T cell memory revisited using single telomere length analysis.

The fundamental basis of T cell memory remains elusive. It is established that antigen stimulation drives clonal proliferation and differentiation, but the relationship between cellular phenotype, replicative history, and longevity, which is likely essential for durable memory, has proven difficult to elucidate. To address these issues, we used conventional markers of differentiation to identify and isolate various subsets of CD8+ memory T cells and measured telomere lengths in these phenotypically defined populations using the most sensitive technique developed to date, namely single telomere length analysis (STELA). Naive cells were excluded on the basis of dual expression of CCR7 and CD45RA. Memory subsets were sorted as CD27+CD45RA+, CD27intCD45RA+, CD27-CD45RA+, CD27+CD45RAint, CD27-CD45RAint, CD27+CD45RA-, and CD27-CD45RA- at >98% purity. The shortest median telomere lengths were detected among subsets that lacked expression of CD45RA, and the longest median telomere lengths were detected among subsets that expressed CD45RA. Longer median telomere lengths were also a feature of subsets that expressed CD27 in compartments defined by the absence or presence of CD45RA. Collectively, these data suggested a disconnect between replicative history and CD8+ memory T cell differentiation, which is classically thought to be a linear process that culminates with revertant expression of CD45RA.

ATRX modulates the escape from a telomere crisis.

Telomerase activity is the principal telomere maintenance mechanism in human cancers, however 15% of cancers utilise a recombination-based mechanism referred to as alternative lengthening of telomeres (ALT) that leads to long and heterogenous telomere length distributions. Loss-of-function mutations in the Alpha Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) gene are frequently found in ALT cancers. Here, we demonstrate that the loss of ATRX, coupled with telomere dysfunction during crisis, is sufficient to initiate activation of the ALT pathway and that it confers replicative immortality in human fibroblasts. Additionally, loss of ATRX combined with a telomere-driven crisis in HCT116 epithelial cancer cells led to the initiation of an ALT-like pathway. In these cells, a rapid and precise telomeric elongation and the induction of C-circles was observed; however, this process was transient and the telomeres ultimately continued to erode such that the cells either died or the escape from crisis was associated with telomerase activation. In both of these instances, telomere sequencing revealed that all alleles, irrespective of whether they were elongated, were enriched in variant repeat types, that appeared to be cell-line specific. Thus, our data show that the loss of ATRX combined with telomere dysfunction during crisis induces the ALT pathway in fibroblasts and enables a transient activation of ALT in epithelial cells.

Symptoms of Prenatal Depression Associated with Shorter Telomeres in Female Placenta.

BACKGROUND: Depression is a common mood disorder during pregnancy impacting one in every seven women. Children exposed to prenatal depression are more likely to be born at a low birth weight and develop chronic diseases later in life. A proposed hypothesis for this relationship between early exposure to adversity and poor outcomes is accelerated aging. Telomere length has been used as a biomarker of cellular aging. We used high-resolution telomere length analysis to examine the relationship between placental telomere length distributions and maternal mood symptoms in pregnancy. METHODS: This study utilised samples from the longitudinal Grown in Wales (GiW) study. Women participating in this study were recruited at their presurgical appointment prior to a term elective caesarean section (ELCS). Women completed the Edinburgh Postnatal Depression Scale (EPDS) and trait subscale of the State-Trait Anxiety Inventory (STAI). Telomere length distributions were generated using single telomere length analysis (STELA) in 109 term placenta (37-42 weeks). Multiple linear regression was performed to examine the relationship between maternally reported symptoms of depression and anxiety at term and mean placental telomere length. RESULTS: Prenatal depression symptoms were significantly negatively associated with XpYp telomere length in female placenta (B = -0.098, p = 0.026, 95% CI -0.184, -0.012). There was no association between maternal depression symptoms and telomere length in male placenta (B = 0.022, p = 0.586, 95% CI -0.059, 0.103). There was no association with anxiety symptoms and telomere length for either sex. CONCLUSION: Maternal prenatal depression is associated with sex-specific differences in term placental telomeres. Telomere shortening in female placenta may indicate accelerated placental aging.

UPF1 promotes the formation of R loops to stimulate DNA double-strand break repair.

