Normal Telomere Research Articles

Tethering telomerase to telomeres increases genome instability and promotes chronological aging in yeast.

Chronological aging of the yeast Saccharomyces cerevisiae is attributed to multi-faceted traits especially those involving genome instability, and has been considered to be an aging model for post-mitotic cells in higher organisms. Telomeres are the physical ends of eukaryotic chromosomes, and are essential for genome integrity and stability. It remains elusive whether dysregulated telomerase activity affects chronological aging. We employed the CDC13-EST2 fusion gene, which tethers telomerase to telomeres, to examine the effect of constitutively active telomerase on chronological lifespan (CLS). The expression of Cdc13-Est2 fusion protein resulted in overlong telomeres (2 to 4 folds longer than normal telomeres), and long telomeres were stably maintained during long-term chronological aging. Accordingly, genome instability, manifested by accumulation of extra-chromosomal rDNA circle species, age-dependent CAN1 marker-gene mutation frequency and gross chromosomal rearrangement frequency, was significantly elevated. Importantly, inactivation of Sch9, a downstream kinase of the target of rapamycin complex 1 (TORC1), suppressed both the genome instability and accelerated chronological aging mediated by CDC13-EST2 expression. Interestingly, loss of the CDC13-EST2 fusion gene in the cells with overlong telomeres restored the regular CLS. Altogether, these data suggest that constitutively active telomerase is detrimental to the maintenance of genome stability, and promotes chronological aging in yeast.

Generation of mice with longer and better preserved telomeres in the absence of genetic manipulations.

Although telomere length is genetically determined, mouse embryonic stem (ES) cells with telomeres of twice the normal size have been generated. Here, we use such ES cells with ‘hyper-long' telomeres, which also express green fluorescent protein (GFP), to generate chimaeric mice containing cells with both hyper-long and normal telomeres. We show that chimaeric mice contain GFP-positive cells in all mouse tissues, display normal tissue histology and normal survival. Both hyper-long and normal telomeres shorten with age, but GFP-positive cells retain longer telomeres as mice age. Chimaeric mice with hyper-long telomeres also accumulate fewer cells with short telomeres and less DNA damage with age, and express lower levels of p53. In highly renewing compartments, such as the blood, cells with hyper-long telomeres are longitudinally maintained or enriched with age. We further show that wound-healing rates in the skin are increased in chimaeric mice. Our work demonstrates that mice with functional, longer and better preserved telomeres can be generated without the need for genetic manipulations, such as TERT overexpression.

Relative telomere lengths in tumor and normal mucosa are related to disease progression and chromosome instability profiles in colorectal cancer.

Telomeric dysfunction is linked to colorectal cancer (CRC) initiation. However, the relationship of normal tissue and tumor telomere lengths with CRC progression, molecular features and prognosis is unclear. Here, we measured relative telomere length (RTL) by real-time quantitative PCR in 90 adenomas (aRTL), 419 stage I-IV CRCs (cRTL) and adjacent normal mucosa (nRTL). Age-adjusted RTL was analyzed against germline variants in telomere biology genes, chromosome instability (CIN), microsatellite instability (MSI), CpG island methylator phenotype (CIMP), TP53, KRAS, BRAF mutations and clinical outcomes. In 509 adenoma or CRC patients, nRTL decreased with advancing age. Female gender, proximal location and the TERT rs2736100 G allele were independently associated with longer age-adjusted nRTL. Adenomas and carcinomas exhibited telomere shortening in 79% and 67% and lengthening in 7% and 15% of cases. Age-adjusted nRTL and cRTL were independently associated with tumor stage, decreasing from adenoma to stage III and leveling out or increasing from stage III to IV, respectively. Cancer MSI, CIMP, TP53, KRAS and BRAF status were not related to nRTL or cRTL. Near-tetraploid CRCs exhibited significantly longer cRTLs than CIN- and aneuploidy CRCs, while cRTL was significantly shorter in CRCs with larger numbers of chromosome breaks. Age-adjusted nRTL, cRTL or cRTL:nRTL ratios were not associated with disease-free or overall survival in stage II/III CRC. Taken together, our data show that both normal mucosa and tumor RTL are independently associated with CRC progression, and highlight divergent associations of CRC telomere length with tumor CIN profiles.

