Investigation of diacylglycerol kinase alpha role in lipid signalling during T cell activation

The diacylglycerol kinases (DGKs) are a conserved family of lipid enzymes able to convert diacylglycerol (DAG) into phosphatidic acid (PA). This reaction attenuates DAG intracellular levels, an important second messenger which regulates several signalling proteins. At the same time, also the product of DGK activity, PA, controls different cell functions such as cell growth, differentiation, and migration. Thus, DGK enzymes play a pivotal role within the cells, linking lipid metabolism to signalling functions. In T cells, DGKα and DGKζ isoforms play a major role as negative regulators of DAG-mediated T cell receptor (TCR) signalling, since their activity limits RasGRP1/Ras/MAPK pathway and interleukin 2 (IL-2) production. The aim of the research efforts described in this thesis is to explore the regulation of DGKα-mediated lipid signalling and the resulting biological effects during T cell activation. The project started from the observation that, upon T-cell receptor stimulation, the adaptor protein SAP mediates DGKα inhibition and this process is essential for a correct restimulation induced cell death (RICD) and cytokines production. However, the molecular pathway that links SAP to DGKα was poorly understood. To clarify the pathway and the mechanisms involved we explored the molecular mechanisms which lead to DGKα inhibition, focusing on the reported SAP interactors and specifically on the Wiskott-Aldrich syndrome protein (WASp). The results obtained demonstrate that WASp interacts with the DGKα recoverin homology domain through its homology 1 domain and this interaction leads to DGKα inhibition. In addition, the role of the adaptor protein NCK-1 and the small G protein CDC42 in this signalling was investigated, indicating an indirect WASp mediated role. A second research topic was the investigation of the DGKα promoted signalling, focusing on lipid binding and regulation by PA and other phospholipids on atypical PKCs, an important class of lipid regulated kinases. Finally, the biological relevance of WASp/DGKα was explored in the context of RICD and cytokine production in primary human T cells. While this pathway does not affect RICD, WASp silencing reduces IL-2 synthesis and DGKα inhibition restores this defect of WASp-deficient lymphocytes. In conclusion, we discovered a novel signalling pathway composed of SAP/CDC42 and NCK-1/WASp that, downstream to strong TCR stimulation, blocks DGKα activity resulting in a full cytokine response but minimally perturbs RICD. These findings could be relevant for the treatment not only of primary immunodeficiencies but also of other T cell hyporesponsive states such as anergy of tumour infiltrating lymphocytes

p130Cas/ BCAR1 and p140Cap/ SRCIN1 Adaptors: The Yin Yang in Breast Cancer?

p130Cas/BCAR1 is an adaptor protein devoid of any enzymatic or transcriptional activity, whose modular structure with various binding motifs, allows the formation of multi-protein signaling complexes. This results in the induction and/or maintenance of signaling pathways with pleiotropic effects on cell motility, cell adhesion, cytoskeleton remodeling, invasion, survival, and proliferation. Deregulation of p130Cas/BCAR1 adaptor protein has been extensively demonstrated in a variety of human cancers in which overexpression of p130Cas/BCAR1 correlates with increased malignancy. p140Cap (p130Cas associated protein), encoded by the SRCIN1 gene, has been discovered by affinity chromatography and mass spectrometry analysis of putative interactors of p130Cas. It came out that p140Cap associates with p130Cas not directly but through its interaction with the Src Kinase. p140Cap is highly expressed in neurons and to a lesser extent in epithelial tissues such as the mammary gland. Strikingly, in vivo and in vitro analysis identified its tumor suppressive role in breast cancer and in neuroblastoma, showing an inverse correlation between p140Cap expression in tumors and tumor progression. In this review, a synopsis of 15 years of research on the role of p130Cas/BCAR1 and p140Cap/SRCIN1 in breast cancer will be presented