DNA-RNA hybrid structures have been detected at the vicinity of DNA double-strand breaks (DSBs) occurring within transcriptional active regions of the genome. The induction of DNA-RNA hybrids strongly affects the repair of these DSBs, but the nature of these structures and how they are formed remain poorly understood. Here we provide evidence that R loops, three-stranded structures containing DNA-RNA hybrids and the displaced single-stranded DNA (ssDNA) can form at sub-telomeric DSBs. These R loops are generated independently of DNA resection but are induced alongside two-stranded DNA-RNA hybrids that form on ssDNA generated by DNA resection. We further identified UPF1, an RNA/DNA helicase, as a crucial factor that drives the formation of these R loops and DNA-RNA hybrids to stimulate DNA resection, homologous recombination, microhomology-mediated end joining and DNA damage checkpoint activation. Our data show that R loops and DNA-RNA hybrids are actively generated at DSBs to facilitate DNA repair.

High-throughput STELA provides a rapid test for the diagnosis of telomere biology disorders.

Telomere biology disorders are complex clinical conditions that arise due to mutations in genes required for telomere maintenance. Telomere length has been utilised as part of the diagnostic work-up of patients with these diseases; here, we have tested the utility of high-throughput STELA (HT-STELA) for this purpose. HT-STELA was applied to a cohort of unaffected individuals (n = 171) and a retrospective cohort of mutation carriers (n = 172). HT-STELA displayed a low measurement error with inter- and intra-assay coefficient of variance of 2.3% and 1.8%, respectively. Whilst telomere length in unaffected individuals declined as a function of age, telomere length in mutation carriers appeared to increase due to a preponderance of shorter telomeres detected in younger individuals (< 20 years of age). These individuals were more severely affected, and age-adjusted telomere length differentials could be used to stratify the cohort for overall survival (Hazard Ratio = 5.6 (1.5-20.5); p < 0.0001). Telomere lengths of asymptomatic mutation carriers were shorter than controls (p < 0.0001), but longer than symptomatic mutation carriers (p < 0.0001) and telomere length heterogeneity was dependent on the diagnosis and mutational status. Our data show that the ability of HT-STELA to detect short telomere lengths, that are not readily detected with other methods, means it can provide powerful diagnostic discrimination and prognostic information. The rapid format, with a low measurement error, demonstrates that HT-STELA is a new high-quality laboratory test for the clinical diagnosis of an underlying telomeropathy.

Tracking telomere fusions through crisis reveals conflict between DNA transcription and the DNA damage response.

Identifying attributes that distinguish pre-malignant from senescent cells provides opportunities for targeted disease eradication and revival of anti-tumour immunity. We modelled a telomere-driven crisis in four human fibroblast lines, sampling at multiple time points to delineate genomic rearrangements and transcriptome developments that characterize the transition from dynamic proliferation into replicative crisis. Progression through crisis was associated with abundant intra-chromosomal telomere fusions with increasing asymmetry and reduced microhomology usage, suggesting shifts in DNA repair capacity. Eroded telomeres also fused with genomic loci actively engaged in transcription, with particular enrichment in long genes. Both gross copy number alterations and transcriptional responses to crisis likely underpin the elevated frequencies of telomere fusion with chromosomes 9, 16, 17, 19 and most exceptionally, chromosome 12. Juxtaposition of crisis-regulated genes with loci undergoing de novo recombination exposes the collusive contributions of cellular stress responses to the evolving cancer genome.

Chromothripsis during telomere crisis is independent of NHEJ, and consistent with a replicative origin.

Telomere erosion, dysfunction, and fusion can lead to a state of cellular crisis characterized by large-scale genome instability. We investigated the impact of a telomere-driven crisis on the structural integrity of the genome by undertaking whole-genome sequence analyses of clonal populations of cells that had escaped crisis. Quantification of large-scale structural variants revealed patterns of rearrangement consistent with chromothripsis but formed in the absence of functional nonhomologous end-joining pathways. Rearrangements frequently consisted of short fragments with complex mutational patterns, with a repair topology that deviated from randomness showing preferential repair to local regions or exchange between specific loci. We find evidence of telomere involvement with an enrichment of fold-back inversions demarcating clusters of rearrangements. Our data suggest that chromothriptic rearrangements caused by a telomere crisis arise via a replicative repair process involving template switching.

Metformin and insulin treatment prevent placental telomere attrition in boys exposed to maternal diabetes.

