Control of de novo Pyrimidine Biosynthesis in Mammalian Tissues. Levels and Turnover of Early Intermediates in Mouse Spleen in vivo

Abstract Background Necrotic foci with surrounding hypoxic cellular pseudopalisades and microvascular hyperplasia are histological features found in glioblastoma (GBM). We have previously shown that monocarboxylate transporter 4 (MCT4) is highly expressed in necrotic/hypoxic regions in GBM and that increased levels of MCT4 are associated with worse clinical outcomes. Methods A combined transcriptomics and metabolomics analysis was performed to study the effects of MCT4 depletion in hypoxic GBM neurospheres. Stable and inducible MCT4-depletion systems were used to evaluate the effects of and underlining mechanisms associated with MCT4 depletion in vitro and in vivo, alone and in combination with radiation. Results This study establishes that conditional depletion of MCT4 profoundly impairs self-renewal and reduces the frequency and tumorigenicity of aggressive, therapy-resistant, glioblastoma stem cells. Mechanistically, we observed that MCT4 depletion induces anaplerotic glutaminolysis and abrogates de novo pyrimidine biosynthesis. The latter results in a dramatic increase in DNA damage and apoptotic cell death, phenotypes that were readily rescued by pyrimidine nucleosides supplementation. Consequently, we found that MCT4 depletion promoted a significant prolongation of survival of animals bearing established orthotopic xenografts, an effect that was extended by adjuvant treatment with focused radiation. Conclusions Our findings establish a novel role for MCT4 as a critical regulator of cellular deoxyribonucleotide levels and provide a new therapeutic direction related to MCT4 depletion in GBM.

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

PLoS Genetics ◽

10.1371/journal.pgen.1009117 ◽

2020 ◽

Vol 16 (11) ◽

pp. e1009117

Author(s):

M. Carmen Lafita-Navarro ◽

Niranjan Venkateswaran ◽

Jessica A. Kilgore ◽

Suman Kanji ◽

Jungsoo Han ◽

...

Keyword(s):

Ribosomal Rna ◽

Poor Prognosis ◽

De Novo ◽

Pyrimidine Biosynthesis ◽

Biosynthesis Pathway ◽

Salvage Pathway ◽

Glioblastoma Cells ◽

Rdna Transcription ◽

Nucleolar Stress

Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH’s activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. 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. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.

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Control of pyrimidine biosynthesis in mammalian tissues. III. Multiple forms of aspartate transcarbamoylase of mouse spleen

Biochimica et Biophysica Acta (BBA) - Enzymology ◽

10.1016/0005-2744(70)90280-9 ◽

1970 ◽

Vol 220 (3) ◽

pp. 491-502 ◽

Cited By ~ 15

Author(s):

Akira inagaki ◽

Masamiti Tatibana

Keyword(s):

Pyrimidine Biosynthesis ◽

Aspartate Transcarbamoylase ◽

Mouse Spleen ◽

Multiple Forms ◽

Mammalian Tissues

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A 13C tracer method for quantitating de novo pyrimidine biosynthesis in vitro and in vivo

Analytical Biochemistry ◽

10.1016/0003-2697(83)90003-9 ◽

1983 ◽

Vol 132 (2) ◽

pp. 243-253 ◽

Cited By ~ 8

Author(s):

John M. Strong ◽

Lawrence W. Anderson ◽

Anne Monks ◽

Christine A. Chisena ◽

Richard L. Cysyk

Keyword(s):

De Novo ◽

Pyrimidine Biosynthesis ◽

Tracer Method ◽

13C Tracer Method ◽

13C Tracer

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The Effect of Reticulocytosis in the Rabbit on the Activities of Enzymes in Pyrimidine Biosynthesis

Blood ◽

10.1182/blood.v19.5.593.593 ◽

1962 ◽

Vol 19 (5) ◽

pp. 593-600 ◽

Cited By ~ 7

Author(s):

MYRON LOTZ ◽

LLOYD H. SMITH

Keyword(s):

