Telomeres are specialized structures providing chromosome integrity during cellular division along with protection against premature senescence and apoptosis. Accelerated telomere attrition in patients with myelodysplastic syndrome (MDS) occurs by an undefined mechanism. Although the MDS clone originates within the myeloid compartment, T-lymphocytes display repertoire contraction and loss of naive T-cells. The replicative lifespan of T-cells is stringently regulated by telomerase activity. In MDS cases, we show that purified CD3+ T-cells have significantly shorter telomere length and reduced proliferative capacity upon stimulation compared with controls. To understand the mechanism, telomerase enzymatic activity and telomerase reverse transcriptase (hTERT), gene expression were compared in MDS cases (n=35) and healthy controls (n=42) within different T-cell compartments. Telomerase activity is greatest in naive T-cells illustrating the importance of telomere repair in homeostatic repertoire regulation. Compared with healthy controls, MDS cases had lower telomerase induction (P<0.0001) that correlated with significantly lower hTERT mRNA (P<0.0001), independent of age and disease stratification. hTERT mRNA deficiency affected naive but not memory T-cells, and telomere erosion in MDS occurred without evidence of an hTERT-promoter mutation, copy number variation or deletion. Telomerase insufficiency may undermine homeostatic control within the hematopoietic compartment and promote a change in the T-cell repertoire in MDS.
Myelodysplastic syndromes (MDS) include a spectrum of age-related hematological neoplasms characterized by dysplasia, cytopenias and potential for AML (acute myeloid leukemia) progression.1 Because complex mutations are acquired within the myeloid progenitor or stem cell pool, the biological characteristics are enormously heterogeneous.2, 3 The early manifestations of MDS, however, are relatively well conserved and include inflammation within the bone marrow microenvironment coupled with excessive proliferation and apoptosis of myeloid progenitors.4, 5 In MDS patients, telomere length is shortened by an unknown process related possibly to proliferative stress or by ineffective telomeric repair.
Telomeres are a repetitive hexanucleotide (TTAGGG) region that protect from chromosomal deterioration.6 T-cells and hematopoietic stem/progenitor cells need telomere replenishment by telomerase, which is a specialized reverse transcriptase composed of the rate-limiting telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT) enzyme, the telomerase RNA component (hTERC) template, and the shelterin stabilizing proteins. hTERT gene expression is transcriptionally regulated and its induction limits replicative senescence and protects regenerating somatic cells from apoptosis and genome instability.7 Mutations in telomere components have demonstrated the role for telomerase in hematopoietic repopulation and in immune homeostasis.8 hTERC, hTERT and mutations in shelterin proteins in patients with aplastic anemia (AA) and dyskeratosis congenita have been informative to demonstrate the consequences of telomerase deficiency in bone marrow failure and predisposition to cancer.6, 9 In patients with mutations in telomere components, T-cells undergo TCR signal transduction but fail to display normal clone size after activation.6 In previous studies, siRNA (small interfering (si) ribonucleic acid (RNA)) silencing of hTERT in T-cells renders them sensitive to apoptosis and hinders their regeneration, indicating a direct role for telomerase in replication potential.10 Compared with myeloid cells, there are no data regarding the length of telomeres or telomerase function within the T-cell compartment in MDS patients.2 Of all somatic cells, T-cells are dependent on telomere repair because of their high rate of turnover. This activity is particularly important for maintenance of naive cells after age-dependent thymic involution.10 In MDS, the T-cells are characterized by skewed repertoire distribution11 and form suppressive lymphoid aggregates within the bone marrow of some patients where they can directly suppress hematopoiesis.12, 13
This study was conducted to examine the mechanism of MDS T-cell deregulation related to telomere function. We report that deficiency in hTERT mRNA expression is important in MDS and reveal a novel mechanistic link between AA and other primary telomere repair disorders.
Materials and methods
Cell isolation in patients and healthy controls
MDS patients were recruited from 2005 to 2010 through a collaborative study involving four centers: the Hematologic Malignancy Program at the Moffitt Cancer Center (‘Moffitt’), the Cleveland Clinic Cancer Center, UCLA Medical Center and Penn State Cancer Center. All study participants signed informed consents approved by the institutional review boards at the participating institutions. All diagnoses were histologically confirmed after review at the participating center and classified according to standard criteria including the World Health Organization (WHO)14 and International Prognostic Scoring System (IPSS).1 Inclusion into this study (n=35) was based on having a sufficient volume of frozen PBMCs (peripheral blood mononuclear) available for T-cell isolation (30 × 106 cells). Details describing the isolation of PBMCs and purification of CD3+ T-cells is provided in Supplementary Material.
