of ATM activation from day 2 to day 3 and a decrease of ATR activation starting at day 2. To investigate 6-thio-dG–induced DDR further, HFF + hTERT cells were pretreated with a specific ATM or ATR inhibitor for 2 hours prior to 6-thio-dG treatment for 48 hours. In the presence of either ATM or ATR inhibitor, we observed a reduction in the number of cells with TIFs as well as the number of TIFs per individual cells (Fig. 4C). Compared with ATR inhibition, ATM inhibition led to lower number of TIFs per individual cell. We interpret these results to suggest that 6-thio-dG–induced telomere damage sequentially activates the ATR pathway followed by ATM activation and the formation of TIFs is primarily induced by ATM pathway in normal cells.
Sequential activation of ATR and ATM in response to 6-thio-dG. A, Western blot analysis of ATR-T1989P, total ATR, ATM-S1981P, and total ATM in HFF, and HFF + hTERT cells at day 1, 2, and 3 posttreatment with 3 μmol/L of 6-thio-dG. d, day. β-Actin served as a loading control. Irradiated HFF cells with 5 Gy were used as a positive control for ATM and ATR activation. Bar diagrams (bottom) show the quantification of ATR-T1989P, and ATM-S-1981P phosphorylation levels relative to the corresponding DMSO. O.D. (optical density) was measured by densitometry analysis using ImageJ software. Band intensities were normalized to total ATR or total ATM as applicable. Average values of two independent experiments were evaluated. B, Quantification of IF-FISH data in HFF + hTERT cells treated or untreated (DMSO) with 3 μmol/L of 6-thio-dG for 1 to 3 days. Representative images of cells (bottom) are shown with TIFs (yellow foci denoted by white arrows), telomeres (red), γH2AX (green), and nucleus (DAPI in blue). C, Immunoblot analyses of ATR-T1989P, ATR, ATM S1981-P, ATM, CHK2 T68-P, CHK2, CHK1 S345-P, and CHK1 in HFF + hTERT cells treated with 10 μmol/L of ATM or 50 nmol/L ATR specific inhibitors for 2 hours prior to and in combination with DMSO (control) or 3 μmol/L of 6-thio-dG for 2 days. IF-FISH was performed to assess the number of TIFs in 200 to 300 cells (right). Error bars are the SD of at least three fields per condition (50 or more cells/field). P values are indicated; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
6-thio-dG induced apoptosis in telomerase-positive cancer cells and senescence in telomerase-positive normal cells
We have previously demonstrated that ATM activation, γH2AX-focus formation, and p53 accumulation increased in presenescent cells (2). Therefore, we sought to investigate the ultimate fate of cancer telomerase-positive cells, HeLa, MB004, CCHMC-DIPG-1, and the primary normal human cells: HFF (telomerase-negative) and HFF + hTERT (telomerase-positive) treated with 6-thio-dG. The cells were treated with 3 μmol/L of 6-thio-dG for 4 or 7 days and were evaluated for apoptosis. Although HeLa (Fig. 5A), MB004, and CCHMC-DIPG-1 (Fig. 5C) cells showed an increase in cleaved caspase-3 signal, HFF and HFF + hTERT did not show any evidence of apoptosis (Fig. 5A and B). We further evaluated the effect of long-term exposure of HFF and HFF + hTERT cells to 6-thio-dG. The cells were incubated with 3 μmol/L of 6-thio-dG for 23 days. Prolonged exposure to 6-thio-dG induced a senescence phenotype in 30% of HFF + hTERT cells as assessed by staining for senescence-associated β-galactosidase activity (SA-β-gal), whereas only 7% of HFF cells were SA-β-gal-positive (Fig. 5D), suggesting that the senescence observed in HFF + hTERT cells was due to 6-thio-dG–induced telomere dysfunction. Thus, short-term treatment with 6-thio-dG causes apoptosis in telomerase-positive cancer cells, and the long-term treatment leads to senescence in telomerase-positive normal cells. These results are reminiscent of replicative senescence caused by persistent telomere damage. Interestingly, unlike cancer telomerase-positive cells, fast growing telomerase-negative cancer cells, U2OS, treated with 6-thio-dG, predominantly did not die (Supplementary Fig. S4A–S4C). After an initial growth inhibition, probably to repair the genomic DNA damage, the cells resumed cell growth several days after drug removal (Supplementary Fig. S4B). Finally, we did not observe any DNA fragmentation after treatment with 6-thio-dG (Supplementary Fig. S4D). These data indicate that the cell death observed in telomerase-positive cancer cells is not due to cell growth kinetics or genomic DNA fragmentation but rather due to telomere damage probably in combination with genomic DNA damage.
