Multiple cancer pathways regulate telomere protection
2019; Springer Nature; Volume: 11; Issue: 7 Linguagem: Inglês
10.15252/emmm.201910292
ISSN1757-4684
AutoresLeire Bejarano, Giuseppe Bosso, Jessica Louzame, Rosa Serrano, Elena Gómez‐Casero, Jorge L. Martı́nez-Torrecuadrada, Sonia Martı́nez, Carmen Blanco‐Aparicio, Joaquı́n Pastor, Marı́a A. Blasco,
Tópico(s)Cancer Research and Treatments
ResumoArticle13 June 2019Open Access Source DataTransparent process Multiple cancer pathways regulate telomere protection Leire Bejarano Leire Bejarano Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Giuseppe Bosso Giuseppe Bosso Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Jessica Louzame Jessica Louzame Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Rosa Serrano Rosa Serrano Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Elena Gómez-Casero Elena Gómez-Casero Experimental Therapeutics Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Jorge Martínez-Torrecuadrada Jorge Martínez-Torrecuadrada Biotechnology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Sonia Martínez Sonia Martínez orcid.org/0000-0003-2230-7794 Experimental Therapeutics Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Carmen Blanco-Aparicio Carmen Blanco-Aparicio Experimental Therapeutics Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Joaquín Pastor Joaquín Pastor Experimental Therapeutics Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Maria A Blasco Corresponding Author Maria A Blasco [email protected] orcid.org/0000-0002-4211-233X Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Leire Bejarano Leire Bejarano Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Giuseppe Bosso Giuseppe Bosso Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Jessica Louzame Jessica Louzame Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Rosa Serrano Rosa Serrano Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Elena Gómez-Casero Elena Gómez-Casero Experimental Therapeutics Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Jorge Martínez-Torrecuadrada Jorge Martínez-Torrecuadrada Biotechnology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Sonia Martínez Sonia Martínez orcid.org/0000-0003-2230-7794 Experimental Therapeutics Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Carmen Blanco-Aparicio Carmen Blanco-Aparicio Experimental Therapeutics Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Joaquín Pastor Joaquín Pastor Experimental Therapeutics Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Maria A Blasco Corresponding Author Maria A Blasco [email protected] orcid.org/0000-0002-4211-233X Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain Search for more papers by this author Author Information Leire Bejarano1,‡, Giuseppe Bosso1,‡, Jessica Louzame1, Rosa Serrano1, Elena Gómez-Casero2, Jorge Martínez-Torrecuadrada3, Sonia Martínez2, Carmen Blanco-Aparicio2, Joaquín Pastor2 and Maria A Blasco *,1 1Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain 2Experimental Therapeutics Program, Spanish National Cancer Centre (CNIO), Madrid, Spain 3Biotechnology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain ‡These authors contributed equally to this work *Corresponding author. Tel: +34 91 732 8031; Fax: +34 91 732 8028; E-mail: [email protected] EMBO Mol Med (2019)11:e10292https://doi.org/10.15252/emmm.201910292 See also: J Cherfils-Vicini & E Gilson (July 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Telomeres are considered as universal anti-cancer targets, as telomere maintenance is essential to sustain indefinite cancer growth. Mutations in telomerase, the enzyme that maintains telomeres, are among the most frequently found in cancer. In addition, mutations in components of the telomere protective complex, or shelterin, are also found in familial and sporadic cancers. Most efforts to target telomeres have focused in telomerase inhibition; however, recent studies suggest that direct targeting of the shelterin complex could represent a more effective strategy. In particular, we recently showed that genetic deletion of the TRF1 essential shelterin protein impairs tumor growth in aggressive lung cancer and glioblastoma (GBM) mouse models by direct induction of telomere damage independently of telomere length. Here, we screen for TRF1 inhibitory drugs using a collection of FDA-approved drugs and drugs in clinical trials, which cover the majority of pathways included in the Reactome database. Among other targets, we find that inhibition of several kinases of the Ras pathway, including ERK and MEK, recapitulates the effects of Trf1 genetic deletion, including induction of telomeric