Critical shifts in lipid metabolism promote megakaryocyte differentiation and proplatelet formation.

During megakaryopoiesis, megakaryocytes (MK) undergo cellular morphological changes with strong modification of membrane composition and lipid signaling. Here we adopt a lipid-centric multiomics approach to create a quantitative map of the MK lipidome during maturation and proplatelet formation. Data reveal that MK differentiation is driven by an increased fatty acyl import and de novo lipid synthesis, resulting in an anionic membrane phenotype. Pharmacological perturbation of fatty acid import and phospholipid synthesis blocked membrane remodeling and directly reduced MK polyploidization and proplatelet formation resulting in thrombocytopenia. The anionic lipid shift during megakaryopoiesis was paralleled by lipid-dependent relocalization of the scaffold protein CKIP-1 and recruitment of the kinase CK2α to the plasma membrane, which seems to be essential for sufficient platelet biogenesis. Overall, this study provides a framework to understand how the MK lipidome is altered during maturation and the impact of MK membrane lipid remodeling on MK kinase signaling involved in thrombopoiesis.

Targeting early stages of cardiotoxicity from anti-PD1 immune checkpoint inhibitor therapy.

AIMS: Cardiac immune-related adverse events (irAEs) from immune checkpoint inhibition (ICI) targeting programmed death 1 (PD1) are of growing concern. Once cardiac irAEs become clinically manifest, fatality rates are high. Cardio-oncology aims to prevent detrimental effects before manifestation of severe complications by targeting early pathological changes. We therefore aimed to investigate early consequences of PD1 inhibition for cardiac integrity to prevent the development of overt cardiac disease. METHODS AND RESULTS: We investigated cardiac-specific consequences from anti-PD1 therapy in a combined biochemical and in vivo phenotyping approach. Mouse hearts showed broad expression of the ligand PDL1 on cardiac endothelial cells as a main mediator of immune-crosstalk. Using a novel melanoma mouse model, we assessed that anti-PD1 therapy promoted myocardial infiltration with CD4+ and CD8+ T cells, the latter being markedly activated. Left ventricular (LV) function was impaired during pharmacological stress, as shown by pressure-volume catheterization. This was associated with a dysregulated myocardial metabolism, including the proteome and the lipidome. Analogous to the experimental approach, in patients with metastatic melanoma (n = 7) receiving anti-PD1 therapy, LV function in response to stress was impaired under therapy. Finally, we identified that blockade of tumour necrosis factor alpha (TNFα) preserved LV function without attenuating the anti-cancer efficacy of anti-PD1 therapy. CONCLUSIONS: Anti-PD1 therapy induces a disruption of cardiac immune homeostasis leading to early impairment of myocardial functional integrity, with potential prognostic effects on the growing number of treated patients. Blockade of TNFα may serve as an approach to prevent the manifestation of ICI-related cardiotoxicity.

Simple Targeted Assays for Metabolic Pathways and Signaling: A Powerful Tool for Targeted Proteomics.

We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms.

A multi-omics analysis reveals the unfolded protein response regulon and stress-induced resistance to folate-based antimetabolites.

Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.

Antiproliferative and Apoptotic Potential of Cyanidin-Based Anthocyanins on Melanoma Cells.

Elderberries are known for their high anthocyanins content, which have been shown to possess anti-proliferative and anti-cancer effects. Anthocyanins enriched extract (AEE) was obtained from elderberries and was characterized by LC/DAD/ESI-MS analysis. Five cyanidin-based anthocyanins were identified, among which Cy-3-O-samb was the major compound (51%). The total anthocyanins content of AEE was 495 mg Cy-3-O-samb/100 g FW. AEE inhibited proliferation of metastatic B16-F10 murine melanoma cells, in a concentration-dependent manner, with an IC50 of 264.3 µg/mL. LDH (lactate dehydrogenase), as a marker of membrane integrity, increased 74% in B16-F10 cells treated with 250 µg/mL AEE, compared to control. It was observed that apoptosis is the mechanism of melanoma cell death after AEE treatment, confirmed morphologically by acridine orange/ethidium bromide double staining and TUNEL analysis. These results indicate that elderberry-derived anthocyanins might be utilized in future applications as topical adjuvant in skin cancer therapy.

Chokeberry Anthocyanin Extract as Pancreatic ß-Cell Protectors in Two Models of Induced Oxidative Stress.

The aim of this study was to investigate the protective effects of a chokeberry anthocyanin extract (CAE) on pancreatic ß-cells (ßTC3) exposed to hydrogen peroxide- (H2O2-) and high glucose- (HG-) induced oxidative stress conditions. In order to quantify individual anthocyanins high performance liquid chromatography (HPLC) coupled to photodiode array (PDA) was used. The identification of the fragment ion pattern of anthocyanins was carried out by electrospray ionization mass spectrometry (LC-ESI-MS). The results showed that physiologically achievable concentrations of CAE (1, 5, and 10 µM) protect ßTC3 against H2O2- and HG-induced cytotoxicity. Antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were increased in pancreatic ß-cells pretreated with CAE compared to cells exposed to the prooxidant agents. GSH levels initially reduced after exposure to H2O2 and HG were restored by pretreatment with CAE. Insulin secretion in ßTC3 cells was enhanced by CAE pretreatment. CAE restored the insulin pool and diminished the intracellular reactive oxygen species level in glucose-induced stress condition in ßTC3 cells. These results demonstrate that anthocyanins from CAE were biologically active, showing a secretagogue potential and an antioxidative protection of enzymatic systems, conferring protection against H2O2 and glucose toxicity in ßTC3 cells.