Measurement of telomere length
DNA was extracted from unstimulated purified CD3+ T-cells using the PureLink Genomic DNA Kits according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA), and relative telomere lengths were measured by a modified version of the quantitative real-time (qRT) PCR-based telomere assay, as described previously.15, 16 Details of the assay are provided in Supplementary Methods and Supplementary Figure 1.
Purified CD3+ T-cells were cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) 10% fetal bovine serum (FBS). Cells were stimulated with Dynabeads (R) Human T-activator CD3/CD28 beads (Invitrogen Life Technologies, Grand Island, NY, USA) for 3 days.
Measurement of TREC DNA in purified T-cells
Signal-joint T-cell receptor excision circles (sjTRECs) are circular extrachromosomal fragments of DNA generated during TCR gene rearrangement in the thymus.17 Since they are non-transferable to daughter cells, expression decreases with each round of cell division in culture and quantification is a reliable estimate of population doubling (PD).18 Genomic DNA was extracted from CD3+ T-cell, as described above. Detailed methods for sjTREC quantification can be found in Supplementary Material.
Measurement of hTERT mRNA expression
Details of qRT–PCR methods is provided in the Supplementary Material.
Telomerase activity was determined using the telomerase PCR-ELISA kit (TRAP) (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer’s instructions. Details are provided in Supplementary Methods. Experiments were carried out at least twice to improve the reliability of the results. The relative telomerase activity within each sample was calculated as follows: (the absorbance of the sample—the absorbance of the heat-treated sample)/the absorbance of the internal standard of the sample.
Flow cytometry to detect BrdU incorporation
To assess entry into S-phase, bromodeoxyuridine (BrdU) incorporation (BrdU flow kit, BD Biosciences, San Diego, CA, USA) was used, as described previously.19 Detailed methods are provided in Supplementary Information.
Naive and memory cell purification and flow assays
T-cells subsets were isolated from purified CD3+ T-cells using CD45RO and CD45RA antibody staining and isolation with the Aria flow sorter (BD Biosciences). Subset purity was >95% in post-sort analyses. Memory (CD45RO+CD45RA−) and naive (CD45RA+CD45RO−)17 cells were stimulated with Dynabeads(R) Human T-activator CD3/CD28 beads (Invitrogen Life Technologies) for 3 days. Telomere length was detected in sorted naive and memory T-cells before stimulation and telomerase activity and hTERT expression was measured using the TRAP assay and qRT–PCR reaction, respectively, before and after the 3 days of stimulation. DAPI exclusion discriminated viable and dead cells during activation and CD69 staining was used to determine the TCR response. Detailed methods are provided in Supplementary Material.
Sequencing of hTERT promoter
The core promoter of hTERT was amplified using genomic DNA from purified CD3+ T-cells for sequence analysis. Detailed methods are provided in Supplementary Material.
Telomere length, proliferation (BrdU and PD) and hTERT mRNA data were compared as continuous variables between MDS cases and controls using the Wilcoxon rank-sum test. Data on telomerase activity was normally distributed and compared as a continuous variable using a t-test. The mean age of controls was 52 (range 17–84 years) and MDS cases was 67 (range 28–87 years) (Wilcoxon, P=0.1). For all variables, the statistical significance of the interaction with age as a continuous variable and sex in relation to case–control status was tested by including an interaction term in the logistic regression model of log-transformed data. However, hTERT mRNA data were square-root transformed to achieve normality because log-transformation failed to achieve a normal distribution. Correlations between telomere length and age and hTERT activity and mRNA expression were assessed using the Spearman correlation coefficient.
