Inhibiting TRF1 upstream signaling pathways to target telomeres in cancer cells.

In the context of tumorigenesis, telomere shortening is associated with apparent antagonistic outcomes: On one side, it favors cancer initiation through mechanisms involving genome instability, while on the other side, it prevents cancer progression, due to the activation of the DNA damage response (DDR) checkpoint behaving as a cell-intrinsic proliferation barrier. Consequently, telomerase, which can compensate for replicative erosion by adding telomeric DNA repeats at the chromosomal DNA extremities, is crucial for cancer progression and is upregulated in nearly 90% of human cancers. Therefore, telomeres are considered potential anti-cancer targets and, to date, most of the studies have focused on telomerase inhibition. However, the development of clinically efficient telomerase targeting therapies is still in its infancy. In this context, the findings reported in this issue of EMBO Molecular Medicine by Bejarano et al (2019) open new avenues for alternative telomere therapies.

Expression of Telomere-Associated Proteins is Interdependent to Stabilize Native Telomere Structure and Telomere Dysfunction by G-Quadruplex Ligand Causes TERRA Upregulation.

Telomere DNA can form specialized nucleoprotein structure with telomere-associated proteins to hide free DNA ends or G-quadruplex structures under certain conditions especially in presence of G-quadruplex ligand. Telomere DNA is transcribed to form non-coding telomere repeat-containing RNA (TERRA) whose biogenesis and function is poorly understood. Our aim was to find the role of telomere-associated proteins and telomere structures in TERRA transcription. We silenced four [two shelterin (TRF1, TRF2) and two non-shelterin (PARP-1, SLX4)] telomere-associated genes using siRNA and verified depletion in protein level. Knocking down of one gene modulated expression of other telomere-associated genes and increased TERRA from 10q, 15q, XpYp and XqYq chromosomes in A549 cells. Telomere was destabilized or damaged by G-quadruplex ligand pyridostatin (PDS) and bleomycin. Telomere dysfunction-induced foci (TIFs) were observed for each case of depletion of proteins, treatment with PDS or bleomycin. TERRA level was elevated by PDS and bleomycin treatment alone or in combination with depletion of telomere-associated proteins.

Inhibition of TRF1 Telomere Protein Impairs Tumor Initiation and Progression in Glioblastoma Mouse Models and Patient-Derived Xenografts.

Glioblastoma multiforme (GBM) is a deadly and common brain tumor. Poor prognosis is linked to high proliferation and cell heterogeneity, including glioma stem cells (GSCs). Telomere genes are frequently mutated. The telomere binding protein TRF1 is essential for telomere protection, and for adult and pluripotent stem cells. Here, we find TRF1 upregulation in mouse and human GBM. Brain-specific Trf1 genetic deletion in GBM mouse models inhibited GBM initiation and progression, increasing survival. Trf1 deletion increased telomeric DNA damage and reduced proliferation and stemness. TRF1 chemical inhibitors mimicked these effects in human GBM cells and also blocked tumor sphere formation and tumor growth in xenografts from patient-derived primary GSCs. Thus, targeting telomeres throughout TRF1 inhibition is an effective therapeutic strategy for GBM.

Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage.

Telomeres are considered anti-cancer targets, as telomere maintenance above a minimum length is necessary for cancer growth. Telomerase abrogation in cancer-prone mouse models, however, only decreased tumor growth after several mouse generations when telomeres reach a critically short length, and this effect was lost upon p53 mutation. Here, we address whether induction of telomere uncapping by inhibition of the TRF1 shelterin protein can effectively block cancer growth independently of telomere length. We show that genetic Trf1 ablation impairs the growth of p53-null K-Ras(G12V)-induced lung carcinomas and increases mouse survival independently of telomere length. This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest. Long-term whole-body Trf1 deletion in adult mice did not impact on mouse survival and viability, although some mice showed a moderately decreased cellularity in bone marrow and blood. Importantly, inhibition of TRF1 binding to telomeres by small molecules blocks the growth of already established lung carcinomas without affecting mouse survival or tissue function. Thus, induction of acute telomere uncapping emerges as a potential new therapeutic target for lung cancer.

Over-expression of telomere binding factors (TRF1 & TRF2) in renal cell carcinoma and their inhibition by using SiRNA induce apoptosis, reduce cell proliferation and migration invitro.

Telomere binding factors viz. TRF1 and TRF2 are a part of sheltrin complex that are present exclusively at the ends of chromosomes. These factors play an important role in maintaining chromosomal integrity at the ends. However, their status and role are not clear in renal cell carcinoma (RCC). Therefore, the present study was conducted to evaluate TRF1 and TRF2 expressions in RCC tissues. Further, the role of these factors involved in tumorigenesis was elucidated by gene silencing using siRNA in RCC cell line (A498). The present study documented a significant over-expression of TRF1 (P = 0.005) and TRF2 (P = 0.0048) mRNAs by real time PCR in RCC tissues as compared with adjacent normal kidney tissues. Immunohistochemistry studies also revealed higher expression of TRF1 and TRF2 proteins in RCC. Moreover, TRF1 or TRF2 gene silencing using siRNA showed marked reduction in proliferation of RCC cells (P = 0.000). Further, significantly induced cell cycle arrest (P = 0.000) and apoptosis of RCC cells (P = 0.000) was documented upon TRF1 or TRF2 gene silencing. Henceforth, the results deduce that TRF1 or TRF2 inhibitions play an important role in the induction of apoptosis in A498 cells, which may serve as a potential therapeutic target in RCC.

Mitotic perturbations induced by Nek2 overexpression require interaction with TRF1 in breast cancer cells.

