Telomere elongation and clinical response to androgen treatment in a patient with aplastic anemia and a heterozygous hTERT gene mutation.

Telomere length (TL) both reflects and limits the replicative life span of normal somatic cells. As a consequence, critically shortened telomeres are associated with a variety of disease states. Telomere attrition can be counteracted by a nucleoprotein complex containing telomerase. Mutations in subunits of telomerase, telomerase-binding proteins as well as in members of the shelterin complex have been described both in inherited and acquired bone marrow failure syndromes. Here, we report on a patient with acquired aplastic anemia and a nonsynonymous variation of codon 1062 of the hTERT gene (p.Ala1062Thr) whose substantial and maintained hematologic response to long-term androgen treatment (including complete transfusion independence) was paralleled by a significant and continued increase in TL in multilineage peripheral blood cells. To our knowledge, this represents the first case of sustained telomere elongation in hematopoietic stem cells induced by a pharmacological approach in vivo (141 words).

Functional p53 is required for effective execution of telomerase inhibition in BCR-ABL-positive CML cells.

OBJECTIVE: In chronic myeloid leukemia (CML), increased cellular turnover of hematopoietic cells driven by the oncogene BCR-ABL leads to accelerated telomere shortening despite increased telomerase activity. It has been postulated that shortened telomeres, particularly in the context of increased telomerase activity, might facilitate accumulation of genetic aberrations and, consequently, disease progression from chronic phase to accelerated phase and blast crisis. Therefore, inhibition of telomerase might be a promising approach in CML therapy. MATERIAL AND METHODS: To investigate the therapeutic potential of telomerase inhibition in this model disorder, we used a small molecule telomerase inhibitor, BIBR1532 as well as expression of a dominant-negative mutant of hTERT (DNhTERT-IRES-GFP) in the p53-negative CML blast crisis cell line K562 and characterized the effects in long-term culture. Furthermore, we expressed an inducible p53 construct (vector pBabe-p53ER(tam)) via retroviral transduction in cells with critically short telomeres and in cells with a normal telomere length to explain the role of the tumor suppressor in response to critical telomere shortening in BCR-ABL-positive cells. RESULTS: BIBR1532-treated bulk cultures did not show altered growth kinetics despite significant telomere shortening to a critical length of approximately 5 kb. In comparison, DNhTERT-expressing clones either lost telomere length, leading to a significant but transient slow down in proliferation but eventually all escaped senescence/crisis (group I) or, alternatively, remained virtually unaffected despite measurable telomerase inhibition (group II). Further analyses of group I clones revealed impaired DNA damage response and an accumulation of dicentric chromosomes. However, upon restoration of p53 in telomerase-negative K562 clones with critically short telomeres, immediate reinduction of apoptosis and complete eradication of cells was observed, whereas vector control cells continued to escape from crisis. CONCLUSIONS: These results suggest that the success of strategies aimed at telomerase inhibition in CML is highly dependent on the presence of functional p53 and should be explored preferentially in chronic phase CML.

Telomeres and telomerase in chronic myeloid leukaemia: impact for pathogenesis, disease progression and targeted therapy.

Telomeres are specialized structures localized at the end of human chromosomes. Due to the end replication problem, each cell division results in a loss of telomeric repeats in normal somatic cells. In germ line and stem cells, the multicomponent enzyme telomerase maintains the length of telomere repeats. However, elevated telomerase activity has also been reported in the majority of solid tumours as well as in acute and chronic leukaemia. Chronic myeloid leukaemia (CML) serves as a model disease to study telomere biology in clonal myeloproliferative disorders. In CML, telomere shortening correlates with disease stage, duration of chronic phase (CP), prognosis measured by the Hasford risk score and the response to disease-modifying therapeutics such as the tyrosine kinase inhibitor Imatinib. In addition, telomerase activity (TA) is already increased in CP CML and further upregulated with disease progression to accelerated phase and blast crisis (BC). Furthermore, a correlation of TA with increased genetic instability as well as a shorter survival of the patients has been reported. Here, we review the current state of knowledge of the role of telomere and telomerase biology in CML and discuss the possible impact of novel treatment approaches.

Novel KRIT1 mutation and no molecular evidence of anticipation in a family with cerebral and spinal cavernous malformations.

BACKGROUND: Cerebral cavernous malformations (CCM) are vascular brain anomalies which can result in a variety of neurological symptoms. Familial CCM is inherited as an autosomal-dominant trait. There is one study in the literature which reports statistical evidence for anticipation in familial CCM. METHODS: We reevaluated the clinical course of the disease and performed molecular analyses in a previously described three-generation CCM family with apparent anticipation. RESULTS: Disease started at a younger age in each generation, strongly suggesting anticipation. The patient in generation I showed no clinical symptoms by the age of 68, whereas his son became wheelchair-bound at the age of 43 due to an intramedullary cavernous malformation at the thoracolumbar transition of the spinal cord. The patient in generation III had a pons hemorrhage at the age of 11 due to a large brainstem cavernoma. The hemorrhage caused facial palsy and hemiparesis, persisting as Millard-Gubler syndrome. Sequencing of KRIT1 identified a novel frameshift mutation in exon 15 (c.1561delC or p.Leu551X) which cosegregated with the phenotype. Flow-FISH analysis of granulocyte and lymphocyte telomere length showed that telomeres were longest in the youngest affected family member. CONCLUSIONS: We could not find any evidence for either of the two currently known molecular mechanisms for genetic anticipation (i.e., expansion of repetitive DNA elements or progressive telomere shortening) in this family. However, the family presented here raises the important question whether surveillance of CCM families with gradient-echo MRI should not only include the cerebrum, but the spinal cord as well.

Hypusination of eukaryotic initiation factor 5A (eIF5A): a novel therapeutic target in BCR-ABL-positive leukemias identified by a proteomics approach.

Inhibition of BCR-ABL tyrosine kinase with imatinib represents a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). However, resistance to imatinib develops frequently, particularly in late-stage disease. To identify new cellular BCR-ABL downstream targets, we analyzed differences in global protein expression in BCR-ABL-positive K562 cells treated with or without imatinib in vitro. Among the 19 proteins found to be differentially expressed, we detected the down-regulation of eukaryotic initiation factor 5A (eIF5A), a protein essential for cell proliferation. eIF5A represents the only known eukaryotic protein activated by posttranslational hypusination. Hypusination inhibitors (HIs) alone exerted an antiproliferative effect on BCR-ABL-positive and -negative leukemia cell lines in vitro. However, the synergistic dose-response relationship found for the combination of imatinib and HI was restricted to Bcr-Abl-positive cells. Furthermore, this synergistic effect was confirmed by cytotoxicity assays, cell-cycle analysis, and CFSE labeling of primary CD34+ CML cells. Specificity of this effect could be demonstrated by cotreatment of K562 cells with imatinib and siRNA against eIF5. In conclusion, through a comparative proteomics approach and further functional analysis, we identified the inhibition of eIF5A hypusination as a promising new approach for combination therapy in BCR-ABL-positive leukemias.