Multiple myeloma causes clonal T-cell immunosenescence: identification of potential novel targets for promoting tumour immunity and implications for checkpoint blockade.

Tumour-induced dysfunction of cytotoxic T cells in patients with multiple myeloma (MM) may contribute to immune escape and be responsible for the lack of therapeutic efficacy of immune checkpoint blockade. We therefore investigated dysfunctional clonal T cells in MM and demonstrated immunosenescence but not exhaustion as a predominant feature. T-cell clones were detected in 75% of MM patients and their prognostic significance was revalidated in a new post-immunomodulatory drug cohort. The cells exhibited a senescent secretory effector phenotype: KLRG-1+/CD57+/CD160+/CD28-. Normal-for-age telomere lengths indicate that senescence is telomere independent and potentially reversible. p38-mitogen-activated protein kinase, p16 and p21 signalling pathways known to induce senescence were not elevated. Telomerase activity was found to be elevated and this may explain how normal telomere lengths are maintained in senescent cells. T-cell receptor signalling checkpoints were normal but elevated SMAD levels associated with T-cell inactivation were detected and may provide a potential target for the reversal of clonal T-cell dysfunction in MM. Low programmed death 1 and cytotoxic T-lymphocyte-associated antigen 4 expression detected on T-cell clones infers that these cells are not exhausted but suggests that there would be a suboptimal response to immune checkpoint blockade in MM. Our data suggest that other immunostimulatory strategies are required in MM.

Mixed Integer Linear Programming based machine learning approach identifies regulators of telomerase in yeast.

Understanding telomere length maintenance mechanisms is central in cancer biology as their dysregulation is one of the hallmarks for immortalization of cancer cells. Important for this well-balanced control is the transcriptional regulation of the telomerase genes. We integrated Mixed Integer Linear Programming models into a comparative machine learning based approach to identify regulatory interactions that best explain the discrepancy of telomerase transcript levels in yeast mutants with deleted regulators showing aberrant telomere length, when compared to mutants with normal telomere length. We uncover novel regulators of telomerase expression, several of which affect histone levels or modifications. In particular, our results point to the transcription factors Sum1, Hst1 and Srb2 as being important for the regulation of EST1 transcription, and we validated the effect of Sum1 experimentally. We compiled our machine learning method leading to a user friendly package for R which can straightforwardly be applied to similar problems integrating gene regulator binding information and expression profiles of samples of e.g. different phenotypes, diseases or treatments.

DNA methylation in PRDM8 is indicative for dyskeratosis congenita.

Dyskeratosis congenita (DKC) is associated with impaired telomere maintenance and with clinical features of premature aging. In this study, we analysed global DNA methylation (DNAm) profiles of DKC patients. Age-associated DNAm changes were not generally accelerated in DKC, but there were significant differences to DNAm patterns of healthy controls, particularly in CpG sites related to an internal promoter region of PR domain containing 8 (PRDM8). Notably, the same genomic region was also hypermethylated in aplastic anemia (AA) – another bone marrow failure syndrome. Site-specific analysis of DNAm level in PRDM8 with pyrosequencing and MassARRAY validated aberrant hypermethylation in 11 DKC patients and 27 AA patients. Telomere length, measured by flow-FISH, did not directly correlate with DNAm in PRDM8. Therefore the two methods may be complementary to also identify patients with still normal telomere length. In conclusion, blood of DKC patients reveals aberrant DNAm patterns, albeit age-associated DNAm patterns are not generally accelerated. Aberrant hypermethylation is particularly observed in PRDM8 and this may support identification and classification of bone marrow failure syndromes.

TINF2 Gene Mutation in a Patient with Pulmonary Fibrosis.