Shortened leukocyte and placental telomeres associated with gestational diabetes mellitus (GDM) suggest this exposure triggers telomere attrition contributing to adverse outcomes. We applied high resolution Single Telomere Length Analysis (STELA) to placenta from GDM pregnancies with different treatment pathways to determine their effectiveness at preventing telomere attrition. Differences in telomere length between control (N = 69), GDM lifestyle intervention (n = 14) and GDM treated with metformin and/or insulin (n = 17) was tested by Analysis of Covariance (ANCOVA) followed by group comparisons using Fisher's least significant difference. For male placenta only, there were differences in mean telomere length (F(2,54) = 4.98, P = 0.01) and percentage of telomeres under 5 kb (F(2,54) = 4.65, P = 0.01). Telomeres were shorter in the GDM lifestyle intervention group compared to both controls (P = 0.02) and medically treated pregnancies (P = 0.003). There were more telomeres under 5 kb in the GDM lifestyle intervention group compared to the other two groups (P = 0.03 and P = 0.004). Although further work is necessary, we suggest that early adoption of targeted medical treatment of GDM pregnancies where the fetus is known to be male may be an effective strategy for ameliorating adverse outcomes for children.

PARP1 is required for preserving telomeric integrity but is dispensable for A-NHEJ.

Poly-ADP ribose polymerase 1 (PARP1) is clinically important because of its synthetic lethality with breast cancer allele 1 and 2 mutations, which are causative for inherited breast and ovarian cancers. Biochemically, PARP1 is a single-stranded DNA break repair protein that is needed for preserving genomic integrity. In addition, PARP1 has been implicated in a veritable plethora of additional cellular pathways and thus its precise contribution(s) to human biology has remained obscure. To help address this deficiency, we utilized gene editing to construct genetically-null PARP1 human cancer cells. We found a minor role for PARP1 in an alternative form of DNA double-strand break (DSB) repair, but only when these cells were deficient for the classical form of DSB repair. Despite being proficient for DSB repair, however, cell cycle progression defects and elevated endogenous DNA damage signaling were observed. These deficiencies were instead linked to telomere defects, where PARP1 -/- cells had short telomeres that co-localized with markers of endogenous DNA damage and were compromised in their ability to escape a telomere-driven crisis. Our data suggest that while PARP1 does not participate significantly in DNA DSB repair itself, it does prevent the incidence of telomeric DSBs, which, in turn, can drive genomic instability.

PARP inhibition prevents escape from a telomere-driven crisis and inhibits cell immortalisation.

Telomeric crisis is the final replicative barrier to cell immortalisation; it is characterised by genome instability and cell death and is triggered when telomeres become critically short and are subjected to fusion. Pre-cancerous lesions, or early stage cancers, often show signs of a telomere crisis, suggesting that escape from telomere crisis is a prerequisite for disease progression. Telomeric crisis therefore represents an attractive, and as yet unexplored, opportunity for therapeutic intervention. Here, we show that two clinically approved PARP inhibitors, selectively eliminate human cells undergoing a telomere-driven crisis. Clonal populations of a colorectal cancer cell line (HCT116), or the plasma cell leukaemia cell line (JJN-3), expressing a dominant-negative telomerase, entered a telomere-driven crisis at defined population doubling points and telomere lengths. The addition of the PARP inhibitors, olaparib or rucaparib prevented these cells from escaping crisis. PARP inhibition did not alter cellular proliferation prior to crisis, rates of telomere erosion or the telomere length at which crisis was initiated, but affected repair of eroded telomeres, resulting in an increased in intra-chromosomal telomere fusion. This was accompanied by enhanced DNA damage checkpoint activation and elevated levels of apoptosis. We propose that PARP inhibitors impair the repair of dysfunctional telomeres and/or induce replicative stress at telomeres to inhibit escape from a telomere crisis. This is the first demonstration that a drug can selectively kill cells experiencing telomeric crisis. We propose that this type of drug, which we term 'crisolytic', has the potential to eliminate pre-cancerous lesions and tumours exhibiting short dysfunctional telomeres.

Telomere length is an independent prognostic marker in MDS but not in de novo AML.

Telomere dysfunction is implicated in the generation of large-scale genomic rearrangements that drive progression to malignancy. In this study we used high-resolution single telomere length analysis (STELA) to examine the potential role of telomere dysfunction in 80 myelodysplastic syndrome (MDS) and 95 de novo acute myeloid leukaemia (AML) patients. Despite the MDS cohort being older, they had significantly longer telomeres than the AML cohort (P < 0·0001) where telomere length was also significantly shorter in younger AML patients (age <60 years) (P = 0·02) and in FLT3 internal tandem duplication-mutated AML patients (P = 0·03). Using a previously determined telomere length threshold for telomere dysfunction (3·81 kb) did not provide prognostic resolution in AML [Hazard ratio (HR) = 0·68, P = 0·2]. In contrast, the same length threshold was highly prognostic for overall survival in the MDS cohort (HR = 5·0, P < 0·0001). Furthermore, this telomere length threshold was an independent parameter in multivariate analysis when adjusted for age, gender, cytogenetic risk group, number of cytopenias and International Prognostic Scoring System (IPSS) score (HR = 2·27, P < 0·0001). Therefore, telomere length should be assessed in a larger prospective study to confirm its prognostic role in MDS with a view to integrating this variable into a revised IPSS.

