Discovery and optimization of 4-pyrazolyl-2-aminopyrimidine derivatives as potent spleen tyrosine kinase (SYK) inhibitors.

Spleen tyrosine kinase (Syk) is a key signal transduction mediator of the B cell receptor (BCR) signaling pathway. Abnormal BCR signaling plays a key role in initiation and development of B-cell-derived hematological malignancies, therefore, Syk represents a potential target for inhibiting the BCR signaling resulting in a therapeutic effect in these cancers. Herein, we describe a novel series of SYK inhibitors with 4-(3'-pyrazolyl)-2-amino-pyrimidine scaffold. Extensive study of structure-activity relationships led to the identification of 1 (NMS-0963), a highly potent Syk inhibitor (IC50 = 3 nM) endowed with high selectivity within a panel of tested kinases and high antiproliferative activity in SYK-dependent BaF3-TEL/SYK cells and in other BCR-dependent hematological tumor cell lines. Additionally, 1 effectively inhibited Syk phosphorylation and downstream signaling mediators of the BCR in treated cells. In in vivo pharmacokinetics studies, 1, displayed good pharmacokinetics properties, with linear exposure with dose and excellent oral bioavailability. These findings suggest that 1 is a promising new Syk inhibitor for treating BCR-dependent hematological cancers.

Characterization of covalent inhibitors that disrupt the interaction between the tandem SH2 domains of SYK and FCER1G phospho-ITAM.

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM, however, these compounds lack selectivity and this limits their utility as chemical tools.

Apolipoprotein C3 (ApoC3) facilitates NLRP3 mediated pyroptosis of macrophages through mitochondrial damage by accelerating of the interaction between SCIMP and SYK pathway in acute lung injury.

Respiratory failure caused by severe acute lung injury (ALI) is the main cause of mortality in patients with COVID-19.This study aimed to investigate the effects and underlying biological mechanism of Apolipoprotein C3 (ApoC3) in ALI. To establish an in vivo model, C57BL/6 mice were exposed by lipopolysaccharide (LPS). For the in vitro model, murine bone marrow-derived macrophages (BMDMs) or RAW264.7 cells were stimulated with LPS + adenosine triphosphate (ATP). Serum levels of ApoC3 were found to be upregulated in patients with COVID-19 or pneumonia-induced ALI. Inhibition of ApoC3 reduced lung injury in an ALI model, while overexpression of ApoC3 promoted lung injury. ApoC3 induced mitochondrial damage-mediated pyroptosis in ALI through the activation of the NOD-like receptorprotein 3 (NLRP3) inflammasome. ApoC3 recombinant protein significantly increased SCIMP expression in the lung tissue of mice models with ALI. ApoC3 also facilitated the interaction between the SLP adapter and CSK-interacting membrane protein (SCIMP) protein and Spleen tyrosine kinase (SYK) protein in the ALI model. Moreover, ApoC3 accelerated calcium-dependent reactive oxygen species (ROS) production in the ALI model. The effects of ApoC3 on pyroptosis were mitigated by the use of a pyroptosis inhibitor or an ROS inhibitor in the ALI model. Furthermore, ApoC3 activated the expression of SYK, which in turn induced NLRP3 inflammasome-regulated pyroptosis in the ALI model. METTL3 was found to mediate the m6A mRNA expression of ApoC3. Overall, our study highlights the crucial role of ApoC3 in promoting macrophage pyroptosis in ALI through calcium-dependent ROS production and NLRP3 inflammasome activation via the SCIMP-SYK pathway, providing a potential therapeutic strategy for ALI and other inflammatory diseases.

An Update on the Emerging Role of Wnt/ß-catenin, SYK, PI3K/AKT, and GM-CSF Signaling Pathways in Rheumatoid Arthritis.

Rheumatoid arthritis is an untreatable autoimmune disorder. The disease is accompanied by joint impairment and anomalies, which negatively affect the patient's quality of life and contribute to a decline in manpower. To diagnose and treat rheumatoid arthritis, it is crucial to understand the abnormal signaling pathways that contribute to the disease. This understanding will help develop new rheumatoid arthritis-related intervention targets. Over the last few decades, researchers have given more attention to rheumatoid arthritis. The current review seeks to provide a detailed summary of rheumatoid arthritis, highlighting the basic description of the disease, past occurrences, the study of epidemiology, risk elements, and the process of disease progression, as well as the key scientific development of the disease condition and multiple signaling pathways and enumerating the most current advancements in discovering new rheumatoid arthritis signaling pathways and rheumatoid arthritis inhibitors. This review emphasizes the anti-rheumatoid effects of these inhibitors [for the Wnt/ß-catenin, Phosphoinositide 3-Kinases (PI3K/AKT), Spleen Tyrosine Kinase (SYK), and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) signaling pathways], illustrating their mechanism of action through a literature search, current therapies, and novel drugs under pre-clinical and clinical trials.