DNA damage, telomere fragility, and inhibition of cancer stemness. We further show that both bRAF and ERK2 kinases phosphorylate TRF1 in vitro and that these modifications are essential for TRF1 location to telomeres in vivo. Finally, we use these new TRF1 regulatory pathways as the basis to discover novel drug combinations based on TRF1 inhibition, with the goal of effectively blocking potential resistance to individual drugs in patient-derived glioblastoma xenograft models. Synopsis This study describes novel pathways that regulate telomere protection by modulating the TRF1 shelterin protein. ERK phosphorylates TRF1, and its inhibition results in TRF1 destabilization and persistent telomere damage, which impairs cancer proliferation and stemness. ERK inhibitors hamper TRF1 stability and induce persistent DNA damage at telomeres. TRF1 is directly phosphorylated by ERK kinase. ERK-dependent phosphorylation of TRF1 at T335 and T330 residues is required for TRF1 stability. ERK inhibitors combined PI3K inhibitors synergistically inhibit glioblastoma growth in vitro and in vivo. Introduction Telomeres are heterochromatic structures at the ends of chromosomes, which are essential for chromosome stability (Blackburn, 1991). They are composed by tandem repeats of the TTAGGG repetitive sequence bound by the so-called shelterin complex, which is formed by six proteins named TRF1, TRF2, RAP1, TPP1, TIN2, and POT1. The shelterin complex constitutes the so-called capping of the telomeres, which is essential for their protection, preventing telomeres from fusion to other chromosome ends, from telomere fragility, and from degradation (De Lange, 2005). Also, the shelterin complex prevents the recognition of telomeres as double-strand DNA breaks (DSB) and the subsequent activation of a persistent DNA damage response (DDR; De Lange, 2005). Telomeres get shorter with aging as cells divide to regenerate tissues, and when they reach a very short length, they can contribute to physiological aging (Hemann & Greider, 2000; Samper et al, 2001). Telomere shortening can be compensated through de novo addition of telomeric repeats by telomerase, a reverse transcriptase composed by a catalytic subunit (TERT) and an RNA component (Terc; Greider & Blackburn, 1985). Telomeres can also be elongated by an alternative mechanism known as alternative lengthening of telomeres (ALT), which is based in homologous recombination between chromosome ends (Bryan et al, 1997). The majority of cancer cells aberrantly activate telomerase or ALT mechanisms to be able to divide indefinitely (Kim et al, 1994; Bryan et al, 1995; Shay & Bacchetti, 1997; Barthel et al, 2017). More than 90% of human tumors aberrantly overexpress telomerase (Kim et al, 1994; Shay & Bacchetti, 1997; Joseph et al, 2010), while the remaining telomerase-negative tumors activate ALT (Bryan et al, 1997; Barthel et al, 2017). For this reason, telomeres have been considered as potential anti-cancer targets. Most studies have focused in telomerase inhibition as therapeutic approach for telomere targeting in cancer. One of the most advanced anti-telomerase therapies is GRN163L, also called Imetelstat (Harley, 2008; Joseph et al, 2010). However, clinical trials for several cancer types had shown that this strategy has some limitations (Parkhurst et al, 2004; Middleton et al, 2014; El Fassi, 2015). In mouse models of cancer, the anti-tumorigenic effect of telomerase inhibition is only achieved when telomeres reach a critically short length, and this effect is lost in the absence of the p53 tumor suppressor gene, which is frequently mutated in cancer (Gonzalez-Suarez et al, 2000; Perera et al, 2008). Also, cancer cells could activate ALT to overcome telomerase inhibition. Thus, alternative therapies of telomere targeting should be developed in order to efficiently target telomeres in cancer. In this regard, our group and others have found that not only telomerase but also shelterin proteins are often mutated in cancer. In particular, POT1 is mutated in several types of sporadic and familial human tumors, including chronic lymphocytic leukemia (CLL; Ramsay et al, 2013), familial melanoma (Robles-Espinoza et al, 2014; Shi et al, 2014), Li–Fraumeni-like families (LFL) with cardiac angiosarcomas (CAS; Calvete et al, 2015), glioma (Bainbridge et al, 2015), mantle cell lymphoma (Zhang et al, 2014), and parathyroid adenoma (Newey et al, 2012). Also, previous studies from our group suggested that targeting the shelterin complex through inhibition of one of its central components, TRF1, leads to a rapid telomere dysfunction independently of telomere length, thus avoiding the shortcomings of