Pulmonary fibrosis is a frequent manifestation of telomere syndromes. Telomere gene mutations are found in up to 25% and 3% of patients with familial disease and sporadic disease, respectively. The telomere gene TINF2 encodes an eponymous protein that is part of the shelterin complex, a complex involved in telomere protection and maintenance. A TINF2 gene mutation was recently reported in a family with pulmonary fibrosis. We identified a heterozygous Ser245Tyr mutation in the TINF2 gene of previously healthy female patient that presented with progressive cough due to pulmonary fibrosis as well as panhypogammaglobulinemia at age 52. Retrospective multidisciplinary evaluation classified her as a case of possible idiopathic pulmonary fibrosis. Telomere length-measurement indicated normal telomere length in the peripheral blood compartment. This is the first report of a TINF2 mutation in a patient with sporadic pulmonary fibrosis, which represents another association between TINF2 mutations and this disease. Furthermore, this case underlines the importance of telomere dysfunction and not telomere length alone in telomere syndromes and draws attention to hypogammaglobulinemia as a manifestation of telomere syndromes.

Inositol pyrophosphates modulate cell cycle independently of alteration in telomere length.

Synthesis of inositol pyrophosphates through activation of Kcs1 plays an important role in the signalling response required for cell cycle progression after mating pheromone arrest. Overexpression of Kcs1 doubled the level of inositol pyrophosphates when compared to wild type cells and 30min following the release from α-factor block further increase in inositol pyrophosphates was observed, which resulted that cells overexpressing Kcs1 reached G2/M phase earlier than wild type cells. Similar effect was observed in ipk1Δ cells, which are unable to synthesize IP6-derived inositol pyrophosphates (IP7 and IP8) but will synthesize IP5-derived inositol pyrophosphates (PP-IP4 and (PP)2-IP3). Although ipk1Δ cells have shorter telomeres than wild type cells, overexpression of Kcs1 in both strains have similar effect on cell cycle progression. As it is known that PP-IP4 regulates telomere length through Tel1, inositol polyphosphates, cell cycle and telomere length were determined in tel1Δ cells. The release of the cells from α-factor block and overexpression of Kcs1 in tel1Δ cells produced similar effects on inositol pyrophosphates level and cell cycle progression when compared to wild type cells, although tel1Δ cells possesses shorter telomeres than wild type cells. It can be concluded that telomere length does not affect cell cycle progression, since cells with short telomeres (ipk1Δ and tel1Δ) progress through cell cycle in a similar manner as wild type cells and that overexpression of Kcs1 in cells with either short or normal telomeres will increase S phase progression without affecting telomere length. Furthermore, IP5-derived inositol pyrophosphates can compensate for the loss of IP6-derived inositol pyrophosphates, in modulating S phase progression of the cell cycle.

Telomere biology: Rationale for diagnostics and therapeutics in cancer

The key step of carcinogenesis is the malignant transformation which is fundamentally a telomere biology dysfunction permitting cells to bypass the Hayflick limit and to divide indefinitely and uncontrollably. Thus all partners and structures involved in normal and abnormal telomere maintenance, protection and lengthening can be considered as potential anti-cancer therapeutic targets. In this Point of View we discuss, highlight and provide new perspectives from the current knowledge and understanding to position the different aspects of telomere biology and dysfunction as diagnostic, preventive and curative tools in the field of cancer.

The hnRNP A1 homolog Hrb87F/Hrp36 is important for telomere maintenance in Drosophila melanogaster

Unlike the telomerase-dependent mammalian telomeres, HeT-A, TART, and TAHRE (HTT) retroposon arrays regulate Drosophila telomere length. Cap prevents telomeric associations (TAs) and telomeric fusions (TFs). Our results suggest important roles of Hrb87F in telomeric HTT array and cap maintenance in Drosophila. All chromosome arms, except 2L, in Df(3R)Hrb87F homozygotes (Hrb87F-null) displayed significantly elongated telomeres with amplified HTT arrays and high TAs, all of which resolved without damage. Presence of FLAG-tagged Hrb87F (FLAG-Hrb87F) on cap and subtelomeric regions following hsFLAG-Hrb87F transgene expression in Df(3R)Hrb87F homozygotes suppressed TAs without affecting telomere length. A normal X-chromosome telomere expanded within five generations in Hrb87F-null background and displayed high TAs, but not when hsFLAG-Hrb87F was co-expressed. Tel (1) /Gaiano line or HP1 loss-of-function mutant-derived expanded telomeres carry Hrb87F on cap and HTT arrays while Hrb87F-null telomeres have HP1 and HOAP on caps and expanded HTT arrays. ISWI, seen only on cap on normal telomeres, was abundant on Hrb87F-null expanded HTT arrays. Extended telomeres derived from Tel (1) (Gaiano) or HP1-null mutation background interact with those from Hrb87F-null, since while the end association frequency was negligible in Df(3R)Hrb87F/+ nuclei, it increased significantly in co-presence of Tel (1) or HP1-null-based expanded telomere/s. Together, these suggest complex interactions between members of the proteome of telomere so that absence of any key member leads to telomere expansion and/or enhanced TAs/TFs. HTT expansion in Hrb87F-null condition is not developmental but a germline event presumably because absence of Hrb87F in germline may deregulate HTT retroposition/replication leading to telomere elongation.

