Persistent CAD activity in memory CD8+ T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge

Memory CD8+ T cells are characterized by their ability to persist long after the initial antigen encounter and their ability to generate a rapid recall response. Recent studies have identified a role for metabolic reprogramming and mitochondrial function in promoting the longevity of memory T cells. However, detailed mechanisms involved in promoting the rapid recall response are incompletely understood. Here we identify a novel role for the initial and continued activation of the trifunctional rate-limiting enzyme of the de novo pyrimidine synthesis pathway CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) as critical in promoting the rapid recall response of previously-activated CD8+ T cells. CAD is rapidly phosphorylated upon T cell activation in an mTORC1-dependent manner yet remains phosphorylated long after initial activation. Previously-activated CD8+ T cells display continued de novo pyrimidine synthesis in the absence of mitogenic signals and interfering with this pathway diminishes the speed and magnitude of cytokine production upon rechallenge. Inhibition of CAD does not affect cytokine transcript levels, but diminishes available pre-rRNA, the polycistronic rRNA precursor whose synthesis is the rate-limiting step in ribosomal biogenesis. CAD inhibition additionally decreases levels of detectable ribosomal proteins in previously-activated CD8+ T cells. Overexpression of CAD improves both the cytokine response and proliferation of memory T cells. Overall, our studies reveal a novel and critical role for CAD-induced pyrimidine synthesis and ribosomal biogenesis in promoting the rapid recall response characteristic of memory T cells.

DHODH and cancer: promising prospects to be explored

Human dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme catalyzing the fourth step in the de novo pyrimidine synthesis pathway. It is originally a target for the treatment of the non-neoplastic diseases involving in rheumatoid arthritis and multiple sclerosis, and is re-emerging as a validated therapeutic target for cancer therapy. In this review, we mainly unravel the biological function of DHODH in tumor progression, including its crucial role in de novo pyrimidine synthesis and mitochondrial respiratory chain in cancer cells. Moreover, various DHODH inhibitors developing in the past decades are also been displayed, and the specific mechanism between DHODH and its additional effects are illustrated. Collectively, we detailly discuss the association between DHODH and tumors in recent years here, and believe it will provide significant evidences and potential strategies for utilizing DHODH as a potential target in preclinical and clinical cancer therapies.

Cellular pyrimidine imbalance triggers mitochondrial DNA–dependent innate immunity

Cytosolic 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.

Mechanisms of feedback inhibition and sequential firing of active sites in plant aspartate transcarbamoylase

Aspartate transcarbamoylase (ATC), an essential enzyme for de novo pyrimidine biosynthesis, is uniquely regulated in plants by feedback inhibition of uridine 5-monophosphate (UMP). Despite its importance in plant growth, the structure of this UMP-controlled ATC and the regulatory mechanism remain unknown. Here, we report the crystal structures of Arabidopsis ATC trimer free and bound to UMP, complexed to a transition-state analog or bearing a mutation that turns the enzyme insensitive to UMP. We found that UMP binds and blocks the ATC active site, directly competing with the binding of the substrates. We also prove that UMP recognition relies on a loop exclusively conserved in plants that is also responsible for the sequential firing of the active sites. In this work, we describe unique regulatory and catalytic properties of plant ATCs that could be exploited to modulate de novo pyrimidine synthesis and plant growth.

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

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

Profiling Ribonucleotide and Deoxyribonucleotide Pools Perturbed by Remdesivir in Human Bronchial Epithelial Cells

Remdesivir (RDV) has garnered much hope for its moderate anti-COVID-19 effects, but its limited amelioration of survival in hospitalized patient causes a huge controversy over the applicability of RDV to COVID-19 treatment. Developing strategies to improve its antivirus efficacy is urgently required. As anticipated, RDV exhibits similar behavior with other nucleotide analogs to disrupt the metabolism of natural endogenous ribonucleotides (RNs) and deoxyribonucleotides (dRNs). Alterations in endogenous RNs and dRNs play a critical role in virus replication as well as other key cellular functions. Thus elucidation of the disturbances of RDV on RNs and dRNs could help to understand its exact mechanism of action. Here, the metabolic profiling determined by liquid chromatography–mass spectrometry method showed a general increase in the abundance of nucleotides and a more than 2-fold increase for specific nucleotides. However, the variation of pyrimidine ribonucleotides was relative slight or even contrary, resulting in obvious imbalance between purine and pyrimidine ribonucleotides, which implied the obstacle of RDV to pyrimidine synthesis and could further block the transcription and replication of viral RNA. Additionally, the extreme disequilibrium between cytidine triphosphate (CTP) and cytidine monophosphate might result from the inhibition of CTP synthase and provide a metabolic target for the treatment of COVID-19 infection. Since nucleotides metabolism pathways are vulnerable to nucleotide analogues and are liable to be the regulation targets, it is promising to enhance the efficacy of RDV through co-administration with CTP synthase inhibitors or de novo pyrimidine synthesis inhibitors to exacerbate the imbalance of nucleotide pools.

