Role of Diacylglycerol Kinases in Acute Myeloid Leukemia.

Diacylglycerol kinases (DGKs) play dual roles in cell transformation and immunosurveillance. According to cancer expression databases, acute myeloid leukemia (AML) exhibits significant overexpression of multiple DGK isoforms, including DGKA, DGKD and DGKG, without a precise correlation with specific AML subtypes. In the TGCA database, high DGKA expression negatively correlates with survival, while high DGKG expression is associated with a more favorable prognosis. DGKA and DGKG also feature different patterns of co-expressed genes. Conversely, the BeatAML and TARGET databases show that high DGKH expression is correlated with shorter survival. To assess the suitability of DGKs as therapeutic targets, we treated HL-60 and HEL cells with DGK inhibitors and compared cell growth and survival with those of untransformed lymphocytes. We observed a specific sensitivity to R59022 and R59949, two poorly selective inhibitors, which promoted cytotoxicity and cell accumulation in the S phase in both cell lines. Conversely, the DGKA-specific inhibitors CU-3 and AMB639752 showed poor efficacy. These findings underscore the pivotal and isoform-specific involvement of DGKs in AML, offering a promising pathway for the identification of potential therapeutic targets. Notably, the DGKA and DGKH isoforms emerge as relevant players in AML pathogenesis, albeit DGKA inhibition alone seems insufficient to impair AML cell viability.

Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction.

Background: Phosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion. Results: Herein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity. Conclusion: We discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response.

Circulating Peptidome Is Strongly Altered in COVID-19 Patients.

Whilst the impact of coronavirus disease 2019 (COVID-19) on the host proteome, metabolome, and lipidome has been largely investigated in different bio-fluids, to date, the circulating peptidome remains unexplored. Thus, the present study aimed to apply an untargeted peptidomic approach to provide insight into alterations of circulating peptides in the development and severity of SARS-CoV-2 infection. The circulating peptidome from COVID-19 severe and mildly symptomatic patients and negative controls was characterized using LC-MS/MS analysis for identification and quantification purposes. Database search and statistical analysis allowed a complete characterization of the plasma peptidome and the detection of the most significant modulated peptides that were impacted by the infection. Our results highlighted not only that peptide abundance inversely correlates with disease severity, but also the involvement of biomolecules belonging to inflammatory, immune-response, and coagulation proteins/processes. Moreover, our data suggested a possible involvement of changes in protein degradation patterns. In the present research, for the first time, the untargeted peptidomic approach enabled the identification of circulating peptides potentially playing a crucial role in the progression of COVID-19.

Specific transcriptional programs differentiate ICOS from CD28 costimulatory signaling in human Naïve CD4+ T cells.

Costimulatory molecules of the CD28 family play a crucial role in the activation of immune responses in T lymphocytes, complementing and modulating signals originating from the T-cell receptor (TCR) complex. Although distinct functional roles have been demonstrated for each family member, the specific signaling pathways differentiating ICOS- from CD28-mediated costimulation during early T-cell activation are poorly characterized. In the present study, we have performed RNA-Seq-based global transcriptome profiling of anti-CD3-treated naïve CD4+ T cells upon costimulation through either inducible costimulator (ICOS) or CD28, revealing a set of signaling pathways specifically associated with each signal. In particular, we show that CD3/ICOS costimulation plays a major role in pathways related to STAT3 function and osteoarthritis (OA), whereas the CD3/CD28 axis mainly regulates p38 MAPK signaling. Furthermore, we report the activation of distinct immunometabolic pathways, with CD3/ICOS costimulation preferentially targeting glycosaminoglycans (GAGs) and CD3/CD28 regulating mitochondrial respiratory chain and cholesterol biosynthesis. These data suggest that ICOS and CD28 costimulatory signals play distinct roles during the activation of naïve T cells by modulating distinct sets of immunological and immunometabolic genes.

Diacylglycerol Kinases in Signal Transduction.

