Leukemia (2007) 21, 2456–2462 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu ORIGINAL ARTICLE Short telomeres and high telomerase activity in T-cell prolymphocytic leukemia ARo¨th1,JDu¨rig1, H Himmelreich2,3, S Bug4, R Siebert4,UDu¨hrsen1, PM Lansdorp5,6 and GM Baerlocher2,3 1Department of Hematology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2Department of Hematology, University Hospital, Bern, Switzerland; 3Department of Clinical Research, University Hospital, Bern, Switzerland; 4Institute of Human Genetics, Christian-Albrechts-University Hospital Schleswig-Holstein, Kiel, Germany; 5Terry Fox Laboratory, BC Cancer Research Centre, Vancouver, Canada and 6Department of Medicine, University of British Columbia, Vancouver, Canada 4 5 To test the role of telomere biology in T-cell prolymphocytic repeats of the sequence T2AG3 and associated proteins folded leukemia (T-PLL), a rare aggressive disease characterized by into a telomere loop structure.6 Telomeres are required to the expansion of a T-cell clone derived from immuno-compe- tent post-thymic T-lymphocytes, we analyzed telomere length maintain chromosomal integrity and prevent end-to-end fusions and telomerase activity in subsets of peripheral blood leuko- of chromosomes. When telomeric ends become too short, DNA cytes from 11 newly diagnosed or relapsed patients with damage signals from telomeres can induce apoptosis or a state sporadic T-PLL. Telomere length values of the leukemic T cells of replicative senescence.7,8 Telomeres shorten with each round (mean7s.d.: 1.5370.65 kb) were all below the 1st percentile of of cell division as a result of failure to completely replicate the 30 telomere length values observed in T cells from healthy age- end of chromosomes9,10 as well as other causes.11 The average matched controls whereas telomere length of normal T- and telomere length in cells from most human tissues decreases with B cells fell between the 1st and 99th percentile of the normal 12,13 distribution. Leukemic T cells exhibited high levels of telomer- age in vivo and with culture in vitro. Disruption of control ase and were sensitive to the telomerase inhibitor BIBR1532 at pathways like Rb or p53 allows cells to bypass senescence. doses that showed no effect on normal, unstimulated T cells. Continued proliferation results in telomere attrition to the point Targeting the short telomeres and telomerase activity in T-PLL where shortened telomeres no longer protect chromosome ends, seems an attractive strategy for the future treatment of this leading to genomic instability facilitating the acquisition of devastating disease. Leukemia (2007) 21, 2456–2462; doi:10.1038/sj.leu.2404968; additional mutations important for cancer progression, for published online 27 September 2007 example, by a process of fusion-bridge breakage that leads to 14 Keywords: T-cell leukemia; telomere length; telomerase; the formation of complex nonreciprocal translocations. telomerase inhibitor The human telomerase complex, a ribonucleoprotein poly- merase, is able to synthesize terminal T2AG3 telomeric repeats de novo and thus extends or maintains telomeres.5,15 In contrast to germ line, stem or progenitor cells, most human somatic cells Introduction have only low or undetectable telomerase activity. Cells in numerous types of cancer and hematological malignancies T-cell prolymphocytic leukemia (T-PLL) is a rare aggressive upregulate telomerase to maintain telomeres in order to prevent leukemia, poorly responsive to conventional chemotherapy, 16,17 replicative senescence or apoptosis. Therefore, telomerase with a median survival of only 7.5 months in historic series.1 seems an attractive target for the development of new cancer This lymphoproliferative disease is characterized by specific 18,19 therapeutics. A variety of different classes of telomerase clinical manifestation, morphology, immunophenotype and 2 inhibitors, which block the catalytic activity of the enzyme or cytogenetics. Most T-PLL cells exhibit a mature post-thymic 20–22 the access of telomerase to telomeres, have been tested. CD2 þ , CD3 þ , CD7 þ , CD4 þ , CD8À immunophenotype but This therapeutic approach was also shown with the use of CD4 þ , CD8 þ and CD4À, CD8 þ cases are also seen. The dominant-negative mutant forms of hTERT, which lead to genetic hallmark of T-PLL is the rearrangement of chromosome 1 continuous telomere shortening and subsequent senescence or 14q32.1. The leukemic cells are generally resistant to alkylat- 23–26 apoptosis. A novel nonpeptidic, non-nucleosidic inhibitor ing drugs and the most promising treatment results are currently of the catalytic activity of telomerase is BIBR1532 (2-[(E)-3- achieved using chemo-immunotherapy protocols