Patient-derived glioblastoma cells show significant heterogeneity in treatment responses to the inhibitor-of-apoptosis-protein antagonist birinapant

Abstract

Background: Resistance to temozolomide (TMZ) greatly limits chemotherapeutic effectiveness in glioblastoma (GBM). Here we analysed the ability of the Inhibitor-of-apoptosis-protein (IAP) antagonist birinapant to enhance treatment responses to TMZ in both commercially available and patient-derived GBM cells.

Methods: Responses to TMZ and birinapant were analysed in a panel of commercial and patient-derived GBM cell lines using colorimetric viability assays, flow cytometry, morphological analysis and protein expression profiling of pro- and antiapoptotic proteins. Responses in vivo were analysed in an orthotopic xenograft GBM model.

Results: Single-agent treatment experiments categorised GBM cells into TMZ-sensitive cells, birinapant-sensitive cells, and cells that were insensitive to either treatment. Combination treatment allowed sensitisation to therapy in only a subset of resistant GBM cells. Cell death analysis identified three principal response patterns: Type A cells that readily activated caspase-8 and cell death in response to TMZ while addition of birinapant further sensitised the cells to TMZ-induced cell death; Type B cells that readily activated caspase-8 and cell death in response to birinapant but did not show further sensitisation with TMZ; and Type C cells that showed no significant cell death or moderately enhanced cell death in the combined treatment paradigm. Furthermore, in vivo, a Type C patient-derived cell line that was TMZ-insensitive in vitro and showed a strong sensitivity to TMZ and TMZ plus birinapant treatments.

Conclusions: Our results demonstrate remarkable differences in responses of patient-derived GBM cells to birinapant single and combination treatments, and suggest that therapeutic responses in vivo may be greatly affected by the tumour microenvironment.

Figure 1

Effect of single and combination treatments on cell survival in a panel of GBM cells. (A–C) The characteristics of TMZ-sensitive cells were observed at 72 h post treatment, (D–F) whereas birinapant-sensitive cells at 96 h post treatment. (G) For combination treatments, additional reduction in cell survival were evident at 48 h post treatment in cells that were sensitive to TMZ and birinapant single treatment, (H) and at 72 h post treatment in TMZ- or birinapant-sensitive cells. (I) Three of the GBM cells that were resistant to single treatments had benefited from combined treatments. (J) Four of the GBM cells remained resistant to the treatments. One-way ANOVA, *P<0.05, **P<0.01, ***P<0.001.

Figure 2

Effects of single and combination treatments on IAPs expression profiling. Birinapant as a single agent causes the degradation of cIAP1 in all selected GBM cells, with U251 cells showing complete degradation only in response to the TMZ single and combination treatments. Exposure to TMZ and TMZ plus birinapant induced degradation of XIAP in U251 cells, whereas exposure to birinapant and birinapant plus TMZ did the same in MZ304 cells. Actin was used as a loading control.

Figure 3

Type A response pattern in TMZ-sensitive U251 cells. (A–D) Flow cytometry analysis identified a significant apoptotic and secondary necrotic cell death in response to TMZ but not birinapant, with partial sensitivity to zVAD treatment. (E, F) Combined treatment with birinapant further enhanced cell death, a finding also confirmed by morphological analysis of nuclear condensation. Two-way ANOVA, *P<0.05, **P<0.01, ***P<0.001 versus control cells. Scale bar, 50 μm. (G) Protein expressions show that the TMZ alone and in combination accelerate caspase-8 cleavage and induced reduction of RIP1 and further increased of FADD expression. Actin was used as a loading control.

Figure 4

Type B response pattern in birinapant-sensitive WK1luc cells. (A–D) Flow cytometry analysis demonstrated that birinapant and birinapant plus TMZ induced significant zVAD-sensitive (‘secondary necrotic') cell death. (E, F) A similar finding was observed by Hoechst staining; data are shown as a boxplot with overlaid data points from at least three independent experiments with technical replicates (two-way ANOVA, ***P<0.001 versus control cells, ⁺⁺⁺P<0.001 versus treated cells. Scale bar, 50 μm. (G) Western blot analysis indicated that birinapant treatment readily activated caspase-8, and lead to an increase in FADD and reduction in RIP1 protein levels. Actin was used as a loading control.

Figure 5

Type C response pattern in RN1luc cells. (A–D) Flow cytometry analysis showed moderate sensitisation with the combined treatment. Cell death was not entirely blocked by zVAD. (E, F) A similar finding was observed by Hoechst staining. Two-way ANOVA, *P<0.05 versus control cells, ⁺P<0.05 versus treated cells. Scale bar, 50 μm. (G) Western blot analysis demonstrated a loss of RIP1 expression following birinapant treatment but addition of TMZ reversed the effect of birinapant. Neither single nor combination treatment induced any loss of FADD expression nor cleavage of caspase-8. Actin was used as a loading control.

Figure 6

In vivo tumour growth (bioluminescence) and survival analysis for intracranially inoculated luciferase-expressing RN1luc orthoxenografts. (A) Drug combination treatment and weekly BLI are presented. (B) Effect of TMZ, birinapant or combination on tumour growth. (C) Images show tumour growth over time in a representative animal from each treatment group at 35 days post treatment commencement. (D) Effect of treatment on survival using Kaplan–Meier analysis and log-rank tests was used to compare treatment groups. Error bars represent mean BLI±s.e.m (n=10 animals per group). *P<0.0001 versus vehicle and ^#P<0.0001 versus birinapant; statistically significant using a Bonferroni-adjusted significance level of 0.83% (P<0.0083).