Regulation of nucleotide metabolism in cancers and immune disorders

Abstract

Nucleotides are the foundational elements of life. Proliferative cells acquire nutrients for energy production and the synthesis of macromolecules, including proteins, lipids, and nucleic acids. Nucleotides are continuously replenished through the activation of the nucleotide synthesis pathways. Despite the importance of nucleotides in cell physiology, there is still much to learn about how the purine and pyrimidine synthesis pathways are regulated in response to intracellular and exogenous signals. Over the past decade, evidence has emerged that several signaling pathways [Akt, mechanistic target of rapamycin complex I (mTORC1), RAS, TP53, and Hippo-Yes-associated protein (YAP) signaling] alter nucleotide synthesis activity and influence cell function. Here, we examine the mechanisms by which these signaling networks affect de novo nucleotide synthesis in mammalian cells. We also discuss how these molecular links can be targeted in diseases such as cancers and immune disorders.

Keywords: cancer metabolism; de novo purine and pyrimidine synthesis; immune disorders; metabolic vulnerability; nucleotide signaling; signaling pathways.

Figure 1.. The de novo pyrimidine and purine synthesis metabolic network.

De novo pyrimidine synthesis: CAD, a trifunctional multi-domain enzyme, catalyzes the first three reaction steps of the de novo pyrimidine pathway in the cytosol, which uses atoms from glutamine, bicarbonate, aspartate, and ATP to synthesize dihydroorotate. The dihydroorotate dehydrogenase reaction step occurs in the mitochondria. Phosphoribosylpyrophosphate (PRPP), generated from the glucose-derived pentose phosphate pathway, is incorporated at the UMPS step in the cytosol. Mitochondrial TCA cycle intermediates such as oxaloacetate could also be used to generate cytosolic aspartate for de novo pyrimidine synthesis. Pyrimidine synthesis enzymes: CAD: carbamoyl-phosphate synthetase 2 (E1), aspartate transcarbamoylase (E2), and dihydroorotase (E3); DHODH: dihydroorotate dehydrogenase; UMPS: uridine monophosphate synthetase. UMPS is a bifunctional enzyme and has two domains: orotate phosphoribosyl transferase (OPRT, E1) and orotidine-5’-phosphate decarboxylase (ODC, E2). De novo purine synthesis: Glucose metabolism produces glycolytic intermediates that can be used by auxiliary pathways including the pentose phosphate pathway to generate PRPP, and the mitochondrial tetrahydrofolate (mTHF) cycle to synthesize glycine, ¹⁰N-formyl-THF for incorporation into the purine ring and ⁵N,¹⁰N-methylene-THF for thymidylate production. The de novo purine pathway requires the coordinated actions of six enzymes to catalyze ten sequential reactions to synthesize the first purine nucleotide inosine 5’-monophosphate (IMP) from PRPP. IMP is the precursor of AMP and GMP. Purine synthesis enzymes: PPAT: phosphoribosyl pyrophosphate amidotransferase; GART: glycinamide ribonucleotide transformylase; PFAS: phosphoribosylformylglycinamidine synthase; PAICS: phosphoribosylaminoimidazole carboxylase and phosphoribosylamino-imidazolesuccinocarboxamide synthase; ADSL: adenylosuccinate lyase; ATIC: 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase.

Figure 2.. Regulation of de novo purine and pyrimidine synthesis by the cell signaling networks.

A. In response to growth factors, mTORC1 is activated downstream of the PI3K/Akt pathway and promotes S6K1-dependent phosphorylation of CAD, thereby increasing de novo pyrimidine synthesis. Moreover, mTORC1 activation fuels de novo purine and pyrimidine synthesis by increasing cellular bicarbonate abundance through the stimulation of SLC4A7 mRNA translation via the S6K-dependent phosphorylation of eIF4B. Downstream of mTORC1, the transcription factor SREBP1 stimulates the oxidative pentose phosphate pathway (PPP) to enhance de novo purine and pyrimidine synthesis via increased availability of PRPP. Additionally, mTORC1 activation enhances ATF4 expression, promoting serine/glycine synthesis and ¹⁰N-formyl-THF production, which increases de novo purine synthesis. B. In response to growth factors, ERK phosphorylates CAD and PFAS, enhancing de novo pyrimidine or purine synthesis, respectively. MYC, a downstream transcription factor of RAS-ERK, controls the expression of genes involved in de novo purine and pyrimidine synthesis. C. Insulin activates the PI3K Akt pathway, which leads to mTORC1 activation. Besides, Akt activates the nonoxidative PPP through TKT phosphorylation, boosting PRPP synthesis for nucleotide synthesis. EGF, epidermal growth factor; eIF4B, eukaryotic translation initiation factor 4B; IGF1, insulin-like growth factor 1; PRPP, 5’-phosphoribosyl-pyrophosphate; PFAS, phosphoribosylformylglycinamidine synthase; RPIA, ribose 5-phosphate isomerase A; SLC4A7, solute carrier family 4 member 7; SREBP1, sterol regulatory element-binding protein 1; TKT, transketolase.

Figure 3.. Mechanisms of action of antimetabolites used in cancer therapy

Methotrexate (MTX) is a folate analog that inhibits dihydrofolate reductase (DHFR) required for de novo nucleotide synthesis and several other metabolic processes. DHFR catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). THF is required for the action of folate-dependent enzymes and is therefore vital for DNA synthesis and methylation reactions. The purine analog 6-mercaptopurine (6MP) inhibits 5-phosphoribosyl-1-pyrophosphate amidotransferase (PPAT), the first enzyme in de novo purine biosynthesis as well as hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), a key enzyme in the purine salvage pathway. The pyrimidine analog 5-fluorouracil (5FU) is a synthetic analog of uracil that inhibits thymidylate synthase, limiting the availability of deoxythymidine nucleotides required for cellular DNA synthesis. Moreover, FDA has recently approved IMPDH inhibitor (MPA) for organ transplants and several clinical trials of cancer studies. PRPP, phosphoribosylpyrophosphate; MTX, methotrexate; HPRT1: hypoxanthine phosphoribosyltransferase 1; IMPDH, inosine 5’-monophosphate dehydrogenase. 6MP, 6-mercaptopurine; 5FU, 5-fluorouracil; MPA, mycophenolic acid. dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate.

Figure 4.. Cellular and phenotypic alterations during therapy with DHODH Inhibitors

DHODH is necessary for the generation of cellular pyrimidine levels, and as such, its inhibitors have been long used to treat various autoimmune diseases, and are in clinical trials for cancer and immune disorders. A DHODH inhibitor quickly depletes cellular pyrimidine levels, forcing cells to limit the synthesis of RNA and DNA. Additionally, functional imbalance in ETC, cell cycle arrest, DNA damage, DNA replication stress could play critical roles in reducing tumor growth or inflammatory lesions. Furthermore, inhibition of DHODH might also increase mitochondrial lipid peroxidation and ferroptosis in GPX4^low cancer cells and suppress GPX4^low tumor growth [90]. DHO, dihydroorotate; UMP, uridine monophosphate; ETC, electron transport chain. GPX4, glutathione peroxidase 4.