Venetoclax Inhibition of Pyrimidine Synthesis Guides Methods for Integration with Decitabine or 5-Azacytidine That Are Non-Myelosuppressive

Venetoclax (Ven) administered daily with pulse-cycled parenteral decitabine (Dec) or 5-azacytidine (5Aza) is standard therapy for acute myeloid leukemia (AML) in the elderly. In practice, toxicity/myelosuppression is frequent, and prompts Ven dose reductions, but by guess-work, because the mechanism downstream of BCL2-inhibition by which Ven augments Dec/5Aza activity is unclear. For the first time, we show that Ven inhibits de novo pyrimidine synthesis, an effect that can guide its integration with Dec/5Aza in a way that enhances anti-AML activity without suppressing normal myelopoiesis. Dec and 5Aza are pro-drugs processed by pyrimidine metabolism into a deoxycytidine analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1), a pharmacodynamic effect that terminates malignant but not normal self-replication. We recently demonstrated that Dec- and 5Aza-resistance emerges automatically from adaptive responses of the pyrimidine metabolism network to Dec/5Aza-induced nucleotide perturbations, such that Dec/5Aza processing into DNMT1-depleting nucleotide is forestalled (Leukemia - https://rdcu.be/b58pS). A key element in this auto-resistance is upregulated de novo pyrimidine synthesis, that out-competes salvaged Dec/5Aza. De novo pyrimidine synthesis has an electron-transport dependent mitochondrial step executed by dihydroorotate dehydrogenase (DHODH): BCL2-inhibition by Ven depolarizes mitochondrial membranes - we examined for the first time Ven impact on DHODH/pyrimidine synthesis (others have focused on apoptosis and other metabolic consequences of mitochondrial depolarization). Consistent with Ven inhibiting DHODH/pyrimidine synthesis, both Ven and the direct DHODH inhibitor teriflunomide, at non-apoptotic concentrations, significantly decreased cytidine- and deoxycytidine triphosphate (CTP, dCTP) in AML cells (Fig1A). To see if this effect of Ven can counter Dec/5Aza resistance, we selected for THP1 AML cells double-resistant to Dec 0.5 μM and 5Aza 5 μM - these cells upregulated expression of de novo pyrimidine synthesis enzymes and contained uridine, cytidine and deoxycytidine at levels up to 7-fold higher than parental cells. The double-resistant AML cells were significantly cytoreduced by Ven at a clinically relevant concentration of 1 μM, even though parental THP1 AML cells were minimally sensitive (Fig1B). Consistent with inhibition of de novo pyrimidine synthesis as the mechanism, this action was significantly abrogated by cytidine supplementation (Fig1B). We previously showed that timed alternation of Dec with 5Aza, and incorporation of the cytidine deaminase inhibitor tetrahydrouridine (THU), counters metabolic Dec or 5Aza-resistance to extend non-cytotoxic DNMT1-depletion and survival in vivo (https://rdcu.be/b58pS). However, AML still eventually progresses, via upregulated de novo pyrimidine synthesis. In considering use of Ven to counter this mode of resistance, we reasoned that concurrent administration risks antagonism, because Ven can cause transient cytostasis, and DNMT1-depletion by Dec/5Aza is S-phase dependent. Prior Ven administration, however, creates effect-time sufficient to deplete endogenous nucleotides from AML cells and hence upregulate pyrimidine salvage (that uptakes Dec/5Aza) (Fig1A), as well as resumed cell cycle. Therefore, in vivo in a patient-derived xenotransplant model (PDX) of AML, we introduced Ven (at human equivalent dose), parsimoniously 2X/week, the day before each Dec or 5Aza administration, and at the time of overt AML progression on the base THU-Dec/5Aza regimen. This minimalist Ven application significantly extended survival (time-to-distress) even though it was initiated at progression (Fig1C). As expected, use of this regimen upfront in a PDX of Dec/5Aza-resistant AML produced even greater (several-fold) survival extension (treatment ongoing) vs the base regimen (Fig1D). A non-cytotoxic/non-myelosuppressive mechanism-of-action was confirmed by serial blood counts on-therapy. In practice, Ven dose in combination with Dec or 5Aza to treat AML is frequently empirically reduced because of toxicity/myelosuppression. Instead, Ven inhibition of de novo pyrimidine synthesis can guide mechanism-based, intermittent administration that avoids toxicity/myelosuppression yet enhances Dec/5Aza anti-AML activity. Disclosures Maciejewski: Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago.