Novel Targeting of Transcription and Metabolism in Glioblastoma

Abstract

Purpose: Glioblastoma (GBM) is highly resistant to treatment, largely due to disease heterogeneity and resistance mechanisms. We sought to investigate a promising drug that can inhibit multiple aspects of cancer cell survival mechanisms and become an effective therapeutic for GBM patients.Experimental Design: To investigate TG02, an agent with known penetration of the blood-brain barrier, we examined the effects as single agent and in combination with temozolomide, a commonly used chemotherapy in GBM. We used human GBM cells and a syngeneic mouse orthotopic GBM model, evaluating survival and the pharmacodynamics of TG02. Mechanistic studies included TG02-induced transcriptional regulation, apoptosis, and RNA sequencing in treated GBM cells as well as the investigation of mitochondrial and glycolytic function assays.Results: We demonstrated that TG02 inhibited cell proliferation, induced cell death, and synergized with temozolomide in GBM cells with different genetic background but not in astrocytes. TG02-induced cytotoxicity was blocked by the overexpression of phosphorylated CDK9, suggesting a CDK9-dependent cell killing. TG02 suppressed transcriptional progression of antiapoptotic proteins and induced apoptosis in GBM cells. We further demonstrated that TG02 caused mitochondrial dysfunction and glycolytic suppression and ultimately ATP depletion in GBM. A prolonged survival was observed in GBM mice receiving combined treatment of TG02 and temozolomide. The TG02-induced decrease of CDK9 phosphorylation was confirmed in the brain tumor tissue.Conclusions: TG02 inhibits multiple survival mechanisms and synergistically decreases energy production with temozolomide, representing a promising therapeutic strategy in GBM, currently under investigation in an ongoing clinical trial. Clin Cancer Res; 24(5); 1124-37. ©2017 AACR.

Figure 1.

Downregulated phosphorylation of CDK9 is a key factor in TG02-mediated cell death. A, The cytotoxic effect of TG02 in GSC923, GSC823, U251, and LN18 cells. Cells were treated with different dosages by serial dilution of TG02 for 48 hours prior to cell viability testing. The dose-response curve (plot a) and the EC₅₀ of TG02 in GBM cells are shown in a and b, respectively. B, TG02 alters transcriptional regulation by inhibiting phosphorylation of CDK9 and RNA Pol II. The protein expressions of CDK7, CDK9, phosho-CDK9, and phospho-Pol II in GSC923 and GSC827 cells were determined by Western blotting following treatment with TG02 for 24 hours. C, TG02 decreases the expressions of antiapoptotic proteins. The protein expressions of Mcl-1, Survivin, and XIAP in GSC923 and GSC827 were determined by Western blotting after treatment with TG02 for 24 hours. D, TG02 induces cell apoptosis. The protein expressions of cleaved caspase-3, cleaved-PARP, and phospho-H2A.X were detected in GSC923 and GSC827 cells by Western blotting after treating TG02 for 24 hours. E and F, The overexpression phosphorylated mimic form of CDK9 blocks TG02-mediated cell death in both GSC923 (E) and GSC827 cells (F). The cells were transiently transfected with either Flag-vector control, Flag-CDK9, or Flag-CDK9 T186E and then were treated with 50 nmol/L of TG02. The cell viability was determined using cell counting method (n = 3 biological replicates). Cell viability of the nontreated cells was set as 100% of survival for each group, and the cells transfected with Flag-vector, Flag-CDK9, and CDK9-T186E are illustrated by circle, triangle, and square, respectively. **, P < 0.01; ****, P < 0.0001.

Figure 2.

TG02 induces cell death and synergizes with TMZ in GBM. A and B, TG02 inhibits cell growth in both MGMT high-expressing (A, plots a–d) and MGMT low-expressing cells (B, plots a–d). Cell viability was determined after treatment with vehicle, TG02, TMZ, or TG02/TMZ for 72 hours. C and D, Combined TG02/TMZ treatment has a synergistic antiglioma effect in GSC923 (C) and GSC827 cells (D). Dose-response curves (plot a) were plotted from the cell viability following 72 hours of treatment. The synergistic effects of TG02/TMZ in the cells were determined by CI in b. CI values were calculated by COMPUSYN software and are shown in c. CI < 1 is a synergistic, CI = 1 is an additive, and CI > 1 is an antagonistic effect of the two compounds combined (20). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 3.

TG02 and TMZ induce mitochondrial dysfunction in GBMs. A, Principal component analysis of the transcriptomic profiles of RNA sequencing of GSC923 that were treated with TG02, TMZ, and TG02/TMZ. B, Supervised HC of mitochondrial complexes I to V. The genes in the heat map were derived from the mitochondrial dysfunction pathway in IPA. C, GSC923 cells were treated with indicated drugs. The NADH dehydrogenase activity was measured using the complex I enzyme activity microplate assay kit (n = 3 biological replicates). D, Western blot of Cyt c in cytosolic and mitochondrial compartments, indicating that Cyt c is released from mitochondria to cytoplasm. GSC923 cells were treated and subjected to cell fractionation separating the cytosolic fraction (left) and particulate fraction (right). We set the intensity of Cyt c/loading control (GAPDH or mitochondria) under no treatment as 1, and the numbers indicate the normalized intensities of Cyt c/loading control. E, The Raman imaging shows the Cyt c location (bottom). Cyt c is shown in red. Top plots are the live images of the cell. F, Flow cytometric analysis of mitochondrial membrane potential changes (∆ym) following TG02, TMZ, and TG02/TMZ treatment in GSC923 cells (a). The statistical analysis was showed in b (n = 3 biological replicates). G, Transmission electron micrographs of TG02-, TMZ-, and TG02/TMZ-treated GSC923 cells. H, TG02 and TG02/TMZ induce cell apoptosis in GSC923. Early apoptosis was determined by staining with Hoechst 33342/PI using flow cytometry (a). The statistical analysis was from three independent experiments (b). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