Abstract 4624: Clinical impact of telomere shortening in normal and leukemia cells in chronic lymphocytic leukemia

Abstract Introduction: Prognosis in CLL is heterogeneous with the elderly, males and those with multiple co-morbidities having the highest risk of poorer outcomes. Whether this reflects differences in the biology of CLL or the result of altered immunosuppression is unknown. In normal individuals, the telomeres in somatic cells shorten with age, predisposing those with shorter telomeres to death from infections, cancer and cardiovascular disease. In chronic lymphocytic leukemia (CLL), short telomere length in the leukemia cells predicts poor prognosis. The contribution of un-modifiable factors of age, sex or comorbidities to telomere shortening in CLL has not been elucidated. In the present study, we evaluated buccal cell (BC) telomere length in patients with CLL, to determine if this is predictive of survival, and can enhance the predictive value of leukemia cell telomere length. Methods: The Manitoba CLL tumor bank contains 168 samples from newly diagnosed CLL patients between 2007-2011; with a median follow-up of 4 years. Half of these patients required chemotherapy and one-tenth have died. Genomic DNA was extracted from purified CLL cells and buccal cells (BC) collected at diagnosis. Telomere length was established by multiplex quantitative real-time PCR. Telomere/standard (t/s) ratio was calculated using the beta-globulin gene as the standard. Statistical analysis was performed using Statistical Analysis Software (SAS) and Prism software. Results: The median adjusted telomere length was shorter in CLL cells than in BCs being 0.53 and 2.01, respectively. In BCs, telomere length significantly shortened with increasing age (p = 0.01) but was not reflective of the number of comorbidities or survival. In contrast, telomere length in CLL cells was independent of age (p = 0.44) and sex (p = 0.75). Short CLL telomere length correlated with other biological and clinical markers of poorer prognosis including un-mutated IGHV status (p<0.0001), Zap70 positivity (p = 0.05), CD38 positivity (p = 0.003), short lymphocyte doubling time (p = 0.004), higher Rai stage (p = 0.02) and an earlier time to treatment (p<0.0001). Patients with short CLL telomeres had also decreased survival at four years (p = 0.05); living 22 months less than those with normal telomeres. Adjusting for BC telomere length improved the prognostic value of CLL telomere length as an independent predictor of survival. Conclusions: These results demonstrate that BC telomere length in CLL patients shorten with age independent of co-morbidities, secondary malignancies and the respective CLL telomere length. CLL telomere shortening is strongly associated with biochemical and clinical markers of disease progression and survival. The predictive value for overall survival is enhanced by correcting for changes in buccal cell telomere length. Note: This abstract was not presented at the meeting. Citation Format: Lin Yang, Sara Beiggi, Yunli Zhang, Robert Schmidt, Spencer B. Gibson, James B. Johnston. Clinical impact of telomere shortening in normal and leukemia cells in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4624. doi:10.1158/1538-7445.AM2015-4624

Telomerase deficiency delays renal recovery in mice after ischemia–reperfusion injury by impairing autophagy