telomerase inhibition (Garcia-Beccaria et al, 2015; Bejarano et al, 2017). In particular, we reported that induction of telomere uncapping by either Trf1 genetic depletion or TRF1 chemical inhibition can effectively block initiation and progression of aggressive tumors in both lung cancer and glioblastoma mouse models, in a manner that is independent of telomere length (Garcia-Beccaria et al, 2015; Bejarano et al, 2017). We further demonstrated that TRF1 abrogation in normal tissues was tolerated and did not result in decreased mouse survival or severe defects in tissues (i.e., TRF1 abrogation did not affect brain olfactory or memory functions nor affected highly proliferative tissues) (Garcia-Beccaria et al, 2015; Bejarano et al, 2017). These findings were recapitulated by using TRF1 chemical inhibitors. In particular, we found that TRF1 is phosphorylated at different residues by AKT and that these modifications regulate TRF1 foci formation in vivo (Mendez-Pertuz et al, 2017). Thus, PI3K inhibitors, as well as inhibitors of the PI3K downstream target AKT, significantly reduced TRF1 telomeric foci and lead to increased telomeric DNA damage and fragility, also impairing the growth of lung and GBM cells (Garcia-Beccaria et al, 2015; Bejarano et al, 2017). Given these results, here we set to discover additional TRF1 modulatory pathways by carrying out a screening with FDA-approved drugs or drugs that are currently in clinical trials, and which cover the majority of known cancer pathways. We found several drugs that inhibit TRF1 independently of the Pi3K pathway, including inhibitors of some of the most deregulated pathways in cancer. Among these pathways, here we demonstrate an unprecedented role of the Ras pathway in regulating telomere protection. Finally, we used these new TRF1 regulatory pathways as a rational to discover novel drug combinations based on TRF1 inhibition, which effectively block resistance to individual drugs in patient-derived glioblastoma mouse models. The results shown here uncover the importance of telomere capping for cancer cells and identify novel therapeutic strategies based on telomere targeting. Results Identification of novel TRF1 regulatory pathways To identify novel pathways that regulate TRF1 protein levels, we used a cell-based high-throughput screening to determine TRF1 foci fluorescence upon treatment with different drugs (Garcia-Beccaria et al, 2015). We screened a CNIO collection of 114 anti-tumoral drugs, which are either approved by the Food and Drug Administration (FDA) or in clinical trials, and which cover 20 of the 26 pathways included in Reactome database (Fig EV1A). To this end, we treated CHA-9.3 mouse lung cancer cells (Garcia-Beccaria et al, 2015) with the compounds at 1 μM concentration during 24 h, followed by immunofluorescence analysis using anti-TRF1 antibody to quantify TRF1 foci fluorescence (Fig 1A). As positive controls, we used PI3K inhibitors (PI3Ki), previously shown by us to inhibit TRF1 telomeric foci (Mendez-Pertuz et al, 2017). Click here to expand this figure. Figure EV1. Validation of the novel pathways of TRF1 regulation A. Representative image of the pathways included in Reactome database. B. Quantification of TRF1 nuclear fluorescence in CHA9-3 lung cancer cells treated with DMSO or structurally different MEK inhibitors for 24 h at 1 μM. Data are representative of n = 2 biological replicates C. Quantification of TRF1 nuclear fluorescence in CHA9-3 lung cancer cells treated with DMSO or structurally different ERK inhibitors for 24 h at 1 μM. Data are representative of n = 2 biological replicates D. Western blot images (up) and p-ERK protein levels (down) of CHA9-3 lung cancer cells treated with the indicated compounds for 24 h at 1 μM. Data are representative of n = 2 biological replicates E. Quantification of TRF1 nuclear fluorescence in CHA9-3 lung cancer cells treated with DMSO or structurally different HSP90 inhibitors for 24 h at 1 μM. Data are representative of n = 2 biological replicates F. Quantification of TRF1 nuclear fluorescence in CHA9-3 lung cancer cells treated with DMSO or different tubulin agents for 24 h at 1 μM. Data are representative of n = 2 biological replicates. Data information: Data are represented as mean ± SEM. Significant differences using unpaired t-test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Identification of novel compounds with the ability to downregulate TRF1 protein levels A. Experimental procedure: 114 compounds approved by the FDA or in clinical trials are assessed by the Opera High Screening system for their ability to reduce TRF1 protein levels in CHA9.3 lung cancer mouse