scholarly journals De Novo Pyrimidine Synthesis is a Targetable Vulnerability in IDH Mutant Glioma

2021 ◽  
Author(s):  
Diana D. Shi ◽  
Milan R. Savani ◽  
Michael M. Levitt ◽  
Adam C. Wang ◽  
Jennifer E. Endress ◽  
...  
Keyword(s):  
De Novo ◽  
Synthetic Lethality ◽  
Treatment Strategies ◽  
Glioma Cells ◽  
Genetically Engineered ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
Genetically Engineered Mouse Model ◽  
Mutant Idh1

Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they appear less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1 mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH mutant gliomas. Mechanistically, this vulnerability selectively applies to de novo pyrimidine, but not purine, synthesis because glioma cells engage disparate programs to produce these nucleotide species and because IDH oncogenes increase DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.

scholarly journals DDRE-29. DE NOVO PYRIMIDINE SYNTHESIS IS A TARGETABLE VULNERABILITY IN IDH-MUTANT GLIOMA

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i12-i13
Author(s):  
Diana D Shi ◽  
Adam C Wang ◽  
Michael M Levitt ◽  
Jennifer E Endress ◽  
Min Xu ◽  
...  
Keyword(s):  
Cell Lines ◽  
De Novo ◽  
Synthetic Lethality ◽  
Nucleotide Metabolism ◽  
Metabolic Inhibitor ◽  
Lower Grade ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
Idh1 Mutations

Abstract 70–90% of lower-grade gliomas and secondary glioblastomas harbor gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1), causing overproduction of the oncometabolite (R)-2-hydroxyglutarate [(R)-2HG]. Although inhibitors of mutant IDH enzymes are effective in other cancers, including leukemia, they have shown guarded efficacy in preclinical and clinical brain tumor studies, thus underscoring the need to identify additional therapeutic targets in IDH mutant glioma. We sought to identify tumor-specific metabolic vulnerabilities induced by IDH1 mutations that could be exploited therapeutically. To uncover such vulnerabilities, we conducted a chemical synthetic lethality screen using isogenic IDH1 mutant and IDH1 wild-type (WT) glioma cell lines and a novel metabolic inhibitor screening platform. We discovered that IDH1 mutant cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). This vulnerability is specific because inhibitors of purine nucleotide metabolism did not score in our screen. We validated that the cytotoxicity of pyrimidine synthesis inhibitors is on-target and showed that IDH1 mutant patient-derived glioma stem-like cell lines are also hyperdependent on de novo pyrimidine nucleotide synthesis compared to IDH1 WT lines. To test pyrimidine synthesis dependence of IDH1 mutant gliomas in vivo, we used a brain-penetrent DHODH inhibitor currently undergoing evaluation in leukemia patients, BAY 2402234. We found that BAY 2402234 displays monotherapy activity against gliomas in an orthotopic xenograft model of IDH1 mutant glioma, with an effect size that compared favorably with radiotherapy. We also developed novel genetically engineered and allograft mouse models of mutant IDH1-driven anaplastic astrocytoma and showed that BAY 2402234 blocked growth of orthotopic astrocytoma allografts. Our findings bolster rationale to target DHODH in glioma, highlight BAY 2402234 as a clinical-stage drug that can be used to inhibit DHODH in brain tumors, and establish IDH1 mutations as predictive biomarkers of DHODH inhibitor efficacy.

scholarly journals Cellular pyrimidine imbalance triggers mitochondrial DNA–dependent innate immunity

2021 ◽  
Author(s):  
Hans-Georg Sprenger ◽  
Thomas MacVicar ◽  
Amir Bahat ◽  
Kai Uwe Fiedler ◽  
Steffen Hermans ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Cultured Cells ◽  
De Novo ◽  
Metabolic Response ◽  
Interferon Response ◽  
Type I ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
De Novo Pyrimidine Synthesis