The selective inhibition of the Syk tyrosine kinase ameliorates experimental autoimmune arthritis.

Autoimmune arthritis - such as rheumatoid arthritis - affect a significant proportion of the population, which can cause everyday joint pain, decreased mobility and reduced quality of life. Despite having more and more therapeutic options available, there are still a lot of patients who cannot reach remission or low disease activity by current therapies. This causes an urgent need for the development of new treatment options. The Syk tyrosine kinase plays an essential role in B cell receptor, Fc receptor and integrin signaling. It has been shown that the hematopoietic cell-specific deletion of Syk resulted in a complete protection against autoantibody-induced experimental arthritis. This prompted us to test the effect of entospletinib, a second generation, Syk-selective inhibitor, which has a tolerable safety profile according to hematological clinical trials, in experimental autoimmune arthritis. We found that entospletinib dose-dependently decreased the macroscopic signs of joint inflammation, while it did not affect the health status of the animals. In line with these findings, local neutrophil accumulation and cytokine levels were reduced compared to the vehicle-treated group, while macrophage accumulation and synovial fibroblast numbers were not significantly altered. Meanwhile, entospletinib dose-dependently decreased the cell responses of immune complex- or integrin ligand-activated neutrophils. Overall, we found that selective Syk inhibition by entospletinib reduced the activity of autoantibody-induced experimental arthritis, which seems to be based mainly on the effect of the inhibitor on neutrophil functions. Our data raise the possibility that entospletinib could be a good drug candidate in the treatment of human autoimmune arthritis.

C-type lectin-like receptor-2 (CLEC-2) is a key regulator of kappa-carrageenan-induced tail thrombosis model in mice.

Kappa-carrageenan (KCG), which is used to induce thrombosis in laboratory animals for antithrombotic drug screening, can trigger platelet aggregation. However, the cell-surface receptor and related signaling pathways remain unclear. In this study, we investigated the molecular basis of KCG-induced platelet activation using light-transmittance aggregometry, flow cytometry, western blotting, and surface plasmon resonance assays using platelets from platelet receptor-deficient mice and recombinant proteins. KCG-induced tail thrombosis was also evaluated in mice lacking the platelet receptor. We found that KCG induces platelet aggregation with α-granule secretion, activated integrin αIIbß3, and phosphatidylserine exposure. As this aggregation was significantly inhibited by the Src family kinase inhibitor and spleen tyrosine kinase (Syk) inhibitor, a tyrosine kinase-dependent pathway is required. Platelets exposed to KCG exhibited intracellular tyrosine phosphorylation of Syk, linker activated T cells, and phospholipase C gamma 2. KCG-induced platelet aggregation was abolished in platelets from C-type lectin-like receptor-2 (CLEC-2)-deficient mice, but not in platelets pre-treated with glycoprotein VI-blocking antibody, JAQ1. Surface plasmon resonance assays showed a direct association between murine/human recombinant CLEC-2 and KCG. KCG-induced thrombosis and thrombocytopenia were significantly inhibited in CLEC-2-deficient mice. Our findings show that KCG induces platelet activation via CLEC-2.

