Annika Scheffold

Telomerase gene mutations are associated with cirrhosis formation

Telomere shortening impairs liver regeneration in mice and is associated with cirrhosis formation in humans with chronic liver disease. In humans, telomerase mutations have been associated with familial diseases leading to bone marrow failure or lung fibrosis. It is currently unknown whether telomerase mutations associate with cirrhosis induced by chronic liver disease. The telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT) were sequenced in 1,121 individuals (521 patients with cirrhosis induced by chronic liver disease and 600 noncirrhosis controls). Telomere length was analyzed in patients carrying telomerase gene mutations. Functional defects of telomerase gene mutations were investigated in primary human fibroblasts and patient‐derived lymphocytes. An increased incidence of telomerase mutations was detected in cirrhosis patients (allele frequency 0.017) compared to noncirrhosis controls (0.003, P value 0.0007; relative risk [RR] 1.859; 95% confidence interval [CI] 1.552‐2.227). Cirrhosis patients with TERT mutations showed shortened telomeres in white blood cells compared to control patients. Cirrhosis‐associated telomerase mutations led to reduced telomerase activity and defects in maintaining telomere length and the replicative potential of primary cells in culture. Conclusion: This study provides the first experimental evidence that telomerase gene mutations are present in patients developing cirrhosis as a consequence of chronic liver disease. These data support the concept that telomere shortening can represent a causal factor impairing liver regeneration and accelerating cirrhosis formation in response to chronic liver disease. (HEPATOLOGY 2011;)

Telomere shortening reduces Alzheimer's disease amyloid pathology in mice

Alzheimers disease is a neurodegenerative disorder of the elderly and advancing age is the major risk factor for Alzheimers disease development. Telomere shortening represents one of the molecular causes of ageing that limits the proliferative capacity of cells, including neural stem cells. Studies on telomere lengths in patients with Alzheimers disease have revealed contrary results and the functional role of telomere shortening on brain ageing and Alzheimers disease is not known. Here, we have investigated the effects of telomere shortening on adult neurogenesis and Alzheimers disease progression in mice. The study shows that aged telomerase knockout mice with short telomeres (G3Terc-/-) exhibit reduced dentate gyrus neurogenesis and loss of neurons in hippocampus and frontal cortex, associated with short-term memory deficit in comparison to mice with long telomere reserves (Terc+/+). In contrast, telomere shortening improved the spatial learning ability of ageing APP23 transgenic mice, a mouse model for Alzheimers disease. Telomere shortening was also associated with an activation of microglia in ageing amyloid-free brain. However, in APP23 transgenic mice, telomere shortening reduced both amyloid plaque pathology and reactive microgliosis. Together, these results provide the first experimental evidence that telomere shortening, despite impairing adult neurogenesis and maintenance of post-mitotic neurons, can slow down the progression of amyloid plaque pathology in Alzheimers disease, possibly involving telomere-dependent effects on microglia activation.

Mutual exacerbation of peroxisome proliferator-activated receptor γ coactivator 1α deregulation and α-synuclein oligomerization

Aggregation of α‐synuclein (α‐syn) and α‐syn cytotoxicity are hallmarks of sporadic and familial Parkinson disease (PD), with accumulating evidence that prefibrillar oligomers and protofibrils are the pathogenic species in PD and related synucleinopathies. Peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α), a key regulator of mitochondrial biogenesis and cellular energy metabolism, has recently been associated with the pathophysiology of PD. Despite extensive effort on studying the function of PGC‐1α in mitochondria, no studies have addressed whether PGC‐1α directly influences oligomerization of α‐syn or whether α‐syn oligomers impact PGC‐1α expression.

Transient telomere dysfunction induces chromosomal instability and promotes carcinogenesis

Telomere shortening limits the proliferative capacity of a cell, but perhaps surprisingly, shortening is also known to be associated with increased rates of tumor initiation. A current hypothesis suggests that telomere dysfunction increases tumor initiation by induction of chromosomal instability, but that initiated tumors need to reactivate telomerase for genome stabilization and tumor progression. This concept has not been tested in vivo, since appropriate mouse models were lacking. Here, we analyzed hepatocarcinogenesis in a mouse model of inducible telomere dysfunction on a telomerase-proficient background, in telomerase knockout mice with chronic telomere dysfunction (G3 mTerc-/-), and in WT mice with functional telomeres and telomerase. Transient or chronic telomere dysfunction enhanced the rates of chromosomal aberrations during hepatocarcinogenesis, but only telomerase-proficient mice exhibited significantly increased rates of macroscopic tumor formation in response to telomere dysfunction. In contrast, telomere dysfunction resulted in pronounced accumulation of DNA damage, cell-cycle arrest, and apoptosis in telomerase-deficient liver tumors. Together, these data provide in vivo evidence that transient telomere dysfunction during early or late stages of tumorigenesis promotes chromosomal instability and carcinogenesis in telomerase-proficient mice.