cells. B. Representative images (top) and quantification (bottom) of TRF1 nuclear fluorescence of patient-derived h676 GSCs cells treated with the indicated compounds for 24 h at 1 μM. Scale bars, 5 μm. Data are representative of n = 3 biological replicates. C. Western blot images (top) and TRF1 protein levels (bottom) of patient-derived h676 GSCs cells treated with the indicated compounds for 24 h at 1 μM. Data are representative of n = 3 (PLKi, HSP90i, and RTKi) and n = 4 (Aurorai, mTOR, CDKi, docetaxel, gemcitabine, ERKi, MEKi) biological replicates. D. Schematic representation of the novel TRF1 regulatory pathways. Asterisk indicates targets of TRF1 inhibitory compounds found in the screening. Data information: Data are represented as mean ± SEM. n represents biological replicates. Significant differences using unpaired t-test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 1 [emmm201910292-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint We found several drugs with the ability to downregulate TRF1 levels, including inhibitors of some of the most deregulated pathways in cancer. In particular, we identified inhibitors of the Ras pathway, including ERK and MEK inhibitors (ERKi and MEKi); compounds related with the cell cycle, such as Aurora inhibitors (Aurorai), CDK inhibitors (CDKi), and PLK1 inhibitors (PLK1i); an inhibitor of the chaperone HSP90 (HSP90i); two chemotherapeutic agents named gemcitabine and docetaxel; and, as expected, several compounds related with the PI3K pathway, including mTOR inhibitors (mTORi) and RTK inhibitors (RTKi) (Fig 1B; see Appendix Table S1 for results with all drugs tested). To test whether the inhibitors found to decrease TRF1 levels also decreased the levels of other shelterin components, we studied RAP1 and TIN2 protein levels (Appendix Fig S1A and B). We found that TIN2 protein levels were not affected by any of the compounds found to decrease TRF1, with the exception of RTKi that increased TIN2 levels (Appendix Fig S1A). In the case of RAP1, we found a significant decrease by MEKi, again docetaxel increased RAP1 protein levels (Appendix Fig S1B). To validate the ERK/MEK pathway as a novel signaling pathway that modulates TRF1 foci formation, we selected 5 structurally different MEK inhibitors and three structurally different ERK inhibitors (see Materials and Methods) and tested these compounds at 1 μM during 24 h in the CHA-9.3 mouse lung cancer cell line followed by TRF1 immunofluorescence analysis. All the MEK and ERK inhibitors showed a clear inhibition of TRF1 levels (Fig EV1B and C) with the exception of the ERK inhibitor GDC-0944, which, in fact, did not inhibit phospho-ERK levels in this cell line, explaining the lack of effect on TRF1 foci (Fig EV1D). The same chemical biology approach was used to validate HSP90 and tubulin agents as TRF1 modulators. We tested a total of five structurally different HSP90 inhibitors and three independent taxol derivatives (Materials and Methods). All of the compounds rendered a significant decrease in TRF1 foci fluorescence (Fig EV1E and F). In summary, we identify here new TRF1 regulatory pathways and validate these novel TRF1 modulators by using several chemically diverse inhibitors of these pathways. As we previously described that TRF1 inhibition effectively blocked glioblastoma growth both in mouse models and in xenograft models of patient-derived glioma stem cells (GSCs; Bejarano et al, 2017), we next tested whether these newly identified TRF1 modulators were also able to downregulate TRF1 protein levels in patient-derived GSCs. To this end, we treated h676 patient-derived primary GSCs (Bejarano et al, 2017) with the different compounds at 1 μM concentration during 24 h and analyzed TRF1 levels by Western blot. All the compounds, with the exception of Aurora and PLK1 inhibitors, were able to downregulate TRF1 protein levels in human GSCs (Fig 1C); thus, we further characterized all the compounds except for Aurora and PLK1 inhibitors. In summary, by using an unbiased screening we identified here several pathways and compounds with the ability to downregulate TRF1 levels in both lung cancer and glioblastoma cells, including inhibitors of the Ras pathway (ERK and MEK), the cell cycle-related CDK inhibitor, the inhibitor of the chaperone HSP90, RTK, and mTOR inhibitors (Fig 1D), and two chemotherapeutic agents (gemcitabine and docetaxel). Novel TRF1 modulators induce telomeric DNA damage in cancer cells Trf1 deletion has been previously shown to induce a persistent DDR at telomeres in different cell lines, which leads to decreased cell viability (Martinez et al, 2009; Sfeir et al, 2009). To address whether