Pivotal role of dihydroorotate dehydrogenase as a therapeutic target in adult T-cell leukemia.

OBJECTIVES: We aimed to determine the role of dihydroorotate dehydrogenase (DHODH) in pathogenesis of adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1) and the effects of its inhibition on the de novo pyrimidine biosynthesis pathway. METHODS: Cell proliferation, viability, cycle, and apoptosis were analyzed using WST-8 assays, flow cytometry, and Hoechst 33342 staining. To elucidate the molecular mechanisms involved in the anti-ATL effects of DHODH knockdown and inhibition, RT-PCR and immunoblotting were conducted. RESULTS: HTLV-1-infected T-cell lines aberrantly expressed DHODH. Viral infection and the oncoprotein, Tax, enhanced DHODH expression, while knockdown of DHODH decreased HTLV-1-infected T-cell growth. In addition, BAY2402234, a DHODH inhibitor, exerted an anti-proliferative effect, which was reversed by uridine supplementation. BAY2402234 induced DNA damage and S phase arrest by downregulating c-Myc, CDK2, and cyclin A and upregulating p53 and cyclin E. It also induced caspase-mediated apoptosis by the upregulation of pro-apoptotic and downregulation of anti-apoptotic proteins. Furthermore, BAY2402234 induced caspase-independent ferroptosis and necroptosis. It decreased phosphorylation of IKK, IκBα, PTEN, Akt, and its downstream targets, suggesting that inhibition of NF-κB and Akt signaling is involved in its anti-ATL action. CONCLUSION: These findings highlight DHODH as a potential therapeutic target for treating ATL.

Cyclin D1 extensively reprograms metabolism to support biosynthetic pathways in hepatocytes.

Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme dihydroorotate dehydrogenase synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.

A druggable addiction to de novo pyrimidine biosynthesis in 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 druggable targets for DMG, we conducted a genome-wide CRISPR screen that reveals a DMG selective dependency on the de novo pathway for pyrimidine biosynthesis. This metabolic vulnerability reflects an elevated rate of uridine/uracil degradation that depletes DMG cells of substrates for the alternate salvage pyrimidine biosynthesis pathway. A clinical stage inhibitor of DHODH (rate-limiting enzyme in the de novo pathway) diminishes uridine-5'-phosphate (UMP) pools, generates DNA damage, and induces apoptosis through suppression of replication forks-an "on-target" effect, as shown by uridine rescue. Matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy imaging demonstrates that this DHODH inhibitor (BAY2402234) accumulates in the brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in vivo, and prolongs survival of mice bearing intracranial DMG xenografts, highlighting BAY2402234 as a promising therapy against DMGs.

A Bioluminescent 3CLPro Activity Assay to Monitor SARS-CoV-2 Replication and Identify Inhibitors.

Our therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CLPro of coronaviruses, so that the luminescent biosensor is turned on upon 3CLPro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development.