MiR-139-5p is a potent tumor suppressor in adult acute myeloid leukemia.

Hematopoiesis depends on a tightly controlled balance of selfrenewal, proliferation, cell death and differentiation. Although the disturbance of this equilibrium creates a predisposition to leukemogenesis, targeted manipulation or modulation can in turn lead to therapeutic advances. In addition to chromosomal aberrations and frequently mutated genes such as NPM1 and FLT3, dysregulation of microRNAs (miRNAs) is now recognized as having an important role in leukemogenesis. Distinct miRNA expression profiles can classify AML subgroups and miRNAs have recently emerged as novel therapeutic targets in hematopoietic malignancies. Building on our previous efforts to resolve potential involvement of miRNAs in hematopoiesis, we identified miR-139-5p as a myeloid-specific miRNA with expression being restricted to neutrophils and macrophages (Supplementary Figure S1A). This distinct expression profile during normal hematopoiesis pointed towards a possible deregulation in malignant hematopoiesis. In order to relate miR-139-5p expression to distinct genetic subgroups and clinical outcome in adult AML patients, we analyzed the published miRNA sequencing dataset from The Cancer Genome Atlas (TCGA). A trend for prolonged overall survival (OS) was observed for patients with miR-139-5p levels above the median independent of cytogenetic subgroup (P= 0.07, Figure 1a left panel), whereas AML patients with normal karyotype (CN-AML) exhibited a significantly better OS (P= 0.02, Figure 1a right panel). Our findings were further corroborated in CN-AML by comparing the expression quartiles, demonstrating that low miR-139-5p expressors (q3 and q4) are associated with an unfavorable OS (Supplementary Figure S1B). In addition, no effect could be demonstrated for the less abundant miR-139-3p (Supplementary Figure S1C), indicating that miR-139-5p is the active strand. Our findings are in line with Emmrich et al., who associated high miR-139-5p levels with a favorable outcome in pediatric AML. Further analysis of the TCGA dataset revealed a significant downregulation of miR-139-5p in CN-AML patients harboring mutated FLT3 (P= 0.03 and P= 0.001, respectively) compared with the total AML population. (Figure 1b left panel). Considering the poor prognosis of CN-AML patients with mutated FLT3, our findings reinforce the unfavorable outcome of CN-AML patients with low miR-139-5p levels. In contrast, miR-139-5p was upregulated in patients carrying an inv(16) (P= 0.001) translocation, a prognostically favorable AML subgroup (Figure 1b left panel). No differences were found for miR-139-5p levels in AML patients with t(15;17) and MLL rearrangements, indicating that miR-139-5p levels are associated with specific genetic subtypes rather than with the degree of differentiation. These findings were validated by qRT-PCR in an independent cohort consisting of 49 adult AML patients and 4 healthy donors of total bone marrow (bm) referred to as the Ulm cohort with uniform treatment procedure. All patient samples from the Ulm cohort were collected from adult patients enroled on German-Austrian AML Study Group (AMLSG) treatment protocols for younger adults (AMLSG-HD98A (NCT00146120) and AMLSG 07-04 (NCT00151242)) and comprised Ficoll gradient purified mononuclear cells mainly from diagnostic bm samples with blast counts of ~ 80% in all analyzed cases. In line with the TCGA dataset, patients harboring mutated FLT3 displayed the lowest miR-139-5p expression among the various cytogenetic subtypes in the Ulm cohort (P= 0.03, Figure 1b right panel). Decreased expression of miR-139 in NPM1-mutated CNAML patients, has been previously reported by Garzon et al. We observed a similar trend within the TCGA dataset (P= 0.06) (Figure 1b left panel), highlighting the robustness of both the datasets. Taken together, we have demonstrated in two independent AML patient cohorts that decreased miR-139-5p levels are associated with mutated FLT3 and with a lower OS in CN-AML in the TCGA dataset. However, it remains to be determined if miR-139-5p is directly or indirectly transcriptionally regulated through activated FLT3. The distinct expression patterns of miR-139-5p in CN-AML imply a tumor suppressor role for this miRNA. In order to further explore the therapeutic potential of miR-139-5p, we used a syngenic bm transplantation AML model based on the transformation of murine bm cells through the combined retroviral overexpression of Hoxa9 and Meis1 (Hoxa9/Meis1), which causes a rapid AML in vivo compared with the pre-leukemic Hoxa9 cells that lead to a long latency AML. Upregulation of HOXA9 and MEIS1 is frequently observed in NPM1-mutated CN-AML and enhances FLT3 signaling. Based on the expression pattern of miR-139-5p in AML patients, we further hypothesized that miR-139-5p would be downregulated in Hoxa9/Meis1 cells compared with untransformed wild-type bm cells. Indeed, miR-139-5p was significantly downregulated in Hoxa9/Meis1 cells compared with untransduced bm cells as measured by qRT-PCR (Supplementary Figure S1D), highlighting the relevance of this in vivo model. To validate the putative tumor suppressor function of miR-139-5p, its expression levels were restored through ectopic lentiviral coexpression of miR-139 and GFP as reported in Hoxa9/Meis1-transformed bm cells. Hoxa9/Meis1/miR-139-5p bm or Hoxa9/Meis1 cells overexpressing an empty control vector (miR-ctrl) were FACS-sorted for GFP, followed by transplantation of 2 × 10 cells per arm into lethally irradiated recipient mice. Restoration of miR-139 levels significantly delayed Hoxa9/Meis1-mediated leukemogenesis (P= 0.0003, Figure 2a) in two independent experiments, highlighting its tumor suppressor activity in a primary transplantation model. Bm cells of deceased mice retained overexpression of miR-139-5p compared with the control arm as quantified by qRT-PCR (Supplementary Figure S1E). Lentiviral expression of miR-139-5p in Hoxa9/Meis1 cells restored miR-139-5p at similar levels compared with untransduced bm cells (Supplementary Figures 1D and E), minimizing possible artefacts of ectopic expression. Notably, we detected no endogenous expression of miR-139-3p in the Hoxa9/Meis1/miR-ctrl bm and overexpression of miR-139 generated lower levels of miR-139-3p in comparison with miR-139-5p, highlighting miR-139-5p as the active strand (Supplementary Figure S1F). Recently, Gibbs et al. demonstrated that Hoxa9/Meis1 cells harbor three, immunophenotypically distinct compartments with varying tumor-initiating activity. Therefore, bm cells of deceased mice were analyzed by flow cytometry for their respective immunophenotype (Figure 2b). We found a significant reduction Citation: Blood Cancer Journal (2016) 6, e508; doi:10.1038/bcj.2016.110

