The novel TERF2::PDGFRB fusion gene enhances tumorigenesis via PDGFRB/STAT5 signalling pathways and sensitivity to TKI in ph-like ALL.

Patients with Philadelphia chromosome-like acute lymphoblastic leukaemia (Ph-like ALL) often face a grim prognosis, with PDGFRB gene fusions being commonly detected in this subgroup. Our study has unveiled a newfound fusion gene, TERF2::PDGFRB, and we have found that patients carrying this fusion gene exhibit sensitivity to dasatinib. Ba/F3 cells harbouring the TERF2::PDGFRB fusion display IL-3-independent cell proliferation through activation of the p-PDGFRB and p-STAT5 signalling pathways. These cells exhibit reduced apoptosis and demonstrate sensitivity to imatinib in vitro. When transfused into mice, Ba/F3 cells with the TERF2::PDGFRB fusion gene induce tumorigenesis and a shortened lifespan in cell-derived graft models, but this outcome can be improved with imatinib treatment. In summary, we have identified the novel TERF2::PDGFRB fusion gene, which exhibits oncogenic potential both in vitro and in vivo, making it a potential therapeutic target for tyrosine kinase inhibitors (TKIs).

The shelterin component TRF2 mediates columnar stacking of human telomeric chromatin.

Telomere repeat binding factor 2 (TRF2) is an essential component of the telomeres and also plays an important role in a number of other non-telomeric processes. Detailed knowledge of the binding and interaction of TRF2 with telomeric nucleosomes is limited. Here, we study the binding of TRF2 to in vitro-reconstituted kilobasepair-long human telomeric chromatin fibres using electron microscopy, single-molecule force spectroscopy and analytical ultracentrifugation sedimentation velocity. Our electron microscopy results revealed that full-length and N-terminally truncated TRF2 promote the formation of a columnar structure of the fibres with an average width and compaction larger than that induced by the addition of Mg2+, in agreement with the in vivo observations. Single-molecule force spectroscopy showed that TRF2 increases the mechanical and thermodynamic stability of the telomeric fibres when stretched with magnetic tweezers. This was in contrast to the result for fibres reconstituted on the 'Widom 601' high-affinity nucleosome positioning sequence, where minor effects on fibre stability were observed. Overall, TRF2 binding induces and stabilises columnar fibres, which may play an important role in telomere maintenance.

ZNF524 directly interacts with telomeric DNA and supports telomere integrity.

Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing nonhomologous end joining (NHEJ) through its subunit TRF2. Here, we describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity, and reveal base-specific sequence recognition by cocrystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.

Irreversible inhibition of TRF2TRFH recruiting functions by a covalent cyclic peptide induces telomeric replication stress in cancer cells.

The TRF2 shelterin component is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, causing telomeric DNA damage. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2TRFH domain. We identify APOD53 as our most promising compound, as it consistently induces a telomeric DNA damage response in cancer cell lines. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2TRFH domain and induces phenotypes consistent with TRF2TRFH domain mutants. These include induction of a telomeric DNA damage response, increased telomeric replication stress, and impaired recruitment of RTEL1 and SLX4 to telomeres. We demonstrate that APOD53 impairs cancer cell growth and find that co-treatment with APOD53 can exacerbate telomere replication stress caused by the G4 stabilizer RHPS4 and low dose aphidicolin (APH).

Drug repositioning strategy for the identification of novel telomere-damaging agents: A role for NAMPT inhibitors.

Drug repositioning strategy represents a valid tool to accelerate the pharmacological development through the identification of new applications for already existing compounds. In this view, we aimed at discovering molecules able to trigger telomere-localized DNA damage and tumor cell death. By applying an automated high-content spinning-disk microscopy, we performed a screening aimed at identifying, on a library of 527 drugs, molecules able to negatively affect the expression of TRF2, a key protein in telomere maintenance. FK866, resulting from the screening as the best candidate hit, was then validated at biochemical and molecular levels and the mechanism underlying its activity in telomere deprotection was elucidated both in vitro and in vivo. The results of this study allow us to discover a novel role of FK866 in promoting, through the production of reactive oxygen species, telomere loss and deprotection, two events leading to an accumulation of DNA damage and tumor cell death. The ability of FK866 to induce telomere damage and apoptosis was also demonstrated in advanced preclinical models evidencing the antitumoral activity of FK866 in triple-negative breast cancer-a particularly aggressive breast cancer subtype still orphan of targeted therapies and characterized by high expression levels of both NAMPT and TRF2. Overall, our findings pave the way to the development of novel anticancer strategies to counteract triple-negative breast cancer, based on the use of telomere deprotecting agents, including NAMPT inhibitors, that would rapidly progress from bench to bedside.