Thrombosis is a serious medical condition that occurs when blood clots form in the blood vessels and can lead to heart attacks or strokes. Animal models are important for evaluating the effectiveness of drugs in thrombosis treatment. Kappa-carrageenan (KCG) is a food thickener and a substance used to induce clot formation in laboratory animals. In this study, we investigated the molecular basis of KCG-induced platelet activation, which is an important step in thrombosis development. We found that KCG activates platelets via a receptor called C-type lectin-like receptor 2 (CLEC-2), leading to a prothrombotic state in mice. We also showed that KCG-induced tail thrombosis (CTT) is significantly inhibited in CLEC-2 deficient mice. Our findings suggest that CLEC-2-mediated platelet activation plays a key role in the pathogenesis of thrombosis and CLEC-2 May participate in innate immunity as a receptor for sulfate-polysaccharide.Abbreviation; CLEC-2: C-type lectin-like receptor 2; CRP: collagen-related peptide; CTT: KCGN-induced tail thrombosis; DIC: disseminated intravascular coagulation; EDTA: ethylenediaminetetraacetic acid; GPVI: glycoprotein VI; HRP: horseradish peroxidase; KCG: Κ-Carrageenan; LAT: linker activated T cells; LDS: lithium dodecyl sulfate; LTA: light-transmittance aggregometry; MFI: mean fluorescence intensity; PFA: paraformaldehyde; PLCγ2: phospholipase C gamma 2; PS: phosphatidylserine; Syk: spleen tyrosine kinase; Co-HP: Cobalt-hematoporphyrin.

Human dermal fibroblast subpopulations and epithelial mesenchymal transition signals in hidradenitis suppurativa tunnels are normalized by spleen tyrosine kinase antagonism in vivo.

Hidradenitis Suppurativa is a chronic inflammatory disease of which the pathogenesis is incompletely understood. Dermal fibroblasts have been previously identified as a major source of inflammatory cytokines, however information pertaining to the characteristics of subpopulations of fibroblasts in HS remains unexplored. Using in silico-deconvolution of whole-tissue RNAseq, Nanostring gene expression panels and confirmatory immunohistochemistry we identified fibroblast subpopulations in HS tissue and their relationship to disease severity and lesion morphology. Gene signatures of SFRP2+ fibroblast subsets were increased in lesional tissue, with gene signatures of SFRP1+ fibroblast subsets decreased. SFRP2+ and CXCL12+ fibroblast numbers, measured by IHC, were increased in HS tissue, with greater numbers associated with epithelialized tunnels and Hurley Stage 3 disease. Pro-inflammatory CXCL12+ fibroblasts were also increased, with reductions in SFRP1+ fibroblasts compared to healthy controls. Evidence of Epithelial Mesenchymal Transition was seen via altered gene expression of SNAI2 and altered protein expression of ZEB1, TWIST1, Snail/Slug, E-Cadherin and N-Cadherin in HS lesional tissue. The greatest dysregulation of EMT associated proteins was seen in biopsies containing epithelialized tunnels. The use of the oral Spleen tyrosine Kinase inhibitor Fostamatinib significantly reduced expression of genes associated with chronic inflammation, fibroblast proliferation and migration suggesting a potential role for targeting fibroblast activity in HS.

Methiothepin downregulates SNAP-23 and inhibits degranulation of rat basophilic leukemia cells and mouse bone marrow-derived mast cells.

In the present study, we found that methiothepin (a nonselective 5-hydroxytryptamine [5-HT] receptor antagonist) inhibited antigen-induced degranulation in rat basophilic leukemia cells and mouse bone marrow-derived mast cells. Although antigen stimulation induces release of histamine and serotonin (5-HT) by exocytosis and mast cells express several types of 5-HT receptor, the detailed role of these receptors remains unclear. Here, pretreatment of cells with methiothepin attenuated increased intracellular Ca2+ concentration, phosphorylated critical upstream signaling components (Src family tyrosine kinases, Syk, and PLCγ1), and suppressed TNF-α secretion via inhibition of Akt (a Ser/Thr kinase activated by PI3K)and ERK phosphorylation. Furthermore, it inhibited PMA/ionomycin-induced degranulation; this finding suggested that methiothepin affected downstream signaling. IκB kinase ß phosphorylates synaptosomal associated protein 23, which regulates the fusion events of the secretory granule/plasma membrane after mast cell activation, resulting in degranulation. We showed that methiothepin blocked PMA/ionomycin-induced phosphorylation of synaptosomal associated protein 23 by inhibiting its interaction with IκB kinase ß. Together with the results of selective 5-HT antagonists, it is suggested that methiothepin inhibits mast cell degranulation by downregulating upstream signaling pathways and exocytotic fusion machinery through mainly 5-HT1A receptor. Our findings provide that 5-HT antagonists may be used to relieve allergic reactions.

Immunomodulatory role of spleen tyrosine kinase in chronic inflammatory and autoimmune diseases.