6-thio-dG induces apoptosis in telomerase-positive cancer cells and senescence in telomerase-positive normal cells. A, Representative IF images of cleaved caspase-3 (green) in HFF, HFF + hTERT, and HeLa cells treated with DMSO or 3 μmol/L of 6-thio-dG for 4 days. DAPI (blue), nucleus staining. B, Corresponding immunoblot analysis of cleaved caspase-3. β-Actin was used as a loading control. C, Representative IF images of cleaved caspase-3 (green) in MB004, and CCHMC-DIPG-1 cells treated with DMSO or 3 μmol/L of 6-thio-dG for 4 and 7 days. Nucleus in blue (DAPI). D, Senescence-associated β-gal assay. Senescent HFF cells (HFF-S) at 70 population doublings (PD 70) were used as positive control for senescence (replicative senescence). Senescent cells are stained in blue. X-gal (+) and (−) indicate X-gal was added or not, respectively. Early-passage HFF (negative control) and HFF + hTERT were treated with 3 μmol/L of 6-thio-dG or DMSO for 23 days. The plot on the right represents the quantification of the number of cells SA-β-gal-positive (senescent). Error bars represent the SD generated from triplicates. P value is indicated; *, P < 0.05. Each experiment was performed at least twice.
6-thio-dG treatment inhibited tumor growth in pediatric high-risk group-3 medulloblastoma xenografts by inducing an increase in in-tumor telomere dysfunction, a decrease of tumor mitotic index, and apoptosis
To evaluate the in vivo activity of 6-thio-dG, we subcutaneously injected primary patient-derived stem-like cells MB004 in athymic nude mice (1 × 104 cells/mouse). We previously observed that these cells form aggressive and fast growing tumors in mice. Tumors were established at 24 days postimplantation, at which point, the tumor volumes ranged from 100 to 200 mm3. A mixture of DMSO and PBS solution was used as vehicle control in 6 mice. To monitor 6-thio-dG toxicity in mice, we weighed both treated and untreated mice. We did not observe a noticeable weight difference between the two groups of mice, and no dehydration or clinical symptom of sickness were observed in the treated group, indicating that this 6-thio-dG regimen is not toxic (Fig. 6A). Tumor growth kinetics showed the majority of treated mice had a slower tumor growth rate compared with the untreated group (Fig. 6B). Four of 6 treated mice had reduced or delayed tumor growth. Two of 6 treated mice showed fast tumor growth, most likely due to the tumor aggressive nature and bigger tumor volume at the start of 6-thio-dG treatment. IHC of cleaved caspase-3 was performed in five tumors from each group to evaluate in-tumor apoptosis. Compared with the control group, we observed a significant increase in apoptosis in the treated group with a higher increase in tumors with slower growth (Fig. 6B and C). Accordingly, hematoxylin and eosin staining (H&E) showed a significant decrease in the number of mitotic figures and an increase in apoptotic bodies in 6-thio-dG–treated mice compared with untreated group (Fig. 6D). FISH staining using a combination of a telomeric probe and 53BP1 immunostaining showed a marked in-tumor increase of TIFs compared with untreated tumors (Fig. 6E). More than 21% of cells showed at least 1 to 3 TIFs in the 6-thio-dG group, whereas only approximately 6% in the control group (Fig. 6E). Moreover, a population of cells with 4 to 6 TIFs per cell was detected only in treated tumors. The probability of animal survival was significantly higher in 6-thio-dG–treated mice compared with the vehicle group upon reaching the tumor volume at a size of 1,500 mm3 (≥6 times of initial volume) considered to be a tumor burden (Supplementary Fig. S5). Together, these data can be interpreted to indicate that 6-thio-dG treatment inhibits the growth of therapy-resistant MB004 tumors by inducing telomere dysfunction, inhibition of cell growth, and apoptosis.