Telomere erosion in NF1 tumorigenesis.

Neurofibromatosis type 1 (NF1; MIM# 162200) is a familial cancer syndrome that affects 1 in 3,500 individuals worldwide and is inherited in an autosomal dominant fashion. Malignant Peripheral Nerve Sheath Tumors (MPNSTs) represent a significant cause of morbidity and mortality in NF1 and currently there is no treatment or definite prognostic biomarkers for these tumors. Telomere shortening has been documented in numerous tumor types. Short dysfunctional telomeres are capable of fusion and it is considered that the ensuing genomic instability may facilitate clonal evolution and the progression to malignancy. To evaluate the potential role of telomere dysfunction in NF1-associated tumors, we undertook a comparative analysis of telomere length in samples derived from 10 cutaneous and 10 diffused plexiform neurofibromas, and 19 MPNSTs. Telomere length was determined using high-resolution Single Telomere Length Analysis (STELA). The mean Xp/Yp telomere length detected in MPNSTs, at 3.282 kb, was significantly shorter than that observed in both plexiform neurofibromas (5.793 kb; [p = 0.0006]) and cutaneous neurofibromas (6.141 kb; [p = 0.0007]). The telomere length distributions of MPNSTs were within the length-ranges in which telomere fusion is detected and that confer a poor prognosis in other tumor types. These data indicate that telomere length may play a role in driving genomic instability and clonal progression in NF1-associated MPNSTs.

Extensive telomere erosion is consistent with localised clonal expansions in Barrett's metaplasia.

Barrett's oesophagus is a premalignant metaplastic condition that predisposes patients to the development of oesophageal adenocarcinoma. However, only a minor fraction of Barrett's oesophagus patients progress to adenocarcinoma and it is thus essential to determine bio-molecular markers that can predict the progression of this condition. Telomere dysfunction is considered to drive clonal evolution in several tumour types and telomere length analysis provides clinically relevant prognostic and predictive information. The aim of this work was to use high-resolution telomere analysis to examine telomere dynamics in Barrett's oesophagus. Telomere length analysis of XpYp, 17p, 11q and 9p, chromosome arms that contain key cancer related genes that are known to be subjected to copy number changes in Barrett's metaplasia, revealed similar profiles at each chromosome end, indicating that no one specific telomere is likely to suffer preferential telomere erosion. Analysis of patient matched tissues (233 samples from 32 patients) sampled from normal squamous oesophagus, Z-line, and 2 cm intervals within Barrett's metaplasia, plus oesophago-gastric junction, gastric body and antrum, revealed extensive telomere erosion in Barrett's metaplasia to within the length ranges at which telomere fusion is detected in other tumour types. Telomere erosion was not uniform, with distinct zones displaying more extensive erosion and more homogenous telomere length profiles. These data are consistent with an extensive proliferative history of cells within Barrett's metaplasia and are indicative of localised clonal growth. The extent of telomere erosion highlights the potential of telomere dysfunction to drive genome instability and clonal evolution in Barrett's metaplasia.

Telomere length is a critical determinant for survival in multiple myeloma.

The variable clinical outcomes of Multiple Myeloma (MM) patients are incompletely defined by current prognostication tools. We examined the clinical utility of high-resolution telomere length analysis as a prognostic marker in MM. Cohort stratification, using a previously determined length threshold for telomere dysfunction, revealed that patients with short telomeres had a significantly shorter overall survival (P < 0·0001; HR = 3·4). Multivariate modelling using forward selection identified International Staging System (ISS) stage as the most important prognostic factor, followed by age and telomere length. Importantly, each ISS prognostic subset could be further risk-stratified according to telomere length, supporting the inclusion of this parameter as a refinement of the ISS.

Escape from telomere-driven crisis is DNA ligase III dependent.