Since T-cell homeostasis is altered by thymic involution, and younger MDS patients (that is, under the age of 65 years old) are suspected of having a different pathobiologic mechanism for impaired hematopoiesis related to an immunological attack, we stratified the analyses by age <65 or ⩾65 years. For these subset analyses, the five youngest healthy controls were omitted to better approximate the age range among younger MDS cases. In these subgroups, the mean age of controls was 47 years (range 26–63 years, n=23) and the mean age of cases was 52 years (n=9, range 28–61 years) (Wilcoxon, P=0.27). The older group was balanced with regard to age with a mean of 73 (n=14, range 65–84 years) in controls and a mean of 73 (n=26, range 65–87 years) in cases (Wilcoxon, P=0.97). All statistical analyses were performed with the SPSS 19.0 software package (SPSS Inc., Chicago, IL, USA).
Clinical characteristics of MDS patients
Characteristics of the 35 MDS patients and 42 controls are summarized in Table 1. Of the 42 controls and 35 cases, 18 (43%) and 24 (69%) were male, respectively (P=0.02). MDS patients were classified as refractory anemia with or without ringed sideroblast (RA, RARS) (n=2, 5.7%), refractory cytopenia with multilineage dysplasia (RCMD) (n=15, 42.9%), refractory anemia with excess blasts (RAEB)-1 (n=5, 14.3%) and RAEB-2 (n=9, 25.7%) and MDS-unclassified (MDS-U, n=4, 11.4%) based on WHO classification criteria.14 Based on IPSS, 12 (34.3%) were low risk, 9 (25.7%) intermediate-1, 8 (22.9%) intermediate-2, and 6 (17.1%) high risk.1 In all, 20 of 35 (57.1%) had identifiable cytogenetic abnormalities by metaphase karyotyping or FISH, and 15 (42.9%) had normal cytogenetics.
Age-independent telomere attrition in purified T-cells in MDS
The mean relative telomere length in purified T-cells was compared relative to hemoglobin and found to be significantly shorter among MDS cases (n=35) (median=1.1, 95% confidence interval (CI), 1.1–1.4) compared with controls (n=42) (median=2.1, 95% CI, 1.9–2.3) after adjustment for age and sex (P<0.0001) (Figure 1a) using log-transformed data. In healthy individuals, T-cell telomere length declined progressively with age (r=−0.447, P=0.003) (Figure 1b) in agreement with previous reports reflecting the proliferative pressure on the T-cell compartment.20 Although not statistically significant, the age-related trend was similar in cases (r=−0.177, P=0.309) (Figure 1b). To further evaluate the effect of age, we compared telomere length between MDS cases and controls stratified into younger (<65 years) and older (⩾65 years) age groups (Figure 1c). As expected, telomere lengths were significantly shorter among older controls compared with younger controls (P=0.001), although no difference in telomere lengths was observed between older and younger MDS cases. MDS cases tended to have shorter telomeres than controls in both the <65 age group (P=0.001) and the 65+ age group (P=0.003). In general, MDS patients within the younger group had telomeres that were shorter than the oldest healthy individuals attesting to the proliferative stress or loss of telomeric repair in the T-cell compartment.
Proliferative defects in MDS T-cells
Replicative potential is closely linked to telomere length and telomerase activation.10, 21 To define the proliferative capacity of T-cells in MDS compared with healthy controls, both the percentage of cells capable of entering the cell cycle (that is, percentage of cells in S-phase as indicated by BrdU incorporation), cell death (percentage of CD69+ DAPI+ cells) and their proliferative burst potential (that is, PD) were assessed after anti-CD3/CD28 bead stimulation (Figure 2a). The percentage of BrdU-positive T-cells is significantly reduced in MDS cases (median=22.3, 95% CI, 16.3–26.3) compared with controls (median=32.0, 95% CI, 30.0–34.6) (P=0.0008) (Figure 2b). However, wide variability was observed in the percentage of BrdU-positive cells in MDS. The discrepancy in cell-cycle regulation was more relevant in older cases compared with younger cases (case:control difference, P=0.03 in older group compared with case:control difference P=0.50 in younger group), but there were patients in both age groups with normal capacity for S-phase transition (Figure 2c).
