Discovery of a new pyrimidine synthesis inhibitor eradicating glioblastoma-initiating cells

Abstract

Background: Glioblastoma-initiating cells (GICs) comprise a tumorigenic subpopulation of cells that are resistant to radio- and chemotherapies and are responsible for cancer recurrence. The aim of this study was to identify novel compounds that specifically eradicate GICs using a high throughput drug screening approach.

Methods: We performed a cell proliferation/death-based drug screening using 10 560 independent compounds. We identified dihydroorotate dehydrogenase (DHODH) as a target protein of hit compound 10580 using ligand-fishing and mass spectrometry analysis. The medical efficacy of 10580 was investigated by in vitro cell proliferation/death and differentiation and in vivo tumorigenic assays.

Results: Among the effective compounds, we identified 10580, which induced cell cycle arrest, decreased the expression of stem cell factors in GICs, and prevented tumorigenesis upon oral administration without any visible side effects. Mechanistic studies revealed that 10580 decreased pyrimidine nucleotide levels and enhanced sex determining region Y-box 2 nuclear export by antagonizing the enzyme activity of DHODH, an essential enzyme for the de novo pyrimidine synthesis.

Conclusion: In this study, we identified 10580 as a promising new drug against GICs. Given that normal tissue cells, in particular brain cells, tend to use the alternative salvage pathway for pyrimidine synthesis, our findings suggest that 10580 can be used for glioblastoma therapy without side effects.Key Points1. Chemical screening identified 10580 as a novel GIC-eliminating drug that targets DHODH, an essential enzyme for the de novo pyrimidine synthesis pathway. 2. Compound 10580 induced cell cycle arrest, apoptosis, and differentiation in GICs.

Keywords: DHODH; O-GlcNAc; Sox2; chemical screening; glioblastoma-initiating cells, GICs.

Fig. 1

Identification of 10580 as a candidate compound for GIC/GICR eradication. (A) Summary of chemical screening. (B) E3R cell viability plots for 10 560 compounds; 319 compounds that cell viability to less than 20% were entered into the second screen. (C) The cytotoxicity of 319 compounds against E3 (gray column, upper panel), E3R (black column, upper panel), E6 (gray column, lower panel), and E6R (black column, lower panel). Then, 302 compounds that decreased both GIC and GICR viability to less than 50% were included in the third screen. (D) The cytotoxicity of 302 compounds against NSCs (black column) and astrocytes (gray column); 9700 (dashed arrow) and 10607 (solid arrow) were selected as candidate compounds by their GI₅₀ effects against GICs/GICRs and structural analysis.

Fig. 2

Identification of the DHODH inhibitor 10580 as a new anti-GIC compound. (A) Chemical structures of 10607, 10580, and traditional DHODH inhibitors. (B) Dose-dependent effects of the candidate compounds, 10607 (open circle), 9700 (open triangle), and 10580 (closed circle) against GICs (E6 and E16 cells) and GICRs (E6R and E16R cells). (C) Dose-dependent effects of the DHODH inhibitors teriflunomide (open circle), leflunomide (open square), vidofludimus (cross), BRQ (open diamond), and 10580 (closed triangle) against GICs (E6 and E16 cells). (D) Pyrimidine biosynthesis pathways. (E) Representative data of metabolites in control (DMSO, white column) and 10580-treated (black column) GICs (E6 and E16 cells). All experiments were repeated at least 3 times with similar results. Error bar: ±SD. Statistical significance was determined by the t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

10580 induces cell cycle arrest, cell death, and differentiation in GICs. (A) Proportion of BrdU+ cells among E6 (black column) and E16 GICs (white column) cultured in the presence of 10580. (B) Proportion of CASP3+ cells among E6 (black column) and E16 GICs (white column) cultured in the presence of 10580. (C) Proportion of cells stained for the NSC markers SOX2 and NESTIN and the differentiation markers βIII tubulin, GFAP, and GC, in E6 (black column) and E16 GICs (white column) cultured in the presence of 10580. (D) Representative photographs of either control (cont)-sh- or DHODH-sh-expressing E6 cells that were immunostained for GFP (green), SOX2 (purple or white), and CASP3 (red). In cont-sh cells (left panels), arrows indicate GFP+/SOX2+/Casp3− cells. In DHODH-sh cells (right panels), arrows indicate GFP+/Sox2+/−/Casp3+ cells, arrowheads indicate GFP+/SOX2−/CASP3− cells. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (blue). Scale bar: 30 µm. (E) Proportion of SOX2+ cells among either cont-sh- or DHODH-sh-expressing GICs (GFP+). (F) Proportion of CASP3+ cells among either cont-sh- or DHODH-sh-expressing GICs (GFP+). All experiments were repeated at least 3 times with similar results. Error bar: ±SD. Statistical significance was determined by the t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4

Antitumorigenic effect of 10580 against GICRs. (A) E6R and E16R tumor volumes in mice treated daily with saline (open circles), 10580 (closed circles), or BRQ (closed triangles) (n = 6). Statistical significance was determined by the t-test. Error bar: ±SEM. (B) Proportion of Sox2+ cells among human mitochondria+ cells in the tumors. Error bar: ±SD. (C) Proportion of CASP3+ cells in the tumors. Error bar: ±SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5

Nuclear export of SOX2 is a part of 10580-dependent GIC death. (A) O-GlcNAcylation of SOX2 through the OGT/UDP-GlcNAc reaction. (B) Proportion of SOX2+ cells among E6 and E16 GICs cultured in the presence of 10580 alone or in combination with UDP-GlcNAc. (C) Dose-dependent effects of UDP (open circle) and UDP-GlcNAc (closed circle) in 10580 (10 µM)-treated E6 and E16 GICs. (D) O-GlcNAcylation analysis of SOX2 in E6 GIC that were cultured in DMSO alone (N), 10580 alone (–), 10580+UDP-GlcNAc (GlcNAc), or 10580+UDP. Immunoprecipitated cell lysates using anti-FLAG M2 antibody were analyzed by western blotting (upper panel). O-GlcNAcylated SOX2 level shown in upper panels was presented as fold change against FLAG (lower panel). Protein quantification was performed using ImageJ software. (E) Proportion of nuclear SOX2-retaining GICs expressing control, SOX2 wt, SOX2 K75A, or SOX2 S248A. (F) Proportion of survival/proliferating 10580-treated GICs in the presence (black column) or absence (white column) of the pan-caspase inhibitor Z-VAD-FMK. (G) Proportion of nuclear SOX2+ GICs that were cultured in DMSO, 10580, or 10580+Z-VAD for 2 days. ns: not significant. All experiments were repeated at least 3 times with similar results. Error bar: ±SD. Statistical significance was determined by t-test. *P < 0.05, **P < 0.01, ***P < 0.001.