Telomere shortening leads to an acceleration of synucleinopathy and impaired microglia response in a genetic mouse model

Parkinson’s disease is one of the most common neurodegenerative disorders of the elderly and ageing hence described to be a major risk factor. Telomere shortening as a result of the inability to fully replicate the ends of linear chromosomes is one of the hallmarks of ageing. The role of telomere dysfunction in neurological diseases and the ageing brain is not clarified and there is an ongoing discussion whether telomere shortening is linked to Parkinson’s disease. Here we studied a mouse model of Parkinson’s disease (Thy-1 [A30P] α-synuclein transgenic mouse model) in the background of telomere shortening (Terc knockout mouse model). α-synuclein transgenic mice with short telomeres (αSYNtg/tg G3Terc-/-) developed an accelerated disease with significantly decreased survival. This accelerated phenotype of mice with short telomeres was characterized by a declined motor performance and an increased formation of α-synuclein aggregates. Immunohistochemical analysis and mRNA expression studies revealed that the disease end-stage brain stem microglia showed an impaired response in αSYNtg/tg G3Terc-/- microglia animals. These results provide the first experimental data that telomere shortening accelerates α-synuclein pathology that is linked to limited microglia function in the brainstem.

Impact of telomere length on the outcome of allogeneic stem cell transplantation for poor-risk chronic lymphocytic leukaemia: results from the GCLLSG CLL3X trial.