Endothelial cell telomere dysfunction induces senescence and results in vascular and metabolic impairments.

In advanced age, increases in oxidative stress and inflammation impair endothelial function, which contributes to the development of cardiovascular disease (CVD). One plausible source of this oxidative stress and inflammation is an increase in the abundance of senescent endothelial cells. Cellular senescence is a cell cycle arrest that occurs in response to various damaging stimuli. In the present study, we tested the hypothesis that advanced age results in endothelial cell telomere dysfunction that induces senescence. In both human and mouse endothelial cells, advanced age resulted in an increased abundance of dysfunctional telomeres, characterized by activation of DNA damage signaling at telomeric DNA. To test whether this results in senescence, we selectively reduced the telomere shelterin protein telomere repeat binding factor 2 (Trf2) from endothelial cells of young mice. Trf2 reduction increased endothelial cell telomere dysfunction and resulted in cellular senescence. Furthermore, induction of endothelial cell telomere dysfunction increased inflammatory signaling and oxidative stress, resulting in impairments in endothelial function. Finally, we demonstrate that endothelial cell telomere dysfunction-induced senescence impairs glucose tolerance. This likely occurs through increases in inflammatory signaling in the liver and adipose tissue, as well as reductions in microvascular density and vasodilation to metabolic stimuli. Cumulatively, the findings of the present study identify age-related telomere dysfunction as a mechanism that leads to endothelial cell senescence. Furthermore, these data provide compelling evidence that senescent endothelial cells contribute to age-related increases in oxidative stress and inflammation that impair arterial and metabolic function.

Non-canonical telomere protection role of FOXO3a of human skeletal muscle cells regulated by the TRF2-redox axis.

Telomeric repeat binding factor 2 (TRF2) binds to telomeres and protects chromosome ends against the DNA damage response and senescence. Although the expression of TRF2 is downregulated upon cellular senescence and in various aging tissues, including skeletal muscle tissues, very little is known about the contribution of this decline to aging. We previously showed that TRF2 loss in myofibers does not trigger telomere deprotection but mitochondrial dysfunction leading to an increased level of reactive oxygen species. We show here that this oxidative stress triggers the binding of FOXO3a to telomeres where it protects against ATM activation, revealing a previously unrecognized telomere protective function of FOXO3a, to the best of our knowledge. We further showed in transformed fibroblasts and myotubes that the telomere properties of FOXO3a are dependent on the C-terminal segment of its CR2 domain (CR2C) but independent of its Forkhead DNA binding domain and of its CR3 transactivation domain. We propose that these non-canonical properties of FOXO3a at telomeres play a role downstream of the mitochondrial signaling induced by TRF2 downregulation to regulate skeletal muscle homeostasis and aging.

EPAS1 prevents telomeric damage-induced senescence by enhancing transcription of TRF1, TRF2, and RAD50.

Telomeres are nucleoprotein structures located at the end of each chromosome, which function in terminal protection and genomic stability. Telomeric damage is closely related to replicative senescence in vitro and physical aging in vivo. As relatively long-lived mammals based on body size, bats display unique telomeric patterns, including the up-regulation of genes involved in alternative lengthening of telomeres (ALT), DNA repair, and DNA replication. At present, however, the relevant molecular mechanisms remain unclear. In this study, we performed cross-species comparison and identified EPAS1, a well-defined oxygen response gene, as a key telomeric protector in bat fibroblasts. Bat fibroblasts showed high expression of EPAS1, which enhanced the transcription of shelterin components TRF1 and TRF2, as well as DNA repair factor RAD50, conferring bat fibroblasts with resistance to senescence during long-term consecutive expansion. Based on a human single-cell transcriptome atlas, we found that EPAS1 was predominantly expressed in the human pulmonary endothelial cell subpopulation. Using in vitro-cultured human pulmonary endothelial cells, we confirmed the functional and mechanistic conservation of EPAS1 in telomeric protection between bats and humans. In addition, the EPAS1 agonist M1001 was shown to be a protective compound against bleomycin-induced pulmonary telomeric damage and senescence. In conclusion, we identified a potential mechanism for regulating telomere stability in human pulmonary diseases associated with aging, drawing insights from the longevity of bats.