Telomerase deficiency primarily impacts replication burst potential as failure to induce the telomere repair machinery leads to apoptosis and premature growth arrest.6 Estimation of replication potential was assessed using sjTREC dilution (Figure 2a) as a measure of PD after in vitro activation with anti-CD3/CD28-coated beads. The change in copy number reflects the dilution of DNA through expansion after activation. Results were compared in younger and older cases and controls. In controls (n=42), the mean sjTREC DNA copy number decreased from 80 to 15 copies per cell after stimulation, indicating that an average of six PDs occurred in the 3-day period of the assay. Fluorescent lipophilic dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining confirmed that T-cell populations in controls progress through roughly 5–7 generations within the 3-day expansion period, which is consistent with the sjTREC dilution assay (Supplementary Figure 2). In the CSFE assay, ∼50% of the cells are able to divide in bead-activated control T-cells. Compared with controls, PD was significantly decreased in MDS cases, in both the younger (P<0.0001) and older (P=0.001) groups (Figure 2d). In MDS, expansion progressed past one generation of division on average in only 15% of cells using the CSFE assay (Supplementary Figure 2). This indicates that there is a defect in the proliferative clonal burst in MDS T-cells after stimulation. Reduced clonal burst potential suggests that the cells undergo premature cell death or apoptosis. The % of DAPI-positive cells prior to and after anti-CD3/CD28 stimulation was assessed in CD69+-activated T-cells. As shown in Figures 2e and f, cell death was significantly higher in activated T-cells from MDS patients compared with controls.
Short telomere length in naive T-cells in MDS patients suggests inherent loss of telomere maintenance
Telomeres may shorten secondary to proliferative stress, as previously hypothesized in MDS myeloid progenitors. In this case, telomere length should be shortest in memory cells that have been exposed to antigenic stimulation. To assess whether telomere dysfunction is a feature of antigen exposure, naive and memory populations were compared in cases and controls. sjTREC expression was examined and found, as expected, to reside primarily within the naive (CD45RA+CD45RO−) not the memory (CD45RO+CD45RA−) population (Figure 3a)17 in sorted populations, as shown in Figure 3c. Using phenotyping, naive T-cells have been shown to be reduced in MDS.19, 22, 23 Since naive T-cells are lost through a normal aging process, sjTREC copy number was compared in unstimulated samples from younger and older groups of cases and controls (Figure 3b). As indicated by sjTREC copies, MDS cases tended to have less naive cells in the 65+ age group (P=0.01). Although decreased in younger MDS cases compared with younger controls, this difference was not statistically significant (P=0.14, Wilcoxon).
Flow sorting was then used to isolate naive and memory cells, as shown in Figure 3c. Purified (sorted) naive and memory T-cells were compared in a subset of MDS cases (n=8) and healthy controls (n=8) with a sufficient number of cells available for sorting. This subgroup of cases and controls were individually age-matched. In healthy controls, the mean relative telomere length was significantly longer in naive T-cells compared with memory cells (P=0.006) reflecting their history of proliferative expansion (Figure 3d).10 The telomere length in sorted naive cells was significantly decreased among MDS cases compared with controls (P=0.018), whereas no case–control difference was observed for telomere length measured in memory cells. Thus, telomere length is shortest within the naive T-cell compartment in MDS patients consistent with an inherent telomere defect. Since it is critical to confirm the naive phenotype of the sorted MDS T-cells, additional surface markers including CD27, CD28 and CCR724 were used and indeed confirmed that the isolated naive cells have no evidence of antigen-activation-associated phenotypic changes (data not shown). These results indicate that shorter telomere length in bulk T-cell populations reflects in part a reduction in naive cells, as well as premature telomere attrition in antigen inexperienced, naive T-cells, indicating that the defect is not dependent on past antigen exposure.
