Transcriptional Regulation of Protein Synthesis by Mediator Kinase in MYC-driven Medulloblastoma.

MYC-driven medulloblastoma (MB) is a highly aggressive cancer type with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening across MB cell lines, we identified the Mediator-associated kinase CDK8 as the top dependence for MYC-driven MB. Loss of CDK8 markedly reduces MYC expression and impedes MB growth. Mechanistically, we demonstrate that CDK8 depletion suppresses ribosome biogenesis and mRNA translation. CDK8 regulates occupancy of phospho-Polymerase II at specific chromatin loci facilitating an epigenetic alteration that promotes transcriptional regulation of ribosome biogenesis. Additionally, CDK8-mediated phosphorylation of 4EBP1 plays a crucial role in initiating eIF4E-dependent translation. Targeting CDK8 effectively suppresses cancer stem and progenitor cells, characterized by increased ribosome biogenesis activity. We also report the synergistic inhibition of CDK8 and mTOR in vivo and in vitro . Overall, our findings establish a connection between transcription and translation regulation, suggesting a promising therapeutic approach targets multiple points in the protein synthesis network for MYC-driven MB.

Uridine 5'-Diphospho-glucuronosyltransferase 1A3 (UGT1A3) Prediction of Hepatic Clearance of Organic Anion Transporting Polypeptide 1B3 (OATP1B3) Substrate Telmisartan by Glucuronidation Using In Vitro-In Vivo Extrapolation (IVIVE).

BACKGROUND AND OBJECTIVE: The prediction of pharmacokinetic parameters for drugs metabolised by cytochrome P450 enzymes has been the subject of active research for many years, while the application of in vitro-in vivo extrapolation (IVIVE) techniques for non-cytochrome P450 enzymes has not been thoroughly evaluated. There is still no established quantitative method for predicting hepatic clearance of drugs metabolised by uridine 5'-diphospho-glucuronosyltransferases (UGTs), not to mention those which undergo hepatic uptake. The objective of the study was to predict the human hepatic clearance for telmisartan based on in vitro metabolic stability and hepatic uptake results. METHODS: Telmisartan was examined in liver systems, allowing to estimate intrinsic clearance (CLint, in vitro) based on the substrate disappearance rate with the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) technique. Obtained CLint, in vitro values were corrected for corresponding unbound fractions. Prediction of human hepatic clearance was made from scaled unbound CLint, in vitro data with the use of the well-stirred model, and finally referenced to the literature value of observed clearance in humans, allowing determination of the essential scaling factors. RESULTS: The in vitro scaled CLint, in vitro by UGT1A3 was assessed using three systems, human hepatocytes, liver microsomes, and recombinant enzymes. Obtained values were scaled and hepatic metabolism clearance was predicted, resulting in significant clearance underprediction. Utilization of the extended clearance concept (ECC) and hepatic uptake improved prediction of hepatic metabolism clearance. The scaling factors for hepatocytes, assessing the in vitro-in vivo difference, changed from sixfold difference to only twofold difference with the application of the ECC. CONCLUSIONS: The study showed that taking into consideration hepatic uptake of a drug allows us to obtain satisfactory scaling factors, hence enabling the prediction of in vivo hepatic glucuronidation from in vitro data.

Novel method of measurement of in vitro drug uptake in OATP1B3 overexpressing cells in the presence of dextran.

BACKGROUND: In predictions about hepatic clearance (CLH), a number of studies explored the role of albumin and transporters in drug uptake by liver cells, challenging the traditional free-drug theory. It was proposed that liver uptake can occur for transporter substrate compounds not only from the drug's unbound form but also directly from the drug-albumin complex, a phenomenon known as uptake facilitated by albumin. In contrast to albumin, dextran does not exhibit binding properties for compounds. However, as a result of its inherent capacity for stabilization, it is widely used to mimic conditions within cells. METHODS: The uptake of eight known substrates of the organic anion-transporting polypeptide 1B3 (OATP1B3) was assessed using a human embryonic kidney cell line (HEK293), which stably overexpresses this transporter. An inert polymer, dextran, was used to simulate cellular conditions, and the results were compared with experiments involving human plasma and human serum albumin (HSA). RESULTS: This study is the first to demonstrate that dextran increases compound uptake in cells with overexpression of the OATP1B3 transporter. Contrary to the common theory that highly protein-bound ligands interact with hepatocytes to increase drug uptake, the results indicate that dextran's interaction with test compounds does not significantly increase concentrations near the cell membrane surface. CONCLUSIONS: We evaluated the effect of dextran on the uptake of known substrates using OATP1B3 overexpressed in the HEK293 cell line, and we suggest that its impact on drug concentrations in liver cells may differ from the traditional role of plasma proteins and albumin.

Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

Activation of the coagulation cascade favours metastatic spread, but antithrombotic therapy might also have detrimental effects on cancer progression. In this study, we characterized the effects of dabigatran, a direct reversible thrombin inhibitor, on the pulmonary endothelial barrier and metastatic spread in a murine model of breast cancer metastasis. Dabigatran etexilate (100 mg kg-1) was administered to mice twice daily by oral gavage. Pulmonary metastasis, pulmonary endothelium permeability in vivo, and platelet reactivity were evaluated after intravenous injection of 4T1 breast cancer cells into BALB/c mice. The effect of dabigatran on platelet-dependent protection of pulmonary endothelial barrier in the presence of an inflammatory stimulus was also verified in vitro using human lung microvascular endothelial cell (HLMVEC) cultures. Dabigatran-treated mice harbored more metastases in their lungs and displayed increased pulmonary endothelium permeability after cancer cell injection. It was not associated with altered lung fibrin deposition, changes in INFγ, or complement activation. In the in vitro model of the pulmonary endothelial barrier, dabigatran inhibited platelet-mediated protection of pulmonary endothelium. In a murine model of breast cancer metastasis, dabigatran treatment promoted pulmonary metastasis by the inhibition of platelet-dependent protection of pulmonary endothelial barrier integrity.