TG02 decreases intracellular ATP levels and suppresses glycolysis as a single agent and in combination with TMZ. A–C, TG02 and TMZ inhibit glycolysis. A, ECAR measurements of U251 cells that were treated with TG02, TMZ, and TG02/TMZ. B, Basal glycolysis (a) and maximal capacity of glycolysis (b) were calculated. C, TG02/TMZ downregulates key enzymes in glycolytic pathway. Western blots of HK2, PKM2, and LDHA in GSC923 and GSC827 cells. D, Intracelluar ATP levels were decreased in TG02 and TG02/TMZ-treated GSC923 cells. ATP levels were determined following TG02, TMZ, and TG02/TMZ treatment. E, Schematic illustrating the mechanisms of the synergism between TG02 and TMZ. TG02 decreased phosphorylation of CDK9 and inhibited RNA Pol II–mediated transcriptional regulation of antiapoptotic proteins, including Mcl-1 and Survivin (also known as BIRC5 gene), leading to release of Cyt c, which induced mitochondrial-mediated apoptosis in GBM cells (shown in red). On the other hand, TG02 also caused disruption of the mitochondrial respiration complexes and inhibition of the glycolysis, resulting in ATP depletion. TMZ induced DNA damage, effectively reducing the level of nicotinamide adenine dinucleotide (NAD⁺) through PARP activity, and thus suppressed glycolysis to reduce energy production in the cells (shown in blue). The combination of TG02/TMZ treatment downregulated protein expressions of HK2, PKM2, and LDHA to decrease ATP production. The synergism between TG02 and TMZ is partially due to suppressed glycolysis and inhibited ATP generation (shown in purple). As a consequence of disruption of ATP production by combination of TG02/TMZ, the cells undergo apoptosis and necrosis. EC, elongation complex. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 5.

TG02, TMZ, and TG02/TMZ induce cytotoxicity in GBMs but less in normal cells. A, TG02, TMZ, and TG02/TMZ induced cytotoxicity in GSC923, GSC827, U251, and LN18 cells (a–d). Survival fractions are derived from the numbers of colony formation 14 days after 72-hour treatments. The control survival fraction was set as 1, and all other values were normalized to the control. B, HAs (a), normal Human Endothelial Cells (b and c), and normal Epithelial cells (d) are less sensitive to TG02 treatment, reflected by cell viability 7 days after 72-hour treatment. C, Flow cytometric detection of mitochondrial membrane potential changes (∆ym) after TG02, TMZ, and TG02/TMZ treatment in HAs (a). The statistical results from 3 biological replicates are shown in b. D, Flow cytometric detection of apoptosis in HAs following TG02, TMZ, and TG02/TMZ treatment (a). The statistical results from 3 biological replicates are shown in b. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Figure 6.

Treatment of combined TG02 and TMZ has an antiglioma effect in GL261 mouse GBM model. A, Combined TG02/TMZ treatment has a synergistic antiglioma effect in GL261 cells. Cells were treated with various concentrations of TG02, TMZ, and TG02/TMZ for 72 hours, and cell viability was determined by cell counting (a). The synergistic effect of TG02/TMZ was evaluated by CI index in b. CI values were calculated by COMPUSYN software and are shown in c. B, TG02 downregulates CDK9 and phospho-CDK9 in vivo. The protein expressions of CDK9, phospho-CDK9, and GAPDH were determined from GL261 tumor tissues with or without TG02 treatment (a). The bar graph represents the normalized relative intensity quantification of each Western blot band. The intensity of each band was relative to the corresponding band of vehicle 1 and then normalized to the relative GAPDH (b). Imaging tumor size of orthotopic glioblastoma xenografts derived from luciferase-expressing GL261 was tracked by bioluminescence at 12 and 19 days (c). Hematoxylin and eosin stain of mice brain tissues in vehicle and TG02 treatment (d). The scale bar represents 1 mm (top) and 100 µm (bottom). C, Combination treatment of TG02/TMZ prolongs mice survival in the allograft model. a,A schematic illustration of drug administration. b, Kaplan–Meier survival plots of C57BL/6 albino mice (n = 5–7 per cohort) with GL261 allografts showing the median overall survivals of 24, 24.5, 27.5, and 32 days in cohorts which received vehicle, TG02, TMZ, or combination treatment, respectively. The result was analyzed using the Log-rank test for trends in GraphPad Prism software (χ² = 9.063; df 1; **, P 0.0026). c, The intensity of BLI of tumors in cohorts received vehicle, TG02, TMZ, or combination treatment. The values were calculated and normalized to the initial intensity at day 5. d, Representative BLI of 2 mice from each cohort that were recorded at days 5, 12, and 20.