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

Beyond Traditional Morphological Characterization of Lung Neuroendocrine Neoplasms: In Silico Study of Next-Generation Sequencing Mutations Analysis across the Four World Health Organization Defined Groups

Lung neuroendocrine neoplasms (LNENs) classes, as proposed by the World Health Organization 2015, do not provide properly prognostic and therapeutic indications. In fact, high-throughput molecular analysis, based on next-generation sequencing, identified novel molecular subgroups, associated with different genomic signatures, that could pave the way for alternative therapeutic approaches. The present review, coupled with in silico molecular analysis, could show the current genomic alterations state in actual LNENS groups. Interestingly our manuscript suggests that the molecular novelties could improve the LNENs therapeutics efficacy. In more detail, we reported the differences of gene alterations and mutational rate between LNENS, confirming the central pathogenetic role given by a different mutational rate in chromatin remodeling genes and tumor suppressors TP53-RB1. In conclusion, our results underlined that a further molecular layer is needed to improve the efficacy of LNENs medical treatment.Lung neuroendocrine neoplasms (LNENs) represent a rare and heterogeneous population of lung tumors. LNENs incidence rate has increased dramatically over the past 30 years. The current World Health Organization LNENs classification (WHO 2015), distinguished four LNENs prognostic categories, according to their morphology, necrosis amount and mitotic count: typical carcinoid (TC), atypical-carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC) and small cell lung cancer (SCLC). At present, due to their rarity and biological heterogeneity there is still no consensus on the best therapeutic approach. Next-generation-sequencing analysis showed that WHO 2015 LNENs classes, could be characterized also by specific molecular alterations: frequently mutated genes involving chromatin remodeling and generally characterized by low mutational burden (MB) are frequently detected in both TC and AC; otherwise, TP53 and RB1 tumor suppressor genes alterations and high MB are usually detected in LCNEC and SCLC. We provide an overview concerning gene mutations in each WHO 2015 LNENs class in order to report the current LNENs mutational status as potential tool to better understand their clinical outcome and to drive medical treatment

Prognostic features of gastro-entero-pancreatic neuroendocrine neoplasms in primary and metastatic sites: Grade, mesenteric tumour deposits and emerging novelties

Updates in classification of gastro-entero-pancreatic neuroendocrine neoplasms better reflect the biological characteristics of these tumours. In the present study, we analysed the characteristics of neuroendocrine tumours that could aid in a more precise stratification of risk groups. In addition, we have highlighted the importance of grade (re)assessment based on investigation of secondary tumour lesions. Two hundred and sixty-four cases of neuroendocrine tumours of gastro-entero-pancreatic origin from three centres were included in the study. Tumour morphology, mitotic count and Ki67 labelling index were evaluated in specimens of primary tumours, lymph node metastases and distant metastases. These variables were correlated with overall survival (OS) and relapse-free survival (RFS). Tumour stage, number of affected lymph nodes, presence of tumour deposits and synchronous/metachronous metastases were tested as possible prognostic features. Mitotic count, Ki-67 labelling index, primary tumour site, tumour stage, presence of tumour deposits and two or more affected lymph nodes were significant predictors of OS and RFS. At the same time, mitotic count and Ki-67 labelling index can be addressed as continuous variables determining prognosis. We observed a very high correlation between the measures of proliferative activity in primary and secondary tumour foci. The presence of isolated tumour deposits was identified as an important determinant of both RFS and OS for pancreatic (hazard ratio [HR] = 7.61, 95% confidence interval [CI] = 3.96-14.6, P < 0.0001 for RFS; HR = 3.28, 95% CI = 1.56-6.87, P = 0.0017 for OS) and ileal/jejunal neuroendocrine tumours (HR = 1.98, 95% CI = 1.25-3.13, P = 0.0036 for RFS and HR 2.59, 95% CI = 1.27-5.26, P = 0.009 for OS). The present study identifies the presence of mesenterial tumour deposits as an important prognostic factor for gastro-entero-pancreatic neuroendocrine tumours, provides evidence that proliferative parameters need to be treated as continuous variables and further supports the importance of grade determination in all available tumour foci