Homology directed telomere clustering, ultrabright telomere formation and nuclear envelope rupture in cells lacking TRF2B and RAP1.

Double-strand breaks (DSBs) due to genotoxic stress represent potential threats to genome stability. Dysfunctional telomeres are recognized as DSBs and are repaired by distinct DNA repair mechanisms. RAP1 and TRF2 are telomere binding proteins essential to protect telomeres from engaging in homology directed repair (HDR), but how this occurs remains unclear. In this study, we examined how the basic domain of TRF2 (TRF2B) and RAP1 cooperate to repress HDR at telomeres. Telomeres lacking TRF2B and RAP1 cluster into structures termed ultrabright telomeres (UTs). HDR factors localize to UTs, and UT formation is abolished by RNaseH1, DDX21 and ADAR1p110, suggesting that they contain DNA-RNA hybrids. Interaction between the BRCT domain of RAP1 and KU70/KU80 is also required to repress UT formation. Expressing TRF2∆B in Rap1-/- cells resulted in aberrant lamin A localization in the nuclear envelope and dramatically increased UT formation. Expressing lamin A phosphomimetic mutants induced nuclear envelope rupturing and aberrant HDR-mediated UT formation. Our results highlight the importance of shelterin and proteins in the nuclear envelope in repressing aberrant telomere-telomere recombination to maintain telomere homeostasis.

In Vitro Characterization of the Physical Interactions between the Long Noncoding RNA TERRA and the Telomeric Proteins TRF1 and TRF2.

RNA-protein interactions drive key cellular pathways such as protein translation, nuclear organization and genome stability maintenance. The human telomeric protein TRF2 binds to the long noncoding RNA TERRA through independent domains, including its N-terminal B domain. We previously demonstrated that TRF2 B domain binding to TERRA supports invasion of TERRA into telomeric double stranded DNA, leading to the formation of telomeric RNA:DNA hybrids. The other telomeric protein TRF1, which also binds to TERRA, suppresses this TRF2-associated activity by preventing TERRA-B domain interactions. Herein, we show that the binding of both TRF1 and TRF2 to TERRA depends on the ability of the latter to form G-quadruplex structures. Moreover, a cluster of arginines within the B domain is largely responsible for its binding to TERRA. On the other side, a patch of glutamates within the N-terminal A domain of TRF1 mainly accounts for the inhibition of TERRA-B domain complex formation. Finally, mouse TRF2 B domain binds to TERRA, similarly to its human counterpart, while mouse TRF1 A domain lacks the inhibitory activity. Our data shed further light on the complex crosstalk between telomeric proteins and RNAs and suggest a lack of functional conservation in mouse.

TRF2 inhibition rather than telomerase disruption drives CD4T cell dysfunction during chronic viral infection.

We investigated the role of telomerase and telomere repeat-binding factor 2 (TRF2 or TERF2) in T-cell dysfunction in chronic viral infection. We found that the expression and activity of telomerase in CD4+ T (CD4T) cells from patients with hepatitis C virus (HCV) infections or people living with HIV (PLWH) were intact, but TRF2 expression was significantly inhibited at the post-transcriptional level, suggesting that TRF2 inhibition is responsible for the CD4T cell dysfunction observed during chronic viral infection. Silencing TRF2 expression in CD4T cells derived from healthy subjects induced telomeric DNA damage and CD4T cell dysfunction without affecting telomerase activity or translocation - similar to what we observed in CD4T cells from HCV patients and PLWH. These findings indicate that premature T-cell aging and dysfunction during chronic HCV or HIV infection are primarily caused by chronic immune stimulation and T-cell overactivation and/or proliferation that induce telomeric DNA damage due to TRF2 inhibition, rather than telomerase disruption. This study suggests that restoring TRF2 presents a novel approach to prevent telomeric DNA damage and premature T-cell aging, thus rejuvenating T-cell functions during chronic viral infection.

The non-telomeric evolutionary trajectory of TRF2 in zebrafish reveals its specific roles in neurodevelopment and aging.