BACKGROUND: The high prevalence of chronic inflammatory diseases or autoimmune reactions is a major source of concern and affects the quality of life of patients. Chronic inflammatory or autoimmune diseases are associated with many diseases in humans, including asthma, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease and cancer. Splenic tyrosine kinase (SYK) is a non-receptor tyrosine kinase that plays an important role in immune receptor signalling in immune and inflammatory responses. METHODS: This is a review article in which we searched for keywords "splenic tyrosine kinase", "inflammation" and "autoimmune diseases" in published literature such as Pubmed and Web of Science to collect relevant information and then conducted a study focusing on the latest findings on the involvement of SYK in chronic inflammatory or autoimmune diseases. RESULTS: This paper reviews the regulation of Fcγ, NF-κB, B cell and T cell-related signalling pathways by SYK, which contributes to disease progression in chronic inflammatory and autoimmune diseases such as airway fibrosis, inflammatory skin disease and inflammatory bowel disease. CONCLUSION: This paper shows that SYK plays an important role in chronic inflammatory and autoimmune diseases. syk targets hematological, autoimmune and other inflammatory diseases and therefore, inhibition of SYK expression or blocking its related pathways may provide new ideas for clinical prevention and treatment of inflammatory or autoimmune diseases.

The orchestrating role of deteriorating neurons and TREM-1 in crosstalk with SYK in Alzheimer's disease progression and neuroinflammation.

Alzheimer's disease (AD) is a progressive type of neurodegenerative disease characterized by successive loss of the conventional structure and functions of neurons. In addition to dead neurons type detected within AD brain tissues, there are a predominantly varying number of deteriorating neurons (DTNs). As the number of deteriorating neurons increases, they exaggerate the release of inflammatory factors and oxidative stress that trigger the cascade of neuroinflammation. Triggering receptor expressed on myeloid cells 1 (TREM-1) which is a transmembrane immune receptor type regularly expressed by phagocytic cells, may act as a stimulating factor for neuroinflammation. Once TREM-1 is activated, it directly activates spleen tyrosine kinase (SYK) downstream signaling cascades, which can be considered an initiating phase for AD pathology and AD progression. Sequentially, SYK activates the pro-inflammatory microglia M1 phenotype which executes several inflammatory actions, leading to neurotoxicity. These released neurotoxins promote neuronal cell death, synaptic dysfunctions, and memory impairments. Thus, the current review outlines the direct etiological and pathologic features of Alzheimer's disease linked with deteriorating neurons, TREM-1, and SYK.

Phosphorylation of spleen tyrosine kinase at Y346 negatively regulates ITAM-mediated signaling and function in platelets.

Spleen tyrosine kinase (Syk) is expressed in a variety of hemopoietic cells. Upon phosphorylation of the platelet immunoreceptor-based activation motif of the glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor, both the tyrosine phosphorylation and activity of Syk are increased leading to downstream signaling events. Although it has been established that the activity of Syk is regulated by tyrosine phosphorylation, the specific roles of individual phosphorylation sites remain to be elucidated. We observed that Syk Y346 in mouse platelets was still phosphorylated when GPVI-induced Syk activity was inhibited. We then generated Syk Y346F mice and analyzed the effect this mutation exerts on platelet responses. Syk Y346F mice bred normally, and their blood cell count was unaltered. We did observe potentiation of GPVI-induced platelet aggregation and ATP secretion as well as increased phosphorylation of other tyrosines on Syk in the Syk Y346F mouse platelets when compared to WT littermates. This phenotype was specific for GPVI-dependent activation, since it was not seen when AYPGKF, a PAR4 agonist, or 2-MeSADP, a purinergic receptor agonist, was used to activate platelets. Despite a clear effect of Syk Y346F on GPVI-mediated signaling and cellular responses, there was no effect of this mutation on hemostasis as measured by tail-bleeding times, although the time to thrombus formation determined using the ferric chloride injury model was reduced. Thus, our results indicate a significant effect of Syk Y346F on platelet activation and responses in vitro and reveal its complex nature manifesting itself by the diversified translation of platelet activation into physiological responses.

TULA Proteins in Men, Mice, Hens, and Lice: Welcome to the Family.