Impaired inducible telomerase enzyme activity in T-cells in MDS patients
Telomeric repeats may be lost in the naive compartment if there is a defect in telomere repair as there is an exorbitant demand for proliferation in the naive compartment after thymic involution.21 In T-cells, telomerase activity is generally absent in resting cells and induced upon TCR activation.21 To assess the activity of telomerase in cases and controls, the TCR was stimulated with anti-CD3/anti-CD28-conjugated beads and the amount of enzyme activity determined by the TRAP assay. Preliminary assays were conducted to confirm the kinetics of telomerase activity and a 3-day stimulation period was found to be optimal (Figure 4a). Results were compared at day 0 (that is, basal activity) and after 3 days of stimulation (that is, inducible activity) in cases and controls. Basal telomerase activity was significantly higher in MDS cases compared with controls, but the levels were still very low compared with that of stimulated cells (Figure 4b). After stimulation, the amount of telomerase activity induced in MDS T-cells was significantly less (median=15.0, 95% CI, 13.4–17.2) than healthy controls (median=34.6, 95% CI, 33.6–39.3) (Wilcoxon P<0.0001) after adjustment for age and sex using log-transformed values of telomerase activity (P<0.0001). AA and MDS are clinically and epidemiologically linked bone marrow failure syndromes6 and large granular lymphocyte (LGL) leukemia is related to MDS by virtue of an association with cytopenias and clonal T-lymphocyte expansion.11 As shown in Supplementary Figure 3, telomere length in purified T-cells from patients with LGL leukemia was significantly shorter than control. We compared inducible telomerase activity in purified T-cells in LGL leukemia, MDS and AA cases and found similar levels of activity in LGL leukemia and controls, as shown in Figure 4b, but low telomerase activity in both of the highly related bone marrow failure diseases.
We then compared the telomerase activity in older and younger MDS case and control groups (Figure 4c) and observed a significant difference in both cases (P<0.0001 younger group and P=0.002 older group). No difference in telomerase activity was observed in the younger and older controls (P=0.50), indicating that telomerase function is preserved although telomere length shortens with age. To ensure that T-cells from patients were adequately responsive to TCR stimulation, we measured the surface expression of CD69; an inducible early activation antigen.25 In contrast to the impairment in telomerase activity, BrdU incorporation (Figure 2c) and PD (Figure 2d), the expression of CD69 was similar in younger (P=0.90) and older (P=0.88) cases and controls on day 3 (Figure 4d), suggesting that the telomerase defect is not due to a generalized loss in TCR signaling responses.
Given the data on telomere length in naive and memory cells, telomerase activity was examined among these cell populations. Inducible telomerase activity was greatest in control naive cells and significantly less in memory cells (P=0.02, Figure 4e). Moreover, the case:control difference in telomerase activity was present in naive cells (P=0.2) while the memory T-cell population showed no difference (P=0.50, Figure 4e) compared with controls, indicating that a primary telomerase deficiency underlies telomere loss in the naive T-cell compartment and that the defect is not related to prior antigen exposure.
hTERT transcriptional deficiency responsible for impaired telomerase function
Transcriptional induction of hTERT mRNA represents the rate-limiting step for telomerase regulation.26 Interstitial deletions on chromosome 5 occur in MDS and refined mapping of the region was recently examined using SNP-A. Abnormalities including deletions and uniparental disomy (UPD) were reported to occur in 142 (12%) of 1155 patients with MDS, MDS/myeloproliferative neoplasms and AML.27 The hTERT gene maps to chromosome 5p15.33, which is non-overlapping with regions spanning commonly deleted regions on 5q (Figure 5a). SNP-A was then performed on purified T-cells with data compared with an internal control series (n=1003) and the Database of Genomic Variants. No CNV or UPD were detected on chromosome 5 in purified T-cells (data not shown). To assess transcriptional regulation, basal and inducible hTERT mRNA expression was examined on day 0 and 3 after stimulation. As expected in control cells, there is little to no hTERT mRNA without stimulation (Figure 5b). Basal hTERT mRNA expression was higher in MDS cases (P<0.0001) compared with controls, but the inducible amount was significantly lower in cases (median=15.7, 95% CI, 14.9–20.5 vs median=46.0, 95% CI, 46.8–64.6 in controls) (P<0.0001) (Figure 5b) and this case–control difference was independent of age and sex using square-root transformed hTERT data. Comparing the younger and older patient and control subset, the hTERT deficiency was observed across both age groups (P<0.0001 in younger vs older cases and controls, Figure 5c).
The amount of inducible hTERT mRNA expression was then correlated to the level of inducible telomerase activity in T-cells, as shown in Figure 5d using square-root transformation to normalize hTERT data (r=0.89, P<0.001). The results suggest that there is a mechanistic link between telomerase deficiency and hTERT transcriptional impairment as indicated by the close correlation between these two events.
