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.