The two members of the UBASH3/STS/TULA protein family have been shown to critically regulate key biological functions, including immunity and hemostasis, in mammalian biological systems. Negative regulation of signaling through immune receptor tyrosine-based activation motif (ITAM)- and hemITAM-bearing receptors mediated by Syk-family protein tyrosine kinases appears to be a major molecular mechanism of the down-regulatory effect of TULA-family proteins, which possess protein tyrosine phosphatase (PTP) activity. However, these proteins are likely to carry out some PTP-independent functions as well. Whereas the effects of TULA-family proteins overlap, their characteristics and their individual contributions to cellular regulation also demonstrate clearly distinct features. Protein structure, enzymatic activity, molecular mechanisms of regulation, and biological functions of TULA-family proteins are discussed in this review. In particular, the usefulness of the comparative analysis of TULA proteins in various metazoan taxa, for identifying potential roles of TULA-family proteins outside of their functions already established in mammalian systems, is examined.

Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets.

Bruton's tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI-Syk-Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis.

Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists.

CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands. Fluorescence correlation spectroscopy (FCS) was used to show that the multivalent nanobodies cluster CLEC-2 in the membrane and that clustering is reduced by inhibition of Syk. Strikingly, the tetravalent nanobody stimulated aggregation of human platelets, whereas the divalent nanobody was an antagonist. In contrast, in human CLEC-2 knock-in mouse platelets, the divalent nanobody stimulated aggregation. Mouse platelets express a higher level of CLEC-2 than human platelets. In line with this, the divalent nanobody was an agonist in high-expressing transfected DT40 cells and an antagonist in low-expressing cells. FCS, stepwise photobleaching and non-detergent membrane extraction show that CLEC-2 is a mixture of monomers and dimers, with the degree of dimerisation increasing with expression thereby favouring crosslinking of CLEC-2 dimers. These results identify ligand valency, receptor expression/dimerisation and Syk as variables that govern activation of CLEC-2 and suggest that divalent ligands should be considered as partial agonists.

IgG:FcγRIIb signals block effector programs of IgE:FcεRI-activated mast cells but spare survival pathways.

BACKGROUND: IgE-induced mast cell (MC) degranulation can be inhibited by IgG antibodies, signaling via FcγRIIb, but the effects of IgG on IgE-induced MC transcription are unknown. OBJECTIVE: We sought to assess inhibitory IgG:FcγRIIb effects on MC responses to IgE using complementary transcriptomic and functional approaches. METHODS: RNA sequencing was performed on bone marrow-derived MCs from wild-type and FcγRIIb-deficient mice to identify genes activated following IgE receptor crosslinking that were further modulated in the presence of antigen-specific IgG in an FcγRIIb-dependent fashion. Parallel analyses of signaling pathways and allergic responses in vivo were performed to assess the impact of these changes in gene expression. RESULTS: Rapid changes in the transcription of 879 genes occurred in MCs activated by IgE, peaking at 1 hour. Surprisingly, only 12% of these were altered by IgG signaling via FcγRIIb, including numerous transcripts involved in orchestrating type 2 responses linked to spleen tyrosine kinase signaling. Consistent with this finding, IgG suppressed IgE-induced phospho-intermediates in the spleen tyrosine kinase signaling pathway. In vivo studies confirmed that the IgG-mediated suppression of both systemic anaphylaxis and MC-driven tissue recruitment of inflammatory cells following allergen challenge was dependent on FcγRIIb. In contrast, genes in the STAT5a cell survival pathway were unaltered by IgG, and STAT5a phosphorylation increased after IgE-induced MC activation but was unaffected by IgG. CONCLUSIONS: Our findings indicate that inhibitory IgG:FcγRIIb signals block an IgE-induced proallergic program but spare a prosurvival program.

Human dendritic cell maturation induced by amorphous silica nanoparticles is Syk-dependent and triggered by lipid raft aggregation.