De Novo ◽

Pyrimidine Biosynthesis ◽

Purine Nucleotide ◽

Nucleotide Biosynthesis ◽

Mature Erythrocyte ◽

Pyrimidine Nucleotide

Abstract Five sequential enzymes leading to the formation of uridine-5'-phosphate were studied in acetophenylhydrazine-induced reticulocytes in the rabbit. All of these enzymes—aspartate carbamyltransferase, dihydroorotase, dihydroorotic dehydrogenase, orotidylic pyrophosphorylase, and orotidylic decarboxylase—decreased markedly in activity during in vivo maturation and aging of the reticulocytes. In analogy to previous studies on purine nucleotide biosynthesis, it is concluded that the reticulocyte, but not the mature erythrocyte, is capable of de novo pyrimidine nucleotide biosynthesis.

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A druggable addiction to de novo pyrimidine biosynthesis in diffuse midline glioma

10.1101/2021.11.30.470644 ◽

2021 ◽

Author(s):

SHARMISTHA PAL ◽

Jakub P Kaplan ◽

Huy Nguyen ◽

Sylwia A Stopka ◽

Michael S Regan ◽

...

Keyword(s):

De Novo ◽

Clinical Stage ◽

Pyrimidine Biosynthesis ◽

De Novo Purine Biosynthesis ◽

Genome Wide ◽

A Genome ◽

Target Effect ◽

Rate Limiting ◽

Diffuse Midline Glioma

Diffuse midline glioma (DMG) is a uniformly fatal pediatric cancer driven by oncohistones that do not readily lend themselves to drug development. To identify therapeutic targets for DMG, we conducted a genome-wide CRIPSR screen for DMG metabolic vulnerabilities, which revealed a DMG selective dependency on the de novo pathway for pyrimidine biosynthesis. The dependency is specific to pyrimidines as there is no selectivity for suppression of de novo purine biosynthesis. A clinical stage inhibitor of DHODH (a rate limiting enzyme in the de novo pathway) generates DNA damage and induces apoptosis through suppression of replication forks--an on target effect, as shown by uridine rescue. MALDI mass spectroscopy imaging demonstrates that BAY2402234 accumulates in brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in vivo, and prolongs survival of mice bearing intracranial DMG xenografts. Our results highlight BAY2402234, a brain-penetrant DHODH inhibitor, as a promising therapy against DMGs.

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Inhibition of pyrimidine biosynthesis targets protein translation in AML

10.1101/2021.09.24.461627 ◽

2021 ◽

Author(s):

Joan So ◽

Alexander C. Lewis ◽

Lorey K. Smith ◽

Kym Stanley ◽

Lizzy Pijpers ◽

...

Keyword(s):

De Novo ◽

Protein Translation ◽

Pyrimidine Biosynthesis ◽

Minimal Impact ◽

Potential Biomarker ◽

Anti Cancer ◽

Differentiation Block ◽

Transcription Factor Yy1 ◽

Rate Limiting

AbstractThe mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) catalyzes one of the rate-limiting steps in de novo pyrimidine biosynthesis, a pathway that provides essential metabolic precursors for nucleic acids, glycoproteins and phospholipids. DHODH inhibitors (DHODHi) are clinically used for autoimmune diseases and are emerging as a novel class of anti-cancer agents, especially in acute myeloid leukemia (AML) where pyrimidine starvation was recently shown to reverse the characteristic differentiation block in AML cells. Herein we show that DHODH blockade rapidly shuts down protein translation in leukemic stem cells (LSCs) by down-regulation of the multi-functional transcription factor YY1, has potent activity against AML in vivo and is well tolerated with minimal impact on normal blood development. Moreover, we find that ablation of CDK5, a gene that is recurrently deleted in AML and related disorders, increases the sensitivity of AML cells to DHODHi. Our studies provide important molecular insights and identify a potential biomarker for an emerging strategy to target AML.

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

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab024.054 ◽

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.

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