Ketoconazole and Posaconazole Selectively Target HK2-expressing Glioblastoma Cells

Abstract

Purpose: Hexokinase II (HK2) protein expression is elevated in glioblastoma (GBM), and we have shown that HK2 could serve as an effective therapeutic target for GBM. Here, we interrogated compounds that target HK2 effectively and restrict tumor growth in cell lines, patient-derived glioma stem cells (GSCs), and mouse models of GBM.Experimental Design: We performed a screen using a set of 15 drugs that were predicted to inhibit the HK2-associated gene signature. We next determined the EC₅₀ of the compounds by treating glioma cell lines and GSCs. Selected compounds showing significant impact in vitro were used to treat mice and examine their effect on survival and tumor characteristics. The effect of compounds on the metabolic activity in glioma cells was also assessed in vitro.

Results: This screen identified the azole class of antifungals as inhibitors of tumor metabolism. Among the compounds tested, ketoconazole and posaconazole displayed the greatest inhibitory effect on GBM both in vitro and in vivo. Treatment of mice bearing GBM with ketoconazole and posaconazole increased their survival, reduced tumor cell proliferation, and decreased tumor metabolism. In addition, treatment with azoles resulted in increased proportion of apoptotic cells.

Conclusions: Overall, we provide evidence that azoles exert their effect by targeting genes and pathways regulated by HK2. These findings shed light on the action of azoles in GBM. Combined with existing literature and preclinical results, these data support the value of repurposing azoles in GBM clinical trials.

Figure 1.

HK2 loss results in suppression of several oncogenic signaling pathways. A, Western blot analysis was performed 72 hours post-siRNA transfection to confirm HK2 knockdown in multiple GBM cell lines and GSC. Western blot analysis on HK1 was performed as a control to confirm HK2 siRNA specificity. B, Hierarchical clustering of 1,087 genes that were up- or downregulated with HK2 siRNA treatment greater than 1.5-fold based on microarray results. Microarray analysis was performed on 2 cell lines (U87, GSC30) with 2 biological replicates for each. R1 and R2 stand for biological replicates 1 and 2, respectively. C, RT-PCR analysis was performed to confirm HK2 mRNA decrease in HK2 siRNA–treated cells. This analysis was performed on 3 biological replicates and 3 technical replicates for each treatment condition indicated and data are presented as the average of decrease in 4 cell lines. D, Top 200 differentially expressed mRNAs with HK2 siRNA treatment in the U87 and GSC30 cell lines were subjected to pathway analysis using Cytoscape. Blue circles denote downregulated pathways, whereas red circles denote upregulated processes including hypoxia, metabolism, and blood vessel formation.

Figure 2.

Gene signature associated with HK2 knockdown identified several small-molecule inhibitors that can target cells expressing high levels of HK2. A, Schematic of C-Map that was utilized for analysis of genes that were up- and downregulated with HK2 knockdown. B, List of top 15 drugs with a negative enrichment score from C-Map analysis that were predicted to inhibit gene signatures associated with HK2 knockdown. Cells were treated with the identified inhibitors at 1 μmol/L (C) and 10 μmol/L (D) and their viability was assessed using an ATP-cell titer GLO viability assay. The average viability of 3 independent experiments is shown and is depicted as a heatmap. E, Results from yeast drug screen on HXK2 (HK2 homolog) and HXK2 knockout (KO) identified ketoconazole as a potential inhibitor of HXK2, as it did not cause a fitness defect in HXK2 knockout yeast.

Figure 3.

Azole drugs selectively target GBM cells but not normal cells in vitro. A, Several azoles were used at varying doses (0–100 μmol/L) to assess viability of GBM cells and normal cells and the results are depicted as a heat-map. EC₅₀ curves for ketoconazole (B) and posaconazole (C) in GBM cell lines and nonimmortalized NHAs. EC₅₀ curves for ketoconazole (D) and posaconazole (E) in GSCs and fetal NSCs. All analyses were performed in triplicates and error bars represent SD of the data mean.

Figure 4.

Ketoconazole and posaconazole inhibit GBM growth in xenograft mouse models. Kaplan–Meier survival analysis of vehicle-treated, ketoconazole-treated (25 mg/kg), and posaconazole-treated (25 mg/kg) mouse GBM models intracranially injected with GSC 8–18 (A) and GSC7–2 (B) cells. C and D, Histopathologic analysis of GBMs extracted from mice treated with azoles by hematoxylin and eosin (H&E), TUNEL, Ki-67, and HK2 staining. E, Quantification of Ki-67–positive cells in azole-treated and vehicle-treated GBM xenografts as an indication of proliferating cells indicated a decrease in proportion of proliferating cells in treated tumors. F, Quantification of TUNEL-positive cells in treated and untreated GBM xenograft models as a measure for proportion of apoptotic cells suggested that treatment with both ketoconazole and posaconazole increased the percentage of apoptotic cells in tumors. G, Quantification of HK2 to determine the effect of azoles of HK2 protein level in tumor sections. All experiments were performed with a minimum of 3 biological replicates and 3 technical replicates. Asterisks on graphs denote a significant difference (*, P < 0.05, **, P < 0.01).

Figure 5.

Ketoconazole and posaconazole reduce metabolism in GBM cells. A, ¹³C glucose–labeled flux analysis in GSC 8–18 cells treated with vehicle (DMSO; V), ketoconazole (K), and posaconazole (P) for 24 hours and quantification of glycolytic intermediates. B and C, Quantification of relative hexokinase (HK) activity and extracellular lactate production in GSC 8–18 cells treated with ketoconazole (Keto) or posaconazole (Posa), compared with vehicle-treated cells (V) at 24 hours. Alamar blue cell viability analysis on drug-treated and vehicle-treated GSC 8–18 (D) and GSC 7–2 (E) cells transfected with HK2 siRNA or scrambled siRNA at 72 hours. All experiments were performed with a minimum of 3 biological replicates and 3 technical replicates. Asterisks on graphs denote a significant difference (*, P < 0.05, **, P < 0.01). KD, knockdown.