The shelterin protein complex is required for telomere protection in various eukaryotic organisms. In mammals, the shelterin subunit TRF2 is specialized in preventing ATM activation at telomeres and chromosome end fusion in somatic cells. Here, we demonstrate that the zebrafish ortholog of TRF2 (encoded by the terfa gene) is protecting against unwanted ATM activation genome-wide. The terfa-compromised fish develop a prominent and specific embryonic neurodevelopmental failure. The heterozygous fish survive to adulthood but exhibit a premature aging phenotype. The recovery from embryonic neurodevelopmental failure requires both ATM inhibition and transcriptional complementation of neural genes. Furthermore, restoring the expression of TRF2 in glial cells rescues the embryonic neurodevelopment phenotype. These results indicate that the shelterin subunit TRF2 evolved in zebrafish as a general factor of genome maintenance and transcriptional regulation that is required for proper neurodevelopment and normal aging. These findings uncover how TRF2 links development to aging by separate functions in gene expression regulation and genome stability control.

Compartmentalization of telomeres through DNA-scaffolded phase separation.

Telomeres form unique nuclear compartments that prevent degradation and fusion of chromosome ends by recruiting shelterin proteins and regulating access of DNA damage repair factors. To understand how these dynamic components protect chromosome ends, we combine in vivo biophysical interrogation and in vitro reconstitution of human shelterin. We show that shelterin components form multicomponent liquid condensates with selective biomolecular partitioning on telomeric DNA. Tethering and anomalous diffusion prevent multiple telomeres from coalescing into a single condensate in mammalian cells. However, telomeres coalesce when brought into contact via an optogenetic approach. TRF1 and TRF2 subunits of shelterin drive phase separation, and their N-terminal domains specify interactions with telomeric DNA in vitro. Telomeric condensates selectively recruit telomere-associated factors and regulate access of DNA damage repair factors. We propose that shelterin mediates phase separation of telomeric chromatin, which underlies the dynamic yet persistent nature of the end-protection mechanism.

TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA.

The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2-TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein-DNA structures, and monitoring of DNA-DNA and DNA-RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA-dsDNA, dsDNA-ssDNA and dsDNA-ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

TRF2 promotes dynamic and stepwise looping of POT1 bound telomeric overhang.

Human telomeres are protected by shelterin proteins, but how telomeres maintain a dynamic structure remains elusive. Here, we report an unexpected activity of POT1 in imparting conformational dynamics of the telomere overhang, even at a monomer level. Strikingly, such POT1-induced overhang dynamics is greatly enhanced when TRF2 engages with the telomere duplex. Interestingly, TRF2, but not TRF2ΔB, recruits POT1-bound overhangs to the telomere ds/ss junction and induces a discrete stepwise movement up and down the axis of telomere duplex. The same steps are observed regardless of the length of the POT1-bound overhang, suggesting a tightly regulated conformational dynamic coordinated by TRF2 and POT1. TPP1 and TIN2 which physically connect POT1 and TRF2 act to generate a smooth movement along the axis of the telomere duplex. Our results suggest a plausible mechanism wherein telomeres maintain a dynamic structure orchestrated by shelterin.

TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress.

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2-ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2-ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244-511), which competitively inhibited the TRF2-ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.

Estrogen Replacement Therapy Induces Antioxidant and Longevity-Related Genes in Women after Medically Induced Menopause.

Females live longer than males in many species, including humans, and estrogens are in part responsible for this protection against aging. We reported previously that estrogens can protect rats against oxidative stress, by inducing antioxidant and longevity-related genes. Thus, this study was aimed at confirming the ability of estrogens to upregulate antioxidant and longevity-related genes in humans. For this purpose, we selected 16 women of reproductive age (18-42 years old) undergoing a fertility treatment that includes a medically induced menopause, at the Valencian Infertility Institute. We took blood samples at each time point of the treatment (basal, induced menopause, estrogen, and estrogen plus progesterone replacement therapy). mRNA expression of antioxidant and longevity-related genes in peripheral blood mononuclear cells (PBMC) was determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Determination of reduced glutathione (GSH) in total blood was carried out using high-performance liquid chromatography (HPLC). As expected, we found that medically induced menopause significantly decreased sexual hormone (estrogens and progesterone) levels. It also lowered glutathione peroxidase (GPx), 16S rRNA, P21, and TERF2 mRNA expression and blood GSH levels. Estrogen replacement therapy significantly restored estrogen levels and induced mRNA expression of manganese superoxide dismutase (MnSOD), GPx, 16S rRNA, P53, P21, and TERF2 and restored blood GSH levels. Progesterone replacement therapy induced a significant increase in MnSOD, P53, sestrin 2 (SENS2), and TERF2 mRNA expression when compared to basal conditions. These findings provide evidence for estrogen beneficial effects in upregulating antioxidant and longevity-related genes in women.

