Thus, we decided to study the effect of each of the single mutants TRF1\T328, TRF1\T330, and TRF1\T335 separately by transducing the mutant alleles into deletion

Thus, we decided to study the effect of each of the single mutants TRF1\T328, TRF1\T330, and TRF1\T335 separately by transducing the mutant alleles into deletion. 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. Pseudohypericin 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 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 and that these modifications are essential for TRF1 location to telomeres 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 option lengthening of telomeres (ALT), which is based in homologous recombination between chromosome ends (Bryan tumor suppressor gene, which is frequently mutated in cancer (Gonzalez\Suarez 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 usually impartial of telomere length (Garcia\Beccaria (Mendez\Pertuz (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 deletion has been previously shown to induce a persistent DDR at telomeres in different cell lines, which leads to decreased cell viability (Martinez 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 genetic deletion significantly reduced stemness in both neural stem cells (NSCs) and glioma stem cells (GSCs; Bejarano (Mendez\Pertuz kinase assays with affinity\purified mouse GST\TRF1 incubated with either mouse\purified ERK2, mouse\purified MEK1, human\purified bRaf, or human\purified mTOR, usually in the presence of [\32P]ATP (Materials and Methods). Importantly, ERK2 and bRaf but not MEK yielded a clear COL5A2 TRF1 phosphorylation signal (Fig?4ACD). Interestingly, an oncogenic mutant of bRaf (V600E; Davies phosphorylation assays with the indicated GST\TRF1 wild\type or Pseudohypericin mutated forms in the presence of mouse ERK2 kinase. Data are representative of ****validation of the ERK phosphorylation sites, we generated the GST\tagged alleles T44, T195, T298, and T358 as singles mutants and T4/S6/S7, T268/T270/T274, and T328/T330/T335 as triple mutants. In all the cases, threonine or serine was mutated to alanine. The affinity\purified GST\TRF1 WT or mutant alleles were incubated with mouse\purified ERK2 Pseudohypericin usually in the presence of [\32P]ATP. We found significantly decreased TRF1 phosphorylation levels in the variants harboring T4/S6/S7, T44, T268/T270/T274, and T328/T330/T335 substitutions compared to wild\type TRF1 (Fig?4M). We extended this analysis with additional TRF1 single mutants in ERK\phosphorylation sites, such as T328A, T330A, and T335A (Fig?4N), all of which resulted in decreased ERK\dependent TRF1 phosphorylation. Furthermore, we demonstrate that, among the AKT\dependent phosphosites of TRF1, S344 (T358 in human) is as also a target for ERK\mediated phosphorylation (Fig?4O). As unfavorable control, we also generated a TRF1 phosphomutant in residue T248 whose phosphorylation is usually mediated by AKT but not ERK (Fig?4O; Mendez\Pertuz role of TRF1 modifications by ERK2, eGFP\tagged wild\type and mutant alleles were transduced into p53\deficient deletion. Overexpression of eGFP\alleles and endogenous deletion were confirmed by Western blot analysis using a specific TRF1 antibody (Fig?5B). Quantification of nuclear eGFP spot fluorescence in whether the different TRF1 Pseudohypericin mutants were able to rescue the proliferation defects of wild\type or mutant alleles. All the single mutants were able to completely or almost completely rescue the proliferation defects associated with deficiency (Fig?5D). We next assessed the triple mutants (T4/S6/S7, T268/T270/T274, and T328/T330/T335), Pseudohypericin and, in agreement with eGFP\TRF1 telomeric foci findings (Fig?5C), we observed that this triple mutant TRF1\T328/T330/T335 showed the more severe proliferation defects (Fig?5D). Thus, we decided to study the effect of each of the single mutants TRF1\T328, TRF1\T330, and TRF1\T335 separately by transducing the mutant alleles into deletion. Overexpression.