the newly identified TRF1 inhibitors were also able to induce DNA damage specifically at telomeres (the so-called telomere-induced DNA damage foci or TIFs), we treated CHA9.3 lung cancer cells with all the selected hits for 24 h at 1 μM followed by a double immunofluorescence of the γH2AX to detect DNA damage and of the shelterin component RAP1 to mark the telomeres. We found that all the TRF1 compounds were able to significantly increase the number of cells with more than 1 TIF, with the exception of mTOR inhibitors, where the TRF1 decreased did not reach statistical significance (Fig 2A). Furthermore, when we normalized the mean number of TIFs per nucleus (Appendix Fig S2A) to total γH2AX DNA damage (Appendix Fig S2B), we observed that in the majority of the cases the DNA damage steams from telomeres (Appendix Fig S2C). Importantly, in patient-derived glioblastoma stem cells (h676 GSCs), we found that all the TRF1 inhibitors induced increased global DNA damage as indicated by increased numbers of cells positive for the γH2AX DNA damage marker (Fig 2B); however, in this case owing to the fact that these cells cannot be attached to the plates, we could not perform TIF analysis. Figure 2. New TRF1 chemical inhibitors induce DNA damage in lung cancer and glioblastoma cells A. Representative images (left) and percentage (right) of cells presenting 1 or more γH2AX and RAP1 colocalizing foci (TIFs) upon treatment of CHA9-3 lung cancer cells with the indicated compounds. White arrowheads point to colocalization of γH2AX and RAP1. Scale bars, 5 μm. Data are representative of n = 6 (DMSO) and n = 3 (mTORi, PI3Ki, RTKi, MEKi, ERKi, HSPO90i, CDKi, docetaxel) biological replicates. B. Representative images (left) and percentage (right) of γH2AX-positive cells per field in DMSO or compound-treated patient-derived h676 GSCs. Scale bars, 50 μm. Data are representative of 6 (DMSO) and 3 (mTORi, docetaxel, ERKi, MEKi, RTKi, HSP90i, gemcitabine, CDKi) biological replicates. C. Quantification of multitelomeric signals (MTS) in patient-derived h676 GSC metaphases upon treatment with the indicated compounds. Representative images of the qFISH in the metaphases (left). Multitelomeric signals are indicated by arrowheads. Scale bars, 1 μm. Data are representative of n = 31 (DMSO), n = 18 (mTORi), n = 11 (MEKi), and n = 24 (RTKi) biological replicates. Data information: Data are represented as mean ± SEM. Significant differences using unpaired t-test are indicated by *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Trf1 inhibition by using genetic deletion has been previously shown to induce the so-called multitelomeric signals (MTS), which are associated with increased telomere fragility and increased telomere damage (Martinez et al, 2009; Sfeir et al, 2009). Thus, we next tested whether our hits had the ability to increase the number of MTS in h676 patient-derived GSCs. To this end, we treated GSCs with the different compounds for 24 h at 1 μM and then performed telomere quantitative fluorescence in situ hybridization (Q-FISH) on metaphase spreads to visualize telomeres (Materials and Methods). We found that mTOR inhibitors, MEK inhibitors, and RTK inhibitors significantly increase the number of MTS (Fig 2C). The rest of the compounds, namely ERK inhibitors, CDK inhibitors, HSP90 inhibitors, docetaxel, and gemcitabine, completely blocked the formation of metaphases, and this prevented determination of MTS upon treatment with these compounds. In summary, the newly identified TRF1 inhibitory compounds recapitulate the telomeric defects associated with TRF1 genetic deletion, such as induction of telomeric DNA damage (TIFs) and induction of telomere fragility (MTS). Novel TRF1 inhibitors reduce stemness of primary patient-derived GSCs We previously demonstrated that TRF1 is overexpressed in both adult and pluripotent stem cells and it is essential for stemness (Schneider et al, 2013) and that Trf1 genetic deletion significantly reduced stemness in both neural stem cells (NSCs) and glioma stem cells (GSCs; Bejarano et al, 2017). Thus, we next set to address whether the novel TRF1 modulators identified here also have the ability to reduce stemness in two independent patient-derived GSCs, h676, and h543 cells (Bejarano et al, 2017). To this end, we performed a dose–response sphere formation assay by plating disaggregated cells in a 96-well plate and treated them with the different compounds at several concentrations (Materials and Methods). We found that all the compounds had the ability to reduce stemness in both patient-derived GSC cell lines as indicated by decreased number of spheres 7 days after plating (Fig 3A–H). It