BACKGROUND: Synthetic amorphous silica nanoparticles (SAS-NPs) are widely employed in pharmaceutics, cosmetics, food and concretes. Workers and the general population are exposed daily via diverse routes of exposure. SAS-NPs are generally recognized as safe (GRAS) by the Food and Drug Administration, but because of their nanoscale size and extensive uses, a better assessment of their immunotoxicity is required. In the presence of immune "danger signals", dendritic cells (DCs) undergo a maturation process resulting in their migration to regional lymph nodes where they activate naive T-cells. We have previously shown that fumed silica pyrogenic SAS-NPs promote the two first steps of the adaptative immune response by triggering DC maturation and T-lymphocyte response, suggesting that SAS-NPs could behave as immune "danger signals". The present work aims to identify the mechanism and the signalling pathways involved in DC phenotype modifications provoked by pyrogenic SAS-NPs. As a pivotal intracellular signalling molecule whose phosphorylation is associated with DC maturation, we hypothesized that Spleen tyrosine kinase (Syk) may play a central role in SAS-NPs-induced DC response. RESULTS: In human monocyte-derived dendritic cells (moDCs) exposed to SAS-NPs, Syk inhibition prevented the induction of CD83 and CD86 marker expression. A significant decrease in T-cell proliferation and IFN-γ, IL-17F and IL-9 production was found in an allogeneic moDC:T-cell co-culture model. These results suggested that the activation of Syk was necessary for optimal co-stimulation of T-cells. Moreover, Syk phosphorylation, observed 30 min after SAS-NP exposure, occurred upstream of the c-Jun N-terminal kinase (JNK) Mitogen-activated protein kinases (MAPK) and was elicited by the Src family of protein tyrosine kinases. Our results also showed for the first time that SAS-NPs provoked aggregation of lipid rafts in moDCs and that MßCD-mediated raft destabilisation altered Syk activation. CONCLUSIONS: We showed that SAS-NPs could act as an immune danger signal in DCs through a Syk-dependent pathway. Our findings revealed an original mechanism whereby the interaction of SAS-NPs with DC membranes promoted aggregation of lipid rafts, leading to a Src kinase-initiated activation loop triggering Syk activation and functional DC maturation.

Luxeptinib interferes with LYN-mediated activation of SYK and modulates BCR signaling in lymphoma.

Luxeptinib (LUX) is a novel oral kinase inhibitor that inhibits FLT3 and also interferes with signaling from the BCR and cell surface TLRs, as well as activation of the NLRP3 inflammasome. Ongoing clinical trials are testing its activity in patients with lymphoma and AML. This study sought to refine understanding of how LUX modulates the earliest steps downstream of the BCR following its activation by anti-IgM in lymphoma cells in comparison to ibrutinib (IB). LUX decreased anti-IgM-induced phosphorylation of BTK at Y551 and Y223 but its ability to reduce phosphorylation of kinases further upstream suggests that BTK is not the primary target. LUX was more effective than IB at reducing both steady state and anti-IgM-induced phosphorylation of LYN and SYK. LUX decreased phosphorylation of SYK (Y525/Y526) and BLNK (Y96) which are necessary regulators of BTK activation. Further upstream, LUX blunted the anti-IgM-induced phosphorylation of LYN (Y397) whose activation is required for phosphorylation of SYK and BLNK. These results indicate that LUX is targeting autophosphorylation of LYN or a step further upstream of LYN in the cascade of signal generated by BCR and that it does so more effectively than IB. The fact that LUX has activity at or upstream of LYN is important because LYN is an essential signaling intermediate in multiple cellular signaling processes that regulate growth, differentiation, apoptosis, immunoregulation, migration and EMT in normal and cancer cells.

Epigenetic upregulation of spleen tyrosine kinase in cancer cells through p53-dependent downregulation of DNA methyltransferase.

Syk is a tumor suppressor gene in some solid tumors. Currently, it remains unknown how Syk gene hypermethylation is controlled by DNA methyltransferase (DNMT) and p53. In colorectal cancer HCT116 cells, we found that protein and mRNA levels of Syk were much higher in WT than in p53-/- cells. Both p53 inhibitor PFT-α and p53 silencing can reduce the protein and mRNA expression of Syk in WT cells, while DNMT inhibitor 5-Aza-2'-dC can increase Syk expression in p53-/- cells. Interestingly, the DNMT expression in p53-/- HCT116 cells was higher than that in WT cells. PFT-α can not only enhance Syk gene methylation but also increase DNMT1 protein and mRNA levels in WT HCT116 cells. In metastatic lung cancer cell lines A549 and PC9, which express WT p53 and gain function of p53, respectively, PFT-α can also downregulate Syk mRNA and protein expression. However, the Syk methylation level was increased by PFT-α in A549 but not in PC9 cells. Likewise, 5-Aza-2'-dC transcriptionally increased Syk gene expression in A549 cells, but not in PC9 cells. In summary methylation of Syk promoter requires DNMT1, and p53 can upregulate Syk expression via downregulation of DNMT1 at the transcriptional level.