AbstractCytosolic mitochondrial DNA (mtDNA) elicits a type I interferon response, but signals triggering the release of mtDNA from mitochondria remain enigmatic. Here, we show that mtDNA-dependent immune signalling via the cyclic GMP–AMP synthase‒stimulator of interferon genes‒TANK-binding kinase 1 (cGAS–STING–TBK1) pathway is under metabolic control and is induced by cellular pyrimidine deficiency. The mitochondrial protease YME1L preserves pyrimidine pools by supporting de novo nucleotide synthesis and by proteolysis of the pyrimidine nucleotide carrier SLC25A33. Deficiency of YME1L causes inflammation in mouse retinas and in cultured cells. It drives the release of mtDNA and a cGAS–STING–TBK1-dependent inflammatory response, which requires SLC25A33 and is suppressed upon replenishment of cellular pyrimidine pools. Overexpression of SLC25A33 is sufficient to induce immune signalling by mtDNA. Similarly, depletion of cytosolic nucleotides upon inhibition of de novo pyrimidine synthesis triggers mtDNA-dependent immune responses in wild-type cells. Our results thus identify mtDNA release and innate immune signalling as a metabolic response to cellular pyrimidine deficiencies.

scholarly journals Pyrimidine nucleotide availability is essential for efficient photosynthesis, ROS scavenging, and organelle development

2021 ◽  
Author(s):  
Leo Bellin ◽  
Michael Melzer ◽  
Alexander Hilo ◽  
Diana Laura Garza Amaya ◽  
Isabel Keller ◽  
...  
Keyword(s):  
De Novo ◽  
Energy State ◽  
Chloroplast Ultrastructure ◽  
Low Energy ◽  
Ros Scavenging ◽  
Nucleotide Synthesis ◽  
Knock Down ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
De Novo Pyrimidine Synthesis

ABSTRACTDe novo synthesis of pyrimidines is an essential and highly conserved pathway in all organisms. A peculiarity in plants is the localization of the first committed step, catalyzed by aspartate transcarbamoylase (ATC), in chloroplasts. By contrast, the third step in the pathway is catalyzed by dihydroorotate dehydrogenase (DHODH) localized in mitochondria in eukaryotes, including plants. To unravel pathway- and organelle specific functions, we analyzed knock-down mutants in ATC and DHODH in detail. ATC knock-downs were most severely affected, exhibiting low levels of pyrimidine metabolites, a low energy state, reduced photosynthetic capacity and accumulated reactive oxygen species (ROS). Furthermore, we observed altered leaf morphology and chloroplast ultrastructure in the mutants. Although less affected, DHODH knock-down mutants showed impaired seed germination and altered mitochondrial ultrastructure. Our results point to an integration of de novo pyrimidine synthesis and cellular energy states via photosynthesis and mitochondrial respiration. These findings highlight the likelihood of further regulatory roles for ATC and DHODH in pathways located in the corresponding organelles.ONE-SENTENCE SUMMARYImpaired pyrimidine nucleotide synthesis results in a low energy state, affecting photosynthesis and organellar ultrastructure, thus leading to reduced growth, reproduction, and seed yield

scholarly journals The effect of glutamine concentration on the activity of carbamoyl-phosphate synthase II and on the incorporation of [3H]thymidine into DNA in rat mesenteric lymphocytes stimulated by phytohaemagglutinin

1989 ◽  
Vol 261 (3) ◽  
pp. 979-983 ◽  
Author(s):  
Z Szondy ◽  
E A Newsholme
Keyword(s):  
De Novo ◽  
Glutamine Concentration ◽  
Carbamoyl Phosphate ◽  
Purine Nucleotides ◽  
Pyrimidine Nucleotides ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
Formation De Novo ◽  
Phosphate Synthase