In recent years, the significant research efforts put into the clarification of the PI3K/AKT/mTOR pathway resulted in the approval of the first targeted therapies based on lipid kinase inhibitors [...].

Immune Checkpoint Receptors Signaling in T Cells.

The characterization of the receptors negatively modulating lymphocyte function is rapidly advancing, driven by success in tumor immunotherapy. As a result, the number of immune checkpoint receptors characterized from a functional perspective and targeted by innovative drugs continues to expand. This review focuses on the less explored area of the signaling mechanisms of these receptors, of those expressed in T cells. Studies conducted mainly on PD-1, CTLA-4, and BTLA have evidenced that the extracellular parts of some of the receptors act as decoy receptors for activating ligands, but in all instances, the tyrosine phosphorylation of their cytoplasmatic tail drives a crucial inhibitory signal. This negative signal is mediated by a few key signal transducers, such as tyrosine phosphatase, inositol phosphatase, and diacylglycerol kinase, which allows them to counteract TCR-mediated activation. The characterization of these signaling pathways is of great interest in the development of therapies for counteracting tumor-infiltrating lymphocyte exhaustion/anergy independently from the receptors involved.

Exosomes in chronic respiratory diseases.

Exosomes are nano-sized vesicles released by almost all cell types, with a central role as mediators of intercellular communication. In addition to physiological conditions, these extracellular vesicles seem to play a pivotal role in inflammatory processes. This assumption offers the opportunity to study exosomes as promising biomarkers and therapeutic tools for chronic respiratory disorders. Indeed, although it is well-known that at the basis of conditions like asthma, chronic obstructive pulmonary disease, alpha-1 antitrypsin deficiency and idiopathic pulmonary fibrosis there is a dysregulated inflammatory process, an unequivocal correlation between different phenotypes and their pathophysiological mechanisms has not been established yet. In this review, we report and discuss some of the most significant studies on exosomes from body fluids of subjects affected by airway diseases. Furthermore, the most widespread techniques for exosome isolation and characterization are described. Further studies are needed to answer the unresolved questions about the functional link between exosomes and chronic respiratory diseases.

Diacylglycerol Kinase alpha in X Linked Lymphoproliferative Disease Type 1.

Diacylglycerol kinases are intracellular enzymes that control the balance between the secondary messengers diacylglycerol and phosphatidic acid. DGKα and DGKζ are the prominent isoforms that restrain the intensity of T cell receptor signalling by metabolizing PLCγ generated diacylglycerol. Thus, their activity must be tightly controlled to grant cellular homeostasis and refine immune responses. DGKα is specifically inhibited by strong T cell activating signals to allow for full diacylglycerol signalling which mediates T cell response. In X-linked lymphoproliferative disease 1, deficiency of the adaptor protein SAP results in altered T cell receptor signalling, due in part to persistent DGKα activity. This activity constrains diacylglycerol levels, attenuating downstream pathways such as PKCθ and Ras/MAPK and decreasing T cell restimulation induced cell death. This is a form of apoptosis triggered by prolonged T cell activation that is indeed defective in CD8+ cells of X-linked lymphoproliferative disease type 1 patients. Accordingly, inhibition or downregulation of DGKα activity restores in vitro a correct diacylglycerol dependent signal transduction, cytokines production and restimulation induced apoptosis. In animal disease models, DGKα inhibitors limit CD8+ expansion and immune-mediated tissue damage, suggesting the possibility of using inhibitors of diacylglycerol kinase as a new therapeutic approach.

Circulating Exosomes Are Strongly Involved in SARS-CoV-2 Infection.

