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. 2013 Jul 29;8(7):e70428.
doi: 10.1371/journal.pone.0070428. Print 2013.

Telomerase Contributes to Fludarabine Resistance in Primary Human Leukemic Lymphocytes

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Free PMC article

Telomerase Contributes to Fludarabine Resistance in Primary Human Leukemic Lymphocytes

May Shawi et al. PLoS One. .
Free PMC article

Abstract

We report that Imetelstat, a telomerase inhibitor that binds to the RNA component of telomerase (hTR), can sensitize primary CLL lymphocytes to fludarabine in vitro. This effect was observed in lymphocytes from clinically resistant cases and with cytogenetic abnormalities associated with bad prognosis. Imetelstat mediated-sensitization to fludarabine was not associated with telomerase activity, but with the basal expression of Ku80. Since both Imetelstat and Ku80 bind hTR, we assessed 1) if Ku80 and Imetelstat alter each other's binding to hTR in vitro and 2) the effect of an oligonucleotide complementary to the Ku binding site in hTR (Ku oligo) on the survival of primary CLL lymphocytes exposed to fludarabine. We show that Imetelstat interferes with the binding of Ku70/80 (Ku) to hTR and that the Ku oligo can sensitize CLL lymphocytes to FLU. Our results suggest that Ku binding to hTR may contribute to fludarabine resistance in CLL lmphocytes. This is the first report highlighting the potentially broad effectiveness of Imetelstat in CLL, and the potential biological and clinical implications of a functional interaction between Ku and hTR in primary human cancer cells.

Conflict of interest statement

Competing Interests: SMG works at Geron Corporation. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Telomerase Activity is associated with in vitro FLU resistance.
A) Basal telomerase activity (y-axis) was lower in FLU sensitive CLL lymphocyte samples (left bar) compared to FLU resistant CLL lymphocyte samples (right bar) (Mann-Whitney Rank Sum Test, *p = 0.02). The cut off to define sensitivity or resistance to FLU was 2.5 µM, the median FLU IC50. B) FLU resistance of lymphocytes from CLL patients (y-axis) was associated with telomerase activity measured after FLU treatment (x-axis). Pearson Product Moment Correlation coefficient of r = 0.75 with a value p = 0.003. The solid line is the regression line. The straight dotted line represents the confidence interval of the population and the curved dotted line the confidence interval of the regression. C) Effect on telomerase activity (y-axis) of the indicated treatments (x-axis). Kruskal-Wallis One Way Analysis of Variance on Ranks indicates that the treatments significantly affected telomerase activity (****p = 0.007) followed by Wilcoxon Signed Rank test for paired samples (p =  *0.006; **p = <0.001 and ***P = 0.002). D) Imetelstat significantly sensitized primary CLL lymphocytes to FLU. The R value obtained with Imetelstat was significantly different from 1, Wilcoxon Signed Rank test of paired samples indicates a significant difference (*p<0.001).
Figure 2
Figure 2. Imetelstat-mediated sensitization to FLU is associated with Ku expression.
A) The plot represents the sensitization values obtained (y-axis) for CLL lymphocytes from each case (x-axis). The arrows indicate the clinically resistant (CR) cases and cases positive for del11 and del17. The ** indicated a del17 case that changed from untreated to clinically resistant with increased percentage of cells positive for del17 (Table S1). B) Comparison of the sensitization R values (y-axis) between telomerase negative (right bar) and telomerase positive samples (left bar) (Mann-Whitney Rank Sum Test p = 0.295). C) Ku80 expression (y-axis) in primary CLL lymphocytes samples segregated by the median R value (1.5) (Mann-Whitney Rank Sum Test *p = 0.03).
Figure 3
Figure 3. A catalytic site inhibitor of telomerase or an oligonucleotide targeting the Ku binding site of hTR sensitize CLL lymphocytes to FLU.
A) Formation of the Ku70/80-hTR complex is completely inhibited by 1 µM Imetelstat (n = 3) but not by the control sense oligonucleotide (n = 2). Lane 1: radiolabeled hTR (denoted by **). Lane 2: The formation of a Ku70/80-hTR complex (denoted by *) occurs upon the addition of 0.2ug Ku70/80. Increasing concentrations of 0.5, 1 and 2 µM Imetelstat (lanes 3–5) or a control sense oligonucleotide (lanes 8–10) were added. B) Addition of Ku resulted in supershifts of the hTR-Imetelstat complex. Lane 1: radiolabeled Imetelstat. Lane 2: The formation of hTR-Imetelstat complex occurs upon addition of 0.02 pmol hTR (denoted by 3 squares). Lanes 3 and 4: Addition of 0.2 or 0.6 µg Ku results in supershift of the hTR-Imetelstat complex (denoted by 1–2 squares). C) The dot plot represents the sensitization R values (y axis) obtained after the indicated treatments (x axis). The open circles represent the values obtained in each sampe and the closed circles represent the mean value of sensitization in each group and the whiskers the standard error. Kruskal-Wallis One Way Analysis of Variance on Ranks (****p = 0.007) followed by Wilcoxon Signed Rank Test ***P = <0.001, Paired t-test **p = 0.001 and *p<0.05.
Figure 4
Figure 4. Imetelstat increases FLU-induced γH2AX and decreases FLU-induced pDNA-PK.
A) The graph represents the mean levels of γH2AX (phosphorylated H2AX) (y-axis) after treatment of CLL lymphocytes as indicated (x-axis) in eight samples. The whiskers represent the standard error for each group of four samples tested. One Way Analysis of Variance, *p<0.001. B) The median sensitization R values (x-axis) in CLL lymphocyte samples where Imetelstat did not affect FLU-induced γ-H2AX (left bar) was significantly lower than in CLL lymphocyte samples where Imetelstat increased FLU-induced γH2AX (right bar) with respective median values of 1.4 and 1.8 (t-test *p<0.05). C) Changes in DNA-PK serine 2056 (pDNA-PK) in ten primary CLL lymphocyte samples treated with FLU alone (IC50) or in combination with 1 µM Imetelstat. The values in the y-axis represent fold change in pDNA-PK with respect to vehicle treated lymphocytes. 1 µM M Imetelstat significantly decreased FLU-induced pDNA-PK (Wilcoxon Signed Rank Test, p = 0.004).

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