The maximum catalytic activities of carbamoyl-phosphate synthase II, a limiting enzyme for pyrimidine nucleotide synthesis, are very much less than those of glutaminase, a limiting enzyme for glutamine utilization, in lymphocytes and macrophages; and the flux through the pathway for pyrimidine formation de novo is only about 0.4% of the rate of glutamine utilization by lymphocytes. The Km of synthase II for glutamine is about 16 microM and the concentration of glutamine necessary to stimulate lymphocyte proliferation half-maximally is about 21 microM. This agreement suggests that the importance of glutamine for these cells is provision of nitrogen for biosynthesis of pyrimidine nucleotides (and probably purine nucleotides). However, the glutamine concentration necessary for half-maximal stimulation of glutamine utilization (glutaminolysis) by the lymphocytes is 2.5 mM. The fact that the rate of glutamine utilization by lymphocytes is markedly in excess of the rate of the pathway for pyrimidine nucleotide synthesis de novo and that the Km and ‘half-maximal concentration’ values are so different, suggests that the glutaminolytic pathway is independent of the use of glutamine nitrogen for pyrimidine synthesis.

scholarly journals DHODH inhibition synergizes with DNA-demethylating agents in the treatment of myelodysplastic syndromes

2021 ◽  
Vol 5 (2) ◽  
pp. 438-450
Author(s):  
Kensuke Kayamori ◽  
Yurie Nagai ◽  
Cheng Zhong ◽  
Satoshi Kaito ◽  
Daisuke Shinoda ◽  
...  
Keyword(s):  
Cell Lines ◽  
De Novo ◽  
Apoptotic Cell ◽  
Therapeutic Option ◽  
Single Agent ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
Gene Sets ◽  
Growth Inhibitory

Abstract Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine nucleotide synthesis. DHODH inhibition has recently been recognized as a potential new approach for treating acute myeloid leukemia (AML) by inducing differentiation. We investigated the efficacy of PTC299, a novel DHODH inhibitor, for myelodysplastic syndrome (MDS). PTC299 inhibited the proliferation of MDS cell lines, and this was rescued by exogenous uridine, which bypasses de novo pyrimidine synthesis. In contrast to AML cells, PTC299 was inefficient at inhibiting growth and inducing the differentiation of MDS cells, but synergized with hypomethylating agents, such as decitabine, to inhibit the growth of MDS cells. This synergistic effect was confirmed in primary MDS samples. As a single agent, PTC299 prolonged the survival of mice in xenograft models using MDS cell lines, and was more potent in combination with decitabine. Mechanistically, a treatment with PTC299 induced intra-S-phase arrest followed by apoptotic cell death. Of interest, PTC299 enhanced the incorporation of decitabine, an analog of cytidine, into DNA by inhibiting pyrimidine production, thereby enhancing the cytotoxic effects of decitabine. RNA-seq data revealed the marked downregulation of MYC target gene sets with PTC299 exposure. Transfection of MDS cell lines with MYC largely attenuated the growth inhibitory effects of PTC299, suggesting MYC as one of the major targets of PTC299. Our results indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells and acts in a synergistic manner with decitabine. This combination therapy may be a new therapeutic option for the treatment of MDS.

scholarly journals DDRE-32. THERAPEUTIC TARGETING OF A NOVEL METABOLIC ADDICTION IN DIFFUSE MIDLINE GLIOMA

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. i13-i13
Author(s):  
Sharmistha Pal ◽  
Jakub P Kaplan ◽  
Sylwia A Stopka ◽  
Michael S Regan ◽  
Bradley R Hunsel ◽  
...  
Keyword(s):  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Biosynthesis Pathway ◽  
Nucleotide Synthesis ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
A Genome ◽  
De Novo Pyrimidine Synthesis ◽  
Diffuse Midline Glioma