Knowledge of the host response to the novel coronavirus SARS-CoV-2 remains limited, hindering the understanding of COVID-19 pathogenesis and the development of therapeutic strategies. During the course of a viral infection, host cells release exosomes and other extracellular vesicles carrying viral and host components that can modulate the immune response. The present study used a shotgun proteomic approach to map the host circulating exosomes' response to SARS-CoV-2 infection. We investigated how SARS-CoV-2 infection modulates exosome content, exosomes' involvement in disease progression, and the potential use of plasma exosomes as biomarkers of disease severity. A proteomic analysis of patient-derived exosomes identified several molecules involved in the immune response, inflammation, and activation of the coagulation and complement pathways, which are the main mechanisms of COVID-19-associated tissue damage and multiple organ dysfunctions. In addition, several potential biomarkers-such as fibrinogen, fibronectin, complement C1r subcomponent and serum amyloid P-component-were shown to have a diagnostic feature presenting an area under the curve (AUC) of almost 1. Proteins correlating with disease severity were also detected. Moreover, for the first time, we identified the presence of SARS-CoV-2 RNA in the exosomal cargo, which suggests that the virus might use the endocytosis route to spread infection. Our findings indicate circulating exosomes' significant contribution to several processes-such as inflammation, coagulation, and immunomodulation-during SARS-CoV-2 infection. The study's data are available via ProteomeXchange with the identifier PXD021144.

Identification of Key Phospholipids That Bind and Activate Atypical PKCs.

PKCζ and PKCι/λ form the atypical protein kinase C subgroup, characterised by a lack of regulation by calcium and the neutral lipid diacylglycerol. To better understand the regulation of these kinases, we systematically explored their interactions with various purified phospholipids using the lipid overlay assays, followed by kinase activity assays to evaluate the lipid effects on their enzymatic activity. We observed that both PKCζ and PKCι interact with phosphatidic acid and phosphatidylserine. Conversely, PKCι is unique in binding also to phosphatidylinositol-monophosphates (e.g., phosphatidylinositol 3-phosphate, 4-phosphate, and 5-phosphate). Moreover, we observed that phosphatidylinositol 4-phosphate specifically activates PKCι, while both isoforms are responsive to phosphatidic acid and phosphatidylserine. Overall, our results suggest that atypical Protein kinase C (PKC) localisation and activity are regulated by membrane lipids distinct from those involved in conventional PKCs and unveil a specific regulation of PKCι by phosphatidylinositol-monophosphates.

Psychotropic Drugs Show Anticancer Activity by Disrupting Mitochondrial and Lysosomal Function.

Background and Purpose: Drug repositioning is a promising strategy for discovering new therapeutic strategies for cancer therapy. We investigated psychotropic drugs for their antitumor activity because of several epidemiological studies reporting lower cancer incidence in individuals receiving long term drug treatment. Experimental Approach: We investigated 27 psychotropic drugs for their cytotoxic activity in colorectal carcinoma, glioblastoma and breast cancer cell lines. Consistent with the cationic amphiphilic structure of the most cytotoxic compounds, we investigated their effect on mitochondrial and lysosomal compartments. Results: Penfluridol, ebastine, pimozide and fluoxetine, fluspirilene and nefazodone showed significant cytotoxicity, in the low micromolar range, in all cell lines tested. In MCF7 cells these drugs caused mitochondrial membrane depolarization, increased the acidic vesicular compartments and induced phospholipidosis. Both penfluridol and spiperone induced AMPK activation and autophagy. Neither caspase nor autophagy inhibitors rescued cells from death induced by ebastine, fluoxetine, fluspirilene and nefazodone. Treatment with 3-methyladenine partially rescued cell death induced by pimozide and spiperone, whereas enhanced the cytotoxic activity of penfluridol. Conversely, inhibition of lysosomal cathepsins significantly reduced cell death induced by ebastin, penfluridol, pimozide, spiperone and mildly in fluoxetine treated cells. Lastly, Spiperone cytotoxicity was restricted to colorectal cancer and breast cancer and caused apoptotic cell death in MCF7 cells. Conclusions: The cytotoxicity of psychotropic drugs with cationic amphiphilic structures relied on simultaneous mitochondrial and lysosomal disruption and induction of cell death that not necessarily requires apoptosis. Since dual targeting of lysosomes and mitochondria constitutes a new promising therapeutic approach for cancer, particularly those in which the apoptotic machinery is defective, these data further support their clinical development for cancer therapy.

