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 Points 1. 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.

Inhibition of the de novo pyrimidine biosynthesis pathway limits ribosomal RNA transcription causing nucleolar stress in glioblastoma cells

“…It is possible that VEGFA expression is regulated by other mechanisms in the LN229 xenograft tumors. Moreover, in agreement with our results in vitro (Fig 2F, 2G, 2K and 2M-2O), the tumors treated with brequinar had higher expression of p53 as well as the neuronal differentiation marker acetyl-tubulin ( Fig 3G), which is supported by recent work showing that inhibition of the de novo pyrimidine synthesis pathway increases the differentiation of glioblastoma tumor cells [34,35].…”

“…Here, we show that the activation of the de novo biosynthesis of pyrimidines is an adaptive mechanism used by glioblastoma cells to sustain the high transcriptional rates exhibited by cancer cells. As others previously demonstrated, DHODH inhibition leads to a decrease in proliferation of glioblastoma cells [34,35]. While previous studies attributed this phenotype to a decrease in stemness of glioblastoma-initiating cells [34,35], our current results indicate that the decrease of glioblastoma cell proliferation upon inhibition of DHODH is caused by a specific decrease in pre-rRNA and rRNA abundance, which leads to nucleolar stress and insufficient protein synthesis (Fig 6).…”

“…As others previously demonstrated, DHODH inhibition leads to a decrease in proliferation of glioblastoma cells [34,35]. While previous studies attributed this phenotype to a decrease in stemness of glioblastoma-initiating cells [34,35], our current results indicate that the decrease of glioblastoma cell proliferation upon inhibition of DHODH is caused by a specific decrease in pre-rRNA and rRNA abundance, which leads to nucleolar stress and insufficient protein synthesis (Fig 6). While inhibition of DHODH causes a decrease in rRNA production in glioblastoma cells, it does not affect ACTIN abundance at short incubation periods.…”

“…Alterations that activate the de novo nucleotide biosynthesis pathways are emerging as key features of glioblastomas [34][35][36][37] and have been shown to be essential to maintain the stemness of glioblastoma-initiating cells and tumor growth [34][35][36]. Recently, inhibition of the purine guanosine monophosphate biosynthesis was shown to decrease the production of rRNA and glioblastoma cell growth [36], and to increase the sensitivity to radiotherapy [37].…”

Elevated DHODH expression promotes cell proliferation via stabilizing β-catenin in esophageal squamous cell carcinoma

“…Ultraviolet radiation could transcriptionally induce DHODH expression by activating STAT3 17 . Inhibition of DHODH leads to the starvation of pyrimidine pool and thereby decreases DNA and RNA synthesis and cell proliferation 18,19 . Meanwhile, DHODH has impacts on mitochondrial Oxidative phosphorylation (OXPHOS), which is also responsible for de novo pyrimidine synthesis pathway 20 .…”