Chronic lymphocytic leukaemia (CLL) is the most common malignancy of the B cell system, affecting predominantly the elderly, with a median diagnostic age around 72 years (Rai, 2015). CLL has a very heterogeneous clinical course ranging from indolent disease to genetically-defined poor prognostic groups with aggressive course under standard chemoimmunotherapy (Zenz et al, 2010; Stilgenbauer et al, 2015). Although novel signalling pathway inhibitors have changed the therapeutic landscape (Rai, 2015; Stilgenbauer et al, 2015), allogeneic stem cell transplantation (allo-SCT) has been shown to be the only curative therapy in patients with high-risk CLL, overcoming the impact of adverse prognostic factors. The German CLL study group (GCLLSG) CLL3X study evaluated allo-SCT in patients with poor risk CLL. Overall survival (OS) was 53% after a median follow-up of 6 years (0 6–10 8 years), (Dreger et al, 2010). The primary factors associated with outcome were prior response to chemotherapy and minimal residual disease clearance after allo-SCT, but not genetic markers (including high-risk characteristics, e.g., 17p-, mutated TP53) (Dreger et al, 2013, 2010). Among the many prognostic factors, short telomere length was shown to be an independent adverse factor in CLL (Roos et al, 2008; Rampazzo et al, 2012; Mansouri et al, 2013; Lin et al, 2014). Short telomeres at diagnosis are associated with poor-risk clinical and biological disease characteristics (Roos et al, 2008; Mansouri et al, 2013; Lin et al, 2014), but the impact of telomere length following allo-SCT has never been studied. We analysed telomere length on samples from CLL3X (N = 58), at baseline prior to allo-SCT and studied associations with disease characteristics and impact on survival. The CLL3X trial was a multicentre phase 2 clinical study, described previously (Dreger et al, 2013, 2010), which included 100 patients (median age, 53 years; range: 27–65 years). A representative cohort of 58 patients was included for analysis of telomere length (Table S1). As the samples were not CD19 enriched, only cases fulfilling at least one of the criteria in Table S2 were included to ensure analysis of CLL tumour cell telomere length. The study was approved by the institutional review boards

Mutual exacerbation of PGC-1α deregulation and α-synuclein oligomerization

Aggregation of α‐synuclein (α‐syn) and α‐syn cytotoxicity are hallmarks of sporadic and familial Parkinson disease (PD), with accumulating evidence that prefibrillar oligomers and protofibrils are the pathogenic species in PD and related synucleinopathies. Peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α), a key regulator of mitochondrial biogenesis and cellular energy metabolism, has recently been associated with the pathophysiology of PD. Despite extensive effort on studying the function of PGC‐1α in mitochondria, no studies have addressed whether PGC‐1α directly influences oligomerization of α‐syn or whether α‐syn oligomers impact PGC‐1α expression.

Substantial telomere shortening in the substantia nigra of telomerase-deficient mice does not increase susceptibility to MPTP-induced dopamine depletion.

The most important risk factor for developing Parkinson’s disease (PD) is age. Aging is ascribed to different mechanisms, including telomere shortening. Telomeres consist of repetitive DNA sequences and stabilize chromosome integrity. Currently, however, the data reported on telomere shortening in PD patients are inconsistent. We investigated the effect of telomere shortening in the MPTP mouse model of PD using late-generation telomerase-deficient mice (G3 Terc−/− mice). G3 Terc−/− mice showed a reduction in telomere length in nigral tyrosine hydroxylase-positive neurons by 40%, as indicated by quantitative fluorescence in-situ hybridization. There was no difference in the total motor activity and striatal tissue concentrations of dopamine, DOPAC (3,4-dihydroxyphenylacetic acid), HVA (4-hydroxy-3-methoxyphenylacetic acid), and 3-MT (3-methoxytyramine) concentrations or dopamine turnover in G3 Terc−/− mice in comparison with controls without MPTP treatment. Low-dose MPTP treatment (four injections, 2 h intervals, 2×5 and 2×7.5 mg/kg) led to a significant decrease in striatal dopamine concentrations that did not differ in G3 Terc−/− mice compared with control mice (19.15±0.44 to 12.81±1.26 ng/mg in control mice in comparison with 19.51±0.59 to 13.56±1.10 ng/mg in G3 Terc−/− mice). In conclusion, telomere shortening does not increase susceptibility to MPTP-induced dopamine depletion in mice. These data indicate that other age-related mechanisms in the brain may play a more important role in enhancing MPTP-induced dopamine depletion.

Telomere length in poor-risk chronic lymphocytic leukemia: associations with disease characteristics and outcome

Abstract Telomere length in chronic lymphocytic leukemia (CLL) is described as an independent prognostic factor based largely on previously untreated patients from chemotherapy based trials. Here, we studied telomere length associations in high-risk, relapsed/refractory CLL treated with alemtuzumab in the CLL2O study (n = 110) of German and French CLL study groups. Telomere length (median 3.28 kb, range 2.52–7.24 kb) was relatively short, since 84.4% of patients had 17p- which is generally associated with short telomeres. Median telomere length was used for dichotomization into short and long telomere subgroups. Telomere length was associated with s-TK (p = .025) and TP53 mutations (p = .050) in untreated patients, while no association with clinical/biological characteristics was observed in relapsed/refractory CLL. Short telomeres had significant association with shorter PFS (p = .018) only in refractory CLL. Presence of short telomeres, loss of genes maintaining genomic integrity (SMC5) and increased incidence of chromothripsis, indicated the prevalence of genomic instability in this high-risk cohort (clinicaltrials.gov: NCT01392079).