NIMA-related kinase 2 (Nek2), a serine-threonine protein kinase, plays a major role in mitotic progression, including timing of mitotic entry, chromatin condensation, spindle organization, and cytokinesis. Nek2 overexpression results in premature centrosome separation, while kinase death Nek2 mutant expression or Nek2-depleted cells lead to centrosome separation failure. In addition, it has been revealed that telomeric repeat binding factor 1 (TRF1) interacts directly with Nek2. TRF1 not only regulates telomere length, but is also associated with cell cycle regulation. However, the interactions and correlations between Nek2 and TRF1 are far from clear. Here, we show that mitotic aberrations through Nek2 overexpression are likely to require TRF1. Our results demonstrate that Nek2 directly binds and phosphorylates TRF1 through multiple sites on TRF1. Nek2 overexpression in breast cancer cells, MDA-MB-231 and MCF7, results in increased numbers of centrosomes and multinucleated cells, which leads to cytokinetic failure and aneuploidization. Additionally, TRF1 depletion by siRNA prevents the phenomenon of unaligned chromosomes by Nek2 overexpression during metaphase. Concurrent Nek2 overexpression and TRF1-depleted cells demonstrated ≤ 2 centrosomes per cell, similar to mock plasmid and negative control siRNA-transfected cells. Interestingly, when exogenous TRF1 was added back in Nek2-overexpressed cells with endogenous TRF1 depletion, cells had re-induced cytokinetic failure. Therefore, we propose that TRF1 is required for overexpressed Nek2 to trigger abnormal mitosis and chromosomal instability.

Computationally designed peptide inhibitors of the ubiquitin E3 ligase SCF(Fbx4).

A structure-based computational approach was used to rationally design peptide inhibitors that can target an E3 ligase (SCF(Fbx4) )-substrate (TRF1) interface and subsequent ubiquitylation. Characterization of the inhibitors demonstrates that our sequence-optimization protocol results in an increase in peptide-TRF1 affinity without compromising peptide-protein specificity.

The BLM helicase contributes to telomere maintenance through processing of late-replicating intermediate structures.

Werner's syndrome (WS) and Bloom's syndrome (BS) are cancer predisposition disorders caused by loss of function of the RecQ helicases WRN or BLM, respectively. BS and WS are characterized by replication defects, hyperrecombination events and chromosomal aberrations, which are hallmarks of cancer. Inefficient replication of the G-rich telomeric strand contributes to chromosome aberrations in WS cells, demonstrating a link between WRN, telomeres and genomic stability. Herein, we provide evidence that BLM also contributes to chromosome-end maintenance. Telomere defects (TDs) are observed in BLM-deficient cells at an elevated frequency, which is similar to cells lacking a functional WRN helicase. Loss of both helicases exacerbates TDs and chromosome aberrations, indicating that BLM and WRN function independently in telomere maintenance. BLM localization, particularly its recruitment to telomeres, changes in response to replication dysfunction, such as in WRN-deficient cells or after aphidicolin treatment. Exposure to replication challenge causes an increase in decatenated deoxyribonucleic acid (DNA) structures and late-replicating intermediates (LRIs), which are visible as BLM-covered ultra-fine bridges (UFBs) in anaphase. A subset of UFBs originates from telomeric DNA and their frequency correlates with telomere replication defects. We propose that the BLM complex contributes to telomere maintenance through its activity in resolving LRIs.

Dicer independent small RNAs associate with telomeric heterochromatin.

Small RNAs play important roles in the establishment and maintenance of heterochromatin structures. We show the presence of telomere specific small RNAs (tel-sRNAs) in mouse embryonic stem cells that are approximately 24 nucleotides in length, Dicer-independent, and 2'-O-methylated at the 3' terminus. The tel-sRNAs are asymmetric with specificity toward telomere G-rich strand, and evolutionarily conserved from protozoan to mammalian cells. Furthermore, tel-sRNAs are up-regulated in cells that carry null mutation of H3K4 methyltransferase MLL (Mll((-/-))) and down-regulated in cells that carry null mutations of histone H3K9 methyltransferase SUV39H (Suv39h1/h2((-/-))), suggesting that they are subject to epigenetic regulation. These results support that tel-sRNAs are heterochromatin associated pi-like small RNAs.

Identification of candidate alternative lengthening of telomeres genes by methionine restriction and RNA interference.

Telomerase-negative cancer cells can maintain their telomeres by a recombination-mediated alternative lengthening of telomeres (ALT) process. We reported previously that sequestration of MRE11/RAD50/NBS1 complexes represses ALT-mediated telomere length maintenance, and suppresses formation of ALT-associated promyelocytic leukemia (PML) bodies (APBs). APBs are PML bodies containing telomeric DNA and telomere-binding proteins, and are observed only in a small fraction of cells within asynchronously dividing ALT-positive cell populations. Here, we report that methionine restriction caused a reversible arrest in G0/G1 phase of the cell cycle and reversible induction of APB formation in most cells within an ALT-positive population. We combined methionine restriction with RNA interference to test whether the following proteins are required for APB formation: PML body-associated proteins, PML and Sp100; telomere-associated proteins, TRF1, TRF2, TIN2 and RAP1; and DNA repair proteins, MRE11, RAD50, NBS1 and 53BP1. APB formation was not decreased by depletion of Sp100 (as reported previously) or of 53BP1, although 53BP1 partially colocalizes with APBs. Depletion of the other proteins suppressed APB formation. Because of the close linkage between ALT-mediated telomere maintenance and ability to form APBs, the eight proteins identified by this screen as being required for APB formation are also likely to be required for the ALT mechanism.