The Bcl-2 inhibitor Obatoclax overcomes resistance to histone deacetylase inhibitors SAHA and LBH589 as radiosensitizers in patient-derived glioblastoma stem-like cells

Abstract

Glioblastoma has shown resistance to histone deacetylase inhibitors (HDACi) as radiosensitizers in cultures with Bcl-XL over-expression. We study the efficacy of SAHA/RTx and LBH589/RTx when manipulating Bcl-2 family proteins using the Bcl-2 inhibitor Obatoclax in patient-derived glioblastoma stem-like cell (GSC) cultures. GSC cultures in general have a deletion in phosphatase and tensin homolog (PTEN). Synergy was determined by the Chou Talalay method. The effects on apoptosis and autophagy were studied by measuring caspase-3/7, Bcl-XL, Mcl-1 and LC3BI/II proteins. The relation between treatment response and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, recurrence and gene expression levels of the tumors were studied. Obatoclax synergized with SAHA and LBH589 and sensitized cells to HDACi/RTx. Over 50% of GSC cultures were responsive to Obatoclax with either single agent. Combined with HDACi/RTx treatment, Obatoclax increased caspase-3/7 and inhibited Bcl-2 family proteins Bcl-XL and Mcl-1 more effectively than other treatments. Genes predictive for treatment response were identified, including the F-box/WD repeat-containing protein-7, which was previously related to Bcl-2 inhibition and HDACi sensitivity. We emphasize the functional relation between Bcl-2 proteins and radiosensitization by HDACi and provide a target for increasing responsiveness in glioblastoma by using the Bcl-2 inhibitor Obatoclax.

Keywords: Bcl-2; Bcl-XL; HDAC inhibitor; LBH589; Obatoclax; SAHA; radiation.

Figure 1A-D. Sensitivity of patient-derived GSC cultures to Obatoclax treatment

(A) The immortalized glioma cell line U373 and the GSC cultures GS102, GS79 and GS257 were incubated with a concentration-range of Obatoclax and the IC₅₀ values were calculated based on viability on day eight by median equation. We show the viable percentage of cells compared to non-treated controls with standard errors. (B) Cells of patient-derived GSC cultures were treated with at least three different concentrations of Obatoclax in the nanomolar range. After eight days, viability was measured by CellTiter-Glo assay and the IC₅₀ values were calculated by median equation. The graph shows the results of nineteen screened patient-derived GSC cultures. (C) MGMT promoter methylation and recurrence status of the patient-derived GSC cultures were related to the IC₅₀ values of Obatoclax. (D) The Bcl-2 and Bcl-XL protein levels were related to IC₅₀ values of the cultures for which this status was known.

Figure 2A-C. The effect of Obatoclax on HDACi and radiation treatment in patient-derived GSC cultures

(A) Left: Concentration-range assays were performed to determine synergy between Obatoclax and the HDACi SAHA and LBH589 on patient-derived GSC culture GS79 (sensitive to HDACi/RTx treatment) and GS257 (resistant to HDACi/RTx). After eight days of incubation, the viability and synergy were determined by CellTiter-Glo assay and median equation calculation. Means of triplicate tests were shown with the standard errors. Right: The combination indices corresponding to the viability graphs in Figure 2A were calculated according to the Chou Talalay method determining synergism between the two drugs. Synergistic effects were observed in the two patient-derived GSC cultures for both HDACi and Obatoclax, with combination indices (CI) lower than 1 (CI<1 is considered as synergy, CI=1 is considered additive effects, CI>1 is considered as antagonism). (B) The MGMT promoter methylation and recurrence status of the patient-derived GSC cultures were related to the effect sizes of the SAHA/Obatoclax, LBH589/Obatoclax or RTx/Obatoclax treatments. No significant relations were found between responses to treatment and those important prognostic parameters. (C) The results on viability at day eight, of two patient-derived GSC cultures treated with Obatoclax combined with SAHA or LBH589, RTx and all possible combinations. RTx = radiation. * Indicates significance at p<0.05 for the different combination treatments compared to as indicated in the graphs by the vertical lines.

Figure 3A-C. The effects of Obatoclax on HDACi in patient-derived GSC cultures

(A) Nine (left, high concentrations) and five (right, low concentrations) patient-derived GSC cultures were treated with two different concentrations of Obatoclax and SAHA (1μM). After eight days viability was measured by CellTiter-Glo assay. Responders (green bars) and non-responders (red bars) were identified. The color blue indicates significance at the p<0.05 level for one of the concentrations. (B) Nine (left, high concentrations) and five (right, low concentrations) patient-derived GSC cultures were treated with two different concentrations of Obatoclax and LBH589 (20nM). After eight days viability was measured by CellTiter-Glo assay. Responders (green bars) and non-responders (red bars) were identified. The color blue indicates significance at the p<0.05 level. (C) Nine (left, high concentrations) and five (right, low concentrations) patient-derived glioblastoma cultures were treated with two different concentrations of Obatoclax (Oba) and RTx (3Gy). After eight days viability was measured by CellTiter-Glo assay. Responders (green bars) and non-responders (red bars) were identified. The color blue indicates significance at the p<0.05 level.

Figure 4A-C. The mechanisms involved in the efficacy of the combination treatments

(A) Cells of the GSC culture GS257 were treated with Obatoclax (225nM), SAHA (1μM) and all possible combinations. After 24 hours, cells were radiated or not (controls) with 3Gy. Caspase-3/7 reagent was added to the wells, the plates were placed in an IncuCyte and followed for 60 consecutive hours. Fluorescence was measured using the IncuCyte software. *Indicates significance at p<0.05 compared to non-treated controls. **/***Indicate significance at p<0.05 of the treatments, compared to the treatments as indicated by the vertical bars (compared to either 2 or 3 treatments). (B) The GSC cultures GS257 were treated as in Figure 4A. Cells were collected either 24 or 48 hours post-treatment with RTx. The conversion of LC3BI/II, indicating autophagy was studied by using the Western blot analysis. (C) The cells of the GSC culture GS257 were treated as in Figure 4A. Cells were collected either 24 or 48 hours post-treatment with RTx. The anti-apoptotic Bcl-2 family proteins Bcl-Xl and Mcl-1 were studied by using the Western blot analysis.

Figure 5A-E. Gene expression profiling of the response to treatments with Obatoclax

(A) The gene expression levels that were associated with the effect sizes to LBH589/Obatoclax treatment. Criteria were a p-value of <0.05, and a correlation coefficient of >0.5 or <-0.5. The gene expression intensity levels are displayed on log-2 scale. (B) The gene expression levels that are associated with the effect sizes to RTx/Obatoclax treatment. Criteria were a p-value of <0.05, and a correlation coefficient of >0.5 or <-0.5. The gene expression intensity levels are displayed on log-2 scale. (C) The gene expression levels that are associated with the effect sizes to SAHA/Obatoclax treatment. Criteria were a p-value of <0.05, and a correlation coefficient of >0.5 or <-0.5. The gene expression intensity levels are displayed on log-2 scale. (D,E) Heat map of the genes that correlated to the effect sizes of the combination treatments of LBH589/Obatoclax, RTx/Obatoclax and SAHA/Obatoclax Gene expression levels: red, up-regulated genes compared with the geometric mean; blue, down-regulated genes compared with the geometric mean. The color intensity correlates with the degree of change. The green-to-red intensity bar correlates with the degree of change in effect sizes of the corresponding GSC cultures based on the viability assays.