Here, we report a new approach to target glioblastoma growth through the inhibition of the de novo biosynthesis of pyrimidines, which preferentially limits ribosomal RNA (rRNA) production. Aims: To investigate the regulation of de novo pyrimidine biosynthesis in the bacterium Pseudomonas resinovorans ATCC 14235. https://doi.org/10.1371/journal.pgen.1009117, Editor: Simon Khagi, University of North Carolina at Chapel Hill, UNITED STATES, Received: May 20, 2020; Accepted: September 14, 2020; Published: November 17, 2020. Total RNA was extracted with TRI Reagent solution (Sigma) following the manufacturer’s instructions, and cDNA was produced with the iScript RT Supermix for RT-qPCR (BIO-RAD). We are grateful to the Sorrell lab members and Dr. Sandra Schmid, Dr. Peter Michaely, Dr. Javier León Serrano, and Dr. Marcos Iglesias Lozano for their valuable input and the Live Cell Imaging Facility at UTSW directed by Dr. Kate Luby-Phelps. While several individual reactions in this pathway are reversible, other irreversible reactions drive the pathway in the direction of UMP biosynthesis in the normal cell. It is possible that the MGMT locus is highly transcriptionally active as a mechanism to increase MGMT levels and DNA repair. These results indicate that the inhibition of the de novo pyrimidine biosynthesis pathway, but not DNA damage, are responsible for the increase in p53 observed in cells treated with brequinar or ML390 (Fig 2G, 2H, 2K and 2M–2O). Table showing the amounts of brequinar, UMP, UDP, UTP and uridine in the LN229 tumor xenografts, brain tissue, liver tissue and serum in each mouse. Secondary antibodies (Alexa Fluor 594 Goat Anti-Mouse IgG H+L and Alexa Fluor 594 Donkey Anti-Goat IgG H+L -Thermo Fisher), were diluted at 1:1000 in 5% BSA-PBS, incubated for 1 h at RT, washed with PBS 3 times for 10 min and mounted with Mowiol mounting media. The blank was subtracted from the standard curve and the subtracted values were used to determine analytical concentration of the compounds. The samples were resuspended in Buffer A containing the internal standards (IS) UMP-13C9,15N2 (UMP-IS) and tolbutamide (Resuspension Solution). Uridine did not cause apparent effects in the nucleolar morphology in the control conditions (Fig 5A and 5B and S6A–S6E Fig). https://doi.org/10.1371/journal.pgen.1009117.s004. Writing – review & editing, Affiliation (C) Representation of de novo and salvage pyrimidine biosynthesis pathways and brequinar. All reactions are thus annotated here only in the forward direction. Both pathways are increased in regenerating tissue or in the human lymphocyte undergoing blast transformation. We propose that glioblastoma cells rely more heavily on the de novo biosynthesis of nucleotides than in the salvage pathways to sustain rRNA production and proliferation, and therefore our work highlights the metabolic vulnerabilities of glioblastoma tumors. The following precursor to fragment ion transitions were optimized for each nucleotide: UMP 325.084 to 97.1; UDP 405.053 to 97.0; UTP 484.984 to 96.9; UMP-IS 336.092 to 102.0. The carbamoyl phosphate synthetase used in pyrimidine biosynthesis is located in the cytoplasm. (N) Western blot of ARPE, LN229, and GBM9 for p53, p21 and cleaved caspase 3 after 72 h of treatment with 2 μM ML390 in the presence or absence of uridine 100 μM. Our results indicate that the p53 response is triggered by the inhibition of ETC at the cytochrome bc1 complex. A Phenomenex (Torrance, CA) Synergi Polar-RP column (2.0 X 150mm, 4 micron packing) was used for chromatography with the following conditions: Buffer A: ddH2O with 0.2% acetic acid. N = 2 for p53, N = 3 for MGMT. N = 2–6. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Consistent with the effects of the DHODH inhibitors, knocking down DHODH expression by siRNA in LN229 and GBM9 cells decreased their proliferation. Writing – original draft, To investigate the need of glioblastoma cells for the de novo pyrimidine biosynthesis pathway, we chose 3 distinct cell lines (S1B Fig): SF188, a commercially available pediatric male cell line expressing p53 that is mutated in the DNA binding domain (G622E); LN229, a commercially available adult female cell line with mutated p53 outside the DNA binding domain (P98L); and the patient-derived GBM9 with unknown p53 status [38]. Similar results were previously reported in HeLa cells [5]. (M) Western blot of ARPE, LN229, SF188 and GBM9 for p53, p21 and cleaved caspase 3 after 72 h of treatment with 0.1 μM brequinar in the presence or absence of 100 μM uridine. N = 2–5. Definition. No, Is the Subject Area "Pyrimidines" applicable to this article? Tables including numerical values of the experiments represented in Fig 2. https://doi.org/10.1371/journal.pgen.1009117.s010. According to the Central Brain Tumor Registry of the United States, based on data collected