Sequencing of hTERT promoter
There are three main regions required for induction of hTERT expression consisting of a sequence from −203 to +55, corresponding to the promoter core, an activating region −1397 and −798, and an inhibitory region between −798 and −400. Several transcription factor binding sites are located in these regions, as shown in Supplementary Figure 4. No mutations were observed by direct sequencing of cloned hTERT promoter DNA from five MDS patients with 4–5 clones tested per patient.
No correlation between clinical classification and telomere repair
Measurements including telomere length, hTERT mRNA and inducible telomerase activity were correlated among patients to IPSS score, WHO subtype and cytogenetics, as shown in Table 2. There was no statistically significant association between these telomere variables and disease stratification, although patients with higher-risk (int−2+high) IPSS classification demonstrated a trend for shorter telomere length (P=0.1), lower induction of telomerase activity (P=0.06) and less hTERT mRNA expression (P=0.12) in T-cells.
Proliferation defect is related to telomerase deficiency not telomere length
Telomere length must be critically short to force cell-cycle arrest. It is possible that telomerase deficiency has important consequences that more broadly impact cell fate as treatment with RNA interference (siRNA) to hTERT resulted in an immediate suppression in replication potential and apoptosis without impacting telomere length.8 The proportion of T-cells in MDS patients that underwent S-phase transition (that is, % BrdU positive) was examined in relationship to telomerase activity and to telomere length. A mechanistic link between replication potential and telomerase activity is suggested by the close correlation (r=0.844, P<0.0001) between BrdU incorporation and telomerase enzymatic activity, but unrelated to telomere length (P=0.65) (Figures 6a and b).
Evidence indicates that accelerated telomere shortening occurs within the myeloid progenitor and stem cell compartments in patients with MDS,16, 28, 29, 30, 31, 32 while telomere length is preserved in stromal cells,33 suggesting that the defect is acquired within the hematopoietic compartment. Few studies have directly examined telomerase activity in MDS, and those that have were limited to unstimulated myeloid cells.29, 30 This study is the first to investigate the mechanism for pre-mature telomere attrition in MDS T-cells. For normal telomerase regulation, we show the need for TCR stimulation and demonstrate how this process is disrupted in all MDS patients compared with controls.
Telomerase plays a complex role during tumorigenesis and in the regulation of normal homeostasis. Normal telomerase activity coupled to short telomeres in LGL leukemia and older control individuals suggests that telomere repair is an incomplete restorative process. Replication in MDS T-cells was closely correlated to the activity of telomerase rather than to telomere length, indicating that telomerase broadly impacts cell fate. Telomere repair is predominantly functional within naive cells so the telomerase deficiency selectively affected the naive compartment and potentially contributed to reduced numbers of naive T-cells. Oligoclonal memory expansion and limited repertoire diversity are prominent, unexplained features of the disease.23 In order to maintain the total number of T-cells in the periphery, expansion of memory clones may fill the void left by the declining naive cell compartment. We found evidence of excessive activation-induced cell death in MDS T-cells consistent with abnormalities in this pathway.
Naive T-cells appear to prematurely reach replicative senescence possibly impacted by age and accelerated proliferation after thymic involution. A telomerase deficiency in naive T-cells hinders their regeneration, which may contribute to the accumulation of senescent cells in MDS. Some MDS patients have increased regulatory T-cells,34, 35 and enhanced liberation of inflammatory cytokines such as tumor necrosis factor-α,36, 37 Fas ligand4, 38 and other proapoptotic cytokines that may impact hematopoiesis and T-cell function.3, 5 Since senescent T-cells remain viable but produce more inflammatory cytokines and demonstrate non-MHC-restricted cytotoxicity through acquired natural-killer receptors,39 the telomere defect in naive cells may contribute indirectly to the inflammatory cytokine milieu in MDS.