Transient Receptor Potential Vanilloid 1 Modulates Central Inflammation in Multiple Sclerosis

Introduction: Disease course of multiple sclerosis (MS) is negatively influenced by proinflammatory molecules released by activated T and B lymphocytes and local immune cells. The endovanilloid system plays different physiological functions, and preclinical data suggest that transient receptor potential vanilloid type 1 (TRPV1) could modulate neuroinflammation in this disorder.Methods: The effect of TRPV1 activation on the release of two main proinflammatory cytokines, tumor necrosis factor (TNF) and interleukin (IL)-6, was explored in activated microglial cells. Furthermore, in a group of 132 MS patients, the association between the cerebrospinal fluid (CSF) levels of TNF and IL-6 and a single nucleotide polymorphisms (SNP) influencing TRPV1 protein expression and function (rs222747) was assessed.Results: In in vitro experiments, TRPV1 stimulation by capsaicin significantly reduced TNF and IL-6 release by activated microglial cells. Moreover, the anti-inflammatory effect of TRPV1 activation was confirmed by another TRPV1 agonist, the resiniferatoxin (RTX), whose effects were significantly inhibited by the TRPV1 antagonist, 5-iodoresiniferatoxin (5-IRTX). Vice versa, BV2 pre-treatment with 5-IRTX increased the inflammatory response induced by LPS. Moreover, in MS patients, a significant association emerged between TRPV1 SNP rs222747 and CSF TNF levels. In particular, the presence of a G allele, known to result in increased TRPV1 protein expression and function, was associated to lower CSF levels of TNF.Conclusions: Our results indicate that TRPV1 influences central inflammation in MS by regulating cytokine release by activated microglial cells. The modulation of the endovanilloid system may represent a useful approach to contrast neuroinflammation in MS

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

BackgroundPhosphorylation 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.ResultsHerein, 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.ConclusionWe discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response

Lipocalin-2 promotes adipose-macrophage interactions to shape peripheral and central inflammatory responses in experimental autoimmune encephalomyelitis

Objective: Accumulating evidence suggests that dysfunctional adipose tissue (AT) plays a major role in the risk of developing multiple sclerosis (MS), the most common immune-mediated and demyelinating disease of the central nervous system. However, the contribution of adipose tissue to the etiology and progression of MS is still obscure. This study aimed at deciphering the responses of AT in experimental autoimmune encephalomyelitis (EAE), the best characterized animal model of MS. Results and methods: We observed a significant AT loss in EAE mice at the onset of disease, with a significant infiltration of M1-like macrophages and fibrosis in the AT, resembling a cachectic phenotype. Through an integrative and multilayered approach, we identified lipocalin2 (LCN2) as the key molecule released by dysfunctional adipocytes through redox-dependent mechanism. Adipose-derived LCN2 shapes the pro-inflammatory macrophage phenotype, and the genetic deficiency of LCN2 specifically in AT reduced weight loss as well as inflammatory macrophage infiltration in spinal cord in EAE mice. Mature adipocytes downregulating LCN2 reduced lipolytic response to inflammatory stimuli (e.g. TNFα) through an ATGL-mediated mechanism. Conclusions: Overall data highlighted a role LCN2 in exacerbating inflammatory phenotype in EAE model, suggesting a pathogenic role of dysfunctional AT in MS

Combined Large Cell Neuroendocrine Carcinomas of the Lung: Integrative Molecular Analysis Identifies Subtypes with Potential Therapeutic Implications

Combined large cell neuroendocrine carcinoma (CoLCNEC) is given by the association of LCNEC with adeno or squamous or any non-neuroendocrine carcinoma. Molecular bases of CoLCNEC pathogenesis are scant and no standardized therapies are defined