from 2001 to 2015, glioblastomas were mainly diagnosed in patients with an average age of 65 years old [25]. The key enzyme of the pathway is deoxycytidylate deaminase (dCMP deaminase) ( EC 3.5.4.12). Overall, our findings suggest new therapeutic possibilities for glioblastoma patients by blocking the production of ribosomes through the specific inhibition of the de novo pyrimidine biosynthesis pathway. However, higher concentrations of brequinar and ML390 (1 μM and 4 μM, respectively), activated apoptosis in LN229, SF188 and GBM9, but not in ARPE. Tissues (xenograft tumors, brain, and liver tissues) were weighed and homogenized in a 3-fold volume of PBS (3 x weight of tumor in g = vol PBS in mL; total homogenate volume (in mL) = 4 X weight of tissue). (E) Correlation of pre-rRNA, mature 28S and 18S rRNA, and ACTIN RNA levels with tumor weight. Tumor volumes were recorded once they reached 100 mm3 at day 37, 44, 51, and 59. Cell proliferation was measured by crystal violet staining and 595 nm absorbance quantification of the solubilized die. The samples were snap-frozen three times in liquid nitrogen. The lack of therapeutic options for treatment of glioblastoma patients have created an interest in identifying metabolic dependencies of glioblastoma cells that can be specifically manipulated [31–33]. Thus, we report here that glioblastoma cells, including TMZ-resistant cells, are specifically vulnerable to pharmacologic inhibition of the enzyme DHODH and that inhibiting de novo pyrimidine biosynthesis effectively decreases the production of rRNA in glioblastoma cells. Numerical values for each of the experiments represented are available in S8 Data. To determine whether the effects of TMZ, brequinar or ML390 on proliferation were cytostatic or cytotoxic, we treated LN229 cells with DMSO, 0.1 μM brequinar, 2 μM ML390 (in the absence or presence of 100 μM uridine) and with 100 μM TMZ for 24 h and analyzed the cell cycle by flow cytometry. RNA expression data for CAD, DHODH, and UMPS in grade II to IV (GBM) gliomas was obtained from the RNA-seq experiments deposited in The Cancer Genome Atlas (TCGA) Program through the Pancancer TCGA project (https://portal.gdc.cancer.gov) and the Chinese Glioma Genome Atlas (http://www.cgga.org.cn). Glioblastomas grow very rapidly and frequently develop resistance to treatment by increasing the expression of DNA repair enzymes such as O-6-Methylguanine-DNA Methyltransferase (MGMT), thus explaining in part the very poor prognosis [28–30]. VENDREDI 8 JANVIER, 14H, SÉMINAIRE IRCM Pierre-Olivier Vidalain Centre International de Recherche en Infectiologie (CIRI), INSERM /CNRS/ENS, Lyon Glycolytic enzymes and the pyrimidine biosynthesis pathway: two examples of functional interactions between metabolism and innate immunity Le séminaire aura lieu dans la salle de séminaire avec une audience limitée pour respecter … For example, PRPP is added to anthranilate during the biosynthesis of tryptophan in bacteria. Importantly, different cancer types, such as acute and chronic myeloid leukemia (AML and CML) [14, 15], multiple myeloma [16], melanoma [17], K-Ras–driven pancreatic cancer [18], PTEN-mutated breast cancer or glioblastoma [19], and chemotherapy-resistant triple-negative breast cancer cells [13], have been found to be vulnerable to the inhibition of DHODH. The 6 enzymatic reactions of the de novo pyrimidine biosynthesis pathway are performed by 3 essential enzymes: 1-Carbamoyl-Phosphate Synthetase 2, 2-Aspartate Transcarbamylase, and 3-Dihydroorotase (CAD); Dihydroorotate dehydrogenase (quinone) (DHODH); and 1-Orotate Phosphoribosyl Transferase and 2-Orotidine-5'-Decarboxylase/Uridine Monophosphate Synthetase … These analyses showed that brequinar and ML390 effectively lowered the concentration of UTP, UDP, UMP, and uridine in treated cells (Fig 1E and 1F, and S1C Fig). Despite this low survival rate, the therapy for glioblastoma has not improved significantly since 2005 when the DNA methylating agent temozolomide was incorporated to the standard therapy regimen of surgical resection followed by radiotherapy [26, 27]. ML390 (21395 Cayman Chemical) dissolved in DMSO. Numerical values for each of the experiments represented are available in S11 Data. Cells from in vitro experiments were harvested and lysed with 80% cold methanol to extract the cell metabolites. 2004 Aug 6;279(32):33035-8. doi: 10.1074/jbc.R400007200. Pyrimidine Biosynthesis 1. e1009117. The 6 enzymatic reactions of the de novo pyrimidine biosynthesis pathway are performed by 3 essential enzymes: 1-Carbamoyl-Phosphate Synthetase 2, 2-Aspartate Transcarbamylase, and 3-Dihydroorotase (CAD); Dihydroorotate dehydrogenase (quinone) (DHODH); and 1-Orotate Phosphoribosyl Transferase and 2-Orotidine-5'-Decarboxylase/Uridine Monophosphate Synthetase (UMPS). 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