Short dysfunctional telomeres are capable of fusion, generating dicentric chromosomes and initiating breakage-fusion-bridge cycles. Cells that escape the ensuing cellular crisis exhibit large-scale genomic rearrangements that drive clonal evolution and malignant progression. We demonstrate that there is an absolute requirement for fully functional DNA ligase III (LIG3), but not ligase IV (LIG4), to facilitate the escape from a telomere-driven crisis. LIG3- and LIG4-dependent alternative (A) and classical (C) nonhomologous end-joining (NHEJ) pathways were capable of mediating the fusion of short dysfunctional telomeres, both displaying characteristic patterns of microhomology and deletion. Cells that failed to escape crisis exhibited increased proportions of C-NHEJ-mediated interchromosomal fusions, whereas those that escaped displayed increased proportions of intrachromosomal fusions. We propose that the balance between inter- and intrachromosomal telomere fusions dictates the ability of human cells to escape crisis and is influenced by the relative activities of A- and C-NHEJ at short dysfunctional telomeres.

Telomere dysfunction accurately predicts clinical outcome in chronic lymphocytic leukaemia, even in patients with early stage disease.

Defining the prognosis of individual cancer sufferers remains a significant clinical challenge. Here we assessed the ability of high-resolution single telomere length analysis (STELA), combined with an experimentally derived definition of telomere dysfunction, to predict the clinical outcome of patients with chronic lymphocytic leukaemia (CLL). We defined the upper telomere length threshold at which telomere fusions occur and then used the mean of the telomere 'fusogenic' range as a prognostic tool. Patients with telomeres within the fusogenic range had a significantly shorter overall survival (P < 0·0001; Hazard ratio [HR] = 13·2, 95% confidence interval [CI] = 11·6-106·4) and this was preserved in early-stage disease patients (P < 0·0001, HR=19·3, 95% CI = 17·8-802·5). Indeed, our assay allowed the accurate stratification of Binet stage A patients into those with indolent disease (91% survival at 10 years) and those with poor prognosis (13% survival at 10 years). Furthermore, patients with telomeres above the fusogenic mean showed superior prognosis regardless of their IGHV mutation status or cytogenetic risk group. In keeping with this finding, telomere dysfunction was the dominant variable in multivariate analysis. Taken together, this study provides compelling evidence for the use of high-resolution telomere length analysis coupled with a definition of telomere dysfunction in the prognostic assessment of CLL.

Activation of p38 MAP kinase and stress signalling in fibroblasts from the progeroid Rothmund-Thomson syndrome.

Rothmund-Thomson fibroblasts had replicative lifespans and growth rates within the range for normal fibroblasts; however, they show elevated levels of the stress-associated p38 MAP kinase, suggestive of stress during growth. Treatment with the p38 MAP kinase inhibitor SB203580 increased both lifespan and growth rate, as did reduction of oxidative stress using low oxygen in some strains. At replicative senescence p53, p21(WAF1) and p16(INK4A) levels were elevated, and abrogation of p53 using shRNA knockdown allowed the cells to bypass senescence. Ectopic expression of human telomerase allowed Rothmund-Thomson fibroblasts to bypass senescence. However, activated p38 was still present, and continuous growth for some telomerised clones required either a reduction in oxidative stress or SB203580 treatment. Overall, the evidence suggests that replicative senescence in Rothmund-Thomson cells resembles normal senescence in that it is telomere driven and p53 dependent. However, the lack of RECQL4 leads to enhanced levels of stress during cell growth that may lead to moderate levels of stress-induced premature senescence. As replicative senescence is believed to underlie human ageing, a moderate level of stress-induced premature senescence and p38 activity may play a role in the relatively mild ageing phenotype seen in Rothmund-Thomson.

STAT1 contributes to the maintenance of the latency III viral programme observed in Epstein-Barr virus-transformed B cells and their recognition by CD8+ T cells.

The transformation of B cells by Epstein-Barr virus (EBV), into lymphoblastoid cell lines (LCLs) results in the upregulation of STAT1, a key transcription factor in the interferon signalling pathway. Although the mechanism of EBV induction of STAT1 protein expression has been intensively studied, there has been little investigation into the function of STAT1 in EBV-transformed LCLs. In this study, we have implemented a novel strategy to investigate the functional role of STAT1 through the introduction of the simian virus 5 (SV5) V-protein into LCLs by retroviral gene transfer. The V-protein is a virally evolved STAT1 inhibitor that specifically targets STAT1 for proteasomal degradation. Using this in vitro model, we have shown that major histocompatibility complex (MHC) class I and class II molecules are downregulated at the cell surface following a reduction in STAT1 protein expression. With regards to MHC class I, the impairment of the antigen processing machinery renders the cells less recognized by the host EBV-specific immunosurveillance. In addition, downregulation of STAT1 increases the expression of LMP2A and lytic cycle antigens and results in a higher proportion of cells entering the lytic cycle. These results suggest that STAT1 is involved in maintaining the latency III viral program observed in transformed B cells and regulating immunorecognition by EBV-specific T cells.