Large-Scale Plasma Analysis Revealed New Mechanisms and Molecules Associated with the Host Response to SARS-CoV-2.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to nearly every continent, registering over 1,250,000 deaths worldwide. The effects of SARS-CoV-2 on host targets remains largely limited, hampering our understanding of Coronavirus Disease 2019 (COVID-19) pathogenesis and the development of therapeutic strategies. The present study used a comprehensive untargeted metabolomic and lipidomic approach to capture the host response to SARS-CoV-2 infection. We found that several circulating lipids acted as potential biomarkers, such as phosphatidylcholine 14:0_22:6 (area under the curve (AUC) = 0.96), phosphatidylcholine 16:1_22:6 (AUC = 0.97), and phosphatidylethanolamine 18:1_20:4 (AUC = 0.94). Furthermore, triglycerides and free fatty acids, especially arachidonic acid (AUC = 0.99) and oleic acid (AUC = 0.98), were well correlated to the severity of the disease. An untargeted analysis of non-critical COVID-19 patients identified a strong alteration of lipids and a perturbation of phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, aminoacyl-tRNA degradation, arachidonic acid metabolism, and the tricarboxylic acid (TCA) cycle. The severity of the disease was characterized by the activation of gluconeogenesis and the metabolism of porphyrins, which play a crucial role in the progress of the infection. In addition, our study provided further evidence for considering phospholipase A2 (PLA2) activity as a potential key factor in the pathogenesis of COVID-19 and a possible therapeutic target. To date, the present study provides the largest untargeted metabolomics and lipidomics analysis of plasma from COVID-19 patients and control groups, identifying new mechanisms associated with the host response to COVID-19, potential plasma biomarkers, and therapeutic targets.

Osteopontin binds ICOSL promoting tumor metastasis.

ICOSL/ICOS are costimulatory molecules pertaining to immune checkpoints; their binding transduces signals having anti-tumor activity. Osteopontin (OPN) is here identified as a ligand for ICOSL. OPN binds a different domain from that used by ICOS, and the binding induces a conformational change in OPN, exposing domains that are relevant for its functions. Here we show that in vitro, ICOSL triggering by OPN induces cell migration, while inhibiting anchorage-independent cell growth. The mouse 4T1 breast cancer model confirms these data. In vivo, OPN-triggering of ICOSL increases angiogenesis and tumor metastatization. The findings shed new light on ICOSL function and indicate that another partner beside ICOS may be involved; they also provide a rationale for developing alternative therapeutic approaches targeting this molecular trio.

Potential role of diacylglycerol kinases in immune-mediated diseases.

The mechanism promoting exacerbated immune responses in allergy and autoimmunity as well as those blunting the immune control of cancer cells are of primary interest in medicine. Diacylglycerol kinases (DGKs) are key modulators of signal transduction, which blunt diacylglycerol (DAG) signals and produce phosphatidic acid (PA). By modulating lipid second messengers, DGK modulate the activity of downstream signaling proteins, vesicle trafficking and membrane shape. The biological role of the DGK α and ζ isoforms in immune cells differentiation and effector function was subjected to in deep investigations. DGK α and ζ resulted in negatively regulating synergistic way basal and receptor induced DAG signals in T cells as well as leukocytes. In this way, they contributed to keep under control the immune response but also downmodulate immune response against tumors. Alteration in DGKα activity is also implicated in the pathogenesis of genetic perturbations of the immune function such as the X-linked lymphoproliferative disease 1 and localized juvenile periodontitis. These findings suggested a participation of DGK to the pathogenetic mechanisms underlying several immune-mediated diseases and prompted several researches aiming to target DGK with pharmacologic and molecular strategies. Those findings are discussed inhere together with experimental applications in tumors as well as in other immune-mediated diseases such as asthma.

Structure activity relationship studies on Amb639752: toward the identification of a common pharmacophoric structure for DGKα inhibitors.