combining the naphtalen-2-yl-but-2-enoylamino]-benzoic acid). BIBR1532 anti-CD52 monoclonal antibody alemtuzumab with fludara- inhibits telomerase activity, comprising the human telomerase bine, anthracyclines and alkylating agents.1,3 reverse transcriptase and human telomerase RNA components, Telomeres are specialized structures at the end of eukaryotic in a dose-dependent manner, with half-maximal inhibitory chromosomes, which consist of 2–15 kb of noncoding tandem concentrations (IC50)of93nM by interfering with the processivity of the enzyme. BIBR1532 is a noncompetitive inhibitor with a drug- Correspondence: Dr A Ro¨th, Department of Hematology, University binding site distinct from the binding sites for deoxyribonucleotides Hospital Essen, Hufelandstr. 55, Essen D-45122, Germany. 27 E-mail: [email protected] or Dr G Baerlocher, Department and the DNA primer. of Hematology and Department of Clinical Research, University One of the problems for the potential clinical use of Hospital Bern, Freiburgstrasse 4, Bern CH-3010, Switzerland. telomerase inhibitors is that the effect is dependent on the E-mail: [email protected] telomere length reserve of the tumor cells. The response could Contributions: AR, JD and GMB designed the research; AR, HH, SB be delayed until chromosomes run out of their telomeric and GMB performed the experiments and collected the data; AR, JD, repeats. Thus, the optimal situation for telomerase inhibition RS and GMB analyzed and interpreted the data; AR, RS, UD, PML and GMB wrote the paper. might be tumor cells with extremely short telomeres and high Received 31 May 2007; revised 20 August 2007; accepted 29 August telomerase activity. Nevertheless, telomere length and levels of 2007; published online 27 September 2007 telomerase activity vary between different hematological Telomeres and telomerase in T-PLL ARo¨th et al 2457 neoplasias, which makes measurement of telomere length and telomerase activity a useful strategy to identify such situations.28 Here, we can demonstrate that T-PLL cells have extremely short telomeres and high levels of telomerase activity. Further- more, we show that inhibition of telomerase activity in T-PLL cells in vitro is cytotoxic, suggesting that telomerase inhibition 1mo should be further explored as a possible therapy for this o devastating disease. Materials and methods pentostatin), 4 mo (alemtuzumab), pentostatin), 1 mo (regimens, time post treatment) Patients and samples Ten T-PLL samples with cytogenetically detectable inv(14)(q11q32) or breakpoints in the TCL1 and TCRAD loci determined by fluorescence in situ hybridization (FISH)29 as well as one T-PLL sample with clonal changes but without evidence of inv(14)/TCRAD-TCL1 fusion diagnosed according to the WHO criteria were selected for this study (Table 1). Cells were obtained from peripheral venous blood samples after informed consent and according to institutional guidelines. First relapse Four regimens and 1 after relapse Peripheral blood mononuclear cells (PBMCs) were separated by density centrifugation using Ficoll-Hypaque (Pharmacia, Frei- burg, Germany). Genetic analyses were possible in all cases. Peripheral blood cells from age-matched healthy donors served as controls. Cell culture, BIBR1532 and cytospin preparation inv(14)(q11q32)F Primary diagnosisinv(14)(q11q32) Untreated Primaryinv(14)(q11q32) diagnosis Primary diagnosis Untreated Untreated Cultures were initiated with 1 Â 106 cells mlÀ1 per well in inv(14)(q11q32)inv(14)(q11q32) Primary diagnosis Primary diagnosisinv(14)(q11q32) One regimen (alemtuzumab), 1 mo Two regimens Primary (chlorambucil, diagnosis Two regimens (bendamustin, 24-well tissue culture plates (Falcon, Becton Dickinson, Heidelberg, Germany) with fresh or thawed samples. T-PLL À À À À cells and normal T cells were cultured in Roswell Park Memorial À À À Institute (RPMI)-1640 medium (Gibco, Karlsruhe, Germany) containing 10% pooled human AB serum (HS, Sigma, Munich, Germany), 100 U mlÀ1 penicillin per 100 mgmlÀ1 streptomycin (Gibco), 2 mML-glutamine (Gibco), 1 mM sodium pyruvate MEM FACS Cytogenetics Disease status Treatment (Gibco), 1 Â MEM non essential amino acids (Gibco), 100 mgmlÀ1 kanamycin (Gibco), 0.05 mM 2-mercaptoethanol À1 ) (Gibco) supplemented with 100 U ml recombinant interleu- 1 À l kin-2 (rIL-2; Proleukin, Chiron, Munich, Germany). T-PLL 6 samples exhibited no active proliferation or expansion in 10 Â short-term culture. BIBR1532 was synthesized by Boehringer ( Ingelheim (Biberach, Germany) and a stock solution of BIBR1532 at a concentration of 5 mM was prepared by dissolving the compound in 50% sterile dimethyl sulfoxide (DMSO) and RPMI 1640 medium (Gibco) and stored at À20 1C until use. Trypan blue exclusion was used to