6-thio-dG treatment inhibits tumor growth in pediatric high-risk group 3 medulloblastoma and induces TIFs in DIPG xenografts. A, Average weight of mice treated with DMSO-PBS (vehicle) or 6-thio-dG. MB004 tumor cells were injected subcutaneously in the mouse flank and intraperitoneally injected when tumor was established with DMSO-PBS (vehicle) or 2.5 mg/kg of 6-thio-dG every 2 days for the indicated period of time. The arrow indicates the time of the tumor establishment and the start of the treatment. Error bars, SD from 6 mice per group. B, Tumor growth kinetics. Each line denotes tumor growth per mouse. Blue and red lines indicate vehicle and 6-thio-dG treatments, respectively. C, Representative IHC images indicating cleaved caspase-3 staining (brown) in FFPE sections from MB004 tumors treated with vehicle or 6-thio-dG. The plot on the right shows percent in-tumor apoptotic cells (cleaved caspase-3 positive). Each dot represents average percent of apoptotic cells per individual tumor per mouse. P value is indicated; *, P < 0.05. Circles in B and C correspond to the same tumors showing slower growth kinetics (B) and higher apoptosis (C). D, Quantification of in-tumor mitotic bodies (left), and apoptotic bodies (right) from FFPE sections stained with H&E. Average values of at least three fields per mouse were evaluated. P values are indicated; ***, P < 0.001. E, The number of in-tumor telomere damage (TIFs) were counted and expressed as percent of cells with TIFs. Vehicle indicates tumor from mice treated with DMSO-PBS, and 6-thio-dG indicates tumor from mice treated with 6-thio-dG. Error bars were generated from SD of at least three fields (50 cells or more/field) per mouse. P value is indicated; ***, P < 0.001. On the right, representative images of TIF analysis in 6-thio-dG and vehicle-treated tumors. Telomeres are in red, 53BP1 (green), and DAPI (blue). White arrows, TIFs (yellow). F, Diagrammatic workflow showing detection of CCHMC-DIPG-1 orthotopic xenograft by luminescence imaging followed by resection and histologic staining with H&E and Ki67 of collected tumors and matched normal tissue. Tumor and normal tissues are indicated. Circles indicate the tumor location in the brain. G, Representative images of tissue IF-TIF analysis in 6-thio-dG and vehicle-treated tumor. White arrows indicate TIFs (yellow), telomeres (red), and 53BP1 (green). DAPI, blue. On the right, quantification of TIFs. Error bars were generated from SD of at least three fields (50 cells or more/field) counted per mouse. Significance between vehicle- and 6-thio-dG–treated tumors is indicated by P value; **, P < 0.01.