The aged population suffers increased morbidity and higher mortality in response to episodes of acute kidney injury (AKI). Aging is associated with telomere shortening, and both telomerase reverse transcriptase (TerT) and RNA (TerC) are essential to maintain telomere length. To define a role of telomerase deficiency in susceptibility to AKI, we used ischemia/reperfusion injury in wild type mice or mice with either TerC or TerT deletion. Injury induced similar renal impairment at day 1 in each genotype, as assessed by azotemia, proteinuria, acute tubular injury score and apoptotic tubular epithelial cell index. However, either TerC or TerT knockout significantly delayed recovery compared to wild type mice. Electron microscopy showed increased autophagosome formation in renal tubular epithelial cells in wild type mice but a significant delay of their development in TerC and TerT knockout mice. There were also impeded increases in the expression of the autophagosome marker LC3 II, prolonged accumulation of the autophagosome protein P62, an increase of the cell cycle regulator p16, and greater activation of the mTOR pathway. The mTORC1 inhibitor, rapamycin, partially restored the ischemia/reperfusion-induced autophagy response, without a significant effect on either p16 induction or tubule epithelial cell proliferation. Thus, muting the maintenance of normal telomere length in mice impaired recovery from AKI, due to an increase in tubule cell senescence and impairment of mTOR-mediated autophagy.

Telomere length alterations unique to invasive lobular carcinoma.

Telomeres are nucleoprotein complexes located at the extreme ends of eukaryotic chromosomes and protect chromosomal ends from degradation and recombination. Dysfunctional telomeres contribute to genomic instability, promote tumorigenesis, and, in breast cancer, have been associated with increased cancer risk and poor prognosis. Short telomere lengths have been previously associated with triple-negative and human epidermal growth factor receptor (Her2)--positive ductal carcinomas. However, these investigations have not specifically assessed invasive lobular carcinomas (ILCs), which accounts for 5% to 15% of all invasive breast cancers. Here, we evaluate telomere lengths within 48 primary ILCs with complete characterization of estrogen receptor (ER), progesterone receptor (PR), and Her2 status, including 32 luminal/Her2- (ER+/PR+/Her2-), 8 luminal/Her2+ (ER+/PR+/Her2+), 3 Her2+ (ER-/PR-/Her2+), and 5 triple-negative (ER-/PR-/Her2-) carcinomas. A telomere-specific fluorescence in situ hybridization assay, which provides single-cell telomere length resolution, was used to evaluate telomere lengths and compare with standard clinicopathological markers. In contrast to breast ductal carcinoma, in which more than 85% of cases display abnormally short telomeres, approximately half (52%) of the ILCs displayed either normal or long telomeres. Short telomere length was associated with older patient age. Interestingly, 3 cases (6%) displayed a unique telomere pattern consisting of 1 or 2 bright telomere spots among the normal telomere signals within each individual cancer cell, a phenotype that has not been previously described. Additional studies are needed to further evaluate the significance of the unique bright telomere spot phenotype and the potential utility of telomere length as a prognostic marker in ILC.

Evaluation of Quantitative Fluorescence in situ Hybridization for Relative Measurement of Telomere Length in Placental Mesenchymal Core Cells

Background: Reduced telomere length in placental mesenchymal core cells has been reported during pregnancies complicated by intrauterine growth restriction. To estimate telomere length, a precise, accurate and reproducible technique must be used. Objective: We evaluated the characteristics of a quantitative fluorescence in situ hybridization (Q-FISH) technique for measuring relative telomere length in placental mesenchymal core cells. Methods: From late chorionic villus samplings, telomere length in placental mesenchymal core cells was estimated by a Q-FISH technique using peptide nucleic acid telomere probes. The main characteristics of the Q-FISH technique, such as precision and reproducibility, were evaluated. Results: The telomere length of the cultured placental mesenchymal cells did not follow a normal distribution. When the Q-FISH technique was performed on interphase nuclei of uncultured mesenchymal core cells, normal telomere length distribution was observed. The precision of the technique when applied to cultured placental mesenchymal core cells was estimated to be <6%, and its reproducibility ranged from to 92.9 to 104.7%. Conclusion: Our results showed that cell culture of placental villi produced a non-normal telomere length distribution, probably related to telomere DNA replication during the cell cycle. Despite the influence of cell culture, the Q-FISH technique reported herein showed good precision and reproducibility.