KA-104, a new multitargeted anticonvulsant with potent antinociceptive activity in preclinical models.

OBJECTIVE: The main objective of the present work was to assess the utility of KA-104 as potential therapy for drug-resistant seizures and neuropathic pain, and to characterize its druglike properties in a series of absorption, distribution, metabolism, excretion and toxicity (ADME-Tox) studies. We also aimed to establish its mechanism of action in electrophysiological studies. METHODS: The activity of KA-104 against drug-resistant seizures was tested in the mouse 6-Hz (44-mA) model, whereas the antinociceptive activity was assessed with the capsaicin- and oxaliplatin-induced pain models in mice. The patch-clamp technique was used to study the influence of KA-104 on fast voltage-gated sodium currents in rat prefrontal cortex pyramidal neurons. The pharmacokinetic profile was determined after intraperitoneal (ip) injection in mice. The in vitro ADME-Tox properties were studied by applying routine testing procedures. RESULTS: KA-104 was effective in the 6-Hz (44-mA) model (median effective dose [ED50 ] = 73.2 mg/kg) and revealed high efficacy in capsaicin-induced neurogenic pain as well as in oxaliplatin-induced neuropathic pain in mice. Patch-clamp technique showed that KA-104 reversibly inhibits voltage-gated sodium currents. KA-104 was rapidly absorbed after the ip injection and showed relatively good penetration through the blood-brain barrier. This molecule was also characterized by high passive permeability, moderate influence on CYP2C9, and negligible hepatotoxicity on HepG2 cells. SIGNIFICANCE: The results reported herein indicate that KA-104 is a new wide-spectrum multitargeted anticonvulsant with favorable in vitro ADME-Tox properties. Importantly, this compound may also prove to become an interesting and hopefully more effective therapeutic option for treatment of neuropathic pain.

Antiplatelet Effect of Carbon Monoxide Is Mediated by NAD+ and ATP Depletion.

OBJECTIVES: Carbon monoxide (CO) produced by haem oxygenases or released by CO-releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO-induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry-based metabolomics were used. CORM-A1-induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1-induced delay of tricarboxylic acid cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD+) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH [glyceraldehyde 3-phosphate dehydrogenase]), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD+-dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1-induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD+ regeneration, pointing out to the key role of NAD+ depletion in the inhibition of platelet bioenergetics by CORM-A1. CONCLUSIONS: The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration-attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis-ascribed to cytosolic NAD+ depletion.

Alterations in arginine and energy metabolism, structural and signalling lipids in metastatic breast cancer in mice detected in plasma by targeted metabolomics and lipidomics.

BACKGROUND: The early detection of metastasis based on biomarkers in plasma may improve cancer prognosis and guide treatment. The aim of this work was to characterize alterations in metabolites of the arginine pathway, energy metabolism, and structural and signalling lipids in plasma in the early and late stages of murine breast cancer metastasis. METHODS: Mice were orthotopically inoculated with 4T1 metastatic breast cancer cells, and plasma was analysed along the pulmonary metastasis progression using LC-MS/MS-based targeted metabolomics and lipidomics. RESULTS: Based on primary tumour growth and pulmonary metastases, 1-2 weeks after 4T1 cancer cell inoculation was defined as an early metastatic stage, and 3-4 weeks after 4T1 cancer cell inoculation was defined as a late metastatic stage. Early metastasis was featured in plasma by a shift of L-arginine metabolism towards arginase (increased ornithine/arginine ratio) and polyamine synthesis (increased putrescine). Late metastasis was reflected in plasma by further progression of changes in the arginine pathway with an additional increase in asymmetric dimethylarginine plasma concentration, as well as by a profound energy metabolism reprogramming towards glycolysis, an accelerated pentose phosphate pathway and a concomitant decrease in tricarboxylic cycle rate ("Warburg effect"). The late but not the early phase of metastasis was also characterized by a different lipid profile pattern in plasma, including a decrease in total phosphatidylcholines, a decrease in diester-bound phospholipid fraction and an increase in lysophospholipids associated with an increase in total sphingomyelins. CONCLUSIONS: The early phase of metastasis in murine 4T1 metastatic breast cancer was associated with plasma metabolome changes characteristic of arginase activation and polyamine synthesis. The late metastasis was reflected in plasma not only by the alterations in arginine pathways but also by a shift towards glycolysis and the pentose pathway, remodelling of structural lipids and activation of lipid signalling, all of which coincided with metastasis progression.

Carbon monoxide shifts energetic metabolism from glycolysis to oxidative phosphorylation in endothelial cells.

Carbon monoxide (CO) modulates mitochondrial respiration, but the mechanisms involved are not completely understood. The aim of the present study was to investigate the acute effects of CO on bioenergetics and metabolism in intact EA.hy926 endothelial cells using live cell imaging techniques. Our findings indicate that CORM-401, a compound that liberates CO, reduces ATP production from glycolysis, and induces a mild mitochondrial depolarization. In addition, CO from CORM-401 increases mitochondrial calcium and activates complexes I and II. The subsequent increase in mitochondrial respiration leads to ATP production through oxidative phosphorylation. Thus, our results show that nonactivated endothelial cells rely primarily on glycolysis, but in the presence of CO, mitochondrial Ca2+ increases and activates respiration that shifts the metabolism of endothelial cells from glycolysis- to oxidative phosphorylation-dependent ATP production.