Telomerase Inhibitor BIBR1532 Induces Telomere Shortening and Growth Inhibition in Extra-Germinal Center (GC)-Derived Lymphoid Cell Lines.

Abstract Background and aim of the study. Telomerase inhibition is a novel promising anticancer strategy which has been pursued using antisense oligonucleotides and more recently pharmacological inhibitors. BIBR-1532 is a mixed-type non-competitive inhibitor of the catalytic subunit of telomerase (h-TERT). In solid tumor cell lines, BIBR-1532 successfully induced telomere shortening and growth inhibition (Damm K et al, The Embo J, 2001). The activity of BIBR-1532 has not been so far evaluated in hematological cancers. Aim of this study is to evaluate the activity of this compound in cell lines derived from B-cell lymphoid tumors. These neoplasms are an interesting model to investigate BIBR1532 since they show marked differences in their telomere lenght according to GC origin (i.e. GC-derived have long telomeres and extra GC-derived have short telomeres)(Ladetto et al Blood 2004). Methods. We employed two extra-GC derived cell lines (the multiple myeloma KMS-11 and the chronic lymphocytic leukemia JVM-2 provided respectively by M. Massaia MD and C Carlo Stella MD) and two GC-derived cell lines (the Burkitt’s Lymphoma CA46 and the follicular lymphoma cell line DHL16 provided by J.G. Gribben MD). All the four cell lines were h-TERT positive. BIBR-1532 (kindly provided by Klaus Damm MD and Jacques van Meel, PhD, Boehringer Ingelheim Pharma, Germany) was administrated thrice a week to the standard concentration of 10μM. Cells were cultured for at least 80 mitotic divisions both in presence and absence of the agent. Cell growth was assessed three times a week. Aliquots of cultured cells were taken approximately every 20 days for telomere length evaluation. Cells were also assessed using the propidium iodide assay to verify whether growth inhibition was due to apotosis or prolipherative arrest. Results. As expected based on their GC-status, the four cell lines had different baseline telomere length (CA46: 6500bp DHL 16: 7000bp KMS11 3200bp, JVM-2 3500). When cells were cultured without BIBR 1532 no significant telomere shortening occurred over the whole culture period. In the presence of BIBR-1532, cell lines approximately lost 32bp at each mitotic division. After 20 mitoses KMS-11 and JVM-2 underwent marked and progressive growth inhibition with a clear plateau effect after 50 to 55 mitoses. In contrast no growth inhibition was noticed in GC-derived cell lines despite a similar degree telomere erosion. Growth inhibition in cell lines responsive to BIBR-1532 was mostly associated with G1 arrest. Conclusions: These results are the first reported experience on the use of BIBR1532 on B-lymphoid cell lines and indicate that: a) BIBR1532 effectively induces telomere shortening in lymphoid tumors; b) in extra-GC telomere erosion quickly lead to pharmacologically-induced senescence with marked growth inhibition; c) GC-derived lymphoid cell lines are effectively protected from pharmacological senescence induced by BIBR 1532 due to their long telomeres. These results indicate that telomerase inhibitors require extensive evaluation as anti-cancer agents in extra-GC-derived lymphoid tumors.