Long-Term Outcomes of Accelerated Telomere Shortening in Acquired Aplastic Anemia

Introduction: Accelerated telomere shortening (TS) in blood cells has been well documented in both dyskeratosis congenita and acquired aplastic anemia (AA). The present study investigated the incidence and long-term outcomes of TS in AA patients treated with ATG plus cyclosporine immunosuppressive therapy (IST).Methods: A total of 53 patients (31 M and 22 F, median age 24, 9-62) with moderate (MAA 15), severe (SAA 22) and very severe (vSAA 16) AA were included in cross-sectional study of telomere length (TEL) between 2008 and 2009 and then followed prospectively for 5 years. Treatment-naïve, recovering after IST and refractory AA were in 13, 35 and 5 patients respectively. There were no cases with physical abnormalities or family history of hematological disorders. TEL in granulocytes was assessed by Flow-FISH method with calculation of absolute and age-adjusted (DeltaTEL) values. TS was defined as absolute TEL value below the normal 99% confidence interval in age matched healthy donors (n=71). Outcome measures were response rate in treatment-naïve patients, cumulative incidence (CI) of relapse and clonal evolution, overall (OS) and failure-free survival (FFS). Adult patients or parents of children signed informed consent.Results: TS was detected in 24 patients (45%) with a median DeltaTEL of -1.715 kbp compared to -0.009 kbp in 29 patients with normal telomere length (TN) (Table 1).Table 1Patient and disease characteristics in cross-sectional studyTS (n=24)TN (n=29)PDeltaTEL, kbp, median (range)-1.715 (-5.100 – -0.983)-0.009 (-0.760 – 4.142)<0.001Age, years, median (range)22.5 (9-37)26 (14-62)0.102Gender, M/F, n15/916/130.780Severity, n (%)MAA10 (42%)5 (17%)0.0485SAA/vSAA14 (58%)24 (83%)Response to prior IST, n (%)Treatment-naïve4 (17%)9 (31%)0.338Partial response7 (29%)9 (31%)Complete response8 (33%)11 (38%)Refractory5 (21%)0 (0%)0.047PNH positive, n/n (%)12/24 (50%)13/28 (46%)1.000Interval between AA onset and IST, days, median (range)92 (17-508)102 (26-585)0.848Interval between AA onset and TEL testing, days, median (range)1093 (49-3792)785 (64-3494)0.173TS was associated with MAA (p = 0.049) and refractory disease (p=0.047) in cross-sectional study. No significant differences in gender, median age, baseline blood counts, PNH positivity or interval from AA onset and IST were observed between the two groups.Median follow-up after IST start and TEL testing were 90 and 67 months respectively. Response rates to IST were similar in TS (3/4, 75%) and TN (6/9, 67%) patients evaluated prospectively (p=1.0). TS and TN patients showed similar 10-year CI of relapse (16% vs 14%, p=0.905) and hemolytic PNH (28% vs 20%, p=0.532). However, all 3 cases of MDS /AML occurred in patients with TS with CI of 15.4% vs. 0% in the TN (p = 0.063). Compared patient groups have similar high probability of 10-year OS (TS 86.9% vs TN 92.6%, p=0.571) due to the large proportion of patients responding to treatment. The FFS rates were much lower (38.5% and 57.5% respectively, p=0.510).Conclusions: TS is a relatively common phenomenon in patients with otherwise typical acquired AA and more frequently detected in MAA and refractory to IST cases in cross-sectional study. Our data confirm the previously reported association of TS and increased risk of MDS/AML, which should be considered for a more careful monitoring and treatment planning, including allogeneic BMT as a curative approach. DisclosuresNo relevant conflicts of interest to declare.

Protective Cytotoxic Clonal T-Cells in Myeloma Have the Characteristics of Telomere-Independent Senescence Rather Than an Exhausted or Anergic Phenotype: Implications for Immunotherapy