The structurally conserved TELR region on shelterin protein TPP1 is essential for telomerase processivity but not recruitment.

The shelterin protein TPP1 is involved in both recruiting telomerase and stimulating telomerase processivity in human cells. Assessing the in vivo significance of the latter role of TPP1 has been difficult, because TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. The Saccharomyces cerevisiae telomerase-associated Est3 protein adopts a protein fold similar to the N-terminal region of TPP1. Interestingly, a previous structure-guided mutagenesis study of Est3 revealed a TELR surface region that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase [T. Rao et al., Proc. Natl. Acad. Sci. U.S.A. 111, 214-218 (2014)]. Here, we show that mutations within the structurally conserved TELR region on human TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1 in a process separable from its role in recruiting telomerase, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term viability of human cells.

Telomere shortening and expression of TRF1 and TRF2 in uterine leiomyoma.

Uterine leiomyoma is a benign smooth muscle tumor of the uterus that can exhibit histopathological traits that mimic malignancy. Telomere shortening is an early event in tumorigenesis and telomerase activation facilitates tumor progression later in the course of carcinogenesis. Telomeric repeat­binding factor (TRF)1 and TRF2 protect telomeres, and their gene expression levels are dysregulated in various cancer types. However, the roles of telomeres and telomere protection proteins in uterine leiomyoma remain largely unknown. In this study, telomere length and the mRNA levels of various telomere­related genes in normal tissues and leiomyoma were determined, and their relationships were evaluated. Uterine leiomyoma and normal myometrium were surgically obtained from 18 and 13 patients, respectively. Telomere length and gene expression were determined by Southern blot analysis and reverse transcription­quantitative PCR, respectively. In matched samples, telomeres were consistently shorter in leiomyoma tissue than in adjacent normal tissue. TRF1, TRF2, PIN2­interacting telomerase inhibitor 1 (PINX1), and telomerase RNA component were expressed at comparable levels in both leiomyoma and normal tissues. None of these genes were associated with telomere length in leiomyoma. All tested tissues were negative for telomerase reverse transcriptase, which encodes the catalytic component of telomerase, indicating that cells in uterine leiomyoma were not immortalized. In summary, telomere erosion, which reflects active proliferation during tumor evolution, was evident in uterine leiomyoma. Steady­state expression of TRF1, TRF2 and PINX1 may be important for maintenance of telomere integrity in leiomyoma, where telomere length is shortened.

Immune Activation Induces Telomeric DNA Damage and Promotes Short-Lived Effector T Cell Differentiation in Chronic HCV Infection.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) leads to a high rate of chronic infection and T cell dysfunction. Although it is well known that chronic antigenic stimulation is a driving force for impaired T cell functions, the precise mechanisms underlying immune activation-induced T cell dysfunctions during HCV infection remain elusive. APPROACH AND RESULTS: Here, we demonstrated that circulating CD4+ T cells from patients who are chronically HCV-infected exhibit an immune activation status, as evidenced by the overexpression of cell activation markers human leukocyte antigen-antigen D-related, glucose transporter 1, granzyme B, and the short-lived effector marker CD127- killer cell lectin-like receptor G1+ . In contrast, the expression of stem cell-like transcription factor T cell factor 1 and telomeric repeat-binding factor 2 (TRF2) are significantly reduced in CD4+ T cells from patients who are chronically HCV-infected compared with healthy participants (HP). Mechanistic studies revealed that CD4+ T cells from participants with HCV exhibit phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling hyperactivation on T cell receptor stimulation, promoting proinflammatory effector cell differentiation, telomeric DNA damage, and cellular apoptosis. Inhibition of Akt signaling during T cell activation preserved the precursor memory cell population and prevented inflammatory effector cell expansion, DNA damage, and apoptotic death. Moreover, knockdown of TRF2 reduced HP T cell stemness and triggered telomeric DNA damage and cellular apoptosis, whereas overexpression of TRF2 in CD4 T cells prevented telomeric DNA damage. CONCLUSIONS: These results suggest that modulation of immune activation through inhibiting Akt signaling and protecting telomeres through enhancing TRF2 expression may open therapeutic strategies to fine tune the adaptive immune responses in the setting of persistent immune activation and inflammation during chronic HCV infection.