Telomerase inhibition abolishes the tumorigenicity of pediatric ependymoma tumor-initiating cells

Abstract

Pediatric ependymomas are highly recurrent tumors resistant to conventional chemotherapy. Telomerase, a ribonucleoprotein critical in permitting limitless replication, has been found to be critically important for the maintenance of tumor-initiating cells (TICs). These TICs are chemoresistant, repopulate the tumor from which they are identified, and are drivers of recurrence in numerous cancers. In this study, telomerase enzymatic activity was directly measured and inhibited to assess the therapeutic potential of targeting telomerase. Telomerase repeat amplification protocol (TRAP) (n = 36) and C-circle assay/telomere FISH/ATRX staining (n = 76) were performed on primary ependymomas to determine the prevalence and prognostic potential of telomerase activity or alternative lengthening of telomeres (ALT) as telomere maintenance mechanisms, respectively. Imetelstat, a phase 2 telomerase inhibitor, was used to elucidate the effect of telomerase inhibition on proliferation and tumorigenicity in established cell lines (BXD-1425EPN, R254), a primary TIC line (E520) and xenograft models of pediatric ependymoma. Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT. Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity. Imetelstat inhibited proliferation and self-renewal by shortening telomeres and inducing senescence in vitro. In vivo, Imetelstat significantly reduced subcutaneous xenograft growth by 40 % (p = 0.03) and completely abolished the tumorigenicity of pediatric ependymoma TICs in an orthotopic xenograft model. Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.

Fig. 1

Telomerase activity predicts progression and survival in pediatric ependymoma, while no cases rely on ALT to maintain telomeres. Kaplan–Meier analysis (n = 36) showed children with ependymomas possessing active telomerase (positive) had reduced progression-free survival (a) and overall survival (b) compared to children whose tumors lacked active telomerase (negative). Telomere FISH showed a lack of ultrabright intranuclear foci (c) in 56 primary ependymomas indicating a lack of ALT, while these foci (arrow) could be observed in an ALT-positive high-grade glioma positive control (d). Significance was determined using log-rank statistics and images were taken at 1,000× magnification. PFS progression-free survival, OS overall survival

Fig. 2

Imetelstat reduced proliferation, inhibited telomerase and shortened telomeres in three pediatric ependymoma cell lines. Prolonged Imetelstat treatment of BXD (a), R254 (b) and E520 (c) cells inhibited proliferation following 8, 6 and 16 weeks, respectively. TRAP assay showed that BXD (d), R254 (e) and E520 (f) cells treated with Imetelstat had a marked reduction in telomerase activity throughout treatment compared to untreated and mismatch control cells as indicated by a reduced banding pattern. TRF assay also showed BXD (g), R254 (h) and E520 (i) cells treated with Imetelstat underwent a progressive decrease in telomere length as determined by lower banding compared to untreated and mismatch control cells as treatment duration increased (weeks). Positive control (+) for TRAP and TRF assays were kit provided telomerase-positive lysate and control DNA, respectively. Negative control (−) for TRAP and TRF assays were lysis buffer and sterile water, respectively. TRF ladder represents kbps. U untreated, M mismatch, I Imetelstat, IC PCR internal control, L ladder

Fig. 3

Imetelstat-treated cells displayed an activated DNA damage response associated with a progressive increase in senescence. Immunofluorescence showed BXD (a), R254 (b) and E520 (c) cells had increased γH2AX staining compared to untreated and mismatch control cells following 27, 17 and 34 weeks of treatment, respectively. β-galactosidase (β-gal) staining showed a progressive increase in cells undergoing senescence in BXD (d), R254 (e) and E520 (f) cells. All images were taken at 200× magnification and scale bars represent either 50 or 100 μm as indicated. Error bars represent ± SD of the mean. Asterick represents significance at p < 0.05 compared to both untreated and mismatch control

Fig. 4

Imetelstat reduced E520 subcutaneous ependymoma growth and shortened telomeres (a). Following 5 weeks of treatment, Imetelstat-treated mice possessed tumors with average volumes 40 % smaller than PBS-treated mice (b). Upon killing (5 weeks post-treatment), Imetelstat-treated tumors appeared smaller than PBS-treated tumors and weighed 35 % less (c). d TRF showed Imetelstat-treated tumors had significantly shorter telomeres than PBS-treated mice, as determined by lower banding. e A significant (p = 0.02) Pearson product-moment correlation (r = −0.65) existed between telomere length and tumor mass. (n = 6 mice/group). Error bars represent ± SEM of the average for a and ± SD for c. Positive control (+) for TRF assay was kit provided DNA, while negative control (−) was sterile water. Ladder (L) is in kbps. Asterick represents significance at p < 0.05 compared to PBS control

Fig. 5

Imetelstat reduced self-renewal and tumorigenicity of pediatric ependymoma cells. a Colony forming assay showed Imetelstat progressively inhibited the self-renewal of R254 ependymoma cells, with complete inhibition by week 17 of treatment. b Sphere-forming assay showed a 75 % increase of cells required to be seeded to generate at least one sphere in each of four wells following 34 weeks of treatment. c Kaplan–Meier survival analysis showed mice injected supratentorially with Imetelstat-pretreated E520 cells (34 weeks) were asymptomatic at 90 days while mice injected with untreated E520 cells all required killing by day 60 (n = 7/group). Upon pathological analysis, all mice injected with untreated cells possessed tumors (d), while no mice injected with Imetelstat-pretreated cells showed evidence of neoplastic growth (e). Images were captured at 40× magnification with 200× inlets shown in bottom left corners. Student’s t test was used to determine significance in a and b while log-rank statistics were used to test significance in c. Error bars represent ± SD of triplicates