Telomerase activity in MDS correlates with the amount of hTERT mRNA and our results are consistent with a deficiency in hTERT mRNA production. Although interstitial deletions of chromosome 5 occur in this disease,27 UPD and deletions in the region of the hTERT gene on 5p were not detected in T-cells. Signaling events leading to the transcriptional induction of CD69 expression are intact, indicating that the hTERT defect is selective. The minimal core promoter (located at −40 to −775) is required for expression and has a NFAT1-binding site flanked by two SP1-binding sites,26 as determined by luciferase and electrophoretic mobility shift assays using serial deletions of the 5′-UTR of the hTERT gene. Mutations in the core promoter region of the 5′-hTERT UTR were not detected in MDS patients (Supplementary Figure 4). Defects in telomere repair have been reported in inflammatory diseases such as rheumatoid arthritis and the defect is manifest by hTERT transcriptional repression.10, 40 In cancer and normal cells, mediators of hTERT transcription include cAMP responsive element binding protein (CREB), estradiol (E2) estrogen receptor α and β (ERα and ERβ) c-Myc, β-catenin/TCF4, HIF-1, signal transducers and activators of transcription (STAT)-3 and interferon regulatory factor 1 (IRF-1).41, 42 These transcription factors act downstream of well-defined signaling cascades such as Ras, PI3K/Akt, NF-κB, MAP kinase and GSK-3β.41 At this point, It is unclear which pathways are blocked in MDS T-cells. Examination of hTERT deficient T-cells in rheumatoid arthritis demonstrated that apoptosis induction is dependent on upregulation of DNA-damage sensing enzymes DNA-dependent protein kinase (DNA-PKcs), activation of pro-apoptotic BH3-only proteins Bim/Bmf and activation of the MAPK family member JNK, but was independent of pATM and p53.43 Increased expression of Bim/Bmf would be expected to induce apoptosis by stimulating the release of pro-apoptotic Bcl-2-family proteins like Bax/Bak, resulting in the activation of downstream caspases.44 Pharmacological inhibition of JNK with TLK199, a glutathione analog that inhibits JNK kinase activity, is now under investigation in MDS.45 Stabilization of apoptosis through inhibition of this pathway should be further verified. A strong case can be made for a mechanism involving suppression of hTERT through changes in specific signaling events that limits the cellular yield of T-cells by inducing cell death.
While multiple congenital and acquired mutations have been reported in hTERT, hTERC and shelterin proteins;6, 46, 47, 48 this is the first report implicating aberrant inducible hTERT transcriptional regulation in MDS or in bone marrow failure. Telomere length in peripheral blood leukocytes from MDS patients was independent of genotype in samples screened for hTERT polymorphisms and mutations in codons 202, 279, 305, 412, 441 and 1062.16 Codons 1062, 279 and 412 polymorphisms were previously shown to be more prevalent in AA.16, 47 Generally, myeloid and lymphoid cells in MDS are considered genetically divergent populations with the clonal population arising exclusively within a multipotent myeloid progenitor that gives rise to abnormal granulocytes, megakaryocytes and erythrocytes.49 A telomere defect within both the myeloid and lymphoid populations could be explained if the disease initiating event originates within a pluripotent stem cell with myeloid and lymphoid populating capacity. The origin of MDS within a true stem cell population is a matter of debate.49 Especially in MDS myeloid cells, critically short telomeres may be recognized as DNA damage, resulting in the recruitment and activation of the DNA-damage response pathway (that is, DNA-PKcs or p53 pathway), stimulating apoptosis and increasing genetic events.50 According to studies in solid tumors and in patients with congenital or acquired mutations in telomere components, individuals with shorter telomeres are at higher risk for developing malignancies due to genomic instability.32
The current study defines MDS as a member of the telomere repair disorders. Opportunities to improve MDS diagnosis and to design novel therapeutic interventions may be achieved from a better understanding of telomere abnormalities in T-cells and in the myeloid hematopoietic/stem cell compartment contributing to malignant transformation in MDS.
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Funding for this project was provided by NCI R01 Grant CA129952. Flow cytometry was supported by the H Lee Moffitt Cancer Center Flow Cytometry Core Facility and statistical analysis was performed with assistance from Jimmy J Fulp and Dr Dung-Tsa Chen from the H Lee Moffitt Cancer Center Biostatistics Program.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website
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Yang, L., Mailloux, A., Rollison, D. et al. Naive T-cells in myelodysplastic syndrome display intrinsic human telomerase reverse transcriptase (hTERT) deficiency. Leukemia 27, 897–906 (2013). https://doi.org/10.1038/leu.2012.300
- myelodysplastic syndrome
- T lymphocyte
- bone marrow failure
Expression and regulation of telomerase in human T cell differentiation, activation, aging and diseases
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