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

Barszczyk M, Buczkowicz P, Castelo-Branco P, Mack SC, Ramaswamy V, Mangerel J, Agnihotri S, Remke M, Golbourn B, Pajovic S, Elizabeth C, Yu M, Luu B, Morrison A, Adamski J, Nethery-Brokx K, Li XN, Van Meter T, Dirks PB, Rutka JT, Taylor MD, Tabori U, Hawkins C - Acta Neuropathol. (2014)

Bottom Line: 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.Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.

View Article: PubMed Central - PubMed

Affiliation: The Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children, Toronto, ON, Canada.

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.

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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
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Fig5: 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

Mentions: Since pediatric ependymomas are highly recurrent, we thought it was critical to determine whether telomerase inhibition attenuates the self-renewal and tumorigenic capacity of cells that may contribute to recurrence. Imetelstat induced a progressive decrease in the self-renewal of R254 cells throughout treatment, achieving complete inhibition of self-renewal following 17 weeks (Fig. 5a; p < 0.05). Similarly, using a sphere-forming assay to survey the self-renewal of E520 TICs, Imetelstat inhibited self-renewal by 75 % compared to untreated or mismatch control cells following 34 weeks of treatment (Fig. 5b; p < 0.01). Finally, untreated and Imetelstat-pretreated E520 cells (34 weeks) were injected intracranially into mice to assess whether telomerase inhibition attenuates tumorigenicity in vivo. Following 90 days, none of the mice injected with Imetelstat-pretreated cells showed any clinical evidence of tumor formation, while all of the mice injected with untreated E520 cells required killing (Fig. 5c). Histopathological examination revealed tumor formation in all mice that received E520 controls, while none of the mice injected with pretreated cells showed evidence of tumor formation (Fig. 5d, e). Therefore, these observations suggest loss of self-renewal and tumor initiating capacity of ependymoma cells in vitro and in vivo following telomerase inhibition.Fig. 5


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

Barszczyk M, Buczkowicz P, Castelo-Branco P, Mack SC, Ramaswamy V, Mangerel J, Agnihotri S, Remke M, Golbourn B, Pajovic S, Elizabeth C, Yu M, Luu B, Morrison A, Adamski J, Nethery-Brokx K, Li XN, Van Meter T, Dirks PB, Rutka JT, Taylor MD, Tabori U, Hawkins C - Acta Neuropathol. (2014)

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
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Related In: Results  -  Collection

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Fig5: 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
Mentions: Since pediatric ependymomas are highly recurrent, we thought it was critical to determine whether telomerase inhibition attenuates the self-renewal and tumorigenic capacity of cells that may contribute to recurrence. Imetelstat induced a progressive decrease in the self-renewal of R254 cells throughout treatment, achieving complete inhibition of self-renewal following 17 weeks (Fig. 5a; p < 0.05). Similarly, using a sphere-forming assay to survey the self-renewal of E520 TICs, Imetelstat inhibited self-renewal by 75 % compared to untreated or mismatch control cells following 34 weeks of treatment (Fig. 5b; p < 0.01). Finally, untreated and Imetelstat-pretreated E520 cells (34 weeks) were injected intracranially into mice to assess whether telomerase inhibition attenuates tumorigenicity in vivo. Following 90 days, none of the mice injected with Imetelstat-pretreated cells showed any clinical evidence of tumor formation, while all of the mice injected with untreated E520 cells required killing (Fig. 5c). Histopathological examination revealed tumor formation in all mice that received E520 controls, while none of the mice injected with pretreated cells showed evidence of tumor formation (Fig. 5d, e). Therefore, these observations suggest loss of self-renewal and tumor initiating capacity of ependymoma cells in vitro and in vivo following telomerase inhibition.Fig. 5

Bottom Line: 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.Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas.

View Article: PubMed Central - PubMed

Affiliation: The Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children, Toronto, ON, Canada.

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.

Show MeSH
Related in: MedlinePlus