6-thio-dG reached the tumor site and induced intratumoral TIFs in an orthotopic patient-derived xenograft model of diffuse intrinsic pontine glioma
To investigate the penetration and activity of 6-thio-dG in patient-derived brain tumors xenografted into mice, we injected intracranially luciferase-positive DIPG patient-derived primary cancer stem-like cells, CCHMC-DIPG-1, into the pons of the mouse brain using a stereotactic device. Tumor growth was monitored by bioluminescence imaging of the mouse brain (Fig. 6F). Upon tumor establishment at 8 to 10 days postimplantation, 6-thio-dG or DMSO-PBS administration was started by intraperitoneal injection. The mice were treated for 7 to 8 days (4–5 doses in total). Mouse brains were collected for analyses after animal euthanasia. The tumors were highly aggressive and histopathologic staining using H&E and Ki67 indicated high cellularity and proliferation of tumor tissue compared with matched normal brain (Fig. 6F). Telomerase activity was retained in the xenograft relative to the patient-derived neurosphere cells (Supplementary Fig. S6A). FISH analyses of 6-thio-dG–treated orthotopic tumors revealed an increase in the number of cells with genomic DNA damage and TIFs as well as higher number of TIFs per cell compared with untreated tumors (Fig. 6G). TIFs and genomic DNA damage was not observed in normal mouse brain (Supplementary Fig. S6B). This indicates that 6-thio-dG reached the tumor in the pons, and induced TIFs and genomic DNA damage with no obvious effect on normal brain tissue. Because of the aggressiveness of the tumors, further in vivo studies in orthotopic models of pediatric brain tumors are warranted to evaluate the long-term effect of 6-thio-dG on tumor growth and mouse overall survival.
Discussion
Telomerase activity is present in most human cancers but is undetectable in the majority of normal human somatic cells, supporting the rationale of targeting telomerase and telomeres to treat cancer. Our previous clinical trial of imetelstat, a potent direct inhibitor of telomerase, proved intolerable and ineffective in children with recurrent CNS malignancies. We believe that this was due, at least in part, to toxicities, such as thrombocytopenia, which led to CNS bleeding that prevented more frequent dosing of imetelstat to allow sustained inhibition of telomerase. Mender and colleagues reported that no animal deaths or weight loss were observed in mice treated with 6-thio-dG. Moreover, the treatment did not cause any toxic effects on hematologic counts, liver, and kidney functions (17).
Using a lung cancer model, the previous study has demonstrated that 6-thio-dG caused both in vitro and in vivo telomere damage (TIFs) and induced rapid cancer cell death, likely due to telomerase-dependent telomere uncapping and dysfunction caused by 6-thio-dG treatment (17). In the current study, we sought to evaluate the effect of 6-thio-dG in telomerase-positive primary pediatric brain tumor cells derived from patients with high-risk and treatment-resistant tumors. We found that treatment with 6-thio-dG caused telomere dysfunction and cell growth inhibition in a telomerase-specific manner within one week. Interestingly, both cell lines derived from the same patient at diagnosis and at recurrence after chemo- and radiotherapy were sensitive to 6-thio-dG, demonstrating that cells from previously treated and recurrent tumors remain sensitive to 6-thio-dG.
Mechanistically, we showed that 6-thio-dG induced G2–M cell-cycle arrest in both telomerase-positive normal (HFF + hTERT) and cancer cells (HeLa and MB004). G2–M arrest was sustained after 6-thio-dG removal and was associated with apoptosis in cancer cells. Long-term exposure of telomerase-positive normal cells to 6-thio-dG induced senescence, probably due to telomere dysfunction previously shown to be also associated with replicative senescence (2). Interestingly, our data can be interpreted to suggest that 6-thio-dG–induced senescence is initiated in the G2–M phase, whereas senescent cells are usually in the G1 phase (32). However, recent publications also support the initiation of senescence from G2 (33–35).