is important to point out that in these graphs, the data are represented as a percentage of spheres normalized to the DMSO cells. In addition, we calculated the growth inhibition 50 (or GI50) for every compound, which indicates the concentration at which the drug causes the 50% of reduction in stemness (Table 1). We observed that the most potent compounds were the HSP90 inhibitor, gemcitabine and docetaxel, which had a GI50 ranging between 0.1 and 1.5 nM. They were followed by mTOR inhibitor, ERK inhibitor, MEK inhibitor, and ERK inhibitor in which the GI50 was between 0.05 and 0.5 μM, and finally, the RTK inhibitors showed the highest GI50, ranging between 1 and 5 μM (Table 1). The fact that the RTK inhibitor, which strongly decreased TRF1 protein levels 24 h after treatment, had a high GI50 may be due to a low stability of this compound in long-lasting experiments. In summary, all the newly identified TRF1 modulators significantly reduce the stemness of two independent primary patient-derived GSCs lines. Figure 3. New TRF1 inhibitory compounds reduce stemness in patient-derived glioma stem cells A–H. Dose–response curves of h543 and h676 patient-derived GSCs treated with the indicated compounds at several concentrations. Data are representative of n = 2 biological replicates. Data are represented as mean ± SEM normalized to DMSO. Download figure Download PowerPoint Table 1. Growth inhibition 50 of the selected compounds in h676 and h543 GSCs Compound CNIO code GI50 (nM) h676 GI50 (nM) h543 HSP90i (Geldanamycin) ETP-50853 0.15 0.25 Docetaxel ETP-45335 0.24 0.46 Gemcitabine ETP-45337 1.25 0.5 mTORi ETP-50537 433 125 ERKi ETP-50728 218 354 MEKi ETP-51677 61 90 CDKi ETP-47306 164 86 RTKi (Dasatinib) ETP-51801 4,400 913 TRF1 is directly phosphorylated by ERK2, mTOR, and bRaf kinases Next, we set to study the mechanisms by which the newly identified compounds regulate TRF1 levels. We had previously reported that the TRF1 telomeric protein is regulated by the PI3K signaling pathway (Mendez-Pertuz et al, 2017). In particular, TRF1 is directly phosphorylated by the PI3K downstream target AKT at different residues (T248, T330, and S344), and this phosphorylation is necessary for TRF1 stability and TRF1 foci formation in vivo (Mendez-Pertuz et al, 2017). Thus, we first addressed here whether the newly identified compounds were acting through the PI3K/AKT pathway by assessing p-AKT levels upon treatment with the different inhibitors. To this end, we treated h676 GSCs with the compounds at 1 μM for 24 h and we checked p-AKT levels by Western blot. From all the tested compounds, RTKi, mTORi, and HSP90i significantly downregulated AKT phosphorylation (Appendix Fig S3A). However, we observed that ERKi, MEKi, CDKi, gemcitabine, and docetaxel did not affect p-AKT, suggesting that these compounds are acting through AKT-independent pathways (Appendix Fig S3A). As the Ras pathway is heavily mutated in cancer, we next set to explore whether TRF1 is also a direct substrate of different kinases of the Ras pathway—including ERK, MEK, and bRaf kinases. In parallel, we also tested whether TRF1 was a target of the mTOR kinase, also found here to modulate TRF1 (Fig 1A and B). To this end, we carried out in vitro kinase assays with affinity-purified mouse GST-TRF1 incubated with either mouse-purified ERK2, mouse-purified MEK1, human-purified bRaf, or human-purified mTOR, always in the presence of [γ-32P]ATP (Materials and Methods). Importantly, ERK2 and bRaf but not MEK yielded a clear TRF1 phosphorylation signal (Fig 4A–D). Interestingly, an oncogenic mutant of bRaf (V600E; Davies et al, 2002) triggered a significantly higher TRF1 phosphorylation compared to the wild-type bRaf kinase (Fig 4C), thus suggesting a potentially important role of the bRaf kinase in TRF1 regulation in cancer. Figure 4. ERK2, bRaf, and mTOR kinases phosphorylate TRF1 in vitro A–D. 1 or 2 μM of GST or GST-TRF1 was incubated with the indicated concentrations of mouse ERK2 kinase (A), human BRaf kinase (WT or V600E) (B, C), or mouse MEK1 kinase (D) in the presence of 5 μCi [γ-32P]ATP. The mixture was resolved by SDS–PAGE followed by autoradiography. E. 1 μM of GST-TRF1 and 0.2 μM of mouse ERK2 kinase were incubated in the presence of ERK and MEK inhibitors. F. 2 μM of GST-TRF1 and 0.1 μM of human BRaf kinase were incubated in the presence of the bRaf inhibitors dabrafenib and vemurafenib. G, H. 1 or 2 μM of GST or GST-TRF1 was incubated with the indicated concentrations of human mTOR kinase (G) in the presence of the mTOR inhibitors rapamycin and Ku0063794 (H). I–K. Phosphopeptide peak intensity normalized to total TRF1 signal in s
Referência(s)