Dysfunctional T-cells associated with human tumors can be identified utilizing multi-parameter flow cytometry and studied for intracellular signalling checkpoints using phospho-flow or time-of-flight mass spectrometry. Whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent has not yet been determined. Anergic T-cells are hypo-responsive with low cytokine production and induce tolerance to protect the host from autoimmune disease. T-cell exhaustion is usually associated with chronic viral infection such as CMV but also with some cancers. Both exhausted and anergic T-cells express PD-1, LAG-3, Tim-3, CD160 and CD28. Hypo-responsive T-cells associated with melanoma have the phenotype of exhausted T-cells which can be reactivated using targeted immune check point blockade, resulting in clinical benefit. Senescent T-cells accumulate in an oligoclonal manner with ageing and chronic antigen exposure. Senescence can be characterised by telomere shortening, the expression of CD57, KLRG-1, CD160 and the absence of CD28. However, not all senescent cells have shortened telomeres as telomere independent senescence involving the p21-p53 and p16-pRb pathways and senescence associated secretory phenotype (SASP) T-cells have been described. The aim of this study was to determine whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent and to identify targetable checkpoints to restore T-cell function.Cytotoxic T-cell clones (CD57+ CD28- TCRVβ restricted) determined by BetaMark TCRVβ analysis were present in 51% of patients with myeloma (n=264), are protective (OS χ2=6.2; p<0.01) and maintain high cytokine secretion. CFSE proliferation assays of flow sorted T-cell clones demonstrated a significant lack of proliferation after stimulation with MACS iBeads (n=12; p<0.0001). They failed to respond in vitro to cytokines such as IL- 2, IL-12, IL-15 and OX40. In contrast, the T-cell clones were proliferative in a group of long term myeloma survivors in whom such T-cells are universally present suggesting that the impaired proliferation may be reversible. Geneset enrichment analysis of mRNA expression from Affymetrix U133 plus 2.0 arrays demonstrated pleiotropic dysfunction in multiple intracellular signalling pathways involved with proliferation and T cell inactivation. Utilizing phospho-flow techniques we demonstrated reduced phosphorylated ERK levels in clonal vs non-clonal T-cells (n=8; p<0.002) as well as elevated levels of phosphorylated Smad2/3 (n=10; p<0.02) and Bcl-xL (n=7; p<0.04) .These findings suggest that the ERK pathway, associated with proliferation, was suppressed, the TGFβ pathway was activated and that the dysfunctional T-cell clones have a survival advantage due to apoptotic resistance. In contrast, normal SHP-2 and phosphorylated ZAP-70 levels suggested there was no defect in upstream TCR signaling.Clonal T-cells lacked CD28, PD-1, CTLA-4, LAG-3, CD160 and Tim-3 expression and therefore are neither exhausted nor anergic. However, telomere assessment by qPCR demonstrated telomere T/S ratios were normal length for age and not significantly less than autologous CD57+TCR-Vβ- cells. T-cell clones also had normal p38 expression suggesting that the senescence was not due to TNF-α inhibiting the p38/MAPK pathway.The cytotoxic T-cell clones in patients with myeloma have the characteristics of telomere-independent senescence. The normal telomere length suggests that the hypo-responsiveness is induced independent of telomeres, and is therefore potentially reversible, though the pleiotropic nature of this dysfunction might require the targeting of multiple intracellular signalling pathways. The lack of PD-1 and CTLA-4 expression suggests that immune checkpoint blockade in myeloma would be unable to reverse the T cell dysfunction. DisclosuresNo relevant conflicts of interest to declare.

Clinical features of ATRX or DAXX mutated neuroblastoma

PurposePreviously, we reported that alternative lengthening of telomere (ALT) may be a biomarker for chemo-sensitivity and late recurrence in neuroblastoma (NBL). In this study, alterations of ATRX or DAXX, which both encode chromatin remodeling proteins in telomeric region, and their relationship to ALT were examined in NBLs. MethodsOur previous report on 121 NBLs revealed 11 NBLs with elongated telomeres by ALT. In these NBLs, ATRX or DAXX gene alterations were identified using next-generation sequencing and compared to clinical and other biological factors. ResultsIn 11 ALT cases, DAXX mutations were detected in one case, and ATRX alterations were detected in 10 cases. Except for one case, no DAXX or ATRX alterations were detected in 110 tumors with normal or shortened telomeres. MYCN amplification was not detected in ATRX altered tumors. In ALT cases, three infants showed ATRX deletions, and all seven cases detected after 18months of age showed poor prognosis. ConclusionsIn NBLs, ALT was caused by ATRX or DAXX alterations. ATRX altered cases without MYCN amplification detected at greater than 18months showed poor prognosis, suggesting that ATRX or DAXX alterations are a particular NBL subtype. Since these tumors showed chemo-resistance and late recurrence, complete resection in a surgical approach should be performed to improve patient prognosis.