6-thio-dG treatment–induced G2–M arrest and TIFs formation was sustained several days after drug withdrawal, suggesting that a short exposure time in a clinical setting may be sufficient to have a therapeutic effect. It would be informative to investigate a combination treatment with 6-thio-dG and G2–M checkpoint inhibitors already tested in clinical trials such as AZD0156 (ATM inhibitor), VX-970 (ATR inhibitor), and AZD1775 (WEE1 inhibitor; ref. 36). The expectation is that the cells will progress to the M-phase, causing mitotic catastrophe and massive cell death. 6-thio-dG treatment sequentially activates ATR and ATM DDR pathways. 6-thio-dG activated the ATR but not the ATM pathway in normal telomerase-negative cells. In contrast, 6-thio-dG activated ATR and then ATM in normal telomerase-positive cells, suggesting that the 6-thio-dG–induced genomic DNA damage activates ATR and then 6-thio-dG–induced telomere damage activates the ATM pathway. However, TIFs could be induced by either pathway if ATR or ATM is inhibited. Thus, it would be informative to evaluate the cell-cycle progression in the presence of 6-thio-dG and ATM or ATR inhibitors. Importantly, 6-thio-dG treatment completely abolished neurosphere formation ability, suggesting that self-renewal and stemness are potential targets of 6-thio-dG. Given the extensive genomic DNA damage in telomerase-positive cells, we are not ruling out the possibility that both genomic and telomeric damage contribute to the ultimate fate of the cells in the context of the presence of 6-thio-dG–induced TIFs. Of note, it is well accepted that unlike genomic DNA damage, replicative senescence or apoptosis due to telomere dysfunction does not necessarily depend on the extent of the damage (number of TIFs), but rather on the telomeric localization of the DNA damage. Future studies are required to investigate the link between 6-thio-dG–induced telomere damage and genomic DNA damage.
Using an orthotopic mouse model for DIPG, we showed that 6-thio-dG provided by intraperitoneal injection reached the tumor site in the pons and induced telomere damage in the tumor, demonstrating that 6-thio-dG crossed the blood–brain barrier. Importantly, we did not observe any adverse effects of 6-thio-dG on normal mouse brain or mouse behavior. In addition to an increase in telomere damage, we also observed an increase in genomic DNA damage, indicating an enhancement of general damage initiated by the 6-thio-dG–induced telomere dysfunction as shown previously (17, 37). In future studies, we will optimize the number of injected brain tumor cells in the pons, the dose, and the timing of 6-thio-dG administration to evaluate the effect of 6-thio-dG on mouse survival. As this compound is not currently in clinical trials, further testing in multiple animal models is required to fully evaluate efficacy and toxicity.
In conclusion, our findings document that 6-thio-dG is a promising novel approach to treat therapy-resistant pediatric brain tumors and provides a rationale for 6-thio-dG testing as a single agent or in combination, with G2–M checkpoint inhibitors already in clinical trials to treat children with high-risk pediatric brain tumors.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: S. Sengupta, R. Drissi
Development of methodology: S. Sengupta, R. Drissi
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Sengupta, K. Lee, S.S. Kumar, C. Fuller, J.W. Shay
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Sengupta, M. Sobo, K. Lee, S.S. Kumar, A.R. White, J.W. Shay, R. Drissi
Writing, review, and/or revision of the manuscript: S. Sengupta, K. Lee, S.S. Kumar, A.R. White, I. Mender, C. Fuller, M. Fouladi, J.W. Shay, R. Drissi
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Sengupta, M. Sobo, A.R. White, L.M.L. Chow, J.W. Shay
Study supervision: J.W. Shay, R. Drissi
Acknowledgments
This work was supported by CancerFree KIDS Pediatric Cancer Research Alliance (to R. Drissi and S. Sengupta), by the Division of Oncology, and by the Brain Tumor Center, Cincinnati Children's Hospital Medical Center. We thank D.D. Bigner (Duke University) for kindly providing us with D-425 and D-458 cell lines, Y.-J. Cho (Oregon Health & Science University) for MB004 cell line, X.-N. Li (Baylor College of Medicine) for R0315-GBM cell line, M. Monje (Stanford University) for SU-DIPG-VI cell line, and N. Oatman and B. Dasgupta (CCHMC) for technical assistance and for luciferase labeling of CCHMC-DIPG-1 cells. We thank the Comprehensive Mouse and Cancer Core, and J. Mulloy laboratory, CCHMC for providing the Athymic Ncr-nu/nu and NRG mice, respectively.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Footnotes
Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).
- Received August 20, 2017.
- Revision received January 3, 2018.
- Accepted March 15, 2018.
- Published first April 13, 2018.
- ©2018 American Association for Cancer Research.
References
Volume 17, Issue 7