Abstract Diffuse midline glioma (DMG) is a uniformly fatal pediatric cancer that is in need of urgent “outside the box” therapeutic approaches. Recent studies show that tumor cells adapt to stresses created by oncogenic mutations and these oncogene-induced adaptations create vulnerabilities that can be exploited to therapeutic ends. To uncover these oncogene-induced vulnerabilities in DMGs we conducted a genome-wide CRIPSR knockout screen in three DMG lines. The top common DMG dependency pathway that we discovered is de novo pyrimidine biosynthesis. Under normal conditions pyrimidine nucleotide needs are met through the salvage pathway. However, in DMG tumorigenesis, pyrimidine nucleotide synthesis is rewired such that the cells become dependent on the de novo biosynthesis pathway. De novo pyrimidine synthesis is catalyzed by CAD, DHODH and UMPS; all three genes are identified as dependencies in our screen and have been validated using shRNA mediated gene knockdown. Interestingly, DMG cells did not exhibit a dependency on the de novo purine biosynthesis pathway. Using a small molecule inhibitor of DHODH, BAY2402234 [currently studied in phase I trial for myeloid malignancies (NCT03404726)], we have demonstrated and validated, (i) efficacy and specificity of de novo pyrimidine synthesis inhibition in vitro in DMG cells; (ii) de novo pyrimidine addiction is not attributable to cell proliferation; (iii) DHODH inhibition induces apoptosis by hindering replication and inciting DNA damage; (iv) DHODH and ATR inhibition act synergistically to induce DMG cell death; and (v) critical in vivo efficacy. The in vivo experiment documents that BAY2402234 crosses the blood-brain barrier, is present in the brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in intracranial DMG tumors in mice, and prolongs survival of orthotopic DMG tumor bearing mice. Taken together, our studies have identified a novel metabolic vulnerability that can be translated for the treatment of DMG patients.

scholarly journals Uridylate trapping, induction of UTP deficiency, and stimulation of pyrimidine synthesis de novo by d-galactosone

1982 ◽  
Vol 206 (1) ◽  
pp. 139-146 ◽  
Author(s):  
Dietrich O. R. Keppler ◽  
Christa Schulz-Holstege ◽  
Joachim Fauler ◽  
Karl A. Reiffen ◽  
Friedhelm Schneider
Keyword(s):  
Rat Liver ◽  
De Novo ◽  
Combined Treatment ◽  
Hepatoma Cells ◽  
Nucleotide Metabolism ◽  
Phosphate Deficiency ◽  
Initial Time ◽  
Pyrimidine Synthesis ◽  
Pyrimidine Nucleotide ◽  
Time Period

d-Galactosone (d-lyxo-2-hexosulose) is phosphorylated and metabolized to the uridine diphosphate derivative in AS-30D hepatoma cells and rat liver. These reactions were catalysed in vitro by galactokinase and hexose-1-phosphate uridylyltransferase. Nucleotide analyses by high-performance liquid chromatography and enzymic assays revealed that this galactose analogue interferes with cellular pyrimidine nucleotide metabolism leading to a deficiency of UTP. [14C]Uridine labelling of hepatoma cells indicated a division of [14C]uridylate from UTP into UDP-galactosone; the latter was formed at a rate of more than 1.7mmol×h−1×(kg AS-30D or liver wet wt.)−1. As a consequence of UTP deficiency, d-galactosone (1mmol/1 or 1mmol/kg body wt.) strongly enhanced the rate of pyrimidine synthesis de novo as evidenced by incorporation of 14CO2 into uridylate and by an expansion of the uridylate pool. This resulted in a doubling of the total acid-soluble uridylate pool within 70min in the hepatoma cells and within 110min in rat liver. Combined treatment of hepatoma cells with d-galactosone and N-(phosphonoacetyl)-l-aspartate, an inhibitor of aspartate carbamoyltransferase, prevented the expansion of the uridylate pool and led to a synergistic reduction of UTP to 10% of the content in control cells. Hepatic UTP deficiency was selective with respect to other nucleotide 5′-triphosphates but was associated with reduced contents of UDP-glucose, UDP-glucuronate, and UDP-N-acetylhexosamines. Isolation of the UDP derivative of d-galactosone revealed an extremely alkali-labile UDP-sugar, probably an isomerization product of UDP-galactosone, that was degraded by elimination of UDP with a half-life of 45min at pH7.5 and 37°C. The instability of UDP-galactosone may contribute in vivo to limit the time period of severe uridine phosphate deficiency in addition to the compensatory role of pyrimidine synthesis de novo. During the initial time period, however, d-galactosone is effective as a powerful uridylate-trapping sugar analogue.

Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide

2021 ◽  
Author(s):  
Yao Li ◽  
Li Yi ◽  
Sipeng Cheng ◽  
Yongshan Wang ◽  
Jiongjiong Wang ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Active Metabolite ◽  
Canine Distemper Virus ◽  
De Novo ◽  
Specific Inhibitor ◽  
Vero Cells ◽  
Canine Distemper ◽  
Nucleotide Synthesis ◽  
Pyrimidine Nucleotide ◽  
Rate Limiting

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

scholarly journals Inhibition of De Novo Pyrimidine Nucleotide Synthesis By the Novel DHODH Inhibitor PTC299 Induces Differentiation and/or Death of AML Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5152-5152
Author(s):  
Marla Weetall ◽  
Kensuke Kojima ◽  
Sujan Piya ◽  
Christopher Trotta ◽  
John Baird ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
De Novo ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Dna Breaks ◽  
Advisory Committees ◽  
Pyrimidine Nucleotides ◽  
Nucleotide Synthesis ◽  
Pyrimidine Nucleotide

Background: Pyrimidine nucleotides are generated either by de novo synthesis or the salvage pathway in which pyrimidine nucleotides are obtained from the diet. Resting cells typically acquire adequate pyrimidine nucleotides from the salvage pathway. Rapidly proliferating cells, however, are dependent on the de novo synthesis of pyrimidine nucleotides. PTC299 is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for de novo pyrimidine nucleotide synthesis that had previously been in clinical trials for treatment of solid tumors. Results: Using 15N-labelled glutamine, we show that PTC299 reduces de novo pyrimidine nucleotide synthesis in PTC299-sensitive AML cell lines resulting in a depletion of total pyrimidine nucleotides. In parallel to reduction in pyrimidine nucleotides, PTC 299 leads to accumulation of DHO, the substrate of DHODH and unexpectedly, an accumulation of N-carbamoyl aspartate the metabolite above DHO in the de novo pyrimidine nucleotide synthesis pathway. PTC299 was broadly active against leukemia and lymphoma lines, with 80% of the AML lines tested showing sensitivity. Treatment of AML cell lines with PTC299 induced differentiation as shown by increased CD14 and/or reduced proliferation. Using isogenic AML lines, we show that PTC299 reduces the proliferation of both p53 wildtype and p53 deficient leukemia calls with similar potency as measured by the concentration of PTC299 required to reduce cell number by 50% (CC50). In cells expressing wildtype p53, PTC299 increases p53 activation. However, p53- wildtype cells undergo increased apoptosis whereas p53-deficience cells undergo necrosis. PTC299 induced a G1/S cell cycle arrest, also independent of p53 status. PTC299 increased H2A.X (a marker of double stranded DNA breaks) in both p53 wildtype and p53 deficient cells. These data suggest that the depletion of nucleotides results in stalling at the replication fork, and subsequent DNA-breaks. Conclusion: De novo pyrimidine nucleotide synthesis is critical for AML survival and proliferation. Depletion of nucleotides results in reduced proliferation, triggering either differentiation and/or cell death. Disclosures Weetall: PTC Therapeutics: Employment. Trotta:PTC Therapeutics: Employment. Baird:PTC Therapeutics: Employment. O'Keefe:PTC Therapeutics: Employment. Furia:PTC Therapeutics: Employment. Borthakur:PTC Therapeutics: Consultancy; Janssen: Research Funding; AbbVie: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; NKarta: Consultancy; AstraZeneca: Research Funding; Xbiotech USA: Research Funding; Incyte: Research Funding; GSK: Research Funding; Oncoceutics, Inc.: Research Funding; Novartis: Research Funding; Agensys: Research Funding; BMS: Research Funding; Oncoceutics: Research Funding; Cantargia AB: Research Funding; Bayer Healthcare AG: Research Funding; Eisai: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Polaris: Research Funding; Merck: Research Funding; Cyclacel: Research Funding; Eli Lilly and Co.: Research Funding; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Arvinas: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Strategia Therapeutics: Research Funding. Spiegel:PTC Therapeutics: Consultancy.

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