Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: I. Isolation of a mutant defective in the early steps of de novo pyrimidine synthesis

1977 ◽  
Vol 3 (5) ◽  
pp. 483-495 ◽  
Author(s):  
David Patterson ◽  
Diane V. Carnright
Keyword(s):  
Genetic Analysis ◽  
Mammalian Cells ◽  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Pyrimidine Synthesis ◽  
Biochemical Genetic ◽  
De Novo Pyrimidine Synthesis

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 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 Function and Evolution of Plasmid-Borne Genes for Pyrimidine Biosynthesis in Borrelia spp

2006 ◽  
Vol 188 (3) ◽  
pp. 909-918 ◽  
Author(s):  
Jianmin Zhong ◽  
Stephane Skouloubris ◽  
Qiyuan Dai ◽  
Hannu Myllykallio ◽  
Alan G. Barbour
Keyword(s):  
Thymidylate Synthase ◽  
De Novo ◽  
Linear Plasmid ◽  
Borrelia Hermsii ◽  
Borrelia Garinii ◽  
E Coli ◽  
Pyrimidine Synthesis ◽  
De Novo Pyrimidine Synthesis

ABSTRACT The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.

scholarly journals Missense mutation of the COQ2 gene causes defects of bioenergetics and de novo pyrimidine synthesis

2007 ◽  
Vol 16 (9) ◽  
pp. 1091-1097 ◽  
Author(s):  
José M. López-Martín ◽  
Leonardo Salviati ◽  
Eva Trevisson ◽  
Giovanni Montini ◽  
Salvatore DiMauro ◽  
...  
Keyword(s):  
Missense Mutation ◽  
De Novo ◽  
Pyrimidine Synthesis ◽  
De Novo Pyrimidine Synthesis

scholarly journals Diversion of aspartate in ASS1-deficient tumours fosters de novo pyrimidine synthesis

Nature ◽  
2015 ◽  
Vol 527 (7578) ◽  
pp. 379-383 ◽  
Author(s):  
Shiran Rabinovich ◽  
Lital Adler ◽  
Keren Yizhak ◽  
Alona Sarver ◽  
Alon Silberman ◽  
...  
Keyword(s):  
De Novo ◽  
Pyrimidine Synthesis ◽  
De Novo Pyrimidine Synthesis
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