Functional characterization of the TERRA transcriptome at damaged telomeres.

Telomere deprotection occurs during tumorigenesis and aging upon telomere shortening or loss of the telomeric shelterin component TRF2. Deprotected telomeres undergo changes in chromatin structure and elicit a DNA damage response (DDR) that leads to cellular senescence. The telomeric long noncoding RNA TERRA has been implicated in modulating the structure and processing of deprotected telomeres. Here, we characterize the human TERRA transcriptome at normal and TRF2-depleted telomeres and demonstrate that TERRA upregulation is occurring upon depletion of TRF2 at all transcribed telomeres. TRF2 represses TERRA transcription through its homodimerization domain, which was previously shown to induce chromatin compaction and to prevent the early steps of DDR activation. We show that TERRA associates with SUV39H1 H3K9 histone methyltransferase, which promotes accumulation of H3K9me3 at damaged telomeres and end-to-end fusions. Altogether our data elucidate the TERRA landscape and defines critical roles for this RNA in the telomeric DNA damage response.

At short telomeres Tel1 directs early replication and phosphorylates Rif1.

The replication time of Saccharomyces cerevisiae telomeres responds to TG1–3 repeat length, with telomeres of normal length replicating late during S phase and short telomeres replicating early. Here we show that Tel1 kinase, which is recruited to short telomeres, specifies their early replication, because we find a tel1Δ mutant has short telomeres that nonetheless replicate late. Consistent with a role for Tel1 in driving early telomere replication, initiation at a replication origin close to an induced short telomere was reduced in tel1Δ cells, in an S phase blocked by hydroxyurea. The telomeric chromatin component Rif1 mediates late replication of normal telomeres and is a potential substrate of Tel1 phosphorylation, so we tested whether Tel1 directs early replication of short telomeres by inactivating Rif1. A strain lacking both Rif1 and Tel1 behaves like a rif1Δ mutant by replicating its telomeres early, implying that Tel1 can counteract the delaying effect of Rif1 to control telomere replication time. Proteomic analyses reveals that in yku70Δ cells that have short telomeres, Rif1 is phosphorylated at Tel1 consensus sequences (S/TQ sites), with phosphorylation of Serine-1308 being completely dependent on Tel1. Replication timing analysis of a strain mutated at these phosphorylation sites, however, suggested that Tel1-mediated phosphorylation of Rif1 is not the sole mechanism of replication timing control at telomeres. Overall, our results reveal two new functions of Tel1 at shortened telomeres: phosphorylation of Rif1, and specification of early replication by counteracting the Rif1-mediated delay in initiation at nearby replication origins.

Oligonucleotides and G-quadruplex stabilizers: targeting telomeres and telomerase in cancer therapy.

Cancer is a leading cause of death worldwide and an estimated 1 in 4 deaths in the United States is due to cancer. Despite recent advances in cancer treatment, adverse effects related to cancer therapy remain a limiting factor for many patients. The ideal cancer treatment would selectively target cancerous cells while sparing normal, healthy cells to offer maximal therapeutic benefit while minimizing toxicity. Telomeres are structurally unique DNA sequences at the end of human chromosomes, which play an integral role in the cellular mortality of normal cells. As telomeres shorten with successive cellular divisions, cells develop chromosomal instability and undergo either apoptosis or senescence. In many cancers, this apoptosis or senescence is avoided as normal telomere length is maintained by a ribonucleoprotein reverse transcriptase called telomerase. Telomerase is expressed in more than 85% of all cancers and confers cancerous cells with a replicative immortality, which is a hallmark of malignant tumors. In contrast, telomerase activity is not detectable in the majority of normal somatic cell populations. Therefore, the targeting of telomerase and telomere maintenance mechanisms represent a potentially promising therapeutic approach for various types of cancer. This review evaluates the roles of GRN163L, T-oligo and small molecule G-quadruplex stabilizers as potential anticancer therapies by targeting telomerase and other telomere maintenance mechanisms.