A series of analogues of Amb639752, a novel diacylglycerol kinase (DGK) inhibitor recently discovered by us via virtual screening, have been tested. The compounds were evaluated as DGK inhibitors on α, θ, and ζ isoforms, and as antagonists on serotonin receptors. From these assays emerged two novel compounds, namely 11 and 20, which with an IC50 respectively of 1.6 and 1.8 µM are the most potent inhibitors of DGKα discovered to date. Both compounds demonstrated the ability to restore apoptosis in a cellular model of X-linked lymphoproliferative disease as well as the capacity to reduce the migration of cancer cells, suggesting their potential utility in preventing metastasis. Finally, relying on experimental biological data, molecular modelling studies allow us to set a three-point pharmacophore model for DGK inhibitors.

DGKα in Neutrophil Biology and Its Implications for Respiratory Diseases.

Diacylglycerol kinases (DGKs) play a key role in phosphoinositide signaling by removing diacylglycerol and generating phosphatidic acid. Besides the well-documented role of DGKα and DGKζ as negative regulators of lymphocyte responses, a robust body of literature points to those enzymes, and specifically DGKα, as crucial regulators of leukocyte function. Upon neutrophil stimulation, DGKα activation is necessary for migration and a productive response. The role of DGKα in neutrophils is evidenced by its aberrant behavior in juvenile periodontitis patients, which express an inactive DGKα transcript. Together with in vitro experiments, this suggests that DGKs may represent potential therapeutic targets for disorders where inflammation, and neutrophils in particular, plays a major role. In this paper we focus on obstructive respiratory diseases, including asthma and chronic obstructive pulmonary disease (COPD), but also rare genetic diseases such as alpha-1-antitrypsin deficiency. Indeed, the biological role of DGKα is understudied outside the T lymphocyte field. The recent wave of research aiming to develop novel and specific inhibitors as well as KO mice will allow a better understanding of DGK's role in neutrophilic inflammation. Better knowledge and pharmacologic tools may also allow DGK to move from the laboratory bench to clinical trials.

Identification of a novel DGKα inhibitor for XLP-1 therapy by virtual screening.

As part of an effort to identify druggable diacylglycerol kinase alpha (DGKα) inhibitors, we used an in-silico approach based on chemical homology with the two commercially available DGKα inhibitors R59022 and R59949. Ritanserin and compound AMB639752 emerged from the screening of 127 compounds, showing an inhibitory activity superior to the two commercial inhibitors, being furthermore specific for the alpha isoform of diacylglycerol kinase. Interestingly, AMB639752 was also devoid of serotoninergic activity. The ability of both ritanserin and AMB639752, by inhibiting DGKα in intact cells, to restore restimulation induced cell death (RICD) in SAP deficient lymphocytes was also tested. Both compounds restored RICD at concentrations lower than the two previously available inhibitors, indicating their potential use for the treatment of X-linked lymphoproliferative disease 1 (XLP-1), a rare genetic disorder in which DGKα activity is deregulated.

Integrins in T Cell Physiology.

From the thymus to the peripheral lymph nodes, integrin-mediated interactions with neighbor cells and the extracellular matrix tune T cell behavior by organizing cytoskeletal remodeling and modulating receptor signaling. LFA-1 (αLß2 integrin) and VLA-4 (α4ß1 integrin) play a key role throughout the T cell lifecycle from thymocyte differentiation to lymphocyte extravasation and finally play a fundamental role in organizing immune synapse, providing an essential costimulatory signal for the T cell receptor. Apart from tuning T cell signaling, integrins also contribute to homing to specific target organs as exemplified by the importance of α4ß7 in maintaining the gut immune system. However, apart from those well-characterized examples, the physiological significance of the other integrin dimers expressed by T cells is far less understood. Thus, integrin-mediated cell-to-cell and cell-to-matrix interactions during the T cell lifespan still represent an open field of research.

Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is Involved in Cell Cycle Progression.

Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550-2557, 2017. © 2016 Wiley Periodicals, Inc.